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
  • A61L27/3804—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 specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
  • A61F2/02—Prostheses implantable into the body
  • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
  • A61F2/06—Blood vessels
  • A61F2/062—Apparatus for the production of blood vessels made from natural tissue or with layers of living cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
  • A61F2/02—Prostheses implantable into the body
  • A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
  • A61F2/2412—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body with soft flexible valve members, e.g. tissue valves shaped like natural valves
  • A61F2/2415—Manufacturing methods
• • 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
  • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
  • A61L27/3625—Vascular tissue, e.g. heart valves
• • 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
  • A61L27/3641—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 characterised by the site of application in the body
  • A61L27/3645—Connective tissue
• • 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
  • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
• • 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
  • A61L27/3843—Connective tissue
• • 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
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels

Definitions

• the invention relates to growing in vitro, human cells such as fibroblasts on a three-dimensional scaffold, comprising porcine aortic leaflets and walls, intact heart valves, other biological scaffolding suitable for reconstructing a valve or valve components, for example, including but not limited to the pericardium or the small intestinal submucosa, and biodegradable frameworks, such that the scaffold is populated with viable human cells having normal function, and fibroblasts are stimulated to produce human matrix proteins to supplement and replace the existing matrix on the scaffold.
• a three-dimensional scaffold comprising porcine aortic leaflets and walls, intact heart valves, other biological scaffolding suitable for reconstructing a valve or valve components, for example, including but not limited to the pericardium or the small intestinal submucosa, and biodegradable frameworks, such that the scaffold is populated with viable human cells having normal function, and fibroblasts are stimulated to produce human matrix proteins to supplement and replace the existing matrix on the scaffold.
• the resulting three-dimensional tissue constructs have a variety of applications ranging from transplantation or implantation in vivo for replacement and/or reconstruction of a single valve component or the entire heart valve, to screening cytotoxic compounds and pharmaceutical compounds in vitro.
• Valve replacement surgical therapy is required for the treatment of various types of valvular heart diseases, including, but not limited to, aortic stenosis, aortic regurgitation, mitral stenosis, mitral reg rgitation, pulmonary valve disease, tricuspid valve disease, multivalvular disease, Marfan syndrome and artificial valve disease.
• Two general types of valve replacement are available: the artificial, mechanical prosthesis or valve, and tissue biological prosthesis or valve.
• mechanical prosthesis such as the ball valve, the tilting disk and the central flow disk.
• tissue prostheses including preserved homografts and stent-mounted, porcine valve heterografts.
• the primary advantage of the mechanical prosthesis is durability, whereas the disadvantage is a requirement that patients be on an anticoagulant therapy to reduce the risk of thromboembolic complications.
• the advantage of the biological prothesis is a lower risk of thromboembolic complications, but may still require anticoagulant therapy in some situations. Moreover, the biological grafts which are currently used are not prone to sudden failure.
• Allogeneic human valves derived from a donor of the same species
• xenogeneic valves derived from a donor of a different species
• allogeneic, transplanted heart valves may be obtained fresh, or may be cryopreserved to maintain viability of cellular components.
• Patients receiving allogeneic transplants usually must undergo immunosuppressive therapy. Despite such therapy, many of the transplants become inflamed and fail within five to ten years.
• Autologous human tissue i.e.. derived from the recipient
• Such use eliminates complications of immunorejection resulting in better graft survival.
• complications ensue with autologous heart valve transplants e.g. , thrombosis and occlusion in the post-implant period and scarring of implant tissue.
• tissue-based heart valves for transplantation is necessary due to unmet patient demands to improve upon existing heart valve technologies, which are mechanical valves requiring the constant use of anticoagulants and glutaraldehyde fixed tissue valves which eventually experience calcification.
• the present invention relates to transplantable cardiac tissue or bioprosthetic grafts composed of human cells grown on three-dimensional frameworks, scaffolds or matrices, a method of culturing human cells on such frameworks and uses of such three-dimensional cell cultures.
• stromal cells including but not limited to human fibroblasts, are inoculated and grown on a three-dimensional frameworks, such as intact heart valves, aortic walls and leaflets, or other biological scaffolding suitable for reconstructing a valve or valve components, including for example, but not limited to the pericardium or the small intestinal submucosa or biodegradable frameworks or matrices.
• the preferred three-dimensional framework may be prepared from intact porcine heart valves, aortic wall tissue, or leaflets which are decellularized (at -20°C to -70°C or with detergents and enzymes) and sterilized by: chemical methods including, but not limited to, ethylene oxide and peracetic acid; irradiation including, but not limited to, gamma and electron beam; and steam sterilization including, but not limited to autoclaving. No viable cells remain in the decellularized/sterilized tissue samples which are used as a scaffold or framework for culturing the stromal cells.
• the stromal cells can be genetically engineered to express a gene product beneficial for successful and/or improved transplantation.
• the stromal cells can be genetically engineered to express anticoagulation gene products to reduce the risk of thromboembolism, or anti- inflammatory gene products to reduce the risk of failure due to inflammatory reactions.
• the stromal cells can be genetically engineered to express tissue plasminogen activator (TPA) , streptokinase or urokinase to reduce the risk of clotting.
• TPA tissue plasminogen activator
• streptokinase or urokinase to reduce the risk of clotting.
• the stromal cells can be engineered to express anti- inflammatory gene products, e.g..
• the three-dimensional culture system of the invention may afford a vehicle for introducing genes and gene products in vivo to assist or improve the results of the transplantation and/or for use in gene therapies.
• genes that prevent or ameliorate symptoms of valvular disease such as thrombus formation, inflammatory reactions, fibr ⁇ sis and calcification, may be underexpressed or overexpressed in disease conditions.
• the level of gene activity in the patient may be increased or decreased, respectively, by gene replacement therapy by adjusting the level of the active gene product in genetically engineered stromal cells.
• the present invention thus, relates to a method of repopulating porcine aortic walls and leaflets with human fibroblasts to produce human matrix proteins in which the porcine aortic leaflets and walls are first sterilized with peracetic acid (or by other chemical means such as ethylene oxide) or by radiation with an electron beam (or by gamma irradiation) or by steam (autoclaving) .
• peracetic acid or by other chemical means such as ethylene oxide
• an electron beam or by gamma irradiation
• steam autoclaving
• Such an approach provides an improved method and means of designing, constructing and utilizing aortic walls and leaflets, intact heart valves other biological scaffolding suitable for reconstructing a valve or valve component (e.g. , pericardium, small intestinal submucosa, etc.) and biodegradable frameworks, as scaffolding for growth and implantation of human fibroblasts in vitro.
• a heart valve consisting of human cells and human tissue matrix proteins made by human dermal or cardiac fibroblasts and a completely or nearly complete bioresorbable/biocompatible polymer scaffolding in the shape of different types of valves or their components, for example, but not limited to aortic, pulmonary, mitral, and tricuspid valves.
• aortic, pulmonary, mitral, and tricuspid valves for example, but not limited to aortic, pulmonary, mitral, and tricuspid valves.
• a valve or valve components e.g. , pericardium, small intestinal submucosa, etc.
• Such an approach provides an in vitro system in which human fibroblast cells retain their morphology and cell function for the secretion of bioactive molecules normally produced in the body by the cells of the aortic walls and leaflets or the intact heart valve or the pulmonary, mitral, and tricuspid valves.
• Figure 1 is a photograph of autoradiographed proteins synthesized by human dermal fibroblasts post seeding onto porcine aortic leaflets and walls.
• Figure 2 is a photograph of hematoxylin and eosin stained tissue sections: a) a fresh porcine leaflet (the cardiac fibroblast nuclei native to the tissue appear purple in coloration) ; b) a detergent and/or enzyme extracted porcine leaflet (no porcine cell nuclei are detected after chemical treatment) ; c) a detergent and/or enzyme extracted porcine leaflet cultured with human fibroblasts for 18 weeks (the dermal human fibroblasts are present in the porcine matrix). (Stained with Hematoxylin/Eosin.) (lOx) .
• Figure 3 is a photograph of a porcine leaflet seeded with human dermal fibroblasts and cultured for 4 weeks. (Stained with Hematoxylin/Eosin.)
• Figure 5 is a SDS gel autoradiograph analysis showing protein bands: non-viable porcine leaflet (lane 1) and wall biopsy (lane 2) seeded with human fibroblasts show protein synthesis, whereas unseeded, non-viable porcine leaflet (lane 3) and porcine wall biopsy (lane 4) show no activity.
• Fresh, viable porcine leaflet (lane 5) and wall biopsy (lane 6) seeded with human fibroblasts have similar patterns to fresh, viable, unseeded porcine leaflet (lane 7) and wall biopsy (lane 8).
• Figure 7 is a photograph of autoradiographed protein incorporation of human fibroblasts after dynamic culture on porcine aortic leaflets.
• the present invention relates to transplantable cardiac tissue constructs or bioprosthetic grafts grown in three-dimensional frameworks, a method of culturing human cells on such frameworks and uses of such three- dimensional, recellularized tissue constructs grown in cultures.
• stromal cells including but not limited to human fibroblasts, are inoculated and grown on a three-dimensional framework or construct of intact heart valves, aortic walls and leaflets or other biological scaffolding suitable for reconstructing a valve or valve components, for example, including but not limited to the pericardium or the small intestinal submucosa or biodegradable frameworks.
• Cells grown on a three-dimensional framework grow to form a cellular tissue-matrix which resembles tissue found in vivo to a greater degree than previously described.
• the three- dimensional cell culture system treated with human stromal cells is applicable to the proliferation of different types of cells and formation of a number of different tissues, including but not limited to aortic walls and leaflets, or intact heart valves, pulmonary, mitral, and tricuspid valves.
• the stromal cells grown in the system may be genetically engineered to produce gene products beneficial to transplantation, e.g. , anti-coagulation factors, e.g. f TPA, streptokinase, etc.
• the stromal cells may be genetically engineered to "knock out” expression of native gene products that promote platelet binding and clot formation, e.g.. fibrinogen, von Willebrands factor, or "knock out” expression of MHC in order to lower the risk of rejection.
• the stromal cells may be genetically engineered for use in gene therapy to adjust the level of gene activity in a patient to assist or improve the results of the transplantation.
• human foreskin fibroblasts in the three- dimensional tissue constructs has a variety of advantages and applications.
• the three-dimensional tissue constructs can be produced at a rapid rate and may itself be transplanted or implanted into a living organism without undue delay.
• the three- dimensional tissue constructs may also be used in vitro for testing the effectiveness or cytotoxicity of pharmaceutical agents, screening compounds for use in treatment of clotting or thromboembolism, as anticoagulants, as anti-inflammatory agents, as anti- calcification agents or as endothelialization agents.
• the three-dimensional tissue construct system may be cellularized within a "bioreactor" to produce a valve or valve component with leaflet mobility and full valve function.
• a bioreactor for example, an intact valve comprising of leaflets attached to the wall, may be assembled as a three-dimensional framework, inoculated with human stromal cells and maintained in recirculating culture medium regulated by a peristaltic or pneumatic pump which also keeps the leaflets or tissue sheets/patches in a dynamic state.
• the bioreactor provides a closed system free from problems of contamination during procedures involving sterilization, seeding, culturing, shipping and/or testing valve function.
• the three-dimensional framework for use in the present invention may be of any material and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it) ; and (b) allows cells to grow in more than one layer. It is preferred that allogeneic and .xenogeneic aortic walls and leaflets or intact heart valves or other biological scaffolding suitable for reconstructing a valve or valve components, for example, but not limited to the pericardium or the small intestinal submucosa or biodegradable frameworks, obtained from a variety of mammals, including but not limited to, man, pig, cow, sheep or dog, may be used.
• porcine leaflets and aortic biopsies may be used in the following forms: irradiated or chemically treated or steam treated (sterilized) ; decellularized (for example, detergent and/or enzyme treated) , extracted and sterilized; and valve tissue with nonviable cells and other biological tissues, for example, but not limited to, pericardium or small intestinal submucosa (accomplished by such procedures as freezing at -20°C to -70°C, or by repeated freezing and thawing) .
• the tissue sample can be subjected to enzymatic digestion and/or extracting with reagents that break down the cellular membranes and allow removal of cell contents.
• detergents include non-ionic detergents (for example, TRITON X-100, o ⁇ tylphenoxy polyethoxyethanol, (Rohm and Haas ⁇ ; BRIJ-35, a polyethoxyethanol lauryl ether (Atlas Chemical Co.), TWEEN 20, a polyethoxyethanol ⁇ orbitan monolaureate (Rohm and Haas) , LUBROL-PX, or polyethylene lauryl ether (Rohm and Haas) ) ; and ionic detergents (for example , sodium dodecyl sulphate, sulfated higher aliphatic alcohol, sulfonated alkane and sulfonated alkylarene containing 7 to 22 carbon atoms in a branched or unbranched chain
• the enzymes used may include nucleases (for example, deoxyribonuclease and ribonuclease) , proteases, phospholipases and lipase ⁇ .
• the tissues in the invention can also be decellularized using physical procedures such as ultrasonic treatment or osmotic shock, or by chemical treatment using peracetic acid.
• Such frameworks or constructs may be molded into the shape of heart valves or repair sheets/patches prior to inoculation of human cells. Where possible, however, it is most preferable to use a three-dimensional construct of the tissue of origin, for example, the aortic walls and leaflets or intact heart valves.
• the invention is based in part, on the discovery that the three-dimensional system supports the proliferation, migration, differentiation, and segregation of cells in culture in vitro to form components of tissues analogous to counterparts found in vivo.
• the human cells added to the scaffolds repopulate the porcine valve without the need for exogenously added growth factors. This is contrary to Orton's teachings which show that leaflet tissue not treated with bFGF remained acellular.
• growth factors for example, but not limited to, ⁇ FGF, ⁇ FGF, insulin growth factor or TGF-betas
• bioactive biological molecules for example, but not limited to, hyaluronic acid or hormones
• the three-dimensional framework provides a greater surface area for protein attachment, and consequently, for the adherence of stromal cells.
• stromal cells Because of the three-dimensionality of the framework, stromal cells continue to actively grow, in contrast to many cells in monolayer cultures, which grow to confluence, exhibit contact inhibition, and cease to grow and divide.
• the elaboration of extracellular matrix proteins and secretion of growth and regulatory factors by replicating stromal cells may be partially responsible for stimulating proliferation, maintaining normal tissue differentiation and regulating differentiation of cells in culture.
• the increase in potential volume for cell growth in the three-dimensional system may allow the establishment of localized microenvironments conducive to cellular maturation.
• the three-dimensional framework maximizes cell- cell interactions by allowing greater potential for movement of migratory cells.
• Stromal cells comprising fibroblasts, with or without other stromal cells and elements described below, are inoculated onto the three-dimensional framework.
• Human fibroblasts may be added to the culture prior to, during or subsequent to inoculation of other stromal cells.
• the concentration of fibroblasts maintained in the cultures can be monitored and adjusted appropriately to optimize growth and to regulate scaffold colonization.
• stromal cells that are genetically engineered to express and produce factors similar to those produced by cells of the heart valve, may be included in the inoculum. These cells could serve as a source of protein factor(s) in the culture. Preferably, the gene or coding sequence for factor(s) would be placed under the control of a regulated promoter, so that production of factor(s) in culture can be controlled.
• the genetically engineered cells will be screened to select those cell types: 1) that bring about amelioration of blood clotting, coagulation, thromboembolism and inflammatory reactions in vivo, and 2) escape immunological surveillance and rejection.
• Stromal tissue comprising dermal fibroblasts, cardiac fibroblasts and cells capable of producing collagen type I and III, elastin and other heart valve matrix proteins, for example, but not limited to fibronectin and glycosaminoglycans, are used to grow ji vitro, transplantable tissue or bioprosthetic heart valves.
• Stromal cells such as fibroblasts can be obtained in quantity rather conveniently from skin, human foreskin, heart tissue or any appropriate organ.
• Fetal and neonatal fibroblasts can be used to form a "generic" three-dimensional stromal tissue construct that will support the growth of a variety of different cells and/or tissues.
• Fibroblasts may be readily isolated by disaggregating an appropriate organ or tissue which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art.
• the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage.
• Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination.
• the suspension can be fractionated into subpopulations from which the fibroblasts and/or other stromal cells and/or elements can be obtained. This also may be accomplished using standard techniques for cell separation including but not limited to cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection) , separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counter- streaming centrifugation) , unit gravity separation, counter current distribution, electrophoresis and fluorescence-activated cell sorting.
• standard techniques for cell separation including but not limited to cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection) , separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal el
• fibroblasts may, for example, be carried out as follows: fresh tissue samples are thoroughly washed and minced in Hanks balanced salt solution (HBSS) in order to remove serum. The minced tissue is incubated from 1-12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After such incubation, the dissociated cells are suspended, pelleted by centrifugation and plated onto culture dishes. Fibroblasts will attach before other cells, therefore, appropriate stromal cells can be selectively isolated and grown. The isolated fibroblasts can then be grown to confluency, lifted from the confluent culture and inoculated onto the three-dimensional support (see, Nau ⁇ hton et al.. 1987, J. Med.
• HBSS Hanks balanced salt solution
• the cultured cells are to be used for transplantation or implantation in vivo it is preferable to obtain the stromal cells from the patient's own tissues. However, it is also possible to use allogeneic compatible human cells, without significant rejection reactions following transplantation.
• the growth of cells in the presence of the three-dimensional stromal support matrix may be further enhanced by adding to the matrix, or coating the matrix support with specific amino acids, proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and/or other materials.
• the three-dimensional matrix should be incubated in an appropriate nutrient medium.
• Many commercially available media such as DMEM, RPMI 1640, Fisher's Iscove's, McCoy's, and the like may be suitable for use.
• the three-dimensional stromal matrix be suspended or floated in the medium during the incubation period in order to maximize proliferative activity.
• the container in this protocol is kept stable in the incubator, i.e.. under static conditions (no circulating or flowing fluid) .
• the culture should be "fed” periodically to remove the spent media, depopulate released cells, and add fresh nutrients. The concentration of fibroblasts may be adjusted during these steps.
• the stromal cells will attach and proliferate along the three-dimensional framework before beginning to migrate into the depths of the matrix.
• One objective is to grow the cells to an appropriate degree which reflects the amount of stromal cells present in the in vivo tissue.
• a second objective is to regulate the number of cells in the inoculum and/or their growth on the scaffold such that the amount of scaffold colonization can be controlled as desired, and reproducibly.
• the openings of the non-tissue framework or constructs should be of an appropriate size to allow the stromal cells to stretch across the openings. Maintaining actively growing stromal cells which stretch across the framework enhances the production of growth factors which are elaborated by the stromal cells, and hence will support long term cultures. For example, if the openings are too small, the stromal cells may rapidly achieve confluence but be unable to easily exit from the mesh; trapped cells may exhibit contact inhibition and cease production of the appropriate factors necessary to support proliferation and maintain long term cultures. If the openings are too large, the stromal cells may be unable to stretch across the opening; this will also decrease stromal cell production of the appropriate factors necessary to support proliferation and maintain long term cultures.
• openings ranging from about 150 ⁇ m to about 220 ⁇ m will work satisfactory. However, depending upon the three-dimensional structure and intricacy of the framework, other sizes may work equally well. In fact, any shape or structure that allows the stromal cells to stretch and continue to replicate and grow for lengthy time periods will work in accordance with the invention.
• the human dermal fibroblasts exhibit a varied affinity for the different types of porcine tissue matrices.
• the greatest fibroblast colonization occurs when using a porcine matrix that is detergent and/or enzyme extracted. Additionally, the amount of fibroblast colonization in the porcine tissue correlates with time.
• collagen types I and III are preferably deposited in the initial matrix.
• the proportions of collagen types deposited can be manipulated or enhanced by selecting fibroblasts or cells which elaborate the appropriate collagen type. This can be accomplished using monoclonal antibodies of appropriate isotypes or subclass that are capable of activating complement, and which define particular collagen type. These antibodies and complement can be used to negatively select the fibroblasts which express the desired collagen type.
• the stroma used to inoculate the matrix can be a mixture of cells which synthesize the appropriate collagen types desired. The distribution and origins of the five types of collagen is shown in Table I.
• fibroblasts are the preferred cells for the present invention. TABLE I
• proliferating cells may be released from the framework. These released cells may stick to the walls of the culture vessel where they may continue to proliferate and form a confluent monolayer. This should be prevented or minimized, for example, by removal of the released cells during feeding, by coating the culture vessel with substances such as silicone to decrease cellular attachment, or by transferring the three- dimensional stromal framework to a new culture vessel. The presence of a confluent monolayer in the vessel will "shut down" the growth of cells in the three-dimensional framework and/or culture. Removal of the confluent monolayer or transfer of the framework to fresh media in a new vessel will restore proliferative activity of the three-dimensional culture system. Such removal or transfers should be done in any culture vessel which has a stromal monolayer exceeding 25% confluency.
• the culture system could be agitated to prevent the released cells from adhering, or instead of periodically feeding the cultures, the culture system could be set up so that fresh media continuously flows through the system.
• the flow rate could be adjusted to both maximize proliferation within the three-dimensional culture, and to wash out and remove cells released from the matrix, so that they will not adhere to the walls of the vessel and grow to confluence.
• the three-dimensional culture system of the invention can be used in a variety of applications.
• Aortic stenosis is the obstruction to flow across the aortic valve during left ventricular systolic ejection. It can be caused by a congenital unicuspid or bicuspid valve, rheumatic fever, or degenerative calcification of the valve in the elderly.
• the incidence of bicuspid aortic valve has been estimated at 4 in 1,000 live births, with males dominating over females at 4:1.
• Leaflets often thicken by age 40 and almost invariably by age 50, but calcium deposits are rarely detected before 40 years of age.
• aortic valvular stenosis Although symptoms generally occur late in the course of aortic stenosis, 3 to 5 percent of patients may die suddenly during an otherwise a symptomatic period. Thus, patients with any sign of congestive heart failure, angina, or exertional syncope in the presence of significant aortic valvular stenosis should undergo aortic valve replacement promptly. In addition, asymptomatic patients with significant aortic valvular stenosis should be advised to have valve replacement therapy.
• Aortic regurgitation is the diastolic flow of blood from the aorta into the left ventricle. It is caused by incompetent closure of the aortic valve which results from intrinsic disease of the cusp or from diseases affecting the aorta. Acquired intrinsic diseases of the aortic valve are either rheumatic or from bacterial origin. In the Marfan syndrome the primary basis for aortic insufficiency usually resides in the aorta, but there may be prolapse of the aortic cusps due to myxomatous changes. Infrequent changes are seen with r rheumatoid arthritis, systemic lupus erythematosus, and trauma.
• Mitral regurgitation occurs when contraction of the left ventricle ejects blood into left atrium as a result of abnormalities in the mitral valve apparatus.
• Acute mitral regurgitation can be created from mechanical disruption of the chordae tendineae, rupture of the papillary muscle, or perforation of the leaflet.
• Rheumatic fever, mitral valve prolapse and coronary artery disease such as left ventricular dilation, calcified mitral annulus, heritable disorders (Marfan syndrome, Ehlers-Danlos, osteogenesis) , congenital heart disease, systemic lupus erythematosus, rupture of papillary muscle and perforation of leaflet, are the predominant mechanisms for the incompetence of the mitral valve.
• mitral valve, valve components and/or other affected parts such as the chordae
• replacement of the mitral valve, valve components and/or other affected parts is required in cases of rheumatic involvement leading to severe mitral regurgitation, mitral stenosis with loss of pliability of the leaflets, and various other causes of mitral regurgitation, such as infective endocarditis, and in some cases in chronic heart disease. Calcification and immobility of the leaflets are also indications for valve replacement.
• Pulmonary stenosis is created by obstruction to systolic flow across the valve and is most commonly congenital. It generally leads to pulmonary regurgitation. Pulmonary valve replacement may be performed for acquired conditions such as carcinoid heart disease and infective endocarditis.
• Tricuspid regurgitation develops when the tricuspid valve allows blood to enter the right atrium during right ventricular contraction.
• Tricuspid stenosis represents obstruction to diastolic flow across the valve during diastolic filling of the right ventricle.
• the main cause of tricuspid and mitral regurgitation is the rupture of one or more of the elements of the tensor apparatus, with disruption of the papillary muscle and rupture of the chordae tendineae. Replacement is necessary if the changes in the leaflets and subvalvular mechanism are advanced, or if severe regurgitation cannot be relieved by annulopolasty.
• Multivalvular disease indicates obstruction and/or incompetence of the aortic, mitral, and tricuspid valves.
• Rheumatic fever, connective tissue diseases, Marfan syndrome, calcification of the mitral valve in the aging patient and bacterial endocarditis remain important causes in combined disease of the mitral and aortic valves.
• both valves are generally repressed by surgery.
• Artificial valve disease includes any abnormality of a surgically implanted device to replace a diseased cardiac valve. Artificial valve disease can result from prosthetic dysfunction, thrombus formation, infection, fibrosis, or calcification. Roberts, W.C. , 1973, Prog. Cardiovasc. Dis. 35:539. Congestive heart failure due to mechanical valve dysfunction is the major indication for replacement of a mechanical artificial valve. Replacement of the prosthesis is indicated if the symptoms cannot be controlled medically or if there is evidence of progressive ventricular dysfunction.
• the second most common operation performed in adults is replacement of the aortic or mitral valve.
• the valves produced in accordance with the invention may be transplanted using similar, if not the same surgical techniques, well known to those skilled in the art.
• the procedure for the replacement of the aortic valve is performed through a median sternum-splitting incision. After cardiopulmonary bypass is begun, a vascular clamp is placed across the distal ascending aorta. A sump suction cannula is placed in the left atrium through an incision in the right superior pulmonary vein to decompress the left heart. A transverse incision is made in the proximal aorta and the diseased valve is excised. Horizontal mattress sutures are placed at the three commissures for traction.
• either portions of the culture or the entire three-dimensional culture could be implanted, depending upon the type of tissue involved.
• three-dimensional heart valve cultures can be maintained in vitro for long periods. Section of tissues or the entire three- dimensional tissue structure can be transplanted in vivo in patients needing new heart valves.
• Three-dimensional tissue culture implants may, according to the inventions, be used to replace or augment existing tissue, to introduce new or altered tissue, to modify artificial prostheses, or to join together biological tissues or structures.
• the three-dimensional culture system of the invention may afford a vehicle for introducing genes and gene products in vivo to assist or improve the results of the transplantation and/or for use in gene therapies.
• the stromal cells can be genetically engineered to express anticoagulation gene products to reduce the risk of thromboembolism, or anti-inflammatory gene products to reduce the risk of failure due to inflammatory reactions.
• the stromal cells can be genetically engineered to express TPA, streptokinase or urokinase to reduce the risk of clotting.
• the stromal cells can be engineered to express anti-inflammatory gene products, for example, peptides or polypeptides corresponding to the idiotype of neutralizing antibodies for TNF, IL-2, or other inflammatory cytokines.
• the cells are engineered to express such gene products transiently and/or under inducible control during the post-operative recovery period, or as a chimeric fusion protein anchored to the stromal cells, for example, a chimeric molecule composed of an intracellular and/or transmembrane domain of a receptor or receptor-like molecule, fused to the gene product as the extracellular domain.
• the stromal cells could be genetically engineered to express a gene for which a patient is deficient, or which would exert a therapeutic effect, e.g.. HDL, apolipoprotein E, etc.
• the genes of interest engineered into the stromal cells need to be related to heart disease.
• the stromal cells can be engineered to express gene products that are carried by the blood; e.g.. cerebredase, adenosine deaminase, ⁇ -1- antitrypsin.
• a genetically engineered valve culture implanted to replace the pulmonary valve can be used to deliver gene products such as ⁇ -l-antitrypsin to the lungs; in such an approach, constitutive expression of the gene product is preferred.
• the stromal cells can be engineered using a recombinant DNA construct containing the gene used to transform or transfect a host cell which is cloned and then clonally expanded in the three-dimensional culture system.
• the three-dimensional culture which expresses the active gene product could be implanted into an individual who is deficient for that product.
• genes that prevent or ameliorate symptoms of various types of valvular heart diseases may be underexpressed or down regulated under disease conditions.
• expression of genes involved in preventing the following pathological conditions may be down-regulated, for example: thrombus formation, inflammatory reactions, and fibrosis and calcification of the valves.
• the activity of gene products may be diminished, leading to the manifestations of some or all of the above pathological conditions and eventual development of symptoms of valvular disease.
• the level of gene activity may be increased by either increasing the level of gene product present or by increasing the level of the active gene product which is present in the three-dimensional culture system.
• the three-dimensional culture which expresses the active target gene product can then be implanted into the valvular disease patient who is deficient for that product.
• “Target gene,” as used herein, refers to a gene involved in valvular disease in a manner by which modulation of the level of target gene expression or of target gene product activity may act to ameliorate symptoms of valvular disease.
• Heart valve constructs or sheets may be designed specifically to meet the requirements of an individual patient, for example, the stromal cells may be genetically engineered to regulate one or more genes; or the regulation of gene expression may be transient or long-term; or the gene activity may be non-inducible or inducible.
• one or more copies of a normal target gene, or a portion of the gene that directs the production of a normal target gene protein product with target gene function may be inserted into human cells that populate the three-dimensional constructs using either non-inducible vectors including, but are not limited to, adenovirus, adeno-associated virus, and retrovirus vectors, or inducible promoters, including metallothionein, or heat shock protein, in addition to other particles that introduce DNA into cells, such as liposomes or direct DNA injection or in gold particles.
• the gene encoding the human complement regulatory protein which prevents rejection of the graft by the host, may be inserted into human fibroblasts. Nature 375: 89 (May, 1995).
• the three-dimensional cultures containing such genetically engineered stromal cells e.g.. either mixtures of stromal cells each expressing a different desired gene product, or a stromal cell engineered to express several specific genes are then implanted into the patient to allow for the amelioration of the symptoms of valvular disease.
• the gene expression may be under the control of a non-inducible (i.e.. constitutive) or inducible promoter.
• the level of gene expression and the type of gene regulated can be controlled depending upon the treatment modality being followed for an individual patient.
• the use of the three-dimensional culture in gene therapy has a number of advantages. Firstly, since the culture comprises eukaryotic cells, the gene product will be properly expressed and processed in culture to form an active product. Secondly, gene therapy techniques are useful only if the number of transfected cells can be substantially enhanced to be of clinical value, relevance, and utility; the three-dimensional cultures of the invention allow for expansion of the number of transfected cells and amplification (via cell division) of transfected cells.
• transkaryotic suggests that the nuclei of the implanted cells have been altered by the addition of DNA sequences by stable or transient transfection.
• the cells can be engineered using any of the variety of vectors including, but not limited to, intergating viral vectors, e.g.. retrovirus vector or adeno-associated viral vectors, or non-integrating replicating vectors, e.g..
• papilloma virus vectors SV40 vectors, adenoviral vectors; or replication-defective viral vectors.
• non-integrating vectors and replication defective vectors may be preferred, since either inducible or constitutive promoters can be used in these systems to control expression of the gene of interest.
• integrating vectors can be used to obtain transient expression, provided the gene of interest is controlled by an inducible promoter.
• the expression control elements used should allow for the regulated expression of the gene so that the product is synthesized only when needed in vivo.
• the promoter chosen would depend, in part upon the type of tissue and cells cultured. Cells and tissues which are capable of secreting proteins (e.g. , those characterized by abundant rough endoplasmic reticulum, and golgi complex) are preferable.
• Hosts cells can be transformed with DNA controlled by appropriate expression control elements (e.g.. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which, in turn, can be cloned and expanded into cell lines.
• This method can advantageously be used to engineer cell lines which express the gene protein product.
• any promoter may be used to drive the expression of the inserted gene.
• viral promoters include but are not limited to the CMV promoter/enhancer, SV 40, papillomavirus, Epstein-Barr virus, elastin gene promoter and 3-globin. If transient expression is desired, such constitutive promoters are preferably used in a non- integrating and/or replication-defective vector.
• inducible promoters could be used to drive the expression of the inserted gene when necessary.
• inducible promoters include, but are not limited to, metallothionein and heat shock protein.
• transcriptional control regions that exhibit tissue specificity for connective tissues which have been described and could be used, include but are not limited to: elastin or elastase I gene control region which is active in pancreatic acinar cells (Swit et al.. 1984, Cell 38:639-646; Ornitz et al.. 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515).
• the deposition of elastin is correlated with specific physiological and developmental events in different tissues, including the heart valves. For example, atrioventricular valve cusps are initially thick and fleshy in an embryo, and later in the development are transformed into thin and fibrous cusps.
• elastin deposition appears to be coordinated with changes in arterial pressure and mechanical activity.
• Animals that contain valves and ligamental structures that are elastic contain elastin.
• the transduction mechanisms that link mechanical activity to elastin expression involve cell- surface receptors. Once elastin-synthesizing cells are attached to elastin through cell-surface receptors, the synthesis of additional elastin and other matrix proteins may be influenced by exposure to stress or mechanical forces in the tissue (for example, the constant movement of the construct in the bioreactor) or other factors that influence cellular shape.
• TPA tumor necrosis factor
• streptokinase or urokinase activity
• urokinase activity can bring about amelioration of platelet aggregation, blood coagulation or thromboembolism.
• This activity is maintained for a limited time only, for example, to prevent potential complications that generally develop during the early phase after valve implantation, such as, platelet aggregation, blood clotting, coagulation or thromboembolism.
• the presence of the anti-inflammatory gene products for example, peptide ⁇ or polypeptides corresponding to the idiotype of neutralizing antibodies for TNF, IL-2, or other inflammatory cytokines, can bring about amelioration of the inflammatory reactions associated with valvular disease.
• the stromal cells used in the three-dimensional culture system of the invention may be genetically engineered to "knock out" expression of factors or surface antigens that promote clotting or rejection at the implant site. Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below. "Negative modulation”, as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
• the expression of a gene native to stromal cell can be reduced or knocked out using a number of techniques, for example, expression may be inhibited by inactivating the gene completely (commonly termed "knockout") using the homologous recombination technique.
• an exon encoding an important region of the protein is interrupted by a positive selectable marker (for example neo) , preventing the production of normal mRNA from the target gene and resulting in inactivation of the gene.
• a gene may also be inactivated by creating a deletion in part of a gene, or by deleting the entire gene. By using a construct with two regions of homology to the target gene that are far apart in the genome, the sequences intervening the two regions can be deleted. Mombaerts, P., et al.. 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084-3087.
• Antisense and ribozyme molecules which inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene activity.
• antisense RNA molecules which inhibit the expression of major histocompatibility gene complexes (HLA) shown to be most versatile with respect to immune responses.
• triple helix molecules can be utilized in reducing the level of target gene activity.
• the expression of fibrinogen, von Willebrands factor, factor V or any cell surface molecule that binds to the platelet a2B ⁇ -3 receptor can be knocked out in the stromal cells to reduce the risk of clot formation at the valve.
• the expression of MHC class II molecules can be knocked out in order to reduce the risk of rejection of the graft.
• a "bioreactor” has been devised which takes advantage of the flow method for feeding the three-dimensional cultures in vitro. Essentially, as fresh media is passed through the three-dimensional culture, the gene product is washed out of the culture along with the cells released from the culture. The gene product is isolated fe.g.. by HPLC column chromatography, electrophoresis, etc.) from the outflow of spent or conditioned media.
• the three-dimensional culture of the present invention provides for the growth of stromal cells such as fibroblasts upon decellularized heart valves in vitro, in a system designed to mimic physiologic conditions in vivo. Importantly, the cells replicated in this system synthesize proteins similar to those produced by the normal aortic wall and leaflet cells.
• Porcine aortic leaflets and walls were seeded in eight well dishes with lxlO 5 human dermal fibroblasts and cultured for one day. The aortic walls and leaflets were transferred into new well dishes and grown for an additional four weeks.
• the eight cultures were made up of: (l) previously frozen leaflet seeded with human fibroblasts; (2) previously frozen wall seeded with human fibroblasts; (3) previously frozen leaflet without seeding; (4) previously frozen wall without seeding; (5) fresh leaflet seeded with human fibroblasts; (6) fresh wall seeded with human fibroblasts; (7) fresh leaflet without seeding; and (8) fresh wall without seeding.
• the cultures were labeled with [ 35 S]-methionine and [ 35 S]- cysteine (Tran 35 S-Label, ICN) for four hours.
• the samples were boiled in Laemmli sample buffer containing
• porcine leaflet or wall tissues were housed in a multi-well dish (one piece of tissue/well) as described above in Section 6.1.2.
• the human dermal fibroblasts were suspended in a nutrient-rich growth medium and were seeded onto the specific types of porcine leaflet or wall tissues such as: 1) frozen leaflets and walls; 2) electron beamed leaflets; 3) detergent and/or enzyme extracted leaflets and walls; and 4) detergent and/or enzyme extracted + electron beamed leaflets.
• Glucose rjofiHiimption As an indicator of fibroblast viability, nutrient consumption (glucose) and metabolic waste products (lactate) contained in the tissue construct are measured as described in Halberstadt, C.R. , et al.. 1994, Biotechnology and Bioengineering 43:740-746. Viable fibroblasts decrease the concentration of glucose over time and increase the concentration of lactate.
• BrdU is a non-radioactive, thymidine analog which incorporates into newly synthesized DNA of dividing fibroblasts. Tissues are incubated in BrdU-containing media for 24-48 hr. The fibroblasts containing BrdU can be visualized in the histology sections of the tissue constructs using a monoclonal antibody to the BrdU, followed by an enzyme-chromogen detection system using the Zymed Kit. (ZYMED Laboratories, Inc. San Francisco, Ca) .
• radiolabelled a ino acids which are added to the nutrient-rich media during the tissue culture process.
• the radiolabelled amino acids are incorporated into newly synthesized proteins in the tissue constructs and can be measured using a scintillation counter and/or extracted and separated on a polyacrylamide (10%) gel by their molecular weights.
• the gel is washed in salicylic acid (IM) , then exposed to an X-ray film (4-16 hr at 4 -25°C) which, upon developing, detects the images of radiolabelled proteins.
• IM salicylic acid
• X-ray film (4-16 hr at 4 -25°C
• Protein production measured as collagen synthesis ( 3 H-proline labeling) indicated that the human dermal fibroblasts were producing collagen and some proteins that are present in porcine leaflets ( 33 S- cysteine/methionine labeling) ( Figure 5) .
• porcine aortic leaflets and walls can be statically or dynamically seeded with human fibroblasts. These human fibroblasts attach and colonize the aortic leaflet and wall scaffolds, and remain metabolically active by secreting extracellular matrix molecules.

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