EP1191955A1 - Remplacement biologique d'un caillot de fibrine a usage intra-articulaire - Google Patents

Remplacement biologique d'un caillot de fibrine a usage intra-articulaire

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Publication number
EP1191955A1
EP1191955A1 EP00943011A EP00943011A EP1191955A1 EP 1191955 A1 EP1191955 A1 EP 1191955A1 EP 00943011 A EP00943011 A EP 00943011A EP 00943011 A EP00943011 A EP 00943011A EP 1191955 A1 EP1191955 A1 EP 1191955A1
Authority
EP
European Patent Office
Prior art keywords
tissue
ligament
cells
collagen
anterior cruciate
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Withdrawn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
EP00943011A
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German (de)
English (en)
Inventor
Martha Meaney Murray
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.)
Brigham and Womens Hospital Inc
Original Assignee
Brigham and Womens Hospital Inc
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 Brigham and Womens Hospital Inc filed Critical Brigham and Womens Hospital Inc
Priority claimed from PCT/US2000/017069 external-priority patent/WO2000078370A1/fr
Publication of EP1191955A1 publication Critical patent/EP1191955A1/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Withdrawn legal-status Critical Current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
    • A61L27/3604—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
    • A61L27/3616—Blood, e.g. platelet-rich plasma
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/043—Mixtures of macromolecular materials
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/102—Collagen
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • 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
    • A61L27/38—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
    • 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

Definitions

  • This invention relates generally to prosthetic devices, and specifically to devices for repairing injured intra-articular tissue.
  • the anterior cruciate ligament is located in the center of the knee and connects the femur and tibia.
  • the anterior cruciate ligament is made up of many individual fascicles that attach from the back of the femur to the front of the tibia, to maintain knee stability. More than 200,000 people rupture their anterior cruciate ligament annually.
  • intra-articular tissues such as the anterior cruciate ligament (ACL)
  • ACL anterior cruciate ligament
  • the meniscus and the articular cartilage in human joints often fail to heal after an injury. Tissues found outside of joints heal by forming a fibrin clot, which connects the ruptured tissue ends and is subsequently remodeled to form scar, which heals the tissue.
  • a fibrin clot either fails to form or is quickly lysed after injury to the knee, thus preventing joint arthrosis and stiffness after minor injury.
  • Joints contain synovial fluid which, as part of normal joint activity, naturally prevent clot formation in joints. This fibrinolytic process results in premature degradation of the fibrin clot scaffold and disruption of the healing process for tissues within the joint or within intra-articular tissues.
  • the current treatment method for human anterior cruciate ligament repair after rupture involves removing the ruptured fan-shaped ligament and replacing it with a point-to-point tendon graft. While this procedure can initially restore gross stability in most patients, longer follow-up demonstrates many post-operative patients have abnormal structural laxity, suggesting the reconstruction may not withstand the physiologic forces applied over time (Dye, 325 Clin. Orthop. 130-139 (1996)).
  • the loss of anterior cruciate ligament function has been found to result in early and progressive radiographic changes consistent with joint deterioration (Hefti et al., 73A(3) J. Bone Joint Surg. 373-383 (1991)). As anterior cruciate ligament rupture is most commonly an injury of a young athletes, early osteoarthritis in this group has difficult consequences.
  • the invention provides devices and methods for promoting a connection between the ruptured ends of the tissue and fibers after injury, by encouraging the migration of appropriate healing cells to form scar and new tissue in the device.
  • the device is a bioengineered substitute for the fibrin clot and is implanted between the ruptured ends of the ligament fascicles.
  • This substitute scaffold is designed to stimulate cell proliferation and extracellular matrix production in the gap between the ruptured ends of the anterior cruciate ligament, thus facilitating healing and regeneration.
  • the device resists premature degradation of the replacement clot by the intra-synovial environment.
  • the invention provides a three-dimensional (3-D) scaffold composition for repairing a ruptured anterior cruciate ligament (ACL) and a method for attaching the composition to the ruptured anterior cruciate ligament.
  • the scaffold composition includes an inductive core, made of collagen or other material, and is surrounded by a layer critical to the attachment of the core to the surrounding tissue, called the adhesive zone.
  • the adhesive zone provides a microenvironment for inducing fibroblast cells from the anterior cruciate ligament to migrate into the scaffold. After migrating into the inductive core of the scaffold, the fibroblast cells conform to the collagen structure between the ligament and heal the gap between the ruptured ends.
  • the invention also includes the use of a collagen-based glue as an adhesive to maintain contact between the torn edges of the meniscus.
  • the torn edges of the meniscus are pretreated to expose selected extracellular matrix components in the meniscus.
  • the glue is introduced into the tear. Bonds are formed between the extracellular matrix of in the meniscal tissue and the material of the glue. The bonds form a bridge across the gap in the meniscus.
  • This adhesive zone bridge can then induce the migration of cells to the bridge, which is then remodeled by the meniscal cells, thus healing the tear.
  • This invention further includes the use of a collagen-based scaffold as an adhesive (as well as a cell migration inducer) to maintain and restore contact between the torn cartilage and the surrounding cartilage and bone.
  • the torn edges are pretreated to expose the extracellular matrix components in the cartilage.
  • a collagen scaffold is then introduced into the tear. Bonds are formed between the extracellular matrix and the material of the glue. The bonds form a bridge across the gap in the articular cartilage. This adhesive zone bridge can then induce the migration of cells to the bridge, which is remodeled by the cartilage cells, thus healing the injured area.
  • FIG. 1 is a schematic drawing of a replacement clot with an inductive core and an adhesive zone.
  • FIG. 2 is a schematic drawing of the bonding between fibers as an attachment mechanism.
  • FIG. 3 is a schematic drawing of bonding between the inductive core and the tissue by maintaining mechanical contact.
  • FIG. 4 is a schematic of tissue allocation for explants for 2-dimensional (2-D) and 3 -dimensional (2-D) migration constructs.
  • FIG. 5 is a schematic of test and control 3-dimensional (3-D) constructs viewed from the top.
  • FIG. 7 is a histogram demonstrating the changes in cell density in the fascicle-collagen-glycosaminoglycan (CG) scaffold construct as a function of time in culture
  • FIG. 8 is a histogram of the maximum cell number density in the collagen-glycosaminoglycan scaffold as a function of weeks in culture (values are mean ⁇ SEM).
  • FIG. 9 is a histogram showing the cell densities in collagen-glycosaminoglycan (GAG) matrices into which cells from explants from femoral, middle, and tibial zones of ruptured anterior cruciate ligaments migrated and proliferated after 1 , 2, 3, and 4 weeks in culture. Mean+SEM.
  • FIG. 10 is a histogram showing migration into a collagen-glycosaminoglycan (CG) scaffold from explants of intact and ruptured human anterior cruciate ligaments.
  • CG collagen-glycosaminoglycan
  • FIG. 11 is a histogram showing the cell number density near the site of rupture in the human anterior cruciate ligament as a function of time after injury.
  • FIG. 12 is a schematic of the gross and histologic appearance of the four phases of the healing response in the human anterior cruciate ligament.
  • FIG. 12A shows the inflammatory phase showing mop-ends of the remnants (a), disruption of the epiligament and synovial covering of the ligament (b), intimal hyperplasia of the vessels (c) and loss of the regular crimp structure near the site of injury (d).
  • FIG. 12B shows the epiligamentous regeneration phase involving a gradual recovering of the ligament remnant by vascularized, epiligamentous tissue and synovium (e).
  • FIG. 12C shows the proliferative phase with a revascularization of the remnant with groups of capillaries (f).
  • FIG. 12D shows remodeling and maturation stage characterized by a decrease in cell number density and blood vessel density (g), and retraction of the ligament remnant (h).
  • FIG. 13 is a histogram demonstrating changes in cell number density near the site of injury as a function of time after complete anterior cruciate ligament rupture and comparison with the cell number density in the proximal unruptured anterior cruciate ligament. Error bars represent the standard error of the mean (SEM).
  • FIG. 14 is a histogram demonstrating the changes in blood vessel density near the site of injury as a function of time after complete anterior cruciate ligament rupture and comparison with the blood vessel density in the proximal unruptured anterior cruciate ligament. Error bars represent the standard error of the mean (SEM).
  • FIG. 15 is a schematic of tissue allocation for explants for 2-dimensional (2-D) and 3 -dimensional (3-D) migration constructs.
  • FIG. 16 is a histogram of the maximum cell number density in the collagen-glycosaminoglycan template as a function of explant harvest location (values are mean + SEM).
  • FIG. 17 is a histogram of the effect of location on outgrowth rate for high and low serum concentration.
  • FIG. 18 is a histogram for outgrowth rates from human anterior cruciate ligament explants as a function of location and TGF- ⁇ concentration.
  • FIG. 19 is a histogram showing maximum cell number density in the collagen-glycosaminoglycan scaffold as a function of time in culture.
  • ACL anterior cruciate ligament
  • Anterior cruciate ligament injuries occur most frequently in planting and cutting sports such as basketball, soccer, and volleyball.
  • a sudden deceleration (coming to a quick stop), combined with a direction change while running, pivoting, landing from a jump, or overextending the knee joint in either direction can cause injury to the anterior cruciate ligament. This injury occurs in 4 out of 1 ,000 people.
  • the device of the invention is useful for treating treat orthopedic surgery patients of any age for a ruptured anterior cruciate ligament, treat a torn knee meniscus, or encourage regeneration of cartilage after injury.
  • the method of the invention can be used in the repair of many tissues within articular joints, including the anterior cruciate ligament, knee meniscus, glenoid labrum, and acetabular labrum.
  • the method could be used to treat intra-articular fractures, scaphoid fractures, talus fractures, and triangular fibrocartilage complex injury (wrist), or any tissue that is intra-articular.
  • the method can be used to repair bone fractures, especially where the bone fractures are located in an intra-articular environment.
  • the device of the invention can be readily tested for sterility, mechanical strength, and function; for ways to freeze the tissue and store the scaffold; and for ways to scale up manufacturing to meet demand.
  • the device of the invention promotes regeneration of the human anterior cruciate ligament. Regeneration offers several advantages over reconstruction, including maintenance of the complex insertion sites and fan-shape of the ligament, and preservation of remaining proprioceptive fibers within the ligament substance.
  • the invention provides a scaffold on which the patient's body can develop a network of capillaries, arteries, and veins. Well-vascularized connective tissues heal as a result of migration of fibroblasts into the scaffold. Wound closure is subsequently facilitated by a contractile cell.
  • the invention also permits the re-enervation of the damaged area by providing a cellular substrate for regenerating neurons.
  • the advantages of the invention also include (1) a less invasive treatment as compared with the current techniques, which involve drilling into the bone; (2) faster surgery (as opposed to current meniscal repair techniques); (3) no donor site morbidity (as is seen with harvesting tendon grafts); (4) a quicker healing time; (5) a greater likelihood of the restoration of the normal function of the ligament (because the collagen scaffold is repopulated by the patient's own ligament cells); and (6) restoration of the meniscal structure (as contrasted with meniscectomy) or the articular cartilage structure (as contrasted with total joint arthroplasty). Implanting a device that facilitates the migration of the patient's own cells to the injured area
  • FIG. 1 a biological replacement fibrin clot of the invention is shown in FIG. 1.
  • the replacement fibrin clot includes a central inductive core surrounded by an adhesive zone.
  • the inductive core is preferably made of a compressible, resilient material which has some resistance to degradation by synovial fluid.
  • the inductive core member may be made of either permanent or biodegradable materials.
  • Scaffolds that make up the inductive core may function either as insoluble regulators of cell function or simply as delivery vehicles of a supporting structure for cell migration or synthesis.
  • Numerous matrices made of either natural or synthetic components have been investigated for use in ligament repair and reconstruction.
  • Natural matrices are made from processed or reconstituted tissue components (such as collagens and GAGs). Because natural matrices mimic the structures ordinarily responsible for the reciprocal interaction between cells and their environment, they act as cell regulators with minimal modification, giving the cells the ability to remodel an implanted material, which is a prerequisite for regeneration.
  • collagen compositions either collagen fiber or collagen gel
  • GAG glycosaminoglycan
  • hyaluran compositions various synthetic compositions.
  • Collagen-glycosaminoglycan (CG) copolymers have been used successfully in the regeneration of dermis and peripheral nerve.
  • Porous natural polymers, fabricated as sponge-like and fibrous scaffolds, have been investigated as implants to facilitate regeneration of selected musculoskeletal tissues including ligaments.
  • An important subset of natural matrices are those made predominantly from collagen, the main structural component in ligament.
  • Type I collagen is the predominant component of the extracellular matrix for the human anterior cruciate ligament.
  • Collagen occurs predominantly in a fibrous form, allowing design of materials with very different mechanical properties by altering the volume fraction, fiber orientation, and degree of cross-linking of the collagen.
  • the biologic properties of cell infiltration rate and scaffold degradation may also be altered by varying the pore size, degree of cross-linking, and the use of additional proteins, such as glycosaminoglycans, growth factors, and cytokines.
  • collagen-based biomaterials can be manufactured from a patient's own skin, thus minimizing the antigenicity of the implant (Ford et al, 105 Laryngoscope 944-948 (1995)).
  • Porous collagen scaffolds of varying composition and architecture have been researched as templates for regeneration of a variety of tissues including bone, skin and muscle.
  • a porous collagen-glycosaminoglycan (CG) scaffold has been used successfully in regeneration of dermis (Yannas et al, 86 Proc. Natl. Acad. Sci. USA 933-937 (1989)) and peripheral nerve (Chamberlain, Long Term Functional And Morphological Evaluation Of Peripheral Nerves Regenerated Through Degradable Collagen Implants (Massachusetts Institute of Technology, 1998)).
  • Synthetic matrices are made predominantly of polymeric materials. Synthetic matrices offer the advantage of a range of carefully defined chemical compositions and structural arrangements. Some synthetic matrices are not degradable. While the non-degradable matrices may aid in repair, non-degradable matrices are not replaced by remodeling and therefore cannot be used to fully regenerate ligament. It is also undesirable to leave foreign materials permanently in a joint due to the problems associated with the generation of wear particles, thus only degradable materials are preferred for work in regeneration. Degradable synthetic scaffolds can be engineered to control the rate of degradation.
  • the inductive core can be composed of foamed rubber, natural material, synthetic materials such as rubber, silicone and plastic, ground and compacted material, perforated material, or a compressible solid material.
  • the inductive core can be made of (1) an injectable high molecular weight poly(propylene fumarate) copolymer that hardens quickly in the body (Peter et al, 10(3) J. Biomater. Sci. Polym. Ed.
  • the inductive core can be any shape that is useful for implantation into a patient's joint, including a solid cylindrical member, cylindrical member having hollow cavities, a tube, a flat sheet rolled into a tube so as to define a hollow cavity, or an amorphous shape which conforms to that of the tissue gap.
  • the inductive core may incorporate several different materials in different phases.
  • the inductive core may be made of a gel, porous or non-porous solid or liquid material or some combination of these. There may be a combination of several different materials, some of which may be designed to release chemicals, enzymes, hormones, cytokines, or growth factors to enhance the inductive qualities of the inductive core.
  • the inductive core and adhesive zone can form a single continuous zone, either before insertion into the intra-articular zone or after insertion.
  • the inductive core may be seeded with cells.
  • the cells can genetically altered to express growth factors or other chemicals. Growth Factors.
  • PDGP-AA platelet derived growth factor-AA
  • PDGF-BB platelet derived growth factor-BB
  • PDGF-AB platelet derived growth factor- AB
  • TGF- ⁇ transforming growth factor beta
  • EGF epidermal growth factor
  • aFGF acidic fibroblast growth factor
  • bFGF basic fibroblast growth factor
  • IGF-1 insulin-like growth factor- 1
  • IL-l ⁇ insulin-like growth factor-alpha
  • Adhesive zone As shown in FIG. 1 , the adhesive zone maintains contact between the inductive core and the patient tissue to promote the migration of cells from tissue into the inductive core.
  • the adhesive zone may be made of permanent or biodegradable materials such as polymers and copolymers.
  • the adhesive zone can be composed, for example, of collagen fibers, collagen gel, foamed rubber, natural material, synthetic materials such as rubber, silicone and plastic, ground and compacted material, perforated material, or a compressible solid material.
  • the adhesive zone can also be any shape that is useful for implantation into a patient's joint.
  • the contact between the inductive core and the surrounding tissue can be accomplished by formation of chemical bonds between the material of the core and the tissue, or by bonding the material of the core to the adhesive zone combined with bonding the adhesive zone to the surrounding tissue (FIG. 2). Mechanical bonds can be formed that interlock the core with the tissue. Alternatively, pressure can be maintained on the core/tissue interface (FIG. 3). Cross-linking.
  • the formation or attachment of the adhesive zone can be enhanced by the use of other methods or agents, such as methods or agents that cross-link the adhesive phase together, or that cross-link the adhesive phase to the tissue, or both.
  • the cross-linking may be by chemical means, such as glutaraldehyde or alcohol, or by physical means, such as heat, ultraviolet (UV) light, dehydrothermal treatment, or laser treatment.
  • Physical cross-linking methods avoid the release of toxic by-products.
  • Dehydrothermal cross-linking is achieved through drastic dehydration which forms interchain peptide bonds.
  • Ultraviolet irradiation is believed to form cross-links between free radicals which are formed during irradiation.
  • the cross-linker may be added as an agent (such as a cross-linking protein) or performed in situ.
  • the cross-linking may be between the collagen fibers or may be between other tissue proteins or glycosaminoglycans.
  • Cross-linking of collagen-based scaffolds affects the strength, biocompatibility, resorption rate, and antigenicity of these biomaterials (Torres, Effects Of Modulus Of Elasticity Of Collagen Sponges On Their Cell-Mediated Contraction In Vitro (Massachusetts Institute of Technology, 1998); Troxel, Delay of skin wound contraction by porous collagen-GAG matrices (Massachusetts Institute of Technology, 1994); Weadock et al, 29 J. Biomed. Mater.
  • Cross-linking can be performed using chemicals, such as glutaraldehyde or alcohol, or physical methods, such as ultraviolet light or dehydrothermal treatment.
  • the degree to which the properties of the scaffold are affected is dependent upon the method and degree of cross- linking.
  • Cross-linking with glutaraldehyde has been widely used to alter the strength and degradation rate of collagen-based biomaterials scaffolds (Kato & Silver, 1 1 Biomaterials 169- 175 (1990), Torres, Effects Of Modulus Of Elasticity Of Collagen Sponges On Their Cell- Mediated Contraction In Vitro (Massachusetts Institute of Technology, 1998); Troxel, Delay Of Skin Wound Contraction By Porous Collagen-GAG Matrices (Massachusetts Institute of Technology, 1994)), and glutaraldehyde-cross-linked collagen products are commercially available for implant use in urologic and plastic surgery applications.
  • DHT dehydrothermal
  • UV ultraviolet
  • Torres Seeded collagen-based scaffolds with calf tenocytes and demonstrated a statistically significant increased rate of calf tenocyte cell proliferation in the glutaraldehyde and ethanol cross-linked scaffolds when compared with the dehydrothermal cross-linked group at 14 and 21 days post-seeding. Additional length of cross-linking in glutaraldehyde lead to increasing stiffness of the collagen scaffold, with values approaching that seen in the ultraviolet and ethanol groups.
  • the ultraviolet cross-linked group demonstrated a statistically significant increase over the dehydrothermal group at 21 days, but not at 14 days post-seeding. This result suggests an influence of cross-linking method with fibroblast proliferation within the collagen-based scaffold.
  • Method of use The methods of the invention may be used to treat injuries to the anterior cruciate ligament, the meniscus, labrum, cartilage, and other tissues exposed to synovial fluid after injury.
  • the intra-articular scaffold is designed for use with arthroscopic equipment.
  • the scaffold is compressible to allow introduction through arthroscopic portals and equipment.
  • the scaffold can also be pre-treated in antibiotic solution prior to implantation.
  • the affected extremity is prepared and draped in the standard sterile fashion.
  • a tourniquet may be used if indicated.
  • Standard arthroscopy equipment may be used.
  • the tissue ends are pretreated, either mechanically or chemically, and the scaffold introduced into the tissue defect.
  • the scaffold is then bonded to the surrounding tissue by creating chemical or mechanical bonds between the tissue proteins and the scaffold adhesive zone. This can be done by the addition of a chemical agent or a physical agent such ultraviolet light, a laser, or heat,
  • the scaffold may be reinforced by placement of sutures or clips.
  • the arthroscopic portals can be closed and a sterile dressing placed. The post-operative rehabilitation is dependent on the joint affected, the type and size of lesion treated, and the tissue involved.
  • a diagnostic arthroscopy is performed and the lesion defined.
  • the knee may be drained of arthroscopic fluid and the glue inserted into the tear under wet or dry conditions, depending on the composition of the glue.
  • the glue is bonded to the surrounding injured tissue and, when the desired bonding has been achieved, the knee is refilled with arthroscopic fluid and irrigated.
  • the arthroscopic portals are closed and a sterile dressing applied.
  • the patient is kept in a hinged knee brace post-operatively, with the degree of flexion allowed dependent on the location and size of the meniscal tear.
  • This EXAMPLE is to confirm the presence of cells expressing a contractile actin isoform alpha-smooth muscle actin ( ⁇ -sm; SMA), in the intact human anterior cruciate ligament, as shown by Murray & Spector, 17(1) J. Orthop. Res. 18-27 (1999).
  • Actin is a major cytoskeletal protein associated with cell motility, secretion, phagocytosis, and cytokinesis. Actin is expressed in mammals as six isoforms which are coded by different genes and differ in their amino acid sequence. Two of the isoforms ( ⁇ and ⁇ ) are found in practically all cells, while the other four ( ⁇ 's) are thought to represent differentiation markers of muscle cells.
  • the ⁇ -sm actin isoform is associated with the contractile phase of healing in several connective tissues, including dermis, cornea, tendon and medial collateral ligament. This isoform has also been associated with cell migration by Yamanaka & Rennard, 93(4) Clin. Sci.
  • the anterior cruciate ligament is a complex tissue composed of structural proteins, proteoglycans, and cells.
  • the histology of the human anterior cruciate ligament is characterized by the specific distribution and density of the fibroblast phenotype as well as by the unique organization of the structural proteins.
  • Three histologically different zones were found to be present along the anteromedial bundle from the femoral to the tibial attachment. Two of the zones (the fusiform and ovoid) were located in the proximal 3 of the bundle. The third zone (the spheroid) occupied the distal 1/3 of the bundle fascicles.
  • the fusiform cell zone had a high number density of longitudinally oriented cells with a fusiform-shaped nucleus, longitudinal blood vessels, and high crimp length.
  • the cytoplasm of the cells in the fusiform zone were intimately attached to the extracellular collagen and followed the crimp waveform of the fibers.
  • Fusiform cells stained positively for the ⁇ -sm actin isoform in the fusiform zone, particularly at areas of crimp disruption.
  • the ovoid cell zone had a high number density of cells with an ovoid-shaped nucleus, longitudinal vessels, and a high crimp length. Ovoid cells stained positively for the ⁇ -sm actin isoform in the ovoid cell zone.
  • the spheroid cell zone had a low density of spheroid cells, few blood vessels, and short crimp length. Cells were found within and among fascicles, as well as within lacunae. In selected areas, as many as 50% of the cells in this region stained positively for the ⁇ -sm actin isoform.
  • ⁇ -sm actin positive, potentially contractile, cells in the human anterior cruciate ligament thus provides a possible explanation for the retraction of ligament remnants seen after rupture.
  • Down-regulation of the myofibroblast phenotype may be useful preventing premature ligament retraction, while up-regulation may be useful in self-tensioning of the healed ligament during the remodelling phase.
  • ⁇ -sm actin positive, potentially contractile, cells in the ruptured human anterior cruciate ligament may provide one possible explanation for the retraction of ligament remnants seen after complete rupture.
  • Down-regulation of the myofibroblast phenotype may be useful preventing premature ligament retraction, while up-regulation may be useful in self- tensioning of the healed ligament during the remodelling phase.
  • Quantifying the degree of expression of the contractile actin and the effect of scaffold cross-linking and growth factors on this expression is a first step towards understanding possible regulation mechanisms.
  • This EXAMPLE was to confirm that human ligament fibroblasts can migrate into collagen-glycosaminoglycan copolymers in vitro.
  • Methods Fifteen intact anterior cruciate ligaments were obtained from total knee arthroplasty patients, ages 54 to 82 years. Four of the ligaments were used solely for histology and immunohistochemistry. The remaining ligaments were sectioned into fascicles that were divided transversely in the midsubstance to make explants.
  • the highly porous collagen-glycosaminoglycan matrix composed of type 1 bovine hide collagen and chondroitin-6-sulfate, was prepared by freeze-drying the collagen-glycosaminoglycan dispension as described by Murray & Spector, in 45 i ' x Annual Meeting, Orthopaedic Research Society, Anaheim, CA (1999).
  • the average pore size of the collagen-glycosaminoglycan scaffold was 100 ⁇ m.
  • This EXAMPLE shows the potential for human anterior cruciate ligament fibroblasts to migrate from their native extracellular matrix into collagen-glycosaminoglycan scaffolds that may ultimately be used as implants to facilitate ligament regeneration.
  • This EXAMPLE was designed to determine if fibroblasts intrinsic to the human anterior cruciate ligament were capable of migrating from their native extracellular matrix onto an adjacent provisional scaffold in vitro. Another objective was to determine whether any of the cells which successfully migrated into the scaffold expressed the contractile actin isoform, ⁇ -sm actin, associated with wound contraction in other tissues. This EXAMPLE demonstrates that the cells intrinsic to the human anterior cruciate ligament are able to migrate into a collagen-glycosaminoglycan scaffold, bridging a gap between transected fascicles in vitro.
  • Explants of human anterior cruciate ligament are useful as the source of cells for migration testing, because the explants provide a known distribution of cells within an extracellular matrix carrier. Thus, any cells which are found in the adjacent collagen-glycosaminoglycan scaffold during the test must have migrated there, as fluid flow during cell seeding is avoided. This method also avoids possible modification of cell phenotype which may occur during cell isolation, expansion in 2-D culture, and seeding of scaffolds. As a result of cell migration and proliferation, areas in the scaffold contained cell number densities similar to that seen in the human anterior cruciate ligament in vivo.
  • This EXAMPLE demonstrates that cells that migrate into and proliferate within the collagen-glycosaminoglycan matrix have contractile potential as reflected in their expression of the ⁇ -sm actin isoform. Moreover, this EXAMPLE demonstrates the potential of cells intrinsic to the human anterior cruciate ligament to migrate into collagen-glycosaminoglycan scaffolds.
  • Outgrowth from the explant b ops es was recorded every 3 days as the surface area covered by contiguous fibroblasts.
  • the area of outgrowth was measured using an inverted microscope and a transparent grid sheet. The number of squares covered by the contiguous cells was counted and the corresponding area determined.
  • the effective radius of outgrowth was calculated by assuming a circular area of contiguous cells.
  • the rate of outgrowth was then calculated by plotting the average effective radius of outgrowth as a function of time from the first observed outgrowth, and the slope from the linear regression analysis was used as the rate of outgrowth. Twenty- four of the 33 samples demonstrated contiguous cell growth for at least 2 consecutive time periods prior to termination of the culture and were included in the calculation of the average rate. All explanted tissue and fibroblasts on the culture wells were fixed in formalin after 4 weeks in culture.
  • Collagen-Glycosaminoglycan Scaffold The porous collagen-glycosaminoglycan scaffold used in this EXAMPLE has been used successfully in regeneration of dermis (Yannas, in Collagen Vol III: Biotechnology, Nimni, ed., p. 87-115 (CRC Press, Boca Raton, FL.,
  • the 3-D culture substrate was a highly porous collagen-glycosaminoglycan matrix, composed of type I bovine tendon collagen (Integra Life Sciences, Inc., Plainsboro, NJ) and chondroitin-6-sulfate (Sigma Chemical, St.. Louis, MO).
  • the scaffold was prepared by freeze-drying the collagen-glycosaminoglycan dispersion under specific freezing conditions described by Yannas et al, 8 Trans. Soc. Biomater. 146 (1985) to form a tube with pore channels preferentially oriented longitudinally.
  • the average pore size of the collagen-glycosaminoglycan scaffold manufactured in this manner has previously been reported by Louie, Effect Of A Porous Collagen-Glycosaminoglycan Copolymer On Early
  • Fascicular Collagen-Glycosaminoglycan Scaffold Constructs The 6 fascicles from each of the 6 patients were divided into test (fascicle-scaffold- fascicle) and control (fascicle-fascicle) groups. This yielded one test and one control construct per patient for examination after 2 weeks, 4 weeks, and 6 weeks in culture, providing 6 test and 6 control constructs at each of the 3 time points.
  • the 18 test constructs were made by suturing each of the 2 fascicle lengths to an open channel cut from silicon tubing such that a 3-mm gap separated the transected ends. A 5-mm length of collagen-glycosaminoglycan scaffold (see, below) was compressed into the gap (FIG. 5).
  • the 18 control constructs were made by reapposing the transected ends and then securing the fascicles to similar open channels (FIG. 5). All of the 36 fascicle constructs were cultured in media containing Dulbecco's DMEMI F12 with 10%o fetal bovine serum, 2% penicillin streptomycin, 1% amphotericin B, 1% L-glutamine and 2%o ascorbic acid. Media were changed 3x a week.
  • the average value for cell number at each position was multiplied by 10 to obtain the number of cells/mm 2 (see, FIG. 19).
  • the fascicular tissue and collagen-glycosaminoglycan scaffolding were examined using polarized light to determine the degree of crimp and collagen alignment.
  • ⁇ -sm actin was determined using a monoclonal antibody.
  • 3-D culture specimens deparaffmized, hydrated slides were digested with 0.1% trypsin (Sigma Chemical, St. Louis, MO, USA) for 20 minutes (min). Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 min. Nonspecific sites were blocked using 20% goat serum for 30 min.
  • the sections were then incubated with the mouse anti- ⁇ -sm actin monoclonal antibody (Sigma Chemical, St. Louis, MO, USA) for 1 hr at room temperature. Negative controls were incubated with mouse serum diluted to an identical protein content.
  • the sections were then incubated with biotinylated goat anti-mouse IgG secondary antibody for 30 min followed by 30 min of incubation with affinity purified avidin.
  • the labeling was developed using the AEC chromagen kit (Sigma Chemical, St. Louis, MO) for ten min. Counterstaining with Mayer's hematoxylin for 20 min was followed by a 20 min tap water wash and coverslipping with warmed glycerol gelatin.
  • the histology of the Ligament Fascicles was as follows: The proximal 1/3 was populated predominantly by fusiform and ovoid cells in relatively high density, and the distal 2/3was populated by a lower density of spheroid cells.
  • ⁇ -sm actin immunohistochemistry of the transected region showed positive staining in 8.3 ⁇ 3.0% of fibroblasts not associated with blood vessels.
  • the percentage of cells expressing the ⁇ -sm actin isoform at the edge of the fascicle decreased with time in culture to 6 ⁇ 4% at week 6, a value not statistically significantly different from that before culture (paired t-test, p > 0.30).
  • the percentage of cells staining positive for ⁇ -sm actin remained low in the bulk of the fascicle, with 2 ⁇ 2% of cells staining positive at 6 weeks.
  • 2-D Culture Outgrowth The outgrowth of cells onto the 2-D culture dishes was observed to occur as early as 6 days and as late as 19 days, with outgrowth first detected after an average of 10 ⁇ 3 days. The time of onset or rate of outgrowth was not found to correlate with explant size. Linear regression analysis of the plot of effective outgrowth radius versus time for all explants that demonstrated contiguous outgrowth had a coefficient of determination of 0.98. The average rate of outgrowth, represented by the slope of this plot, was 0.25 mm/day (FIG. 6).
  • fibroblasts were noted to migrate from the human anterior cruciate ligament fascicles into the scaffolds at the earliest time point (2 weeks). Migration into the scaffold was seen in 5 of 6 constructs at 2 weeks, 5 of 6 constructs at 4 weeks, and in all 5 of the 6-week constructs. While the average cell number density in the fascicle decreased with time, the average cell number density in the scaffold increased with time in culture (FIG. 7). Initially, cells were noted predominantly at the edge of the scaffold. With time, the average cell number density at the edge of the scaffold increased from 57 ⁇ 22 cells/mm 2 at 2 weeks and to 120 ⁇ 41 cells/mm 2 at six weeks.
  • the average migration distance at the 2-week time period was 475 micrometers. At the 4-week time point, cells had migrated as far as 1.5 mm toward the center of the scaffold. In areas where a gap greater than 50 microns was present between the explant and collagen-glycosaminoglycan scaffold, no cell migration into the scaffold was seen.
  • This EXAMPLE demonstrates that the cells intrinsic to the human anterior cruciate ligament were able to migrate into the gap between transected fascicles, eventually attaining selected areas with cell number densities similar to that seen in the human anterior cruciate ligament in vivo, if a provisional scaffold was provided. No extracellular matrix formation was seen between transected ends directly apposed without provisional scaffold. A gap between the explant and scaffold, even, as small as 50 ⁇ m, prevented cell migration to the scaffold at the site of loss of contact. Cells with all three previously described ligament fibroblast morphologies - fusiform, ovoid and spheroid -were noted to migrate into the scaffold. The cell density within the scaffold and maximum migration distance increased with time.
  • Outgrowth from explants likely has two components - migration and proliferation. Previous results assumed minimal contribution from the proliferation component and reported outgrowth rates as migration rates (Geiger et al, 30(3) Connect Tissue Res. 215-224 (1994)); the migration rate from rabbit anterior cruciate ligament explants was 0.48 mm/day. Using this same approach, the migration rate from human anterior cruciate ligament explants In this EXAMPLE is 0.25 mm/day. Previous studies did not report the cell number density of the explants (see also, Deie et al., 66(1) Acta Orthop. Scand. 28-32 (1995)), so one cannot predict whether differences in reported results are due to species differences or to differences in the cell number density or phenotype.
  • This EXAMPLE demonstrates the chronology of expression of this phenotype in explants of ligament tissue in culture, as well as in cells which successfully migrate onto a 3-D scaffold.
  • the percentage of ⁇ -sm actin-positive cells increases at the periphery of the explants from 8 to 30% after 2 weeks in culture. All ligament cells which migrated into the collagen-glycosaminoglycan matrix at 2 weeks contained ⁇ -sm actin, suggesting a role for this contractile actin isoform in cell migration. Moreover, most of these cells displayed a unipolar distribution of the contractile actin isoform.
  • This EXAMPLE shows the potential of human anterior cruciate ligament fibroblasts to migrate from their native extracellular matrix into collagen-glycosaminoglycan scaffolds that may ultimately be investigated as implants to facilitate ligament healing.
  • the EXAMPLE allows for the analysis of the migration of fibroblasts out of human tissues directly onto a porous 3-D scaffold.
  • the purpose of this EXAMPLE is to demonstrate the process of fibroblast-mediated tissue regeneration, to determine the effect of cross-linking of a collagen-based scaffold on (a) the rate of fibroblast migration; (b) the rate of fibroblast proliferation; (c) expression of a contractile actin; and (d) the rate of type I collagen synthesis by fibroblasts in the collagen-based scaffold.
  • This EXAMPLE is also intended to determine the effect of addition of selected growth factors on these same outcome variables.
  • the results of this EXAMPLE can be used to determine how specific alterations in scaffold cross-linking and the addition of specific growth factors alter the fibroinductive properties of a collagen-based scaffold.
  • the fibroinductive potential of the scaffold is defined as its ability to promote fibroblast infiltration, proliferation and type I collagen synthesis.
  • the fibroinductive properties of the collagen-based scaffold may be regulated by the choice of cross-linking method.
  • the addition of growth factors to the collagen-glycosaminoglycan scaffold alters (a) the rates of fibroblast migration from an anterior cruciate ligament explant to a collagen-based scaffold; (b) the rates of fibroblast proliferation; (c) the expression of a contractile actin; and (d) the type I collagen synthesis within the scaffold.
  • the bases for this rationale are (a) the alteration in fibroblast migration rates onto 2-D surfaces, (b) synthesis of type I collagen in vitro when growth factors are added to the culture media, and (c) alterations in rates of incisional wound healing.
  • human anterior cruciate ligament tissue are obtained from 6 patients and 10 explant/scaffold constructs made for each of the four types of cross-linked collagen (a total of 40 constructs per patient).
  • human anterior cruciate ligament tissue are obtained from 6 additional patients and 10 explant/scaffold constructs made for each of the four types of cross-linked collagen (a total of 40 constructs/patient).
  • test constructs used in this EXAMPLE are explants of human tissue placed into culture directly onto 3-D fibrous collagen-glycosaminoglycan scaffolds (see, EXAMPLE 3). Human anterior cruciate ligament explants are obtained from patients undergoing total knee arthroplasty.
  • This construct allows for the analysis of the migration of fibroblasts out of human tissues directly onto a 3-D fibrous scaffold in a controlled in vitro environment and obviates several confounding factors, such as modulation of cell phenotype, which may occur dunng cell extraction or 2-D cell culture
  • This construct also allows for investigation of human fibroblasts and tissue, thus avoiding interspecies variability. Careful control of growth factor concentration and substrate selection are also possible with this in vitro model. Preparation of the collagen-based scaffold.
  • Type I collagen from bovine tendon is combined with chondroitm 6 sulfate from shark cartilage to form a co-precipitate slurry
  • the slurry is lyophihzed m a freeze drier and minimally cross-lmked with dehydrothermal treatment for 24 hr at 105°C and 30 mtorr.
  • All of the 3-D collagen-glycosaminoglycan scaffolds are minimally cross-linked using dehydrothermal treatment at 105°C and 30 mtorr for 24 hr Additional cross-linking is performed for the glutaraldehyde, ultraviolet, and ethanol groups.
  • Glutaraldehyde cross-linking are performed by rehydratmg the collagen-based scaffolds in acetic acid, treating in 0 25% glutaraldehyde for thirty minutes, ⁇ nsing and storing m a buffer solution.
  • Ethanol cross-linking is performed by soaking the collagen scaffolds in 70% ethanol for 10 mm, nnsmg, and sto ⁇ ng in buffer.
  • Ultraviolet light cross-linking is performed by placing the scaffold 30 cm from an ultraviolet lamp rated at 5.3 W total output, 55.5 W/cm 2 at 1 m.
  • the scaffolds is cross-linked for 12 hr, 6 hr on each side as previously desc ⁇ bed by Tones, Effects Of Modulus Of Elasticity Of Collagen Sponges On The Cell-Mediated Contraction In Vitro (Massachusetts Institute of Technology, 1998). Addition of growth factors .
  • the 4 growth factors are added to the cell culture media m concentrations based on those previously reported to be successful in the literature.
  • EGF at 10 ng/ml
  • bFGF at 0 6 ng/ml
  • TGF- ⁇ at 0.6 ng/ml
  • PDGF-AB PDGF-AB at 10 ng/ml
  • Each growth factor is added individually to the control cell culture media containing DMEM-F12, 0.5% fetal bovine serum, 2% penicillin/streptomypin, 1% amphotericin B, 1% L-glutamme and 25 ⁇ g/ml of ascorbic acid.
  • Hematoxylin and eosin staining are performed to facilitate light microscopy examination of cell morphology in both explant and scaffold, maximum migration distance into the collagen-glycosaminoglycan scaffold and maximal number density of fibroblasts in the scaffold.
  • DNA Assay for Cell Proliferation Specimens allocated for analysis of DNA content are fluorometrically. Specimens are rinsed in phosphate-buffered saline and the explant separated from the scaffold. The scaffold is stored at -70°C. The scaffolds is digested in 1 ml of 0.5% papain/buffer solution in a 65°C water bath. A 200 ⁇ l aliquot of the digest is combined with 40 ⁇ l of Hoechst dye no. 33258 and evaluated fluorometrically. The results are extrapolated from a standard curve using calf thymus DNA. For one run of the DNA assay, a standard curve based on a sample of human ligament cells are used to estimate the cell number from the DNA measurement.
  • Negative control specimens consisting of the scaffold material alone are also assayed to assess background from the scaffold. Additionally, a tritiated thymidine assay can be evaluated. Then, the specimens used for proliferation can be fixed and serially sectioned, with sections at regular intervals examined for cell number density. Maximum number density is recorded for each specimen type. Associated histology is used to estimate the percentage of dead cells.
  • Type I collagen is measured using SDS-PAGE techniques. Specimens allocated for analysis of synthesis of type I collagen are cultured with tritiated proline for specific time periods after selected time in culture. Proline uptake studies is performed for scaffolds from each group. Biochemical determination of collagen types in both the scaffold and conditioned media is eluted with Triton and assayed by PAGE. Immunohistochemistry. Immunohistochemistry is used to determine the distribution of cells producing the ⁇ -sm actin isoform in both the explanted tissue and the scaffold (see EXAMPLE 3). An additional benefits of this construct is that serial sections can be stained immunohistochemically for any protein for which an antibody is available. Therefore, additional investigation into the expression of the other subtypes of actin, or members of the integrin family during cellular migration may be performed, if time allows.
  • Transmission Electron Microscopy is used to evaluate morphologic features of the migrating cells, as well changes in the extracellular matrix. Processing of specimens for transmission electron microscopy analysis begins with fixation for 6 hr in Kamovsky's fixative, followed by post-fixation with osmium tetraoxide, dehydration through graded alcohols, infiltration with graded propylene oxide/epon, embedding in epon, ultramicrotomy (70 angstroms) and post-staining with uranyl acetate.
  • Characteristics of migrating cells to be examined in the TEM include characteristics of cytoplasm (such as the presence of abundant rough endoplasmic reticulum and presence of microfilaments consistent with ⁇ -sm actin) and characteristics of extracellular matrix (such as the presence of pericellular fine fibrils consistent with new collagen formation).
  • the principal variables evaluated are the number of cells populating the scaffold, the production of type I, II and III collagen, and the expression of the contractile actin isoform.
  • the control group are the minimally cross-linked scaffolds with no growth factor addition. Assuming a standard deviation of 30%, to detect a difference between groups of 30%, with an " ⁇ " of 0.05 and a " ⁇ " of 0.1 (i.e., a power of 90%) has a sample size of 13 for each group. Therefore, to investigate 4 growth factors at 4 time points uses 208 constructs each for the histology and TEM, DNA testing, and SDS-PAGE analysis, a total of 624 constructs.
  • the objective of this EXAMPLE was to evaluate the migration of cells from explants from selected zones within ruptured human anterior cruciate ligaments into collagen-glycosaminoglycan matrices in vitro. The proliferation of cells in the matrices and their contractile behavior were also assessed.
  • the collagen-glycosaminoglycan matrix was prepared by freeze-drying a coprecipitate of type I bovine tendon collagen (Integra Life Science, Plainsboro, N.J.) and shark chondroitin 6-su ate gma em. o., t. Lou s, . e mat x was cross- n e or 24 r. us ng a dehydrothermal treatment.
  • the scaffolds had a pore diameter of approximately 90 ⁇ m.
  • the diameter of the sponges was measured with time in culture. Matrices without explants were cultured under the same conditions as controls. The cell density within the matrices was determined by dividing the number of cells evaluated histologically by the area of analysis, and immunohistochemistry using a monoclonal antibody was performed to determine the percentage of cells containing a contractile actin isoform, ⁇ -smooth muscle actin ( ⁇ -sm). The results were compared with cells migrating from explants obtained from intact human anterior cruciate ligament specimens. Results. Cells from the explants migrated into, and proliferated within, the collagen-glycosaminoglycan matrices resulting in an increase in the cell density in the scaffolds with time (FIG. 9).
  • FIG. 9 The cell density resulting from explants from the femoral zone of the ruptured anterior cruciate ligaments was greater than that from intact human anterior cruciate ligament explants after 2 (1 10 ⁇ 38 cells/mm 2 ; mean ⁇ SEM) and 4 weeks (170 ⁇ 71). Immunohistochemistry revealed the presence of ⁇ -sm in the ligament cells in the scaffolds. There was a significant decrease in the diameter of the matrices with time in culture to approximately 70% of the original diameter evidencing the contractile behavior of the ⁇ -sm -positive cells.
  • the objective of this EXAMPLE was to determine whether anterior cruciate ligament cells would continue to migrate after complete rupture, and to determine what effect the location of cells in the ruptured human anterior cruciate ligament had on their ability to migrate.
  • This EXAMPLE was performed to determine if two of the biologic responses required for regeneration of tissue (revascularization and fibroblast proliferation) occur in the human anterior cruciate ligament after injury.
  • Histomorphometric analysis was performed to determine cell number density, blood vessel density, nuclear aspect ratio and the percentage of ⁇ -sm positive, non-vascular cells at 1-2 mm increments along the length of the ligament section.
  • Blood vessel density was determined by measuring the width of the section and counting the number of vessels crossing that width.
  • Two-way ANOVA was used to determine the significance of time after injury, distance from the site of injury, and patient age on the cell number density, blood vessel density, nuclear morphometry and ⁇ -sm positive staining within the ligaments. Bonferroni-Dunn post-hoc testing was used to generate specific p values between groups.
  • Phase II Epiligamentous regeneration. Between three and eight weeks after rupture, gradual overgrowth of epiligamentous tissue with a synovial sheath was noted to form over the ruptured end of the ligament remnant. Histologically, this phase was characterized by a relatively unchanging blood vessel density and cell number density within the remnant. P ase I . ro i eration. etween g t an twenty wee s a ter rupture, t e pro erat ve response in the epiligamentous tissue subsided and a marked increase in cell number density and blood vessel density within the ligament remnant was noted. Fibroblasts were the predominant cell type. Vascular endothelial capillary buds were noted to appear at the beginning of this phase, and loops from anastomoses of proximal sprouts began to form a diffuse network of immature capillaries within the ligament remnant.
  • Cell number density in the ligament in the ligament after rupture was dependent on time after injury and distance from the injury site. The cell number density within the ligament remnant peaked at 16 to 20 weeks (FIG. 1 1, p ⁇ 0.005), and was highest near the site of the injury at all time points (TABLE 1). Patient age was not found to significantly affect cell number density (p>0.80). Blood vessel density was dependent on time after injury, with a peak at 16 to 20 weeks (p ⁇ 0.003). Age did not have a significant effect on vessel density. Cells straining positive for the contractile actin isoform, ⁇ -sm, were present throughout the intact and ruptured anterior cruciate ligaments. Time after injury and age of the patient were not found to significantly effect the percentage of cells straining positive.
  • Weeks post-rupture Ruptured 1 mm from 2 mm from 4 mm from edge edge edge edge
  • Vessel density (#/mm) 2.1 ⁇ 2.0 1.5 ⁇ 1.3 1.2 ⁇ 0.7 1.3 ⁇ 0.6
  • the human anterior cruciate ligament undergoes a process of revascularization and fibroblast proliferation after complete rupture.
  • the healing response can be described in four phases, with a peak in activity at 4 to 5 months after rupture.
  • This response is similar to that seen in other dense, organized, connective tissues which heal, such as the medial collateral ligament, with two exceptions: (1) the lack of any tissue bridging the rupture site after injury, and (2) the presence of an epiligamentous regeneration phase.
  • the results of this EXAMPLE showing that fibroblast proliferation and angiogenesis occur within the human anterior cruciate ligament remnant, are important to the development of future methods of facilitating anterior cruciate ligament healing. Harnessing the neovascularization and cell proliferation, and extending it into the gap between ruptured ligament ends provides guidance for a method of anterior cruciate ligament repair.
  • the objectives of this EXAMPLE were to investigate the effects of enzymatic treatment on the potential for cell outgrowth from adult human articular cartilage and to determine if ⁇ -sm is present in chondrocytes in articular cartilage and in the outgrowing cells.
  • Samples of articular cartilage were obtained from 15 patients undergoing total joint arthroplasty for osteoarthrosis. While the specimens were obtained from patients with joint pathology, areas of cartilage with no grossly noticeable thinning, fissuring, or fibrillation were selected. Using a dermal punch, cylindrincal samples (4.5mm diameter and 2-3 mm thick), were cut from the specimens. Explants were cultured in 6-well culture dishes and oriented so that deep zone of the tissue contracted the culture dish. In the first test, 20 cartilage samples were obtained from each of the 9 patients. Four plugs of cartilage were allocated to one of five groups that received collagenase treatment for 0, 1 , 5, 10, or 15 min.
  • Explants allocated for immunohistochemistry were fixed in 10% formalin, paraffin embedded and cut to 7 ⁇ m sections. Sections were stained with a ⁇ -sm monoclonal antibody (Sigma Chemical, St.. Louis, MO). Statistical analysis was performed by ANOVA with Fisher's PLSD post-hoc test.
  • This EXAMPLE was designed to determine: (a) whether the ruptured ante ⁇ or cruciate ligament remnant was capable of maintaining cells within its substance after rupture, in the intrasynovial environment; (b) whether an increase in cell number density would occur in the ante ⁇ or cruciate ligament after complete rupture; and (c) whether the ruptured ligament would revascula ⁇ ze after injury. Another objective was to determine if cells with a contractile actin isoform, ⁇ -sm actin were present in the healing human anterior cruciate ligament.
  • the ligaments were marked with a suture at the site of tibial transection, and fixed in neutral buffered formalin for one week. After fixation, specimens were embedded longitudinally in paraffin and 7 ⁇ m thick longitudinal sections were microtomed and fixed onto glass slides. Representative sections from each ligament were stained with hematoxylin and eosin and with a monoclonal antibody to ⁇ -sm actin (Sigma Chemical, St Louis, MO, USA). In the immunohistochemical procedure, deparaffmized, hydrated slides were digested with 0.1% trypsin (Sigma Chemical, St. Louis, MO, USA) for 20 minutes.
  • Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 minutes. Nonspecific sites were blocked using 20% goat serum for thirty minutes.
  • the sections were then incubated with the mouse monoclonal antibody to ⁇ -sm actin for 1 hr at room temperature.
  • a negative control section on each microscope slide was incubated with non-immune mouse serum diluted to the same protein content, instead of the ⁇ -sm actin antibody, to monitor for non-specific staining.
  • the sections were then incubated with a biotinylated goat anti-mouse IgG secondary antibody for thirty minutes followed by thirty minutes of incubation with affinity purified avidin.
  • the labeling was developed using the AEC chromogen kit (Sigma Chemical, St Louis, MO) for 10 minutes. Counterstaining with Mayer's hematoxylin for twenty minutes was followed by a 20-minute tap water wash and coverslipping with warmed glycerol gelatin.
  • 0.1 mm areas were evaluated by determining the total cell number density and the predominant nuclear morphology, and by calculating the percentage of cells positive for the ⁇ -sm actin isoform. Between 20 and 230 cells were counted at each of the three areas. At each location, the total number of cells was counted and divided by the area of analysis to yield the cell number density, or cellularity. The cell morphology was classified based on nuclear shape: fusiform, ovoid, or spheroid.
  • Fibroblasts with nuclei with aspect ratios i.e., length divided by width
  • nuclei with aspect ratios i.e., length divided by width
  • the total number of blood vessels crossing the section at each location was divided by the width of the section at each location to obtain a blood vessel density for each location.
  • Smooth muscle cells surrounding vessels were used as internal positive controls for determination of ⁇ -sm actin positive cells. Positive cells were those that demonstrated chromogen intensity similar to that seen in the smooth muscle cells on the same microscope slide and that had significantly more intense stain than the perivascular cells on the negative control section. Any cell with a questionable intensity of stain (e.g., light pink tint) was not counted as positive.
  • the ⁇ -sm actin positive cell density was reported as the number of stained cells divided by the area of analysis and the percentage of ⁇ -sm actin positive cells was determined by dividing the number of stained cells by the total number of cells in a particular histologic zone.
  • Polarized light microscopy was used to aid in defining the borders of fascicles and in visualizing the crimp within the fascicles. Measurement of the crimp length was performed using a calibrated reticule under polarized light.
  • the ligament remnants retrieved in this time period were populated by fibroblasts and several types of inflammatory cells: polymorphonuclear neutrophils, lymphocytes, and macrophages.
  • the inflammatory cells were found in greatest concentration around blood vessels near the site of injury. Macrophages appeared to be actively phagocytosing cell and tissue debris. Arterioles near the site of injury were noted to be dilated, with intimal hyperplasia
  • FIG. 12A consisting of dramatic smooth muscle cell wall proliferation and thickening. Venules were noted to be dilated, with less evident smooth muscle cell hyperplasia. Capillaries appeared congested, with rouleaux and thrombus formation noted in their lumens.
  • the collagenous extracellular matrix appeared disorganized and edematous near the site of injury. Loss of the regular organization of the collagen fibers was evident (FIG. 12 A) and replacement with disorganized, less dense, amorphous tissue was seen.
  • the cells populating this amo ⁇ hous tissue consisted of both fibroblasts and inflammatory cells.
  • At the site of rupture several adjacent ruptured distal fascicles were bridged by a fibrin clot at ten days, and several of the ruptured fascicle ends were covered by a twenty- to fifty micrometer thick fibrin clot. However, no gaps larger than 700 micrometers contained any bridging material.
  • the epiligamentous regeneration phase was characterized by a relatively unchanging cell number density and blood vessel density in the ligament remnant.
  • the number of inflammatory cells decreased, and fibroblasis became the dominant cell type.
  • the cell number density of fibroblasts was similar to that seen in the uninjured ligament and the remaining blood vessels displayed near normal mo ⁇ hologies, with little intimal hype ⁇ lasia. No neovascularization was noted within the ligament fascicles. Most of the changes occured in the epiligament that displayed an increase in cell number density and blood vessel density.
  • the vascular epiligamentous tissue was noted to gradually extend over the ruptured ligament end, encapsulating the mop-ends of the individual capsules. Thickening of the epiligament and fibroblast proliferation were seen to occur during this time period. A synovial layer, similar to that seen covering the epiligamentous tissue in the intact anterior cruciate ligament, was noted to form over the extending neoepiligamentous tissue. Phase III. Proliferation. By eight weeks, the distal anterior cruciate ligament remnants were completely encapsulated by a synovial sheath, and few remaining mop-ends were seen grossly (FIG. 12C). No tissue was visible between the proximal and distal ligament remnants.
  • the collagenous material of the ligament fascicles remained disorganized near the site of injury. No preferential orientation was seen; however, bands of parallel collagen fibers were noted to begin to form and develop a waveform similar to the crimp seen in the intact human anterior cruciate ligament. These areas were a small component of the remnant, and the longitudinal axis of the waveform was rarely aligned with the longitudinal axis of the ligament remnant.
  • the epiligamentous tissue remained vascular and was relatively unchanged in appearance throughout this phase.
  • the synovial layer persisted as a two-cell layer continuous over the epiligamentous tissue.
  • Immunohistochemistry revealed ⁇ -sm actin containing cells distributed throughout the intact and ruptured ligaments, albeit in relatively low percentages
  • the fibroblast nuclei were increasingly fusiform with the long axis of the nucleus aligned with the longitudinal axis of the ligament. There was decreased blood vessel density within the ligament remnant. The epiligamentous tissue continued to decreased n t c ness; owever, t e synov a s eat pers ste . more ax al alignment of the collagen fascicles was seen. The cell number density decreased to a level similar to that seen in the intact human anterior cruciate ligament.
  • Two-way ANOVA demonstrated that the cell number density in the human ruptured anterior cruciate ligament was significantly affected by location in the ligament remnant and time after rupture. The cell number density was highest near the site of injury at all time points. This cellularity increased significantly to a maximum at sixteen to twenty weeks (FIG. 13; Bonferroni-Dunn post-hoc testing, p ⁇ 0.005) and decreased between twenty and fifty-two weeks after injury(Bonfe ⁇ Oni-Dunn post-hoc testing, p ⁇ 0.005). With the number of ligaments available, age and gender were not found to significantly affect cell number density (two-way ANOVA, p> 0.80 and p ⁇ 0.40, respectively).
  • the mo ⁇ hology of the cell nuclei was also significantly affected by the location in the ligament remnant, but not by time after injury, gender or age. Using two-way ANOVA, the proximal part of the ligament remnant was found to have cells with a higher nuclear aspect ratio when compared with cells in the more distal remnants (Bonferroni-Dunn post-hoc testing, p ⁇ 0.0005). This pattern was also observed in the intact ligaments. Two-way ANOVA demonstrated that the mo ⁇ hology of the cell nuclei was significantly affected by the location in the ligament remnant (p ⁇ 0.003), but with the numbers available, not by time after injury
  • the blood vessel density was found to be significantly affected by the time after injury with two-way ANOVA.
  • the blood vessel density reached its highest value at sixteen to twenty weeks (Bonferroni-Dunn post-hoc testing, p ⁇ 0.003) and decreased after that time point
  • the response to injury is similar to that reported in other dense connective tissues with two exceptions: the presence of a epiligamentous regeneration phase which lasts eight to twelve weeks, and the lack of any tissue bridging the rupture site.
  • Other characteristics reported in dense connective tissue healing such as fibroblast proliferation, expression of ⁇ -sm actin and angiogenesis are all seen to occur in the human anterior cruciate ligament.
  • the finding of a epiligamentous regeneration phase distinguishes the ruptured human anterior cruciate ligament from other connective tissues which heal successful and reconciles the other findings in this EXAMPLE of a productive response to injury with previous reports of failure of the anterior cruciate ligament cells to respond to rupture.
  • the presence of the epiligamentous regeneration phase in this EXAMPLE illustrates the importantance of analyzing the results of primary repair or augmentation techniques. These procedures may have different results depending on the timing of repair after injury. Repair done in the first few weeks after injury may result in filling of the gap with the proliferative epiligamentous vascular tissue which is active at that time. Repair performed months after injury, when the endoligamentous tissue is proliferating, may result in a different mode of repair. This EXAMPLE also demonstrates the lack of any tissue seen in the gap between the ligament remnants. In extra-articular tissues which successfully heal, the fibrin clot forms and is invaded by fibroblasts and gradually replaced by collagen fibers.
  • This EXAMPLE provides guidance for the analysis of human tissue that has been ruptured and maintained in an in vivo, intrasynovial environment until the time of retrieval.
  • the overall object of the invention is to restore only the ligament tissue which is damaged during rupture, while retaining the rest of the ligament.
  • the model used in this EXAMPLE involves filling the gap between the ruptured ligament ends with a bioengineered regeneration bridge, or template, designed to facilitate cell ingrowth and guided tissue regeneration.
  • a bioengineered regeneration bridge, or template designed to facilitate cell ingrowth and guided tissue regeneration.
  • This EXAMPLE focuses on whether the cells of the human anterior cruciate ligament cells are able to migrate to a template after the anterior cruciate ligament has been ruptured.
  • the site closest to the rupture, or injury zone contains a higher cell number density than that of the more distal remnant, which resembles the histology of the intact anterior cruciate ligament. Therefore, the more distal remnant (normal zone) was used as an age and gender matched control for the tissue obtained at the site of injury (injury zone) and 0.5 cm distal to the site of injury (middle zone).
  • the 3-D culture substrate used in this EXAMPLE was a highly porous collagen-glycosaminoglycan matrix, composed of type I bovine hide collagen and chondroitin-6-sulfate, prepared by freeze-drying the collagen-glycosaminoglycan dispersion under specific freezing conditions (Yannas et al, 8 Trans Soc Biomater. 146 (1985)) to form a tube with pore orientation preferentially oriented, longitudinally.
  • the average pore size of the collagen-glycosaminoglycan scaffold manufactured in this manner has previously been reported as 100 gm (Chamberlain, Long Term Functional And Morphological Evaluation Of Peripheral Nerves Regenerated Through Degradable Collagen Implants.
  • the sections were then incubated with biotinylated goat anti-mouse IgG secondary antibody for 30 minutes followed by thirty minutes of incubation with affinity purified avidin.
  • the labeling was developed using the AEC chromagen kit (Sigma Chemical, St. Louis, MO) for ten minutes. Counterstaining with Mayer's hematoxylin for 20 minutes was followed by a 20 minute tap water wash and coverslipping with warmed glycerol gelatin.
  • Histology of the Ligament Fascicles The proximal one-third was populated predominantly by fusiform and ovoid cells in relatively high density, and the distal two-thirds was populated by a lower density of spheroid cells.
  • ⁇ -sm actin immunohistochemistry of the ruptured ligaments showed positive staining in 2 to 20% of fibroblasts not associated with blood vessels.
  • 2-D Culture Outgrowth The outgrowth of cells onto the 2-D culture dishes was observed to occur as early as 3 days and as late as 21 days, with outgrowth first detected at an average of 6.6 ⁇ 2.0 days after explanting. Explant size was not found to co ⁇ elate with the time of onset or rate of outgrowth. Linear regression analysis of the plot of effective outgrowth radius versus time for all explants that demonstrated confluent outgrowth had a coefficient of determination of 0.98. The average rate of outgrowth, represented by the slope of this plot, was 0.25 mm/day.
  • 3-D Culture Outgrowth In the constructs with inte ⁇ osed collagen-glycosaminoglycan scaffolding, fibroblasts migrated from the human anterior cruciate ligament explants into the templates at the earliest time point (1 week). At one week, migration into the templates was seen in 4 of 4 of the templates cultured with explants from the injury zone, I of 4 templates cultured with explants from the middle zone, and 1 of 4 of the templates cultured with explants from the normal zone. By four weeks, cells were seen in 3 of 3 templates cultured with the injury zone explants (the fourth template had been completely degraded) and in 3 of four of the templates cultured with the normal zone explants. Five of the explants completely degraded the template prior to the collection time.
  • the difference between the template cell density for templates cultured with explants from the middle and tibial of the twelve explants demonstrated confluent growth for at least two consecutive time periods prior to termination and were included in the calculation of the average rate.
  • CG collagen-glycosaminoglycan
  • This EXAMPLE demonstrated that the cells intrinsic to the ruptured human anterior cruciate ligament were able to migrate into a regeneration template, eventually attaining small areas with cell number densities similar to that seen in the human anterior cruciate ligament in vivo. Explants from the transected region demonstrated outgrowth onto a 2-D surface with a linear increase in outgrowth radius as a function of time in culture. Cells which migrated into the collagen-glycosaminoglycan scaffold differed significantly from the populations of the ruptured anterior cruciate ligament in that while an average of 2 to 20% of cells are positive for ⁇ -sm actin in the ruptured anterior cruciate ligament, 100% of cells noted to migrate at the early time periods were positive for this actin isoform.
  • the cellular response to injury appears to be the appropriate one in the anterior cruciate ligament; however, no regeneration of the tissue in the gap between ruptured ends is noted.
  • Cells from the human anterior cruciate ligament are capable of migrating into an adjacent regeneration template in vitro. Cells migrate in the greatest density from the zone nearest the site of rupture, or injury zone when compared with tissue taken far from the site of injury. This suggests the approach of developing a ligament regeneration template, or "bridge", which reconnects the ruptured ligament ends, may be successful in facilitating ligament regeneration after rupture.
  • the potential advantages of this approach over anterior cruciate ligament reconstruction include preservation of the proprioceptive innervation of the anterior cruciate ligament, retention of the complex shape and footprints of the anterior cruciate ligament, and restoration of the pre-injury knee mechanics. Successful regeneration of the anterior cruciate ligament may lead to similar advances for meniscal and cartilage regeneration after injury.
  • This EXAMPLE shows the potential of cells from the ruptured human anterior cruciate ligament fibroblasts to migrate into collagen-glycosaminoglycan templates that may ultimately be used to facilitate regeneration anterior cruciate ligament after rupture.
  • the model used here allows for the analysis of the migration of fibroblasts out of human tissues directly onto a porous 3-D scaffold in a controlled, in vitro, environment. This construct obviates several possible confounding factors, such as modulation of cell phenotype, which may occur during cell extraction or 2-D cell culture.
  • EXAMPLE 1 1 EFFECTS OF LOCATION IN THE HUMAN ACL ON CELLULAR OUTGROWTH AND
  • the pu ⁇ ose of this EXAMPLE was to determine how cells in selected locations in the human anterior cruciate ligament varied in certain behavior that might affect their potential for repair. Specifically, in this EXAMPLE the outgrowth of cells in vitro from explants different locations in the anterior cruciate ligament, at two concentrations of fetal bovine serum (FBS) and three concentrations of TGF- ⁇ 1 were measured. Methods. Fifteen intact human anterior cruciate ligaments were retrieved from patients undergoing TKA. The ligaments were cut transversely into four 2-3 mm thick sections. Each section was divided into six explants, two of which were reserved for histological analysis and four of which were placed in 2-D culture wells.
  • FBS fetal bovine serum
  • Explants from the proximal and distal sections were cultured in 10% FBS, 0.5% FBS , and 0.5% FBS with 006 ng/ml.
  • Media were changed 3x a week, and cell outgrowth area measured at each medium change. Cultures were terminated after four weeks.
  • This EXAMPLE demonstrates that explants taken from proximal and distal sites in human anterior cruciate ligament respond differently to low-serum conditions, as well as to the addition of TGF- ⁇ 1. Because these differences do not co ⁇ elate with the cell number density or nuclear mo ⁇ hology, other features of the cellular heterogeneity and fibroblast phenotype within the human anterior cruciate ligament may be associated with the differences in cell behavior.
  • the pu ⁇ ose of this EXAMPLE is to determine if any histological differences are present between the anterior cruciate ligament in women and men. Another objective of this EXAMPLE was to determine if exogenous estrogen had any significant effect on the measured parameters by examining ligaments from two groups of women, those on and off estrogen replacement therapy. Methods. Intact anterior cruciate ligaments were obtained from 22 patients undergoing total knee arthroplasty. Patients with rheumatoid arthritis or on non-steroidal anti-inflammatory medication were excluded from the EXAMPLE.
  • This EXAMPLE demonstrates that the histology of the human anterior cruciate ligament is similar in men and women, with the exception of the cell number density in the proximal region, which is higher in women than men. This EXAMPLE also demonstrates that exogenous estrogen does not have an effect on cell number density, blood vessel density, cell nuclear mo ⁇ hology, or presence of ⁇ -sm actin.
  • This EXAMPLE was performed to determine if two of the biologic responses required for regeneration of tissue, namely revascularization and fibroblast proliferation, occur in the human anterior cruciate ligament after injury.
  • Inflammation Phase I. Inflammation. Inflammatory cells, dilated arterioles and intimal hype ⁇ lasia was seen between 1 and 3 weeks after rupture. Loss of the regular crimp pattern was noted near the site of injury, but maintained 4-6 mm from the site of injury.
  • Phase II Epiligamentous regeneration. Growth of epiligamentous tissue over the ruptured end of the ligament remnant was noted between 3 and 8 weeks. Histologically, this phase was characterized by an unchanging blood vessel density and cell number density within the remnant.
  • Phase III Proliferation. Between 8 and 20 weeks after rupture, a marked increase in cell number density and blood vessel density within the ligament remnant was noted. Vascular endothelial capillary buds were noted to appear at the beginning of this phase, and loops from anastomoses of proximal sprouts began to form a diffuse network of immature capillaries.
  • Phase IV Remodeling and Maturation. After one year from ligament rupture, the ligament ends were dense and white. Histologically, the fibroblast nuclei were increasingly uniform in shape and orientation. Decreased cell number density and blood vessel density were seen during this phase, to a level similar to that seen in the intact human anterior cruciate ligament s.
  • Cell number density in the ligament after rupture was dependent on time after injury and distance from the injury site. The cell number density within the ligament remnant peaked at 16 to 20 weeks (p ⁇ 0.005), and was highest near the site of injury at all time points. Blood vessel density was dependent on time after injury, with a peak at 16 to 20 weeks (p ⁇ 0.003). Cells staining positive for the contractile actin isoform, -sm, were present throughout the intact and ruptured anterior cruciate ligaments, but were not significantly effected by time after injury.
  • the pu ⁇ ose of this EXAMPLE is to determine the process of fibroblast-mediated connective tissue healing and how specific alterations in the extracellular environment alter this process.
  • ⁇ -sm smooth muscle actin
  • This EXAMPLE provides improved rates of migration, proliferation, and type I collagen synthesis of anterior cruciate ligament fibroblasts by altering the degree and type of cross- linking of the scaffold and by adding four different growth factors to the scaffold.
  • the specific aims for this EXAMPLE are (1 ) to determine the effect of cross-linking of a collagen-based scaffold on (a) the rate of fibroblast migration, (b) the rate of fibroblast proliferation, (c) expression of a contractile actin, and (d) the rate of type I collagen synthesis by fibroblasts in the collagen-based scaffold, and (2) to determine the effect of addition of selected growth factors on these same outcome variables.
  • this EXAMPLE determines how specific alterations in scaffold cross-linking and the addition of specific growth factors alter the fibroinductive properties of a collagen based scaffold.
  • the fibroinductive potential of the scaffold is defined as its ability to promote fibroblast infiltration, proliferation and type I collagen synthesis.
  • the addition of growth factors to the CG scaffold alters the rates of fibroblast migration from an anterior cruciate ligament explant to a collagen-based scaffold as well as the rates of fibroblast proliferation, expression of a contractile actin, and type I collagen synthesis within the scaffold.
  • the rationale for this hypothesis is the alteration in fibroblast migration rates onto 2-D surfaces and synthesis of type I collagen in vitro when growth factors are added to the culture media, as well as alteration in rates of incisional wound healing with the addition of growth factors.
  • Validation of this hypothesis shows how the fibroinductive properties of the collagen-based scaffold may be regulated by the addition of a specific growth factor.
  • the growth factors to be studied in this EXAMPLE include TGF- ⁇ , EGF, bFGF and PDGF-AB. Constructs of human anterior cruciate ligament explants and collagen-based scaffolds cultured in media containing growth factors are used to determine the rates of cell migration, proliferation, expression of a contractile actin and type I collagen synthesis in these constructs.
  • the control wells contain only 0.5% fetal bovine serum, a protocol which has been reported previously by DesRosiers et al, 14 J. Orthop. Res. 200-208 (1996). We correlate growth factor presence with the regulation of the fibroinductive properties of the scaffold.
  • the assay design is similar to that of EXAMPLE 4.
  • Human anterior cruciate ligament explants are obtained from patients undergoing total knee arthroplasty. Ligaments which are grossly disrupted or demonstrate gross signs of fatty degeneration are excluded from the analysis. A fairly uniform distribution of cells occurs in the distal 2/3 of the ligament fascicles, so this section is used for all assays.
  • the preparation of the collagen-based scaffold is as described in EXAMPLE 4 and previously reported by Tones, Effects Of Modulus Of Elasticity Of Collagen Sponges On Their Cell-Mediated Contraction In Vitro (Massachusetts Institute of Technology, 1998).
  • the cross-linking of the scaffolds is as described in EXAMPLE 4 and as previously described by Tones, Effects Of Modulus Of Elasticity Of Collagen Sponges On Their Cell-Mediated Contraction In Vitro (Massachusetts Institute of Technology, 1998).
  • the cross-linking of the scaffolds is as described in EXAMPLE 4 and as previously described by
  • the growth factors are added to the cell culture media as described in EXAMPLE 4.
  • Culture, histology for analysis of cell migration, DNA assay for ce pro erat on, mmuno stoc em stry or e contract e act n so orm, an - analysis for the synthesis of type I collagen are as described in EXAMPLE 4.
  • a pilot assay is performed to assess the DNA content with the DHT cross-linked scaffold with the addition of no growth factors.
  • a tritiated thymidine assay can be evaluated or the specimens used for proliferation can be fixed and serially sectioned, with sections at regular intervals examined for cell number density. Maximum number density is recorded for each specimen type.
  • Associated histology is used to estimate the percentage of dead cells.
  • This EXAMPLE uses of a provisional scaffold to encourage tissue regeneration in the gap between the ends of the ruptured anterior cruciate ligament without removal of the ligament. This has the advantages of retaining the complex anterior cruciate ligament geometry and proprioceptive innervation of the ligament.
  • the objective of this EXAMPLE is to show the in vivo effect of placement of a provisional scaffold between the ruptured ends of the anterior cruciate ligament.
  • a rabbit model is chosen because of its previous establishment as a mechanical and biochemical model for the human anterior cruciate ligament.
  • a CG scaffold is chosen as the provisional scaffold, given its success in dermis and tendon and in the human anterior cruciate ligament in vitro model.
  • the goal of this EXAMPLE is to evaluate a novel method of treatment of anterior cruciate ligament rupture which would facilitate ligament healing and regeneration after complete rupture.
  • the potential advantages of regeneration over reconstruction include retention of the complex footprints of the human anterior cruciate ligament, preservation of the proprioceptive nerve endings within the anterior cruciate ligament tissue, less invasive surgery with no graft harvest required, and maintenance of the complex fascicular structure of the anterior cruciate ligament.
  • Effective, minimally invasive, treatment of anterior cruciate ligament rupture would be particularly beneficial to women engaged in military training, as they are at an especially high risk for this injury.
  • the problem to be investigated in this EXAMPLE is the development of an implant to be used for anterior cruciate ligament regeneration after complete rupture of the ligament. Loss of the function of the anterior cruciate ligament leads to pain, joint instability and swelling. Left untreated, a knee with instability secondary to anterior cruciate ligament rupture leads to joint degeneration and osteoarthritis.
  • the objective of this EXAMPLE is to compare immediate primary repair with primary repair and scaffold augmentation in the treatment of anterior cruciate ligament rupture in a rabbit model.
  • the technique of primary repair involves reapproximation of the ruptured ligament ends with sutures passed both through ligament and bone to stabilize the tissue.
  • the specific aim of this EXAMPLE is to evaluate the effect of a provisional collagen sponge-like implant to facilitate anterior cruciate ligament regeneration of the ligament at 3 weeks, 3 months, 6 months, and 1 year after injury, resulting in a change in the relative percentage of various tissue types in the defect.
  • anterior cruciate ligament rupture may be a career-ending injury, as many patients can not return to their previous level of activity, even after repair or reconstruction (Marshall et al. , 143 Clin Orthop 97-106 (1979); Noyes et al, 68B J. Bone Joint Surg. 1125-1136 (1980)). Development of new methods of treatment of the ruptured anterior cruciate ligament, including ligament regeneration, may lead to quicker recovery times and improved rates of return to high levels of physical training for both women and men.
  • An anterior cruciate ligament rupture can be a devastating, if not career-ending, injury for women engaged in competitive athletics, and it is likely to be an event of similar magnitude in women in the military engaged in heavy physical activity.
  • Our biological implant treats the defect in the ruptured anterior cruciate ligament.
  • Such treatment may prevent the progression of joint deterioration seen in anterior cruciate ligament deficient knees, and in knees after anterior cruciate ligament reconstruction. It provides a less invasive method of treatment for this common injury, and potentially retain the complex anatomy and innervation of the anterior cruciate ligament. To facilitate the continuance of women in physically demanding careers, a new method of treatment of anterior cruciate ligament rupture is necessary, one which is minimally invasive, can restore the original structure and function of the anterior cruciate ligament, and has the potential to minimize the progression to premature osteoarthritis.
  • Experimental Design and Rationale The following tests are provided to achieve the specific aim. TABLE 7 shows the 3 test groups.
  • Collagen-glycosaminoglycan (CG) scaffold synthesis The scaffold used in this EXAMPLE is the same scaffold used in EXAMPLE 3.
  • the 3-D culture substrate is a highly porous CG matrix, composed of type I bovine hide collagen and chondroitin-6-sulfate. This is prepared by freeze-drying the collagen-glycosaminoglycan dispersion under specific freezing conditions (Louie, Effect of a porous collagen-glyosaminoglycan copolymer on early tendon healing in a novel animal model (Massachusetts Institute of Technology 1997)).
  • the average pore size of the CG scaffold manufactured in this manner is 100 ⁇ m.
  • Knees undergoing primary repair with the placement of the scaffold in the defect between ruptured ligament ends have sutures placed in an identical manner to that in the primary repair group.
  • the CG scaffolds is placed into the defect prior to tensioning of the sutures.
  • the specific tissue types filling the defect are determined by evaluating the percentage of the area of the central section through the defect occupied by each tissue type: (1) dense, crimped co agenous t ssue, (2) dense, unorgan zed collagenous t ssue, (3) synovial tissue, and (4) no tissue. Cell number density, blood vessel density and nuclear mo ⁇ hology of the fibroblasts are determined at each point along the length of the ruptured ligament.
  • Radiographic Analysis All knees have anteroposterior and lateral x-rays taken pre- operatively to assess for the presence of degenerative joint disease. Any animals demonstrating degenerative joint disease are disqualified from the analysis. At the time of sacrifice, all knees are radiographed a second time to assess the development of radiographic changes consistent with degenerative joint disease. Conelation between radiographic findings and histologic changes in the articular cartilage of the knee is made.
  • the biologic replacement for fibrin clot for intra-articular use of the invention is prepared and analyzed, such as is set forth in Guidance Document For Testing Biodegradable
  • composition and material structure e.g., phases, reinforcement, matrix, coating
  • analyses can include the following:
  • composition and molecular structure (a) main ingredients (such as collagen and glycosaminoglycan); (b) trace elements (e.g., heavy metals are low); (c) catalysts; (d) low molecular weight (MW) components (separate components which have and have not chemically reacted with the polymer, e.g., crosslinking agents); (e) polymer stereoregularity and monomer optical purity (if the monomer is optically active; not applicable for collagen or glycosaminoglycan); (f) polydispersity, (g) number average molecular weight (M n ) (h) weight average molecular weight (M w ); (i) molecular weight distribution (MWD); (j) intrinsic (or inherent) viscosity (specify solvent, concentrations and temperature; not applicable for collagen or glycosaminoglycan); (k) whether the polymer is linear, crosslinked or branched (1) copolymer conversion (e.g., block, random, graft
  • the inherent viscosity logarithmic viscosity number
  • some other justifiable method e.g., GPC
  • Composite structure (a) laminate structure; (b) thickness of each ply; (c) number of plies; (d) orientation and stacking sequence of plies; (e) symmetry of the layup; (f) position of reinforcement within the matrix; (g) location within the part; (h) 3 dimensional orientation; (i)f ⁇ ber density (e.g., distance between reinforcement components or reinforcement:matrix volume and weight ratios); (j) fiber contacts and cross-overs per mm; (k) reinforcement structure; (1) cross-sectional shape (m) surface texture and treatment; (n) dimensions; (o) fiber twist; (p) denier; (q) weave; (r) coating; (s) total number of coating layers; (t) thickness of each layer; (u) voids; (v) mean volume percent; (w) interconnections; (x) penetration depth and profile; and (y) drawing or photographs of the product illustrating the position of the coating and any variation in coating thickness (for example, see, FIGS.) The anatomical location and attachment mechanism for the
  • Physical properties (a) dimensional changes of the material as a function of time; (b) densities of reinforcement, matrix and composite; (c) mass of the smallest and largest sizes; (d) roughness of all surfaces; (e) surface area of the smallest and largest sizes; (f) dimensioned engineering drawings of any nonrandom surface structure patterns (e.g., machined structures).
  • Mechanical properties are important because they determine whether the fracture site is adequately fixed to avoid loosening, motion and nonunion. Weight loss and inherent viscosity measurements may be helpful in screening different materials and in understanding degradation mechanisms, though they may not directly address the mechanical properties of the device. For weight loss testing, test samples are weighed to an accuracy of 0.1 % of the total sample weight prior to placement in the physiological solution.
  • each sample is removed and dried to a constant weight. Drying conditions may include enclosure in a desiccator at standard temperature and pressure, use of a partial vacuum or the use of elevated temperatures. The weight is recorded to an accuracy of 0.1 % of the original total sample weight. Elevated temperatures can be used for drying of the sample provided that the temperature used does not change the sample (such as for collagen and glycosaminoglycan). The drying conditions used to achieve a constant weight are noted.
  • Samples are discarded if the measured pH is outside the specified value of more than ⁇ 0.2.
  • Each sampling container should be sealable against solution loss by evaporation.
  • Each test specimen is kept in separate containers and isolated from other specimens to avoid cross contamination of degradation byproducts. The solution is kept sterile and properly buffered or changed periodically.
  • Samples are fully immersed in the physiological solution at 37°C for the specified period of time.
  • One group of samples are stressed during the entire time in solution to simulate clinical worst case conditions, while another group of samples are set-up in the same environment, without stressing.
  • the amount of sample agitation, solution flow past test specimens, frequency that the solution is replaced, and the clinical significance of these factors are recorded and analyzed.
  • In vitro degradation rates are compared to the in vivo degradation rates so the in vitro test results can be extrapolated to clinical conditions.
  • Samples are implanted in an animal model and mechanically tested to determine if there are any significant difference in the outcome of test samples degraded in vitro and in vivo.
  • the degradation of the mechanical properties of the test device is compared to a device known in the art.
  • the biological replacement of the invention is compared for the determination of substantial equivalence to a device such as is known in the art (.see. BACKGROUND OF THE INVENTION).
  • a comparison of the similarities and differences of the known device to the biological replacement of the invention is made in terms of design, materials, intended use, etc.
  • Both devices are implanted either at the site of actual loaded use (for example, the anterior cruciate ligament) or at a nearby site.
  • a range of healing time for the indicated repair is provided from the literature (see, BACKGROUND OF THE INVENTION).
  • the implantation time should be at least twice as long the longest time over which healing of the repair is expected to occur. Data for this set of tests may be from the same animals used in other tests.
  • For mechanical testing the degradation of the mechanical properties of the biological replacement of the invention over time is compared to the same changes for a device known in the art. The degradation values are validated to in vivo results. At time period throughout the duration of the immersion/loading time, samples are removed and tested. Samples are tested in a non-dried or 'wet' condition.
  • Biocompatibility The biologic replacement of the invention is tested for biological response in an appropriate animal model. As part of the analysis, the degradation by-products and their metabolic pathways are identified.
  • the implantation time are at least twice as long the longest time over which healing of the repair is expected to occur.
  • a histological analysis of biocompatibility at the implant site determines the tissue response, normal and abnormal, to the presence of the biologic replacement of the invention and its breakdown products.
  • the biologic replacement of the invention is implanted into an animal model such that it experiences loading.
  • Sterilization information See the Sterility Review Guidance. U.S. Food & Drug Administration (July 3, 1997).
  • the sterilization method that was used [radiation, steam, EtO] is provided. If the sterilization method is radiation, then the radiation dose that was used is provided. If the sterilization method is EtO, then the maximum residual levels of ethylene oxide, ethylene chlorohydrin and ethylene glycol that were met is provided. These levels are below those limits proposed in the Federal Register FR-27482 (June 23, 1978).

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Abstract

L'invention concerne, d'une part, une structure de support tridimensionnelle permettant de réparer un ligament croisé antérieur déchiré et, d'autre part, un procédé permettant de fixer cette structure audit ligament. La structure de support est pourvue d'un noyau inductif et d'une zone adhésive. Après insertion de la structure support dans la région située entre les extrémités déchirées du ligament croisé antérieur et après fixation par collage auxdites extrémités, la zone adhésive procure un micro-environnement permettant de produire des fibroblastes à partir du ligament croisé antérieur qui migrent dans le noyau inductif. Après leur migration dans le noyau inductif, les fibroblastes s'associent à la structure collagène située entre les ligaments et referment l'espace vide entre les extrémités déchirées. L'invention concerne également l'utilisation d'une colle à base de collagène comme adhésif permettant de maintenir le contact entre les bords déchirés du ménisque ; et l'utilisation d'une structure de support à base de collagène comme adhésif (ainsi que comme inducteur de migration des cellules) permettant de maintenir et de rétablir le contact entre le cartilage déchiré et le cartilage et l'os qui sont autour.
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