EP2187837A1

EP2187837A1 - Graft collar system for inducing formation of fibrocartilage and related methods

Info

Publication number: EP2187837A1
Application number: EP08831799A
Authority: EP
Inventors: Helen H. Lu; Jeffrey P. Spalazzi; Moira C. Vyner; Kristen L. Moffat
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2007-09-21
Filing date: 2008-09-22
Publication date: 2010-05-26
Also published as: WO2009038808A1; EP2187837A4; WO2009038808A9

Abstract

Apparatuses and methods are provided for inducing formation of fibrocartilage. For example, an apparatus is provided which comprises a graft collar having a hollow central portion along a longitudinal axis, wherein an outer surface of the graft collar is wrapped with a polymer-fiber mesh, or wherein an outer surface of the graft collar is clamped by a clamp, to apply compression or static loading to the graft collar. A method for making said apparatus and for inducing formation of fibrocartilage is also provided.

Description

GRAFT COLLAR SYSTEM FOR INDUCING FORMATION OF FIBROCARTILAGE AND RELATED METHODS

Throughout this application, certain publications are referenced by Arabic numerals. Full citations for these publications, as well as additional related references, may be found immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entirety into this application in order to more fully describe the state of the art as of the date of the methods and apparatuses described and claimed herein.

Background

The ACL is the most frequently injured ligament of the knee³¹, with over 300,000 ACL injuries reported²² and more than 100,000 reconstruction procedures performed annually¹ in the United States. Primary ACL reconstruction has traditionally been based on autologous bone-patellar tendon-bone (BPTB) grafts, with a shift in recent years toward the utilization of semitendinosus or hamstring tendon grafts²¹'^{" 66; 75} due to the high incidence of donor site morbidity and complications related to the harvest of BPTB grafts. Allografts are also routinely utilized for ACL reconstruction²⁵' ³⁰, especially with advancements in allograft processing_^ and comprehensive studies demonstrating comparable clinical outcomes between allogeneic and autologous grafts²⁶'^{" 45; 56; 57; 65}. Examples of allogeneic grafts used include the patellar, Achilles, anterior or posterior tibialis, semitendinosus or gracilis, and quadriceps tendons, with the tibialis and Achilles tendons being the most common²⁵'^{' 26; 56; 57; 65; 72}. The long term performance of ACL reconstruction grafts is dependent on several factors, including the structural and material properties of the graft, the initial graft tension⁶'^{" 7; 16; 17; 24; 64}, the intra-articular position of the graft³⁷'^{' 43}, and graft fixation³³'^{' 59}. Increased emphasis has been placed on graft fixation since post-surgical rehabilitation regimens require the immediate ability to regain the full range of motion, re-establish neuromuscular function, and bear weight⁹'^{' 61}. The BPTB graft has been the gold standard for ACL reconstruction in part due to its ability to integrate with subchondral bone via the bony ends. Moreover, it possesses intact insertion sites which can serve as functional transitions between soft tissue and bone. In contrast, the autologous hamstring tendon graft and tendon allografts are fixed mechanically within the femoral bone tunnel by passing the tendon around a transfemoral pin, while an interference screw with a washer or staple is used to fix the graft within the tibial bone tunnel. Currently, the primary cause of failure for these tendon-based grafts is their inability to integrate with subchondral bone through an anatomic soft tissue-to-bone interface²'^{' 8; 10; 19; 23; 31; 36; 53;} ^{6i; 71,}- ^{78; 83} _ _{It has been} reported that the lack of graft integration within the bone tunnels contributes to the sub-optimal clinical outcome of semitendinosus grafts¹⁸'^{" 27;} ⁸¹. Post-operative tendon-to-bone healing does not result in the complete re-establishment of the normal transition zones of the native ACL-to-bone enthesis²'^{" 3; 8; 10; 11; 14; 23; 36;} ^42,- ^53,- ^{6i; 67,}- ^{7i; 83} _^ Rather, a non~anatomic fibrovascular scar tissue forms at the graft and bone junction within the bone tunnel^60"62. Consequently, the tendon graft-to-bone interface represents the weak link of the reconstructed ACL graft³³. Therefore, developing biological fixation methods that promote the regeneration of the native interface on soft tissue-based autografts or allografts will be critical for expediting reconstruction graft healing and achieving long term functionality.

The ACL inserts into subchondral bone through a fibrocartilage interface, which can be subdivided into non-mineralized and mineralized regions¹²'^{' 46; 52; 55; 63; 76; 77}. The principal function of this complex interface is to minimize stress concentrations and to facilitate load transfer between two distinct tissue types⁴'^{' 44; 49; 70; 79; 80}. While the mechanism governing, the formation of the fibrocartilage interface is not well understood, it has been postulated that fibrocartilage forms due to metaplasia of tendon or ligament²⁰. Nawata et al.⁵¹ examined the development of ACL insertions in a rodent model, and reported that insertion site fibrochondrocytes are derived from ligament fibroblasts. Benjamin and Ralphs observed that the amount of non-mineralized fibrocartilage at an enthesis may be related to the degree of motion at the tendon- or ligament-to-bone interface, suggesting that mechanical signaling is responsible for fibroblast differentiation into fibrochondrocytes and subsequent fibrocartilage formation⁵. These reports, coupled with the observation that fibrocartilage develops in regions where the tendon is subjected to compression⁵' ⁷⁴, collectively suggest that compressive loading is necessary for inducing tendon fibroblast trans-differentiation and fibrocartilage formation on tendon grafts.

Vogel et al.^{32: 41; 54; 58; 73} have conducted extensive studies investigating the effects of compressive loading in fibrocartilage formation in flexor tendons, and have reported that compression may induce metaplasia of tendinous matrix to fibrocartilage. The presence of this fibrocartilage region is believed to enable tendons to - A -

resist compressive loading via the accumulation of proteoglycans⁷⁴. For example, while gene expression for aggrecan was absent in the wrap-around region of fetal and neonatal bovine deep flexor tendons, the proteoglycan was strongly expressed in mature animals, suggesting post-natal remodeling of fibrocartilage with physiological loading⁵⁴. In addition, anterior translocation of the rabbit flexor digitorum profundus tendon to remove compressive loading led to a decrease in the size of the fibrocartilage region, breakdown of the collagen fiber network, and lower matrix glycosaminoglycan content⁴¹. Moreover, in vitro dynamic compressive loading of fibrocartilaginous regions of bovine deep flexor tendon resulted in increased expression of aggrecan, biglycan, and versican after 72 hours⁵⁸. Recently, Wang et al. observed that the fibrocartilage interface of the ACL-to-bone insertion undergoes significant structural changes during post-natal development, after the onset of physiological loading⁷⁶. Spalazzi et al. demonstrated the existence of compressive strains at the ACL insertion sites when the tibiofemoral joint is loaded in tension⁷⁰. Therefore, compressive loading may be important for fibrocartilage formation and remodeling at the ACL-to-bone enthesis.

To address the challenge of achieving biological fixation of soft tissue-based ACL reconstruction grafts, functional methods to regenerate an anatomic fibrocartilage transition on tendon grafts need to be developed. To this end, a novel scaffold system was designed which can directly apply compressive mechanical loading to tendon grafts . Specifically, this scaffold system combines a degradable graft collar³⁸'^{' 69} with nanofiber meshes fabricated from poly(lactic-co-glycolic acid) (PLGA)³⁵'^{' 50}. It is anticipated that with the inherent contraction of the nanofiber meshes^{48; 84}, this biphasic scaffold system can be used to apply compressive mechanical loading to tendon grafts and induce fibrocartilage formation. To test this hypothesis, experiments were conducted with a number of objectives, beginning with the characterization of the contractile properties of the nanofiber mesh as well as the mesh+graft collar scaffold complex. The second objective evaluates the effect of scaffold-induced compression on fibrocartilage development on a tendon graft, focusing on matrix remodeling and the development of fibrocartilage- related markers. Another objective is to compare effects of scaffold-induced dynamic and static compression on a tendon graft. This is the first reported study decribing scaffold-mediated mechanical loading and its potential to promote fibrocartilage formation on soft tissue grafts. It is envisioned that the mechano-active scaffold complex can be used clinically to apply both biochemical and mechanical stimuli to induce metaplasia of the tendinous matrix, ultimately facilitating the formation of an anatomic fibrocartilage interface on these grafts. This approach offers significant promise as the functional transition between soft tissue and bone would be re-established, with the potential to ensure long-term graft stability and improve clinical outcome through biological fixation.

Summary

This application provides an apparatus for inducing formation of fibrocartilage, said apparatus comprising a graft collar having a hollow central portion along a longitudinal axis, wherein an outer surface of the graft collar is wrapped with a polymer-fiber mesh configured to apply compression to the graft collar.

This application further provides a method for making a device for inducing formation of fibrocartilage comprising forming a graft collar and wrapping the graft collar with a polymer-fiber mesh, to form said device.

This application also provides a method for inducing formation of fibrocartilage comprising enclosing a tendon within a hollow central portion of a polymer-fiber mesh- wrapped graft collar configured to apply compression to the tendon.

This application further provides an apparatus for inducing formation of fibrocartilage, said apparatus comprising a graft collar having a hollow central portion along a longitudinal axis wherein an outer surface of the graft collar is clamped by a clamp to apply static loading to the graft collar.

Brief Description of the Figures

Figure 1. Characterization of Nanofiber Mesh Contraction.

A) As-fabricated nanofiber mesh with preferential fiber alignment at low (left) and high (right) magnification as shown by scanning electron microscopy (low: x500, high: x2000). B) Percent contraction of the aligned nanofiber mesh in the direction along (y-axis) and normal to (x- axis) fiber alignment (*p<0.05). Significant mesh contraction was the greatest along the direction of fiber alignment, and contraction stabilized after 24 hours.

Figure 2. Compression of Graft Collar Scaffold with Nanofiber Mesh. A) Microsphere scaffold wrapped with nanofiber mesh before (top) and after (bottom) 24 hours of mesh contraction. B) Changes in scaffold inner diameter due to compression induced by the nanofiber mesh. While the scaffold-only control swelled (4%), nanofiber mesh contraction induced over 15% decrease in scaffold diameter after 24 hours.

Figure 3. Compression of Tendon Graft with Nanofiber Mesh. A) Nanofiber mesh wrapped around a patellar tendon sample before (top, day 0) and after (bottom, day 1) mesh contraction. B) Effects of mesh contraction on tendon matrix organization. After five days of culture, the compressed tendon matrix exhibited greater cell density and is morphologically distinct from the unloaded control. After 14 days, however, no difference was observed between the groups. (H&E, xlO, arrows denote the direction of compressive loading applied by the mesh) .

Figure 4. Compression of Tendon Graft with Graft Collar Scaffold and Nanofiber Mesh. A) Wrapping of the tendon graft with graft collar scaffold and mesh (top) and the tendon graft with mesh+scaffold complex after 24 hours (bottom) . B) Effects of compression on tendon matrix organization. Within 24 hours of loading, the tendon matrix no longer exhibits the crimp pattern evident in the unloaded control. In addition, local cell density increased and there is evidence of matrix remodeling, and this organization is maintained after two weeks of static compression. (H&E, x20, arrows denote the direction of compressive loading applied by the scaffold) .

Figure 5. Effects of Compression on Collagen Organization. Scaffold-induced compression modulated collagen organization. Collagen organization was affected by scaffold-mediated loading at (A) control, Day 1, (B) loaded, Day 1, (C) control, Day 14, and (D) loaded, Day 14. In addition, fiber diameter was smaller in the compressed group. Disruption of the collagen matrix was evident only in the control group after 14 days (Stain, picrosirius red as viewed under polarized light; original magnification, x20) .

Figure 6. Effects of Compression on Tendon Cellularity and Matrix Composition. A) Cells proliferated in the unloaded group and cell number was significantly higher in the control tendons compared to the compressed tendons after 24 hours of loading (p<0.05). B) Glycosaminoglycan content in the mesh was significantly higher in the compressed group after 24 hours of loading (*p<0.05) .

Figure 7. Effects of Compression on the Expression of Fibrocartilage-Related Markers. Scaffold-induced compression of the tendon graft resulted in significant up-regulation of type II collagen, aggrecan, and TGF-β3 after 24 hours ( *p<0.05) . All three fibrocartilage interface-related markers increased in the tendon after scaffold-induced compression. Figure 8. Manufacturing of the Polymer-Fiber Mesh .

Figure 9. Graft Collar Scaffold Fabrication.

Figure 10. Comparison of Scaffold-Induced Dynamic and Static Compression on a Tendon Graft. (A) Experimental Design. (B) Photogrphs of the compressed group and the control group.

Figure 11. Cross Section of Scaffold + Mesh Complex Applied to Tendon Graft.

Figure 12. Effects of Compression on Tendon Graft Matrix Morphology. (A) Control Group. (B) Dynamic Compression Group. (C) Static Compression Group.

Figure 13. Effects of Compression on Tendon Graft Collagen Fiber Diameter. (A) Control Group. (B) Dynamic Compression Group. (C) Static Compression Group.

Figure 14. Effects of Compression on Matrix Proteoglycan Content. Greater retention of GAG in the loaded groups.

Figure 15. Effects of Compression on Cell Number. Cell number constant in the loaded group x 10⁶.

Figure 16. Effects of Compression on Gene Expression. Gene expression for fibrocartilage markers up-regulated in static compressed group over seven days (Collagen, Aggrecan, TGF-B3) .

Figure 17. Effects of Compression on Cell Viability. Cell viability and migration onto the graft collar was observed in the compressed groups.

Figure 18. Effects of Compression on Tendon Matrix- Preliminary In Vivo Study. Little fiber diameter change at day 1 while notable fiber diameter decrease by day 14. Figure 19. Schematic of Graft Collar + Mesh Complex Applied to Graft .

Detailed Description

In order to facilitate an understanding of the material which follows, one may refer to Freshney, R. Ian. Culture of Animal Cells - A Manual of Basic Technique (New York: Wiley-Liss, 2000) for certain frequently occurring methodologies and/or terms which are described therein.

However, except as otherwise expressly provided herein, each of the following terms, as used in this application, shall have the meaning set forth below.

As used herein, "aligned fibers" shall mean groups of fibers which are oriented along the same directional axis. Examples of aligned fibers include, but are not limited to, groups of parallel fibers .

As used herein, "allogenic", in regards to a biopolymer mesh, shall mean a biopolymer mesh derived from a material originating from the same species as the subject receiving the biopolymer mesh.

As used herein, "bioactive" shall include a quality of a material such that the material has an osteointegrative potential, or in other words the ability to bond with bone. Generally, materials that are bioactive develop an adherent interface with tissues that resist substantial mechanical forces.

As used herein, "biomimetic" shall mean a resemblance of a synthesized material to a substance that occurs naturally in a human body and which is not rejected by (e.g., does not cause an adverse reaction in) the human body.

As used herein, "biopolymer mesh" shall mean any material derived from a biological source. Examples of a biopolymer mesh include, but are limited to, collagen, chitosan, silk and alginate.

As used herein, "BFGF" shall basic fibroblast growth factor .

As used herein, "BMP" shall mean bone morphogenic protein.

As used herein, "BMSC" shall mean bone marrow-derived stem cells .

As used herein, "chondrocyte" shall mean a differentiated cell responsible for secretion of extracellular matrix of cartilage.

As used herein, "clamp" shall mean a device which statically compresses the soft tissue graft. The clamp can be made of metal, ceramic, polymers, composites thereof, or other material that can compress a soft tissue graft. The material can be porous, permeable, or degradable .

As used herein, "fibroblast" shall mean a cell of connective tissue, mesodermally derived, that secretes proteins and molecular collagen including fibrillar procollagen, fibronectin and collagenase, from which an extracellular fibrillar matrix of connective tissue may be formed.

As used herein, "functional" shall mean affecting physiological or psychological functions but not organic structure.

Generally, "glass transition temperature" is the temperature at which, upon cooling, a noncrystalline ceramic or polymer transforms from a supercooled liquid into a rigid glass. The noncrystalline ceramic or polymer may be of multiple form and composition, and may be formed as microspheres. In the context of a sintering process, such as discussed in this application, the polymer chains from adjacent microspheres typically entangle, effectively forming a bond between the microspheres upon cooling. As the polymer is heated above its glass transition temperature, long range polymer chain motion begins.

As used herein, "graft" shall mean a device or material to be implanted during medical grafting, which is a surgical procedure to transplant tissue without a blood supply, including but not limited to soft tissue graft, synthetic grafts, and the like.

As used herein, "graft collar" shall mean a device embodying a graft and configured like a collar, that is, having a hollow cylindrical body in a longitudinal direction. A graft collar can be permeable, so the tissue can survive. As indicated by the results of the experiment described in this disclosure, the tissues can survive despite the presence of compression.

As used herein, "hydrogel" shall mean any colloid in which the particles are in the external or dispersion phase and water is in the internal or dispersed phase. For example, a chondrocyte-embedded agarose hydrogel may be used in some instances. As another example, the hydrogel may be formed from hyaluronic acid, chitosan, alginate, collagen, glycosaminoglycan and polyethylene glycol (degradable and non-degradable) , which can be modified to be light- sensitive. It should be appreciated, however, that other biomimetic hydrogels may be used instead.

As used herein, "lyophilized", in regards to a graft collar, shall mean a graft collar that has been rapidly frozen and dehydrated. As used herein, "osteoblast" shall mean a bone-forming cell that is derived from mesenchymal osteoprognitor cells and forms an osseous matrix in which it becomes enclosed as an osteocyte. The term is also used broadly to encompass osteoblast-like, and related, cells, such as osteocytes and osteoclasts.

As used herein, "osteointegrative" shall mean ability to chemically bond to bone.

As used herein, "PDGF" shall mean platelet-derived growth factor.

As used herein, "polymer" shall mean a chemical compound or mixture of compounds formed by polymerization and including repeating structural units. Polymers may be constructed in multiple forms and compositions or combinations of compositions.

As used herein, "particle reinforcer" shall mean a composite with a higher strength than the original material .

As used herein, "porosity" shall mean the ratio of the volume of interstices of a material to a volume of a mass of the material.

As used herein, "sintering" shall mean densification of a particulate polymer compact involving a removal of pores between particles (which may be accompanied by equivalent shrinkage) combined with coalescence and strong bonding between adjacent particles. The particles may include particles of varying size and composition, or a combination of sizes and compositions. As used herein, "soft tissue graft" shall mean a graft which is not synthetic, and can include autologous grafts, syngeneic grafts, allogeneic grafts, and xenogeneic graft.

As used herein, "synthetic" shall mean that the material is not of a human or animal origin.

As used herein, "TGF" shall mean transforming growth factor .

As used herein, "VEGF" shall mean vascular endothelial growth factor.

As used herein, "xenogenic", in regards to a biopolymer mesh, shall mean a biopolymer mesh derived from a material originating from a species other than that of the subject receiving the biopolymer mesh.

This application describes an apparatus for inducing formation of fibrocartilage, comprising a graft collar having a hollow central portion along a longitudinal axis, wherein an outer surface of the graft collar is wrapped with a polymer-fiber mesh, to apply compression to the graft collar. This application further describes a method for making said apparatus and a method for inducing formation of fibrocartilage .

The following exemplary embodiments and experimental details sections are set forth to aid in an understanding of the subject matter of this disclosure but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

In one embodiment, the graft collar has a cylindrical body. In another embodiment, the graft collar includes a sliced cut parallel to a longitudinal axis of the cylindrical body. This embodiment would permit encasing a soft tissue graft, such as a tendon, on all sides. In another embodiment, the outer surface of the graft collar is wrapped in its entirety.

In one embodiment, the polymer-fiber mesh comprises nanofibers . In another embodiment, the nanofibers are aligned. In another embodiment, the nanofibers are aligned perpendicular to the longitudinal axis of the graft collar. In another embodiment, the nanofibers are unaligned.

In one embodiment, the graft collar includes at least one of the following substances: anti-infectives, antibiotics, bisphosphonate, hormones, analgesics, antiinflammatory agents, growth factors, angiogenic factors, chemotherapeutic agents, anti-rejection agents, and RGD peptides. Examples of growth factors include, but are not limited to, TGFs, BMPs, IGFs, VEGFs, BFGFs and PDGFs. In one embodiment, the TGF is TGF-β. In another embodiment, the BMP is BMP-2.

In one embodiment, the graft collar includes one or more of the following types of cells: chondrocytes, osteoblasts, osteoblast-like cells and stem cells. In another embodiment, the graft collar includes at least one of the following: osteogenic agents, osteogenic materials, osteoinductive agents, osteoinductive materials, osteoconductive agents, osteoconductive materials and chemical factors.

In one embodiment, the graft collar can comprise multiple phases. In one embodiment, the graft collar comprises first through third phases, wherein the first phase comprises a material which promotes growth and proliferation of fibroblasts, (ii) the second phase adjacent to the first phase comprises a material which promotes growth and proliferation of chondroblasts, and (iii) the third phase adjacent to the second phase comprises a material which promotes the growth and proliferation of osteoblasts. In this embodiment, the graft collar can comprise a degradable cell barrier, such as a nanofiber mesh, inserted between the adjacent phases.

In one embodiment, the graft collar has multiple phases joined by a gradient of properties. In another embodiment, the multiple phases of the graft collar are processed through one or more sintering stages. In another embodiment, the gradient of properties across the multiple phases of the graft collar includes mechanical properties. In another embodiment, the gradient of properties across the multiple phases of the graft collar includes chemical properties. In another embodiment, the gradient of properties across the multiple phases of the graft collar includes mineral content. In another embodiment, the gradient of properties across the multiple phases of the graft collar includes structural properties. In another embodiment, the gradient of properties across the multiple phases of the graft collar includes porosity. In another embodiment, the gradient of properties across the multiple phases of the graft collar includes geometry.

In one embodiment, the polymer-fiber mesh is selected from the group comprising aliphatic polyesters, poly (amino acids), copoly (ether-esters ), polyalkylenes oxalates, polyamides, poly (iminocarbonates) , polyorthoesters, polyoxaesters, polyamidoesters, poly ( ε-caprolactone) s, polyanhydrides, polyarylates, polyphosphazenes, polyhydroxyalkanoates , polysaccharides, and biopolymers, and a blend of two or more of the preceding polymers. In another embodiment, the polymer-fiber mesh comprises at least one of the poly (lactic-co-glycolic acid), poly (lactide) and poly (glycolide) . In another embodiment, the polymer-fiber mesh is 35% poly (DL-lactide-co-glycolic acid) 85:15, 55% N, N-dimethylformamide, and 10% ethanol .

In one embodiment, the polymer-fiber mesh comprises particulate reinforcers. In another embodiment, the particulate reinforcers comprise nanoparticles .

In one embodiment, the graft collar is porous. In another embodiment, the graft collar is lyophilized. In another embodiment, the graft collar is biodegradable. In another embodiment, the graft collar is osteointegrative . In one embodiment, the graft collar is permeable, so the tissue can survive despite the presence of compression. As evidence by the cell number and matrix production results described in this application, tissue can survive despite the presence of compression.

In one embodiment, the graft collar is composed of microspheres. In another embodiment, the microspheres comprise poly (DL-lactide-co-glycolic acid). In another embodiment, microspheres comprise poly (DL-lactide-co- glycolic acid) and bioactive glass.

In one embodiment, the apparatus further comprise a device which applies static loading to the graft collar. In another embodiment, the device is a clamp.

In one embodiment, the degree of strain of said graft collar is adjusted based on polymer composition. In another embodiment, the degree of strain of said graft collar is adjusted based on nanofiber composition. In one embodiment, the graft collar comprises (a) a first region comprising a biopolymer mesh and hydrogel and (b) a second region adjoining the first region and comprising polymer microspheres.

In one embodiment, the first region supports the growth and maintenance of an interfacial zone between tendon and bone, and the second region supports the growth and maintenance of bone tissue. In another embodiment, the hydrogel is photopolymerized, thermoset or chemically cross-linked. In another embodiment, the hydrogel is polyethylene glycol.

In one embodiment, the biopolymer mesh comprises aligned fibers .

In one embodiment, the first region contains TGF, such as TGF-β.

In one embodiment, the first region contains chondrocytes. The chondrocytes can be, but are not limited to, BMSC- derived chondrocytes.

In one embodiment, the first region contains stem cells. The stem cells can be, but are not limited to, BMSCs.

In one embodiment, the biopolymer mesh is derived from at least one of collagen, chitosan, silk and alginate. In another embodiment, the biopolymer mesh is allogenic or xenogenic .

In one embodiment, the second region contains at least one of the following growth factors: BMP, IGF, VEGF, BFGF and PDGF. The BMP can be, but is not limited to, BMP-2. In one embodiment, the second region includes osteoblasts and/or osteoblast-like cells. The osteoblasts and/or osteoblast like cells can be BMSC-derived.

In one embodiment, the second region includes at least one of the following: osteogenic agents, osteogenic materials, osteoinductive agents, osteoinductive materials, osteoconductive agents, osteoconductive materials and chemical factors.

In one embodiment, the second region contains nanoparticles of calcium phosphate. Examples of calcium phosphate include, but are not limited to, tricalcium phosphate, hydroxyapatite or a combination thereof.

In one embodiment, the second region contains nanoparticles of bioactive glass. In another embodiment, the graft collar is biodegradable. In another embodiment, the graft collar is osteointegrative .

In one embodiment, the graft collar comprises (a) a first region comprising a polymer-fiber mesh and hydrogel and (b) a second region adjoining the first region and comprising polymer microspheres.

In one embodiment, the first region supports the growth and maintenance of an interfacial zone between tendon and bone, and the second region supports the growth and maintenance of bone tissue.

In one embodiment, the graft collar includes at least one of the following substances: anti-infectives, antibiotics, bisphophonate, hormones, analgesics, antiinflammatory agents, growth factors, angiogenic factors, chemotherapeutic agents, anti-rejections agents, and RGD peptides In one embodiment, the hydrogel is photopolymerized, thermoset or chemically cross-linked. In another embodiment, the hydrogel is polyethylene glycol.

In one embodiment, the polymer-fiber mesh comprises aligned fibers.

In one embodiment, the first region contains TGF. The TGF can be, but is not limited to, TGF-β.

In one embodiment, the first region contains chondrocytes. The chondrocytes can be BMSC-derived chondrocytes.

In one embodiment, the second region contains at least one of the following growth factors: BMP, IGF, VEGF, BFGF and PDGF. The BMP can be, but is not limited to, BMP-2.

In one embodiment, the second region includes osteoblasts and/or osteoblast-like cells. The osteoblasts and/or osteoblast like cells can be BMSC-derived.

In one embodiment, the microspheres comprise poly (DL- lactide-co-glycolic acid) . In another embodiment, the microspheres comprise poly (DL- lactide-co-glycolic acid) and bioactive glass.

This application further discloses a method for making a device for inducing formation of fibrocartilage comprising (a) forming a graft collar and (b) wrapping the graft collar prepared in step (a) with a polymer-fiber mesh, to form said device.

In one embodiment, said step (a) comprises (al) processing a plurality of microspheres, (a2) laying the microspheres processed in step (a) in a mold and (a3) sintering together the microspheres in the mold above a glass transition temperature.

In one embodiment, the microspheres further comprise bioactive glass. In another embodiment, the polymer-fiber mesh comprises nanofibers .

In one embodiment, the polymer-fiber mesh is selected from the group comprising aliphatic polyesters, poly (amino acids), copoly (ether-esters) , polyalkylenes oxalates, polyamides, poly (iminocarbonates) , polyorthoesters, polyoxaesters, polyamidoesters, poly ( ε-caprolactone) s, polyanhydrides , polyarylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, and biopolymers, and a blend of two or more of the preceding polymers.

In one embodiment, the polymer-fiber mesh comprises at least one of the poly (lactic-co-glycolic acid), poly (lactide) and poly (glycolide) . In another embodiment, the polymer-fiber mesh is 35% poly (DL-lactide-co-glycolic acid) 85:15, 55% N, N-dimethylformamide, and 10% ethanol .

In one embodiment, the nanofibers wrapped around the graft collar are perpendicular to the longitudinal axis of the graft collar.

In one embodiment, the method further comprises incubating the polymer-fiber mesh-wrapped graft collar at a suitable temperature, time and humidity to allow sintering of the polymer-fiber mesh to the graft collar. In one embodiment, the polymer-fiber-mesh-wrapped graft collar is incubated at or around 37 °C and at or around 5% CO₂.

This application further describes a method for inducing formation of fibrocartilage comprising enclosing a tendon within a polymer-fiber mesh-wrapped graft collar configured to apply compression to the tendon. Any of the aforementioned graft collar systems can be utilized in this method.

This application further describes an apparatus for inducing formation of fibrocartilage, said apparatus comprising a graft collar having a hollow central portion along a longitudinal axis wherein an outer surface of the graft collar is clamped by a clamp to apply static loading to the graft collar.

The specific embodiments described herein are illustrative, and many variations can be introduced on these embodiments without departing from the spirit of the disclosure or from the scope of the appended claims. Elements and/or features of different illustrative embodiments may be combined with each other and/or substituted for each other within the scope of this disclosure and appended claims.

Further non-limiting details are described in the following Experimental Details section which is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the claims which follow thereafter.

Experimental Details

Biological fixation of soft tissue-based grafts utilized for Anterior Cruciate Ligament (ACL) reconstruction poses a significant clinical challenge. The ACL integrates with subchondral bone through a fibrocartilage interface, which serves to minimize stress concentrations and facilitate load transfer between two distinct types of tissue. Functional integration thus requires the re-establishment of this fibrocartilage region on the reconstructed grafts. To this end, this study focuses on the design and evaluation of a mechano-active scaffold system based on a composite of poly-α-hydroxyester nanofiber mesh and sintered microspheres. Specifically, the effects of scaffold-induced compression on tendon matrix remodeling and the development of fibrocartilage-related markers are evaluated over a two-week period. Scaffold contraction resulted in over 15% compression of the patellar tendon graft and up-regulated the expression of fibrocartilage- related markers such as type II collagen, aggrecan and transforming growth factor-β3. In addition, proteoglycan content was significantly higher in the compressed tendon group after one day of loading. Further, the effects of scaffold-induced dynamic and static compression on a tendon graft were compared. It was found that static compression produced greater compression in the fibers, greater retention of matrix proteoglycan, higher cell number count, and greater expression of fibrocartilage-related markers. This is the first reported study describing scaffold- mediated mechanical loading, and the findings of this study demonstrate the potential of the mechano-active scaffold to promote the formation of an anatomic fibrocartilage transition on tendon-based ACL reconstruction grafts, which is critical for achieving biological fixation and extending graft functionality.

MATERIALS AND METHODS

Tendon Graft Isolation

Patellar tendon grafts were isolated from neonatal bovine tibiofemoral joints (1-7 days old) obtained from a local abattoir (Green Village Packing, Green Village, NJ) . Briefly, the joints were first cleaned in an antimicrobial bath. Under antiseptic conditions, midline longitudinal incisions were made through the subcutaneous fascia to expose the patellar tendon. The paratenon was removed, and the patellar tendon dissected from the underlying fat pad. Sharp incisions were made through the patellar tendon at the patellar and tibial insertions, and the insertions were completely removed from the graft.

Nanofiber Mesh Fabrication and Characterization

Aligned nanofiber meshes (Fig. IA, B) were fabricated by electrospinning¹³. A viscous polymer solution consisting of 35% poly (DL-lactic-co-glycolic acid) 85:15 (PLGA, I. V. = 0.70 dL/g, Lakeshore Biomaterials, Birmingham, AL), 55% N, N-dimethylformamide (Sigma, St. Louis, MO), and 10% ethanol (Commercial Alcohol, Inc., Toronto, Ontario) was loaded into a syringe fitted with an 18-gauge needle (Becton Dickinson, Franklin Lakes, NJ) . Aligned fibers R 9 were obtained using an aluminum drum with an outer diameter of 10.2 cm rotating with a surface velocity of 20 m/s. A constant flow rate of 1 mL/hr was maintained using a syringe pump (Harvard Apparatus, Holliston, MA) , and an electrical potential was applied between the needle and the grounded substrate (distance=10 cm) using a high voltage DC power supply (Spellman, Hauppauge, NY, 8-1OkV) . (Fig. 8) Fiber morphology, diameter and alignment of the as-fabricated mesh samples were analyzed using scanning electron microscopy (SEM) . Briefly, the samples were sputter-coated with gold (LVC-76, Plasma Sciences, Lorton, VA) and subsequently imaged (JSM 5600LV, JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV.

Graft Collar Scaffold Fabrication

A tendon graft collar based on a sintered microsphere scaffold was fabricated following published methods³⁸'^{' 69}. Specifically, the scaffold is composed of composite microspheres consisting of PLGA (85:15, I. V. = 3.42 dl/g, Purac, Lincolnshire, IL) and 45S5 bioactive glass (BG, 20 μm, MO-SCI Corporation, Rolla, MD) . The microspheres were formed following the methods of Lu et al.^3β, where the polymer was first dissolved in dichloromethane (Acros Organics, Morris Plains, NJ) and then BG particles were added (20 wt%) . After vortexing, the suspension was poured into a 1% solution of polyvinyl alcohol (Sigma, St. Louis, MO) to form the microspheres. The microspheres were subsequently sintered at 7O⁰C for 5 hours in a custom mold to form cylindrical scaffolds with an outer diameter of 0.7 cm and an inner diameter of 0.3 cm. (Fig. 9)

Characterization of Nanofiber Mesh Contraction Mesh contraction was evaluated using digital image analysis. Briefly, the nanofiber meshes were cut into 10 mm x 10 mm squares and immersed in Dulbecco's Modification of Eagle's Medium (DMEM, Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Norcross, GA) and incubated at 37⁰C and 5% CO2. The meshes were imaged using stereomicroscopy at 0, 2, 24, and 72 hours. Mesh dimensions (n=5) were measured by image analysis (ImageJ 1.34s, NIH, Bethesda, MD), and contraction was calculated based on percent change in length both in the x-axis and along the direction of fiber alignment (y-axis) .

Compression of Graft Collar Scaffold with Nanofiber Mesh

In addition to mesh contraction, the nanofiber mesh- mediated compression of the microsphere-based graft collar was also evaluated in vitro. Briefly, strips of nanofiber mesh (15.5 cm x 1.5 cm) were wrapped around the graft collar scaffold, with the fibers aligned perpendicular to the scaffold long axis. The mesh+scaffold was then incubated in PBS at 37°C and 5% CO₂, and changes in scaffold diameter (n=6) due to mesh contraction were monitored over 24 hours using image analysis (ImageJ) .

Compression of Tendon with Nanofiber Mesh

The potential of utilizing nanofiber mesh contraction to directly apply compression to the tendon graft was evaluated over time. Briefly, the aligned electrospun meshes were cut into 10 cm x 2 cm strips, with fiber alignment oriented along the long axis of the mesh. The patellar tendon graft was bisected along its long axis, and one half of the tendon was wrapped with the nanofiber mesh while the other half served as the unloaded control

(Fig. 3A) . The samples were cultured in DMEM supplemented with 1% non-essential amino acids, 1% antibiotics, and 0.1% antifungal (all from Mediatech) and 10% FBS (Atlanta Biologicals) . At days 5 and 14, the effects of compression on tissue morphology and cellularity were characterized by histology⁶⁸. The samples were rinsed with phosphate buffered saline (PBS, Sigma) , fixed with 10% neutral buffered formalin (Fisher Scientic and Sigma) and embedded in paraffin (Fisher Scientific, Pittsburgh, PA). The samples were then cut into 7-μm thick sections and stained with hematoxylin and eosin (H&E) .

Compression of Tendon Graft with the Graft Collar Scaffold and Nanofiber Mesh

The potential of the graft collar scaffold and nanofiber mesh complex to apply static compression to the patellar tendon graft was also evaluated in vitro. Specifically, the patellar tendon graft was dissected into 2 cm x 0.3 cm segments and the cylindrical scaffold was halved along its long axis. Each tendon segment was inserted between the two scaffold halves (Fig. 4A, top). For the experimental group, the tendon+graft collar was wrapped with the aligned nanofiber mesh (15.5 cm x 1.5 cm), while the control scaffolds were wrapped with pre-contracted electrospun mesh (n=2) . In addition, to ensure static compression of the tendon graft, the experimental group was wrapped with new mesh strips on every other day during the two week culturing period. The scaffold+tendon graft complex (Fig. 4A, bottom) was cultured in fully supplemented media at 37⁰C and 5% CO₂.

Effects of Compression on Tendon Graft Cellularity and Matrix Content

The effects of static compression on tendon matrix organization (n=2) were analyzed at 1 and 14 days via histology (H&E) . In addition, since most of the mesh compression occurs within the first 24 hours, total cell number (n=5) and proteoglycan content in the tendon graft were evaluated at day 1. For the biochemical assays ' ⁶⁹, both the wet and dry weights of the tendon samples were determined at day 0 and day 1, and the tissue was subsequently digested for 16 hours in 2% papain (Sigma) buffer at 60°C. Total DNA content of the digest was determined with the PicoGreen dsDNA assay (Molecular Probes), following the manufacturer's suggested protocol. Sample fluorescence was measured using a microplate reader (Tecan, Research Triangle Park, NC) , with excitation and emission wavelengths set at 485 and 535 nm, respectively. The total number of cells in the sample was calculated using the conversion factor of 8 pg DNA/cell⁴⁰.

Sulfated glycosaminoglycan (GAG) content was quantified using a colorimetric 1, 9-dimethylmethylene blue (DMMB) assay. Tissue digest from the cell quantitation assay was combined with DMMB dye, and the concentration of GAG- DMMB complexes was determined using a plate reader at 540 and 595 nm and correlated to a standard prepared with chondroitin-6-sulfate .

Cell Phenotype

Gene expression for fibrocartilage markers (n=2) such as collagen I, II, aggrecan, and Transforming Growth Factor- Beta 3 (TGF-β3) was determined at day 1 using reverse- transcription polymerase chain reaction (RT-PCR) . Briefly, after removing the graft collar and nanofiber mesh, total RNA of the tendon graft was obtained using the Trizol extraction method (Invitrogen, Carlsbad, CA) . The isolated RNA was reverse-transcribed into cDNA using the Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) and the cDNA product was amplified using recombinant Platinum Taq DNA polymerase (Invitrogen) . GAPDH was used as the housekeeping gene, and expression band intensities were measured (ImageJ) and normalized against GAPDH.

Statistical Analysis

Results are presented in the form of mean ± standard deviation, with n equal to the number of samples analyzed. Two-way analysis of variance (ANOVA) was first performed to assess if differences exist among the means. Fisher's LSD post-hoc test was subsequently performed for all pair- wise comparisons and statistical significance was attained at p<0.05. For gene expression, a one-way ANOVA and Fisher' s LSD post-hoc test were performed to determine the effect of compression. All statistical analyses were performed using the JMP statistical software package (SAS Institute, Cavy, NC) .

Comparison of Effects of Scaffold-Induced Dynamic and Static Compression on a Tendon Graft

Three groups of scaffold and graft apparatus were used: the dynamic compression group, the static compression group and the control group. For the dynamic compression group, scaffold and graft were compressed by mesh every 48 hours and relaxation for 24 hours is allowed between compressions. For the static compression group, scaffold and graft were compressed by mesh every 48 hours with no relaxation allowed between compressions. For the control group, no compression was applied. (Fig. 10A-B) RESULTS

Nanofiber Characterization and Mesh Contraction

The nanofiber mesh exhibited a high degree of alignment with an average fiber diameter of 0.9 ± 0.4 μm (Fig. IA). Anisotropic mesh contractile behavior was observed in the mesh, with significantly higher contraction found in the direction of nanofiber alignment. Specifically, the mesh contracted over 57% along the aligned fiber direction (y- axis) by 2 hours, with less than 13% reduction in the x- axis (Fig. IB) . Mesh contraction continued over time, exhibiting over 70% contraction in the y-axis and 20% in the x-axis by 24 hours and stabilizing thereafter, with no significant differences found between the 24- and 72-hour groups .

Compression of Graft Collar Scaffold with Nanofiber Mesh

After the nanofiber mesh was wrapped around the graft collar scaffold, mesh contraction resulted in a significant decrease in scaffold inner diameter, averaging 15% strain within 24 hours (Fig. 2) . In contrast, the control scaffold without mesh cultured under similar conditions expanded and measured an increase in inner diameter (4%), although the difference was not statistically significant (p<0.05).

Compression of Tendon with Nanofiber Mesh

When the nanofiber mesh was used to compress the tendon graft, mesh contraction resulted in an approximately 30% decrease in graft diameter by 24 hours (Fig. 3A) . After five days of explant culture, the compressed tendon exhibits less of the crimp structure evident in the control group, and remodeled into a dense matrix with high cellularity (Fig. 3B) . However, by day 14, the crimp pattern was restored in the compressed group, with ultrastructure and cellularity indistinguishable from the unloaded control group.

Compression of Tendon with the Graft Collar Scaffold and Nanofiber Mesh

In order to apply a physiological level of loading (10- 15%), the tendon graft was compressed by a complex of the graft collar scaffold and nanofiber mesh. It was observed that at 24 hours post-compression (Fig. 4B, top), the tendon graft matrix organization was distinct from that of the unloaded control, with increased matrix density and less of the characteristic crimp of the tendon. After 14 days of compression by the scaffold+mesh complex, it was found that the matrix remodeling visible 24 hours following the onset of loading was maintained over time (Fig. 4B, bottom) . In contrast, the control tendon retained its characteristic crimp, with evident disruption of the matrix ultrastructure. Further, compression distinctly changed matrix collagen organization. The color of collagen fibers stained with Pricrosirius red and viewed under polarized light is reported to correlate with fiber diameter,^85'87 progressing from green, yellow, orange to red wxth increasing fiber diameter. While no change in fiber diameter was observed in the unloaded control group (Fig. 5A), the collagen fiber diameter of the group compressed with the mesh + collar scaffold became smaller (in green) after 24 hours of loading (Fig 5B) . Moreover, disruption of the tendon collagen matrix was evident in the control group by day 14 (Fig. 5C) . In general, collagen fibers remained perpendicular to the direction of loading after 24 hours, and this effect was maintained over 14 days with the mesh + collar scaffold complex (Fig. 5D) . In addition to changes in tendon matrix organization, total cell number in the tendons remained relatively constant in the compressed group, with a significantly higher number of cells found in the control tendons by day 1 (Fig. 6A) . Interestingly, matrix glycosaminoglycan (GAG) content was found to be significantly higher in compressed tendon group after one day of culture (Fig. 6B) .

Effects of Compression on the Expression of Fibrocartilage-Related Markers

The expression of fibrocartilage markers such as types I and II collagen, aggrecan and TGF-β3 were evaluated after compression with the graft collar scaffold and nanofiber mesh. As shown in Fig. 6, after 24 hours of compression, gene expression of type II collagen, aggrecan and TGF-β3 were all up-regulated in the loaded group when compared to non-compressed tendons (Fig. I)₁ with significant differences found in aggrecan and TGF-β3 expression.

The morphology of the control group is maintained from day 1 to day 14. Crimp in the tissue was maintained. In both the dynamic and the static compression group, fiber morphology was compressed after day 1 and continues to compress to day 14. (Fig. 12)

Graft Collagen Fiber Diameter did not change in the control group. For the dynamic compression group, there was no change after day 1 but fiber diameter decreases to day 14. For the static compression group, there was little fiber diameter change at day 1 and notable fiber diameter decrease by day 14. (Fig 13) Further, there was greater retention of matrix proteoglycan content in the compressed group, especially in the static compression group through day 14. (Fig. 14)

In addition, the cell number is greater in the static compression group than in the dynamic compression group.

(Fig. 15) Additionally, gene expression for fibrocartilage markers up-regulated in static compressed group over seven days. (Fig. 16)

Finally, cell viability and migration onto the graft collar was observed in the compressed groups but not in the control group. (Fig. 17)

Effects of Compression on Tendon Matrix -Preliminary in Vivo Study

After 1 week of implantation, the re-organization of the tendon due to mesh contraction was maintained without re- wrapping. There was little fiber diameter change at day 1. However, there was notable fiber diameter decreases by day 14. (Fig. 18) DISCUSSION

The long term goal is to achieve biological fixation by engineering a functional and anatomical fibrocartilage interface on biological and synthetic soft tissue grafts used in orthopaedic repair³⁹. To this end, the current study focuses on the design and evaluation of a novel graft collar scaffold system capable of applying mechanical loading and inducing fibrocartilage formation on tendon grafts. Specifically, scaffold-mediated compression of a patellar tendon graft was evaluated over time, focusing on the effects of loading on tendon matrix organization and cell response. In addition, effects of scaffold-induced dynamic and static compression on a tendon graft were compared. It was found that the complex of the nanofiber mesh and graft collar was able to apply a physiological range of compressive loading to tendon grafts. Moreover, scaffold-mediated compression promoted matrix remodeling, maintained graft glycosaminoglycan content and, interestingly, induced gene expression for fibrocartilage markers, including type II collagen, aggrecan, and TGF-β3. Further, static compression was found to be more effective in producing changes in graft collagen fiber diameter, increasing matrix proteoglycan content, cell number and gene expression for fibrocartilage markers. These promising results demonstrate that compressive loading can be incorporated into scaffold design and used to promote fibrocartilage formation on tendon grafts.

Two scaffold-based loading systems were described in this study. The first design involved using a nanofiber mesh to directly load the tendon graft. The pre-designed alignment of the nanofiber mesh results in anisotropic mesh contractile behavior, effectively translating contractile force into compression, which has been utilized in this study to apply compressive loading to the tendon grafts. Histological analysis of the grafts revealed that the scaffold-mediated compression induced extensive remodeling of the tendon ultrastructure, with the compressed graft exhibiting a denser matrix with increased local cell density. This matrix modulation effect, however, diminished over time, with the control and loaded groups nearly indistinguishable by day 14. As mesh contraction stabilizes after 24 hours, it is likely that the tendon graft is no longer experiencing mechanical stimulation in long term cultures. These observations suggest that it is necessary to incorporate extended mechanical stimulation into scaffold design.

The short-term effect of mesh-induced compressive loading on graft matrix organization and the high magnitude of compression (approximately 30%) initiated the development of the second mechano-active scaffold system. Specifically, the nanofiber mesh was combined with a degradable microsphere-based graft collar system in order to achieve a physiological range of loading (15%) . Moreover, to maintain static compression, the tendon- scaffold complex was wrapped with new nanofiber mesh every other day. It was observed that under static compression, the remodeled tendon matrix with cells embedded in a dense matrix was maintained over time, with marked differences observed between control and the loaded groups . These observations demonstrate the potential of this scaffold system to provide continuous mechanical stimulation and promote sustained tissue remodeling. Proteoglycan content of the tendon matrix was also significantly higher in the compressed group compared to the control at day 1, further indicating that the scaffold-induced compression influences matrix maintenance and remodeling.

Scaffold-mediated compression also resulted in the up- regulation of fibrocartilage markers including type II collagen, aggrecan, and Transforming Growth Factor-β3 (TGF-β3) . It is well known that fibrocartilage in tendons is largely comprised of types I and II collagen, as well as proteoglycans⁵'^{' 15; 32; qi} . Moreover, compressive loading of fibrocartilaginous regions of tendons has been reported to increase the synthesis cf Transforming Growth Factor-βl (TGF-βl)⁵⁸ and large proteoglycans, as well as enhancing aggrecan gene expression¹⁵'^{' 32}. Compression of the non- fibrocartilaginous regions of the deep flexor tendon has also been reported to promote proteoglycan synthesis¹⁵. The findings of this study are in agreement with these published studies on the effects of compressive loading, and demonstrate the feasibility of implementing a degradable scaffold system for fibrocartilage interface formation on tendon grafts. In addition to applying continuous compressive loading to the graft within a physiological range, it is anticipated that this novel scaffold system also can be used to deliver cells and growth factors. These design optimizations will be critical for allograft recellularization and exercising biochemical stimulation to direct cellular differentiation as well as transformation of the tendon matrix into fibrocartilage .

Contraction of PLGA meshes has been previously reported in the literature⁸⁴, although the phenomenon has been discredited as a shortcoming rather than promoted as an advantageous attribute of the system. Currently, the mechanism underlying mesh contraction is not known. Zong et aJ.⁸⁴ have observed that electrospun nanofiber mesh comprised of crystalline polyesters contract significantly less than amorphous polyester co-polymers such as PLGA 75:25. It was proposed that when nanofiber meshes comprised of crystalline polymers are incubated at 37°C, the polymer glass transition temperature is approached and crystallization rapidly occurs, resulting in a lamellar structure that constrains the relaxation of the polymer chains and in turn prevents contraction⁸⁴. The polyester co-polymer utilized in this study has a high D,L-lactide content (85%) and is non-crystalline, thus the above mechanism may explain the high degree of contraction observed. Although not the focus of the current study, fiber alignment-related scaffold anisotropy may be controlled to modulate mesh contraction, and consequently, the magnitude and direction of compressive loading on the graft may be controlled by customizing the degree of fiber alignment. Future studies will focus on elucidating the mechanism of mesh contraction as well as exploring methods to control this process for mechanical stimulation.

This is the first study to incorporate mechanical loading into scaffold design and to demonstrate the potential of using this mechano-active scaffold system to induce fibrocartilage formation on soft tissue grafts. The mesh- collar system is intended to be applied clinically as a degradable graft collar, and will be used to initiate and direct regeneration of an anatomical fibrocartilage interface at the insertion of tendon-based ACL reconstruction grafts. In addition to providing a three- dimensional environment for matrix development and growth factors for guided cell differentiation, the innovative scaffold system described here can also apply physiologic mechanical stimulation crucial for directing cellular function and tissue remodeling. For utilization with viable autografts, it is envisioned that the graft would be inserted through the collars immediately prior to implantation, and compression of the graft and subsequent fibrocartilage formation would occur in vivo. Allografts, which do not contain viable cells necessary for remodeling the tendon matrix, would need to be repopulated with fibroblasts or stem cells delivered either from the scaffold in vitro prior to graft implantation. It has been reported that mesenchymal stem cell (MSC) -seeded type I collagen sponges inserted into excised sheep patellar tendons and loaded using an ex vivo wrap-around system results in an up-regulation of chondrogenic markers such as Sox9 and Fos²ⁱ . A similar response by a cell-populated tendon allograft is anticipated following scaffold-mediated compressive loading. Moreover, the mesh-scaffold system is based on degradable poly-α-hydroxyester polymers, thus it is expected that the mechano-active scaffold will be replaced by newly formed tissue after a functional fibrocartilage interface has been formed on the graft.

This application claims the benefit of U.S. Provisional Application No. 60/994,745, filed September 21, 2007, the entire content of which is hereby incorporated by reference herein.

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Claims

What is claimed is :

1. An apparatus for inducing formation of fibrocartilage, said apparatus comprising a graft collar having a hollow central portion along a longitudinal axis, wherein an outer surface of the graft collar is wrapped with a polymer-fiber mesh configured to apply compression to the graft collar.

2. The apparatus of claim 1, wherein the graft collar has a cylindrical body.

3. The apparatus of claim 1, wherein the graft collar includes a sliced cut parallel to a longitudinal axis

4. The apparatus of claim 1, wherein the outer surface of the graft collar is wrapped in its entirety.

5. The apparatus of claim 1, wherein the polymer-fiber mesh comprises nanofibers.

6. The apparatus of claim 5, wherein the nanofibers are aligned .

7. The apparatus of claim 6, wherein the nanofibers are aligned perpendicular to the longitudinal axis of the graft collar.

8. The apparatus of claim 1, wherein the nanofibers are unaligned.

9. The apparatus of claim 1, wherein the graft collar includes at least one of the following substances: anti-infectives, antibiotics, bisphosphonate, hormones, analgesics, anti-inflammatory agents, growth factors, angiogenic factors, chemotherapeutic agents, anti-rejection agents, and RGD peptides.

10. The apparatus of claim 9, wherein the growth factors are selected from the group consisting of TGFs, BMPs, IGFs, VEGFs and PDGFs.

11. The apparatus of claim 10, wherein the TGF is TGF-β.

12. The apparatus of claim 10, wherein the BMP is BMP-2.

13. The apparatus of claim 1, wherein the graft collar includes one or more of the following types of cells: chondrocytes, osteoblasts, osteoblast-like cells and stem cells.

14. The apparatus of claim 1, wherein the graft collar includes at least one of the following: osteogenic agents, osteogenic materials, osteoinductive agents, osteoinductive materials, osteoconductive agents, osteoconductive materials and chemical factors.

15. The apparatus of claim 1, wherein the polymer-fiber mesh is selected from the group comprising aliphatic polyesters, poly (amino acids), copoly (ether-esters) , polyalkylenes oxalates, polyamides, poly (iminocarbonates) , polyorthoesters, polyoxaesters, polyamidoesters, poly(ε- caprolactone) s, polyanhydrides, polyarylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, and biopolymers, and a blend of two or more of the preceding polymers.

16. The apparatus of claim 15, wherein the polymer comprises at least one of the poly (lactic-co-glycolic acid), poly (lactide) and poly (glycolide) .

17. The apparatus of claim 1, wherein the polymer-fiber mesh is 35% poly (DL-lactide-co-glycolic acid) 85:15, 55% N, N-dimethylformamide, and 10% ethanol .

18. The apparatus of claim 1, wherein the polymer-fiber mesh comprises particulate reinforcers .

19. The apparatus of claim 18, wherein the particulate reinforcers comprise nanoparticles .

20. The apparatus of claim 1, wherein the graft collar is porous .

21. The apparatus of claim 1, wherein the graft collar is lyophilized .

22. The apparatus of claim 1, wherein the graft collar is biodegradable .

23. The apparatus of claim 1, wherein the graft collar is osteointegrative .

24. The apparatus of claim 1, wherein the graft collar is composed of microspheres.

25. The apparatus of claim 24, wherein the microspheres comprise poly ( DL-lactide-co-glycolic acid).

26. The apparatus of claim 24, wherein the microspheres comprise poly (DL-lactide-co-glycolic acid) and bioactive glass .

27. The apparatus of claim 1, wherein the apparatus further comprises a device which applies static loading to the graft collar.

28. The apparatus of claim 27, wherein the device is a clamp .

29. The apparatus of claim 1, wherein the degree of strain of said graft collar is adjusted based on polymer composition.

30. The apparatus of claim 1, wherein the degree of strain of said graft collar is adjusted based on nanofiber composition.

31. The apparatus of claim 1, wherein the graft collar comprises :

(a) a first region comprising a polymer-fiber mesh and hydrogel; and

(b) a second region adjoining the first region and comprising polymer microspheres.

32. The apparatus of claim 31, wherein the first region supports the growth and maintenance of an interfacial zone between tendon and bone, and the second region supports the growth and maintenance of bone tissue.

33. The apparatus of claim 31, wherein the graft collar includes at least one of the following substances: anti-infectives , antibiotics, bisphophonate, hormones, analgesics, anti-inflammatory agents, growth factors, angiogenic factors, chemotherapeutic agents, anti-rejections agents, and RGD peptides.

34. The apparatus of claim 31, wherein the hydrogel is photopolymerized, thermoset or chemically cross- linked .

35. The apparatus of claim 31, wherein the hydrogel is polyethylene glycol.

36. The apparatus of claim 31, wherein the polymer-fiber mesh comprises aligned fibers.

37. The apparatus of claim 31, wherein the first region contains TGF.

38. The apparatus of claim 37, wherein the TGF is TGF-β.

39. The apparatus of claim 31, wherein the first region contains chondrocytes.

40. The apparatus of claim 39, wherein the chondrocytes are BMSC-derived.

41. The apparatus of claim 31, wherein the first region contains stem cells.

42. The apparatus of claim 41, wherein the stem cells are BMSCs.

43. The apparatus of claim 31, wherein the second region contains at least one of the following growth factors: BMP, IGF, VEGF and PDGF.

44. The apparatus of claim 43, wherein the BMP is BMP-2.

45. The apparatus of claim 31, wherein the second region includes osteoblasts and/or osteoblast-like cells.

46. The apparatus of claim 45, wherein the osteoblasts and/or osteoblast like cells are BMSC-derived.

47. The apparatus of claim 31, wherein the second region includes at least one of the following: osteogenic agents, osteogenic materials, osteoinductive agents, osteoinductive materials, osteoconductive agents, osteoconductive materials and chemical factors.

48. The apparatus of claim 31, wherein the microspheres comprise poly (DL-lactide-co-glycolic acid).

49. The apparatus of claim 30, wherein the microspheres comprise poly (DL-lactide-co-glycolic acid) and bioactive glass .

50. The apparatus of claim 31, wherein the second region contains nanoparticles of calcium phosphate.

51. The apparatus of claim 50, wherein the calcium phosphate is selected from the group comprising tricalcium phosphate, hydroxyapatite and a combination thereof.

52. The apparatus of claim 31, wherein the second region contains nanoparticles of bioactive glass.

53. The apparatus of claim 31, wherein the graft collar is biodegradable.

54. The apparatus of claim 31, wherein the graft collar is osteointegrative .

55. A method for making a device for inducing formation of fibrocartilage comprising:

(a) forming a graft collar; and

(b) wrapping the graft collar prepared in step (a) with a polymer-fiber mesh, to form said device .

56. The method of claim 54, wherein said step (a) comprises :

(al) processing a plurality of microspheres;

(a2) laying the microspheres processed in step (a) in a mold; and

(a3) sintering together the microspheres in the mold above a glass transition temperature.

57. The method of claim 56, wherein the microspheres further comprise bioactive glass.

58. The method of claim 55, wherein the polymer-fiber mesh comprises nanofibers .

59. The method of claim 55, wherein the polymer-fiber mesh is selected from the group comprising aliphatic polyesters, poly (amino acids), copoly (ether-esters) , polyalkylenes oxalates, polyamides, poly ( iminocarbonates) , polyorthoesters, polyoxaesters, polyamidoesters, poly(ε- caprolactone) s, polyanhydrides, polyarylates, polyphosphazenes, polyhydroxyalkanoates, polysaccharides, and biopolymers, and a blend of two or more of the preceding polymers.

60. The method of claim 55, wherein the polymer-fiber mesh comprises at least one of the poly (lactic-co- glycolic acid), poly (lactide) and poly (glycolide) .

61. The method of claim 55, wherein the polymer-fiber mesh is 35% poly (DL-lactide-co-glycolic acid) 85:15, 55% N, N-dimethylformamide, and 10% ethanol .

62. The method of claim 55, wherein the polymer-fiber mesh comprises particulate reinforcers.

63. The method of claim 62, wherein the particulate reinforcers comprise nanoparticles .

64. The method of claim 55, wherein the nanofibers wrapped around the graft collar are perpendicular to the longitudinal axis of the graft collar.

65. The method of claim 55, further comprising incubating the polymer-fiber mesh-wrapped graft collar at a suitable temperature, time and humidity to allow sintering of the polymer-fiber mesh to the graft collar .

66. The method of claim 65, wherein the polymer-fiber- mesh-wrapped graft collar is incubated at or around 37⁰C and at or around 5% CO₂.

67. A method for inducing formation of fibrocartilage comprising enclosing a tendon within a polymer-fiber mesh-wrapped graft collar configured to apply compression to the tendon.

68. An apparatus for inducing formation of fibrocartilage, said apparatus comprising a graft collar having a hollow central portion along a longitudinal axis wherein an outer surface of the graft collar is clamped by a clamp to apply static loading to the graft collar.