JP2002506691A - Biopolymer mats used for tissue repair and reconstruction - Google Patents

Abstract

  (57) [Summary] Biopolymer mats, biopolymer mat composites, biopolymer mat compositions, and methods of making the mats and composite mats will be described. In addition, biopolymer constructs containing extracellular matrix macromolecules and methods of making these constructs are described. The mats, mat compositions and biocompatible constructs of the present invention can be used for tissue repair and reconstruction.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Collagenous scaffolds have been used for tissue repair and tissue reconstruction.
Typically, crosslinking this backbone will provide the necessary wet strength for these applications,
Provides measurement resistance to lysis. In general, cross-linking of sponges or foams with collagen reduces or degrades the cells and the normal binding sites to which certain molecules secreted by the cells bind. Furthermore, collagen sponges, gelatin sponges or collagen gels are biologically active, but lack the information that binds to collagen or to components other than collagen due to the lack of components other than collagen. Therefore, they lack the biological activity typically present in the extracellular matrix environment. Due to this deficiency, collagen skeletons cross-linked in a known manner induce little in vivo regeneration or poorly serve as tissue and organ typical models in vitro.

Therefore, there is a need for a superior biopolymer type that overcomes or minimizes the above problems and maintains the natural structure of collagen.

SUMMARY OF THE INVENTION The present invention is directed to a membrane or thick tissue for tissue repair and tissue reconstruction, such as a resorbable biopolymer mat, having high strength per unit volume even before crosslinking. It is characterized by a biopolymer mat or a biopolymer mat composite material, or a biopolymer skeleton in the form of a filler. The present invention further provides a biopolymer mat composition comprising a biopolymer mat and various layers of biopolymer foam; a biocompatible construct comprising a biopolymer mat and an extracellular matrix macromolecule; , A biopolymer mat composite, a biopolymer mat composition, and a method for making and utilizing a biocompatible mat construct.

The present biopolymer mat and biopolymer mat composition can be used, for example, in vitro as a model system for research, or as a prosthesis or implant to replace damaged or diseased tissue, or as host cells. Can be remodeled when occupied by other cells to form a skeleton that becomes a functional tissue. In any case, the mat, the mat composite, and the mat composition, the mat, the mat composite,
Alternatively, the mat composition may be used to inoculate cells of the same type as the tissue to be repaired, reconstructed, or replaced, eg, mammalian cells such as human cells. Examples of tissues that can be repaired and / or reconstructed using the present mats, mat composites, and mat compositions described herein include nerve tissue, skin, vascular tissue, heart tissue, pericardial tissue, muscle tissue,
Connective tissue such as eyeball tissue, periodontal tissue, bone, cartilage, tendon, and ligament; organ tissue such as kidney tissue and liver tissue; gland tissue such as pancreatic tissue, breast tissue, and adrenal tissue; bladder tissue and ureteral tissue And digestive tissue such as intestinal tissue.

In one aspect of the invention, the present mat, a mat composite, inoculated with tissue-specific cells,
And the mat composition is introduced into the recipient, eg, a mammal such as a human. Alternatively, the inoculated cells that have been organized into tissues in vitro to provide the opportunity to secrete tissue-specific biosynthetic products that bind to the mat and mat composition, such as extracellular matrix proteins and / or growth factors, The mat or mat composition is removed prior to implantation into the recipient.

Accordingly, the present invention relates to a biopolymer mat having predetermined properties.
In another aspect of the present invention, the biopolymer mat comprises a closely aligned random arrangement of biopolymer fibrils, has a high strength per unit volume, Maintains natural structure. Examples of molecules capable of forming biopolymer fibrils that can be used in the present biopolymer mat include collagen, laminin, elastin, fibronectin, fibrinogen, thrombospondin, gelatin, polysaccharide, poly-l-amino acid and Combinations of biopolymers. A preferred molecule for making biopolymers is collagen, such as fetal porcine collagen.
In other embodiments, the biopolymer mat can also include macromolecules required for cell growth, morphogenesis, differentiation, or tissue construction and combinations thereof, extracellular matrix microparticles, and / or cells.

[0007] The biopolymer mat of the present invention comprises a step of forming a biopolymer solution in which fibril formation such as formation of collagen fibrils occurs, and capturing and / or embedding the fibrils to form a semi-solid. Pouring this solution onto a porous structure that forms a fibril-gel structure, and drying the semi-solid fibril-gel structure forms a thin dense fibrous membrane, such as a mat. You. In another embodiment, sodium chloride is added to the neutralization buffer to reduce gel components and to increase fibril concentration.

[0008] In another aspect of the invention, a mat composite can be formed by collecting a continuous layer of fibril slurry on a porous support.

In yet another aspect of the invention, the porosity of the mat may be manipulated in various physical or chemical ways. In still another embodiment of the present invention, the strength of the biopolymer mat,
It may operate in various physical or chemical ways.

In yet another aspect of the invention, the biopolymer mat, mat composite, or mat composition may be further conditioned by cells prior to use in vitro or in vivo. Cell conditioning hastened the integration of the mat, mat composite, or mat composition into its new function, hastening the recovery of repaired tissue and directing the genuine replacement of damaged or lost tissue. This is an application-specific method used for The biopolymer mat, biopolymer mat composite, or biopolymer mat composition can also be used as a substrate for cell growth suitable for the site to be used. For example, in the case of a biopolymer mat, biopolymer mat composite, or biopolymer mat composition used to repair bone defects as periosteum, the conditioned cells include, for example, osteoblasts. Would. In the case of a biopolymer mat, biopolymer mat composite, or biopolymer mat composition used as a pericardial membrane, conditioned cells would include, for example, mesothelial cells.

During refining, cells residing on a biopolymer mat, biopolymer mat composite, or biopolymer mat composition are added to the mat, mat composite, or mat composition by the mat, mat composite. The deposition of macromolecules, such as protein products, which can be recognized by cells adjacent to the defect at the location of the material or mat composition. Two things can be determined by the choice of cells and thus the protein product. These direct the movement of adjacent cells to the mat, mat composite, or mat composition, or are remodeled to the mat, mat composite, or mat composition material to replace the genuine coating. Or other cells stimulate the desired tissue regrowth under the mat, mat composite, or mat composition while remodeling the mat, mat composite, or mat composition from the other side. Replacement with a likely cell product can be directed. After a period of time in which the conditioned cells have sufficient signaling molecules and extracellular matrix molecules deposited on the mat, mat composite, or mat composition, the mat, mat composite, or mat composition is replaced with a living tissue equivalent or It may be used as a living implant that serves as a model tissue system. Alternatively, the cells of the mat, mat composite, or mat composition may be killed by freezing or freeze-drying the construct. Although lyophilization causes the loss of vital materials, the deposited proteins will remain in their natural state.

The biopolymer mat can also be used alone, for example, as a peri-tooth barrier, or a collagen-like membrane for a periosteal barrier to aid bone repair. Further, the biopolymer mat can be used as a biopolymer composite by collecting successive layers of different fibril slurries on a porous support and fusing these layers together. Further, the present biopolymer mat or biopolymer mat composite can be used as a mat composition containing a biopolymer mat and a biopolymer foam, for example, in the case of tissue repair of the dura of the central nervous system. is there. For example, casting a single-density foam into a mat of final shape, and having two distinct properties having a high density, low porosity mat layer and a low density, high porosity foam layer A structure consisting of layers may be produced. Single density and double density biopolymer foams are described in U.S. Patent Application Serial No. 08 / 754,818, filed November 21, 1996, the entire contents of which are incorporated herein by reference. Such an implant site constituting a composite tissue may be treated with a mat composition comprising epithelial cells, mesothelial cells or endothelial cells on the upper surface of the mat and mesenchymal cells in the foam skeleton. In these applications, the low porosity side of the mat can reduce adhesions or fluid loss, while the high porosity side can attract cells needed for healing and enhance cell growth and differentiation. I can help. In order to further prevent adhesion and fluid disappearance in these applications and applications that require the use of the mat alone, the surface of the mat may be improved. Improvements can be achieved biologically by growing and differentiating keratinocytes on one side of the mat to give the stratum corneum. A mat composition comprising one or more layers of a biopolymer mat or a biopolymer mat composite, and a mat composition comprising two or more layers of a single or double density biopolymer foam are also specifically described herein. It is about to be considered.

As mentioned above, the mat may include a fiber structure, such as a single fiber, braid, or fabric, to achieve relative reinforcement, directional reinforcement, or directional reinforcement. Cell growth can be achieved. Examples of implants requiring such a structure include skeletal replacement or temporary reinforcement structures. The mat may be cast into a shape other than the sheet. Cast into a circular shape such as a tube or a sphere,
A membrane structure that can contain materials or liquids for specialized functions may be made. Examples of implants made from mats, mat composites, or mat compositions include, for example, blood vessels, ducts, ureters, bladder, and bone implants formed from mat cylinders filled with skeletal replacement materials.

Using a mat composition, with or without cell inoculation, with and without freeze-drying, and a single density foam, a biological tissue equivalent or model tissue system can be constructed. An example of this is the growth of dermal fibroblasts in a single-density foam and the differentiated growth of keratinocytes on a porous surface mat layer for a skin model or a bioimplant system. Alternatively, bioimplant systems are a rapid replacement for lost functionality in critical locations and can be lyophilized for stockpiling. If this is not desirable as a bioimplant system, the complex in which the cells are embedded and grown can be lyophilized for use as an implant at a later date, from which the cultured cells are deposited on the structure prior to lyophilization. Information can direct regrowth to the host organization.

[0015] In another aspect, the invention includes a pericardial patch that includes biopolymer fibrils. The biopolymer fibrils include collagen, laminin, elastin, fibronectin, fibrinogen, thrombospondin, gelatin, polysaccharide, poly-l
-Selected from any of amino acids and combinations thereof. In one preferred embodiment, the pericardial patch comprises collagen which is porcine fetal collagen. In another embodiment, the pericardial patch comprises epithelial cells and / or mesothelial cells, eg, inoculated with exogenous growth factors.

The features of the Detailed Description of the Invention The present invention and other details, more specifically described, it is assumed that depict the point in the claims. It will be understood that the specific embodiments of the present invention have been set forth by way of illustration and not by way of limitation. The basic features of the invention can be used in various embodiments without departing from the scope of the invention.

The present invention provides a biopolymer mat, a biopolymer mat composite, a biopolymer mat composition including a biopolymer mat and a biopolymer foam, a biopolymer mat and an extracellular matrix macromolecule. It features biocompatible constructs and methods of making and using the present mats, mat composites, and mat compositions. The present biopolymer mats, mat composites, and mat compositions contain information that, in their native fibril structure, induces the repair or regeneration of damaged, diseased, or lost tissue. Further information for repair or regeneration may be added by mixing other information macromolecules into the biopolymer mat, mat composite, and mat composition. The present biopolymer mats, mat composites, and mat compositions are fully absorbable, unless reinforced with non-absorbable fibers, and can be replaced over time with new, normal host tissue. This biopolymer mat,
The mat composite, and the mat composition, are more resistant to enzymatic degradation than other informative collagen products, such as, for example, gels or foams. The biopolymer mat, mat composite, and mat composition can be made under physiological conditions so that living cells can be incorporated throughout the structure and final form, thus forming a living implant. it can. Information contained in biopolymer mats, mat composites, and mat compositions can trigger genuine healing and repair. For example, a mat with live cells can immediately replace lost host tissue and its function, yet be progressively remodeled by genuine host tissue without any interference with tissue function. The present biopolymer mats, mat composites, and mat compositions can be made in such a manner that they are stronger than other collagen products with a high information content. Therefore, they can be used in situations requiring high strength implants.

Biopolymers are naturally occurring macromolecules formed from individual molecules in biological systems or organisms. Furthermore, biomacromolecules can be made by manipulating individual molecules once obtained outside their biological system or organism. The biopolymer is suitable for introduction into living organisms such as mammals such as humans. The biopolymer is non-toxic and bioabsorbable when introduced into an organism, and any degradation products of the biopolymer must also be non-toxic to the organism. The biomacromolecules of the present invention can be used in any biocompatible form, with or without all other extracellular matrix macromolecules, such as biocompatible foams, biocompatible gels, biocompatible fibers such as collagen fibers, etc. And mats, mat composites, and mat compositions containing biocompatible constructs, including collagen and collagen fabrics. Examples of molecules that can form biopolymers and can be used in the present invention include collagen, laminin, elastin, fibronectin, fibrinogen, thrombospondin, gelatin, polysaccharides,
There are poly-l-amino acids and combinations thereof. In one embodiment, combinations or mixtures of one or more biopolymers can be used to form the biocompatible shapes, such as the fibers, mats, and mat compositions of the present invention. For example, a combination of laminin and type IV collagen may be used to form the biopolymer fibers described herein. A preferred molecule for biopolymer production is collagen.

Suitable sources of molecules that form biopolymers include, for example, mammals such as pigs, for example, near-term pig fetuses, sheep, sheep fetuses, cattle and bovine fetuses. Other sources of molecules capable of forming biopolymers include both terrestrial and marine vertebrates and invertebrates. In one embodiment, collagen can be obtained from the skin of a fetal pig from a perinatal livestock, which has been intactly collected in an amniotic membrane. Collagen or a combination of collagen types may be used in the mats or mat compositions described herein. Examples of collagen or combinations of collagen types include collagen type I, collagen type II, collagen type III, collagen type IV, collagen type V, collagen type VI, collagen type VII, collagen type VIII, collagen type IX, and collagen type X. , Collagen type XI, collagen type XII, collagen type XIII,
And collagen type XIV. Suitable combinations of collagen types include collagen type I, collagen type III, and collagen type IV. Suitable mammalian tissues for extracting molecules capable of forming biopolymers include any mammalian fetus, such as fetal porcine, dermis, tendons, muscles, and connective tissue. Fetal tissue is advantageous as a source of collagen because fetal tissue collagen is not as cross-linked as adult tissue. Thus, when collagen is extracted using an acid extract, a higher percentage of intact collagen molecules than in adult tissue is obtained from fetal tissue. Fetal tissue also contains various molecular factors that are present in normal tissue at different stages of animal development.

In one preferred embodiment, the adult polymeric mat, mat composite, or mat composition is a collagen mat, collagen mat composite, or collagen mat composition. The collagen solution can be produced by salt extraction, acid extraction, and / or pepsin extraction from the starting material. In a preferred embodiment, the collagen used is produced by sequentially purifying two forms of collagen from the same collagen-containing starting material. First, the intact collagen is acid extracted from the starting material,
The extract is collected to prepare collagen as a collagen solution, for example, by precipitating collagen with sodium chloride and solubilizing the collagen in a medium having an acidic pH. In the meantime, the truncated collagen, i.e., the telopeptide was cleaved or partially cleaved to a helical portion only, or a helical portion leaving only some telopeptides from the starting material, such as pepsin, Acidic pH
Extract using an enzyme extract such as an enzyme that is functional in Next, the collagen from the pepsin extract is separately purified by a method similar to the first extraction.

By using the biopolymer, a mat, mat composite, or mat composition that can be formed into any shape such as a string, a sheet, a tube, and the like can be produced. In addition, the biopolymer can be used with a tissue culture insert of a multi-well plate that can be made into a mat that can be supported by a polymer mesh, such as a Teflon mesh, or used as a template. The mat, mat composite, and mat composition of the present invention can be formed in the form of a polycarbonate film of the insert. The use of a polymer mesh in conjunction with the mats, mat composites, and mat compositions of the present invention provides mats, mat composites,
And the cells contained or contained on the mat composition can be exposed to the environment, for example, when the mat, mat composite, and mat composition are used as skin equivalents that stimulate the formation of stratum corneum. . Both such meshes and culture inserts have the advantage of providing a means of handling the mat, mat composite, and mat composition without having to actually contact the mat, mat composite, and mat composition. The shapes and shapes from which the mats, mat composites, and mat compositions are made may mimic the tissue or body part to be replaced, thus facilitating the regeneration of the recipient's replacement tissue by remodeling the tissue cells. It can be used as a prosthesis or implant.

Further, when macromolecules necessary for cell growth, morphogenesis, differentiation, and tissue construction are added to the molecules of the biopolymer or the fibrils of the biopolymer, cell implantation in a mat and tissue development and organization Can be promoted. The phrase "macromolecules required for cell growth, morphogenesis, differentiation, and tissue construction" refers to molecules that participate in tissue growth, such as macromolecules such as proteins. Such molecules contain biological, physiological and structural information for the development or regeneration and function of tissue structures. Examples of these macromolecules include, but are not limited to, growth factors, extracellular matrix proteins, proteoglycans, glycosaminoglycans and polysaccharides. Alternatively, the biopolymer mats, mat composites, and mat compositions of the present invention may include extracellular matrix macromolecules in particulate form, or extracellular matrix molecules with cells or viable cells deposited.

The term “growth factor” is art-recognized and refers to platelet-derived growth factor (PD
GF), such as PDGF AA, PDGF BB, insulin-like growth factor (I
GF), for example, IGF-I, IGF-II, fibroblast growth factor (FGF) such as acidic FGF, basic FGF, β-endothelial growth factor, FGF4, FGF5, FGF6, FGF7, FGF8, and FGF9. Transforming growth factor (TGF), such as TGF-β1, TGF-β1.2, TGF-β2, TGF-β3, TGF-
β5, bone morphogenetic protein (BMP), such as BMP1, BMP2, BMP3,
BMP4, vascular endothelial growth factor (VEGF) such as VEGF, placental growth factor, epidermal growth factor (EGF) such as EGF, amphiregulin, betacellulin, heparin-binding EGF, interleukins such as 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, IL-14, colony stimulating factor (CSF
), For example, CSF-G, CSF-GM, CSF-M, nerve growth factor (NGF),
Includes, but is not limited to, one or more of stem cell factor, hepatocyte growth factor, and ciliary neurotrophic factor. The term encompasses currently unknown growth factors that may be discovered in the future, because their properties as growth factors can be readily determined by those skilled in the art.

The term “extracellular matrix protein” is recognized in the art,
Fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, ancholine, chondronectin, link protein, bone charoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, unjurin, epiligrin, And one or more of kalinin. The term encompasses currently unknown extracellular matrix proteins that may be discovered in the future, because their properties as extracellular matrix proteins can be readily determined by one skilled in the art. It is.

The term “proteoglycan” is recognized in the art and includes decorin and dermatan sulfate proteoglycans, keratin or keratan sulfate proteoglycans, aggrecan or chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, biglycan, syndecan, perlecan, or serglycine. Is intended to include one or more of the following. The term encompasses currently unknown proteoglycans that may be discovered in the future because their properties as proteoglycans can be readily determined by one of ordinary skill in the art.

The term “glycosaminoglycan” is recognized in the art and is intended to include one or more of heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid. . The term encompasses currently unknown glycosaminoglycans that may be discovered in the future because their properties as glycosaminoglycans can be readily determined by those skilled in the art. It is.

The term “polysaccharide” is recognized in the art and is intended to include one or more of heparin, dextran sulfate, chitin, alginic acid, pectin, and xylan. The term encompasses currently unknown polysaccharides that may be discovered in the future, because their properties as polysaccharides can be readily determined by those skilled in the art.

Suitable living cells include epidermal cells, such as keratinocytes, adipocytes, hepatocytes, neurons, glial cells, astrocytes, podocytes, breast epidermal cells, islet cells, endothelial cells, aortic capillaries and venous endothelium Cells, and mesenchymal cells, such as skin fibroblasts, mesothelial cells, stem cells, osteoblasts, smooth muscle cells, striated muscle cells, ligament fibroblasts, tendon fibroblasts, chondrocytes, and fibroblasts. Including, but not limited to.

As used herein, the term “mat” refers to a biopolymer scaffold comprising a closely aligned, random array of biopolymer fibrils or bundles or particles of fibrils, such as collagen fibrils. As mentioned above, the dried mat has a wet tensile strength of at least 0.02 MPa, but the preferred strength is greater than 1 MPa, and 1 mg of collagen at a concentration of 10 units of collagenase per cm ^{2 of} product. Have collagenase resistance of at least 20 minutes per hour. Typically, the fibrils or bundles of fibrils have a diameter between about 0.01 μm and 50 μm and a length between about 0.0002 and 5.0 mm, but preferably have a width of The thickness is preferably from 0.1 μm to 20 μm and the length is from 0.01 mm to 3 mm. The mat has the following properties when dried or not. (1) It is non-toxic to living organisms. (2) It can serve as a substrate for cell adhesion and growth. And (3) about 0.01 mm to 20 mm in thickness, preferably 0.1 to 5.0 mm in thickness.
In addition, the dried mat is physically stable in aqueous solution.

The term “fibrils” as used herein refers to ordered multimers of molecules that give rise to a fibrous overall structure. In the case of collagen fibrils, collagen molecules are arranged in a quarter twisted form, and the binding of each adjacent molecule is regularly 25%.
(One collagen molecule's head is
Arranged 25% below the chain of the molecule). Fibrils, especially those made of collagen, often have a characteristic appearance by electron microscopy. The fibrils are bundled together. Fibers with a higher number of fibril bundles are fibers.

The term “mat composite” refers to a biopolymer form that includes a continuous layer of biopolymer adhered to one another.

The term “mat composition” refers to mats, for example, which are preferably resorbable,
A biopolymer composition comprising one or more biopolymer foams, depending on the choice, such as a biopolymer mat that is a single or double density foam. Single density and double density biopolymer foams are incorporated by reference herein in their entirety, US Ser. No. 08 / 754,8, filed Nov. 21, 1996.
No. 18 explains.

The biopolymer foam may be a single density or double density foam. As used herein, the term "foam" refers to a network of communicating microcompartments in which biopolymer molecules and / or biopolymer filaments are interspersed within the walls of the microcompartments. The phrase "single density foam" refers to a biopolymer foam having at least two of the following properties: That is, 1) approximately equal x = length, y
= Micro-compartments with volume dimensions of x, y and z, where = width and z = height.
Typically, the range of x, y, and z is from about 1 μm to about 300 μm, preferably from about 20 μm to about 200 μm, more preferably from about 40 μm to about 150 μm, and most preferably from about 50 μm to about 100 μm ,. 2) about 10μ
It has microcompartments with an average wall thickness of less than m. 3) It has microcompartments with wall surfaces containing biopolymer fibers and / or filaments. 4) Physically stable in aqueous solution. 5) Non-toxic to living organisms. And 6) can serve as a substrate for cell adhesion and growth. Single-density foams maintain their structure when hydrated, such as in aqueous buffers or tissue culture media. In addition, the three-dimensional structure of single-density foams helps to organize the cells inoculated into them. When a single density foam is made from collagen without cells,
For example, it can be easily digested with collagenase such as 0.1% collagenase. Examples of molecules that can form biopolymers that can be used for single-density biopolymer foams include collagen, alginic acid, polyvinyl alcohol, elastin, chondroitin sulfate, laminin, fibronectin, fibrinogen,
And combinations of these biopolymers. A preferred biopolymer is collagen, such as porcine fetal collagen. In another embodiment, the single density biopolymer foam can include extracellular matrix microparticles and / or cells.

The phrase “dual density foam” as used herein refers to a biopolymer foam having at least two of the following properties: That is, 1) it has a micro compartment with volume dimensions of x, y and z, where x = length, y = width, and z = height, two of which are approximately equal, and the third is From about 1 μm to about 300 μm, preferably from about 20 μm to about 200 μm, with a factor of at least about 10, and more preferably at least about 20 or more compared to the same size of the single density foam. , More preferably from about 40 μm to about 150 μm
And most preferably in the range from about 50 μm to about 100 μm. 2) having microcompartments with an average wall thickness of less than about 10 μm; 3) Biopolymer It has a microcompartment having a wall surface containing fibers and / or filaments. 4) Physically stable in aqueous solution. 5) Non-toxic to living organisms. And 6
) Can serve as a substrate for cell adhesion and growth. Dual density foams made from collagen are more resistant to collagenase digestion than single density foams made from collagen, for example, about 3 to about 5 times or more, and have a 0-fold higher density than single density foams. More resistant to 1% collagenase. Dual density foams made from collagen also have a higher collagen density per unit than the collagen content per unit volume of single density foams. When hydrated, the dual density foam typically has a height of about 0
. It is between 2 mm and about 0.4 mm. Any surface of this dual density foam is a suitable substrate for plating epithelial, endothelial, and mesothelial cells that can form a sheet. Mesenchymal cells may also be inoculated into this dual density foam. Double-density foams can be made the same size and shape as single-density foams, e.g., in any shape and any combination, and adhered to a polymer mesh, or as multi-well plate inserts. it can. Both cells grown in the single and dual density forms of the invention have the morphological characteristics of the cells of the three-dimensional tissue and have a normal intercellular relationship, i.e., from which they were derived or obtained. A similar intercellular relationship can be formed with that of a damaged tissue. Suitable biopolymers for use in the dual density foam are as described for the single density foam. In another example, the dual density biopolymer foam can include extracellular matrix microparticles and / or cells. Making the surface of any mat, mat composite, or mat composition a substrate that can be formed into a sheet or other material suitable for plating epithelial, endothelial, and mesenchymal cells Can be. In addition, cells may be seeded in single or double density foam. The biopolymer mat and cells grown on the biopolymer mat composition have the morphological characteristics of the cells of the three-dimensional tissue and have a normal intercellular relationship, i.e., from or derived from them. Intercellular relationships similar to those of tissues can be formed.

The biopolymer mat of the present invention comprises a molecule capable of forming a biopolymer, such as collagen, elastin, laminin, fibronectin, poly-l-amino acid, fibrinogen, gelatin, polysaccharide, thrombospondin, and the like. It can be prepared by diluting a solution such as a combination of biopolymers. Preferably, biopolymeric collagen, more preferably porcine fetal collagen, is dissolved in a buffer, for example 1 to 50 mM HEPES, TES or tris, pH 7.4, in 5 to 400 mM sodium or potassium phosphate, pH 7.4. With 0. Dilute to 1 to 10 mg / ml, with or without the addition of 1 to 150 mM sodium chloride, standard balanced saline or serum-free tissue culture medium to the above buffer. This pH is then adjusted to pH using a solution of a dilute base such as sodium, ammonium or potassium hydroxide, preferably 5% ammonium hydroxide.
Finely adjust to 7.4. The solution is incubated at a constant temperature (between 37 and 4 ° C., preferably 22 ° C.) while simultaneously mixing gently to form fibrils. The resulting fibrils are between about 0.01 and 50 μm wide and between about 0.0002 and 5.0 mm long. The fibril solution may have a porous structure, for example, a porosity of about 800 to about 18 mesh, preferably about 50 to 500 mesh, or between about 15 to about 1000 μm, preferably about 25 to 280 μm. Or a knitted or nonwoven fabric or, for example, a single density foam. Unpolymerized molecules pass through this porous structure and the restricted mesh porosity prevents the passage of fibrils. As the fibrils are trapped at and slightly below the upper surface, these layered structures become denser and the flow of the solution through the mesh becomes slower. The flow of this solution can be facilitated by the application of an absorbent on the opposite side of the porous structure, by means of a light vacuum or capillary action. As the speed of the solution is reduced, the remaining solution above the porous structure is allowed to gel and capture the fibrils, from about 0.01 to about 2.0 centimeters, and preferably from about 0.01 to 0. A 5 cm thick semi-solid fibril-gel structure can be formed.

In one embodiment of the present invention, the addition of sodium chloride to the neutralization buffer reduces gel components and allows more fibrils to be collected. To accelerate fibril collection, the incubated neutral collagen-fibril solution may be slowed, e.g.
The centrifugation may be carried out at a pressure of from about 5,000 × g, preferably about 1500 × g, for about 2 to 30 minutes, preferably 5 minutes. The supernatant is discarded and the pelleted fibrils are resuspended to form a slurry. As a result of this improvement, a mat structure can be constructed without depending on the drainage properties of the screen. Thus, the fibril slurry can be transferred to a solid bottom mold or dual density foam, or applied to a dish or test tube by applying centrifugal or rotational motion.

In another embodiment of the invention, fibrils are formed or harvested in a physiological buffer, such as phosphate buffered saline, balanced saline, or serum-free tissue culture media. Cells may be included. These cells are mixed with fibrils or fibril slurries and collected on a porous support in a mat. The mat may then be incubated in an environment in which cells can survive and condition the mat based on its properties.

In another embodiment of the present invention, a mat composite can be made. This is achieved by collecting successive layers of slurry of different fibrils / fibril bundles on a porous support. This may be done in such a way that the layers adhere to each other and bring new properties to the mat.

In yet another embodiment of the invention, an object can be embedded in a mat to change its texture or tear properties. In some applications, the handling and use of the mat may require a particular texture or tear resistance. Texture can be manipulated by surface features of the porous support used to collect fibrils, or by embedding textured objects within the mat structure during mat formation. Tear resistance can be manipulated by the material embedded in the mat or by the nature of the mat composite. Using biocompatible particles of any shape that will resist solubilization in physiological buffers, such as calcium phosphate, calcium sulfate beads, calcium sulfate fibers, resorbable polymer fragments, gelatin or agarose beads, etc. You may. Although the use of calcium sulphate creates pores based on its solubility, as described below, the particular reason for using calcium sulphate to manipulate the tear properties of the mat is that the water solubility used to create the pores is Large, but physiological buffers, such as phosphate buffered saline,
Is that the solubility of calcium sulfate is low. The presence of particles in the finished mat acts as a stop in which the tear must travel around to propagate. Because of the presence of particles, the tear must follow a wavy route, reducing the likelihood of tear propagation.

If direct cell inoculation is not preferred, the material entrapped on the porous structure may be deposited between porous absorbents or dried when pressed to flatten,
The finished mat may be peeled off as a thin, dense fibrous membrane.

In another embodiment, the fibril slurry can be bubbled with foam prior to drying to increase the porosity of the mat. In a further embodiment, the porosity may be increased by placing them vertically at desired intervals through a support, such as a screen, fine inert filaments, such as wire filaments. Since the fibrils are deposited around the filaments, they can be removed after the mat is dried to leave holes. In yet another embodiment, the semi-solid fibril-gel structure is treated with a solution, such as an ethanol solution or a slightly less neutral buffer, to wash away the collagen contributing to the gel through the screen and to reduce the fibril on the screen. Leaving only fibrils,
The distance between fibrils may be made porous. In yet another embodiment, the semi-solid fibril-gel is lyophilized to maintain this semi-solid three-dimensional structure,
Porosity may be increased by removing water and leaving gaps where water was. In yet another embodiment, the porosity may be increased by physically perforating the finished mat with a pin or laser beam.

In yet another embodiment, a fibril slurry is mixed with a material having a diameter of about 0.002 to about 2.00 mm, preferably about 0.01 to about 1.0 mm, and the material is removed from the finished mat. Can be removed to leave pores in the space previously occupied by this material, thereby increasing porosity. Examples of these substances include, for example, ferrous particles,
Anything that can be removed by contacting a magnet with a re-wetted mat, such as a minute ball bearing or an iron bead, or a dry mat, such as calcium sulfate, salt, or sugar particles, that is removed by immersing it in water Such as a bead of gelled alginic acid, a bead that can be removed by immersing a dried mat in EDTA at a concentration of about 2 to 1000 mM, preferably 20 to 200 mM, or a bead of agarose or wax. A process that can occur over a period of time on a finished mat product through the action of living cells, such as one that can be removed by exposing the mat to a gradual temperature increase or washing with alcohol, or a bead of gelatin Non-specific protease And those that can be removed. Other methods of providing porosity to the mat are known to those skilled in the art.

The use of alginic acid to increase the porosity of the mat results in a porous material that can retain water and also become a network connecting pores in a sponge-like structure. When various macromolecules are added to the collagen or fibril slurry, they can bind to the collagen or become trapped in the fibril network, giving the mat porosity and water retention. Proteins such as gelatin, pectin, xylan, heparin, dextran sulfate, chitin, polysaccharides such as alginic acid, glycosaminoglycans, especially heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate, and hyaluronic acid, decorin, keratan, heparan Glycosaminoglycan-containing proteoglycans, including sulfuric acid, syndecan, biglycan, perclean, aggrecan, serglycine, and the like, are examples of materials that exhibit these properties for the above applications. Suitable materials include gelatin and / or glycosaminoglycan containing about 0.1 to 4.000%, preferably 1 to 500% by weight of gelatin and / or glycosaminoglycan per dry weight of collagen. There is noglycan.

In another embodiment, the strength of the mat can be increased by placing the reinforcing material in the fibril collection fluid. Such reinforcing materials include fibers, yarns, eg, knitted yarns, and / or fabrics, eg, woven, made from biopolymers, resorbable polymers, or non-resorbable polymers, eg, by a weaving process. Fabric or non-woven fabric. Biopolymer fibers can be made by extruding a biopolymer in a solution into a coagulation bath and transferring the biopolymer to a bath containing ethanol or acetone or other dewatered solution. Alternatively, the fibers may be dehydrated by performing vacuum drying. The biopolymer fiber may then be crosslinked, for example, by the method described in US Ser. No. 08 / 754,818, filed Nov. 21, 1996, which is incorporated herein by reference in its entirety. Good. collagen·
An example of an apparatus for spinning and processing biopolymer fibers, such as fibers, is incorporated by reference herein in its entirety, US Ser. No. 08 / 333,414, filed Nov. 2, 1994. Is described in the issue. The fiber may then be dried, for example, by pulling the moving fiber over more rollers, stretching, drying it, and then winding it, such as by winding it on a spool. The fibers may be woven, knitted, bundled or twisted into fabrics or other complex shapes, or may be constructed by weaving for use as described herein.

The nonwoven biopolymer typically comprises a population of entangled biopolymer fibers of a predetermined size and density. Typically, biopolymer nonwovens are made by winding dried biopolymer fibers onto a drum having a perimeter equal to the length of the individual fiber elements. Winding continues until the number of fibers wound on the drum equals the number of pieces of fiber required for the fabric. So cut across the wound fiber in a direction parallel to the axis of the drum,
Remove the fiber from the drum. Thus, the fiber can be cross-linked if it has not been previously cross-linked. The fiber is then dispersed in a volume of phosphate buffer for a period of time to lower its pH and soften the fiber. The fiber is transferred to a volume of water and mechanically agitated to entangle the fiber chains. The entangled fiber strands are sieved with this water onto a collection screen until they become a mat of uniform density and form a coating on the screen. The nonwoven is then dried on a screen or transferred to another surface, screen or cell culture instrument and then dried. If desired, the nonwoven mat may be cut into smaller shapes after drying or punched.

Examples of suitable materials for the reinforcing fibers, yarns, strings, bundles of fibers, fabrics and / or nonwovens of the mat are those which form biopolymers, resorbable and non-resorbable polymers. . Materials for biopolymers include collagen, alginic acid,
There are laminin, elastin, gelatin, fibronectin, fibrinogen, thrombospondin, polysaccharides, poly-l-amino acids and combinations thereof. Resorbable polymer materials include, for example, poly-α-hydroxy esters such as poly-l-lactic acid and poly-l-glycolic acid, polydioxynone, polyvinyl alcohol, surgical gut, and combinations thereof. Examples of non-resorbable polymeric materials include silk, nylon, polytetrafluoroethylene, polypropylene, polyester, polyurethane, and combinations thereof. Other combinations of fibers include casting a resorbable polymer fiber onto a non-resorbable polymer, or casting a biopolymer fiber onto a resorbable or non-resorbable fiber. Thus, fabrication is possible. A preferred material is collagen, preferably porcine fetal collagen.

Examples of implants requiring such a reinforced structure include structures for skeletal replacement, hernia repair or temporary reinforcement. For example, to repair a rotator cuff, a tough fiber or suture, preferably a resorbable suture, is embedded in the mat structure as the fibril slurry is deposited and a high tension is applied in the direction of reinforcement. It may be applied to the finished product. These structures will be described further below.

In another embodiment, after drying the reinforced mat structure, individual thin fibers of the indicative biopolymer can be cast into the outer surface. The addition of these fibers is indicative rather than structural, and determines the direction of cell growth and tissue repair from the movement of cells along the fibers.

In one embodiment, when the biopolymer is collagen, the collagen may be an enzyme,
For example, it can be treated with lysyl oxidase which initiates crosslinking of collagen. Lysyl oxidase is used, for example, in calf aorta, human placenta, chicken embryonic epiphyseal cartilage, pig skin (Shackleton, DR and Hulmes, DJS (1990) Biochem.
J. 266: 917-919) and can be purified from a variety of sources, including from various locations in porcine embryos, but convert ε-amino groups of lysine to aldehydes. This aldehyde is a reactive functional group that spontaneously binds to other lysine ε-amino groups or other aldehydes on other collagen molecules to form irreversible covalent crosslinks. As a result, the collagen becomes insoluble.
Lysyl oxidase can be added to the collagen solution under conditions that allow aldehyde conversion of lysine. The lysyl oxidase is thus removed from the collagen solution, and the collagen is treated as described herein while spontaneous crosslinks are formed. For example, during processing of the collagen mat, for example, during the polymerization step, as the collagen concentration per unit volume increases, crosslinks are spontaneously formed. This lysyl oxidase-mediated cross-linking is tough, irreversible, and the bond found naturally in collagen. Collagen crosslinked in this manner is insoluble and only undergoes specific enzymatic attacks during tissue remodeling.
Sensitive. Furthermore, lysyl oxidase can be used to crosslink collagen used as mats and mat compositions, convoluted fibers, gels, and the like.

In yet another embodiment, the strength of the mat can be, for example, UV, dehydrothermal,
Alternatively, it can be improved by a standard collagen crosslinking method using a chemical crosslinking agent. Typical chemical crosslinkers include, for example, glutaraldehyde, formaldehyde, acrylamide, carbodiimide, those known in the art, for example, 1-ethyl-3- (
Dimethylaminopropyl) carbodiimide, diones known in the art, such as 2,5-hexanedione, diimidates, such as dimethylsuberimidate, or bisacrylamides, such as N, N'-methylenebisacrylamide. In yet another embodiment, the mat's load bearing capacity can be manipulated by varying the volume or concentration of the fibril-containing solution that is poured onto the screen to produce mats of various thicknesses.

A biocompatible construct comprising a biopolymer mat or biopolymer mat composition of the invention and an extracellular matrix macromolecule will now be discussed more specifically. A mat having extracellular matrix macromolecules or microparticles by further applying a soluble or microparticle type extracellular matrix macromolecule dispersed or suspended in a biopolymer solution on and / or within the mat and mat composition of the present invention , A mat composite, or a mat composition. As used herein, the phrase "particulate extracellular matrix" refers to a process that once had living cells, but which were removed by processing, e.g., growth factors necessary for cell growth, morphogenesis, and differentiation. , An extracellular matrix portion derived from a tissue source that maintains noncellular extracellular matrix factors such as protein, proteoglycan, and glycosaminoglycan. A method of forming extracellular matrix microparticles to produce a transplant is described in US patent application Ser. No. 07 / 926,885, filed Aug. 7, 1992, 19
US patent application Ser. Nos. 08 / 302,087, filed Sep. 6, 1994, and 199
No. 08 / 471,535, filed Jun. 6, 1993. U.S. Patent Application Nos. 07 / 926,885, 08 / 302,087 and 08
No./471,535, which is incorporated herein by reference.

The method of forming the extracellular matrix microparticles includes freezing a tissue source having live cells, such as a connective tissue source, and disrupting the live cells to form components such as cytoplasm and nucleus. Forming a cell remnant consisting of This tissue source is then processed, for example, by grinding, washing, and sieving, to remove cytoplasmic and nuclear components without removing extracellular matrices containing macromolecules required for cell growth, migration, differentiation, and morphogenesis. remove. The extracellular matrix is lyophilized and fragmented, such as by cryomilling to produce fine particles,
Generate extracellular matrix microparticles.

The extracellular matrix microparticles can include extracellular matrix proteins. For example, extracellular matrix microparticles obtained from skin include transforming growth factor β1, platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor,
GF1, bFGF, syndecan-1, decorin, fibronectin, collagen,
Includes laminin, tenascin, and dermatan sulfate. Extracellular matrix microparticles from the lung include PDGF, TGFβ1, bFGF, VEGF, syndecan-1,
Includes fibronectin, laminin, and tenascin. Further, the extracellular matrix microparticles may contain cytokines such as growth factors necessary for tissue development. The term "cytokine" includes, but is not limited to, growth factors, interleukins, interferons and colony stimulating factors.
These factors are present in normal tissues at different stages of tissue development, marked by cell division, morphogenesis and differentiation. Among these factors are stimulatory molecules that provide the necessary signals for tissue repair in vivo. These cytokines can stimulate the conversion of the implant into a functional replacement for the tissue to be replaced. This conversion can be caused by mobilization of tissue cells from similar adjacent tissues, such as from the circulatory system or stem cell reservoirs. Cells can adhere to bioabsorbable prostheses and they can be remodeled into replacement tissue.

The growth factors required for cell growth adhere to structural elements of the extracellular matrix. This structural element includes proteins such as collagen and elastin, as well as glycoproteins, proteoglycans and glycosaminoglycans. Growth factors originally produced and secreted by cells bind to this extracellular matrix and regulate cell behavior in a number of ways. These factors include platelet-derived growth factor (PD
GF), for example, insulin-like growth factors such as PDGF AA, PDGF BB (
IGF), for example, IGF-I, IGF-II, fibroblast growth factor (FGF), for example, acidic FGF, basic FGF, β-endothelial cell growth factor, FGF4, FGF5,
FGF6, FGF7, FGF8, and FGF9, transforming growth factor (TGF),
For example, TGF-β1, TGF-β1.2, TGF-β2, TGF-β3, TGF
-Β5, bone morphogenetic protein (BMP) such as BMP1, BMP2, BMP
3, BMP4, vascular endothelial cell growth factor (VEGF) such as VEGF, placental growth factor, epidermal growth factor (EGF) such as EGF, amphiregulin, betacellulin, heparin-binding EGF, interleukin such as IL-1, IL -2, I
L-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL
-10, IL-11, IL-12, IL-13, IL-14, colony stimulating factor (CSF) such as CSF-G, CSF-GM, CSF-M, nerve growth factor (N
GF), stem cell factor, hepatocyte growth factor, and ciliary neurotrophic factor,
It is not limited to these. Adams et al. "Regulation of Development
and Differentiation by the Extracellular Matrix "Development Vol. 117, p
1183-1198 (1993) (hereinafter "Adams et al") and "Book Guide to the
Extracellular Matrix and Adhesion Proteins, "Oxford University Press (
The author of Kreis et al. (1993) (hereinafter “Kreis et al.”) Describes extracellular matrix components that regulate differentiation and development. Adams et al. Further noted that growth factors bind extracellular matrix proteins and that extracellular matrix is an important part of the microenvironment and play a central role in regulating growth and differentiation in cooperation with growth factors. It discloses what it is doing. The teachings of Adams et al. And Kreis et al. Are incorporated herein by reference.

[0055] Extracellular matrix microparticles may be obtained from a particular tissue. These particles have two types of information properties. The first is their molecular diversity and the second is their microstructure, both of which are conserved in the preparation of the microparticle. Suitable linkages among the various extracellular matrix molecules are also preserved in the microparticle preparation.

The extracellular matrix plays an indicating role, guiding the activity of the cells that it surrounds or has gathered on. Since the execution of cell programs for cell division, morphogenesis, differentiation, tissue construction and regeneration depends on signals emitted from the extracellular matrix, a three-dimensional scaffold such as collagen mat and collagen mat composition is It is enriched with the actual and matrix components that exhibit the sexual and genetic extracellular matrix microstructure and the extracellular matrix microstructure derived from the particular cell.

Additional cell binding and molecular binding sites are provided on the surface of the mat and mat composition
For example, to provide a replacement for a binding site that has disappeared as a result of a cross-linking procedure, before or simultaneously with applying the extracellular matrix macromolecules or microparticles to the collagen mat.
A coating treatment can be provided. In addition, typically about 5 to 500 μm in size
m, an artificial microstructure composed of a matrix polymer such as collagen in combination with other proteins, proteoglycans, glycosaminoglycans, extracellular matrix enzymes, cytokines (including growth factors), and glycosides. And the fine particles may be applied to the surface of the collagen mat or the collagen mat composition by a coating solution. The selected components may be chemically or electrostatically bound to the biopolymer, or may be contained within a microparticle lattice, or may be contained within a dehydrated form of the lattice. Thus, the present invention further relates to a mat coated with collagen and a method for preparing a mat coated with extracellular matrix macromolecules or microparticles. These methods typically involve forming a biopolymer mat of a given type, as described herein, and applying the collagen solution or extracellular matrix macromolecule or microparticle solution to the mat, mat composite, or Applying to the mat composition to form a biopolymer mat, mat composite, or mat composition coated with collagen or coated with extracellular matrix macromolecules or microparticles. In one embodiment, the collagen solution further comprises extracellular matrix macromolecules or microparticles.

The mats, mat composites and mat compositions of the present invention can be used as substrates for ex vivo and in vivo cell growth to establish research model systems. For example, in one embodiment, the mat, mat composite, and mat composition can be inoculated with abnormal cells to study disease states, including cancer. In another embodiment,
A diagnostic test model that determines the chemotherapeutic strategy by screening the mat, mat composite, and mat composition for the ability to kill cancer cells cultured in or on the mat, mat composite, or mat composition It may be useful as. In another embodiment, the mat, mat composite, and mat composition can be utilized to determine the toxicity of various substances that expose cells in or on the mat.

The biopolymer mat, mat composite, and mat composition may be used as formed or may be cell conditioned. Cell conditioning can speed the integration of the mat, mat composite, or mat composition into its new function, speed recovery, and direct the real replacement of damaged or lost tissue. This is an application-specific method used for The mat, mat composite, or mat composition material is utilized as a cell growth substrate appropriate for the site to be used. For example, in the case of a mat, mat composite, or mat composition used as a periosteum to repair bone defects, conditioned cells include:
For example, osteoblasts would be included. In the case of a mat, mat composite, or mat composition used as a pericardial membrane, conditioned cells will include, for example, mesothelial cells or epidermal cells. In the case of a mat, mat composite, or mat composition used for the abdomen, conditioned cells will include mesothelial cells.

For example, in one embodiment, a mat, mat composite or mat composition can be used in or as a pericardial patch. Preferably,
The mat, mat composite, or mat composition may be conditioned with mesothelial cells or epidermal cells. Pericardial patches could be reinforced with biodegradable and biocompatible fibers to enhance the mat, mat composite, or mat composition.
These fibers can be formed from a variety of biocompatible materials, such as polylactic acid, polyamino acids, or more preferably collagen, and most preferably fibrous collagen.

Further, the pericardial patch may be strengthened by cross-linking the present mat, mat composite or mat composition in a manner known in the art. Ultimately, the strength of the pericardial patch must be on the order of 3 to 5 MPa. The pericardial patch is maintained generally intact under normal physiological conditions for a period sufficient to serve as a platform to facilitate regeneration of the pericardial lesion, for example, about 6 to 8 weeks. In one example, the pericardial patch can be formed on a support, such as, for example, a biocompatible and / or biodegradable support. This support can serve as a means of adding strength to the patch or as a means to function as a scaffold on which the patch can be built. In another embodiment, the pericardial patch may be porous, for example, having a pore size of between about 0.01 and 10 microns, preferably between about 0.2 and about 0.5 microns. Good.

During tempering, the cells on the mat, mat composite, or mat composition are those mats that are recognizable by cells adjacent to the defect at the site to be replaced by the mat, mat composite, or mat composition. Deposits on mat composites or mat composition protein products. Cell selection, and thus protein product, can dictate two things in the context of an implant. These direct the movement of adjacent cells to the mat, mat composite, or mat composition, or the mat, mat composite, or mat composition material undergoes remodeling to provide a genuine coating structure, or other A cell product that will stimulate a desired tissue regrowth under the mat, mat composite, or mat composition while the cells of the remodel the mat, mat composite, or mat composition from the other side To be replaced. After a period of time in which the conditioned cells deposit sufficient signaling molecules and extracellular matrix molecules on the mat, mat composite, or mat composition, the mat, mat composite, or mat composition is replaced with a living tissue equivalent. Alternatively, it may be used as a living implant that serves as a model tissue system. Alternatively, cells of the mat, mat composite, or mat composition may be killed by lyophilizing the construct. Although lyophilization causes the loss of vital materials, the deposited proteins will remain in their natural state. Before or after this, the mat, mat composite, or mat composition can be prepared in a variety of gentle methods, as required or desired, using dilute acid (0.001-0.1 M hydrochloric acid or acetic acid) or base (0.001 M). -0.1 sodium hydroxide), diluted nonionic (0.01-1.0% Triton X-100) or zwitterionic surfactant (0.005-0.10 M CHAPS, 3- ( [3-Cholamidopropyl] -dimethylammonio) -1-propane-sulfonate) to eliminate intracellular components. If this optional washing step is performed after lyophilization, the mat, mat composite, or mat composition will again be lyophilized and packaged as a dry product.

The mat, mat composite, and mat composition may further be utilized as a prosthesis that can be introduced or implanted into a recipient, such as a mammalian recipient, such as a human. For example, mats, mat composites, and mat compositions may be used as prostheses or, for example, to reconstruct the following types of tissues. That is, connective tissues such as nerve tissue, skin, vascular tissue, muscle tissue, bone, cartilage, tendon, and ligament, kidney tissue, liver tissue, and pancreatic tissue. The tissue cells inoculated into the mat, mat composite, and mat composition can be obtained from mammals, such as humans. If the tissue cells are not added during mat formation, the tissue cells are first delivered to the mat, mat composite, and mat composition by suspending the cells in a small amount of tissue culture medium. This tissue culture medium containing the cells can then be added dropwise to the mat, mat composite, or mat composition. Alternatively, a mat, mat composite, or mat composition,
Place the tissue culture medium and cells in a container containing the suspension, and place the container in such a way that the tissue culture medium containing cells is distributed throughout the mat, mat composite, or mat composition.
Shake. In another embodiment, the tissue cells may be suspended in a biopolymer solution, such as a low concentration collagen solution, at a temperature of about 4 ° C. to 10 ° C. and a pH of about 7.0. Thus, this solution containing the cells may be delivered to the mat, mat composite, and mat composition. When the mat is warmed to 37 ° C., a biopolymer solution such as a collagen solution forms a gel in the mat. As used herein, the term "gel" refers to a network or mesh or biopolymer filament with an aqueous solution trapped within the biopolymer backbone by biopolymer fibrils. Alginate gels used as vehicles for delivering cells to the mats, mat composites, or mat compositions of the present invention can be produced by the addition of calcium which causes polymerization at room temperature and neutral pH. Thus, selected epidermal, endothelial, or mesothelial cells may be plated on the surface of a gel-filled mat, mat composite, or mat composition.

The biopolymer mat may be used alone, for example, as a collagen membrane to form a root barrier or a periosteal barrier to aid bone repair. Alternatively, biopolymer mats can be utilized in biopolymer mat compositions, including biopolymer mats and biopolymer foams, such as in the case of tissue repair of the dura of the central nervous system, for example, single density Cast into a finished mat with two layers with different properties, namely
A structure having a high density and low to zero porosity mat layer and a low density and high porosity foam layer may be produced. Single and double density biopolymer foams
It is set forth in U.S. Ser. No. 08 / 754,818, filed Nov. 21, 1996, the entire contents of which are incorporated herein by reference. An implant site that requires a complex tissue, such as skin consisting of two tissues, the epidermis and the dermis, is a mat composition that includes epidermis, mesothelium or endothelial cells on the mat surface and mesenchymal cells within the foam skeleton. May be processed. In these applications, the low porosity mat side can reduce adhesions or fluid loss on one surface, and the high porosity side can attract and support the cell growth and differentiation needed for healing. it can. Biological improvements can also be made, for example, keratinocytes can be grown and differentiated on one side of the mat to produce a stratum corneum. Mat compositions comprising one or more layers of a biopolymer mat or biopolymer mat composite, and two or more layers of single or double density biopolymer foam are also specifically contemplated herein. is there.

As described above, the mat includes one fiber, a string, a bundle of fibers,
Or, including fiber structures such as cloth, relative or directional reinforcement may be achieved or directional cell growth may be achieved. Examples of implants requiring such a structure include, for example, a bone replacement structure or a temporary reinforcement structure.

The mat, mat composite, or mat composition may be cast into shapes other than sheets. It may be cast into a circular shape, such as a tube or a sphere, to produce a membrane structure that can contain materials or liquids for specialized functions. Examples of mats, mat composites, or implants made from mat compositions include, for example, blood vessels, ducts, ureters, bladders,
And bone implants formed from mat cylinders filled with skeletal replacement materials. As used herein, the term "bone replacement material" refers to a material that can fill the interstitial space within a bone and assist in bone repair. Examples of bone replacement materials include, but are not limited to, autologous bone grafts, bone powder, demineralized bone, calcium sulfate, and calcium phosphates such as hydroxyapatite, brushite and octacalcium phosphate. Using a mat composition with or without cells, with or without the addition of a dried mat and a single density foam, a biological tissue equivalent or model tissue system can be constructed. An example of this is the growth of dermal fibroblasts in a single-density foam and the differentiated growth of keratinocytes on a porous surface mat layer for a skin model or a bioimplant system. Alternatively, bioimplant systems are a rapid replacement for lost functionality in critical locations and can be lyophilized for stockpiling. If this is not desirable as a bioimplant system, the complex in which the cells have been implanted and grown can be lyophilized for use as an implant at a later date, as described above for cell conditioning, and the structure should be Regrowth can be directed to the host tissue through information derived from the material deposited by the cultured cells.

Further, the mats, mat composites, and mat compositions of the present invention may be formed into a vascular prosthesis in the form of a tube, and smooth muscle cells supplied in a neutralized collagen solution that gels after delivery. Internally, or externally with adventitia fibroblasts, or endothelial cells on the luminal surface. For example, a tubular mat can be formed by dispensing a biopolymer fibril slurry into a tube whose inside diameter is the outside diameter of the desired mat. The tube is continuously rolled while the slurry dries. During drying, fibrils settle on the tube. Applying more slurry to the tube can make the mat thicker. When all fibrils are dry, remove the mat from the tube as a seamless product. If reinforcement of the tubular mat is preferred, fibers, bundles of fibers, or cloth may be placed in the tube before or during the application of the slurry to the tube. These methods also apply when casting the mat into other shapes, such as spheres, where rolling will occur in more than one direction.

In a vascular prosthesis, the mat will be the inner layer into which the endothelial cells will be seeded. A single density foam would be cast around the mat to provide a luminal matrix for smooth muscle cells and adventitia fibroblasts.

The biopolymer multifilament ligament implant of the present invention may be enhanced with the mat, mat composite, and mat composition of the present invention to facilitate cell inoculation. For example, a continuous ligament multifilament structure can be made with or without extracellular matrix microparticles to provide certain properties. The ligament cells may then be delivered to the ligament, which may be embedded within the mat casing. The ligament may then be attached to a tubular tissue ripening chamber. Once the ligament cells attach to the ligament, stress is applied by planning the periodic stretching of the ligament in the axial direction, and the stress is increased as the ligament matures. The aged biopolymer ligament can be used, for example, as a ligament prosthesis.

[0070] Dental implants can be formed from the mats, mat composites, and mat compositions of the present invention. For example, the present mat, mat composite, and mat composition may be made as specialized dental implants for root ligament repair and bone reconstruction. In one embodiment, the mat, mat composite, and mat composition of the present invention can be fabricated as an apron-type implant and tied around the teeth around the apron to secure the teeth. In another embodiment, the mat, mat composite, and mat composition are designed as a molding of the ridge, filled with a bone replacement material.

Apron-type mats for promoting root ligament tissue repair and bone remodeling can also be low porosity mats or folded double or quad density foams, ie double density foams , Can also be produced as a mat composition containing
It can be placed between the gingival piece and the alveolar area where root ligament repair and bone reconstruction is required. The mat may be designed to prevent invasion by the adherent epithelium of a band of cleaned and flattened teeth. Thus, the root ligament cells migrate to the mat, mat composite, or mat composition, bind to the mat, mat composite, or mat composition, and transfer extracellular matrix products within the mat, mat composite, or mat composition. Can be secreted. The mat, mat composite, or mat composition can further penetrate capillary endothelial cells and immune cells that provide protection from microbial attack. By eliminating epidermal cells and stimulating root ligament cells, the present mat, mat composite, or mat composition can promote regeneration of root ligament and alveolar bone. The apron-type dental implant may be further modified to include the bone replacement material described herein. In one embodiment, the material may be included in the outer pocket formation of the apron that can be placed on eroded alveolar bone. This bone replacement material provides a pathway for invading bone cells. In addition, the apron-type dental implant can include extracellular matrix microparticles generated from tooth tissue. These extracellular matrix microparticles will provide suitable growth factors, such as bone and ligament specific growth factors, to promote the growth of root ligament cells and bone cells into implants.

Alternatively, mats, mat composites, and mat compositions according to the present invention may be made as fossil lids after tooth extraction. By using this mat, it is possible to cover the fossa filling material inserted into the fossa after tooth extraction. These fossa fillers promote bone regeneration in the fossa and, at a minimum, provide the basis for metal, e.g., titanium, fasteners, and subsequent placement of the crown. Immediately after tooth extraction, a fixture of titanium or other material may be reinforced, covered, or "tented" with a calcium phosphate bone replacement material reinforced with one of the mats, mat composites, or mat compositions described herein as aprons. Can be fixed.
The fossa filler may further include extracellular matrix microparticles arising from bone tissue or tooth papillae. These extracellular matrix microparticles provide a suitable growth factor, such as bone-specific growth factor, for promoting bone cell growth within the implant. In addition, if the bone base for a metal dental implant does not provide sufficient support for the metal implant, the bone base may be reinforced or reinforced with the foams and foam compositions of the present invention. Can be reinforced.

In yet another embodiment, the mat, mat composite, and mat composition can be designed as a ridge replacement or ridge molding. Ridge substitutes are used to underpin dentures. Typically, the ridge replacement is designed as a biopolymer mat tube of appropriate length, and the mat tube is filled with a non-absorbable calcium phosphate bone substitute to provide a mineralized platform along the ridge. To build
It promotes the development of the bone and connective tissue framework around the calcium phosphate particles.
The ridge ridge according to the invention has the same design as the ridge replacement, with the exception that
The mat tube is filled with a resorbable bone replacement material to promote bone development. The composition of the crest ridge promotes bone cell and blood capillary penetration and can cause ridge regrowth and recovery, for example, prior to implanting a denture or metal implant. The mat tube of the molding of the ridge may further contain extracellular matrix fine particles that promote bone regeneration of the ridge.

Similarly, a book enriched with extracellular matrix microparticles derived from organs such as heart tissue, bladder tissue, small intestine-derived tissue, lung tissue, pancreatic tissue, liver tissue, skin tissue, and other organ tissues Mats, mat composites, and mat compositions according to the invention include, for example, pancreatic endocrine glands, such as pancreatic islet cells, or those of the liver, such as hepatocytes.
Inoculated as a means of promoting cell proliferation before and / or after transplantation, such that a functional replacement organ develops after transplantation and after vascularization of the mat implant in which the cells are implanted. Is also good.

Examples of cell types that have been successfully grown in and on the mats and mat compositions of the present invention include mesenchymal cells, dermal fibroblasts, keratinocytes, osteoblasts, gingival fibroblasts, and Includes tendon cells as well as ligament cells.

The present invention will be further described in the following examples, which should not be construed as further limiting. It should be noted that the contents of all references, including written references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are hereby incorporated by reference. Specify it explicitly.

Example 1: Preparation of a mat from extracts of fetal pig skin 5 ml of 5 mg / ml collagen was added to 20 ml of 30 mM sodium phosphate, pH
Diluted on ice at 7.4. Adjust the pH to 7.4 with 5% ammonium hydroxide,
The final volume was adjusted to 30 ml with phosphate buffer. The solution was alternately inverted for 2 hours at 4 ° C. The solution was poured in batches onto a 270-mesh stainless steel screen (〜50 μm pore size) and the resulting 2 mm thick fibril-gel was dried at 30 ° C. for 16 hours. The mat peeled from this screen, without further manipulation, has a wet tensile strength of 0.03 Mpa, a piece of 1 cm ² can withstand more than 22 hours of digestion with 10 U of collagenase, and 6. Digestion with 4 U elastase was tolerated at 37 ° C. for more than 25 hours. Addition of 0.1 M NaCl to this dilution buffer resulted in a mat of mainly fibrils (slight gel) with a strength of 0.5 Mpa.

Example 2: Collagenase resistance of the mat The collagenase resistance of the mat was calculated by calculating the digestion time per mg of collagen skeleton. The mat was digested in collagenase at a rate of 87 minutes per mg of collagen, but the two types of dual density foams were both 1 mg
Were digested at a rate of 25 minutes per collagen scaffold. Thus, collagen mats are more than three times more resistant to collagenase digestion than similar density collagen scaffolds, and this similar density collagen scaffold itself is three times more resistant to digestion than a single density foam. And resistant.

Example 3 Formation of a Mat Composite Collagen fibrils were formed and their collection was accelerated by centrifugation. The collagen fibril slurry was resuspended and deposited on a screen. The remaining solution was drained from the screen over 15 minutes, and then a 1% gelatin solution was spread over the collagen collection. The material was allowed to dry, during which time the two biopolymer component layers melted. After drying, the mat composite was removed from the screen and examined for its tensile properties. The strength of this mat was lower than the control mat, but the strain was greater than the control mat. The order of application may be reversed. In other words, this example shows how the elasticity of the mat can be manipulated.

Example 4: Manipulation of the tearing properties of the mat 5 ml of collagen fibril / fibril bundle slurry from 140 μm to 400 μm in size
m3 with 0.03 g of calcium sulfate particles. The blended slurry was applied to a porous support and dried. The resulting mat had single calcium sulphate particles or agglomerates of two to five different particles spaced 200 to 700 μm from each other.

[0081] Example 5: suspended in a growth human dermal fibroblasts in the skin fibroblasts on the mat 5 × 10 ⁵ cells / ml in Dulbecco's minimal essential medium plus 5% bovine serum . The mat was hydrated in this medium.
The cell suspension was added dropwise to a 1 cm ² mat, and the cells were allowed to adhere. After cell attachment, additional media was added and the mat was incubated at 37 ° C for 24 hours. When observed with a fluorescence microscope, the cells had a normal spindle-shaped fibroblast morphology. In addition, the arrangement of the cells reflected the texture of the mat surface. When the mat surface was flat, the fibroblasts were evenly distributed on the mat surface. When the mat surface had texture, the distribution of fibroblasts reflected the pattern of texture. Cells were aligned in and along the furrow.

Example 6 Cell Growth of Keratinocytes on a Mat Keratinocytes were inoculated on a mat into a keratinocyte growth medium to a density of 1 × 10 ⁵ cells per cm ² . After culturing in this medium for 3 days, the mat was fixed and analyzed histologically. The keratinocytes were attached, healthy, and proliferated to form multiple layers characteristic of this cell type.

Example 7: Evaluation of the cytotoxicity of mats Extracts are made from sterilized and non-sterilized mats in cell culture medium and given to small confluent cultures of dermal fibroblasts to extract from the mats. Was evaluated for cytotoxicity. Extracts were prepared by placing a 60 cm ² sample in 30 ml of medium (DMEM 5% serum) and incubating for 24 hours at 37 ° C. using a method based on USP guidelines. The extract-medium was sterilized by passing through a 0.22 micron filter. This extract-medium is applied to a small confluent (about 80% confluent) fibroblast culture,
Checked 48 hours later. In all cultures, cells grew to confluence (cell growth was not inhibited by the extract) and cell morphology was in control cultures (DMEM + 5%
Sera). These results indicated a non-cytotoxic substance.

Example 8: Effect of UV treated mat The dried mat on the screen was treated by exposing it to 254 nm of UV light for 20 minutes per side. The wet tensile strength of the treated mat is 2.6 MPa,
In contrast to the control sample, which was not treated with UV light, 0.27 MPa.

Example 9: Incorporation of live cells during production Biopolymer fibrils were diluted in Dulbecco's minimal basal medium (DME) at 1 mg / ml collagen and the pH was adjusted using 5% ammonium hydroxide. Adjusted to 7.4 and formed by mixing the solution for 3.5 hours at 4 ° C. The fibril solution was used to resuspend dermal fibroblasts at a concentration of 3 × 10 ⁶ cells / ml. The cell / fibril solution was placed in a tissue culture insert containing only polycarbonate membrane in 0.4 μm pores or even single density foam. The insert was placed in a multi-well tissue culture dish with additional DME, and the dish was incubated for cell growth. When the inserts were removed, processed and histologically analyzed on days 1 and 7, fibrils and healthy cells were found entangled in layers on the insert membrane or in the foam. Was done.

Example 10 Continuous Extraction of Acids and Enzymes from Collagen from Porcine Fetal Skin The preferred form of collagen used to make biopolymer fibrils for biopolymer mats is described in Example 11 And fetal porcine collagen extracted with acid. Furthermore, in order to purify a less preferred form of fetal porcine collagen for use in preparing a biopolymer mat, the skin remaining after collecting the acid collagen extract of Example 11 may be enzymatically digested. Most of the collagen has one or both telopeptides removed, some of which have a triple helix or one telopeptide and a triple helix. To extract such collagen, the remaining skin is extracted in an extraction buffer supplemented with 0.017% pepsin (at low temperature, as in the subsequent steps) for two of the five to seven day periods. And mix at a ratio of 2 volumes of solution to 1 volume of skin. The two types of pepsin extracts were collected, combined and 40 ml at 13,000 rpm.
/ Min by continuous flow centrifugation. Next, the pepsin-extracted collagen is purified in the same manner as the acid-extracted collagen, except that the solution volume is not reduced before dispersing the first collagen precipitate and the second NaCl addition is 0%.
. Up to 7 M, filtration is only up to 3.0 μm, the hollow fiber pore size has a cutoff of 100,000 Da and the concentration reached with hollow fibers is 6 to 9 mg / ml.

As determined by polyacrylamide gel electrophoresis, the collagen extracted by acid is a mixture of fetal collagen including collagen type I, collagen type III, and collagen type IV. Pepsin-extracted collagen, that is, lightly truncated collagen, contains the core of the triple helix, but is a mixed collagen telopeptide, with the exception of a small amount of type IV, which has been degraded by enzymatic treatment. Do not have. Further, the collagen concentration is determined with a hydroxyproline assay standard. The identity and integrity of the protein is confirmed by polyacrylamide gel electrophoresis and the viscosity is normalized to 5 mg / ml with a minimum of 50 centipoise. EXAMPLE 11 Both the acid-extracted and pepsin-extracted porcine fetal collagens of 11 and 10 were neutral p
H can form a gel. The absence of denatured contaminants is measured by polarity (values below -350 degrees are acceptable). In addition, quality is tested by the ability of collagen to form fibers.

Example 11: Acid extraction of collagen from fetal pig skin The preferred molecule used to make biopolymer fibrils for mats is collagen. A preferred source of collagen is fetal pig skin. Collagen is purified from such skin by methods known to those skilled in the art as follows. Porcine fetal skin was partially thawed, bleached, and sterilized 7 times after removing them from the rinsed pig uterus.
The fetal skin of the near-term fetus immersed in 0% ethanol is peeled off to obtain fetal pig skin. The skin and collagen solution are kept cool throughout the subsequent procedure.
After crushing the skin, it was washed in batches of 5 kg with multiple 5 liters of 1/3 × PBS, and excess washing solution was removed by filtering the skin through a layer of cheesecloth. Collagen is extracted from the washed skin for 7 days with extraction buffer, 0.5 M acetic acid and 2 mM EDTA at pH 2.5 at a rate of 150 g skin per liter of extraction buffer. On day 8, the extract is collected after filtering the skin through cheesecloth and sodium chloride is added to the extract to bring the solution to 0.9 M NaCl. The collagen is precipitated from the solution over the next two hours while stirring the solution. The precipitated collagen is collected in pellet form by continuous flow centrifugation at 300 rpm / min at 12,000 rpm. Discard the supernatant and disperse the pellet in 60% of the original volume of extraction buffer. After stirring overnight in this solution to redissolve the pellet, the collagen is re-precipitated with NaCl to 0.9 M over 2 hours and collected again as a pellet by continuous flow centrifugation. The pellet is dispersed and redissolved in the same volume of extraction buffer as the original pellet. The solution is stirred overnight to allow the pellets to adopt and then clarified by continuous flow centrifugation at 15,000 rpm at 40 ml / min. Discard the pellet and filter the collagen supernatant to 2.0 μm. Next, the collagen concentration is determined by the hydroxyproline assay. The solution is then dialyzed in a continuous concentration step with a 0.1 μm hollow fiber filter, diluted with deionized water and repeated until the acetic acid concentration reaches 0.05M. When this acetic acid concentration is reached, the collagen solution is concentrated to 5 to 7 mg / ml with hollow fibers. The final concentration of collagen is then confirmed by the hydroxyproline assay.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.
Such equivalents are intended to be encompassed by the following claims.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 9, 2000 (200.3.9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Shiosat Tracy M. United States 02026 Reading, Massachusetts, Bothwell Road 26 (72) Inventor Big Gray Michael Jay. United States 02143 Somerville, Mass., Sunborn Avenue 18 F-term (reference) 4C081 AA02 AA13 AA14 AB02 AB06 AB11 AB13 AB18 AC03 BA16 CA052 CA242 CC05 CD012 CD042 CD072 CD112 CD141 CD172 CD27 CD34 DA02 DA04 DA05 DA06 DB03 DB04 DC02 DC03

Claims (21)

[Claims] 1. A biopolymer mat containing biopolymer fibrils. 2. The biopolymer fibril is selected from any of collagen, laminin, elastin, fibronectin, fibrinogen, thrombospondin, gelatin, polysaccharide, poly-l-amino acid and a combination thereof. The biopolymer mat according to claim 1, wherein 3. The biopolymer mat according to claim 1, wherein the biopolymer fibrils are derived from collagen. 4. The biopolymer mat according to claim 3, wherein the collagen is porcine fetal collagen. 5. The biopolymer mat of claim 1, further comprising macromolecules required for cell growth, morphogenesis, differentiation, or tissue construction, and combinations thereof.
A biopolymer mat according to item 1. 6. The biopolymer mat according to claim 1, wherein the biopolymer fibrils are cross-linked. 7. The biopolymer mat according to claim 1, further comprising a fiber, a string, a bundle of fibers, a cloth or a non-woven fabric made of an absorbable polymer. 8. The biopolymer mat according to claim 1, wherein the biopolymer further comprises pores. 9. The biopolymer mat of claim 1, wherein said mat is in or on a support. 10. The biopolymer mat according to claim 9, wherein said support is porous. 11. The biopolymer mat according to claim 10, wherein the porous support is an absorbable polymer cloth. 12. The biopolymer mat is bonded to a single-density biopolymer foam, wherein the single-density biopolymer foam binds biopolymer molecules and / or biopolymer filaments. A network of interconnecting microcompartments interspersed within the wall of the microcompartment, wherein said microcompartments have approximately equal volume dimensions in a range from about 1 μm to about 300 μm, x = length, y = width, and z = The biopolymer mat of claim 10, having a height, and having an average wall thickness of less than about 10 μm. 13. The biopolymer mat of claim 12, wherein said biopolymer foam is derived from porcine fetal collagen. 14. The biopolymer mat of claim 1, wherein said biopolymer mat further comprises cells. 15. A step of forming a solution containing biopolymer fibrils, applying the solution to a porous support, and wherein the density of the fibrils is increased when liquid is excluded from the support. Forming the biopolymer mat by concentrating the solution on the porous support and / or permeating through the porous support so as to increase on the body or in the support A method for producing a biopolymer mat, comprising: 16. The biopolymer fibril is derived from porcine fetal collagen,
The method according to claim 15. 17. The method of claim 15, further comprising cross-linking said biopolymer fibrils or mat. 18. The method of claim 15, wherein the solution containing biopolymer fibrils is further treated with cells or macromolecules required for cell growth, morphogenesis, differentiation, or tissue construction, and combinations thereof. The described method. 19. The method of claim 19, further comprising treating the solution containing the biopolymer fibrils with cells or macromolecules required for cell growth, morphogenesis, differentiation, or tissue construction, and combinations thereof. 16. The method according to 15. 20. A network of interconnecting microcompartments interspersed with biopolymer molecules and / or biopolymer filaments within the wall of the microcompartment, said microcompartment having a size of about 1 μm to about 300 μm. Casting a single density biopolymer foam having substantially equal volumetric dimensions in the range, x = length, y = width, and z = height, and having an average wall thickness of less than about 10 μm. The method of claim 15, comprising: 21. The method of claim 20, wherein the biopolymer foam is selected from any of collagen, alginic acid, polyvinyl alcohol, chondroitin sulfate, laminin, elastin, fibronectin, fibrinogen, and combinations thereof. Method.