JP2010163435A - Electroprocessed collagen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a collagen material which can be used in forming a biocompatible graft which possesses structural perfection and maintains such perfection after implantation. <P>SOLUTION: Micropipettes 10 filled with a collagen-containing solution are arranged above a grounded target 11 and a wire 12 is placed in the solution to charge the solution in each pipette tip 13 to a high voltage, and a collagen stream 14 is directed towards the grounded target to produce an electroprocessed collagen. The electroprocessed collagen can be constituted or formed from, for example, natural collagen, collagen produced by genetic engineering or collagen modified by conservative amino acid substitutions or non-conservative amino acid substitutions or non-naturally occurring amino acid substitutions. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

[Field of the Invention]
The present invention relates to electroprocessed collagen, compositions comprising electroprocessed collagen, the use of electroprocessed collagen as an extracellular matrix for a number of functions, and the production and use of electroprocessed collagen Relates to the new method. Further, the present invention includes combining electroprocessed collagen and cells to form engineered tissue. Modified tissue can include specific organs, or synthetic products of “organ-like” tissue.

[Background of the invention]
There is a continuing need in biomedical sciences for biocompatible compositions that can be used in manufacturing devices for implantation in or on the body of an organism. Much of the traditional focus has been on the use of synthetic polymers. However, many such polymers suffer from the disadvantages associated with their chemical and structural differences from natural materials. Fibrous encapsulation, lack of cellular infiltration, and rejection are problems encountered by such grafts. Efforts to overcome these problems have focused in part on using biodegradable synthetic polymers as scaffolding to manipulate prosthesis constructs to improve biocompatibility. However, many such polymers suffer from the disadvantage of producing major degradation byproducts, which, when in close contact with individual cells, result in an inflammatory response, resulting in pH in the cellular microenvironment. Can be reduced. Therefore, when using biodegradable materials, a process must be performed to ensure that by-products are properly removed from the tissue-modified construct. Another problem is that the bioabsorbable structural material degrades over time, resulting in the structural weakness of the implant. Problems also remain with the fibrous encapsulation and lack of cellular infiltration.

To overcome the drawbacks associated with synthetic grafts, attention has been directed to the use of collagen grafts. Collagen is a protein family that is widely distributed throughout the body. This scaffold-forming material is one of the most prominent proteins present in animal tissues. Collagen is a basic structural element of most extracellular matrix and is therefore an important structural element of most tissues. There are several forms of collagen that are present in different tissues and organs.

To date, most efforts with collagen have been focused on the use of collagen gels or solid collagen constructs (eg, films). The problem with using these constructs is that they lack structural strength (similar to collagen gels) or lose strength after implantation. Harder collagen grafts are destroyed because their construction and orientation are different from those of natural tissue. The cells adapt the transplanted collagen to match normal extracellular matrix construction and fiber orientation. This process loses the post-implantation integrity of the structurally strong graft and eventually decays.

Thus, there is a need in the art for collagen materials that can be used to form biocompatible grafts that retain structural integrity and retain such integrity after implantation. Preferably, such materials should be able to mimic the chemistry and structure of the extracellular matrix and promote cell invasion.

[Summary of Invention]
The composition of the present invention is electroprocessed collagen. The present invention includes collagen that has been electroprocessed by any means. Electroprocessed collagen can be composed or formed of, for example, natural collagen, genetically engineered collagen, or collagen modified by conservative amino acid substitution, non-conservative amino acid substitution, or substitution with a non-naturally occurring amino acid. it can. The collagen used in electroprocessing can be derived from natural sources, can be produced synthetically, or can be produced by any other means. Numerous methods for producing collagen and other proteins are known in the art. Synthetic collagen can be prepared to contain a particular desired amino acid sequence. Electroprocessed collagen can also be formed from collagen itself or any other material that, when electroprocessed, forms a collagen structure. Examples include, but are not limited to, amino acids, peptides, modified peptides such as gelatin derived from modified collagen, polypeptides, and proteins. Collagen can be formed before, during or after electrical processing. Thus, electroprocessed collagen formed by combining procollagen with procollagen peptidase before, during, or after electroprocessing is within the scope of the present invention.

In some embodiments, the compositions of the present invention include additional electroprocessed materials. Other electroprocessed materials can include natural materials, synthetic materials, or combinations thereof. Some preferred examples of natural materials include amino acids, peptides, modified peptides such as gelatin derived from modified collagen, polypeptides, proteins, carbohydrates, lipids, nucleic acids, glycoproteins, lipoproteins, glycolipids, glucosaminoglycans , And proteoglycans. Some preferred synthetic matrix materials for electroprocessing with collagen include polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA, polycaprolactone, poly (ethylene-co-vinyl acetate), ( EVOH), poly (vinyl acetate) (PVA), polyethylene glycol (PEG), and poly (ethylene oxide) (PEO).

In many desirable embodiments, the electroprocessed collagen is combined with one or more substances. Such materials can include any type of molecule, cell, object, or combination thereof. The electroprocessed collagen composition of the present invention can further comprise one substance, or any combination of substances. Some particularly desirable embodiments can use any cell, including the use of cells as a material in combination with an electroprocessed collagen matrix. Cells that can be used include, but are not limited to, stem cells, committed stem cells, and differentiated cells. The molecules can exist in any phase or form, and combinations of molecules can be used. Examples of the types of desirable molecules that may be used include human or veterinary therapeutics, cosmetics, nutritional supplements, agricultural substances such as herbicides, pesticides and fertilizers, vitamins, amino acids, peptides, polypeptides, proteins Carbohydrates, lipids, nucleic acids, glycoproteins, lipoproteins, glycolipids, glycosaminoglycans, proteoglycans, growth factors, hormones, neurotransmitters, pheromones, calones, prostaglandins, immunoglobulins, monokines and other cytokines, wet Agents, metals, gases, plasticizers, minerals, ions, electrically and magnetically responsive materials, photosensitive materials, antioxidants, molecules that can be metabolized as cellular energy sources, antigens, and cellular or physiological responses Any molecule that can cause Examples of objects include cell fragments, cell debris, organelles, and other cellular components, extracellular matrix components, tablets, viruses, and vesicles, liposomes, capsules, nanoparticles, and inclusions for molecules Other structures that can be used include, but are not limited to: Magnetic or electrically reactive materials are also examples of substances that are optionally included within the compositions of the present invention. Examples of electroactive materials include, but are not limited to, carbon black or graphite, carbon nanotubes, and various suspensions of conductive polymers. Examples of magnetically active substances include, but are not limited to, ferrofluids (colloid suspensions of magnetic particles).

The present invention also includes a method of making the composition of the present invention. Methods for making the compositions include, but are not limited to, electrotreating collagen and optionally electrotreating other materials, substances or both. Using one or more electroprocessing techniques, such as electrospin, electrospray, electroaerosol, electrosputter, or any combination thereof, the present invention The electroprocessed collagen material and matrix may be made. In its most basic sense, an electroprocessing device for electroprocessing materials includes an electrodeposition mechanism and a target. In a preferred embodiment, the electrodeposition mechanism includes one or more reservoirs that hold one or more solutions to be electroprocessed or electrodeposited. The reservoir (s) have at least one opening, nozzle, or other means to allow solution flow from the reservoir. Electrical processing occurs due to the presence of charge at either the opening or the target, while others are grounded. The substrate can be used as a variable feature in electroprocessing the materials used to make the electroprocessed composition. Specifically, the target can be the actual substrate for the material used to make the electroprocessed matrix or is deposited on the electroprocessed matrix itself. Alternatively, the substrate can be placed between the target and the nozzle. Also, the target can specifically be charged along a preselected pattern or grounded so that the solution flowing from the opening is directed in a specific direction. The electric field can be controlled by a microprocessor to create an electroprocessed matrix having the desired structure. The target and nozzle (s) can be designed to be movable with respect to each other, thereby allowing further control over the structure of the electroprocessed matrix to be formed. The present invention makes it possible to form a matrix having a defined shape.

The method of the present invention involves pre-selecting a mold that is adapted to produce a defined shape, filling the mold with electroprocessed material, or placing material on the outer surface of the mold. Including electrodeposition. Further shaping can be achieved by manual processing of the formed matrix. For example, a plurality of formed matrices can be stitched, sealed, stapled, or otherwise attached to one another to form a desired shape. it can. The electroprocessed matrix can be ground into a powder or it can be ground and prepared as a hydrated gel composed of striped fibrils. Alternatively, the physical flexibility of many matrices allows the matrix to be manually shaped into the desired structure. The electroprocessed collagen can be further processed, for example, by cross-linking or shaping or placing it in a bioreactor for cell culture. In this way, cells can be grown on an electroprocessed matrix.

The compositions of the present invention also include a number of uses for electroprocessed collagen compositions. The compositions of the present invention have a number of potential uses. Uses include, but are not limited to, the manufacture of modified tissues and organs, including the following: patches of matrix material, plugs or structures such as tissue. These and other constructs can replenish cells or can be used without cell replenishment. Further uses include: prostheses and other implants; tissue scaffolding; wound repair or bandages; hemostatic devices; suture materials, adhesives, surgical and orthopedic screws, and surgical and orthopedic Devices or structures used for tissue repair and support such as plates; natural coatings or components for synthetic implants; cosmetic implants and supplements; repair or structural support for organs or tissues; material delivery; Bioengineering platforms; platforms for testing the effects of substances on cells; cell cultures; and numerous other uses.

Accordingly, it is an object of the present invention to overcome the aforementioned limitations and drawbacks by providing a composition comprising electroprocessed collagen.

A further object of the present invention is to provide a composition comprising electroprocessed collagen and other electroprocessed materials.

Another object of the present invention is to provide a composition comprising electroprocessed collagen and non-electroprocessed material.

It is a further object of the present invention to provide a composition comprising electroprocessed collagen and cells, molecules, objects, or combinations thereof.

Yet another object of the present invention is to provide an electroprocessed collagen composition in a matrix.

It is a further object of the present invention to provide a composition comprising an electroprocessed collagen matrix similar in structure and composition to the extracellular matrix.

Yet another object of the present invention is to provide a method of making a composition comprising electroprocessed collagen.

A further object of the present invention is to provide a construct comprising an electroprocessed collagen matrix.

Yet another object of the present invention is to provide bioengineered tissue comprising the constructs of the present invention.

A further object of the present invention is to provide a biomodified organ comprising the construct of the present invention.

Yet another object of the present invention is to provide a method of making the constructs of the present invention.

A further object of the present invention is to provide a method of making the construct of the present invention.

Another object of the present invention is to provide a substance delivery method.

It is a further object of the present invention to provide a method for testing and studying cells and tissues in vitro.

A further object of the invention provides methods for cell and tissue culture.

Another object of the present invention is to provide a biomechanical platform comprising the composition of the present invention.

These and other objects, features and advantages of the present invention will become apparent after reference to the following detailed description of the disclosed embodiments and the accompanying drawings.

SEM micrograph of type I / elastin 80:20 (size bar = 1 μm, magnification 4000 ×). SEM micrograph of electrospun I / III elastin 45:35:20 matrix (size bar = 1 μm, magnification 4,300). 1 is a schematic diagram of an embodiment of an electrical processing device including an electrical processing apparatus and a rotating wall bioreactor. FIG. 1 is a schematic diagram of an embodiment of an electrical processing device including an electrical processing apparatus and a rotating wall bioreactor. FIG. FIG. 3 is a schematic diagram of another embodiment of an electrical processing device including an electrical processing apparatus and a rotating wall bioreactor. An example of an electrospun matrix before cell seeding (left) and an arterial segment developed from scaffold formation (right). Representative release profile of VEGF from electrospun collagen. VEFG was co-electrospun at a concentration of collagen 0, 25, 50, or 100 ng / ml. The material was then immersed in PBS and the sample was isolated and subjected to ELISA. Release of VEGF from collagen electrospun and glutaraldehyde vapor fixed. VEFG was co-electrospun at a concentration of collagen 0, 25, 50, or 100 ng / ml. The electrospun material was subjected to glutaraldehyde vapor fixation for 15 minutes. The material was then immersed in PBS and the sample was isolated and subjected to ELISA. Transmission electron micrograph of electrospun type I collagen showing 67 nm stripe formation (white graticule bar indicates 100 nm). Electrospun 50:50 blend SEM of collagen type I and type II (human placenta). This matrix was electrospun from a single reservoir source composed of a mixture of type I and type III collagen suspended in HFIP (final protein concentration was equal to 0.06 g / ml). This heterogeneous network is composed of fibers having an average fiber diameter of 390 ± 290 nm (magnification: 4,300 times). SEM micrograph showing marked smooth muscle cell attachment and migration after 1 week of culture with collagen type I / elastin 80:20 matrix. Lumen surface of the prosthesis (scale size = 10 μm, magnification 4,300 times). Scanning electron micrograph of a fixed type II collagen matrix before sowing (scale size = 10 μm, magnification 1,600 times). Sectional drawing of the type II collagen mat 1 week after culture using the seeded chondrocytes. Nearly complete coverage on the seeding side (right) and some reconstruction in the mat (immediately left) can be seen (scale size = 10 μm, magnification 700 ×). Stent before collagen coating (scale size = 100 μm, magnification 43 times). Stent (scale size = 100 μm, magnification 55 times) after coating with collagen nanofibers (before cell seeding).

[Detailed Description of Preferred Embodiments]
Definitions The terms “electroprocessing” and “electrodeposition” refer to all methods of electrospinning, electrospraying, electroaerosol aerosoling, and electrosputtering of materials, combinations of two or more of such methods, and materials Broadly defined to encompass any other method that flows, sprays, sputters, or drops through an electric field and against a target. The electroprocessed material can be electroprocessed from one or more grounded reservoirs in the direction of the charged substrate or from a charged reservoir to a grounded target. “Electrospinning” refers to the process of forming fibers from a solution or melt by flowing a charged solution or melt from an opening. “Electrical aerosol treatment” means the process of forming droplets from a solution or melt by flowing a charged polymer solution or melt from an opening. The term electroprocessing is not limited to the particular embodiments described herein, and the term encompasses any means that uses an electric field to deposit material on a target.

The term “material” refers to any compound, molecule, substance, or group or combination thereof that forms any type of structure or group of structures during or after electrical processing. Materials include natural materials, synthetic materials, or combinations thereof. Naturally occurring organic materials include plants or other materials, regardless of whether the material is produced or can be produced, or whether the material is synthetically altered or can be altered. Any substance found naturally in the body of an organism can be mentioned. Synthetic materials include any material prepared by any means of artificial synthesis, processing, or manufacturing. Preferably the material is a biocompatible material.

One preferred type of material for electroprocessing for making the compositions of the present invention is protein. Extracellular matrix proteins are a preferred type of protein of the present invention. Examples include, but are not limited to collagen, fibrin, elastin, laminin, and fibronectin. A particularly preferred group of proteins in the present invention is any type of collagen. Further preferred materials are other components of the extracellular matrix, such as proteoglycans. In each case, their names, in their broadest definition, are used throughout this application to encompass various isoforms that are generally recognized to exist within different families of proteins and other molecules. There are multiple types of each of these naturally occurring proteins and molecules, as well as types that can be produced or manufactured synthetically, or can be produced or produced by genetic engineering. For example, collagen exists in many forms and types, and all of these types and subsets are encompassed by the present invention.

The term protein and any term used to define a particular protein or protein type further includes fragments, analogs, conservative amino acid substitutions, non-conservatives with respect to the protein or protein type or type. Amino acid substitutions and substitutions with non-naturally occurring amino acids are included, but are not limited to these. Thus, for example, the term collagen includes fragments, analogs, conservative amino acid substitutions, and substitutions with non-naturally occurring amino acids or residues for any type or type of collagen, It is not limited to. The term “residue” is used herein to refer to an amino acid (D or L) or amino acid mimetic that is incorporated into a protein by an amide bond. Thus, a residue may be a naturally occurring amino acid or, unless limited, a known analog of a natural amino acid that functions in a manner similar to a naturally occurring amino acid (ie, an amino acid mimetic). Can be included. In addition, amide bond mimetics include peptide backbone modifications known to those skilled in the art.

In addition, one of ordinary skill in the art can modify individual additions or deletions of a single amino acid or a small number of amino acids (preferably less than 10%, more preferably less than 1%) in the encoded sequence, as described above. It will be appreciated that substitutions, deletions or additions are conservatively altered changes in which this change results in a substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art. Each of the following six groups contains amino acids that are conservative substitutions for each other:
1) Alanine (A), Serine (S), Threonine (T),
2) Aspartic acid (D), glutamic acid (E),
3) Asparagine (N), glutamine (Q),
4) Arginine (R), lysine (K),
5) Isoleucine (I), leucine (L), methionine (M), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The term protein, polypeptide or peptide further encompasses fragments that may be 90-95% of the total amino acid sequence, and extensions of the entire amino acid sequence that are 5-10% longer than the amino acid sequence of the protein, polypeptide or peptide. It will be understood.

If the length of the peptide is relatively short (ie, less than about 50 amino acids), the peptide is often synthesized using standard scientific peptide synthesis techniques. Solid phase synthesis, in which the C-terminal amino acid of a sequence is attached to an insoluble support and then the remaining amino acids are sequentially added to the sequence, is a preferred method for chemical synthesis of antigenic epitopes as described herein. Techniques for solid phase synthesis are known to those skilled in the art.

Alternatively, proteins or peptides that can be electroprocessed are synthesized using recombinant nucleic acid methodology. In general, this involves creating a nucleic acid sequence that encodes a peptide or protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, and expressing the peptide or protein in a host; Isolating the expressed peptide or protein and optionally regenerating the peptide or protein. Techniques sufficient to guide one skilled in the art to such procedures are found in the literature.

Where some desired protein fragment or peptide is encoded by a nucleotide sequence incorporated into a vector, one skilled in the art will recognize that a protein fragment or peptide is a spacer molecule consisting of one or more amino acids (eg, a peptide It will be understood that they may be separated by In general, spacers are linked with the desired protein fragment or peptide, or only preserve some minimum distance or other spatial relationship between them, and have no specific biological activity. . However, the constituent amino acids of the spacer may be selected to affect some properties of the molecule such as folding, net charge, or hydrophobicity. Nucleotide sequences that encode for the production of residues that may be useful in the purification of the expressed recombinant protein may be constructed in the vector. Such sequences are known in the art. For example, a nucleotide sequence encoding a polyhistidine sequence may be added to the vector to facilitate purification of the expressed recombinant protein on a nickel column.

Once expressed, recombinant peptides, polypeptides, and proteins can be purified according to standard procedures known to those skilled in the art including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. A substantially pure composition with about 50-99% uniformity is preferred, with 80-95% or higher uniformity being most preferred for use as a therapeutic agent.

Also, molecules capable of forming some of the designated proteins can be mixed with other polymers during electroprocessing to obtain the desired properties for use of the forming protein in the matrix.

Another type of synthetic material, preferably a biocompatible synthetic material, includes a polymer. Such polymers include the following: poly (urethane), poly (siloxane) or silicone, poly (ethylene), poly (vinyl pyrrolidone), poly (2-hydroxyethyl methacrylate), poly (N-vinyl pyrrolidone), poly (Methyl methacrylate), poly (vinyl alcohol), poly (acrylic acid), polyacrylamide, poly (ethylene-co-vinyl acetate), poly (ethylene glycol), poly (methacrylic acid), polylactide (PLA), polyglycolide (PGA), poly (lactide-co-glycolide) (PLGA), polyanhydrides, and polyorthoesters, or any other similar synthetic polymer that can be developed that is biocompatible, It is not limited to. The term “biocompatible synthetic polymer” also encompasses copolymers and blends, and generally any other combination of the aforementioned polymers together or with other polymers. The use of these polymers depends on the specific application and the required specifications. A more detailed description of these polymers and polymer types can be found in Brannon-Peppas, Lisa, “Polymer”.
in Controlled Drug Delivery, "Medical Plastics and Biomaterials, November 1997, which is hereby incorporated by reference as if fully set forth herein.

“Material” also encompasses electroprocessed materials that can be converted to different materials during or after electroprocessing. For example, procollagen forms collagen when combined with procollagen peptidase. Procollagen, procollagen peptidase, and collagen are all within the definition of materials. Similarly, the protein fibrinogen forms fibrin when combined with thrombin. The fibrinogen or thrombin that is electroprocessed, as well as the fibrin that is subsequently formed, are included within the definition of materials.

In a preferred embodiment, the electroprocessed material forms a matrix. The term “matrix” refers to any structure composed of electroprocessed material. The matrix is composed of fibers, or droplets of material, or a blend of fibers and droplets of any size or shape. A matrix is a single structure or group of structures and can be formed by one or more one or more electroprocessing methods using one or more one or more materials. The matrix is designed to possess a certain porosity. The material may be deposited within the matrix, or may be anchored or placed on the matrix. A cell is a substance that can be deposited in or on a matrix.

The term “substance” is used throughout this application in its broadest definition. The term substance encompasses one or more molecules, objects, or cells of any type or size, or combinations thereof. The substance can be in any form including, but not limited to, a solid, semi-solid, wet or dry mixture, gas, solution, suspension, combinations thereof. Substances include molecules of any size and any combination of molecules. Cells include all types of prokaryotic and eukaryotic cells, whether in the natural state or altered by genetic engineering or any other process. The cells can be derived from natural sources or can be cultured in vitro and can be alive or dead. A combination of different types of cells can be used. The object can be of any size, shape, and composition that can be combined with or bonded to the electroprocessed material. Examples of objects include cell fragments, cell fragments, cell wall fragments, extracellular matrix constituents, virus wall fragments, organelles, and other cell components, tablets, viruses, vesicles, liposomes, capsules, nanoparticles As well as other structures that act as inclusions for molecules. The composition of the present invention may comprise one substance or any combination of substances.

Throughout this application, the term “solution” is used to describe a liquid in a reservoir of an electroprocessing method. The term is broadly defined to encompass any liquid that contains a material to be electroprocessed. It will be understood that any solution capable of forming a material during electroprocessing is within the scope of the present invention. In this application, the term “solution” also refers to a suspension or emulsion containing the material or something to be electroprocessed. “Solutions” can be in organic or biocompatible form. This broad definition is appropriate in terms of a number of solvents or other liquids and carrier molecules (eg, poly (ethylene oxide) (PEO)) that can be used in many variations in electroprocessing. In this application, the term “solution” also refers to the material to be electroprocessed, the melt containing the substance or something, the hydrated gel and the suspension.

Any solvent that allows delivery of the material or substance to the solvent opening or syringe tip or other site where the substance is electroprocessed may be used. The solvent can be used to dissolve or suspend the material or substance to be electroprocessed. Solvents useful for dissolving or suspending the material or substance depend on the substance or material. Electrospinning techniques often require more specific solvent conditions. For example, collagen is water, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol (also known as hexafluoroisopropanol or HFIP) or Can be electrodeposited as a solution or suspension in a combination. Fibrin monomers can be electrodeposited or electrospun from solvents such as urea, monochloroacetic acid, water, 2,2,2-trifluoroethanol, HFIP, or combinations thereof. Elastin can be electrodeposited as a solution or suspension in water, 2,2,2-trifluoroethanol, isopropanol, HFIP, or combinations thereof such as isopropanol and water. In one desirable embodiment, elastin is electrospun from a solution of 70% isopropanol and 30% water containing 250 mg / ml elastin. Other lower alcohols, particularly halogenated alcohols, may be used. Other solvents that may be used in electroprocessing natural matrix materials or that may be combined with other solvents include acetamide, N-methylformamide, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) ), Dimethylacetamide, N-methylpyrrolidone (NMP), acetic acid, trifluoroacetic acid, ethyl acetate, acetonitrile, trifluoroacetic anhydride, 1,1,1-trifluoroacetone, maleic acid, hexafluoroacetone.

Proteins and peptides associated with membranes are often hydrophobic and cannot be readily dissolved in aqueous solutions. Such proteins can be dissolved in organic solvents such as methanol, chloroform, and trifluoroethanol (TFE), and emulsifiers. Any other solvent known to those skilled in the art of protein chemistry may be used, such as those useful in chromatography, particularly high performance liquid chromatography. Proteins and peptides are also soluble in, for example, HFIP, hexafluoroacetone, chloroalcohol combined with aqueous mineral acid, dimethylacetamide containing 5% lithium chloride, and highly diluted acids such as acetic acid, hydrochloric acid and formic acid It is. N-methylmorpholine N-oxide is another solvent that can be used with many polypeptides. Other examples used alone or in combination with organic acids or salts include: triethanolamine, dichloromethane, methylene chloride, 1,4-dioxane, acetonitrile, ethylene glycol, diethylene glycol, ethyl acetate, glycerin, Examples include propane-1,3-diol, furan, tetrahydrofuran, indole, piperazine, pyrrole, pyrrolidone, 2-pyrrolidone, pyridine, quinoline, tetrahydroquinoline, pyrazole, and imidazole. Combinations of solvents can also be used.

Synthetic polymers include, for example, HFIP, methylene chloride, ethyl acetate, acetone, 2-butanone (methyl ethyl ketone), diethyl ether, ethanol, cyclohexane, water, dichloromethane (methylene chloride), tetrahydrofuran, dimethyl sulfoxide (DMSO), acetonitrile, methyl formate And can be electrodeposited from various solvent mixtures. HFIP and methylene chloride are desirable solvents. The choice of solvent depends on the properties of the synthetic polymer to be electrodeposited.

The choice of solvent is somewhat based on consideration of the secondary forces that stabilize the polymer-polymer interactions and the ability of the solvent to replace these interactions with strong polymer-solvent interactions. In the case of polypeptides such as collagen, and in the absence of covalent cross-linking, the basic secondary forces between chains are (1) due to the attractive forces of fixed charges on the backbone and depend on the primary structure. Coulomb forces (eg, lysine and arginine residues will be positively charged at physiological pH and aspartic acid or glutamic acid will be negatively charged), (2) due to permanent dipole interactions Polypeptides due to dipole-dipoles (hydrogen bonds commonly found in polypeptides are the strongest of such interactions), and (3) non-polar species with a low tendency to interact favorably with polar water molecules It is a hydrophobic interaction resulting from the association of nonpolar regions. Thus, a solvent or combination of solvents that can advantageously compete with these interactions can dissolve or disperse the polypeptide. For example, HFIP and TFE possess a highly polar OH bond adjacent to a very hydrophobic fluorinated region. Although not wishing to be bound by the following theory, it is believed that the alcohol moiety can hydrogen bond with the peptide and also solvate the charge on the backbone, thus reducing intermolecular Coulomb interactions. It is done. Furthermore, the hydrophobic portion of these solvents can interact with hydrophobic domains in the polypeptide, helping to resist the tendency of the polypeptide to aggregate via hydrophobic interactions. Furthermore, solvents such as HFIP and TFE are thought to compete less for intramolecular hydrogen bonds that stabilize secondary structures such as α-helices due to their lower total polarity compared to water. Therefore, the α-helix in these solvents is thought to be stabilized by stronger intramolecular hydrogen bonds. Stabilization of polypeptide secondary structure in these solvents may be desirable to protect the proper formation of collagen fibrils during electroprocessing, especially in the case of collagen and elastin.

Furthermore, it is often desirable but not necessary to have a relatively high vapor pressure to promote stabilization of the electrospinning jet to create fibers as the solvent evaporates. Relatively volatile solvents are also desirable for electrospray processing to minimize droplet coalescence during and after spray processing and formation of dry electroprocessed material. In embodiments involving high boiling solvents, it is desirable to promote solvent evaporation by warming the spinning or spray processing solution, and optionally the electroprocessing vapor itself, or by electroprocessing under reduced pressure. There is a case. It is also possible that the creation of a stable jet that produces fibers is facilitated by the low surface tension of the polymer / solvent mixture. Solvent selection can also be guided by this consideration.

In functional terms, the solvent used for electroprocessing plays a major role in creating a mixture with the collagen and / or other materials to be electroprocessed so that the electroprocessing is feasible. The concentration of a given solvent is often an important consideration in determining the type of electroprocessing to be performed. For example, in an electrospray process, the solvent should facilitate a dispersion of droplets of the electroprocessed material so that the initial jet of liquid breaks up into droplets. Thus, the solvent used in the electrospray process should not create the power to stabilize the unrestricted liquid column. In electrospinning, interactions between molecules of electroprocessed material stabilize the jet and cause fiber formation. For electrical span embodiments, the solvent should sufficiently dissolve or disperse the polymer to prevent the jet from breaking up and forming droplets, thereby forming a stable jet in fiber form. Should be possible. In some embodiments, the transition from electrospraying to electrospinning can be determined by examining viscosity measurements (using a Brookfield viscometer) on the polymer solution as a function of concentration. As the concentration of the polymer or other material to be electroprocessed increases, the viscosity increases. However, above the critical concentration, which involves entanglement of the material's extended chains, Brookfield viscosity increases more rapidly with concentration, as opposed to increasing more slowly and linearly at lower concentrations. Deviations from linearity are roughly consistent with the transition from electrospray processing to electrospinning.

The solubility of any electroprocessed material in the solvent may be increased by modifying the material. Any method of modifying the material to increase the solubility of the material may be used. For example, US Pat. No. 4,164,559 (Miyata et al.) Discloses a method of chemically modifying collagen to increase solubility.

Composition of the Invention Electroprocessed Collagen The composition of the present invention comprises electroprocessed collagen. The present invention includes collagen that has been electroprocessed by any means. Electroprocessed collagen can be composed or formed from any collagen within the full meaning of the term described above in the definition of “protein”. Thus, collagen can include collagen fragments, analogs, conservative amino acid substitutions, non-conservative amino acid substitutions, and substitutions with non-naturally occurring amino acids or residues for any type or type of collagen. . The collagen used in electroprocessing can be derived from natural sources, can be produced synthetically, can be produced by genetic engineering, or produced by any other means or combinations thereof can do. Natural sources include, but are not limited to, collagen produced in or contained within tissues of living organisms. For example, the electroprocessed collagen to be implanted into the matrix can include, but is not limited to, autologous collagen, allogeneic collagen, or collagen from another species. Some collagens that can be used include collagen type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, XIII Type, XIV type, XV type, XVI type, XVII type, XVIII type, and XIX type, but are not limited thereto. Some preferred collagens include type I and type II. Synthetic collagen can also include synthetic collagen produced by any artificial means. Numerous methods for producing collagen and other proteins are known in the art. Synthetic collagen can be prepared using specific sequences. For example, genetically engineered collagen can be prepared with specific desired amino acid sequences that differ from natural collagen. Modified collagen may be produced by any means including, for example, peptide, polypeptide, or protein synthesis. For example, cells can be genetically engineered in vivo or in vitro to produce collagen, molecules capable of forming collagen, or collagen subdomains, and collect desired collagen. . In one exemplary embodiment, a desired sequence that forms a binding site on a collagen protein for a cell or peptide can be included in an amount higher than that found in native collagen. The electroprocessed collagen may also be formed from the collagen itself or any other material that forms a collagen structure upon electroprocessing. Examples include, but are not limited to, amino acids, peptides, modified peptides such as gelatin derived from modified collagen, polypeptides, and proteins. Collagen can be formed before, during or after electrical processing. For example, electroprocessed collagen formed by combining procollagen with procollagen peptidase before, during or after electroprocessing is within the scope of the present invention.

The electroprocessed collagen is made using any electroprocessing technique including, but not limited to, electrospinning techniques, electroaerosol techniques, electrospray processing techniques, or electrosputtering techniques, or any combination thereof. Can be done. Thus, electroprocessed droplets Electroprocessed droplets, particles, fibers, fibrils, or combinations thereof are all included in the electroprocessed collagen composition of the present invention. In one desirable embodiment, the collagen is electrospun to form collagen fibers. In some embodiments, the electrospun collagen has a repetitive striped pattern when examined with an electron microscope. This striped pattern is characteristic of collagen fibrils produced by cells in the extracellular matrix. In some embodiments, the collagen type I, type II and / or type III stripes have a spacing of about 65-67 mm, a natural pattern characteristic of these collagens. In some embodiments, the collagen type I, type II and / or type III striped pattern is about 67 nm. In a preferred embodiment, the striped pattern characteristic of electrospun collagen is an important attribute because it brings the cells closer to the active site within the collagen molecule that promotes or regulates the activity of the property. In other embodiments, including some embodiments that include electroprocessed modified collagen derived from gelatin, there is no characteristic horizontal stripe pattern. In some embodiments, the denatured collagen is electrospun into a fiber structure lacking a horizontal stripe pattern.

Several desirable sequences can be incorporated into synthetic collagen. Any sequence that can be incorporated into a collagen molecule may be used. For example, the P-15 site, a 15 amino acid sequence within the collagen molecule, promotes osteoblasts to produce and secrete hydroxyapatite, a bone component. Another example of a specific site and sequence within a collagen molecule that can be manipulated and processed in a similar manner is the RGD binding site characteristic of integrin molecules. The RGD site is a sequence of three amino acids (Arg-Gly-Asp) present in many extracellular matrix materials that act as binding sites for cell adhesion. RGD sites are recognized and bound by, for example, integrins. Furthermore, the electroprocessed collagen can enrich certain desired sequences before, during, or after electroprocessing. The array can be added in a linear or other form. In some embodiments, the RGD sequence is arranged in a cyclic form called cycloRGD.

Other electroprocessed materials In some embodiments, the electroprocessed collagen composition contains additional electroprocessed materials. As defined above, other electroprocessed materials can include natural materials, synthetic materials, or combinations thereof. Examples include amino acids, peptides, modified peptides such as gelatin derived from modified collagen, polypeptides, proteins, carbohydrates, lipids, nucleic acids, glycoproteins, minerals, lipoproteins, glycolipids, glucosaminoglycans, and proteoglycans. However, it is not limited to these.

Some preferred materials for electroprocessing with collagen are naturally occurring extracellular matrix materials and blends of naturally occurring extracellular matrix materials, such as fibrin, elastin, laminin, fibronectin, hyaluronic acid, Examples include, but are not limited to, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparin sulfate, heparin, and keratan sulfate, and proteoglycan. These materials may be produced or isolated by any means including isolation from humans or other animals or cells, or synthetic production. Some particularly preferred natural matrix materials for combination with electroprocessed collagen are fibrin, elastin and fibronectin. For example, FIG. 1 is a scanning electron micrograph of an electrospun matrix of type I collagen / elastin (80:20). FIG. 2 is a scanning electron micrograph of an electrospun matrix of type I collagen / type III collagen / elastin (55:35:20). Tissues, crude extracts of extracellular matrix material, non-natural tissues or extracts of extracellular matrix material are also included, alone or in combination. Extracts of biological material, including but not limited to cells, tissues, organs and tumors, may also be electroprocessed.

It will be appreciated that these electroprocessed materials may be combined with other materials and / or substances in forming the compositions of the present invention. For example, the electroprocessed peptide may be combined with an adjuvant to enhance immunogenicity when implanted subcutaneously. As another example, an electroprocessed collagen matrix containing cells may be combined with electroprocessed biocompatible polymers and growth factors to stimulate cell growth and division in the collagen matrix.

Synthetic electroprocessed materials include any material prepared by any method of artificial synthesis, processing, or manufacturing. The synthetic material is preferably biocompatible for administration in vivo or in vitro. Such polymers include the following: poly (urethane), poly (siloxane) or silicone, poly (ethylene), poly (vinyl pyrrolidone), poly (2-hydroxyethyl methacrylate), poly (N-vinyl pyrrolidone), poly (Methyl methacrylate), poly (vinyl alcohol), poly (acrylic acid), polyacrylamide, poly (ethylene-co-vinyl acetate), poly (ethylene glycol), synthetic polycations (eg, poly (ethyleneimine) )), Synthetic polyanions (eg, poly (styrene sulfonate) and poly (methacrylic acid)), poly (methacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA), poly (lactide-co-glycolide) ) (PLGA), Niro , Polyamides, polyanhydrides, poly (ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly (vinyl acetate) (PVA), poly (ethylene oxide) (PEO), and polyorthoesters, or biocompatible Include, but are not limited to, any other similar synthetic polymer that can be developed. Some preferred synthetic matrix materials include PLA, PGA, PLA and PGA copolymers, polycaprolactone, poly (ethylene-co-vinyl acetate), EVOH, PVA, and PEO. The matrix may be formed from electrospun fibers, electroaerosols, electrosprayed or electrosputtered droplets, or combinations of the above.

In embodiments using natural materials, these materials can be derived from natural sources, can be produced synthetically, or can be produced by genetically engineered cells. For example, genetically engineered proteins can be prepared with specific desired amino acid sequences that differ from the native protein.

By selecting various materials, or combinations thereof, for combination with electroprocessed collagen, many properties of the electroprocessed material can be manipulated. The properties of a matrix composed of electroprocessed collagen may be prepared. Electroprocessed collagen and other electroprocessed materials can provide a therapeutic effect when applied. Furthermore, the choice of matrix material can affect the persistence of the implanted matrix. For example, a matrix made from fibrin degrades rapidly, while a matrix made from collagen is more durable and a synthetic matrix material is more durable. The use of a matrix made from natural materials such as proteins also minimizes rejection or immunological response to the implanted matrix. Thus, the choice of materials for electroprocessing and use in substance delivery is influenced by the desired use. In one embodiment, a transdermal patch of electroprocessed fibrin or collagen combined with a healing promoter, analgesic and / or anesthetic, and anti-rejection agent may be applied to the skin and subsequently dissolved into the skin. Good. In another embodiment, an electroprocessed collagen graft for delivery to bone may be constructed from materials useful for promoting bone growth, osteoblasts and hydroxyapatite, so that it will last for a long time. You may design it. In embodiments where the matrix contains a substance to be released from the matrix, the release of the substance from the electroprocessed composition is adjusted by incorporating an electroprocessed composition such as a biocompatible substance. Can do. For example, layered or laminated structures can be used to control the substance release profile. Non-lamellar structures can also be used, where release is controlled by the relative stability of each component of the construct. For example, a layered structure composed of alternating electroprocessed materials can be prepared by sequentially electroprocessing different materials on the target. The outer layer can be adapted to dissolve faster or slower than for the inner layer, for example. Multiple agents can be delivered by this method, optionally at different release rates. The layers can be adapted to provide a complex multi-kinetic release profile of a single agent over time. Using the combination described above provides for the release of multiple release materials, each released with a complex profile.

Structure layering is used in some embodiments to more closely mimic the composition of natural materials. For example, manipulating the amount of type I collagen, type II collagen, and elastin in the continuous layer can be used to mimic distribution gradients or other patterns across the depth of structures such as vessel walls. Used in the embodiment. Other embodiments achieve such a pattern without layering. For example, by changing the supply rate of type I collagen, type II collagen, and elastin to the electric processing apparatus during the electric processing, it is possible to create a continuous gradient and pattern without layering.

Synthetic materials can be electroprocessed from various solvents. This can be important for the delivery of some materials. In some embodiments, an agent that is insoluble in the solvent used to electroprocess collagen is soluble in the solvent used to electroprocess the synthetic material. In some embodiments, the composite is used to increase the number of materials that can be combined with the electroprocessed collagen matrix. The polymer may be derivatized in such a way as to provide such sensitivity. These properties provide flexibility in making and using electroprocessed materials designed to deliver a variety of substances in vivo and in vitro.

An electroprocessed collagen composition such as a matrix can also be composed of specific subdomains of a matrix component and can be prepared with a synthetic backbone that can be derivatized. For example, RGD peptide sequences, and / or heparin binding domains and / or other sequences can be chemically linked to the synthetic material. Synthetic polymers having binding sequence (s) can be electroprocessed with collagen into a construct. This results in a matrix with unique properties. In these examples, the RGD site provides a cellular site for binding to and interacting with a synthetic component of the matrix. The heparin binding site can be engineered to be used as a site for scaffolding peptide growth factors for the synthetic backbone. Angiogenic peptides, genetic material, growth factors, cytokines, enzymes, and drugs are examples of other non-limiting materials that can be attached to the backbone of the electroprocessed material to provide functionality. Another embodiment of a matrix material having a therapeutic effect is electroprocessed fibrin. Fibrin matrix material helps stop bleeding. Peptide side chains can also be used to attach molecules to functional groups on the polymer backbone. Molecules and other materials can be bound to the material to be electroprocessed by techniques known in the art.

Materials Combined with Electroprocessed Collagen Composition In many desirable embodiments, the electroprocessed collagen is combined with one or more one or more materials. As mentioned above, the word “substance” in the present invention is used in its broadest definition. In embodiments where the electroprocessed collagen composition of the present invention includes one or more substances, the substances can include any type or size of molecule, cell, object, or combinations thereof. The composition of the present invention may comprise one or more substances, or any combination of substances.

A preferred embodiment includes cells as a material in combination with an electroprocessed collagen matrix. Any cell can be used. Some preferred examples include, but are not limited to, stem cells, committed stem cells, and differentiated cells. Examples of stem cells that can be used include, but are not limited to, embryonic stem cells, bone marrow stem cells, and umbilical cord stem cells. Other examples of cells used in various embodiments include osteoblasts, myoblasts, neuroblasts, fibroblasts, glioblasts, germ cells, hepatocytes, chondrocytes, keratinocytes, smooth muscle Examples include, but are not limited to, cells, cardiomyocytes, connective tissue cells, glial cells, epithelial cells, endothelial cells, hormone secreting cells, immune system cells, and neurons. In some embodiments, since many types of stem cells can be induced to differentiate in an organ-specific pattern once delivered to a given organ, the type of stem cell to be used can be pre-selected. It is unnecessary. For example, stem cells delivered to the liver can be induced to become liver cells simply by being placed within the biochemical environment of the liver. The cells in the matrix may be suitable for the purpose of providing scaffolding or inoculation, the purpose of producing a particular compound, or both.

Embodiments in which the substance includes cells include cells that can be cultured in vitro, derived from natural sources, genetically engineered, or produced by any other means. Any natural source of prokaryotic or eukaryotic cells may be used. Embodiments in which the matrix is implanted into an organism can use cells from the recipient, cells from allogeneic or different species, or bacterial or microbial cells. Also included are cells collected from a source and cultured prior to use.

In some embodiments, genetically engineered cells are used. Manipulation includes programming a cell to express one or more genes, showing expression of one or more genes, or both. An example of a genetically engineered cell useful in the present invention is a genetically engineered cell that produces and secretes one or more desired molecules. When an electroprocessed collagen matrix containing genetically engineered cells is implanted into an organism, the molecules produced can produce local or systemic effects and include the molecules identified above as possible substances. it can. Cells can also produce antigenic material in embodiments where one of the uses of the matrix is to generate an immune response. The cells have the following non-comprehensive list of objectives: inhibiting or stimulating inflammation, promoting healing, resisting immune rejection, providing hormone replacement, replacing neurotransmitters, cancer Inhibit or destroy cells, promote cell growth, inhibit or stimulate angiogenesis, augment tissue, and the following tissues, neurons, skin, synovial fluid, tendons, cartilage, ligaments, bones, Substances can be produced to help replenish or augment muscles, organs, dura mater, blood vessels, bone marrow, and extracellular matrix.

Genetic manipulation can include, for example, adding genetic material to a cell or removing genetic material from a cell, altering existing genetic material, or both. Embodiments in which cells are transfected or otherwise engineered to express a gene use a transiently transfected gene, a permanently transfected gene, or both Can do. The gene sequence may be full length or partial length and may be cloned or naturally occurring.

In embodiments where the substance is a molecule, any molecule can be used. Molecules can be, for example, organic or inorganic, and can be solid, semi-solid, liquid, or gas phase. Molecules may be present in combination or mixture with other molecules and may be in solution, suspension, or any other form. Examples of types of molecules that can be used include human or veterinary therapeutics, cosmetics, nutritional supplements, agricultural substances such as herbicides, pesticides and fertilizers, vitamins, salts, electrolytes, amino acids, peptides, Polypeptide, protein, carbohydrate, lipid, nucleic acid, glycoprotein, lipoprotein, glycolipid, glycosaminoglycan, proteoglycan, growth factor, hormone, neurotransmitter, pheromone, callone, prostaglandin, immunoglobulin, monokine and others Cytokines, wetting agents, metals, gases, minerals, plasticizers, ions, electrically and magnetically responsive materials, photosensitive materials, antioxidants, molecules that can be metabolized as cellular energy sources, antigens, and cellular responses Or any molecule that can cause a physiological response. Any combination of molecules can be used, as well as agonists or antagonists of these molecules.

Some preferred embodiments include the use of any therapeutic molecule, including but not limited to any pharmaceutical agent or drug. Examples of pharmaceuticals include anesthetics, hypnotics, sedatives and sleep inducers, antipsychotics, antidepressants, antiallergic drugs, antianginal drugs, antiarthritic drugs, asthma drugs, antidiabetic drugs, diarrhea Antispasmodic, anticonvulsant, anti-ventilant, antihistamine, antipruritic, antiemetic, antiemetic, antispasmodic, appetite suppressant, neurostimulant, neurotransmitter agonist, antagonist, receptor blocker, and Reuptake regulator, beta-adrenergic blocker, calcium channel blocker, disulfiram and disulfiram-like drug, muscle relaxant, analgesic, antipyretic, stimulant, anticholinesterase, parasympathomimetic, hormone, anticoagulant, Antithrombotic, thrombolytic, immunoglobulin, immunosuppressant, hormone agonist / antagonist, vitamin, antibacterial, antitumor, antacid, digestive, laxative, laxative, antiseptic , Diuretics, antiseptics, antifungals, parasite eradications, anthelmintics, heavy metals, heavy metal antagonists, chelants, gases and vapors, alkaloids, salts, ions, otachoids, digitalis, cardiac glycosides, antiarrhythmic drugs, Antihypertensive, vasodilator, vasoconstrictor, antimuscarinic, ganglion stimulant, ganglion blocker, neuromuscular blocker, adrenergic nerve inhibitor, antioxidant, vitamin, cosmetics, anti-inflammatory drug, Wound healing products, antithrombogenic agents, antitumor agents, antiangiogenic agents, anesthetics, antigenic agents, wound healing agents, plant extracts, growth factors, emollients, wetting agents, rejection / anti-rejection agents, Examples include spermicides, conditioners, antibacterial agents, antifungal agents, antiviral agents, antibiotics, tranquilizers, cholesterol-reducing agents, antitussives, histamine blockers, monoamine oxidase inhibitors Including, but not limited to, the use of any therapeutic molecule. All substances listed in the United States Pharmacopeia are also encompassed within the scope of the substances of the present invention.

Another preferred embodiment involves the use of growth factors. Growth factors useful in the present invention include transforming growth factor-α (“TGF-α”), transforming growth factor-β (“TGF-β”), platelet-derived growth factor (AA, AB and BB isoforms). ("PDGF"), fibroblast growth factor ("FGF") (including FGF acidic isoforms 1 and 2, FGF basic type 2, and FGF4, 8, 9, and 10), nerve growth factor (“NGF”) (including NGF2.5s, NGF7.0s, and βNGF and neurotrophin), brain-derived neurotrophic factor, cartilage-derived factor, bone growth factor (BGF), basic fibroblast growth factor, Insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), insulin-like growth factor (IGF) ) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factor (TGF) (TGFα, β, β1, β2, (including β3), skeletal growth factors, bone matrix-derived growth factors, and bone-derived growth factors, and mixtures thereof.

The cytokines useful in the present invention include cardiotrophin, stromal cell-derived factor, macrophage-derived chemokine (MDC), melanoma growth stimulating activity (MGSA), macrophage inflammatory protein 1α (MIP-1α), 2 3α, 3β, 4 and 5, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL -11, IL-12, IL-13, TNF-α, and TNF-β include, but are not limited to. Immunoglobulins useful in the present invention include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGF (nerve growth factor), PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

Other molecules useful as substances in the present invention include growth hormone, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor α and β, endostatin, angiostatin, thrombospondin, osteogenic protein-1. Bone morphogenic proteins 2 and 7, osteonectin, somatomedin-like peptide, osteocalcin, interferon alpha, interferon alpha A, interferon beta, interferon gamma, interferon 1 alpha, and interleukins 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 15, 16, 17 and 18 are not limited thereto.

Embodiments encompassing amino acids, peptides, polypeptides, and proteins can include any type of such molecules having any size and complexity and combinations of such molecules. Examples include, but are not limited to, structural proteins, enzymes and peptide hormones. These compounds can serve various functions. In some embodiments, the matrix may contain peptides containing sequences that inhibit enzyme activity due to competition for the active site. In other applications, antigenic agents that promote immune responses and induce immunity can be incorporated into the construct.

In the case of substances such as nucleic acids, any nucleic acid may be present. Examples include, but are not limited to, deoxyribonucleic acid (DNA), ent-DNA, and ribonucleic acid (RNA). Embodiments that include DNA include, but are not limited to, cDNA sequences, natural DNA sequences from any source, and sense or antisense oligonucleotides. For example, the DNA can be naked (eg, US Pat. Nos. 5,580,859, 5,910,488), can form a complex, or can be encapsulated (eg, US (Patent Nos. 5,908,777 and 5,787,567). The DNA can be present in any type of vector, such as a viral vector or a plasmid vector. In some embodiments, the nucleic acid used serves to promote or inhibit expression of the gene in the cell, inside and / or outside the electroprocessed matrix. The nucleic acid can be in any form effective to enhance cellular uptake.

Substances in the electroprocessed collagen composition of the present invention also include objects. Examples of objects include cell fragments, cell debris, organelles, and other cell components, tablets, and viruses, and other structures that act as inclusions for vesicles, liposomes, capsules, nanoparticles, molecules However, it is not limited to these. In some embodiments, the objects are vesicles, liposomes, capsules, or other inclusions containing compounds that are released at some point after electroprocessing (eg, upon implantation or subsequent stimulation or interaction). is there. In one exemplary embodiment, a transfection agent, such as a liposome, contains the desired nucleotide sequence incorporated into cells that are placed in or on the electroprocessed material or matrix. In other embodiments, cell fragments, specific cell fractions or cell debris are incorporated into the matrix. The presence of cell fragments is known to promote healing in some tissues.

Magnetic or electrically responsive materials are also examples of substances optionally included within the electroprocessed collagen composition of the present invention. Examples of electroactive materials include, but are not limited to, ferrofluids (colloid suspensions of magnetic particles) and various suspensions of conductive polymers. Ferrofluid containing particles having a diameter of about 10 nm, polymer-encapsulated magnetic particles having a diameter of about 1-2 microns, and polymers having glass transition temperatures below room temperature are particularly useful. Examples of electroactive materials include conductive polymers such as polyaniline and polypyrrole, ion conductive polymers such as sulfonated polyacrylamide as related materials, and carbon black, graphite, carbon nanotubes, metal particles, and metal films. Polymers including but not limited to electrical conductors such as plastics or ceramic materials.

In other embodiments, some substances in the electroprocessed collagen material or matrix supplement or enhance the function of other substances. For example, if the composition includes cells that express a particular gene, the composition can contain oligonucleotides that are taken up by the cells and affect gene expression in the cells. Fibronectin is optionally incorporated into the matrix to increase the cellular uptake of the oligonucleotide by pinocytosis.

As detailed above, the electroprocessed material itself can provide a therapeutic effect. Accordingly, the present invention encompasses embodiments that include methods that provide a therapeutic effect by delivering electroprocessed material to a location without incorporating additional substances into the electroprocessed material. Embodiments in which the matrix material alone is delivered, as well as embodiments in which other substances are included in the matrix, are within the scope of the invention.

Stability and Storage of Electroprocessed Collagen Composition The stability of the electroprocessed collagen composition of the present invention, including electroprocessed collagen, also allows for long-term storage of the composition between formulation and use And Stability allows greater flexibility for the user in embodiments where the material is applied after forming a material that has been electroprocessed, for example by dipping and spraying. The formed electroprocessed matrix can be manufactured and stored, and the exact material composition added for a particular application can be prepared and adapted to specific needs immediately prior to implantation or application. This attribute allows the user greater flexibility in both treatment options and inventory management. In many embodiments, the electroprocessed collagen is dry once it is electroprocessed and is essentially dehydrated, thereby facilitating storage in a dry or frozen state. Furthermore, the electroprocessed collagen composition is substantially sterile upon completion, thereby providing further advantages in therapeutic and cosmetic applications.

The storage conditions for the electroprocessed collagen composition of the present invention will depend on the electroprocessed materials and substances in the composition. For example, in embodiments comprising proteins, it is necessary or desirable to store the composition at a temperature below 0 ° C., under vacuum, or under lyophilized conditions. Other storage conditions can be used, for example, at room temperature, in the dark, under vacuum or reduced pressure, under an inert atmosphere, at freezer temperature, in aqueous or other liquid solutions, or in powder form. One skilled in the art can understand the appropriate storage conditions for the materials and substances contained in the composition and select appropriate storage conditions.

The electroprocessed collagen compositions of the present invention, and formulations containing these compositions, may be sterilized by conventional means known to those skilled in the art. Such means include, but are not limited to, filtration, irradiation, and exposure to bactericidal chemical agents such as peracetic acid or ethylene oxide gas. In embodiments where the application of heat does not denature the collagen, heat may be used. The compositions of the invention may also be combined with bacteriostatic agents such as thimerosal to inhibit bacterial growth.

Formulations comprising the electroprocessed collagen composition of the present invention may be provided in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be provided with a sterile liquid carrier (eg, injectable distillation) just prior to use. It may be stored in a freeze-dried (freeze-dried) state that requires only the addition of (water). Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by those skilled in the art. Preferred unit dosage formulations are those containing a dose or unit of the administered ingredient, or an appropriate fraction thereof. In addition to the ingredients specifically described above, it should be understood that the formulations of the present invention may include other agents commonly used by those skilled in the art.

The electroprocessed collagen composition of the present invention can be packaged in a variety of ways, depending on the method used to administer the composition. In general, a distribution article includes a container containing a composition or formulation comprising the composition in a suitable form. Suitable containers are known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders and the like. The container may also include a tamper proof assembly to prevent indiscriminate access to the contents of the package. In addition, the container has a label on the container that describes the contents of the container. The label may also include appropriate attention.

Other Features of the Electroprocessed Collagen Composition The electroprocessed collagen composition of the present invention has many beneficial features. Some features allow for use as a graft within the body of an organism or as a replacement for body tissue or organ. In many preferred embodiments, the electroprocessed material forms a matrix, preferably a matrix similar to the extracellular matrix. For example, the type of collagen selected can be based on similarity to the tissue into which the composition is implanted, or in the case of a prosthesis, the type of tissue, structure or organ is replaced, repaired, or augmented. In such embodiments, the electroprocessed material is combined with other extracellular matrix to more closely mimic the tissue. Such combinations can be made before, during or after the formation of the matrix. Some extracellular material is electroprocessed into the matrix along with collagen or formed by other means. In some embodiments, once the matrix is manufactured, the matrix material is added to the electroprocessed collagen.

The electroprocessed collagen compositions of the present invention have many features that make them suitable for the formation of extracellular matrix. Electrospun fibril structure and stripe formation are similar to naturally occurring collagen. The density and structure of the matrix formed by this method is superior to that achieved by known methods and is similar to that of the natural extracellular matrix.

In embodiments involving electrospun collagen, fibers are produced that are significantly smaller in diameter than can be produced by known manufacturing processes. Electrospun collagen fibers have been observed to have cross-sectional diameters ranging from a few microns to less than 100 nanometers. Electrospun fiber diameter can be manipulated, for example, by changing the composition (in terms of collagen source and type, as well as blending with other materials) and the concentration of collagen and other materials to be electrospun can do. In some embodiments, the addition and removal of molecules that prepare fiber formation or affect fiber formation can be added to manipulate collagen fiber formation. For example, many proteoglycans are known to modulate fiber formation (including affecting fiber diameter). A wide range of fiber diameters can be achieved. Some examples of electrospun fiber diameters in various embodiments include type I collagen with individual filament diameters of 100-730 nm, type I collagen fibers with an average diameter of 100 ± 40 nm, Type II collagen fibers with an average diameter of 0 μm, Type II collagen fibers with an average diameter of 3 ± 2.5 microns, Type III collagen production fibers with an average diameter of 250 ± 150 nm, I with an average diameter of 390 ± 290 nm Electrospun blends of type I and type III collagen producing fibers, and blends of type I collagen / type III collagen / elastin (45:35:20) having a diameter of 800 ± 700 nm. In many embodiments, the electrospun material is formed as a continuous fiber, so that the spun material does not show free end evidence in microscopy.

The use of an electrospun collagen matrix in the graft promotes cell infiltration of the graft. Indeed, constructs comprising the matrix of the present invention show a trend with respect to cell migration that has not been previously known to be achievable with the transplanted construct. It is known that the pore size in the graft affects the device / material interaction with the surrounding host tissue. For porous structures, the interaction depends on the size, size distribution, and continuity of the pores in the device structure. It has previously been thought that the pore size must be greater than about 10 microns in order for cells to be able to migrate to, through or through the structure. However, it has been observed that this restriction does not apply to grafts composed of electrospun nanofibers of at least some natural proteins. In one embodiment, significant cell migration occurred to electrospun collagen / elastin with an average pore size of 3.7 microns. Infiltration can also be achieved with grafts having smaller pore sizes. The pore size of the electroprocessed collagen matrix can be easily controlled by changing the concentration of the solution (and hence the fiber size) by controlling the process parameters, for example by controlling the fiber deposition rate and the movement of the mandrel by the electrolytic strength. Can be operated. Porosity can also be manipulated by mixing porogenic materials such as salts or other extractants, which will leave a defined size of pores in the matrix. If desired, the degree to which cells infiltrate the matrix can be controlled by the amount of cross-linking present in the matrix. Highly cross-linked matrix material is not as rapidly infiltrated as a matrix with a low degree of cross-linking. Adding synthetic material to the matrix can also limit the degree to which cells infiltrate the material.

Electroprocessed collagen has the additional advantage of having higher structural strength than known collagen grafts and maintaining structural strength after implantation. The electroprocessed matrix has a higher structural integrity than the collagen gel used in current implants. The electroprocessed matrix is also less sensitive to post-implantation remodeling and resorption than known collagen matrix techniques. Furthermore, the present invention encompasses a method for controlling the degree to which electroprocessed collagen is resorbed. In some embodiments, electrospun collagen can be resorbed very rapidly in a period of 7-10 days or less. In other embodiments, features such as extensive cross-linking of collagen fibrils are used to allow the matrix to be very stable and to last for months to years. Cross-linking changes also provide an additional ability to mimic natural tissue. Natural collagen in the body exhibits varying degrees of cross-linking and biological stability. The degree of crosslinking with natural collagen can vary as a function of age, physiological condition, and in response to various disease processes.

The ability to combine the electroprocessed collagen composition with other electroprocessed materials provides a number of additional advantages. Preparing a composition or construct comprising electroprocessed collagen and additional electroprocessed extracellular matrix material can further enhance the ability to mimic the extracellular matrix. In some preferred embodiments, the electroprocessed collagen is in a relative amount suitable to mimic the composition of the extracellular matrix material, fibrin, elastin, laminin, fibronectin, integrin, hyaluronic acid, chondroitin 4-sulfate. , Chondroitin 6-sulfate, dermatan sulfate, heparin sulfate, heparin, and keratan sulfate, and electroprocessed materials such as proteoglycans. Where appropriate, substances containing extracellular material can be prepared by means other than electroprocessing and combined with electroprocessed collagen. In some embodiments, multiple crude extracts of collagen isolated from connective tissue can be electroprocessed. In such embodiments, the matrix contains various structural and regulatory elements that may be required to promote activities such as healing, regeneration, and cell differentiation.

Other electroprocessed materials can be included in the matrix to provide other matrix properties. One example is the ability to control the persistence and biodegradation of the transplant matrix. Fibrin as a matrix material tends to degrade faster than collagen when implanted, while some synthetic polymers tend to degrade more slowly. Controlling the relative content of these materials affects the rate at which the matrix degrades. As another example, a material may be included to increase the sensitivity of a matrix, or a construct formed from a matrix, to applying heat sealing, chemical sealing, and mechanical pressure, or combinations thereof. . It has been observed that the inclusion of synthetic polymers increases the ability of the matrix to be cauterized or heat sealed. Inclusion of an electrically or magnetically reactive polymer in the matrix material is another example. In some embodiments, such polymers provide a piezoelectric effect or prepare a conductive matrix that changes the shape, porosity and / or density of the electroprocessed material in response to an electric or magnetic field. Used to. Another example is the use of matrix materials that are known to have therapeutic effects. For example, the fibrin matrix helps stop bleeding. Fibrin is a component of the transient matrix that occurs during early stage healing and can promote the growth of vasculature in adjacent regions, and in many other ways is a natural healing promoter.

The ability to incorporate substances into the electroprocessed composition allows for further benefits. One such benefit is a more stringent tissue mimicry and better graft biocompatibility. In some preferred embodiments, stem cells, committed stem cells that differentiate into the desired cell type, or desired type of differentiated cells are incorporated to more closely mimic the tissue. Furthermore, methods that can be used to encapsulate cells with electroprocessed collagen or otherwise combine cells with electroprocessed materials are more in the matrix than can be achieved by known methods. This results in a large cell density. This density is further increased by the improved cell invasion described above.

The ability of the compositions of the present invention to mimic natural materials minimizes the risk of immune rejection of the implanted matrix. For example, a self-system material can be used. However, the close resemblance to natural materials has made it possible to avoid an immune response, even in some embodiments using dissimilar materials. For example, electrospun bovine collagen type I cylinders (25 mm long and 2 mm wide) implanted in the rat lateral vastus muscle showed no immune response for 7-10 days. A similar construct composed of electrospun electrospun type I collagen was supplemented with myoblasts and transplanted. Similar results showed no evidence of inflammation or rejection and the grafts were dense. Furthermore, in some embodiments of the matrix, it has been observed to avoid graft encapsulation by recipient tissue, a common problem with grafts. In embodiments where encapsulation is desired, the matrix structure is modified to promote inflammation and encapsulation.

Substances that can provide suitable matrix properties include drugs and other substances that can have a therapeutic or physiological effect on cells and tissues in and around the graft. Any material can be used. Many embodiments include substances in the electroprocessed collagen matrix that improve the performance of the electroprocessed matrix being implanted. Examples of substances that can be used include, but are not limited to, peptide growth factors, antibiotics, and / or anti-rejection drugs. Chemicals that affect cell function, such as oligonucleotides, promoters, cell adhesion inhibitors, hormones, and growth factors are further examples of substances that can be incorporated into electroprocessed collagen. Release of those substances from the material can provide a means to control the expression or other function of cells in the electroprocessed material. Alternatively, cells that have been modified to produce the desired compound can be included. The entire construct is cultured, for example, in a bioreactor or conventional culture, or placed directly in vivo. For example, angiogenesis can be stimulated by angiogenic factors and growth promoting factors administered as peptides, proteins or as gene therapy. Angiogenic agents can be incorporated into the electroprocessed collagen matrix. Alternatively, if angiogenesis is not desired, an angiogenic material such as angiostatin may be included in the electroprocessed collagen matrix. Nerve growth factor can be electrospun into a collagen matrix that has been electroprocessed to promote the growth of neurons into the matrix and tissue. In a degradable electroprocessed collagen matrix, progressive degradation / disintegration of the matrix will release these factors and accelerate the growth of the desired tissue. In order to regulate cell differentiation in the matrix, substances can be incorporated into the electroprocessed collagen matrix. Oligonucleotide and peptide drugs such as retinoic acid are examples of such substances. Oligonucleotide DNA or messenger RNA sequences encoding specific proteins in the sense and antisense orientation can also be used. For example, if protein expression is desired, the sense oligonucleotide can be provided for cellular uptake and expression. Antisense oligonucleotides can be released, for example, to suppress the expression of a given gene sequence. The implant can be designed such that the substance affects cells contained within the matrix, outside the matrix, or both.

There are several methods for studying and quantifying certain properties of the matrices of the present invention. Fiber diameter and pore size (porosity) for a collagen-based matrix can be determined by SEM micrographs digitized and analyzed, for example, by UTHSCSA ImageTool 2.0 (NIH Shareware). Water permeability, a property different from porosity, can also be studied using standard methods. Atomic force microscopy is also
It can be used to create a three-dimensional image of the surface topography of a biological specimen over a wide range of temperatures in an ambient liquid or gaseous environment. This tool makes it possible to determine the relationships and interactions between matrix components. Examples of construct composition analysis include histology analysis for determining the degree of cell distribution in the construct interstitial space. To facilitate this analysis, cells may be stained using any known cell staining technique (eg, hematoxylin and eosin, and Masson tricolor). Cell proliferation activity is determined by, for example, labeling cells biosynthetically with a label that is incorporated into cells that actively undergo DNA synthesis (eg, using bromodeoxyuridine) and determining the extent to which the cells undergo nuclear division. Can be studied by using anti-labeled antibodies. Cell density can be determined, for example, by measuring the amount of DNA in an enzyme digested sample using known techniques. The degree of degradation or remodeling of the collagen matrix by the cells can be determined, for example, by measuring the expression and activity of matrix metalloproteinases from the cells. One method for measuring the functionality of cells in an electroprocessed collagen matrix is by measuring the properties of various physiological indicators of the tissue. For example, to determine the contractility of the construct, muscle cells may be stimulated with an electrical signal or attacked with a chemical agent or agent (eg, carbachol). Cell function in endocrine constructs can be determined by measuring the desired hormone production. Those skilled in the art will appreciate that the above list is not exhaustive and that many parameters and indicators can be used in characterizing tissues and matrices using existing methods.

Method of making an electroprocessed collagen composition A method of making an electroprocessed collagen composition includes electrotreating collagen and optionally electrotreating other materials, substances, or both. Including, but not limited to. As defined above, electroprocessing in the compositions of the present invention using one or more electroprocessing techniques such as electrospinning, electrospraying, electroaerosol, electrosputtering, or any combination thereof. Collagen and matrix may be made. In its most basic sense, an electroprocessing device for electroprocessing materials includes an electrodeposition mechanism and a target substrate. The electrodeposition mechanism includes a reservoir or reservoirs that hold one or more solutions to be electroprocessed or electrodeposited. The reservoir (s) have at least one opening or nozzle to allow solution flow from the reservoir. The terms “opening” and “nozzle” are used throughout, but these terms are not intended to be limiting and collectively refer to places where the solution can flow during electrical processing. One or more nozzles may be disposed in the electroprocessing device. Where there are multiple nozzles, each nozzle is attached to one or more reservoirs containing the same or different solutions. Similarly, there can be a single nozzle connected to multiple reservoirs containing the same or different solutions. Multiple nozzles can be connected to a single reservoir. Because various embodiments include single or multiple nozzles and / or reservoirs, any references herein to one or more nozzles or reservoirs are single nozzles, reservoirs, and related devices. As well as embodiments including multiple nozzles, reservoirs, and related devices. The size of the nozzle can be varied to provide an increased or decreased flow of solution from the nozzle. One or more pumps used in connection with the reservoir can be used to control the flow of solution flowing from the reservoir to the nozzle (s). The pump can be programmed to increase or decrease flow at various points during electrical processing. The present invention does not necessarily require a pump, but provides a useful way to control the rate at which material is delivered to the processing electric field. The material can be actively delivered to the electric field as a preformed aerosol using a device such as an airbrush, thereby increasing the electrodeposition rate and providing a new combination of materials. The nozzles may be programmed to deliver material simultaneously or sequentially.

Electrical processing occurs due to the presence of charge at either the opening or the target, the others are grounded. In some embodiments, it is known that the nozzle or opening is charged and the target is grounded. Those skilled in the electroprocessing arts will recognize that the nozzle and solution can be grounded and the target can be charged. The creation of an electric field that forms an electroprocessed composition, and the effect of the electric field on the electroprocessed material or substance occurs whether the charge is found in solution or at the grounded target. In different embodiments, the space between the target and the nozzle or material source can contain air or a selective gas. In various embodiments, the space can be maintained under vacuum or below atmospheric pressure or above normal atmospheric pressure. Solvents used in electroprocessing usually evaporate during the process. This is considered preferred because it ensures that the electroprocessed material is dry. In embodiments using water or other less volatile solvents, the electroprocessing may optionally be performed in a vacuum or other controlled atmosphere (eg, an atmosphere containing ammonia) to facilitate evaporation. Good. The electroprocessing can be oriented in various directions with respect to gravity or can be performed in a weightless environment.

The substrate can be used as a variable feature in the electroprocessing of materials used to make the electroprocessed composition. Specifically, the target can be the actual substrate for the material used to make the electroprocessed matrix or is deposited on the electroprocessed matrix itself. Alternatively, the substrate can be placed between the target and the nozzle. For example, a Petri dish can be placed between the nozzle and the target, and the matrix can be formed in the dish. Other variations include, but are not limited to, a non-stick surface between the nozzle and the target and a tissue or surgical area between the target and the nozzle. Also, the target can specifically be charged along a preselected pattern or grounded so that the solution flowing from the opening is directed in a specific direction. The electric field can be controlled by a microprocessor to create an electroprocessed matrix having the desired structure. The target and nozzle (s) can be designed to be movable with respect to each other, thereby allowing further control over the structure of the electroprocessed matrix to be formed. The entire process can be controlled by a microprocessor programmed with specific parameters to obtain a specific preselected electroprocessed matrix. It will be appreciated that any electroprocessing technique may be used alone or in combination with another electroprocessing technique to make the compositions of the present invention.

Forms of electroprocessed collagen include, but are not limited to, suspensions or solutions, gelatin, microparticle suspensions, or pretreated collagen in hydrated gels. When fibrin is also electroprocessed, it is used as a preformed gel that is electroprocessed by applying pressure, for example by using a syringe or airbrush device with a pressure head behind it to extrude the fibrin gel into an electric field. Can be. In general, when producing fibers using electroprocessing techniques, particularly electrospinning, it is preferred to use monomers of the polymer fibers to be formed. In some embodiments, it is desirable to use monomers to produce fine filaments. In other embodiments, it may be desirable to include partial fibers to add material strength to the matrix and provide additional sites for incorporation of substances. A matrix material such as collagen in the form of gelatin may be used to improve the material's ability to dissolve. Acid extraction methods can be used in preparing such gels to maintain the structure of the monomer subunits. Subsequently, the unit can be treated with enzymes to alter the structure of the monomer.

In embodiments where the two materials are combined to form a third material, the solution containing these components can be mixed together just prior to flowing from the opening in the electrical processing procedure. In this way, the third material is formed such that very fine fibers or microdroplets are formed by an electrospinning process. Alternatively, such a matrix is necessary to electrospray the molecules that can form the matrix material, allowing the moisture, otherwise the formation of the matrix to form filaments in the electric field. It can be formed in a controlled atmosphere of other molecules.

Alternatively, in embodiments where two or more matrix materials are combined to form a third matrix material, the matrix materials can be electroprocessed together with each other or separately from each other. In some desirable embodiments, this is done under conditions that do not allow the two molecules to form a third until the desired time. This can be achieved in several ways. Alternatively, the molecules can be mixed with carriers such as PEO or other synthetic or natural polymers such as collagen. The carrier acts to hold the reactants in place until the reactants are initiated.

As mentioned above, it can be appreciated that the carrier can be used in combination with a matrix material. Various materials, such as extracellular matrix proteins, and / or substances can be mixed with PEG or other known carriers that form filaments. For example, fibrinogen and collagen can be mixed with PEG or other known carriers that form filaments. This gives rise to “hairy filaments”, whose hair is collagen, fibrin, or other matrix material. “Hair” crosslinks the surrounding matrix carrier into a gel or provides reactive sites where cells interact with substances in the matrix carrier, such as immunoglobulins. This approach can be used to gel molecules that form a matrix or do not normally gel.

Alternatively, the electroprocessed collagen can be sputtered to form a sheet. Other molecules that form the sheet include PGA, PLA, copolymers of PGA and PLA, collagen, and fibronectin. In some embodiments, the sheet is formed with two or more materials that can be combined to form a third material. This sheet can be placed in a moist environment allowing conversion to a third material.

In addition to the multiple device changes and variations that can be made to achieve the desired results, the electroprocessed collagen solution can also be varied to obtain different results. For example, any solvent or liquid in which the material is dissolved, suspended, or otherwise combined without deleterious effects on the matrix process or safe use can be used. The material, or compound that forms the material, can be mixed with other molecules, monomers or polymers to achieve the desired result. In some embodiments, a polymer is added to modify the viscosity of the solution. In yet another embodiment, when multiple reservoirs are used, the components in those reservoirs are electrosprayed separately or connected with a nozzle so that the components in the various reservoirs are applied to the electric field of the solution. Can react with each other simultaneously with flow. Also, if multiple reservoirs are used, different components in different reservoirs can be temporarily tuned during processing. These components may include substances.

Embodiments that include modifications to the electroprocessed collagen itself are within the scope of the present invention. Some materials can be altered directly, for example, by altering their carbohydrate profile or the amino acid sequence of a protein, peptide or polypeptide. Also, other materials can be bonded to the matrix material using known techniques such as chemical cross-linking or by specific binding interactions before, during or after electroprocessing. In addition, process temperatures and other physical properties can be varied to obtain different results. The matrix may be compressed or stretched to produce new material properties. Further chemical changes are possible.

Preparing a library of biomaterials for rapid screening using electroprocessing with multiple jets of the same solution with different polymer solutions and / or different types and amounts of substances (eg, growth factors) Can do. Such libraries are desired in the catalyst industry using combinatorial synthesis techniques for the rapid preparation of pharmaceuticals, advanced materials, and large quantities (eg, libraries) of compounds that can be screened. For example, the minimum amount of growth factor released and the optimal release rate from a fibrous collagen scaffold to promote differentiation of certain types of cells can be studied using the compositions and methods of the invention. Other variables include collagen type and fiber diameter. Electroprocessing allows access to a sample array where cells can be cultured in parallel and studied to determine a selection composition that acts as a promising cell growth substrate.

Various effective conditions can be used to electroprocess the collagen matrix. A preferred method is described below, but other protocols can be followed to achieve the same result. Referring to FIG. 3 for electrospinning collagen fibers, a micropipette 10 is filled with a solution containing collagen and placed on a grounded target 11, eg, a metal grounded screen located within the central cylinder of the RCCS bioreactor. Hanging on. Although this embodiment includes two micropipettes that act as a source of material, the present invention includes embodiments that include only one source, or more than two sources. The fine wire 12 is put in the solution, and the solution is charged to a high voltage at each pipette tip 13. The suspended solution in each pipette tip is directed toward the grounded target at a specific voltage determined for each solution and device configuration. This material stream 14 can form a continuous filament, for example when collagen is the material, and when it reaches the grounded target, it is collected and dried to form a three-dimensional ultra-thin interconnect matrix of electroprocessed collagen fibers. Form. Depending on the reaction conditions, a single continuous filament may be formed and deposited with a nonwoven matrix.

As noted above, electroprocessing techniques and material combinations are used in some embodiments. Referring to FIG. 4, each micropipette tip 13 is connected to a micropipette 10 that contains a different material or substance. The micropipette is suspended on the ground target 11. Further, the solution is charged using the fine wire 12. One of the micropipettes produces a collagen fiber 14 flow. Another micropipette produces an electrospun PLA fiber 16 stream, and a third micropipette produces a cellular electroaerosol 17. The fourth micropipette produces an electrospray 18 of PLA droplets. The micropipette is attached to the same voltage supply 15, but PLA is electrosprayed rather than electrospun from the fourth micropipette due to variations in PLA concentration in the solution. Alternatively, a separate voltage supply (not shown) can be attached to each micropipette to allow the application of different potentials to change the electrical processing method to be used.

Similarly, now referring to FIG. 5, collagen material was electrosprayed from collagen fibers 19 electrospun from one of two micropipettes, and the other micropipette, placed at different angles with respect to ground substrate 11. It can be applied as a collagen droplet 20. In addition, the micropipette tip 13 is attached to the micropipette 10 containing various concentrations of material, and thus the fine wire 12 produces different types of electroprocessing flow despite using the same voltage supply 15.

Since minimal current is involved in this process, electroprocessing, in this case electrospinning, causes the collagen to be electroprocessed material because the current causes little or no temperature rise of the solution during the procedure. Or other materials are not denatured. In melt electrospinning, there is some temperature rise associated with the melting of the material. In such embodiments, care is taken to ensure that the materials or substances are not denatured or otherwise exposed to temperatures that would damage or harm them.

An electroaerosol treatment process can be used to produce a dense mat-like matrix of electroprocessed droplets of material. The electroaerosol process is a variation of the electrospinning process in that it utilizes a low concentration of matrix material or molecules that form the electroprocessed material during the procedure. Instead of producing a fiber or single filament spray at the charged tip of the nozzle, droplets are formed. These droplets then move from the charging tip to the substrate to form a sponge-like matrix composed of fused droplets. In some embodiments, the droplet is less than 10 microns in diameter. In other embodiments, constructs formed from fibrils with droplets, such as “beads on a string” may result. The droplets can range in size from 100 nanometers to 10 microns, depending on the polymer and solvent.

Similar to the electrospinning process described earlier, the electroaerosol process can be performed using a variety of effective conditions. The same equipment used in the electrospinning process, for example as shown in FIG. 5, is utilized for the electroaerosol process. Differences from electrospinning include the concentration of the material or substance that forms the matrix material into the solution in the micropipette reservoir and / or the voltage used to create the droplet stream.

One skilled in the art will appreciate that changing the concentration of a material or substance in solution requires a specific voltage change to obtain droplet formation and flow from the tip of the pipette.

The electroprocessing process can be manipulated to meet specific requirements for any given application of the electroprocessed composition electroprocessed composition made using these methods. In one embodiment, the micropipette can be deployed in a frame that moves in the x, y, and z planes with respect to the grounded substrate. The micropipette can be deployed around a grounded substrate, such as a tubular mandrel. In this way, the materials or molecules that form the material that is flowed from the micropipette can be specifically targeted or can form a pattern. Although the micropipette can be moved manually, the frame in which the micropipette is deployed is preferably controlled by a microprocessor and motor that allows the flow pattern of collagen to be defined by the person making the particular matrix . Such microprocessors and motors are known to those skilled in the art. For example, matrix fibers or droplets can be oriented in a particular direction, they can be layered, or they can be programmed to be completely random and not oriented.

In the electrospinning process, the flow (s) can be branched to form fibers. The degree of branching includes many factors including, but not limited to, voltage, grounding structure, distance from the micropipette tip to the substrate, diameter of the micropipette tip, and the concentration of the material or compound that forms the electroprocessed material. Can be changed. As noted above, not all reaction conditions and polymers produce true multifilaments under conditions where a single continuous filament is produced. The materials and various combinations can also be delivered to the electric field of the system by injecting the material into the electric field from the device that causes them to be aerosolized. This process includes voltage (eg, in the range of 0-30,000 volts), distance from the tip of the micropipette to the substrate (eg, 0-40 cm), relative position of the micropipette tip and the target (ie, top, bottom, Horizontal, etc.), and the diameter of the micropipette tip (about 0-2 mm), but can be more varied of many factors, but not limited thereto. Some of these variables are known to those skilled in the art of electrospinning fine fiber woven fabrics.

The structure of the ground target can be modified to produce the desired matrix. For example, by changing the grounding substrate having a planar or linear or multi-point grounding, the direction of the flowable material can be changed and customized for a particular application. For example, a grounded target that includes a series of parallel lines can be used to orient the electrospun material in a particular direction. The grounded target may be a cylindrical mandrel, thereby forming a tubular matrix. Most preferably, the ground is a variable surface that can be controlled by a microprocessor that directs the particular ground structure to be programmed into the particular ground structure. Alternatively, for example, the ground can be deployed on a frame that moves in the x, y, and z planes with respect to a stationary micropipette tip that allows collagen to flow.

The substrate on which the material is flowed, sprayed or sputtered can be the grounded target itself, or it can be placed between the micropipette tip and the grounded target. The substrate can be a specific shape, such as a nerve guide, a skin patch, a fascia sheath, or a vascular graft for subsequent use in vivo. The electroprocessed composition can be shaped to fit the defect or site to be filled. Examples include sites where tumors have been removed, sites of trauma in the skin (cuts, biopsy sites, holes or other defects), and missing or shattered bone. The electroprocessed composition can be used in materials useful for substance delivery, such as skin patches, ingestion lozenges, intraperitoneal implants, subcutaneous implants, stent inner or outer layers, cardiovascular valves, tendons, corneas, ligaments, dental prostheses. May be shaped into muscle grafts, or nerve leads. Electroprocessing allows great flexibility and allows the construction to be customized to virtually any shape that is required. Many matrices are flexible enough to allow them to be shaped into virtually any shape. In shaping the matrix, a portion of the matrix may be sealed, for example, by heat sealing, chemical sealing, and application of mechanical pressure, or a combination thereof. An example of heat sealing is the use of the crosslinking techniques described herein to form a bridge between two parts of the matrix. Sealing can also be used to close openings in the build matrix. Suture can also be used to join portions of the matrix together or to close openings in the matrix. The inclusion of synthetic polymer was observed to enhance the ability of the matrix to be heat sealed.

Other variations of electroprocessing, particularly electrospinning and electroaerosol processing include, but are not limited to:

1. Using different solutions to produce two or more different fibers or droplets simultaneously (fiber or droplet array). In this case, the single component solution can be maintained in a separate reservoir.

2. Using mixed solutions (eg, materials with substances such as cells, growth factors, or both) in the same reservoir (s), the electroprocessed material as well as one or more substances (fiber composition) Production of fibers or droplets composed of the product “blend”). Non-biological but biocompatible materials can be mixed with biological molecules.

3. Utilize multiple potentials applied for different solutions or the same solution.

4. Supply two or more structurally different ground targets (ie large and small mesh screens).

5. Place the mold or mandrel or other non-grounded target in front of the grounded target.

6). Applying an agent such as Teflon on the target to facilitate removal of the electroprocessed material from the target (ie, so that the electroprocessed material does not stick to the target, Make the material more slippery).

7). Forming an electroprocessed material including a material applied using a plurality of electroprocessing methods; For example, electrospun fibers and electroaerosol droplets in the same composition can be beneficial for some applications, depending on the specific structure desired. This fiber and droplet combination can be used to change the polymer concentration in the reservoir by changing the distance from the grounded substrate by changing the charge using the same micropipette and solution. It can be obtained by using micropipettes (some for fiber flow, others for droplet flow) or by any other variation of methods envisioned by those skilled in the art. The fibers and droplets can be layered or mixed together in the same layer. In applications involving multiple micropipettes, the micropipettes can be placed in the same or different directions and distances with respect to the target.

8). Use multiple targets.

9. During electroprocessing, the target or mandrel is rotated to cause the electroprocessed material to have a specific polarity or arrangement.

All of these variations can be performed separately or in combination to produce a wide range of electroprocessed materials and substances.

Various properties of the electroprocessed materials can be prepared according to the needs and specifications of the cells to be suspended and grown in them. For example, the porosity can be varied according to the method of making the electroprocessed material and matrix. For example, a specific matrix can be electroprocessed by varying the fiber (droplet) size and density. A loose matrix can be created if the cells to be grown in the matrix require a large amount of handling of nutrient streams and waste removal. On the other hand, if the tissue to be produced requires a very high density environment, a high density matrix can be designed. Porosity can be manipulated by mixing salts or other extractable agents. Removal of the salt will leave pores with a defined size in the matrix.

In one embodiment of electroprocessed collagen, suitable approximate ranges are voltage (0-30,000 volts, pH 7.0-8.0, temperature 20-42 ° C., and collagen 0-5 mg / ml). One embodiment for electrotreating collagen is acid extraction of type I collagen (carbskin) dissolved in HFIP electroprocessed from a syringe at 25 kV at a distance of 127 mm from the target and a syringe pump speed of 10 ml / hr. Use a concentration of 0.008 g / 1.0 ml of collagen at which the collagen does not show any evidence of electrospinning (fibrosis) and the polymer solution is the syringe tip regardless of the input voltage. Electrosprayed droplets and leaks were formed from an embodiment for elastin of 250 mg / ml Use elastin from the ligament of the neck ligament dissolved in 70% isopropanol / water, then stir the solution to ensure mixing and fill into a 1 ml syringe. Place on pump and set flow rate of 10 ml / hr Place mandrel 7 inches from syringe tip and rotate at selected speed, then turn on pump and power, voltage 24, An electroprocessed collagen matrix with various properties can move pH, change ionic strength (eg, addition of organic salts), or add additional polymer matrix or cationic material. It can be operated by adding.

Methods for Combining Substances with Electroprocessed Materials Substances can be combined with collagen that has been electroprocessed by various means. In some embodiments, the substance includes molecules to be released from, or contained within, the electroprocessed collagen and other materials, and thus is added to the matrix of the electroprocessed material. Or incorporated within it. The substance can be mixed in a solvent carrier or solution of the electroprocessing material. In this system, the material can be mixed with various substances and directly electroprocessed. The resulting electroprocessed matrix and composition comprising the substance can be applied topically to a specific site, and the substance can be released from the material as a function of the material undergoing collapse in the surrounding environment. The material may also be released from the electroprocessed composition of the present invention by diffusion.

The state of the electroprocessed collagen and other electroprocessed materials with respect to the incorporated material is governed and can be controlled by the chemistry of the system, and the choice of matrix material, the solvent (s) used, and these Depending on the solubility of the matrix material in the solvent. These parameters can be manipulated to control the release of the substance (or other elements to the surrounding environment). If the substance to be incorporated into the electroprocessed material is not miscible with the material, separate solvent reservoirs for different components can be used. Thus, substances that are not miscible with the collagen solution can be mixed, for example with collagen from a separate reservoir, in a solvent carrier for other materials to be electroprocessed. Mixing in such embodiments is performed before, during and / or after deposition on the target, or a combination thereof. The substance may be encapsulated or engaged within the electroprocessed material, may be bonded to the material before the material undergoes electroprocessing, or may be bonded to a specific site within the matrix material. It will be understood.

In a variation of this embodiment, the substance is a particle or agglomerate comprising a matrix of a compound or polymer, such as alginate, which sequentially contains one or more compounds released from the electroprocessed material. Substances such as drugs or cells can be combined with alginate, for example by combining a drug suspension or drug microparticles in alginate in the presence of calcium. Alginates are carbohydrates that form aggregates when exposed to calcium. Aggregates can be used to capture drugs. Aggregates dissolve over time, releasing the material trapped in the alginate. The particles are subsequently incorporated into a larger electroprocessed matrix and are biocompatible but relatively stable and will gradually degrade. In some embodiments, the electroprocessed material is like a series of pearls. This is a physical aspect of electroprocessing. When the concentration of the material to be electroprocessed is low, the beads are electrosprayed. As the concentration increases, there are some beads and some fibers. Further increases in the concentration of the material being electroprocessed cause most or all fibers. Thus, the appearance of pearls on the string is a transition phase.

If the substance does not bind or interact with the electroprocessed matrix material, the drug can be encapsulated in PGA or PLA pellets, or an electroaerosol to produce pellets in the electrospun material Can be processed. Some drugs (eg penicillin) can be captured in this way. Pellets or electroaerosol-treated droplets containing the substance begin to dissolve after administration and deliver the encapsulated material. Some agents can be covalently attached to synthetic or natural polymers before or after spinning.

In other embodiments, the material is electroprocessed. The substance can be electroprocessed from the same opening as the collagen and / or other material being electroprocessed or from a different opening. The material can also be subjected to the same or different type of electrical treatment as the material. Molecules can be bound to collagen or other materials that have been electroprocessed with a collagen matrix, either directly or by linking to molecules that have an affinity for the material. An example of this embodiment includes binding a polypeptide substance to heparin that has an affinity for collagen. This embodiment can control the release rate, for example by controlling the degradation rate of the material by enzymatic or hydrolytic degradation.

In other embodiments, the electroprocessed collagen can encapsulate materials during the electrodeposition process. This can be achieved by placing the substance in the space between the source of the electroprocessed stream and the target for the electroprocessed material. Placing such material in the space between the source and target can include suspending, dripping, spraying, or electrotreating the material in air or other gases, but these It can be achieved by a number of methods not limited to: The substance can be present in the space, for example in the form of microparticles, aerosols, colloids or vapors. In these embodiments, the electroprocessed material or matrix physically encapsulates the substance. This embodiment can also be used to encapsulate larger particles (eg, cells, giant particles, or tablets). For example, if the tablet is dropped into the matrix as it forms, the tablet is wrapped with the matrix. If a small object, such as a cell, is dropped into the matrix as it is formed or placed in an aerosol inside the matrix, the object can be trapped between, within the filaments, or outside the filaments Can be attached. For example, by suspending the object in solution or within the matrix, the object can become part of the electrospun matrix during manufacture of the filament. Alternatively, the encapsulation can be done by dropping the substance onto or into the matrix material stream as the matrix forms. Thus, the object becomes encased by a matrix of electroprocessed material. These embodiments may be used to incorporate substances that do not dissolve and / or that are too large to form a suspension in the solvent used to produce the material into the interior of the matrix substance. it can. For example, in the mist or vapor form, the physical and chemical properties of the electroprocessed material, as well as the electroprocessed, are used to control the distribution and composition of matter in the space between the source and target The distribution pattern of substances in the material can be changed. For all of the above embodiments, the substance can be placed in an electroprocessed material in a capsule, vesicle, or other container for subsequent release. Since the solvent carrier often evaporates with electroprocessing techniques when an electroprocessed material such as a filament is formed, the substance may be placed in an electroprocessed matrix, and the solvent toxicity is greatly enhanced. Reduced or eliminated.

In many embodiments, the substance comprises a cell. Cells can be combined with a collagen matrix electroprocessed by any means described above to combine small objects with the matrix. The cells can be suspended, for example, in a solution or other liquid containing collagen, placed in the area between the solution and the target, or separated before, during, or after electroprocessing. It can be delivered from a source to a target or substrate. Cells can be dripped onto the matrix as it is deposited on the target, or suspended within an aerosol as a delivery system for cells to the electroprocessed material. Cells can be delivered in this manner while a matrix is formed. For example, cardiac fibroblasts were suspended in phosphate buffered saline (PBS) at a concentration of about 1 million cells per milliliter. The cell suspension was placed in a Paasche airbrush reservoir. To test the efficiency of delivering cells using this type of device, the cell suspension was first sprayed onto a 100 mm culture dish. Some of the cells survived, attached to the dish, and spread across the substrate. In the second trial, the experiment was repeated with the culture dish placed further away from the airbrush. Cells were observed on the dish. They appeared to be flattened by impact and partially spread over the surface of the base layer. Medium was added to the dish and the cells were placed in an incubator. After 1 hour of culture, the cells were inspected again and many of them were found to spread further across the substrate. These results demonstrate that a simple airbrush device can be used to place cells in aerosol droplets and deliver them to a predetermined surface or site as required. Cell viability limits this technique to cells that are resistant to the shear forces produced by the technique, develops cell suspensions with additives that soften the cells, or a thinner laminar flow It can be improved by refining the aerosol processing device that produces In addition, directing cell aerosol into the matrix material is to form a matrix in the space between the target or the core axis, and the source (s) of molecules being electroprocessed have the effect of tempering the cells. Arise. Although not wishing to be bound by the following claims, cells are thought to be trapped and pulled onto the mandrel by filaments or other body storms produced by electrospinning or electroprocessing. . This situation will have less impact on the cell than spraying the cell directly onto the solid surface.

In some embodiments, cells are added before or simultaneously with collagen and other materials to be electroprocessed. In this way, the cells are suspended throughout the three-dimensional matrix.

Cells can be added as the filament is produced in the space between the target and the polymer source. This includes dropping cells onto the target, dropping cells into the electroprocessed collagen matrix as the electroprocessed collagen matrix is formed, aerosolizing the cells onto the collagen matrix or onto the target. Alternatively, it can be accomplished by electrospraying the cells onto the collagen matrix as the collagen matrix aggregates and forms near or on the grounded target. In another embodiment, cells are sprayed or dropped onto the electroprocessed material or matrix being formed so that the electroprocessed material crosses the air gap between the source solution and the target. Captured.

An alternative method of delivering cells to electroprocessed collagen involves electroaerosol delivery to the cells. Cells can be deposited, for example, by electrostatic spraying directly at 8 kV onto standard polystyrene culture dishes, suggesting that electrostatic cell spraying is a viable approach. Cardiac fibroblasts in phosphate buffered saline (PBS) have been electroaerosol treated with a potential difference of up to 20 Kv. In another example, Schwann cells (rats) were electrosprayed on PS Petri dishes by conventional methods one day later. Schwann cells were also electrosprayed on a PS Petri dish with a metal ground plane on the back of the dish at 10 kV after one day. Both samples grew to near confluence after one week. The electric aerosol approach offers several distinct advantages. First, the shear forces that occur during the delivery phase (ie, the production of aerosols) appear to have much less impact on the cells. Second, the aerosol direction can be controlled with a high degree of fidelity. Essentially cell aerosol can be applied on a given surface. This allows cells to target specific sites. In electroaerosol delivery, cells are suspended in a suitable medium (eg, media, physiological salts, etc.), charged, and directed toward a grounded target. This process is very similar to the process used in electroprocessing, especially electrospinning. This process results in a fine mist of cells trapped inside the droplet as the droplet is produced and directed to a grounded target.

Cells can be delivered on electroprocessed collagen using aerosol and electroaerosol techniques. The cell's electroaerosol can be delivered in parallel (ie, side-by-side) with the electroprocessing material or from a separate site. Cells can be produced in an air gap during the electrodeposition process or delivered to a target-guided filament or particle storm. Cells and electroprocessed material can also be delivered alternately to the target. That is, the cells and the electroprocessed material are electrodeposited, the cells are aerosolized, the material is electrodeposited, and the cells are aerosolized. This allows for separate stratification of constructs in separate layers. Further, a vapor source can be provided by directing water onto the target mandrel used to collect the cells. Providing this moisture improves cell viability by not dehydrating the cells during processing. The cells can be added to the electroprocessed collagen in any aerosol strategy, at any time, or from any orientation. Furthermore, the advantage of this process is generally that, for example, collagen is collected dry on the target mandrel. Thus, some solvents for collagen may be toxic, but they are lost from the system before the filaments collect on the target.

Cells can also be trapped within the carrier prior to producing the aerosol. For example, the cells can be encapsulated inside a material such as alginate. Encapsulated cells are physically protected from shear and impact during processing. Cells delivered to electroprocessed material in this form will have a higher viability when sprayed or seeded electrostatically.

The electroprocessed collagen can also be delivered directly to the desired location. For example, electroprocessed materials can be produced directly on skin wounds with or without substances such as molecules or cells. Additional cells or material can then be aerosolized onto or into the wound site. Other surgical sites can also accommodate for delivery of materials using various electrodeposition techniques, or a combination of these methods.

Magnetic and electroactive materials can be electroprocessed (including, for example, preparing conductive polymer fibers produced by electrospinning). In addition, conducting polymers can be prepared in situ in the matrix, for example by incorporation of monomers (eg pyrrole) followed by treatment with a polymerization initiator and an oxidizing agent (eg FeCl ₃ ). Finally the conducting polymer can be grown in the material after electroprocessing, for example by using a matrix coated conductor as an anode for the electrochemical synthesis of polypyrrole or polyaniline. Collagen or other material to be electroprocessed can be added to an aqueous solution of pyrrole or aniline to create a conductive polymer at the anode with encapsulated electroprocessed material forming compound, followed by It can be treated with other compounds to cause material formation.

More than one method of combining a substance with electroprocessed collagen can be used in a single embodiment or application. The combined method may be particularly useful in embodiments where electroprocessed collagen releases one or more substances, and where the released substances are intended to have complex release kinetics. Such combinations are not limited to these embodiments.

Electroprocessed Material and Matrix Shape The present invention also provides a method for producing a collagen-containing extracellular matrix having a defined shape. The method includes pre-selecting a mold that is adapted to produce a defined shape and filling the mold with collagen or collagen-forming molecules using electroprocessing techniques. In another example of embodiment, the method includes pre-selecting a mold or mandrel adapted to create a defined shape, wherein the mold includes a grounded target substrate and the shape of the matrix Depends on the outer dimension of the mandrel. Thus, a charged solution containing one or more collagens or molecules capable of forming collagen is flowed over a grounded target substrate under conditions effective to deposit the collagen on the substrate. Forming an extracellular matrix having a defined shape. Collagen that is flowed onto the substrate can include electrospun fibers, or electrosprayed droplets. The formed matrix having the shape of the substrate can then be cured and removed from the mandrel. The substrate may have a specific shape, such as nerve induction, skin or muscle patch, fascia.
sheet), vertebral plate, knee meniscus, ligament, tendon, or vascular graft for subsequent use in vivo. The collagen matrix can be shaped to fit the defect or site to be filled. For example, to recreate or restore a site where the tumor has been removed, a trauma site in the skin (cutting, biopsy site, hole or other defect), or missing or shattered bone. Electroprocessing allows great flexibility and allows the construct to be customized to virtually any shape that is required. Some preferred examples include a cylindrical shape, a flat oval shape, a rectangular envelope shape (eg, an envelope), or any other shape. Collagen can be formed into virtually any shape. Complex shapes such as pouch-like organs can be formed. The overall three-dimensional structural shape of the foundation is determined by the final design and type of tissue that can be bioengineered.

There are several ways to prepare a specifically shaped mold. For example, the specific type of organ or tissue that is desired to be replaced has a specific shape, such as a skin patch that fits over a large scalp area associated with a wide area that is removed after discovery of a biopsy site or malignant melanoma. The shape is duplicated and created inside a mold designed to mimic the shape. The mold can be filled by electrodepositing collagen on the mold. In this way, the collagen matrix accurately mimics the mold shape. Creating an extracellular collagen matrix with a specific shape can be very important in creating new organs. The shape of the matrix can induce cells seeded in the collagen matrix to differentiate in a specific manner. Growth factors or other substances may be incorporated as described elsewhere herein. This results in a more effective and more natural organ or tissue being created. A hollow matrix to be filled with a desired substance, such as a cell, or a hollow matrix to replace a hollow organ or structure can also be made. For a cylindrically shaped biomanipulating base, or any other shape of construction where a sealed area is desired, a suture, glue, staple, or heat seal, or some other, to seal one end of the biomanipulating base Other methods may be used. This produces a hollow substrate that is closed at one end and open at the other end. The electrodeposited collagen-containing substrate can now be filled with cells or other materials, or cells or other materials may be placed on the outer surface of the construct. For example, a mixture of collagen from an electroprocessing procedure, other materials such as cells, or molecules such as drugs or growth factors may be placed in the substrate. The free open end of the envelope used to fill the construct with material can be sutured, glued or heat sealed to produce a sealed biomanipulation base. be able to. Mixing the cells with the material during electroprocessing results in the cells being distributed throughout the matrix so that the cells need not migrate to the gel. However, it has been found that collagen electroprocessed as described above promotes infiltration.

Further shaping can be achieved by manual processing of the formed matrix. For example, a plurality of formed matrices can be stitched, sealed, stapled, or otherwise attached to one another to form a desired shape. it can. Alternatively, the physical flexibility of many matrices allows the matrix to be manually shaped into the desired structure.

Electroprocessed collagen and other electroprocessed materials and substance distribution patterns Many embodiments of the present invention manipulate the pattern or distribution of electroprocessed collagen and / or substances within the electroprocessed material. Includes means. For example, the electroprocessing target can also be specifically charged or grounded along a preselected pattern so that the electroprocessed material that is flowed against the target is the target or substrate. Guided in a specific direction or distribution on the material. The electric field can be controlled by a microprocessor to create a matrix having the desired structure. The target and electroprocessing nozzle (s) can be movable relative to each other and relative to the target, thereby allowing further control over the structure of the electroprocessed material to be formed. In embodiments where the material is electroprocessed, this operation also allows the distribution of the material within the electroprocessed material to be controlled. For example, an electroprocessed collagen matrix can be prepared on the mandrel, and materials from separate reservoirs can be sprayed, dripped, electroprocessed in a specific pattern across the existing matrix. This can also be achieved by simultaneously electrospraying a matrix from one source and a substance from another source. In this example, the matrix source may be fixed and the substance source is moved relative to the target mandrel.

Other forms that allow the establishment of such a pattern include the ability to deposit multiple layers of the same or different materials, a combination of different electroprocessing methods, and the use of multiple openings with different contents for electroprocessing , And the existence of numerous methods for combining materials with materials. For example, a gradient of material can be created along the electroprocessed material. For example, in embodiments where the matrix is shaped into a cylindrical structure, the gradient can be created along the long axis (from left to right) or the vertical axis (from inside to outside) of the structure. This arrangement is used to provide a chemoattractant gradient for inducing cell migration into the designated site. Thus, for example, in embodiments where the angiogenic agent is prepared with a vertical axis gradient along the collagen-based construct, the agent can be agglomerated on the back of the material sheet. The ventral side can be placed against the wound and a higher concentration of angiogenic material on the back of the construct will increase endothelial cell migration to electrospun material. Also, embodiments with complex patterns can be used to obtain specific preselected electroprocessing patterns of one or more electroprocessed polymers, optionally with one or more materials. A microprocessor programmed with can be used.

Further Processing of Electroprocessed Collagen Material Electroprocessed collagen and other electroprocessed materials may be further processed to affect various properties. In some embodiments, the electroprocessed material is crosslinked. In some embodiments, cross-linking increases structural rigidity and delays subsequent dissolution of the electroprocessed material, for example, the rate at which the electroprocessed material decomposes, or the substances contained within the matrix are electrically Varying the rate of release from the processed material. Electroprocessed collagen and other materials can be cross-linked simultaneously with their formation by forming them in the presence of a cross-linking agent, or can be treated with a cross-linking agent after electrodeposition. Any technique known to those skilled in the art of cross-linking materials may be used. Examples of techniques include the application of cross-linking agents and the application of specific cross-linking irradiation. Examples of cross-linking agents that work with one or more proteins include condensing agents such as aldehydes (eg, glutaraldehyde), carbodiimide EDC (1-ethyl-3- (3-dimethylaminopropyl)), Examples include, but are not limited to, photosensitive materials that crosslink upon exposure to specific wavelengths, osmium tetroxide, carbodiimide hydrochloride, and NHS (n-hydroxysuccinimide), and Factor XIIIa. Glutaraldehyde is a desirable cross-linking agent for collagen. Ultraviolet radiation is an example of radiation used to crosslink the matrix material in some embodiments. Natural materials can be cross-linked with other natural materials. For example, collagen can be cross-linked and / or stabilized by the addition of fibronectin and / or heparin sulfate. For some polymers, heat can be used to alter the matrix to crosslink the elements of the matrix by fusing adjacent components of the construct. In another example, collagen may be cross-linked using a natural process mediated by cellular elements. For example, electrospun collagen can be cross-linked by a lysyl oxidase enzyme cascade. Synthetic polymers may also be partially solubilized to alter the structure of the material, for example, short exposures of some compounds to alcohols or bases can partially dissolve adjacent filaments. , Both can be annealed. Some polymers may be cross-linked using chemical or thermal fusion techniques. Synthetic polymers can generally be crosslinked using high energy radiation (eg, electron beam, gamma radiation). These usually act on the creation of free radicals on the polymer backbone, followed by bonding and cross-linking. Skeletal free radicals can also be generated by peroxides, azo compounds, aryl ketones, and other radical producing compounds in the presence of heat or light. Redox reactions that produce radicals (eg, peroxides in the presence of transition metal salts) can also be used. In many cases, functional groups on the polymer backbone or side chain can react to form a crosslink. For example, polysaccharides can be treated with diaryl chloride to form diester bridges. Crosslinking may also occur after application of the matrix if desired. For example, the matrix applied to the wound may be crosslinked after application to enhance the adhesion of the matrix to the wound.

One preferred cross-linking agent for electroprocessed collagen is glutaraldehyde. In some embodiments using a type I collagen / type III collagen / elastin (45:35:20) matrix, the matrix exposure to glutaraldehyde vapor under suitable conditions for at least about 10 minutes is satisfactory. Provided a degree of crosslinking. In general, longer intervals of glutaraldehyde cross-linking increase matrix stability but decrease cell infiltration. A desirable range is about 10 to about 20 minutes of exposure. Exposure was achieved by preparing a gas chamber made by placing it in a sterile 10 cm ² Petri dish with the top removed in the center of the 35 cm ² Petri dish with the top remaining. Approximately 4 ml of 3% glutaraldehyde solution was placed in the smaller dish and the collagen mat was placed on the larger dish towards the edges. A 3% glutaraldehyde solution was made by mixing 50% glutaraldehyde with distilled water and 0.2M sodium cacodylate buffer.

Additional substances are applied to the electroprocessed material after formation, for example by immersing the electroprocessed material in the substance or a solution containing the substance or by spraying the solution or substance onto the electroprocessed material can do. A collagen matrix placed in contact with cells in vitro or in vivo is infiltrated by cells that migrate to the matrix. Any in vitro method of seeding cells in the matrix can be used. Examples include, for example, US Pat. No. 6,010,573 (Bowlin et al.), US Pat. No. 5,723,324 (Bowlin et al.), And US Pat. No. 5,714,359 (Bowlin). et al.), such as the placement of bioreactors, or the use of electrostatic cell seeding techniques. The electroprocessed matrix may also be sterilized by known sterilization methods. For example, the electroprocessed material can be immersed in a 70% alcohol solution. Another preferred sterilization method is the peracetic acid sterilization procedure known for collagen-based tissues.

A physical treatment of the formed electroprocessed material is also possible. The electroprocessed matrix may be ground into a powder or may be ground and prepared as a hydrated gel composed of striped fibrils. In some embodiments, a mechanical force, such as compression applied to the electroprocessed material, can speed up the destruction of the matrix by changing the crystal structure of the material. Thus, the structure of the matrix is another parameter that can be manipulated to affect the release kinetics. Polyurethane and other elastic materials such as poly (ethylene-co-vinyl acetate), silicones, and polydienes (eg, polyisoprene), polycaprolactone, polyglycolic acid, and related polymers have a release rate that is mechanically strained. It is an example of a material that can be changed.

Further Processing of Modified Tissue Containing Electroprocessed Collagen Once an electromodified tissue containing electroprocessed collagen and cells has been constructed, the tissue can be inserted into a recipient. Alternatively, the structure can be placed in culture to enhance cell growth. Various types of nutrients and growth factors can be added (or administered to the recipient) to the culture to promote specific types of growth of the modified tissue. In one example, in conjunction with the preparation of a specifically modified muscle tissue, an electrical modified tissue containing collagen and cells to stimulate cell alignment to form a more functional muscle graft. Can be mechanically or passively stained or electrically preconditioned (stimulate electrically sensitive cells such as cardiac muscle or skeletal muscle cells that contract by electrical depolarization). Adding strain increases the tensile strength of the graft. For example, strong stretching or stretching of cells causes hypertrophy as if the cells were subjected to stretching. In skin patches, mechanical stress can be applied to promote the orientation of the skin for use in areas such as the scalp exposed to significant stretch forces. Other tissues that may be beneficial by applying strain include, but are not limited to, muscle tissue, ligament tissue, and tendon tissue. Passive strain in this context refers to a process in which strain is induced by the cell itself as if the cell contracted and reorganized the matrix. This is usually induced by fixing the ends of the electromodified collagen matrix. As the cells shrink and change the matrix, the fixed end of the matrix remains in place, thereby tensioning the cells as they are “pulled” against isometric loads. Strain not only aligns cells, but strain sends signals to cells regarding growth and development. The construct can also be tensioned externally, i.e., after constructing the construct, it can be stretched to cause mechanical alignment. Stretching is usually applied in a gradual manner over time. Collagen can also be stretched to cause alignment in the matrix before the cells are added to the construct (ie, the cells can be added after forming the matrix and stretching the matrix). Any known method of applying mechanical or passive physical strain can be used.

A further method of combining the electroprocessed collagen matrix with cells for implantation is to add the cells to the construct after the construct is prepared. The cells can be placed in a lumen or space within the construct, or can be transplanted adjacent to the graft to promote growth. Alternatively, the implant can be placed in a bioreactor. There are several commercially available bioreactors that are designed devices that provide a low shear, high nutrient perfusion environment. Until recently, most available bioreactors have delivered nutrients and oxygen by maintaining cells in suspension and sprinkling using impellers or other agitation means. These methods result in a high shear environment that can damage cells or inhibit the formation of large scale constructs. The RCCS bioreactor (Synthecon) is a rotating wall bioreactor. It is composed of a small inner cylinder that can be used as a substrate for an electrospinning process, arranged in a large outer cylinder. The electrical span or electroaerosol matrix can be manufactured on the inner cylinder, but other locations within the bioreactor can be used for placement of the seeding matrix. A gap between the inner cylindrical body and the outer cylindrical body functions as a cell culture container space. The medium is oxygenated through the outer hydrophobic membrane. The low shear environment of the RCCS bioreactor from Synthecon is due to nutrient damage or “washing” that occurs with active stirring or spreading.
promotes cell-cell and cell-extracellular matrix (ECM) interactions. Typically, the RCCS device is used to maintain cells in suspension when the RPM speed of the RPM is from 8 to up to 60, and 8 RPM for media immobilized along the central shaft of the container. Operating at less than (preferably 2-3 RPM). Synthecon bioreactors can be used in standard tissue culture incubators. These values and other parameters for spin speed can vary depending on the particular tissue being created.

In other applications, electrospun structures may be manufactured and placed in RCCS bioreactors and subjected to a continuous free-fall, floating environment that facilitates the formation of large multi-layered structures. obtain. Prior to placing the cells in the bioreactor, the cells may be added to the construct. Alternatively, the bioreactor may be used as a platform for seeding cells onto an electrospinning matrix. For example, a cylindrical construct can be placed in a bioreactor vessel. Cells may be added to the container to interact with the electrospun construct during free fall. The speed required to maintain the construct in suspension depends on the size and density of the material present in the construct. Larger constructs (2-4 mm in diameter and 10-12 mm in length) may require rotational speeds close to 15-20 rpm. Larger constructs, such as cartilage, may require even faster rotational speeds.

An electroprocessed collagen material such as a matrix is useful for forming a prosthesis. One application of electroprocessed matrices is the formation of medium diameter and small diameter vascular prostheses. Some preferred materials for this embodiment are collagen and elastin, especially collagen type I and collagen type III. Some examples include, but are not limited to, coronary vessels for bypass or transplantation, femoral artery, popliteal artery, brachial artery, tibial artery, radial artery, aortic bifurcation, or corresponding vein. The electroprocessed material is particularly useful in combination with endothelial cells and smooth muscle cells, such as collagen tubes, on the inside of the vascular prosthesis, and is particularly useful in combination with fibroblasts on the outside of the collagen tube. More complex shapes including tapered containers and / or branch containers can also be constructed. Different shaped mandrels are necessary to wrap large fibers around the electrical span / electric aerosol polymer or to orient the electrical span / electric aerosol polymer.

A combination of electroprocessed collagen and other fibers, such as large diameter (eg, 50-200 μm) collagen or other fibers, can provide optimal growth conditions for the cells. The large diameter fibers form a basic structural matrix that provides mechanical support to the construct, and the electroprocessed matrix is used as a scaffold to deliver and / or support cells. This promotes cell attachment on the structural matrix. Large scale collagen fibers can be incorporated into other biomodified organs and tissues to provide additional mechanical strength as needed. For example, large fibers can be placed in an electrospun matrix designed as a scaffolding for the manufacture of skeletal muscle, myocardium and other smooth muscle-based organs such as the intestine and stomach. In an alternative manufacturing strategy, the cylindrical construct is electrospun onto a suitable target, such as a cylindrical mandrel. Other shapes may be used based on the shape of the site where the implant is placed if desired. The matrix in this embodiment includes electroprocessed collagen and other components such as fibrin, PGA, PLA, and PGA-PLA blends, PEO, PVA or other blends. The relative proportions of the different components of the construct are adapted for specific applications (eg, increased fibrin and less collagen for enhanced angiogenesis in skin grafts). To produce cylindrical muscle, the construct is filled with muscle or stem cells, or other cell types, and the distal end of the electrospun construct is sutured or sealed. The In some embodiments, the cells are mixed with various matrix materials to increase their distribution within the construct. For example, the cells can optionally be mixed with electroprocessed collagen and fibrin prior to insertion into the construct. The purpose of this strategy is to provide additional mechanical support for the construct and to provide cells with a three-dimensional matrix within the construct to promote growth. This also helps maintain cells with a uniform distribution within the construct. This method can be used to enhance the alignment of cells within the construct. This filling material can be extruded directly into a cylindrical construct, and alignment occurs when the filling is extruded. Mixing endothelial cells with other cells (or other cell types) inserted into the construct can be done to accelerate angiogenesis. Another way to achieve this goal is to electrodeposit endothelial cells directly onto an electroprocessed collagen matrix that helps form a cylindrical sheath. The alignment of the fibers within the electroprocessed matrix containing the construct is optionally controlled by controlling the relative movement of the target and source solution with respect to each other. Other cell types, such as tendon fibroblasts, are optionally electrospun to the outer surface of the construct or onto the outer surface of the construct to enhance the formation of the outer connective tissue sheath that forms the construct.

In another example, a sheet of electroprocessed material is prepared, wound into a cylinder, and inserted into the electroprocessed cylinder. The construct is filled with cells as described above, sutured closed and placed in the bioreactor or directly in situ. By aligning the fibrils of the electrospun material sheet parallel to the long axis of the outer cylinder, a scaffold formation for the production of a muscle-like electroprocessed composition is produced. Cells that are in contact with fibrils arranged along the long axis of the sheet develop parallel to the fibrils of the sheet and are aligned and stratified in an organized pattern similar to that present in vivo. Form a construct. This basic design can be adapted to produce many different tissues, including but not limited to skeletal and myocardium. The cylindrical tissue construct is then implanted or placed in an RCCS bioreactor. The rotational speed to maintain this type of construct in suspension ranges from 4-20 rpm, depending on the excess mass of the tissue and the specific material used to produce the outer cylinder.

Angiogenesis of a modified tissue containing an electroprocessed collagen matrix occurs in situ several days after surgery, alone or with other materials. In some embodiments, angiogenesis of modified constructs containing electroprocessed material is enhanced by mixing endothelial cells into the construct during manufacture. Another alternative for supplying modified tissue containing electroprocessed material with a vascular supply is to temporarily implant the tissue into the mesh. The net has an extensive and abundant vascular supply that can be used to support modified tissue such as existing incubators. The modified tissue is removed from the bioreactor, wrapped in a net and supported by nutrient and oxygen diffusion from surrounding tissues in the net. Alternatively or in addition to this approach, the modified tissue is directly connected to the network's endogenous vascular supply. The blood vessel can be partially pierced or can be cut away or cut off from the net. Depending on the construct, wrap around the vessel with modified tissue containing electroprocessed collagen, fibrin, or other material. The modified tissue is supported by nutrients that leak from the penetrated blood vessels or, if the blood vessels remain intact, simply by nutrient diffusion. Regardless of strategy, the modified tissue is surrounded by the net and its rich vascular supply. This procedure can be performed using vessels outside the mesh.

Tissue containing electroprocessed collagen and any other material can be manipulated by the endogenous vasculature. The vasculature can be composed of artificial blood vessels or blood vessels excised from a donor site in a transplant recipient. Next, the modified tissue containing the electroprocessed matrix material is collected around the blood vessel. By wrapping such blood vessels with tissue during or after the modified tissue assembly, the modified tissue has blood vessels that can be connected to the recipient's vasculature. In this example, a blood vessel or other tissue in the network is cut and the modified tissue blood vessel is connected to the two free ends of the network blood vessel. Blood enters the vascular system of the modified tissue from the vascular network to the tissue and is discharged into the vascular system. By wrapping the tissue of the mesh and connecting the tissue to the blood vessels of the mesh, the modified tissue is supported by the diffusion of nutrients from the mesh and blood vessels incorporated into the tissue during its manufacture. After an appropriate amount of time, the tissue is removed from the mesh and placed at the correct site on the recipient. Using this strategy, modified tissue containing electroprocessed material is supported in a nutrient-rich environment during the first few days after removal from the bioreactor. The network environment also promotes the formation of new blood vessels in the transplanted tissue. This network incubator strategy can be combined with other strategies such as combining angiogenic factors in matrix materials during electroprocessing. Several options are available. For example, the graft can be seeded with hemangioblasts and / or endothelial cells to accelerate the formation of vascular elements once the modified tissue is in place. As another example, an angiogenic peptide can be introduced into modified tissue by an osmotic pump. Combinations of methods can also be used. Through the use of an osmotic pump, it is possible to deliver a peptide, or an angiogenic peptide or growth factor as described above, directly to a predetermined site in a biologically effective and cost-effective manner. VEGF delivered to the ischemic hind limb of rabbits accelerated capillary bed growth, increased vascular branching, and improved muscle performance for ischemic control. An alternative approach is to seed fully differentiated tissue constructs containing electroprocessed matrix material with additional endothelial cells and / or vascular fibroblasts just prior to transplantation in situ.

In some embodiments, the stem cells or other cells used to construct the graft are isolated from a subject in need of tissue reconstitution, or other compatible donor. This provides the advantage of using cells that do not elicit an immune response because the cells are from a subject that requires reconstitution (autologous tissue). A relatively small biopsy can be used to obtain a sufficient number of cells to construct a graft. This minimizes functional defects and damage to the endogenous tissue that functions as a donor site for the cell.

In some embodiments, the electroprocessed collagen matrix of the present invention includes a material in the matrix that improves the performance of the electroprocessed matrix to be implanted. Examples of substances that can be used include peptide growth factors, antibiotics, and / or anti-rejection drugs, anesthetics, analgesics, anti-inflammatory drugs. Alternatively, cells that have been modified to produce the desired compound can be included. The entire construct is cultured, for example, in a bioreactor or conventional culture, or placed directly in vivo. For example, angiogenesis can be stimulated by angiogenic factors and growth promoting factors administered as peptides, proteins or as gene therapy. Angiogenic agents can be incorporated into the electroprocessed matrix. Nerve growth factor can be incorporated into the matrix to promote the growth of neurons into the matrix and tissue. Various methods can be used to control the release of these factors into the transplant environment. In a degradable matrix, progressive degradation / disintegration of the matrix will release these factors and accelerate the growth of the desired tissue.

The electroprocessed collagen matrix can also be used in connection with other matrix construction processes. In other words, the extruded tube can have an outer layer electrospun onto it, where the different layers complement each other to provide a suitable matrix to promote specific types of cell growth. For example, a vascular graft composed primarily of collagen tubes can have an electrical span layer of both collagen and cells that are added to promote graft acceptance in a particular recipient. The second example is by growing fibroblasts in one layer, covering the first layer with electroprocessed collagen, and then growing a second layer composed of epidermal cells in a fibrin matrix. The in vitro skin preparation that is formed. This stratification technique can be used to create a variety of tissues.

Uses for Compositions of the Invention The electroprocessed collagen compositions of the invention have a number of potential uses. Uses include the manufacture of modified tissues and organs, including structures such as patches or plugs of tissue or matrix material, prostheses and other implants, tissue scaffolding, wound repair or bandages, hemostatic devices, sutures Materials, surgical and orthopedic screws, and devices for use in tissue repair and support, such as surgical and orthopedic plates, natural coatings or components for synthetic implants, cosmetic implants and aids Including, but not limited to, repair or structural support for organs or tissues, substance delivery, biomechanical platforms, platforms for testing the effects of substances on cells, cell culture, and numerous other uses . This discussion of possible uses is not intended to be comprehensive and there are many other embodiments. Although many specific examples are provided below regarding the combination of collagen with other electroprocessed materials and / or specific substances, many other combinations of materials and substances may be used.

Use of electroprocessed composition as tissue or organ augmentation or replacement The ability to combine cells in electroprocessed collagen material allows the composition of the present invention to be used to construct a tissue, organ, or organ-like tissue. Provide the ability to use. Cells contained in such tissues or organs can include cells that serve the function of delivering substances, seeded cells that provide the source of replacement tissue, or both. Many types of cells can be used to create tissues or organs. Stem cells, committed stem cells, and / or differentiated cells are used in various embodiments. Examples of stem cells used in these embodiments include, but are not limited to, embryonic stem cells, bone marrow stem cells, and umbilical cord stem cells used to make organs or organ-like tissues such as liver, kidney, etc. . In some embodiments, the shape of the electroprocessed composition grows and propagates in a particular type of desired manner, helping to send a signal to the cell. Other substances (eg, differentiation inducers) can be added to the electroprocessed matrix to promote specific types of cell growth. Further, various mixtures of cell types are incorporated into the composition in some embodiments. The ability to use electroprocessed collagen materials and matrices to biomanipulate tissue or organs creates a wide range of biomodified tissue replacement applications. Examples of bioengineered components include bone, dental structures, joints, cartilage, skeletal muscle, smooth muscle, myocardium, tendon, meniscus, ligament, blood vessel, stent, heart valve, cornea, tympanic membrane, nerve induction, Tissue or organ patches or sealants, other missing tissue fillings, cosmetic repair sheets, skin (sheets with cells added to make skin equivalent), pharynx such as trachea, epiglottis and vocal cords Other cartilage structures such as nasal cartilage, mortarboard, tracheal cartilage, thyroid cartilage, and arytenoid cartilage, connective tissue, vascular grafts and their components, and sheets for topical application, and liver, This includes, but is not limited to, repair or replacement of organs such as the kidney and pancreas. In some embodiments, such a matrix is combined with the drug and the substance delivery electroprocessed matrix of the invention to improve the function of the implant. For example, antibiotics, anti-inflammatory agents, local anesthetics, or combinations thereof can be added to the matrix of biomodified organs to speed up the healing process and reduce discomfort.

Electroprocessed collagen matrices have a number of orthopedic applications. Bone can be made by combining electroprocessed collagen with, for example, osteoblasts and bone growth factors. In some embodiments, the matrix can also contain a conductive material that allows an electric current to be applied to the implantation site to promote growth and healing. Optionally, the collagen may be genetically engineered to contain more P-15 sites than in naturally occurring collagen to accelerate the production of hydroxyapatite. Such bone prostheses can be used, for example, in joint repair and replacement, such as hip replacement, or to replace lost or deteriorated bone tissue. Cartilage may be manipulated by combining type II collagen with other matrix materials such as chondroblasts and proteoglycans. In some embodiments, synthetic hyaluronic acid that is not disrupted is used to promote hydration of the modified tissue. Optionally, an angiogenesis inhibitor can be included in the matrix to prevent neovascularization in cartilage. Such engineered cartilage can be used, for example, in spinal disc repair or replacement, cardiac fibrotic skeleton reconstruction, nasal or ear replacement or augmentation, or hip joint repair. The matrix can also be used to manipulate the dentin, for example by incorporating dentine blasts into the matrix. Ligaments (eg, including the knee meniscus, patella ligament, collateral ligament, cruciate ligament, rotator cuff, and hip acetabulum lip) can be prepared using fibroblasts in a matrix of elastin and collagen . In some embodiments, the extruded central core is prepared and modified by ammonia driven fibrillogenesis. The electroprocessed matrix can then be applied to the outer surface. Alternatively, the entire ligament can be formed by electrical processing. Optionally, slight twists can be created by means such as a rotating nozzle or air vortex. In some embodiments, a patient's damaged ligament can be ground in a crude mixture and subsequently sprayed as a new ligament, allowing the use of autologous tissue. Tendons (including, for example, the rotator cuff, the Achilles tendon, and the chord tendon), and muscle can also be prepared using an appropriate combination of cells and matrix materials. Combinations of tendons and muscles are possible. In some joint replacement applications, a synthetic graft may be coated with modified tissue to improve graft compatibility. In embodiments where growth into bone and / or cartilage joints is not desired, the matrix can be engineered to contain agents that inhibit growth.

In neurological applications, the matrix can be used as a filler for, for example, nerve induction, dural patches, seals of damaged nerves, damaged / removed areas of the brain lost during an accident or disease, and for cerebrospinal fluid leakage. It can be used to manufacture brain constructs as “dura” or “arachnoid” patches. Optionally, stem cells and nerve growth factors may be included in the matrix. The construct can also be supplemented with myelinated cells. In some neurological embodiments, it is desirable to use a PGA layer in or on the construct to limit cell infiltration to minimize or avoid fibrosis.

In cardiovascular applications, the matrix can be used to manufacture stents or to coat coated stents composed of synthetic materials, thus providing a hybrid stent that includes synthetic materials surrounded by natural materials To do. Natural materials may contain, for example, collagen and smooth muscle cells. Optionally, the DNA vector may be placed in such a matrix so that it migrates into the matrix upon remodeling of the scaffold formation or is taken up by cells seeded in the matrix. The matrix can be secondary processed into, for example, heart valves, leaflets, and prosthetic vessels for use as, for example, coronary artery bypass grafts. In many embodiments, the vascular prosthesis is prepared “simulated microgravity” in a bioreactor using an electroprocessed matrix material. For example, FIG. 6 shows an electrical spin prior to cell seeding. A photograph of a tempered matrix (left) and an arterial segment developed from scaffold formation (right) This approach provides a method of seeding cells into a construct in a low shear environment that provides high nutrient delivery. In some of the embodiments, the endothelial lining is seeded on the cells to create a graft having a stratified structure that is characteristic of natural blood vessels, which prevents the coagulation and blockage of small diameter arteries. The matrix is also used to replace damaged or cracked myocardium.Myocardial patches can be prepared. In particular, it has been observed that smooth muscle cells transplanted into the heart convert to myocardium, hi some embodiments, the matrix used as a myocardial patch allows for the vascularization of new tissue. Optionally, include nerve growth factor to cause tissue innervation, so that new tissue contraction is coordinated with other myocardium. The heart valve can be constructed with electroprocessed collagen, with or without cells, with the desired growth factors, but without cells. Built for in vitro, for example, a bioreactor, for valve leaflet preconditioning In some embodiments, to mimic the structure of a natural valve and provide a means of attachment and structural support, Make the ring around the edge thicker.

The matrix also has numerous uses on the skin and cosmetic applications include facial muscles and connective tissue. The matrix may be used as a dressing for any type of wound or trauma, including but not limited to skin abrasion or chemical peeling. Such a matrix electrotreated with fibrin can be included to promote hemostatic function and promote healing. The matrix material can also be injected as a wrinkle, scar, or wound filler. Stem cells, fibroblasts, epithelial cells, and / or endothelial cells can be included to provide tissue growth. By adding such a tissue plug, scarring is reduced. The matrix can also be used to prepare live augmentations, repairs and contourings such as lips, tympanic membrane, cornea, eyelid plate, forehead, eyelid, cheek, ear, nose, and chest. The use of a matrix may be combined with other methods of treatment, repair, and contouring. For example, collagen matrix injection can be used in conjunction with Botox (botulinum toxin) for the treatment of wrinkles. The matrix can also be shaped to act as a sling to support the sagging neck and sagging cheeks.

The electroprocessed collagen matrix can also be used to manufacture prosthetic organs or parts of organs. Mixing committed cell lines in a three-dimensional electroprocessed matrix can be used to produce structures that mimic complex organs. The ability to shape the matrix allows the preparation of complex structures to replace organs such as liver lobes, pancreas, other endocrine glands, and kidneys. In such cases, the cells are transplanted to exhibit cell function in the organ. Preferably autologous cells or stem cells are used to minimize the possibility of immune rejection. Alternatively, the cells can be placed in a matrix with a pore size that is small enough to protect the cells from immune surveillance while allowing nutrients to migrate to the cells.

In some embodiments, the matrix is used to prepare partial substitutions or enhancements. For example, in certain disease states, organs are scarred to the extent that they become dysfunctional. A typical example is cirrhosis. In cirrhosis, normal hepatocytes are trapped in the fibrous band of scar tissue. In one embodiment of the present invention, a biopsy is performed on the liver, live liver cells are obtained, cultured in an electroprocessed matrix, and a bridge or replacement for routine liver transplantation. Re-transplant to the patient.

In another example, glucagon-secreting cells, insulin-secreting cells, somatostatin-secreting cells, and / or pancreatic polypeptide-secreting cells, or combinations thereof, are grown in separate cultures and then together with materials that have been electroprocessed by electroprocessing By mixing these, an artificial islet is created. These structures are then placed under the skin, retroperitoneally, in the liver, or other desired location as an implantable long-term treatment for diabetes.

In other embodiments, hormone-producing cells replace anterior pituitary cells, for example, to affect the synthesis and secretion of growth hormone secretion (especially luteinizing hormone, follicle stimulating hormone, protactin and thyroid stimulating hormone). Used for. Gonadal cells such as Leydig cells and follicular epithelial cells are used to supplement testosterone or estrogen levels. Specially designed combinations are useful for hormone replacement therapy in postmenopausal and postmenopausal women, or men after endogenous testosterone secretion has declined. Dopaminergic neurons are used and transplanted in the matrix to recruit defective or damaged dopamine cells in the substantia nigra. In some embodiments, stem cells from a recipient or donor are mixed with slightly damaged cells, such as islet cells or hepatocytes, and placed in an electroprocessed matrix to stem cells to the desired cell type. Can later be collected to control differentiation. In other embodiments, thyroid cells can be seeded and grown to form small thyroid hormone secreting structures. This procedure is performed in vitro or in vivo. Newly formed differentiated cells are introduced into the patient. There are many other embodiments. Collagen is a critical structural element and numerous anatomical elements, and structures can be created, repaired or augmented using the matrix of the present invention. Several types of grafts can be prepared using information regarding tissue structure, histology, and molecular composition, all of which is available to those skilled in the art.

Other uses in medical devices and procedures Other uses of electroprocessed collagen constructs include occlusion or strengthening for occlusion against leakage. For example, an electroprocessed collagen matrix can be used to seal lung openings after lung volume reduction (partial removal). This use is important not only for hemostatic purposes, but also to prevent air leakage and pneumothorax into the pleural space. Electroprocessed collagen can also be formed as an aneurysm reinforcement or in a sleeve for use at the anastomosis site. Such a sleeve is placed over the area where reinforcement is desired and is sutured, sealed, or otherwise attached to the blood vessel. The matrix can also be used as a hemostatic patch and plug for cerebrospinal fluid leakage. Yet another use is as a punctal occlusion for patients suffering from dry eye syndrome. Another use is as a method of pregnancy by injecting a matrix into a duct or tube, such as the vas deferens or fallopian tube.

The matrix can also be used to support or bond tissue or structures that have undergone trauma, surgery or degradation. For example, the matrix can be used in a bladder neck suspension procedure for patients suffering from postpartum incontinence. Rectal support, vaginal support, hernia patches, and repair of uterine prolapse are other exemplary uses. The matrix can be used to repair or strengthen weakened or dysfunctional sphincters such as esophageal sphincters in the case of esophageal reflux. Other examples include strengthening and replacing tissue in the vocal cords, epiglottis, and trachea after removal, eg, in removing cancerous obstructive tissue.

Several uses are possible in the field of surgical repair or construction. For example, the matrices of the present invention are also used to make tissue or orthopedic screws, plates, suture systems, or syrants made of the same material as the tissue in which the device is used. In some embodiments, instead of other binding devices, it is used to apply the electroprocessed matrix directly to the body part of the organism to attach or bond tissue.

Diagnostic and Research Uses The electroprocessed collagen constructs of the present invention also allow in vitro culture of cells for research. The ability to mimic extracellular matrix and tissue conditions in vitro provides a new basis for cell research and manipulation. In some embodiments, selected cells are grown in a matrix and exposed to a selected agent, substance, or treatment. For example, cultures using tumor cells from cancer patients can be used to identify susceptibility to various types of chemotherapy and radiation therapy in vitro. In this way, alternative chemotherapy and radiotherapy treatments are analyzed to estimate the best treatment for a particular patient. For example, a modified tissue can be produced that contains collagen and cancer cells, preferably the patient's own cancer cells. Multiple samples of this tissue can then be subjected to multiple different cancer treatments. The results from different treatments can then be directly compared to each other for efficacy assessment.

Another use of an electroprocessed collagen matrix is as a biological manipulation platform for manipulation of cells in vitro. This application is similar to its use as a research and test platform in that it provides for the placement of cells in a matrix and treating the cells to manipulate them in a specific way. For example, stem cells can be placed in a matrix under conditions that control their differentiation. Differentiation is controlled by the use of matrix materials or substances in the matrix that affect differentiation. For example, an agent such as retinoic acid that plays a role in promoting differentiation may be placed in the matrix. In other embodiments, gene sequences associated with the differentiation process may be electrospun into the matrix. For example, when the transcription factor MYO D is transfected into fibroblasts or stem cells, the transfected cells begin to express muscle-specific gene sequences and differentiate the cells into skeletal muscle. The P15 site of type I collagen is associated with the induction of bone-specific gene sequences.

The compositions of the present invention are also useful for testing and applying various gene therapies. By linking with the composition of the present invention in vitro, various types of gene therapy and manipulation can be achieved by inserting a preselected DNA into a suspension of cells, materials, etc. For example, non-viral techniques such as electroporation are used to treat cultured cells prior to insertion into the matrix of the present invention. In other embodiments, the cells are treated in a matrix before the composition is inserted into the recipient. In vitro gene transfer avoids recipient exposure to viral products, reduces the risk of inflammation due to residual viral particles, and avoids the potential for germline virus integration. It avoids the problem of finding or manipulating viral envelopes that are large enough to allow large genes such as those related to factor VIII (antihemophilic factor). However, in vitro gene therapy is achieved in some embodiments by incorporating DNA into the electroprocessed material, for example when the electroprocessed material is created by the electroprocessing techniques of the present invention, thereby Some DNA will be incorporated into in vivo cells in contact with the composition after the composition is applied to the recipient. This is especially true for small gene sequences such as sense and / or antisense oligonucleotides. Another example is the use of matrices for cell culture and growth. Cells can be electroprocessed or otherwise inserted into a collagen matrix and placed in conditions that allow cell regeneration and tissue growth. Optionally, the matrix can be placed in a bioreactor.

Use of electroprocessed collagen matrix in substance delivery One use of the electroprocessed collagen composition of the present invention is the delivery of one or more substances to a desired location. In some embodiments, the electroprocessed material is simply used to deliver the material itself. In other embodiments, the electroprocessed material is used to deliver a substance contained in the electroprocessed material, or a substance produced or released by the substance contained in the electroprocessed material. Is done. For example, an electroprocessed material containing cells can be implanted into the body and used to deliver molecules produced by the cells after implantation. The composition can be used to deliver substances to an in vivo location, to an in vitro location, or to other locations. The composition can be administered to these locations in any manner.

In the field of substance delivery, the compositions of the present invention have many attributes that allow for the delivery of substances using a wide range of release profiles and release kinetics. For example, the choice of substance and how the substance is combined with the electroprocessed material affects the substance release profile. To the extent that the material is not immobilized on the electroprocessed collagen, the release from the electroprocessed collagen is a function of diffusion. An example of such an embodiment is where the substance is sprayed onto electroprocessed collagen. In the extent that the substance is immobilized on the electroprocessed material, the release rate is closely related to the rate at which the electroprocessed material decomposes. An example of such an embodiment is one in which the substance is covalently bound to electroprocessed collagen. If the substance is trapped within an electrospun aggregate or filament, the release kinetics will be determined by the rate at which the surrounding material degrades or collapses. Yet another embodiment is a substance that is bonded to the electroprocessed material by a photosensitive bond. Exposure of such bonds to light releases material from the electroprocessed material. Conversely, in some embodiments of the invention, the material can be exposed to light to cause binding of the agent in vivo or in vitro. Combining the compound with the electroprocessed material in solution rather than in suspension results in a different pattern of release, thereby providing yet another level of control over the process. Furthermore, the porosity of the electroprocessed collagen can be adjusted, which affects the release rate of the substance. Increased porosity facilitates release. The release of the substance is also increased by milling, fragmenting or grinding the electroprocessed collagen. The ground material can be applied, for example, to the wound site, ingested, or formed into another shape such as a capsule or tablet. In embodiments where the substance is present in the form of a large particle, such as a tablet encapsulated in an electroprocessed material, or a molecule trapped inside an electroprocessed filament, the release may dissolve or degrade the particle. Depending on the speed of the process and the complex interaction of the breakdown or decomposition of the electroprocessed material structure. In embodiments in which the substance includes cells that express one or more desired compounds, factors that affect cell function and viability and the timing, strength, and persistence of expression all affect release kinetics. Can affect. Chemicals that affect cell functions such as oligonucleotides, promoters, cell adhesion inhibitors, hormones, and growth factors can be incorporated into the electroprocessed materials and those substances from the electroprocessed materials The release of can provide a means of controlling cellular expression or other functions in the electroprocessed material.

The release kinetics in some embodiments is manipulated by crosslinking the electroprocessed collagen material by any means. In some embodiments, cross-linking increases structural stiffness and delays subsequent dissolution of the electroprocessed material, for example, the rate at which the electroprocessed collagen matrix degrades, or the compound is electroprocessed. Change the rate at which it is released. The electrotreated collagen matrix can be formed in the presence of a crosslinker or can be treated with a crosslinker after electrodeposition. Any technique for cross-linking materials, as known to those skilled in the art, may be used. Examples of techniques include the application of cross-linking agents and the application of specific cross-linking irradiation. Examples of cross-linking agents that work with one or more proteins include condensing agents such as aldehydes (eg, glutaraldehyde), carbodiimide EDC (1-ethyl-3- (3-dimethylaminopropyl)), Examples include, but are not limited to, photosensitive materials that crosslink upon exposure to specific wavelengths, osmium tetroxide, carbodiimide hydrochloride, and NHS (n-hydroxysuccinimide), and Factor XIIIa. Ultraviolet radiation is an example of radiation used to crosslink the matrix material in some embodiments. Natural materials can be cross-linked with other natural materials. For example, collagen can be cross-linked and / or stabilized by the addition of fibronectin and / or heparin sulfate. For some polymers, heat can be used to alter the matrix to crosslink the elements of the matrix by fusing adjacent components of the construct. The polymer may also be partially solubilized to alter the structure of the material, for example, short exposures of some compounds to alcohol or base will partially dissolve adjacent filaments and Both can be annealed. Some polymers may be cross-linked using chemical or thermal fusion techniques. Synthetic polymers can generally be crosslinked using high energy radiation (eg, electron beam, gamma radiation). These usually act on the creation of free radicals on the polymer backbone, followed by bonding and cross-linking. Skeletal free radicals can also be generated by peroxides, azo compounds, aryl ketones, and other radical producing compounds in the presence of heat or light. Redox reactions that produce radicals (eg, peroxides in the presence of transition metal salts) can also be used. In many cases, functional groups on the polymer backbone or side chain can react to form a crosslink. For example, polysaccharides can be treated with diaryl chloride to form diester bridges. Crosslinking may also occur after application of the matrix if desired. For example, the matrix applied to the wound may be cross-linked after application to enhance the adhesion of the matrix to the wound.

The release kinetics of the substance is also controlled by manipulating the physical and chemical composition of electroprocessed collagen and other electroprocessed materials. For example, PGA fibrils are more susceptible to hydrolysis than PGA's larger diameter fibers. Agents delivered within electroprocessed materials composed of small PGA fibers are released more rapidly than if prepared within materials composed of larger diameter PGA fibers.

In some embodiments, substances such as peptides can be released in a controlled manner at the localization domain. Examples include embodiments in which a substance is bonded to a chemically or covalently electroprocessed material. Peptide gradient formation is an important regulatory element of many biological processes, for example in angiogenesis. Physical treatment of the formed electroprocessed collagen matrix is another way to manipulate the release kinetics. In some embodiments, a mechanical force, such as compression applied to the electroprocessed material, can speed up the destruction of the matrix by changing the crystal structure of the material. Thus, the structure of the matrix is another parameter that can be manipulated to affect the release kinetics. Polyurethane and other elastic materials such as poly (ethylene-co-vinyl acetate), silicones, and polydienes (eg, polyisoprene), polycaprolactone, polyglycolic acid, and related polymers have a release rate that is mechanically strained. It is an example of a material that can be changed. Thus, the matrix containing these materials is controlled by physical manipulation.

Release kinetics can also be controlled by preparing a laminate comprising layers of electroprocessed material having different properties and materials. For example, a layered structure composed of electroprocessed collagen alternating with other electroprocessed materials can be prepared by sequentially electroprocessing different materials on the target. The outer layer can be adapted to dissolve faster or slower than for the inner layer, for example. Multiple agents can be delivered by this method, optionally at different release rates. The layers can be adapted to provide a complex multi-kinetic release profile of a single agent over time. Using the combination described above, it is possible to provide for the release of multiple release materials, each released with a complex profile.

Suspending a substance in particles incorporated into electroprocessed collagen or other electroprocessed material provides another means of controlling the release profile. The selection of these small particle matrix compositions provides yet another way to control the release of compounds from the electroprocessed material. The release profile can be adapted depending on the composition of the materials used in the process.

There are also embodiments in which the substance is contained in liposomes or other vesicles in an electroprocessed matrix. Vesicles that release one or more compounds are preferred when placed in a liquid at a specific pH range, temperature range, or ionic concentration. Methods for preparing such vesicles are known to those skilled in the art. The electroprocessed material can be immediately delivered to a given site, or stored in a dry state or at a pH at which release does not occur, and then delivered to a location containing a liquid having a pH at which release occurs. . An example of this embodiment is an electroprocessed material containing vesicles that release the desired compound at the pH of blood or other fluid released from the wound. The matrix is placed over the wound and releases liquid upon release of liquid from the wound.

Incorporating magnetically sensitive or electrically sensitive components into the electroprocessed collagen material provides another means of controlling the release profile. A magnetic or electric field can then be applied to some or all of the matrix to alter the shape, porosity and / or density of the electroprocessed collagen. For example, the field can stimulate matrix movement or structural changes due to the movement of magnetically or electrically sensitive particles. Such movement can affect the release of the compound from the electroprocessed matrix. For example, changing the structure of the material can increase or decrease the extent to which the material is suitable for compound release.

In some embodiments, a magnetically or electrically sensitive component treated or co-treated with electroprocessed collagen can be implanted subcutaneously to allow release of the drug over an extended period of time. Drug release is induced by passing a magnetic or electric field through the material. The electroprocessed collagen structure is stable and does not change substantially without electromagnetic stimulation. Such embodiments provide controlled drug delivery over an extended period of time. For example, an electroprocessed collagen material with magnetic or electrical properties and insulin can be manufactured and placed subcutaneously at an inconspicuous site. Insulin release can be induced by passing a magnetic or electric field through the composition. A similar strategy may be used to release compounds from constructs with photosensitive elements, and exposure of these materials to light will cause destruction of the materials themselves, or electroprocessed by photosensitive parts. It will cause the release of substances bound to the material.

In other embodiments, the substance comprises a vesicle encapsulated in a collagen material that has been electroprocessed with an electrical or magnetic material. The vesicle contains the compound to be released from the vesicle. When an electric or magnetic field is passed through an electroprocessed material, the compound in the vesicle is released, for example, by deforming the vesicle until it ruptures or by changing (possibly reversing) the permeability of the vesicle wall. obtain. Examples of these embodiments include transfection agents (eg, liposomes) that contain nucleic acids that increase the efficiency of the gene delivery process into the cell.

In other embodiments, the composition comprising electroprocessed collagen and substance is used as a transdermal patch for topical delivery of drugs, or topical delivery of components of a transdermal patch. In some of these embodiments, the conductive material is incorporated into such a composition and subsequently used as a component of an iontophoresis system in which one or more substances are delivered in response to the passage of current. The The conductive material can have a direct healing effect on bone damage. For example, passing a small current through the fracture site promotes healing. An electroprocessed bone mimetic that conducts or generates current can be created and placed in the fracture. The application of current promotes healing at a faster rate than the addition of the electroprocessed composition alone.

In other embodiments, the electroprocessed collagen material or portion thereof containing electromagnetic properties is stimulated upon exposure to a magnet and moves, thereby pressure sensitive containing molecules to be released from the material. Apply or release physical pressure to the capsule or other envelope. Depending on the embodiment, movement affects the release rate of the encapsulated molecule.

The response of the composition to electric and magnetic fields can be controlled by characteristics such as the composition of electroprocessed collagen and any other electroprocessed material, the size of the filaments, and the amount of conductive material added. it can. The electromechanical response from polyaniline is the result of doping induced volume changes, but the ionic gradient that causes the osmotic gradient is field induced in ionic gels such as poly (2-acrylamido-2-methylpropanesulfonic acid). Invite deformation. In each case, ion transport kinetics suppress the response and light transport is observed in the fibrils. Gel swelling and shrinkage kinetics has been found to be proportional to the square of the gel fiber diameter. Electromechanical response times for fiber bundles of less than 0.1 s are possible with typical muscles.

Embodiments involving delivery of molecules produced by cells provide many means by which rejection and immune responses against the cells can be avoided. Thus, embodiments with cells from the recipient avoid the problems associated with rejection of cells and inflammation and immunological responses. In embodiments where cells from organisms other than the recipient are used, the matrix can sequester the cells from immune surveillance by the recipient's immune system. Controlling parameters such as the pore size of the electroprocessed material or matrix allows nutritional support for cells trapped in the matrix, while protecting the cells from detection and response by the recipient's immune system Is done. For example, islet cells that produce insulin collected from a donor can be encapsulated in an electroprocessed matrix and transplanted to a recipient who cannot make insulin. Such an implant can be placed, for example, subcutaneously, in the liver, or intramuscularly. For some immune responses, permanent sequestration from the host system may not be necessary. The electroprocessed collagen material can be designed to shield the implanted material for a predetermined period and begin to break. In yet another embodiment, bacteria or other microbial agents modified to produce the desired compound can be used. This embodiment provides the advantage of using cells that are easier to manipulate than cells from the recipient or donor. The electroprocessed collagen material can also act to shield bacteria from the immune response in this embodiment. The advantage of using a bacterial carrier is that these microorganisms are easier to manipulate to express a wide range of products. Embodiments in which cells are transiently transfected can limit expression to a defined period of time. Transient genetic manipulation can restore the cell to its original state in embodiments where such reversion is desirable to minimize the risk of complications.

In some embodiments, the cells may be genetically engineered such that expression of a particular gene can be promoted or inhibited by various means known in the art. For example, a tetracycline sensitive promoter can be engineered into a gene sequence. The sequence is not expressed until tetracycline is present. Cell markers or bacterial markers can also be used to identify the insert material. For example, a green fluorescent protein placed in engineered genetic material emits a green color when expressed. Embodiments using this property allow confirmation of the viability of cells, bacteria or gene sequences in the matrix. The visibility of such markers also helps to recover the electroprocessed composition to be implanted.

While the present invention provides versatility in release kinetics, there are also embodiments in which one or more substances are not released at all from the electroprocessed collagen. The substance can function at the desired site. For example, in some embodiments, specific molecule antibodies are immobilized on an electroprocessed collagen matrix and the composition is placed at the desired site. In this embodiment, the antibody acts to bind molecules in the vicinity of the composition. This embodiment is useful for isolating molecules that bind to antibodies. One example is an electroprocessed collagen matrix that contains an immobilizing material that irreversibly binds to an undesirable enzyme, thereby inactivating the enzyme.

The compositions of the invention can be administered as a composition in vitro or in vivo in combination with a pharmaceutically acceptable or cosmetically acceptable carrier. Administration forms include injections, solutions, creams, gels, implants, pumps, ointments, emulsions, suspensions, microspheres, particles, microparticles, nanoparticles, liposomes, pastes, patches, tablets, transdermal delivery devices, These include, but are not limited to, sprays, aerosols, or other means familiar to those skilled in the art. Such pharmaceutically acceptable or cosmetically acceptable carriers are generally known to those skilled in the art. The pharmaceutical formulations of the invention can be prepared by procedures known in the art using known readily available ingredients. For example, the compound can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Suitable excipients, diluents, and carriers for such formulations include the following: fillers and bulking agents (eg, starch, sugar, mannitol, and silicate derivatives), binders (eg, carboxymethylcellulose and others) Cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone), humidifiers (eg, glycerol), disintegrants (eg, calcium carbonate and sodium bicarbonate), dissolution retardants (eg, paraffin), absorption enhancers (eg, , Quaternary ammonium compounds), surfactants (eg cetyl alcohol, glycerol monostearate), adsorbent carriers (eg kaolin and bentonite), emulsifiers, preservatives, sweeteners, stabilizers, colorants, flavoring agents, Flavors, lubricants (eg talc, calcium stearate Fine magnesium stearate), solid polyethyl glycols, and mixtures thereof.

The term “pharmaceutically acceptable or cosmetically acceptable carrier” or “pharmaceutically acceptable or cosmetically acceptable vesicle” refers to a living animal or human without causing an adverse physiological or cosmetic response. Water or saline, gels, creams, ointments, solvents, diluents, liquid ointments that are suitable for use in contact with tissue and do not interact with other components of the composition in a harmful manner Used herein to mean any liquid, solid or semi-solid (including but not limited to) bases, ointments, pastes, grafts, liposomes, micelles, giant micelles, etc. The Other pharmaceutically acceptable or cosmetically acceptable carriers or vesicles known to those skilled in the art may be used to make compositions for delivering the molecules of the invention.

The formulation may be configured to release the active ingredient alone, preferably at a specific location, possibly over an extended period of time. Such a combination provides an additional mechanism for controlling the release kinetics. The coating, envelope, and protective matrix can be made from polymeric materials or waxes, for example.

Methods for in vivo administration of the compositions of the invention, or formulations comprising such materials and other materials such as the carrier of the invention particularly suitable for various forms, include oral administration (eg, buccal or sublingual administration). ), Anal, rectal, suppository, topical, aerosol, inhalation, intraperitoneal, intravenous, transdermal, intradermal, subcutaneous, intramuscular, intrauterine, vaginal Administration, administration to a body cavity, surgical administration at the location of a tumor or internal injury, administration to the lumen and parenchyma of an organ, and parenteral administration. Techniques useful for the various forms of administration described above include topical application, ingestion, surgical administration, injection, spray, transdermal delivery device, osmotic pump, direct electrodeposition on the desired site, and familiar to those skilled in the art. Other means, but are not limited to these. The application site can be external, such as the epidermis, or internal, such as a gastric ulcer, a surgical area, or other location.

The electroprocessed collagen composition of the present invention is in the form of a cream, gel, solution, suspension, liposome, particle or other means known to those skilled in the art of formulation and delivery of therapeutic and cosmetic compounds. Can be applied. The size of the ultrafine particles of electroprocessed collagen material can be used for inhalation delivery of therapeutic agents. Some examples of formulations suitable for subcutaneous administration include, but are not limited to, implants, depots, needles, capsules, and osmotic pumps. Some examples of formulations suitable for vaginal administration include, but are not limited to, creams and rings. Some examples of formulations suitable for oral administration include, but are not limited to, pills, liquids, syrups, and suspensions. Some examples of formulations suitable for transdermal administration include, but are not limited to, gels, creams, pastes, patches, sprays, and gels. Some examples of delivery mechanisms suitable for subcutaneous administration include, but are not limited to, implants, depots, needles, capsules, and osmotic pumps. Formulations suitable for parenteral administration include aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostats, and solutes that are isotonic with recipient blood intended for the formulation, as well as non-aqueous. Sterile injectable solutions, as well as aqueous sterile suspensions and non-aqueous sterile suspensions that may contain suspending agents and thickeners are included, but are not limited to. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by those skilled in the art.

Embodiments in which the composition of the invention is combined with, for example, one or more “pharmaceutically acceptable or cosmetically acceptable carriers” or excipients can be conveniently provided in unit dosage form. Can also be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the composition containing the active ingredient and the pharmaceutical carrier (s) or excipient (s). In general, formulations are prepared by uniformly and intimately bringing a liquid carrier into association with an active ingredient. Preferred unit dosage formulations are those containing a dose or unit of the administered ingredient, or an appropriate fraction thereof. In addition to the ingredients specified above, it should be understood that formulations containing the compositions of the present invention may include other agents commonly used by those skilled in the art. The dose volume varies depending on the route of administration. For example, intramuscular injection can be in a volume range of about 0.1 ml to 1.0 ml.

The compositions of the invention can be administered to a human or animal to provide the substance at any dose range that produces the desired physiological or pharmacological result. The dosage depends on the substance (s) to be administered, the desired treatment endpoint, the desired effective concentration at the site of action or in the body fluid, and the type of administration. Information regarding appropriate doses of the substance is known to those skilled in the art and is described in L.L. S. Goodman and A.M. Gilman, eds, The Pharmacological Basis of Therapeutics, Macmillan Publishing, New York, and Katzung, Basic & Clinical Pharmacology, Appleton & Lang, Norwalk, Connecticut, may be found in references such as ⁽⁶ th Ed. 1995) . One desirable dosage range is 0.01 μg to 100 mg. Another desirable dosage range is 0.1 μg to 50 mg. Another desirable dosage range is 0.1 pg to 1.0 μg. A skilled clinician in the desired therapeutic art can select a particular dosage and dose range, as well as the frequency of administration, depending on the circumstances and substance to be administered. For example, a skilled clinician in the field of hormone replacement therapy may determine the specific dosage and dose range for a substance such as progesterone to be administered in combination with estrogen molecules and estrogen modulating molecules, and the frequency of administration, as the situation requires. Can be selected. For example, progesterone, and other progestins known to those skilled in the art, can be administered in amounts ranging from about 50 μg to 300 mg, preferably 100 μg to 200 mg, more preferably 1 mg to 100 mg. The particular dosage and combination of estrogen and estrogen modulating molecule and progesterone dosages will depend on the route and frequency of administration and the condition to be treated. For example, if the composition is formulated for oral use, preferably in a dosage unit form such as a capsule, each dosage unit may preferably contain 1 μg to 5 mg of estrogen molecules and estrogen modulating molecules, and 50 μg to 300 mg of progesterone. . U.S. Pat. No. 4,900,734 provides further examples of acceptable dose combinations of estrogen molecules and progestins.

Other Uses Including Electrically or Magnetically Active Ingredients The compositions of the present invention have many more applications besides substance delivery. There are embodiments in which the incorporation of an electrically or magnetically active component in the electroprocessed material causes the electroprocessed material to move periodically in response to an oscillating electric or magnetic field. Such electroprocessed materials can be used in left ventricular assist devices, for example by providing a pumping action or ventricular massage for the patent. Vibration can be achieved by passive motion of a magnetic or electric field with respect to a conductive material, or vice versa. By manipulating material selection, the electroprocessed material can be designed to remain permanently placed or dissolve over time, once the heart is fully restored, Eliminates the need for surgery to retrieve the device.

There are also embodiments that use the implanted electroprocessed material to conduct charge or current to the tissue. For example, an electrically active component promotes nerve ingrowth, stem cell differentiation, or manipulation muscle contraction, or an orthopedic application where the electroprocessed material is used as a carrier to reconstitute bone Can be electrically stimulated to promote bone formation. In one embodiment, for example, the electroprocessed material is applied to a bone injury site and used to apply an electrical current to the material to facilitate and promote healing. Application of small currents to damaged bone is known to accelerate or promote healing of bone damage.

In other embodiments involving magnetically responsive materials, the magnetic field places the electroprocessed material containing the substance by a relatively non-invasive means, for example by inducing movement of the material in the peritoneum. Used to let In other embodiments, compositions containing electrically active compounds are used to produce electric field driven cell migration. This approach accelerates the healing process and minimizes the risk of bacterial colonization. In one example, the orthopedic implant is coated with an ultrathin (less than 100 microns) layer of electrically active polymer. With a very thin electrode attached to the coating, an electric field can be applied via an external electrode so that after transplantation, the electric field induced cell migration is directed to the graft surface. If desired, the direction can be reversed. The field orientation depends on the implant and the structure of the external electrode.

In surgical applications, anti-vascular peptides or antisense oligonucleotides can be incorporated into electroprocessed materials, enclosing tumor perimeters that are difficult with conventional treatments to allow localized release and effect Or placed inside the tumor. The release of antivascular substances suppresses tumor growth. Antisense oligonucleotides can be released from the construct into the tumor and used to suppress a given expressed gene sequence. In another example, antisense sequences to gene sequences that control growth can be delivered inside a stent coated with an electroprocessed matrix. The stretches normally associated with stent placement initiate smooth muscle proliferation and antisense sequences designed to inhibit cell division reduce the deleterious effects of smooth muscle cell proliferation associated with the procedure. In another embodiment, the electroprocessed material delivers sense and antisense oligonucleotides to promote or inhibit the function of localized cells for a period of time. For example, antisense oligonucleotides are released from the electroprocessed material to suppress the expression of harmful enzymes in the wound. An example of such an enzyme is matrix metalloproteinase (MMP), which is often overexpressed in chronic wounds. In another example, the electroprocessed material applied to the wound releases a plasmid containing a nucleotide sequence encoding a tissue inhibitor of metalloproteinase (TIMP). Cells in the wound express TIMP resulting in local delivery of TIMP that inhibits MMP function.

Example 1
Fibroblast growth factor (FGF) release fibroblast growth factor from a graft composed of type I collagen, PGA and PLA (obtained from FGF, Chemicon, Temecula, Calif.) Type I collagen (80%), Dissolved in a solution of matrix material composed of PGA (10%) and PLA (10%). Ratio refers to the weight of materials relative to each other. These materials were dissolved in HFIP at a final concentration of 0.08 gm / ml. Sufficient FGF was added to 1 ml of the solution to provide a 50 ng / ml FGF concentration of the collagen / PGA / PLA electrospinning solution. The material was electrospun in the form of a cylinder onto the outer surface of a grounded spinning 16 gauge needle about 25-35 mm long. At the end of electrospinning, the material is removed from the needle and the electrospun cylinder is sutured closed by tightening the suture around the outside of the construct in a loop and enclosing the end by pulling tightly. It was. Similar results can be obtained by using hot forceps to pinch the ends together and just heat seal the ends. A hollow encapsulation construct was formed. The construct was then surgically implanted into the rat outer vastus muscle. The construct was left in place for 7 days and collected for inspection. The FGF in the matrix accelerated muscle formation in the electrospun matrix by promoting muscle formation inside the walls of the electrospun cylinder.

Example 2
Vascular endothelial growth factor (VEGF) -releasing vascular endothelial growth factor (obtained from VEGE, Chemicon, Temecula, Calif.) From graft material composed of type I collagen, PGA and PLA, as described in Example 1 Dissolved in a solution of matrix material composed of type I collagen (80%), PGA (10%), and PLA (10%). These materials were dissolved in HFIP at a final concentration of 0.08 gm / ml. Sufficient VEGF was added to 1 ml of the solution to provide a 50 ng / ml VEGF concentration of the collagen / PGA / PLA electrospinning solution. The material was electrospun to form a cylindrical construct and implanted into the rat muscle using the same procedure as described in Example 1. The construct was left in place for 7 days and collected for inspection. VEGF present throughout the construct increased the density of functional capillaries. This was evident from the presence of capillaries containing red blood cells (RBC).

Example 3
Release of VEGF from a construct composed of electroprocessed collagen, PGA and PLA VEGF-spun electroprocessed collagen and PGA: PLA copolymer constructs spun into VEGF-spun matrix, 80% collagen and 20% PGA: Prepared using PLA. Collagen and PGA: PLA were dissolved in HFIP at a final combined concentration of 0.08 gm / ml. Solutions were prepared by adding different amounts of VEGF to 1 ml of collagen and PGA: PLA copolymer solution. Separate electrospinning solutions were prepared containing 0 ng, 25 ng, 50 ng, and 100 ng of VEGF in 1 ml of electrospinning solution, respectively. Constructs were prepared for each solution by electrospinning 1 ml of material onto a cylindrical construct (2 mm diameter). The construct was removed from the target needle and cut in half. Next, one half of each electrospun sample was exposed to glutaraldehyde vapor fixation to crosslink the collagen fibers. Crosslinking was achieved by exposing the construct to glutaraldehyde vapor for 15 minutes. A sample of the electroprocessed construct was placed in a 100 mm tissue culture dish for vapor fixation. A 35 mm tissue culture dish containing 1 ml of 50% glutaraldehyde was placed inside the 100 mm tissue culture dish. The lid of the 100 mm tissue culture dish was replaced and the sample was left at room temperature for 15 minutes. Next, a vapor-fixed sample and a sample of unfixed electrospun construct were immersed in PBS and the amount of VEGF for uncrosslinked and cross-linked samples (expressed in picograms per mg of electrospun material). Measured at various times and are shown in FIGS. 7 and 8, respectively. The release of VEGF in PBS was measured as a function of time by ELISA. An ELISA kit for VEGF was purchased from Chemicon International (part number cyt214) and the ELISA was performed according to the instructions provided in the kit. Samples were centrifuged to remove particulate matter and stored at −20 ° C. until use.

7 and 8, the white diamonds represent release from fibers electrospun from a solution containing PGA: PLA copolymer and collagen without added VEGF. Open squares represent release from fibers electrospun from a solution containing PGA: PLA copolymer plus collagen with 25 ng of VEGF. Open circles represent the release from fibers electrospun from a solution containing PGA: PLA copolymer and collagen supplemented with 50 ng VEGF. Open triangles represent release from fibers electrospun from a solution containing PGA: PLA copolymer plus collagen with 100 ng of VEGF. The results demonstrate that not only does the matrix release VEGF in PBS, but cross-linking with glutaraldehyde delays release from the matrix.

Example 4
Electroprocessed collagen matrix: A mixture of islet cultured insulin secreting cells is seeded into an electroprocessed collagen matrix to form an electroprocessed collagen-containing tissue. The electroprocessed matrix containing insulin secreting cells is implanted into a diabetic recipient in need of insulin. This electroprocessed collagen or fibrin containing tissue optionally contains blood vessels. The matrix is implanted into the retroperitoneal space and the blood vessels are anastomosed to the hepatic portal circulation. Insulin is released from insulin-containing cells and transmitted to the circulation.

The electroprocessed matrix containing insulin-secreting cells is optionally supplemented with cells that synthesize and secrete glucagon, somatostatin, pancreatic polypeptide, or combinations thereof to mimic the hormone complementation of islets.

Optionally, heterologous cells (eg, engineered bacteria or cells from allogeneic donors) allow nutrients to diffuse into the cells, but the cells cannot be detected or inhibited by the recipient's immune system Alternatively, it is placed in a matrix having a delayed pore size.

Example 5
Keratinocytes are collected from healthy sites of patients suffering from an electroprocessed collagen matrix chronic wound for wound healing. Cells are grown in culture and transfected by electroporation to express VEGF. The transfected cells are then mixed or prepared in an electrospun collagen matrix. Antisense oligonucleotides for matrix methanoproteinase (MMP) are also spun into the matrix. A matrix is applied topically to the wound surface. Cells near or in the graft will incorporate antisense sequences and express their transfected gene sequences, reducing MMP production. In other applications, the cells may be genetically engineered to secrete VEGF, thereby promoting healing. Release of the antisense oligonucleotide suppresses the expression of MMP, which is normally overexpressed in chronic wounds. Thus, the wound site is repaired by an implant that simultaneously promotes the natural healing process. Optionally, the matrix is composed of fibrin or a mixture of fibrin and collagen. Fibrin helps stop bleeding and promotes healing.

Example 6
Electrospun collagen matrix for bone repair Osteoblasts from patients with bone injury are cultured and incorporated into an electrospun matrix containing type I collagen. The matrix is shaped into a cavity or defect shape at the damaged site. Bone growth factor (BGF), bone morphogenetic protein (BMP) or gene sequences encoding these proteins are electrospun into the matrix and optionally incorporated into the electrospun matrix. The matrix promotes new bone growth, and BGF or BMP in the matrix promotes bone growth.

Optionally, the collagen used is produced in vitro by genetically engineered cells that express a collagen polymer having more P-15 sites than in normal collagen. Excess P-15 sites promote osteoblasts to produce and secrete hydroxyapatite, further promoting bone growth.

Optionally, the matrix is further electroprocessed with polypyrrole, which is an electroactive material. An electrode is connected to each end of the implantation matrix. A charged electrode is later applied to the entire electrode surface to create a small current through the implant to further promote healing of bone damage. In another embodiment, the piezoelectric elements may be electrospun into the matrix to produce an electrical discharge that promotes healing.

Example 7
Electroprocessed collagen matrix: heart patch In this example, a heart patch is prepared. A sheet of electroprocessed material having aligned filaments of collagen is prepared. The sheet is folded into a pleated sheet in the desired shape and / or wound into a cylinder. The second construct is electrospun with the desired shape, eg, a rectangle. A pleat sheet mimicking an intact heart cell layer is inserted into an electroprocessed rectangular form. The construct is filled with cells, sutured closed and placed directly in the bioreactor or in situ. By aligning the fibrils of the pleated sheet of material that has been electrospun in parallel along the major axis of the outer rectangular shape, a muscle-like structure of the heart is obtained. Natural heart tissue is composed of a layer of cells aligned along a normal axis with an adjacent cell layer slightly spaced from the axis with an overcoat layer and an underlay layer. This construct is more accurately mimicked by the method described below in which the matrix is prepared and the cells are electroprocessed, dripped or sprayed directly onto the matrix as the matrix is prepared. Cells in contact with collagen fibrils aligned along the long axis of the sheet develop parallel to the underlying fibrils of the sheet and are aligned and layered in an in vivo-like organized pattern Form. The construct can be implanted directly or placed in an RCCS bioreactor. Rotational speeds for maintaining this type of construct in suspension in the RCCS bioreactor range from 4-20 rpm, depending on the tissue mass and the specific material used to produce the outer cylinder. is there. Variations on this design include the addition of angiogenic factors in the matrix, gene sequences, and agents that suppress inflammation and / or rejection. Other cell types, such as microvascular endothelial cells, may be added to the construct to accelerate the formation of the capillary system within the construct. Other variations on this design principle can be used. For example, cells may be electroprocessed into the matrix as the matrix is deposited on a grounded target. By changing the fiber pitch during the spinning and spraying process, dripping or electrotreating the cells onto the fibers as the fibers are deposited very accurately controls the positioning of the cells within the construct.

Example 8
Electrospinning of type I collagen Type I collagen was used (calf skin, Sigma Chemical Co.). Collagen was suspended in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) at a concentration of 0.1181 g in 3 ml of HFIP. Once in solution or suspension (milky white solution), the solution was filled into a 1 ml syringe plunger. Next, a 15 gauge luer stub adapter was placed in the syringe to act as an electric spinning nozzle and charging point for the contained collagen solution. The filled syringe was placed in a syringe pump (KD Scientific) set, and the solution was distributed at 18 ml / hr using a 1.0 ml syringe plunger (Becton Dickinson). A positive lead from a high voltage supply was attached to the metal part of the lure stub adapter. The syringe pump was turned on. The high voltage supply was activated and set to 20 kV. The grounding target was a 303 stainless steel mandrel (width (W) 0.6 cm × height (H) 0.05 cm × length (L) 4 cm) disposed approximately 6 inches from the tip of the adapter. During the spinning process, the mandrel was rotated at approximately 500 rpm. About 1 ml of the collagen solution was electrospun to form a white mat on the ground axis. After electrospinning, the collagen mat was removed from the mandrel and processed for a scanning electron microscope. The mat produced was approximately 200 microns thick.

Transmission electrons revealed a collagen fiber diameter of approximately 100 nm and a typical 67 nm stripe formation characteristic of natural collagen polymerization.

Example 9
Electrospinning of collagen and elastin The method of this example was the same as Example 8 except for the solution to be electrospun. In this case, the electrospinning solution consisted of 0.1155 g collagen, 0.1234 g elastin from Fluka and 5 ml HFIP. Approximately 2 ml of the suspension was electrospun to form a mat. The mat produced was approximately 50 microns thick.

Example 10
Electrospinning of Type I Collagen, Type III Collagen, and Elastin The method of this example was the same as Example 8 except for the solution that was electrospun. In this example, the electrospun solution consisted of 0.067 g type I collagen, 0.067 g type III collagen, 0.017 g elastin from the ligament of the ligament, and 2 ml HFIP (44% type I, type III 44%, elastin 12%). This ratio is similar to that found in natural artery wall tissue. The mat produced was approximately 100 microns thick.

Example 11
Collagen (type I and type III) electrospinning, crosslinking, structural analysis, and strength testing acid soluble lyophilized collagen was used in all experiments. Unless otherwise noted, all reagents were purchased from Sigma Chemical Company (St. Louis, MI). For this study, type I collagen from calf skin, type I and type III collagen isolated from human placenta were used. Collagen was dissolved at various concentrations in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The collagen suspension was placed in a 1 ml syringe equipped with a syringe pump (Model 100, KD Scientific Inc., New Hope, Pa.). The syringe was capped with an 18 gauge smooth end needle. A positive lead from a high voltage supply (Spellman CZE1000R, Spellman High Voltage Electronics Corp.) was attached to the outer surface of the metal syringe needle via an alligator clip. A cylindrical (width (W) 0.6 cm x height (H) 0.05 cm x length (L) 4 cm) grounding target made from 303 stainless steel was deployed 4-6 inches from the syringe tip. At the start of electrospinning, a syringe pump was set up to deliver the source solution at various rates from 0 to 25 ml / hr. At the same time, a high voltage was applied (15-30 kilovolts (kV)) through the source solution and grounded target mandrel. Unless otherwise noted, the mandrel is approximately 500 r. p. rotated at m. In summary, during the electrospinning process, collagen isotype and concentration, imposed voltage, air gap distance, and flow rate were examined. Type I collagen derived from calf skin was electrospun onto a 4 mm diameter cylindrical culture substrate (length = 1 cm). The construct was cross-linked in glutaraldehyde for 24 hours at room temperature and then rinsed with several modifications of phosphate buffered saline.

Example 12
Preliminary testing of various conditions for electrospinning collagen identified HFIP as the preferred solvent for electrospinning collagen. HFIP is a volatile solvent having a boiling point of 61 ° C. Electrospinning of collagen fibers showed concentration dependence on the final fiber diameter produced. For example, it was readily soluble in HFIP at a concentration of 0.008 g / 1.0 ml acid extract of type I collagen (calf skin). However, at this concentration, collagen did not show any evidence of electrospinning (fiber formation), and regardless of the input voltage, the polymer solution formed droplets and leaked from the syringe tip. Increasing the collagen concentration 10-fold to 0.0083 g / ml resulted in a hazy solution, resulting in fiber formation during electrospinning. These fibers assembled as a non-woven mat on the target mandrel. Further increases in collagen concentration in the source solution did not significantly change fiber formation.

Next, the voltage input parameters were examined. After suspending type I collagen in HFIP at 0.083 g / ml, a voltage varying from 15 to 30 kV in increments of 2.0 kV was applied. Fiber formation was most pronounced at 25 kV (electric field scale = 2000 V / cm). Changing the air gap distance between the source solution and the grounded target at this input voltage significantly affected the electrospinning process. The optimum air gap distance was approximately 125 mm. Collagen fibrils can be spun over substantially shorter air gap distances, but these fibrils retained significant solvent and assembled on the target in a wet state. When the air gap distance exceeded the critical spacing of 250 mm, the spun fibers could not collect on the target mandrel.

The electric field generated by the electrospinning process was sufficient to extract the collagen source solution from the syringe reservoir. However, it is possible to produce a more uniform collagen mat by metering the rate at which the collagen source solution is delivered to the electric spinning field by the syringe pump. Fibrosis was optimal when the collagen source solution was delivered to the electric field at a rate of approximately 5.0 ml / hr. At higher delivery rates, fiber formation was not consistent.

The rotation of the target mandrel also affected the collagen deposition. 500r. p. m. Collagen fibrils electrospun onto a mandrel rotating at a speed of less than resulted in a random porous matrix of filaments. The rotational speed of the mandrel is 4,500 r. p. m. Increasing (the mandrel surface moving at approximately 1.4 m / sec) resulted in the deposition of fibrils in a linear parallel array along the axis of rotation. Transmission electron microscopic analysis of aligned and non-aligned fibrils revealed that all of these filaments exhibited 67m streaking that is characteristic of natural collagen.

The stress strain profile of this type of electrospun collagen scaffold was measured. Type I collagen derived from calf skin was converted to 4,500 r. p. m. Electrospinning was performed under optimal conditions on a rectangular target mandrel rotating in The scaffold formation was removed from the target and made into a 25 mm (length) x 25 mm (width) sheet. Under the conditions used to produce this matrix, scaffold formation averaged a cross-sectional area diameter of 0.187 mm. Duplicate samples were cut into strips that were parallel or perpendicular to the basic axis of rotation of the mandrel. This approach allowed examination of how to adjust the material properties of the matrix where the local fiber orientation was electrospun. A phase material test parallel to the basic axial fibril alignment showed an average load of 1.17 ± 0.34 N at 1.5 ± 0.2 MPa beak stress. The average modulus for the vertical axis sample was 52.3 ± 5.2 MPa. In the transverse fiber orientation, the peak load at break was 0.75 ± 0.04 N, and the peak stress was 0.7 ± 0.1 MPa. The modulus across the fiber long axis was 26.1 ± 4.0 MPa. These data show that the local orientation of the fibers that make up the electrospun scaffold formation directly adjusts the material properties of the manipulation matrix. The incorporation of varying degrees of crosslinking into this type of nonwoven matrix can be used to further adapt the material properties of the matrix to a specific application.

The uniqueness and source of collagen was studied for its impact on the electrospinning process. Type I collagen isolated from human placenta was electrospun using conditions optimized for type I collagen in calf skin. For calf skin collagen, electrospinning of this material resulted in a less uniform fiber matrix. Individual filaments ranged from 100 to 730 nm in diameter. 0.0 used in these experiments
83 g / ml of collagen appears to represent the critical transition concentration for type I collagen when isolated from placenta. Increasing the concentration of human placental collagen present in the source solution (increasing the viscosity of the electrospinning source solution) and keeping all other variables equal would favor the formation of larger diameter fibers It is. Conversely, decreasing the concentration of the source solution (decreasing the viscosity of the electrospinning source solution) decreases the average filament diameter, resulting in a matrix composed primarily of 100 nm fibers.

The primary sequence of collagen directly affects the formation of this polymer in the electrospinning process. A preliminary attempt to electrospin type III collagen was at an approximate concentration of 0.04 g / ml (HFIP) where the optimal concentration of this peptide was less than 50% of the optimal concentration of type I (calf skin). It showed that there is. Using all other conditions optimized for electrospinning calf skin type I collagen, electrospinning type III collagen at 0.04 g / ml (HFIP) yields an average diameter of 250 ± 150 nm. Resulting in a fiber with The relative ratio of type I collagen to type III collagen in natural ECM affects the structural and functional properties of collagen-based networks. Blending optimal concentrations of type I human placental collagen (0.08 g / ml (HFIP)) and type III human placental collagen (0.04 g / ml (HFIP)) in a 50:50 ratio (final collagen concentration 0. 06 g / ml) affected the formation of fibrils during the electrospinning process. Under electrospinning conditions optimized for calf skin type I collagen, the blended material formed a scaffold composed of fibers averaging 390 ± 290 nm in diameter (FIG. 10).

Cross-linking was also studied. Regardless of the amount of time the collagen mat was exposed to UV light, all samples dissolved immediately when placed in warm media. Glutaraldehyde exposure resulted in somewhat different results. While mats of 1, 2, and 5 minutes dissolved in warm media, the rate at which dissolution occurred was slower than any UV-exposed mat at the time interval studied. The 10 and 20 minute mats became somewhat clear when placed in the medium, but did not dissolve. The mat exposed to glutaraldehyde overnight appeared to be somewhat darker than when it was first placed in the chamber. It had negligible compliance and did not dissolve when placed in the medium or showed signs of any low thickening. Exposure of 45:35:20 (collagen type I, type III, elastin) mat to glutaraldehyde for more than 10 minutes results in sufficient crosslinking to render the mat insoluble.

Example 13
Biological properties of electrospun collagen The biological properties of electrospun collagen were examined in tissue culture experiments. Aortic smooth muscle cells were suspended in an RCCS bioreactor and plated on various formulations of electrospun collagen. The low shear, high nutrient environment provided by the RCCS bioreactor promotes cell-matrix interactions and the formation of large-scale tissue mass in vitro. Microscopic examination of these cultures revealed that the scaffolds were confluent with smooth muscle cells within 7 days. Cross-sectional analysis showed that electrospun collagen promoted extensive cellular infiltration into the fibril network. Smooth muscle cells were observed deep within the matrix and were completely entangled within the electrospun collagen fibrils.

Example 14
In vivo biocompatibility of electrospun collagen-elastin matrix To study in vivo material biocompatibility, electrospun 80:20 type I collagen / elastin matrix was treated with SD rats. (Charles Riv
er) were implanted in both hind legs for 2 weeks. Female rats used in this study weighed approximately 200 g. Cylindrical constructs were implanted to test the performance of this particular manufacturing base. Prior to surgery, rats were anesthetized by intravenous injection of pentobarbital (5-7 mg / 100 g body weight). An incision was made with a length of 10-15 mm along the side of the hind leg just above the lateral vastus. A small channel was prepared in the muscle belly by smooth incision. An electrospun collagen / elastin construct was placed in the channel. Endogenous muscles were re-stitched together on the graft material, the skin incision was repaired, and the area was irrigated with betadine. Fourteen days later, the animals were sedated by intravenous injection of pentobarbital and sacrificed by bilateral pneumothorax. Lung collapse was confirmed visually and the transplanted tissue was harvested for histological evaluation. For histological specimens, the tissue was fixed in half strength Calfonsky fixative for 24 hours, embedded and cut thick.

In order to confirm cell invasion in vivo, blood vessels were observed under SEM. Removal of blood vessels from a rat model after 1 week revealed cell migration, invasion, and coverage similar to blood vessels after 2 weeks in this culture. There was no inflammation (swelling) in or around the transplant area. Rats also showed fluid movement around the cage. There was no visible encapsulation of collagen fibers in the matrix, but rather almost the entire vascular matrix was covered with rat-derived skeletal muscle cells.

Example 15
SEM examination of a three-layered type I / elastin 80:20 matrix cultured with cell infiltrating smooth muscle cells in vitro in an electroprocessed matrix revealed significant cell attachment and including three-dimensional cell migration to the matrix Maturity was revealed. Only one week later, there was visible cell migration and attachment as shown by scanning electron microscopy (FIG. 11).

After 2 weeks in the bioreactor, cell maturation became much more pronounced. The cells appeared to form a confluent layer across the luminal area of the tube. SEM of the cross section of the tube showed cell migration and remodeling in the central region of the wall. No cell migration to the outer wall region of the scaffold was observed in the 2 week tube. However, the central region of the wall was completely infiltrated prior to covering the luminal region.

After 3 weeks in culture, a fully confluent smooth muscle cell layer was formed across the luminal surface of the blood vessel. Cell migration towards the outer region of the blood vessels increased over the 2 week sample. Some immature cells were still visible on the luminal surface, but no collagen fibers were seen.

Under histological staining (H & E, and three colors), cell migration was seen in the 2nd and 3rd week samples. While matrix reconstruction was evident in the 2 week sample, matrix reconstruction was much more pronounced in the 3 week sample. This reconstruction appeared to be more luminal, as some layered layers of matrix were observed in the area. In addition, the 3 week sample showed smooth muscle cell (SMC) infiltration throughout, as evidenced by equal cell density across the sample wall.

Example 16
Demonstration of ligament design To determine the feasibility of ligament design, 4.0 ml of type I collagen 96% and elastin 4% were electrospun onto a 25 x 25 mm square mandrel. The sample was electrospun using the same method as described in Example 8. The resulting mat was then fixed in a seeding chamber and seeded with fibroblasts for 3 hours. Next, the seeded mats were subjected to cell culture for one week to infiltrate the cells. After one week of culture, the mat was held at both ends using a sterile Teflon clamp and rotated one end clockwise until resistance was felt. Additional fibroblasts were seeded on twisted mats for 3 hours in the seeding chamber. The matrix was then removed and spun cell culture for another week, after which the matrix was fixed for SEM and histology.

Demonstration of cartilage design For the study of cartilage design, the feasibility of chondrocyte growth on electrospun type II collagen was determined. First, a 100 mg type II collagen sample was electrospun into 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a concentration of 0.10 g / ml. The sample was electrospun using the same method as described in Example 8. Once the entire 1 ml volume of type II collagen solution has been spun, the resulting electrospun mat is removed from the mandrel and fixed in 3% glutaraldehyde gas and shown by scanning electron microscopy. A fixed type II collagen matrix was formed as shown in FIG. Next, a solution of chondrocytes suspended in the medium (1,000,000 cells) was added to the matrix and placed in the seeding chamber for 3 hours. The mat was then removed from the chamber and cultured for 1 week and 2 weeks, respectively, in a rotating culture cell system. The week 1 sample showed almost complete coverage on the seeded side and some remodeling in the mat (FIG. 13).

Demonstration of Trilayer Prosthesis Design In an attempt to closely mimic the physiological protein distribution of a small diameter vascular matrix, 3 ml of a collagen type I / elastin 80:20 mix was produced at a concentration of 0.083 g / ml and tubular (inner diameter) 4 mm) was electrospun onto the mandrel, removed and fixed with 3% glutaraldehyde gas. The sample was electrospun using the same method as described in Example 8. Similar to the physiological vessel wall, this layer acts as the outer wall of the prosthesis to be seeded with fibroblasts and smooth muscle cells. The tube was closed with cut gut sutures closed at both ends, and the outer surface was seeded with fibroblasts and placed in a rotating cell culture system. After 4 days of culture, the tube was removed, one end was unwound, and the suspended smooth muscle cell solution was injected into the lumen. The resealed end was then tied with suture and the tube was cultured for 4 days. While the first tube remained cultured, a second tube composed of 70% elastin and 30% type I was electrospun onto a mandrel having an inner diameter of 2 mm. After fixation, a tube with an inner diameter of 2 mm was slid into a tube with an inner diameter of 4 mm, and the two tubes were subjected to rotary cell culture for 3 days. This was done to ensure SMC migration to the smaller tube. After 3 days of culture, human umbilical vein endothelial cells were injected inside a tube with an inner diameter of 2 mm and further cultured for 2 days. The resulting prosthesis was then removed and fixed for histology.

Example 17
Aqueous electroprocessed matrix of nano-sized fibers In this embodiment, electroprocessed rat tail collagen was delivered using water as the solvent carrier. In order to increase the volatility of water, electrospinning was performed in vacuum. The device consisted of a standard electrospinning device consisting of a source solution (water / collagen), a high voltage source (30,000 V), and ground.

The vacuum flasks were arranged in a line and connected to a direct pump. The chamber used for electrospinning was capped with a rubber stopper. A 1 ml syringe equipped with a 30 gauge needle was passed through the port prepared in the stopper. A two-way stopcock was placed between the syringe source and the syringe. The syringe port was closed and the pump was started. The chamber was pumped to vacuum for 5-10 minutes. The total volume of the two vacuum flasks was approximately 6.25 liters. Once equilibrium was established, the hose connecting the pump to the first vacuum chamber was sealed with a clamp. The stopcock on the syringe was opened, and the collagen solution (5 mg / ml collagen) was charged to 20 to 25 kV. The solvent initially dropped from the syringe source, and when the voltage reached a critical level, a transient formation of the tailor cone was seen followed by the formation of fibers. The electrospinning process stopped when the water evaporated and the vacuum dissipated. SEM has demonstrated that the fibers appear to show some fine substructure and striped periodicity evidence. The selected fibers had a total linear length close to mm, and the fibers were on a scale of less than a micron in diameter.

Example 18
Development of tissue engineered heart valves and leaflets utilizing electrospun matrices A polymeric matrix is formed directly on the heart axis (mold) to produce a heart valve or leaflet. A reservoir with attached micropipette tip (nozzle) is filled with collagen and polymer solutions and placed at a distance from the grounded target. The ground target is a metal mandrel (non-stick surface). A fine wire is placed in the solution in each pipette tip (spray nozzle) to charge the polymer solution or melt to a high voltage. At a specific voltage, the polymer solution or suspended melt in the pipette tip is directed from the pipette tip towards the grounded target (cardiac axis). This solution flow (spray) begins as a monofilament, which is converted into a multifilament between the pipette tip and the grounded target (electric field driven phenomenon). This allows the production of “web-like” structures and accumulates at the target site. Upon reaching the ground target, the multifilaments assemble and dry to form a three-dimensional continuous polymer matrix (fabric). In experiments to test the effectiveness of this approach, this technique was used to biodegradability of polylactic acid / polyglycolic acid (PLA / PGA, 50/50) polymer and poly (ethylene-co-vinyl acetate). Filaments were produced, both of which were dissolved in methylene chloride.

Electrospinning of polymer scaffolding for heart valves and heart leaflets can be random in orientation, but is usually produced in a specific orientation as described below. If the desired fiber orientation is longitudinal, the mandrel / ground target is moved perpendicular to the polymer spray. This arrangement allows the direct production of a tubular matrix composed mainly of fibers that are oriented specifically (unidirectional).

The specific orientation in this case is any desired pitch with respect to the main axis of the ground target. This orientation is controlled by simultaneous rotation and longitudinal movement of the mandrel / ground surface. This allows the production of a matrix composed of a specific pitch or a series of multiple pitches (cross-hatched arrangement).

When utilizing biodegradable materials, substances such as nucleic acids (vectors) are optionally added to the electrospinning polymer solution for incorporation into the scaffold. Upon consumption / reconstruction of scaffold formation by seeded cells, the cells incorporate the vector into their DNA (ie, genetic engineering) to produce the desired effect. Growth factors or other substances are optionally incorporated into the electrospun matrix to facilitate tissue regeneration / healing.

Example 19
Electrospin coating of stents with polymer solution For this example, polymers in 1/7 wt / vol HFIP (95% PGA and 5% PCL) were used. The applied voltage was 24.5 kV at the syringe tip to the stent surface (Palmaz-Schatz stent) at a distance of 7 inches. Spinning was performed for approximately 20-30 seconds. For spinning, the stent was deployed with a 25 gauge needle through the center and bent at 90 ° at the end to hold the stent in place on the needle during electrospinning and rotation of the stent / cardiac axis. In order to obtain a uniform distribution of the coating, a plastic base of the syringe was fastened to the end of the 4 mm diameter mandrel on the rotating spinning device during electrospinning. Stent deployment was performed by placing two 25 gauge needles through the stent lumen and physically pulling the stent open. The stent was uniformly coated with PGA-PCL fibers, and its diameter was 83% larger than before deployment.

Example 20
Electrospun collagen coated stent procedure A 25 gauge needle was inserted through the center of the stent (Palmaz-Schatz stent). The tip of the needle was bent 90 ° to ensure that the stent was maintained on the needle during rotation of the mandrel. Subsequently, the needle and attached stent were attached to the end of a metal mandrel extension (inner diameter 2 mm). After preparing the stent, 0.080 g of collagen (rat tail type I) was placed in 1 ml of HFIP solution. Next, the collagen solution was put into a 1 ml syringe, and an 18 gauge smooth needle was attached. The sample was electrospun using the same method as described in Example 8. Approximately 400 μl of the solution was electrospun onto the stent. The stent was then removed from the needle and studied under SEM. The results were compared in FIGS. 14 and 15. Electrospun collagen fibers coated the stent.

Example 21
Vascular tree manipulation:
The electrospun fiber is formed on an arbitrarily shaped mandrel. Therefore, a blood vessel tree having a plurality of branches that are one continuous structure that is seamless is produced. An example is electrospinning of a coronary bypass vascular tree for a quadruple bypass procedure. The mold may be custom made (3D custom made biomimetic structure) or the vascular tree may be designed to average population specificity with various available sizes. it can.

Seamless rat aorta-iliac bifurcation grafts were produced from polyethyl vinyl acetate. In this example, the mold was formed from a stainless steel rod and a paper clip. The polymer was then electrospun to form a seamless bifurcation around the mold. The bifurcation was then slid from the mandrel as one seamless construct.

Example 22
Electrospinning for biomedical manipulation applications: muscle biomanipulation electrospinning studies PGA, PLA, PGA: PLA, and collagen were prepared by suspension in HFIP (Sigma, St. Louis) (per volume). 7-8% by weight). Various polymer solutions were filled into 1 ml syringes, charged to 20 kV, and directed to a rotating grounded mandrel through an air gap of 25-30 cms. The construct used for this study was prepared on a 14 gauge needle and its length was 20-25 mm. The cylindrical construct was removed from the mandrel and sterilized in 70% alcohol before use.

Cell isolated newborn rats were sacrificed and the skin removed. Skeletal muscles were excised from the limbs and thorax, subdivided, and subjected to 12 hours collagenase digestion (200 units / ml collagenase activity, Worthington Biochemical, NJ). At the end of digestion, the tissue is cannulated and filtered through a 100 micron mesh filter and diluted in DMEM-F12 supplemented with 10% horse serum (Sigma, St. Louis) and 5% fetal calf serum (Mediatech, VA). did. Fibroblasts were partially purified from the isolate by a 1 hour cycle of two differential adhesions. Partially purified myoblasts were suspended in collagen (1 mg / ml) and placed on electrospun cylindrical constructs. Isolated cells in cylindrical constructs were cultured for 24 hours in a Synthecon rotating wall bioreactor (Houston, TX) prior to transplantation.

Surgical Procedure Adult SD rats (150-200 gm) were anesthetized with pentobarbital. In addition, the second half was taken out. The skin was washed with betadine and an incision was made in the lateral vastus abdomen. A cylindrical construct was placed in the blind channel. One side of the muscle was sutured over the graft to repair the skin incision. The entire area was then wiped twice with betadine. After 7 days, the grafts were harvested and prepared for histological and ultrastructural examination. All tissues were routinely fixed in Calfonsky fixative for 24 hours prior to routine processing for optical or transmission electron microscopy.

Pre-graft experiments were performed to characterize the biophysical properties of electrospun PGA, PLA, PGA: PLA, or type I collagen and PGA: PLA blends. Adult SD rats (150-200 gm) were anesthetized and a small channel was prepared in the outer vastus medius. Cylindrical constructs composed of electrospun PGA, PLA, PGA: PLA blend (50:50), or type I collagen: PGA: PLA blend (80:10:10) (electric on 14 gauge needles) Spinned, approximately 25 mm long) was prepared and placed in the channel. The distal end of the electrospun construct was sutured closed to form a sealed cylinder. The construct was then placed in the prepared channel. Blending electrospun collagen with a small amount of PGA: PLA produced a matrix of substantial material strength that allowed for suturing. The electrospun matrix simultaneously functions as a fascia sheath and tendon insert for bioengineered muscle. Once the graft was in place, the muscle was sutured over the graft and closed to repair the skin incision. The purpose of these experiments was to determine the extent to which the various materials integrate and allow infiltration of cells from the surrounding tissue into the construct. One week later, samples were collected and prepared for microscopic evaluation.

The PGA construct was not well integrated into the host tissue. Fiber capsules were evident at the interface between the graft and the host's surrounding muscle tissue. Large circular multinucleated cells considered to be macrophages were evident in the domain just below the fiber capsule. Some cells invaded beyond the perimeter of the construct. The central domain of the graft was occupied by the rest of the matrix and was almost devoid of cells. Constructs composed of PLA did not promote the formation of significant fiber capsules that characterize PGA grafts. These grafts showed a smooth transition zone due to the interface between the construct and the host tissue. Cells were scattered throughout the cylindrical graft. Small diameter vessels were observed within the PLA-based matrix. The subpopulation of cells within the PLA-based graft appeared to be intensely stained with osmium tetroxide and accumulate lipids. The biophysical properties of the construct composed of a 50/50 mixture of PGA: PLA were intermediate. The fiber zone at the edge of the graft was evident. Although several large multinucleated cells were present in the transition zone, the PGA: PLA mixture did not appear to promote the accumulation of these cells to the same extent as the PGA-based construct. A few cells invaded the central core of the construct and there were no large osmium positive cells.

In contrast to these results, grafts made from type I collagen: PGA: PLA fiber blend (80:10:10) were confluent with cells from the surrounding tissue. There were no signs of fiber capsules in the transition zone. A number of blood vessels were present throughout the construct. Cells within the electrospun matrix were stretched and oriented parallel to the local axis of the electrospun matrix. The central domain of the graft was crowded with cells. A collagen-based matrix was fully incorporated into host tissue and supported the formation of capillaries and small arteries.

Next, the type I collagen-based matrix was studied to determine whether the type I collagen-based matrix would serve as a basis for delivery of endogenous cells to the host tissue. Cylindrical constructs composed of a 50:50 mixture of electrospun type I collagen: PGA: PLA (80:10:10) or PAG: PLA were prepared. The construct was closed and closed at one end and filled with a cell suspension enriched in myoblasts. After myoblast replenishment, the open end of the construct was sealed with a second suture set to form a closed cylinder. The construct was then placed in a channel prepared in the rat outer vastus muscle. As described above, the muscle covering one side was sutured over the graft. After 7 days, the collagen-based graft was fully integrated into the host tissue. There were no signs of inflammation or fiber encapsulation. Overall, the construct was dense with cells. Small arteries and capillaries were found throughout the graft. As described above, the cells in the electrospun matrix were oriented parallel to the local orientation of the collagen filaments. Differentiating myotubes were enriched in (but not limited to) these domains. In some restricted domains, myotubes were organized in a parallel arrangement. However, myotubes in other areas of the graft are often oriented along completely different axes. The central core of the graft was filled with cells and differentiating myotubes were occasionally observed. Functional blood vessels were intermingled with cells in the central core domain. In contrast to these results, bioengineered muscle produced on a PGA: PLA substrate was encapsulated with fiber capsules and was not well integrated into the host's surrounding tissues. Large multinucleated cells lined up in many of the border zones along the interface between the graft and the endogenous tissue. The cell mass present in the cylindrical construct appeared to delaminate from the inner wall of the graft.

In a short-term graft study, the modified tissue was placed unloaded in the rat lateral vastus abdominal cavity. To examine how activity-related mechanical forces can affect the differentiation and incorporation of biomodified tissue muscle, a tendon-to-tendon implantation study was conducted. An electrospun construct composed of a blend of type I collagen: PGA: PLA (80:10:10) was prepared and filled with a myoblast-enriched suspension and sealed. The modified tissue was then passed deep into the outer vastus abdominal plane in a plane separating the outer vastus muscle from the lower quadriceps muscle. The proximal end of the graft was sutured to the tendon of the lateral vertical muscle origin. The incision was repaired and the rats were allowed to recover for 8 weeks. During this period, the animals were routinely observed and there were no signs of unpleasant abnormal behavior or movement.

Upon recovery, the transplanted tissue showed classical light microscopy and ultrastructural characteristics of differentiated muscle. Tissue samples taken at the midpoint between the origin and the insertion site showed a parallel arrangement of myotubes with dense myofibrils. The adjacent Z band was a lateral registry in many myotubes. Several internal subdomains of these myotubes were stained with toluidine blue / crystal violet staining more intensely than adjacent subdomains. In contrast to these results, the myotubes located at the distal end of the graft did not show a parallel arrangement. In the longitudinal section, the individual myotubes showed an intertwined profile. In any given myotube, myofibrils were dense with muscle traits and the Z band was a lateral arrangement. The intertwined nature of these myotubes can reflect the manufacturing process. These domains are in close proximity to the sutures used to seal the construct, and subsequently anchor the graft to the outer vastus tendon joint. The mechanical forces placed through these domains can be very complex, resulting in differentiation, but myotube arrangement is frightening. At the ultrastructural level, the modified tissue showed a parallel arrangement of myofibrils scattered in the mitochondria. The sarcoplasmic reticulum coated these filaments and terminated with T tubules at the Z-band level. A small percentage of myotubes showed signs of structural abnormalities, and sarcomere with a sub-lateral stripe pattern were evident. An electronic high density band was occasionally observed at one end of the Z band, near the AI boundary. Double M and H bands were also observed in several myotubes.

Example 23
Electrospinning of collagen from 2,2,2-trifluoroethanol The collagen used was type I (rat tail, acid extraction). Collagen was suspended in 2,2,2-trifluoroethanol (TFE) at a concentration of 0.100 g in 4 ml of HFIP. Once in solution or suspension (milky white solution), the solution was filled into a 10 ml syringe plunger. Next, an 18 gauge stub needle was placed on the syringe to act as an electric spinning nozzle and charging point for the contained collagen solution. Place the filled syringe in a syringe pump (KD Scientific) set and use a 10 ml syringe plunger made by Becton Diegoson 11
The solution was dispensed at a rate of ml / hr. A positive lead from a high voltage supply was attached to the metal part of the stub adapter. The syringe pump was activated and the high voltage supply was activated and set to 20 kV. The grounding target was a 303 stainless steel mandrel (width (W) 0.6 cm × height (H) 0.05 cm × length (L) 4 cm) disposed approximately 6 inches from the tip of the adapter. In the experiment, the collagen solution was electrospun to form a fine white mat on the ground core. After electrospinning, the collagen mat was removed from the mandrel and processed for scanning electron microscope evaluation. The mat produced was approximately 100 microns thick. The average fiber size of this mat was 0.11 ± 0.06 microns. TFE did not modify the secondary structure of collagen, as shown by Fourier transform infrared spectroscopy.

Example 24
A scaffold for the development of smooth muscle cell migratory small-diameter vascular grafts in electrospun poly (lactic acid) and collagen / elastin was constructed using electrospun collagen / elastin nanofibers and smoothed to this scaffold Myocyte (SMC) migration was examined against electrospun poly (lactic acid) scaffolds. A 4 mm diameter tube with a wall thickness of 300-400 microns and a length of 1 cm was electrospun from poly (lactic acid) (PLA) (Alkermes, Inc.) and collagen (type I) / elastin 80:20, respectively. The sample was electrospun using the same method as described in Example 8. These cylindrical constructs were placed in 55 ml STLV tubes (Rotary Cell Culture System, Synthecon, Inc.) along with aortic smooth muscle cells for seeding (500,000 SMC / ml). The medium was changed every 2 days without adding additional cells. The scaffold was removed from the STV tube and processed for scanning flag electron microscopy (SEM) and histological evaluation. Upon examination of the scaffold, the electrospun collagen / elastin matrix showed an average pore size of 3.7 ± 1.6 microns and a fiber size of 0.08 ± 0.02 microns. SMC migration was demonstrated through the scaffold wall after 1 week, while cell migration was much more pronounced after 2 weeks. After 3 weeks of culture, a fully confluent SMC layer was seen on the outer surface with a high density of SMC across the scaffold wall (uniform distribution throughout). Examination of the PLA scaffold showed an average pore size of 26 ± 4 microns and a fiber size of 10 ± 1 microns. PLA grafts showed limited SMC transport to the wall core. Even after 111 days, the cells formed a predominantly confluent layer on the outer surface of the cylindrical scaffold with scattered cells found in the cross section of the scaffold.

The results show that a scaffold composed of electrospun collagen / elastin nanofibers with a pore size of a few microns compared to the current view that pore sizes of 10-30 microns do not allow cell invasion / migration. Demonstrate that cells migrate. Electrospun collagen / elastin scaffold formation is evident from a 3.7 micron average pore size result with little or no expected cell migration to the core of the structure , Do not follow the accepted paradigm. Thus, with an order of magnitude decrease in pore size (relative to PLA), SMC immediately began to migrate to this micropore size structure. These results support a paradigm in which cells migrate to electrospun (nanostructured) collagen-based scaffolds with applications throughout the area of tissue manipulation.

Example 25
Electrospun structures composed of electrospun collagen / polymer nanofibers and polymer nanofibers with nanopore paradigms are produced to provide a new paradigm for medical applications. The sample was electrospun using the same method as described in Example 8. SEM of collagen type I electrospun matrix revealed complex branching of filaments with diameter average 0.1 ± 0.04 microns. The hole diameter of this scaffold is 0.74 ± 0.56 microns,
The range was 0.2 to 2.3 microns. Smooth muscle cells were cultured with the matrix in a rotating culture system (Synthecon, Inc.). Electrospun collagen scaffold formation after 21 days in culture showing extensive cellular infiltration of smooth muscle cells into the collagen nanostructure matrix with uniform distribution of cells across the entire cross section of the nanostructure scaffold Has occurred.

In contrast, electrospun PLA was capable of slight (mostly cell monolayer on the outer surface) migration of SMC to the core of the structure even after 111 days. The results demonstrate that cells migrate to submicron pore size structures created using electrospun nanometer collagen fibers.

Example 26
Electrospinning of layered laminates A method of forming a multilayered material composed of fibers distributed along different orientations is described. This method provides the ability to form a template for the construction of multi-layered organ constructs that does not require electrospinning of cells directly into the matrix.

A matrix material, biocompatible polymer, blend or other material is electrospun onto the rotating target mandrel. The mandrel is cylindrical or any other shape. Although rectangular target mandrel and collagen are used in this example, other materials and other shapes of mandrel may be used and are considered within the scope of the present invention. A first layer of collagen is electrospun onto the mandrel to form an aligned filament layer distributed along the axis of rotation. Next, in a second separate layer, water soluble filaments such as PEG, PVOH or other matrix are prepared on the first layer. This intermediate layer is sprayed from a separate source. To avoid collapsing the collagen layer, the collagen layer may be stabilized by UV crosslinking, chemical treatment, or other means before applying the water soluble layer. Next, a second layer of collagen is electrosprayed onto the intermediate water-soluble layer (layer 2 in this example). This third layer may be in the same orientation as the first layer of collagen, or it may be in a different orientation than the first layer of collagen. In making the heart or blood vessel, this third layer is slightly balanced with respect to the first layer (although not exactly along the same axis as the first layer, but with complete heart structural alignment). Slightly balance the axes to mimic). The collagen layer is then stabilized by using vapor fixation or other stabilizing agents. The stabilization process is performed at the completion of the manufacturing process after each continuous layer to stabilize each continuous layer to a different extent, or on or off the target mandrel. . The selected collagen layer, or all of the collagen layers, are optionally supplemented with growth agents, or other agents such as cDNA sequences, pharmaceuticals, other peptides, as described herein. Post-treatment of the construct may also be performed. The number of layers to be prepared varies depending on the application and is not inherently limited.

Next, the construct is optionally removed from the mandrel and immersed in water. This causes selective removal of the water-soluble layer. Other solvent combinations are also possible with this design strategy and are not limited to the combinations described in this description. If the collagen layer is prepared along different paths, the final construct is composed of two different collagen layers with slightly different or very different polarities.

The construct is now prepared for cell seeding. In this example, the distal end of the construct is sealed and the cells infiltrate the central lumen (site where the mandrel was present). The cells here are separate and distinct layers. This type of construct can be used in many applications such as the manufacture of nerve leads. Cylindrical constructs selectively infiltrate cells (or other materials) into the outer layer. Schwann cells that surround and protect the natural nerves may be used. This device can be used as nerve induction because Schwann cells infiltrate the inner cell layer and are present at the location necessary to coat the nerve as it regenerates. The rate of infiltration of cells across the inner collagen layer is arbitrarily delayed using PGA: collagen formation or PGA laminates, or accelerated by creating an inner layer derived from collagen alone. In nerve induction, the addition of laminin and other basement membrane materials is optionally used to accelerate, promote or support nerve regrowth. A growth factor gradient along the length of the construct is optionally used.

In another application of this method, cell alignment along a single axis can be controlled within the solid construct. This type of construct is desirable for the production of skeletal muscle, but is not limited to this application. Although an outer sheath is prepared and described in this example for a cylindrical construct composed of collagen, the invention is not limited to this type of design. Next, a flat sheet of material is electrospun onto the rotating rectangular mandrel. A sheet composed of aligned collagen is produced and arranged parallel to the axis of rotation. The sheet is removed from the mandrel and wrapped parallel to the fibril axis, or folded into a pleat (or as desired). Now the sheet is inserted (slid) into the outer cylindrical sheath. The resulting structure is comprised of a cylindrical sheath that is “filled” with a pleated or rolled sheet of collagen. Seal the edges and fill with cells. The net result is a semi-solid cylinder with an inner core of collagen fibrils (or other fibrils) arranged along the long axis of the cylinder. Cells seeded in this type of construct develop in parallel with the lower fibrils of the pleated sheet. The invention is useful as neural induction, in the formation of blood vessels, in the formation of smooth muscle-based organs, and in other applications.

All patents, publications and excerpts cited above are hereby incorporated by reference in their entirety. The foregoing is merely illustrative of the preferred embodiments of the invention, and numerous modifications or changes may be made in the invention without departing from the spirit and scope of the invention as defined in the appended claims. Should be understood.

Claims (17)

Electroprocessed collagen. The electroprocessed collagen of claim 1, wherein the electroprocessed collagen is electrospun. The electroprocessed collagen according to claim 1 or 2 in a matrix. 4. The electroprocessed collagen matrix of claim 3, further comprising one or more substances. 5. The electroprocessed collagen matrix according to claim 4, wherein the one or more substances are cells. 5. The one or more substances are therapeutic agents, cosmetics, growth factors, differentiation derivatives, antioxidants, vitamins, hormones, nucleic acids, peptides, emollients, wetting agents, or conditioners. Of electroprocessed collagen matrix. An electroprocessed collagen matrix further comprising said further electroprocessed material, wherein said further electroprocessed material is one or more natural materials, one or more synthetic materials, or a combination thereof 4. The electroprocessed collagen matrix according to claim 3, which is a combination. A modified tissue comprising the electroprocessed collagen matrix of claim 3 and cells. A construct comprising an electroprocessed collagen matrix according to any one of claims 3-7. A method of delivering a substance to a desired location comprising:
A method comprising combining the material with electroprocessed collagen according to any one of the preceding claims and placing the electroprocessed collagen in combination with the material at the desired location. A method for producing an electroprocessed collagen according to claim 1 or 2 or an electroprocessed collagen matrix according to claim 3 comprising:
One or more charged solutions comprising collagen or molecules capable of forming collagen are subjected to conditions effective to electrodeposit collagen or molecules capable of forming collagen on a substrate. Electrodepositing onto a grounded target substrate, thereby forming the electroprocessed collagen or electroprocessed collagen matrix. A method for producing the modified tissue of claim 8 comprising:
Conditions effective to deposit one or more charged solutions containing collagen or molecules capable of forming collagen into the electroprocessed collagen or molecules capable of forming collagen And electrodepositing on a grounded target substrate, and adding cells onto or into the substrate. A method for assessing the biological response of a cell to a substance comprising: applying the substance to the electroprocessed collagen matrix and cells of claim 5; and
Assessing the biological response of the cells. Use of electroprocessed collagen according to any one of claims 1 to 7 for the delivery of a substance or electroprocessed material to a desired location. 15. Use according to claim 14, wherein the substance comprises one or more molecules, cells, objects, or combinations thereof. 15. Use according to claim 14, wherein the substance comprises a therapeutic substance, a cosmetic substance, or a combination thereof. Use of electroprocessed collagen according to any one of claims 1 to 7 for the preparation of a medicament useful for the delivery of substances or electroprocessed materials.