EP1069822A1 - Creation de tissus tridimensionnels - Google Patents

Creation de tissus tridimensionnels

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Publication number
EP1069822A1
EP1069822A1 EP99917384A EP99917384A EP1069822A1 EP 1069822 A1 EP1069822 A1 EP 1069822A1 EP 99917384 A EP99917384 A EP 99917384A EP 99917384 A EP99917384 A EP 99917384A EP 1069822 A1 EP1069822 A1 EP 1069822A1
Authority
EP
European Patent Office
Prior art keywords
cells
vascularized
providing
constructs
dimensional tissue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99917384A
Other languages
German (de)
English (en)
Inventor
Craig R. Halberstadt
Walter D. Holder, Jr.
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Charlotte Mecklenburg Hospital
Original Assignee
Charlotte Mecklenburg Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Charlotte Mecklenburg Hospital filed Critical Charlotte Mecklenburg Hospital
Publication of EP1069822A1 publication Critical patent/EP1069822A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • This invention relates to methods of tissue engineering, and particularly to a method of creating a vascularized three-dimensional tissue in mammal.
  • tissue loss remains a serious problem. Tissue damage can be caused by various reasons such as surgical removal of cancerous tissues and trauma. After the removal of the damaged tissue, reconstruction is often desired. Another reason relates to organ transplantation where it is possible to replace a damaged organ with a healthy donor organ.
  • organ transplantation approach has drawbacks. First, organs for transplantation are scarce and one may have to wait for a long time before a suitable organ becomes available. Second, the immunological incompatibility between the donor and donee tissues limits the ability of the transplanted organ to survive and function, and further aggravates the availability problem. Moreover, the transplantation process itself is complex, invasive, and costly.
  • U.S. Patent No. 5,716,404 discloses a method for reconstruction or augmentation of breast tissue by implanting isolated cells with a polymeric matrix.
  • the polymeric fibrous matrix used therein has a structure similar to the silicone implants now used. Complex and invasive surgery has to be performed to insert the matrix into breast tissue.
  • a major challenge in mammalian cell transplantation and developing vascularized three-dimensional tissue is promoting long-term cell survival of the transplanted cells.
  • the current approaches rely on transplanting cells on surfaces that have diffusional distances greater than 250 ⁇ m for nutrients which are provided by the surrounding host vasculature.
  • a necrotic core of tissue develops within the construct because of an inadequate supply of nutrients and gases. Therefore, there is need for an effective and substantially non-invasive method of creating vascularized three-dimensional tissue in vivo.
  • the discrete tissue construct acts as a functional unit, e.g., the growth of the transplanted cells and the vascular ingrowth of the cells from the surrounding tissue forms small cellular "islands", interconnected with the surrounding vasculature. As a result, a vascularized three-dimensional tissue is formed.
  • the invention is directed to methods for providing a vascularized three-dimensional tissue in which transplanted cells are no greater than 250 ⁇ m away from a nutrient source, i.e., a developing vascular bed.
  • a porous construct containing cells for transplantation is delivered into an area of interest in a living subject where it is desired to build a vascularized three-dimensional tissue.
  • the implanted porous construct supports the adhesion, growth and migration of the transplanted cells as well as the ingrowth of the tissue cells surrounding the construct, thus forming a three dimensional discrete tissue.
  • the delivery of the porous construct is performed in a non invasive manner, for example, by injection, or by delivering in a biocompatible pouch having an arteriole and venule connection from the outer pouch into the inner pouch. It is also preferred that a plurality of the porous constructs are delivered.
  • the porous construct is made from at least one biocompatible material which supports cell adhesion, growth, and migration. Such biocompatible materials can be bioresorbable or non-resorbable.
  • the cells to be transplanted can be seeded in the porous construct.
  • the porous construct has a dimension in which the distance is about 50 ⁇ m to about 500 ⁇ m from at least one point on the outer surface to the center of the construct, such that when cells are seeded within the construct, the innermost cell within the construct is no greater than about 250 ⁇ m away from an outer surface of the construct. In an average tissue, the maximum distance found between a metabolically active cell and a capillary is approximately 250 ⁇ m.
  • the construct also has interconnected pores having a pore size of from about 10 ⁇ m to about 300 ⁇ m.
  • the porous construct may also contain a signal (e.g., a growth factor or an extracellular matrix protein) for modifying, preferably promoting cell adhesion, growth, or migration.
  • the porous construct containing cells to be transplanted is delivered directly to the area of itnerest.
  • a vascular bed is formed in the area of interest by implanting, before the delivery of the porous construct, a second construct which promotes the formation of a vascularized bed in vivo. The porous construct containing cells to be transplanted is then delivered onto the vascularized bed for formation of a three-dimensional tissue.
  • the method of this invention can be used in the reconstruction of many different tissues, such as breast tissue, pancreatic tissue, liver tissue, neural tissue, kidney tissue, muscle tissue and skin tissue.
  • the present invention provides a minimally invasive procedure and effective method for development of three- dimensional tissues.
  • Figure 1A shows a trichrome stain of alg-RGD (IX concentration of RGD) beads implanted in the subcutaneous space of a rat and harvested after one week of implantation illustrating tissue ingrowth between the beads (40X magnification);
  • Figure IB shows a formation of a vascular bed at the interface of alg- RGD beads and tissue (200X magnification);
  • Figure 2A shows tissue ingrowth between the beads (trichrome stain of alg-RGD (100X concentration of RGD)) implanted in the subcutaneous space of a rat, harvested after two weeks of implantation (40X magnification);
  • Figure 2B shows the formation of a vascular bed at the interface of the beads and the tissue (200X magnification);
  • Figure 3 shows Factor VIII localization in tissue that has grown between alginate-RGD beads
  • Figure 4 illustrates cross sections of collagen beads that were seeded with smooth muscle cells
  • Figure 5 A shows a trichrome stain of histo logical section (40X and
  • Figure 5B illustrates two Factor VIII stains from the same section as Figure 5A indicating that vascular endothelial cells are located throughout the collagen bead implant. (Top picture is 40X magnification and bottom picture is 200X magnification); and
  • Figure 6 illustrates the growth of aortic smooth muscle cells implanted in collagen beads harvested after two weeks (400X magnification).
  • Figure 7 illustrates the cell growth of endothelial cells stained for Factor VIII on a collagen bead harvested after two weeks.
  • Figure 8 is a bar graph comparing histo logical scoring of 2-week subcutaneous implants of alginate rods or beads or collagen beads.
  • Figure 9 is a bar graph comparing histo logical scoring of alginate- RGD bead subcutaneous implants at 2, 4 and 8 weeks from implant.
  • Figure 10 is a bar graph comparing area of alginate-RGD bead implants at various time intervals.
  • Figure 11 is a bar graph comparing width of alginate-RGD bead implant at different times.
  • Figure 12 is a bar graph comparing thickness of Alginate-RGD bead implants at different times.
  • Figure 13 is a bar graph comparing thickness of the capsule surrounding alginate-RGD bead implants at different times.
  • This invention provides a method for developing vascularized three- dimensional tissue in a living subject such as a human.
  • a porous construct seeded with cells for transplantation is created and is delivered into an area of interest in a living subject where it is desired to build a vascularized three-dimensional tissue.
  • the implanted porous construct supports the adhesion, growth and migration of the transplanted cells as well as the ingrowth of the tissue cells surrounding the construct, thus forming a three-dimensional discrete tissue.
  • the porous construct of this invention can be in any three- dimensional shape, so long as it has a dimension in which the distance between an outer surface of the construct and the center of the construct is from about 10 ⁇ m to about 500 ⁇ m, preferably from about 25 ⁇ m to about 400 ⁇ m, more preferably from about 50 ⁇ m to about 250 ⁇ m.
  • the porous construct can be in the shape of spheres, particle beads, rods, triangles, threads, and cubes.
  • the construct has a dimension such that the distance from each and every surface to the center of the construct has a length of about 250 ⁇ m.
  • the construct is configured so that the innermost cell situated in the center of the construct is no greater than 250 ⁇ m from at least one surface of the construct. In this manner, it becomes more probable that, after the construct having cells therein is delivered to an area of interest in a living subject, all the cells within the construct are no greater than 250 ⁇ m from a nutrient source, e.g., a vascularized bed.
  • a nutrient source e.g., a vascularized bed.
  • the distance into the construct at which cells are found is determined primarily by the presence of nutrients and oxygen which diffuse into the porous construct to nourish the cells.
  • the maximum diffusion distance will depend in part on cell type and metabolic state, as well as the porosity of the construct, and may be up to about 400 ⁇ m, or more typically 250-300 ⁇ m. For highly metabolic cells, the maximum diffusion distance may be about 150 ⁇ m. For particles with linear dimensions larger than the maximum diffusion distance, cells will only populate the portion of the particle which may be reached by diffusion of oxygen and nutrients. Areas of the particle that are further from the surface than the maximum diffusion distance will be substantially free of cells. For particles with irregular shapes, the area populated by cells may include all portions of the particle which are within the maximum diffusion distance of at least one surface of the particle.
  • the porous construct has an interconnected macroporous structure formed by one or more biocompatible materials which constitutes the skeleton of the structure. There should be a plurality of macropores on the surfaces of the construct connecting the macropores within the construct so that exchange of molecules and cell migration can occur between inside of the construct and the surrounding environment.
  • the diameter of the pores ranges from about 10 ⁇ m to about 300 ⁇ m, preferably from about 25 ⁇ m to about 200 ⁇ m, more preferably from about 50 ⁇ m to about 100 ⁇ m.
  • macroporous constructs have pores of sufficient dimension to permit the passage of cells by migration through the pores.
  • vascular ingrowth proceeds into the large diameter pores, as well as the spaces between discrete constructs.
  • the vascular bed formed by the ingrowth can provide nutrients and oxygen to cells within particles having overall diameters much larger than the 250 ⁇ m suggested by the diffusion in microporous spherical particles.
  • the preferred constructs of the present invention are microscopic particulates with macroporous structure. A plurality of these particles forms a mass with a abundance of channels both within and between particles, through which nutrient-containing medium (plasma, lymph, cell culture medium, etc.) may reach cells in the particles and through which vascular ingrowth may occur.
  • the overall dimensions of these preferred particulates are typically on the order of 1 millimeter, 8
  • biocompatible is intended that the material used for making the porcus construct does not substantially adversely affect any desired characteristics of the cells to be seeded within the construct and of the cells and tissues in the area of a living subject where the construct is to be delivered. It is also intended that the material used does not cause any substantially medically undesirable effect in any other area of the living subject.
  • the materials are chosen such that the porous construct supports the adhesion, growth, or migration of cells. Thus, the biocompatible material used must allow cell adhesion, growth, and migration.
  • the biocompatible material should also be mechanically strong enough to support cells and to substantially maintain a dimension.
  • the biocompatible material may be synthetic or natural.
  • the biocompatible material may be a material that is non-resorbable such that it will remain once delivered inside the body of a living subject. More preferably the biocompatible material is bioresorbable and will be gradually degraded after the porous construct is delivered to an area of interest inside the body of a living subject. Such degradation should be in a slow and gradual fashion and preferably should take place after sufficient cells have grown within the construct to form an organized three-dimensional tissue.
  • the degradation of the bioresorbable material does not interfere with the formation of a three-dimensional tissue.
  • the bioresorbable material degrades within about 1 to 3 months after transplantation.
  • suitable bioresorbable materials include but are not limited to polyesters, polyethylene glycols, and hydrogels.
  • suitable non-resorbable materials include but not limited to polytetrafluoroethylenes (Teflon), nylon, polycarbonates, and polyethylenes.
  • Suitable examples also include but not limited to polysaccharides such as dextran, dextrin, starch, cellulose, agarose, carrageenan, alginate (a carboxylated seaweed polysaccharide), and the like; synthetic polymers such as polyvinyl alcohols, polyacrylamides, polyamides, polyacrylates, polyesters, polymethacrylates, polyurethanes, polyphosphazene, copolymers of lactic acid and glycolic acid, copolymers of lysine and lactic acid, copolymers of lysine RGD and lactic acid, and the like. Proteins such as collagens, copolymers of collagen and chondroitin sulfate (a proteoglycan component), and the like can also be used.
  • polysaccharides such as dextran, dextrin, starch, cellulose, agarose, carrageenan, alginate (a carboxylated seaweed polysaccharide), and the like
  • synthetic polymers such
  • the construct may also contain a signal for modifying cell adhesion, growth, or migration, preferably stimulating or promoting the adhesion, growth, or migration of the desired cells, and/or inhibiting the adhesion, growth, or migration of the undesired cells.
  • the signals may be selected from growth factors, hormones, extracellular matrix proteins and other cellular adhesion peptides identified in the extracellular matrix protein.
  • Growth factors include for example, epithelial growth factor (EGF), acidic or basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), heparin binding growth factor (HGBF), transforming growth factor (TGF), nerve growth factor (NGF), muscle morphogenic factor (MMP), and platelet derived growth factor.
  • EGF epithelial growth factor
  • FGF acidic or basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • HGBF heparin binding growth factor
  • TGF nerve growth factor
  • MMP muscle morphogenic factor
  • platelet derived growth factor examples include fibronectin, collagens, laminins, and vitronectins; and the tri-peptide RGD (arginine-glycine-aspartate) that is found in many of the extracellular matrix proteins.
  • a signal can also be included to induce the ingrowth of the desired cells, e.g., smooth muscle cells and epithelial cells, surrounding the construct in the area within the body of a living subject.
  • compounds which inhibit undesired cells such as cancerous cells and inflammatory cells, can also be included.
  • the signals can be covalently linked to a biocompatible material in the porous construct or associated with the porous construct by affinity or linked to a material that can be covalently linked to or associated by affinity with a biocompatible material in the construct. See, e.g., U.S. Pat. Nos. 4,517,686; 4,792,525; and 5, 330,911, incorporated herein by reference. 10
  • the method of providing the porous constructs has been successfully applied to the development of many different three-dimensional tissues in mammals.
  • the selection of the cells contained in the porous construct for delivery into an area of interest in the body of a living subject depends on the location and the type of three-dimensional tissue desired. For example, if a three-dimensional pancreatic tissue is desired, the porous construct may contain pancreatic cells.
  • cells such as vascular endothelial cells and smooth muscle cells can be used.
  • Other cells such as mesenchymal cells which include fibroblasts, chondrocytes, and adipocytes can also be used alone or in conjunction with endothelial or smooth muscle cells.
  • Other cell types that may be used in this invention include hepotocytes, renal tubular cells, Sertoli cells, thyroid cells, islet cells, skeletal muscle cells, neural cells, cardiac muscle cells, osteocytes, stem cells, and the like.
  • the cells used in this invention are preferably autologous, i.e., obtained from the living subject itself.
  • the cells can also be allogeneic, i.e., obtained from a subject of same species as the subject of interest or xenogeneic, i.e., from a subject of different species.
  • the cells can also be treated in vivo or in vitro, and before or after being incorporated into the porous construct.
  • the cells may be cultured to expand in number or modified to change one or more characteristics.
  • the cells to be incorporated in the porous construct of the invention can be homogeneous cells from one source, or from different sources.
  • the porous construct may have two or more biocompatible materials and two or more signals discussed above, each supporting a different type of cell in the construct.
  • the number of cells to be incorporated into the porous construct can range from 10 cells/cm 2 to 1x10 s cells/cm 2 .
  • porous construct can be made in many methods known in the art for making a porous scaffold supporting cell growth, with certain modifications of these methods to obtain the distinct features of the porous construct of the present invention as specified above. Such modifications will be apparent to an ordinarily skilled person in the art apprised of the present disclosure.
  • the macroporous lattice structure of the porous construct is formed during the process of polymerization of one or more polymers using conventional methods for making macroporous structures.
  • the macropores in the construct are formed by dissolution of a polymer or removal of one material after polymerization.
  • the construct is formed from a porous matrix of polymers in which the pores are formed by dissolution of one of the polymers.
  • two polymerization precursors e.g., collagen, fibrin, etc.
  • a reversible gel polymer precursor e.g., alginate, gums, agarose, etc.
  • the reversible gel polymer is then dissolved and removed to form an insoluble, porous matrix containing viable cells.
  • This methodology is used to construct small discrete tissue constructs as described herein with the requisite size constraints and the appropriate macroporous materials.
  • An alternative method is described in U.S. provisional application entitled: "Methods of Preparing Porous Hydrogel Materials and Products of the Methods", filed on even date herewith under Attorney Docket No. UMICH 8VI, and incorporated herein by reference. 12
  • the porous construct is made by any known technique of producing small beads or other discrete structures. Suitable techniques include but are not limited to pressure or air shear spraying, extrusion, emulsification, and droplet formation techniques such as electrostatic droplet formation, droplet formation by gravity, droplet formation by centrifugal forces, droplet formation using Raleigh liquid jet instability techniques, and droplet formation using inertial forces.
  • suitable porous constructs are prepared from collagen materials by solidifying a collagen solution or dispersion into dry beads using any known drying techniques including but not limited to spray drying and freeze drying. Also, beads having larger sizes are reduced to small sizes by, for example, grinding.
  • smooth muscle cells can be seeded and grown on porous polyglycolide beads or other porous biodegradable or naturally occurring materials (such as porous type I collagen beads).
  • a coating of a hydrogel for example an alginate RGD containing vascular endothelial cells is polymerized on the beads.
  • the alginate with endothelial cells encompasses the beads, and intercalates within the beads. This provides a close contact between the endothelial cells and the smooth muscle cells causing the necessary cell communication to occur.
  • the smooth muscle beads provide the physical structure necessary to create this tissue.
  • the porous construct should be sterilized before use. Examples of such techniques include but are not limited to UV irradiation, gamma irradiation, e- beam sterilization and sterilization using chemicals such as ethylene oxide.
  • the sterilization method used must not substantially adversely affect the structure of the porous construct and its ability to support the formation of a vascularized three- dimensional tissue.
  • porous construct it is also preferred to coat the porous construct with materials that promote cell adhesion and attachment to the structure of the porous construct.
  • materials include but not limited to fibronectin, vitronectin, 13
  • Such coating can be done at any time, for example, coating on polymer precursors, coating of a prepared construct before seeding cells in vitro, or after seeding in vitro but before delivering the construct into the body of a living subject of interest.
  • An example of coating is to use porous beads such as porous polyglycolide beads and porous collagen beads, prepared as described above and coated with a hydrogel, for example, an alginate RGD, by polymerizing the hydrogel onto the beads.
  • the hydrogel not only encompasses the beads, but also intercalates within the beads.
  • the cells to be transplanted with the porous construct can be incorporated therein while the porous construct is formed, for example, during the process of polymerization. Methods of incorporating cells during the formation of a porous structure are well known in the art. Alternatively, the cells can be seeded into a pre-formed porous construct. Since the pores of the porous construct are fairly large as described above, cell seeding can be easily done, for example, by immersing the porous construct for a period of time in a cell culture medium having cells to be transplanted floating therein. The cell density in the medium and the period of time can be easily controlled to allow a desired number of cells to attach within the porous construct.
  • the cells in the porous construct are allowed to grow in vitro for a period of time before they are transplanted along with the porous construct into the body of a subject of interest.
  • a porous construct with cells attached therein can be conveniently immersed in a suitable culture medium.
  • suitable signal molecules which modify cell adhesion, growth, and migration such as growth factors and extracellular matrix proteins, are added to the culture medium.
  • the porous construct also allows the cells in the area surrounding the construct to migrate into the construct, promoting the formation of a three-dimensional tissue.
  • at least one porous construct having cells therein is delivered into an area inside the body of a living subject where a three-dimensional tissue is desired to be built.
  • a plurality of porous constructs from 0.1 ml of volume to 500 ml of volume may be placed in the body. Each such discrete construct will develop and become interconnected with the surrounding vasculature and with other developed constructs, mimicking the functional units in natural tissue, thus facilitating the development of a three- dimensional tissue.
  • the delivery of the porous constructs is preferably performed in a substantially non-invasive manner, avoiding any complex surgical procedures.
  • the constructs could be fashioned to be easily injected into the tissue of interest. More than one injection may be desired.
  • these constructs are delivered in a biocompatible pouch that has an arteriole and venule connection from the outer pouch into the inner pouch.
  • a second construct is also used which may or may not contain cells. Once delivered into an area in the body of a living subject of interest, this second construct is capable of stimulating the formation of a vascularized bed surrounding it. Once the vascularized bed is formed, the porous construct as described above having therein cells to be transplanted is delivered onto or near the vascularized bed. The pre-developed vascularized bed can better supply the necessary nutrients required by the transplanted cells.
  • a porous construct as described herein is used as the second construct for pre-formation of a vascularized bed.
  • the second construct can be made from any biocompatible material and in virtually any shape or dimension, and is not limited by the features of the porous construct. Preferably, small spheres of about 5 15
  • the second construct need not be macroporous.
  • the second construct contains signals which stimulate the formation of a vascularized bed.
  • An example of such signal is the tri- peptide arginine-glycine-aspartate (RGD). This tri-peptide can be optimally linked to the second construct.
  • Such second construct is solid sodium alginate-RGD macro-beads of about 1 to 2 mm in diameter. These beads were shown to be capable of inducing the development of a vascularized bed inside the body of a living subject in a relatively short period.
  • the second construct may be delivered by the same methods used for the delivery of the porous construct as described above.
  • the present invention can be used in many different applications in mammals, particularly in human.
  • Many types of vascularized tissues can be developed using the method of this invention, including but not limited to three-dimensional breast tissue, pancreatic tissue, liver tissue, kidney tissue, muscle tissue, skin tissue, etc.
  • This method can be used in reconstructive therapy, reconstructing a body part that is injured or from which undesired tissue has been removed.
  • the method of this invention may be used for reconstruction or repair of soft tissue defects.
  • a plurality of discrete, macroporous constructs seeded with cells selected from chondrocytes, fibroblasts, endothelial cells, smooth muscle cells, etc. will be implanted to replace missing soft tissue, forming a tissue mass to fill a depression or produce a protrusion, or to otherwise provide a bulky mass of tissue where it is needed.
  • a soft vascularized breast tissue can be built in the area where cancerous breast tissue has been removed, thereby reconstructing the breast.
  • porous constructs containing both normal vascular endothelial cells and smooth muscle cells isolated from the patient can be created and delivered to the pocket of the expanded breast tissue in accordance with the present invention.
  • the method of the present invention can also be used to treat certain diseases in mammals.
  • pancreatic islets which release a desirable level of insulin can be transferred into relatively vascular sites such as the subcutaneous space and in combination with the creation of a three dimensional vascular bed using these discrete tissue constructs.
  • a plurality of macroporous constructs are constructed from biocompatible materials and contain cells to be transplanted for the development of the three-dimensional tissue.
  • the porous constructs are delivered into the desired area in a substantially non-invasive manner.
  • the dimension of porous constructs and the size of their macropores are made such that the transplanted cells are no greater than 250 ⁇ m apart from a nutrient source in the body.
  • alg-RGD Sodium alginate covalently coupled with arginine, glycine and aspartic acid tri-peptide (RGD)
  • alg-RGD Sodium alginate covalently coupled with arginine, glycine and aspartic acid tri-peptide (RGD)
  • Gibco/BRL calcium-free, magnesium free phosphate buffered saline
  • the alg-RGD solution was then aseptically dropped into a sterile filtered solution of 1.5% calcium chloride solution (1.5 ⁇ m of calcium chloride mixed with 100 ⁇ m of Milipore reverse osmosis water) using a syringe pump set at a flow rate of approximately 50 mL/hr. Beads of approximately 1 to 2 mm in diameter were formed during this process.
  • a subcutaneous pocket was then created on the back of a Fisher Rat (female) by using a purse string suturing technique (a 50 mL conical tube cap was used to mark the 39 mm circumference of the suturing plane, a 4.0 prolene suture was used to follow the outline of the circle, and the skin in the center of the circle was picked up using forceps), and one mL ofa bead slurry was transplanted into this subcutaneous space.
  • a purse string suturing technique a 50 mL conical tube cap was used to mark the 39 mm circumference of the suturing plane, a 4.0 prolene suture was used to follow the outline of the circle, and the skin in the center of the circle was picked up using forceps
  • Figure 1A shows the histological results of a trichrome stain after one week of implantation.
  • Figure IB shows the formation of a vascular bed at the interface of the beads and the tissue that forms. The results indicate that a vasculature can develop in a relatively short time period and may be induced by the shape of the materials. However, this material was not designed to be macroporous and did not provide a means for cellular in growth into the tissue construct. When larger solid implants were used in a similar animal model, a capsule was formed around the entire perimeter of the implant without the formation of a vascular bed that would support cellular survival. 18
  • the alg-RGD solution was then aseptically dropped into a sterile-filtered solution of 1.5% calcium chloride solution (1.5 gm of calcium chloride mixed with 100 Ml of reverse osmosis water) using a syringe pump set at a flow rate of approximately 50 mL/hour. Beads of approximately 1 to 2 mm in diameter were formed during this process.
  • FIG. 2A and 2B show the histological results after two weeks of implantation.
  • Figure 3 is histological section stained for Factor VIII which is a protein that is located on vascular endothelial cells. The material was implanted in a purse string subcutaneous space and harvested after two weeks. The dark open circles in the tissue have been positively identified as vascular endothelial cells.
  • FIG. 4 is a hematoxylin and eosin section of the collagen beads that were seeded with rat aortic smooth muscle cells. Note the highly porous structure of the beads (dark bands in the figure) and how the cells are growing within the beads after a six day period (lighter stained area).
  • Figures 5 A and 5B show the histological results after two weeks of implantation.
  • Figure 5 A is a histological section of collagen stain of the collagen beads implanted in a purse string subcutaneous space after two weeks implantation.
  • Figure 5B is histological section of the collagen beads staining for Factor VIII which is an antigen expressed on the surface of endothelial cells. This is a marker for vascularization. Note the staining that has occurred within the collagen bead.
  • tissue and organs are organized as discrete tissue segments. These segments are generally arranged as islands of parenchymal or stromal cells that are surrounded by an intricate capillary bed. The size of these discrete tissue constructs is dictated by the metabolic need of the cells and diffusional distances for nutrients, gasses, and waste products to and from the cells.
  • Alginate is a natural hydrogel that has previously been used for cell encapsulation and wound dressings. However, this material does not support cellular adhesion, as is required of any tissue engineering scaffold materials. Hence, a process was developed to couple the cellular adhesion tripeptide RGD to the alginate. The next task was to process this material into constructs of varying morphologies and therefore diffusional constraints.
  • Type I bovine collagen or sodium alginate was implanted into the subcutaneous spaces of Lewis rats.
  • Type I bovine collagen was acquired as porous spheres (0.4 to 0.7 mm in diameter), whereas sodium alginate coupled with RGD tripeptide was fashioned either into rods, solid beads (1.0 to 3.0 mm in diameter), or porous beads (2.7 to 3.2 mm in diameter).
  • the volume of each implanted material was one mL.
  • the implants were harvested after two weeks implantation and characterized as to capsule formation, vascular ingrowth, tissue ingrowth, and inflammatory response. Table I outlines the experiment.
  • IACUC Institutional Animal Care and Use Committee
  • the animals were perfused with either Bouins fixative or Z-fix formalin to preserve the tissue biomaterial interface. After at least 24 hours in a formalin fixative, the tissue samples were carefully cut in half at the mid-line to preserve the tissue biomaterial interface. These small tissue blocks were then processed using a paraffin embedding method. Four micron sections were cut, and the tissues were stained with either hematoxylin and e ⁇ sin, Masson's trichrome for the detection of collagen bundles, or stained with an antibody against Von willebrand's factor VIII (Dako, Cupertino, CA) for the detection of endothelial cells. Three to six animals were evaluated for each implanted material type.
  • At least two independent readers histogically scored the samples using 0 as a minimal and 4 as a maximal response.
  • the histology samples were evaluated for vascular ingrowth, capsule formation, tissue ingrowth, and inflammation.
  • the mean and standard deviations were calculated for all implanted materials, and an ANOVA analysis using Fisher's criteria was calculated using StatView 4.5 (Aacus, Berkeley, CA)
  • Table 1 Experimental layout for biomaterials implanted into a Lewis rat model. The number of samples analyzed for the implanted alginate rods was reduced due to the difficulty in preserving the tissue material interface during histological processing.
  • Alginate rods were formed by injecting a 1.2% alg-RGD solution into Spectra/Por dispodialyzers. These dialyzer tubes were 5ml volume and 10mm width, with a molecular weight cut-off of 300,000 and were pre-filled with sterile water. The water in the dispodialyzer case was poured out and replaced with sterile 1.5% CaCl 2 and then 3.3 mis of alg-RGD (in PBS) was placed inside the dialysis tube which was embedded in the case. The alginate-RGD was allowed to gel for one hour, and then the CaCl 2 was replaced with DMEM.
  • the dispodialyzer tube membrane was cut away with small scissors, and the cylinder of alginate was placed into a petri dish. A ruler was placed under the dish, and the alginate was cut into 1-cm rods with a sterile scalpel. The diameter was also 1 cm, making the implant approximately 1 ml, similar to the volume of the bead implants.
  • an incision was made on the flank of the rat, blunt dissection used to form a pocket, the rod was placed inside the pocket, and the incision was stapled.
  • sodium alginate (Pronova, Lot #411-256-05, city/state) was covalently coupled with arginine, glycine, and aspartic acid tripeptide (RGD).
  • the alginate-RGD (alg-RGD) was mixed in calcium-free, magnesium-free phosphate buffered saline (Gibco/BRL) at a concentration of 2 gm/100 mL (2%). After thorough mixing, the alg-RGD was filtered using a 0.45 mm filter (Nalgene). The alg-RGD solution was then aseptically dropped into a sterile-filtered solution of 1.5% calcium chloride solution (Sigma Chemicals, St.
  • a subcutaneous pocket was created on the back of a female Lewis rat using a purse string suturing technique for the beads.
  • the purse string was formed by using a 50-mL conical tube cap to mark a 39-mm circumference of the suturing plane, and then a 4.0 prolene suture was used to follow the outline of the circle.
  • a 1.5 cm incision was made slightly off center in order to bluntly dissect the subcutaneous space before implanting a 1 -mL bead slurry (either alginate or collagen beads), the incision was closed with Vicryl suture (Ethicon, Inc., Cincinnati, Ohio), and the prolene suture was tightened and tied to form a one mL subcutaneous pocket. All materials were pretreated overnight in a DMEM media containing (2%) fetal bovine serum.
  • Porous Collagen Beads A porous bovine Type I collagen bead was chosen to determine if a three-dimensional vascular bed could be established with the requisite dimensions to support transplanted cells.
  • Type I collagen beads were purchased from Cellex (Minnesota). The beads were glutaraldehyde fixed, with an average diameter of 0.4 - 0.7 mm and pore size ranging from 0.05 to 1 mm. The beads were highly porous, ranging in diameter from 0.4 to 0.7 mm, with a pore size range of 0.05 to 0.1 mm, and they promoted cellular attachment.
  • Type I collagen beads were implanted on the back of female Lewis rats using the purse string implant technique described above.
  • a macroporous alginate-RGD bead was developed with a diameter range between 2.7 and 3.2 mm, and an undetermined pore size range. Porous alginate-RGD beads were made using 3% alginate-RGD 25
  • the beads were sterilized using gamma irradiation.
  • the porous alginate-RGD beads were implanted on the back of female Lewis rats using the purse string implant technique described above. Histological cross-section of a porous alginate-RGD bead after 2 weeks implantation showed that a vascular bed is present throughout the bead implant.
  • the alginate-RGD beads had a minimal presence of giant cells and PMNs, while the Type I collagen beads showed a moderate to severe inflammatory reaction.
  • the inflammatory response to the Type I collagen beads may have been due to the glutaraldehyde fixation method used to covalently modify the material.
  • the animal model employed in this experiment provided the ability to test whether a vascular bed could be established in a large defect.
  • the distance from the host vascular bed to the center of the bead implant was 2.0 mm post-histological processing.
  • the ability to create a three-dimensional vascular bed with this thickness will allow for soft tissue reconstruction. It is also feasible through multiple step implants of a similar thickness to serially increase tissue volumes. This approach could be used to develop the larger tissue masses needed for breast reconstruction following a lumpectomy.
  • Macroporous alginate-RGD beads were prepared and implanted as described in Example 4, and histological observations were taken as described at 2, 4, and 8 weeks after the implants. The results are shown in Figures 9-13.
  • the porous alginate-RGD beads allowed tissued ingrowth with the presence of a vascular bed throughout the implanted material.
  • Figure 13 shows that the maximum thickness of the surrounding capsule (corresponding to scar tissue) occurs at two weeks (on the side of the implant facing muscle) or four weeks (on the side of the implant facing skin) and decreases by eight weeks.

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Abstract

L'invention concerne un procédé d'apport d'un tissu tridimensionnel vascularisé dans un sujet vivant. Le procédé consiste à) à créer à partir d'une matière biocompatible capable de support l'adhésion, la croissance et la migration cellulaires, une construction poreuse contenant des cellules à transplanter et b) à administrer la construction dans une zone d'intérêt dans le sujet vivant pour former un tissu tridimensionnel vascularisé. La construction préférentielle présente une dimension dont la distance de la surface la plus externe au centre de la construction mesure entre environ 50 νm et environ 500 νm. La construction préférentielle présente aussi une structure poreuse interconnectée dont la granulométrie des pores est de l'ordre d'environ 10 νm ne dépassant pas 300νm. Les cellules contenues dans la construction préférentielle ne dépassent pas une distance de 250 νm à partir de la surface externe de la construction.
EP99917384A 1998-04-09 1999-04-09 Creation de tissus tridimensionnels Withdrawn EP1069822A1 (fr)

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