WO2008154030A2 - Multi-phased scaffold for fixation of musculoskeletal soft tissue to bone - Google Patents

Abstract

Scaffold apparatuses are provided for musculoskeletal tissue engineering. In one example, a scaffold apparatus can be configured as an interference screw comprising multiple phases for fixing musculoskeletal soft tissue to bone. In another example, a degradable cell barrier is inserted between adjacent phases of the scaffold apparatus. In another example, the scaffold apparatus, is coupled to a synthetic graft for a ligament. In another example, the scaffold apparatus can comprise a graft collar and a polymer-fiber mesh, and the polymer-fiber mesh applies compressive mechanical loading to the graft collar.

Description

MULTI-PHASED SCAFFOLD FOR FIXATION OF MUSCULOSKELETAL SOFT TISSUE TO BONE

Background Throughout this application, certain publications are referenced. 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 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.

This application relates to musculoskeletal tissue engineering. For example, a scaffold apparatus is discussed below which can serve as a functional interface between multiple tissue types. Methods for preparing a multi-phase scaffold are also discussed. Some exemplary embodiments which include a soft tissue-bone interface are discussed.

As an example of a soft tissue-bone interface, the human anterior cruciate ligament (ACL) is described below. The ACL and ACL-bone interface are used in the following discussion as an example and to aid in understanding the description of the methods and apparatuses of this application. This discussion, however, is not intended to, and should not be construed to, limit the claims of this application.

The ACL consists of a band of regularly oriented, dense connective tissue that spans the junction between the femur and tibia. It participates in knee motion control and acts as a joint stabilizer, serving as the primary restraint to anterior tibial translation. The natural ACL-bone interface consists of three regions: ligament, fibrocartilage (non-mineralized and mineralized) and bone. The natural ligament to bone interface is arranged linearly from ligament to fibrocartilage and to bone. The transition results in varying cellular, chemical, and mechanical properties across the interface, and acts to minimize stress concentrations from soft tissue to bone.

The ACL is the most often injured ligament of the knee. Due to its inherently poor healing potential and limited vascularization, ACL ruptures do not heal effectively upon injury, and surgical intervention is typically needed to restore normal function to the knee.

Clinically, autogenous grafts based on either bone-patellar tendon-bone (BPTB) or hamstring-tendon (HST) grafts are often a preferred grafting system for ACL reconstruction, primarily due to a lack of alternative grafting solutions. Current ACL grafts are limited by donor site morbidity, tendonitis and arthritis. Synthetic grafts may exhibit good short term results but encounter clinical failure in long-term follow-ups, since they are unable to duplicate the mechanical strength and structural properties of human ACL tissue. ACL tears and ruptures are currently commonly repaired using semitendinosus grafts. Although semitendinosus autografts are superior, they often fail at the insertion site between the graft and the bone tunnel. One of the major causes of failure in this type of reconstruction grafts is its inability to regenerate the soft-tissue to bone interface.

Despite their distinct advantages over synthetic substitutes, autogenous grafts have a relatively high failure rate. A primary cause for the high failure rate is the lack of consistent graft integration with the subchondral bone within bone tunnels. The site of graft contact in femoral or tibial tunnels represents the weakest point mechanically in the early post-operative healing period. Therefore, success of ACL reconstructive surgery depends heavily on the extent of graft integration with bone .

ACL reconstruction based on autografts often results in loss of functional strength from an initial implantation time, followed by a gradual increase in strength that does not typically reach the original magnitude. Despite its clinical success, long term performance of autogenous ligament substitutes is dependent on a variety of factors, including structural and material properties of the graft, initial graft tension, intrarticular position of the graft, as well as fixation of the graft. These grafts typically do not achieve normal restoration of ACL morphology and knee stability.

There is often a lack of graft integration with host tissue, in particular at bony tunnels, which contributes to suboptimal clinical outcome of these grafts. The fixation sites at the tibial and femoral tunnels, instead of the isolated strength of the graft material, have been identified as mechanically weak points in the reconstructed ACL. Poor graft integration may lead to enlargement of the bone tunnels, and in turn may compromise the long term stability of the graft.

Increased emphasis has been placed on graft fixation, as post surgery rehabilitation protocols require the immediate ability to exercise full range of motion, reestablish neuromuscular function and weight bearing. During ACL reconstruction, the bone-patellar tendon-bone or hamstring- tendon graft is fixed into the tibial and femoral tunnels using a variety of fixation techniques. Fixation devices include, for example, staples, screw and washer, press fit EndoButton® devices, and interference screws. In many instances, EndoButton® devices or Mitek® Anchor devices are utilized for fixation of femoral insertions. Staples, interference screws, or interference screws combined with washers can be used to fix the graft to the tibial region.

Recently, interference screws have emerged as a standard device for graft fixation. The interference screw, about 9 mm in diameter and at least 20 mm in length, is used routinely to secure tendon to bone and bone to bone in ligament reconstruction. Surgically, the knee is flexed and the screw is inserted from the para-patellar incision into the tibial socket, and the tibial screw is inserted just underneath the joint surface. After tension is applied to the femoral graft and the knee is fully flexed, the femoral tunnel screw is inserted. This procedure has been reported to result in stiffness and fixation strength levels which are adequate for daily activities and progressive rehabilitation programs. While the use of interference screws have improved the fixation of ACL grafts, mechanical considerations and biomaterial-related issues associated with existing screw systems have limited the long term functionality of the ligament substitutes. Screw-related laceration of either the ligament substitute or bone plug suture has been reported. In some cases, tibial screw removal was necessary to reduce the pain suffered by the patient. Stress relaxation, distortion of magnetic resonance imaging, and corrosion of metallic screws have provided motivation for development of biodegradable screws based on poly-α-hydroxy acids. While lower incidence of graft laceration was reported for biodegradable screws, the highest interference fixation strength of the grafts to bone is reported to be 475 N, which is significantly lower than the attachment strength of ACL to bone. When tendon- to-bone fixation with polylactic acid-based interference screws was examined in a sheep model, intraligamentous failure was reported by 6 weeks. In addition, fixation strength is dependent on quality of bone (mineral density) and bone compression.

Two insertion zones can be found in the ACL, one at the femoral end and another located at the tibial attachment site. The ACL can attach to mineralized tissue through insertion of collagen fibrils, and there exists a gradual transition from soft tissue to bone. The femoral attachment area in the human ACL was measured to be 113 ± 27 mm² and 136 ± 33 mm² for the tibia insertion. With the exception of the mode of collagen insertion into the subchondral bone, the transition from ACL to bone is histologically similar for the femoral and tibial insertion sites.

The insertion site is comprised of four different zones: ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone. The first zone, which is the ligament proper, is composed of solitary, spindle-shaped fibroblasts aligned in rows, and embedded in parallel collagen fibril bundles of 70-150 μm in diameter. Primarily type I collagen makes up the extracellular matrix, and type III collagen, which are small reticular fibers, are located between the collagen I fibril bundles. The second zone, which is fibro-cartilaginous in nature, is composed of ovoid-shaped chondrocyte-like cells. The cells do not lie solitarily, but are aligned in rows of 3-15 cells per row. Collagen fibril bundles are not strictly parallel and much larger than those found in zone 1. Type II collagen is now found within the pericellular matrix of the chondrocytes, with the matrix still made up predominantly of type I collagen. This zone is primarily avascular, and the primary sulfated proteoglycan is aggrecan. The next zone is mineralized fibrocartilage. In this zone, chondrocytes appear more circular and hypertrophic, surrounded by larger pericellular matrix distal from the ACL. Type X collagen, a specific marker for hypertrophic chondrocytes and subsequent mineralization, is detected and found only within this zone. The interface between mineralized fibrocartilage and subjacent bone is characterized by deep inter-digitations . Increasing number of deep inter-digitations is positively correlated to increased resistance to shear and tensile forces during development of rabbit ligament insertions. The last zone is the subchondral bone and the cells present are osteoblasts, osteocytes and osteoclasts. The predominant collagen is type I and fibrocartilage-specific markers such as type II collagen are no longer present.

For bone-patellar tendon-bone grafts, bone-to-bone integration with the aid of interference screws is the primary mechanism facilitating graft fixation. Several groups have examined the process of tendon-to-bone healing.

Blickenstaff et al . (1997) evaluated the histological and biomechanical changes during the healing of a semitendinosus autograft for ACL reconstruction in a rabbit model . Graft integration occurred by the formation of an indirect tendon insertion to bone at 26 weeks. However, large differences in graft strength and stiffness remained between the normal semi-tendinosus tendon and anterior cruciate ligament after 52 weeks of implantation.

In a similar model, Grana et al . (1994) reported that graft integration within the bone tunnel occurs by an intertwining of graft and connective tissue and anchoring of connective tissue to bone by collagenous fibers and bone formation in the tunnels. The collagenous fibers have the appearance of Sharpey's fibers seen in an indirect tendon insertion.

Rodeo et al . (1993) examined tendon-to-bone healing in a canine model by transplanting digital extensor tendon into a bone tunnel within the proximal tibial metaphysis. A layer of cellular, fibrous tissue was found between the tendon and bone, and this fibrous layer matured and reorganized during the healing process. As the tendon integrated with bone through Sharpey's fibers, the strength of the interface increased between the second and the twelfth week after surgery. The progressive increase in strength was correlated with the degree of bone in growth, mineralization, and maturation of the healing tissue.

In most cases, tendon-to-bone healing with and without interference fixation does not result in the complete re- establishment of the normal transition zones of the native ACL-bone insertions. This inability to fully reproduce these structurally and functionally different regions at the junction between graft and bone is detrimental to the ability of the graft to transmit mechanical stress across the graft proper and leads to sites of stress concentration at the junction between soft tissue and bone.

Zonal variations from soft to hard tissue at the interface facilitate a gradual change in stiffness and can prevent build up of stress concentrations at the attachment sites.

The insertion zone is dominated by non-mineralized and mineralized fibrocartilage, which are tissues adept at transmitting compressive loads. Mechanical factors may be responsible for the development and maintenance of the fibrocartilagenous zone found at many of the interfaces between soft tissue and bone. The fibrocartilage zone with its expected gradual increase in stiffness appears less prone to failure.

Benjamin et al . (1991) suggested that the amount of calcified tissue in the insertion may be positively correlated to the force transmitted across the calcified zone .

Using simple histomorphometry techniques, Gao et al . determined that the thickness of the calcified fibrocartilage zone was 0.22 ± 0.7 mm and that this was not statistically different from the tibial insertion zone.

While the ligament proper is primarily subjected to tensile and torsional loads, the load profile and stress distribution at the insertion zone is more complex.

Matyas et al. (1995) combined histomorphometry with a finite element model (FEM) to correlate tissue phenotype with stress state at the medial collateral ligament (MCL) femoral insertion zone. The FEM model predicted that when the MCL is under tension, the MCL midsubstance is subjected to tension and the highest principal compressive stress is found at the interface between ligament and bone.

Calcium phosphates have been shown to modulate cell morphology, proliferation and differentiation. Calcium ions can serve as a substrate for Ca²⁺-binding proteins, and modulate the function of cytoskeleton proteins involved in cell shape maintenance.

Gregiore et al . (1987) examined human gingival fibroblasts and osteoblasts and reported that these cells underwent changes in morphology, cellular activity, and proliferation as a function of hydroxyapatite particle sizes. Culture distribution varied from a homogenous confluent monolayer to dense, asymmetric, and multi-layers as particle size varied from less than 5 μm to greater than 50 μm, and proliferation changes correlated with hydroxyapatite particles size.

Cheung et al . (1985) further observed that fibroblast mitosis is stimulated with various types of calcium- containing complexes in a concentration-dependent fashion.

Chondrocytes are also dependent on both calcium and phosphates for their function and matrix mineralization. Wuthier et al . (1993) reported that matrix vesicles in fibrocartilage consist of calcium-acidic phospholipids- phosphate complex, which are formed from actively acquired calcium ions and an elevated cytosolic phosphate concentration .

Phosphate ions have been reported to enhance matrix mineralization without regulation of protein production or cell proliferation, likely because phosphate concentration is often the limiting step in mineralization. It has been demonstrated that human foreskin fibroblasts when grown in micromass cultures and under the stimulation of lactic acid can dedifferentiate into chondrocytes and produce type II collagen.

Cheung et al . (1985) found a direct relationship between β- glycerophosphate concentrations and mineralization by both osteoblasts and fibroblasts. Increased mineralization by ligament fibroblasts is observed with increasing concentration of β-glycerophosphate, a media additive commonly used in osteoblast cultures. These reports strongly suggest the plasticity of the fibroblast response and that the de-differentiation of ligament fibroblasts is a function of mineral content in vitro.

Progressing through the four different zones which make up the native ACL insertion zone, several cell types are identified: ligament fibroblasts, chondrocytes, hypertrophic chondrocytes and osteoblasts, osteoclasts, and osteocytes. The development of in vitro multi-cell type culture systems facilitates the formation of the transition zones .

No reported studies on either the co-culture of ligament fibroblasts with osteoblasts, nor on the in vitro and in vivo regeneration of the bone-ligament interface are known.

No reported studies which examine the potential of multi- phased scaffolds in facilitating the fixation of ligament or tendon to bone are known. As the interface between graft and bone is the weakest point during the initial healing period, recent research efforts in ACL tissue engineering have concentrated on design of multi-phased scaffolds in order to promote graft integration.

Goulet et al . (2000) developed a bio-engineered ligament model, where ACL fibroblasts were added to the structure and bone plugs were used to anchor the bioengineered tissue. Fibroblasts isolated from human ACL were grown on bovine type I collagen, and the bony plugs were used to promote the anchoring of the implant within the bone tunnels. Cooper et al. (2000) and Lu et al. (2001) developed a tissue engineered ACL scaffold using biodegradable polymer fibers braided into a 3-D scaffold. This scaffold has been shown to promote the attachment and growth of rabbit ACL cells in vitro and in vivo. However, no multiphased scaffolds for human ligament-to-bone interface are known.

Summary

This application describes apparatuses for musculoskeletal tissue engineering.

A scaffold apparatus, according to one preferred embodiment, is multi-phasic and can support growth, maintenance and differentiation of multiple tissue and cell types. The multi-phasic scaffold apparatus is biomimetic, biodegradable and/or osteointegrative.

This application also provides a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said apparatus comprising two portions, wherein each portion comprises a scaffold, including first through third phases, wherein (i) 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.

This application further provides a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a first phase comprising a material which promotes growth and proliferation of fibroblasts, (ii) a second phase adjacent to the first phase comprising a material which promotes growth and proliferation of chondroblasts, and (iii) a third phase adjacent to the second phase comprising a material which promotes the growth and proliferation of osteoblasts, wherein a degradable cell barrier is inserted between the adjacent phases.

This application further provides a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a first phase comprising a material which promotes growth and proliferation of fibroblasts, (ii) a second phase adjacent to the first phase comprising a material which promotes growth and proliferation of chondroblasts , and (iii) a third phase adjacent to the second phase comprising a material which promotes the growth and proliferation of osteoblasts, wherein said first phase of the apparatus is coupled to a soft tissue graft.

This application further provides a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a graft collar and (ii) a polymer-fiber mesh coupled to the graft collar to apply compressive mechanical loading to the graft collar.

Brief Description of the Figures

Figure 1. I and II: ACL-to-bone insertion (Trichrome, 5x) III: Biomimetic Triphasic scaffold (0 7.5 x 6.5 mm).

Figure 2. Potential clinical applications of the triphasic scaffold.

Figure 3. Clinical application as a bioactive interference screw.

Figure 4. Schematic summary of experimental approach.

Figure 5. I. Multi-phased scaffold design with nanofiber mesh sintered between phases to localize cell seeding. II.

Tracking of fibroblasts (Phase A), chondrocytes (Phase B) and osteoblasts (Phase C) on the multi-phased scaffold (Day

1, 1Ox) . Phase specific cell distribution was maintained, which successfully localized fibroblasts (Fb) , chondrocytes (CH) and osteoblasts (Ob) on Phase A, B and C, respectively.

Figure 6. Jn vivo model. I. Schematic of reconstruction model. II. Reconstruction using flexor tendon graft. III. Bone tunnel formed in the femur and tibia. IV. Microsphere scaffold inserted into the two bone tunnels.

Figure 7. Experimental design for tracking the three types of implanted cell populations in vivo and determining their presence over a 4-week implantation period. Figure 8. Experimental design for interface regeneration on the tri-cultured triphasic scaffold in an intraarticular ACL reconstruction model.

Figure 9. A schematic view of a triphasic scaffold with degradable cell barrier inserted between adjacent phases.

Figure 10. A schematic view of a triphasic scaffold coupled to a synthetic graft for a ligament.

Figure 11. A schematic view of a scaffold-mesh apparatus coupled with a soft tissue 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, "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, "chondrocyte" shall mean a differentiated cell responsible for secretion of extracellular matrix of cartilage.

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.

As used herein, "graft" shall mean the device 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 fixation device" shall mean a device that is useful for affixing a tissue graft to a bone or other body surface, including but not limited to staples, interference (screws with or without washers), press fit EndoButton® devices and Mitek® Anchor devices .

As used herein, "interference screw" shall mean a type of graft fixation device which anchors a flexible transplant like a tendon or a ligament in an opening in a bone. The screw generally has a screw body, a head at one end of said screw body and a penetrating end at an opposite end of said screw body. The device may be used in, for example, anterior cruciate ligament surgery. The device may be metallic or bioabsorbable and may include, but is not limited to, titanium cannulated interference screws, PoIy- L-Lactide (PLLA) interference screws, etc. As used herein, "matrix" shall mean a three-dimensional structure fabricated from biomaterials . The biomaterials can be biologically-derived or synthetic.

As used herein, "nanofiber" shall mean fibers with diameters no more than 1000 nanometers.

As used herein, "nanofiber mesh" shall mean a flexible netting of nanofibers, oriented such that at least some of the nanofibers are not parallel to others of the nanofibers .

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, "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, "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.

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 subject matter as set forth in the claims which follow thereafter.

In an exemplary embodiment (Fig. 2), a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, comprises two portions, each portion including first through third phases, wherein (i) the first phase of the scaffold 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 the exemplary embodiment of Fig. 2, the two portions (for example, portions 31 and 32) encase respective portions of a soft tissue graft on all sides (for example, halves 31a and 31 b) of the scaffold apparatus.

In another embodiment (Fig. 3), two portions combine to encase a portion (35) of a soft tissue graft on all sides.

In another embodiment (for example, Fig. 2), the first phase is exposed to the joint cavit. In another embodiment (for example Fig. 2) the second phase contacts articular cartilage. In another embodiment (for example, Fig. 2), the third phase is encased in bone. In another embodiment, the interference screw is biomimetic. In another embodiment, the interference screw is biodegradable. In another embodiment, the interference screw is osteointegrative . In another embodiment, a degradable cell barrier is inserted between the adjacent phases. In another embodiment, the degradable cell barrier comprises a nanofiber mesh. In another embodiment, the nanofiber mesh comprises polylactide-co-glycolide (PLGA) . In another embodiment, the nanofiber mesh is electrospun.

The application further provides an interference apparatus comprising a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said apparatus comprising two portions, wherein each portion comprises a scaffold, including first through third phases, wherein (i) 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 one embodiment, the interference apparatus is an interference screw.

In another exemplary embodiment (Fig. 9), a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, comprises (i) a first phase (91) comprising a material which promotes growth and proliferation of fibroblasts, (ii) a second phase (92) adjacent to the first phase comprising a material which promotes growth and proliferation of chondroblasts, and (iii) a third phase (93) adjacent to the second phase (92) comprising a material which promotes the growth and proliferation of osteoblasts, wherein a degradable cell barrier (94a, 94b) is inserted between adjacent phases of the scaffold apparatus.

In one embodiment, the first phase is for supporting growth and maintenance of soft tissue, the second phase is for supporting the growth and maintenance of fibrocartilage, and the third phase is for supporting the growth and maintenance of bone tissue. In another embodiment, the first phase is seeded with at least one of fibroblasts and stem cells. In another embodiment, the stem cells are mesenchymal stem cells. In another embodiment, the first phase includes fiber mesh. In another embodiment, the fiber mesh is electrospun.

In another embodiment, the second phase is seeded with at least one of chondrocytes and stem cells. In another embodiment, the stem cells are mesenchymal stem cells. In another embodiment, the third phase is seeded with at least one of osteoblasts, osteoblast-like cells, and stem cells. In another embodiment, the stem cells are mesenchymal stem cells. In another embodiment, said third phase contains at least one of osteogenic agents, osteogenic materials, osteoinductive agents, osteoinductive materials, osteoconductive agents, osteoconductive materials, growth factors and chemical factors.

In one embodiment, said scaffold apparatus is integrated in a graft collar. In another embodiment, said graft collar is adapted for hamstring tendon-to-bone healing. In another embodiment, said first phase is seeded with human hamstring tendon fibroblasts. In another embodiment, said graft collar is adapted for peridontal ligament repair. In another embodiment, said graft collar is adapted for spinal repair. In another embodiment, at least one of said first phase and said third phase is seeded with one or more agents by using a microfluidic system.

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

In one embodiment, the first phase comprises polymer and the third phase comprises one of bioactive glass and calcium phosphate. In another embodiment, the calcium phosphate is selected from a group comprising tricalcium phosphate, hydroxyapatite, and a combination thereof. In another embodiment, the polymer is selected from a 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 comprises at least one of poly (lactide-co- glycolide) , poly ( lactide) and poly (glycolide) .

In one embodiment, the apparatus is biomimetic. In another embodiment, the apparatus is biodegradable. In another embodiment, the apparatus is osteointegrative.

In one embodiment, the apparatus additionally include one or more agents selected from a group comprising antiinfectives , hormones, analgesics, anti-inflammatory agents, growth factors, chemotherapeutic agents, anti- rejection agents and RGD peptides. In one embodiment, the degradable cell barrier is a nanofiber mesh. In another embodiment, the nanofiber mesh comprises polylactide-co-glycolide (PLGA) . In another embodiment, the nanofiber mesh is electrospun.

In another exemplary embodiment, a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, comprises (i) a first phase (101) comprising a material which promotes growth and proliferation of fibroblasts, (ii) a second phase (102) adjacent to the first phase comprising a material which promotes growth and proliferation of chondroblasts , and (iii) a third phase (103) adjacent to the second phase comprising a material which promotes the growth and proliferation of osteoblasts, wherein said first phase (101) of the apparatus is coupled to a soft tissue graft (104). In one embodiment, the soft tissue graft is a graft of a ligament and the ligament is an anterior cruciate ligament.

A scaffold apparatus, according to one preferred embodiment, is multi-phasic, including phases A, B, and C, and preferably can support growth, maintenance and differentiation of multiple tissue and cell types.

The Phase A comprises a first material adapted for integration and growth of a second tissue type seeded with a second type of cells (for example, fibroblasts, chondrocytes, stem cells, etc.). Phase A may include a composite of materials, including, but not limited to, microspheres, a fiber mesh, degradable polymers, etc. Phase C comprises a second material adapted for integration and growth (for example, by including one or more osteogenic agents, osteogenic materials, osteoinductive agents, osteoinductive materials, osteoconductive agents, osteoconductive materials, growth factors, chemical factors, etc.) of a first tissue type and is seeded with a first type of cells (for example, osteoblasts, osteoblast- like cells, stem cells, etc.). The material of the first phase may include, but is not limited to, microspheres, foams, sponges and any other three dimensional (3-D) scaffold construct consisting of polymer and/or ceramic. Polymers may include, but is not restricted to, any biodegradable polymer such as any of the poly- (α-hydroxy acids), or natural polymers such as silk, collagen, or chitosan. Ceramics may include but are not limited to bioactive glass, hydroxyapatite, beta tricalcium phosphate, or any other calcium phosphate material.

Phase B is an interfacial zone between the first and third phases. In one embodiment, Phase B is seeded with choondrocytes, such that a fibrocartilage interface can be formed and maintained with interactions between these three cell types.

The multi-phasic scaffold apparatus preferably is preferably biomimetic, biodegradable (that is, each phase is degradable) and/or osteointegrative.

The scaffold may provide a functional interface between multiple tissue types (for example, soft tissue and bone). Figure 2 shows an example of a multi-phased scaffold apparatus in the form of a graft collar comprising phase A, phase B, and phase C. It should be apparent to one skilled in the art that although the apparatus shown in Figure 2 has three phases, the apparatus can be integrated in a scaffold with four or more phases.

The scaffold apparatus can promote growth and maintenance of multiple tissue types. The scaffold may support growth, maintenance and differentiation of multiple tissue and cell types. The multi-phased scaffold may mimic the inhomogeneous properties of the insertion zone between soft tissue and bone, resulting in desired growth, phenotypic expression, and interactions between relevant cell types.

The phases of the scaffold may be inhomogeneous in properties. The phases may have zonal differences in mineral content and matrix morphology designed to mimic the tissue-bone interface and to facilitate the growth and maintenance of different tissues. The phases may differ in morphology. For example, phase A can include a porous fibrous mesh, while phases B and C include microspheres. According to another embodiment, the scaffold may include a composite of microspheres and a fiber mesh.

The scaffold preferably includes multiple phases. According to one embodiment, one phase (for example, phase A) supports growth and maintenance of soft tissue, another phase (for example, Phase B) is an interfacial zone between the first and second phases and another phase (for example, phase C) supports growth and maintenance of bone. Phase A for supporting growth and maintenance of the soft tissue may be seeded with at least one of fibroblasts, chondrocytes and stem cells. Phase C for supporting growth and maintenance of the bone may be seeded with at least one of osteoblasts, osteoblast-like cells and stem cells. Phase C can contain at least one of osteogenic agents, osteogenic materials, osteoinductive agents, osteoinductive materials, osteoconductive agents, osteoconductive materials, growth factors and chemical factors.

Further, at least one of said Phase A and Phase C may be seeded with one or more agents by using a microfluidic system.

The scaffold may include a composite of microspheres and a fiber mesh. The fiber mesh may be a degradable polymer. For example, the first phase may include a fiber mesh. The fiber mesh of the first phase and the microspheres of the third phase may be sintered together. The fiber mesh may be electrospun.

The mesh can include one or more desired agents and/or compound. For example, at least one of bioactive agents and peptides may coat the surface of the mesh. The bioactive agents and peptides can enhance differentiation, proliferation and attachment of cells and specific cell types. Also or alternatively, at least one of bioactive agents and peptides can directly be incorporated into the mesh.

According to one embodiment, the scaffold may include multiple phases joined by a gradient of properties. The multiple phases joined by the gradient of properties may be processed through one or more sintering stages. The gradient of properties across the multiple phases of the scaffold can include mechanical properties, chemical properties, mineral content, structural properties, porosity and/or geometry.

The scaffold apparatus can include plural phases of microspheres. For example, Phase A of the microspheres can comprise polymer and Phase C of the microspheres can comprise one of bioactive glass and calcium phosphate. Varying concentrations of calcium phosphate can be incorporated into the microspheres. The calcium phosphate can be selected from a group comprising tricalcium phosphate, hydroxyapatite, and a combination thereof. The polymer can be selected from a 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 . The polymer can comprise at least one of poly(lactide-co-glycolide) , poly (lactide) and poly (glycolide) .

The microspheres may comprise one or more of CaP, bioactive glass, polymer, etc. The microspheres may be processed through one or more sintering stages.

The microspheres may comprise one or more desired agents or compounds. For example, at least one of bioactive agents and peptides may coat the surface of at least some of the microspheres. The bioactive agents and peptides can enhance at least one of differentiation, proliferation and attachment of cells and specific cell types. Also or alternatively, at least one of bioactive agents and peptides can directly be incorporated into at least some of the microspheres . The microspheres can additionally include one or more agents selected from a group comprising anti-infectives , hormones, analgesics, anti-inflammatory agents, growth factors, chemotherapeutic agents, anti- rejection agents and RGD peptides.

The apparatus is preferably biomimetic, biodegradable and/or osteointegrative .

According to one exemplary embodiment, the apparatus may be integrated in a graft fixation device. The graft fixation device may be used, for example, for graft fixation at the bone tunnels during anterior cruciate ligament reconstruction .

According to another embodiment, the apparatus may be integrated in an interference screw.

In addition, the scaffold apparatus, according to another exemplary embodiment, may be integrated in a graft collar.

The graft collar has many applications. For example, the graft collar may be adapted for hamstring tendon-to-bone healing. As another example, the graft collar can be adapted for peridontal ligament repair. Further, the graft collar may be adapted for spinal repair. This application further provides a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a graft collar and (ii) a polymer-fiber mesh coupled to the graft collar to apply compressive mechanical loading to the graft collar.

In one embodiment, the polymer-fiber mesh wraps around the graft collar. In another embodiment, the surface of the graft collar is wrapped in its entirety. (Figure 11)

In one embodiment, the graft collar is bi-phasic. In another embodiment, the polymer-fiber mesh comprises nanofibers. In yet another embodiment, the nanofiber mesh comprises polylactide-co-glycolide (PLGA) . In yet another embodiment, the nanofiber mesh is electrospun.

According to one embodiment, the scaffold apparatus is coupled to a soft tissue graft. (Figure 11) According to another embodiment, the soft tissue graft is graft for a ligament. In another embodiment, the ligament is an anterior cruciate ligament.

In another exemplary embodiment (Fig. 11), an interference apparatus comprising a scaffold apparatus is provided for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a graft collar 101 and (ii) a polymer-fiber mesh (105) coupled to the graft collar to apply compressive mechanical loading to the graft collar. In one embodiment, the interference apparatus is an interference screw. In one embodiment, the polymer-fiber mesh wraps around the graft collar. In another embodiment, an outer surface of the graft collar is wrapped in its entirety by the polymer- fiber mesh.

In one embodiment, the graft collar is bi-phasic. In another embodiment, the bi-phasic graft collar includes a first phase comprising a material which promotes growth and proliferation of fibroblasts, and a second phase adjacent to the first phase comprising a material which promotes the growth and proliferation of osteoblasts.

In another embodiment, the polymer-fiber mesh comprises nanofibers. In yet another embodiment, the nanofiber mesh comprises polylactide-co-glycolide (PLGA). In yet another embodiment, the nanofiber mesh is electrospun.

In one embodiment, the scaffold apparatus is coupled to a soft tissue graft. In another embodiment, the soft tissue graft is a graft for a ligament of the subject. In yet another embodiment, the ligament is an anterior cruciate ligament of the subject.

This application further provides a graft-fixation apparatus comprising a scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject comprising (i) a graft collar and (ii) a polymer-fiber mesh coupled to the graft collar to apply compressive mechanical loading to the graft collar.

In an exemplary embodiment, the graft fixation apparatus is an interference screw. This application further provides a scaffold apparatus for fixing musculoskeletal soft tissue to bone, said scaffold apparatus being configured to apply mechanical loading to a soft tissue graft to promote regeneration of a fibrocartilage interface between said soft tissue and said bone.

In one embodiment, the scaffold apparatus comprises a nanofiber mesh configured to apply said mechanical loading to said soft tissue graft.

In another embodiment, mechanical loading is applied by said scaffold apparatus dynamically or intermittently to said soft tissue graft .

In another embodiment, mechanical loading is applied by said scaffold apparatus statically to promote regeneration of a fibrocartilage interface between said soft tissue and said bone in a subject.

In another embodiment, the scaffold apparatus comprises a material that promotes growth and proliferation of chondroblasts .

In another embodiment, the scaffold apparatus comprises first and second phases, wherein (i) the first phase comprises a material that promotes growth and proliferation of chondroblasts, (ii) the second phase adjacent to the first phase comprises a material that promotes growth and proliferation of osteoblasts.

In yet another embodiment, the scaffold apparatus comprises first, second and third phases, wherein (i) the first phase comprises a material that promotes growth and proliferation of fibroblasts, (ii) the second phase adjacent to the first phase comprises a material that promotes growth and proliferation of chondroblasts , and (iii) the third phase adjacent to the second phase comprises a material that promotes the growth and proliferation of osteoblasts.

The specific embodiments and examples described herein are illustrative, and many variations can be introduced on these embodiments and examples without departing from the spirit of the disclosure or from the scope of the appended claims. Elements and/or features of different illustrative embodiments and/or examples 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 subject matter but is not intended to, and should not be construed to, limit in any way the claims which follow thereafter.

Experimental Details

Clinically, the hamstring tendon graft is mechanically fixed extra-articularly by looping the graft around a transfemoral pin in the femoral bone tunnel, while a screw with a washer or a staple is used to fix the graft to the tibia. Interference screws have been used in the bone tunnel, but with limited success due to graft laceration and poor fixation strength. With mechanical fixation, the fibrocartilage interface is not regenerated after ACL reconstruction. A non-physiologic, fibrovascular scar tissue is instead formed within the bone tunnel as part of the healing process. The presence of this partially mineralized layer within the tunnel renders the graft-bone fixation site the weakest point mechanically [7]. This problem is exacerbated by the active lifestyle of ACL injury patients (15-35 years old), which necessitates higher fixation strength and expedited healing. Thus, graft-to-bone fixation remains a significant clinical problem.

The subject approach to addressing the challenge of biological fixation is original and represents a significant departure from the conventional focus on tendon-to-bone healing within the bone tunnel. It is emphasized here that the native anatomical fibrocartilage interface is orthogonal to the subchondral bone and continuous with surrounding articular cartilage. In addition, the neo-fibrocartilage formed within the bone tunnel represents the mechanical weak link for tendon-to- bone integration. Biological fixation therefore requires that the anatomical fibrocartilage insertion is regenerated between graft and bone, accompanied by the complete mineralization of the tendon within the bone tunnel.

It is envisioned that the triphasic scaffold may be used clinically as either as a graft collar or an interference screw during ACL reconstruction surgery. The ultimate goal is to facilitate the formation of the anatomic fibrocartilage interface directly on the soft tissue graft. As a graft collar, the scaffold will be fabricated as a hollow cylinder through which the ACL graft can be inserted. As shown in Fig. 2, the collar can be sutured or secured to the ends of the tendon graft. Fixation is achieved by inserting the collar-graft complex into the bone tunnel, with Phase C positioned inside the bone tunnel, Phase B flush with articular cartilage, and only Phase A directly exposed to the joint cavity. It is anticipated that the designed heterogeneity and optimized interaction between MSC-derived cells will induce the formation of a fibrocartilage interface directly onto the graft. Graft integration within the bone tunnel will be facilitated by Phase C, the osteointegrative polymer- ceramic composite, and with the eventual addition of growth factors (e.g., bone morphogenetic proteins), which will induce osteointegration and mineralization of the tendon graft within the bone tunnel.

For use as an interference screw, the triphasic scaffold can be fabricated as matching portions of the hollow cylinder, with each portion containing the three scaffold phases. As shown in Fig. 3, the two matching portions will encase the soft tissue graft on all sides. The relative position of each phase of the triphasic scaffold would be in the anatomical position, i.e., with Phase A (soft tissue) exposed to the joint cavity, Phase B

(fibrocartilage interface) flush with articular cartilage, and Phase C (bone) encased within the bone tunnel. There are several advantages to this novel interference screw design: 1) the biomimetic triphasic screw design enables the regeneration of the relevant tissue types on the scaffold system, 2) the partitioned design permits the application of mechanical loading to the graft, which has been known to induce fibrocartilage formation, and 3) the tendon graft is in contact with the triphasic scaffold on all sides. Any applied mechanical and chemical stimulation would be uniformly experienced by the graft.

The optimal outcome scenario post-degradation of the screw or graft collar is to have a completely mineralized tissue within the bone tunnel, accompanied by the formation of a physiologically equivalent fibrocartilage insertion directly outside the bone.

For ligament tissue engineering, the triphasic scaffold may be coupled with synthetic grafts for ACL replacement. The future design of ACL replacement grafts must take into consideration the integration of the graft with bone. In this integrative ACL prosthesis design, the ACL prosthesis will contain three regions, a bony end consisting of Phase C, followed by Phase B, then by polymer fiber-based ACL portion. The triphasic scaffold can also be incorporated into any existing ACL prosthesis design, as the soft tissue graft shown in Figs. 2 and 3 can easily be replaced by any synthetic ACL reconstruction scaffold. For example, in the case of a degradable polymer-based ACL prosthesis [13] , the triphasic scaffold can be sintered onto the polymer scaffold and implanted for ACL reconstruction.

One common feature in the above examples of clinical application is the focus on engineering soft tissue-to-bone integration ex vivo, which would reduce the complexity of graft reconstruction to just bone-to-bone integration in vivo. This is more feasible clinically as it is much more difficult to integrate soft tissue with bone compared to bone-to-bone integration.

Extensive characterization of the chemical and mechanical properties of the interface [8-10] has been conducted and novel in vitro co-culture [5] and tri-culture [8] models have been developed to examine the role of cell-cell interactions in interface formation. In combination with knowledge of in vivo models of tendon-bone healing [11 , 12] , there is a solid foundation and clear rationale for the described approach.

From a broader impact perspective, this approach is also unique in that previous tissue engineering methods have focused predominantly on the design of a single type of tissue (e.g., only ligament or bone) on a scaffold with uniform properties, when the application may have involved more than one tissue type. Moreover, the novel scaffold design and co-culture methods described here can be applied to treat other clinical conditions (e.g., rotator cuff, osteoarthritis) and will enable the design of a new generation of integrative fixation devices. The described studies will also provide fundamental insights into the mechanism of soft tissue-bone interface regeneration. Clinical feasibility of the scaffold was determined by testing the hypothesis that the biomimetic matrix heterogeneity engineered on the triphasic scaffold will be maintained in vivo in an intra-articular model. A summary schematic of this research approach is presented below in Fig. 4. It was determined that modifications to the scaffold design were necessary to achieve distinct cell and matrix regions in vivo.

Scaffold Design Optimization

Based on the outcomes of in vitro and in vivo co-culture and tri-culture experiments, the multi-phased scaffold design has been improved upon, with the goal of localizing the interface-relevant cells within Phase B without compromising the scaffold design requirements (higher porosity and pore diameter) necessary for Phase A. Specifically, a degradable cell barrier between adjacent phases has been incorporated. This barrier is based on a polylactide-co-glycolide (PLGA) electrospun nanofiber mesh (Fig. 5-1), which, based on porosimetry analysis, has an average pore diameter of 5.2±0.9 μm. This nanofiber mesh will prevent unwanted cell migration and gel infiltration into Phase A or Phase C. Cell localization is important as 3-D co-culture results demonstrate that cell-specific distribution is required for the development of the biomimetic, controlled matrix distribution on the multi- phased scaffold.

Preliminary cell tracking results of fibroblasts and osteoblasts tri-cultured with chondrocytes loaded in hydrogel for 24 hours on the modified scaffold are shown in Fig. 5-11. Fibroblasts, chondrocytes, and osteoblasts were detected only in their respective phases as determined by fluorescence confocal microscopy. The nanofiber mesh served as an effective barrier to gel infiltration and unwanted cell cross-migration. It is anticipated that the mesh will degrade over time, having ensured the establishment of cell-specific regions in tri-culture.

Mechano-Actuve Scaffold Induces Remodeling and Expression of Fibrocartilage Markers on Tendon Grafts

Based on the hypothesis that mechanical loading, in addition to multi-phasic scaffold design and heterotypic cellular interactions, will be required for interface generation, this experiment focuses on the design and evaluation of a novel mechano-active scaffold that is capable of applying compression to tendon grafts and inducing metaplasia of tendon into fibrocartilage. Specifically the novel scaffold system combines a degradable graft collar with nanofiber meshes fabricated from poly (latic-co-glycolic acid) (PLGA) . One objective of the experiment is to characterize the contractile properties of the nanofiber mesh as well as the mesh and scaffold complex. A second objective of the experiment is to evaluate the effect of scaffold induced compression on fibrocartilage development on tendon graft, focusing on matrix remodeling and the development of fibrocartilage- related markers .

First, aligned nanofiber meshes were fabricated by electrospining. A viscous polymer solution consisting of

35% poly(D,L-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 were obtained using an aluminum drum with an outer diameter of 10.2 cm rotating with a surface velocity of 20 m/2. 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). Fiber morphology, diameter and alignment of the as-fabricated mesh samples were analyzed using scanning electron microscopy (SEM) . 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 gkv.

The nanofiber mesh exhibited a high degree of alignment with an average fiber diameter of 0.9 ± 0.4 μm.

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. 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.

The said nanofiber is then wrapped around a tendon graft collar based on a sintered microsphere scaffold fabricated following published methods. In addition, patellar tendon grafts were isolated from neonatal bovine tibiofemoral joints obtained from a local abattoir. The compression of the nanofiber mesh, the graft collar scaffold with nanofiber mesh, the tendon with nanofiber mesh, and finally the tendon graft with the graft collar scaffold and the nanofiber mesh were evaluated. Further, the effects of compression on graft cellularity, organization, matrix content, and cell phenotype were evaluated.

It was found that complex of nanofiber mesh and graft collar was able to apply a physiological range of compressive loading on the tendons. Moreover scaffold- mediated compression promoted matrix remodeling, maintained graft glycosaminoglycan content and induced gene expression for fibrocartilage markers, including type II collagen, aggrecan core protein, and TGF-β3.

Mesenchymal Stem Cells and Differentiation into Interface- Relevant Cell Populations

The experiments will also utilize fibroblasts, chondrocytes, and osteoblasts derived from adult mesenchymal stem cells (MSCs) originated from human bone marrow. The MSCs are chosen because they are ideal for tissue engineering applications. These cells can be harvested from the patient prior to surgery, expanded, and pre-differentiated into desired cell types, and then seeded onto 3-D scaffolds. In addition to being autologous, MSCs can differentiate into fibroblasts [14,15], chondrocytes [14, 16] , and osteoblasts [ 14 , 17] which are the relevant cell types found at the soft tissue-bone interface. This versatility will simplify the tissue harvest process to a single procedure instead of the normal three required to obtain the three types of cells. Successful implementation of MSC-derived cells will significantly enhance the clinical feasibility and translational potential of the triphasic scaffold.

Specifically, MSCs purchased from Cambrex will be pre- differentiated into fibroblasts (Fb) , chondrocytes (Ch) , and osteoblasts (Ob) based on well-established protocols. The fibrσgenic media will contain 1 ng/mL of basic fibroblast growth factor, 5 ng/mL of transforming growth factor-beta (TGF-β3) and 50 μg/ml of L-Ascorbic Acid-2- Phosphate (AA) [15,18] . The chondrogenie media will contain 5 ng/mL TGF-β3, 0.1 mM non-essential amino acids, 50 μg/ml AA, 10 nM dexamethasone (Dex) , and 5 μg/ml of insulinlβ] . The osteogenic media will contain 10 nM Dex, 10 mM of β- glycerophosphate, and 50 μg/ml AA[17] .

Intra-Articular ACL Reconstruction Model The study will use male athymic rats (Charles River Laboratories, mean weight 300 grams) to demonstrate unilateral ACL reconstruction [6] using a flexor digitorum longus tendon graft from the ipsilateral limb, as shown in Fig 6-1. The rats will be anesthetized with a mixture of ketamine hydrochloride 80mg/kg and xylazine 5mg/kg, administered intraperitoneally. Ampicillin 25 mg/kg subcutaneous injection will be used for antibiotic prophylaxis. After appropriate anesthesia, the rat will be prepared for sterile surgery. The flexor digitorum longus tendon will be harvested via a longitudinal incision made on the medial aspect of the distal leg and ankle. The full length of the flexor digitorum longus tendon (average length 20mm) will be harvested. An incision will be made over the rat knee, and a lateral parapatellar arthrotomy will be performed. The ACL, PCL, MCL, and LCL will be excised. Sectioning these ligaments causes minimal trauma to the knee and is not expected to affect the overall biologic response that will already occur from the knee arthrotomy. Using a needle with outer diameter of 2.5 mm, a bone tunnel will be made in the proximal tibia and the distal femur, entering the joint at the attachment sites of the ACL. We will measure the total length of the femur- tendon-tibia complex to determine the amount of displacement required to apply 1% and 10% strain.

The triphasic scaffold fabricated in the form of the graft collar will be used for implantation. After incorporating the graft collar onto the flexor tendon graft, the graft- scaffold complex will be passed through the bone tunnels to replace the ACL. Both ends of the grafted tendon will be secured to the surrounding periosteum at the extra- articular tunnel exit sites at the distal femur and well as proximal tibia using 4-0 Ethibond suture. Post-operative activity will be controlled using an external fixator that we have designed and fabricated for rat knees [6] .

Cell Tracking In Vivo

A further objective of these experiments is to track the three types of implanted cell populations in vivo and to determine their presence over a 4-week implantation period. Cell Labeling - After pre-differentiation of MSCs into Fb, Ch, and Ob, cells will be seeded based on the optimal cell seeding density (cells/cm³) on their designated phase of the triphasic scaffold based on results from Phase I. As shown in Fig. 5, the Fb will be pre-labeled with Vybrant DiD dye

(green) , Ch with Vybrant DiO (red) , and Ob with Vybrant DiI

(yellow). All dyes can be purchased from Molecular Probes.

The pre-label cells will be seeded on their respective phases of the triphasic scaffold collar, and tricultured for 2 days following established protocols [4] . As summarized in Fig. 7, the scaffold (n=3 per group) will be implanted for 1, 2, and 4 weeks, and the presence of the cells will be tracked over time and correlated to the formation of fibrocartilage tissue on the triphasic scaffold. At each time point, the scaffold collar+graft complex will be excised and cryosectioned for fluorescence microscopy (cell imaging) and histological analysis

(fibrocartilage formation). Specifically, development of interface-relevant markers will be determined: proteoglycan and mineral deposition, as well as immunohistochemistry for collagen types I, II, III, IX, and X. Acellular scaffolds and unoperated contralateral insertion sites will serve as additional controls. A total of 45 animals (15 per time point) will be needed for this experiment.

In Vivo Evaluation for interface Regeneration

This experiment further focuses on interface regeneration on the tri-cultured, triphasic scaffold in an intra- articular ACL reconstruction model. Specifically, MSC- derived fibroblasts, chondrocytes and osteoblasts will be seeded on their respective phases of the tri-phasic scaffold, and cultured in vitro for 2 days [4]. The scaffold will be implanted following the methods described in Section E.3 and the experimental design outlined in Fig. 8. Each animal will receive one scaffold (randomly selected) and will be sacrificed at 4, 8, and 12 weeks. Outcomes will be evaluated using histomorphometric, micro- CT, and biomechanical analyses. Quantitative histomorphometric measurements will be made using the Bioquant Image Analysis system (R&M Biometrics, Inc., Nashville, TN) available in the Analytical Microscopy Laboratory (Director, Dr. S. Doty) . The implant evaluation methods successfully utilized in the previously described in vivo studies will also be used here. Specifically, the development of a fibrocartilage-like tissue and interfacial markers (n=3, see Section E.4) will be determine. Scaffold mechanical properties (n=6) will also be determined over time. Mineralization (total bone mineral content, bone volume fraction, and mineral distribution) will be analyzed by micro-CT prior to mechanical testing, so an additional sample is not needed. A push-out test [19] will be performed on week 12 samples (tri-culture only, n=6) in order to determine the osteointegration potential of Phase C within the bone tunnel. A total of 168 male athymic nude rats (54 animals each for weeks 4 & 8, and 60 animals for week 12) will be used in this experiment.

Expected Outcomes

It is anticipated that for the in vivo cell tracking experiment, all three cell types will persist at the implantation site for up to 4 weeks, and that the seeded chondrocytes will contribute to the formation of a fibrocartilage-like region on the interface phase (Phase B) of the triphasic scaffold. For the in vivo evaluation of interface regeneration experiment, it is expected that an interface-like region will form on the scaffold post-ACL reconstruction. In these experiments, the formation of a fibrocartilage-like tissue on the interface phase of the triphasic scaffold has been focused on for several reasons. The long term role of the scaffold as a graft collar is to induce fibrocartilage formation on the reconstructed graft. After establishing the stability of the triphasic scaffold in the intra-articular model, and the viability of application of controlled mechanical stimulation to induce fibrocartilage formation on the graft, the next stage of the project will focus on the application of controlled chemical stimulation to induce fibrocartilage formation on the graft. For example, phase- specific growth factor delivery can be incorporated to provide chemical stimuli for interface regeneration. It is however critical to first establish the feasibility of the tri-culture, triphasic scaffold in a physiologically relevant intra-articular model.

References

1. Johnson RJ, Int J Sports Med 3:71-79, 1982.

2. Beynnon BD et al., J Bone Joint Surg Am 84-A: 1503-1513 , 2002.

3. Woo SL et al., J Orthop Res 1:22-29, 1983.

4. Spalazzi JP et al . , Tissue Engineering, 12(12) :3497- 3508, 2006.

5. Wang IE and Lu HH, ORS, 2005.

6. Rodeo et al., ORS, 2006.

7. Kurosaka M et al . , Am J Sports Med 15:225-229, 1987.

8. Wang IE et al . , J Orthop Res 24 (8) : 1745-1755, 2006.

9. Spalazzi JP et al., J Orthop Res 24 (10) : 2001-2010 , 2006.

10. Moffat KL et al., ISTL-V, 2005.

11. Rodeo SA et al . , J Bone Joint Surg Am 75:1795-1803, 1993.

12. Kawamura S et al., ORS 2005.

13. Cooper JA et al . , Biomaterials , 26 (13 ): 1523-1532 , 2005. 14. Pittenger MF et al., Science 284:143-147, 1999.

15. Moreau JE et al . , J Orthop Res 23:164-174, 2005.

16. Meinel L et al., Biotechnol Bioeng 88:379-391, 2004.

17. Mauney JR et al . , Biomaterials 26:3173-3185, 2005.

18. Altman GH et al . , Biomaterials 23:4131-4141, 2002.

19. Knowles JC et al . , Biomaterials 13 (S) : 491-496, 1992.

20. Abate, J.A., Fadale, P. D., Hulstyn, M.J. & Walsh, W. R., "Initial fixation strength of polylactic acid interference screws in anterior cruciate ligament reconstruction," Arthroscopy 14, 278-284 (1998).

21. Allum, R. L., "BASK Instructional Lecture 1: graft selection in anterior cruciate ligament reconstruction," Knee 8, 69-72 (2001).

22. Altman, G. H., et al . "Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering, " Journal of Biomechanical Engineering 124, 742-749 (2002).

23. Altman, G. H., et al . , "Silk matrix for tissue engineered anterior cruciate ligaments," Biomaterials 23, 4131-4141 (2002) . 24. American Academy of Orthopaedic Surgeons, "Arthoplasty and Total Joint Replacement Procedures: United States 1990 to 1997," (Report, United States) (1997) .

25. Anderson, K. et al . , "Augmentation of tendon healing in an intraarticular bone tunnel with use of a bone growth factor," Am. J Sports Med. 29, 689-698 (2001).

26. Batycky, R. P., Hanes , J., Langer, R. & Edwards, D. A. , "A theoretical model of erosion and macromolecular drug release from biodegrading microspheres," J. Pharmaceutical Sciences 86, 1464-1477 (1997).

27. Bellincampi, L. D., Closkey, R. F., Prasad, R., Zawadsky, J. P. & Dunn, M. G. , "Viability of fibroblast- seeded ligament analogs after autogenous implantation," J. Orthop. Res. 16, 414-420 (1998).

28. Benjamin, M., Evans, E.J., Rao, R. D., Findlay, J. A. & Pemberton, D.J., "Quantitative differences in the histology of the attachment zones of the meniscal horns in the knee joint of man," J. Anat . 177, 127-134 (1991) .

29. Berg, E. E., "Autograft bone-patella tendon-bone plug comminution with loss of ligament fixation and stability," Arthroscopy 12, 232-235 (1996).

30. Beynnon, B. et al . , "A sagittal plane model of the knee and cruciate ligaments with application of a sensitivity analysis," J. Biomech. Eng. 118, 227-239 (1996) . 31. Beynnon, B. D. et al., "The effect of functional knee bracing on the anterior cruciate ligament in the weightbearing and nonweightbearing knee," Am. J. Sports Med. 25, 353-359 (1997).

32. Blickenstaff , K. R., Grana, W.A. & EgIe, D., "Analysis of a semitendinosus autograft in a rabbit model," Am. J. Sports Med. 25, 554-559 (1997) .

33. Bolton, CW. & Bruchman, W. C, "The GORE-TEX expanded polytetrafluoroethylene prosthetic ligament. An in vitro and in vivo evaluation," Clin. Orthop . 202-213 (1985) .

34. Borden, M., Attawia, M., Khan, Y. & Laurencin, CT. Tissue engineered microsphere-based matrices for bone repair: design and evaluation. Biomaterials 23, 551- 559 (2002) .

35. Bonfield, W., "Composites for bone replacement," J. Biomed. Eng. 10, 522-526 (1988).

36. Boskey, A. L. et al . , "The mechanism of beta- glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures," J. Bone Min. Res. 11, 1694-1702 (1996).

37. Brand, J., Jr., Weiler, A., Caborn, D.N. , Brown, CH., Jr. Sc Johnson, D. L. , "Graft fixation in cruciate ligament reconstruction," Am. J. Sports Med. 28, 761- 774 (2000). 38. Brody, G. A., Eisinger, M., Arnoczky, S. P. & Warren, R. F., "In vitro fibroblast seeding of prosthetic anterior cruciate ligaments. A preliminary study," Am. J. Sports Med. 16, 203-208 (1988).

39. Bromage, T. G., Smolyar, I., Doty, S. B., Holton, E. & Zuyev, A.N. , "Bone growth rate and relative mineralization density during space flight," Scanning 20, 238-239 (1998).

40. Burkart, A., Imhoff, A. B. & Roscher, E., "Foreign-body reaction to the bioabsorbable suretac device, " Arthroscopy 16, 91-95 (2000) .

41. Butler, D. L., Goldstein, S. A. & Guilak, F., "Functional tissue engineering: the role of biomechanics," J. Biomech. Eng . 122, 570-575 (2000).

42. Chen, CH. et al . , "Enveloping the tendon graft with periosteum to enhance tendon-bone healing in a bone tunnel: A biomechanical and histologic study in rabbits," Arthroscopy 19, 290-296 (2003).

43. Cheung, H. S. & McCarty, D.J., "Mitogenesis induced by calcium-containing crystals. Role of intracellular dissolution," Exp. Cell Res. 157, 63-70 (1985).

44. Clark, J.M. & Sidles, J. A. , "The interrelation of fiber bundles in the anterior cruciate ligament, " J. Orthop. Res. 8, 180-188 (1990). 45. Cooper, J. A., "Design, optimization and in vivo evaluation of a tissue-engineered anterior cruciate ligament replacement," Drexel University (Thesis/Dissertation) (2002).

46. Cooper, J. A., Lu, H. H. & Laurencin, CT. , "Fiber-based tissue engineering scaffold for ligament replacement: design considerations and in vitro evaluation, " Proceedings of 5th World Biomaterial Congress, 208 (Abstract) (2000) .

47. Daniel, D. M. et al . , "Fate of the ACL-injured patient. A prospective outcome study," Am. J. Sports Med. 22, 632-644 (1994) .

48. Deitzel et al . , Polymer 42, (2001).

49. Deitzel et al . , Polymer 43, (2002).

50. Ducheyne, P., "Bioceramics : material characteristics versus in vivo behavior," J. Biomed. Matls. Res. 21, 219-236 (1987).

51. Dunn, M. G., Liesch, J. B., Tiku, M. L., Maxian, S. H. & Zawadsky, J. P., "The Tissue Engineering Approach to

Ligament Reconstruction," Matls. Res. Soc . 331, 13-18 (1994) .

52. El-Amin, S. F. et al . , "Human osteoblast integrin expression on degradable polymeric materials for tissue engineered bone," J. Orthop . Res. (In Press) (2001) . 53. Erben, J.Histochem.Cytochem. 45, 307-314 (1997).

54. Fleming, B.C., Abate, J. A., Peura, G. D. & Beynnon, B. D., "The relationship between graft tensioning and the anterior-posterior laxity in the anterior cruciate ligament reconstructed goat knee," J. Orthop. Res. 19, 841-844 (2001) .

55. Fleming, B., Beynnon, B., Howe, J., McLeod, W. & Pope,

M. , "Effect of tension and placement of a prosthetic anterior cruciate ligament on the anteroposterior laxity of the knee," J. Orthop. Res. 10, 177-186

(1992) .

56. Fong et al . , Polymer, (1999).

57. Fridrikh et al . , PhysRevLet (2003).

58. Fu, F. H., Bennett, CH. , Ma, CB. , Menetrey, J. & Lattermann, C, "Current trends in anterior cruciate ligament reconstruction. Part II. Operative procedures and clinical correlations," Am. J. Sports Med. 28, 124 130 (2000) .

59. Fujikawa, K., Iseki, F. & Seedhom, B. B., "Arthroscopy after anterior cruciate reconstruction with the Leeds- Keio ligament, " J. Bone Joint Surg. Br. 71, 566-570 (1989) .

60. Gao, J. & Messner, K., "Quantitative comparison of soft tissue-bone interface at chondral ligament insertions in the rabbit knee joint," J. Anat . 188, 367-373 (1996) .

61. Gao, J., Rasanen, T., Persliden, J. & Messner, K., "The morphology of ligament insertions after failure at low strain velocity: an evaluation of ligament entheses in the rabbit knee," J. Anat. 189, 127-133

(1996) .

62. Gotlin, R. S. & Huie, G., "Anterior cruciate ligament injuries. Operative and rehabilitative options," Phys . Med. Rehabil. Clin. N. Am. 11, 895-928 (2000).

63. Goulet, F. et al . , "Principles of Tissue Engineering," Lanza, R. P., Langer, R. & Vacanti, J. P. (eds.), pp.

639-645 Academic Press (2000).

64. Grana, W.A. , EgIe, D. M. , Mahnken, R. & Goodhart, CW. , "An analysis of autograft fixation after anterior cruciate ligament reconstruction in a rabbit model," Am. J. Sports Med. 22, 344-351 (1994).

65. Gregoire, M., OrIy, I., Kerebel, L.M. & Kerebel , B., "In vitro effects of calcium phosphate biomaterials on fibroblastic cell behavior," Biol. Cell 59, 255-260 (1987) .

66. Harner, CD. et al . , "Quantitative analysis of human cruciate ligament insertions," Arthroscopy 15, 741-749 (1999). 67. Hench, L. L., "Bioceramics : from concept to clinic," J. Am. Cera. Soc. 74(7), 1487-1510 (1991).

68. Jackson, D.W. , American Academy of Orthopaedic Surgeon Bulletin 40, 10-11 (1992) .

69. Jackson, D.W. , Grood, E. S., Arnoczky, S. P., Butler, D. L. & Simon, T.M. , "Cruciate reconstruction using freeze dried anterior cruciate ligament allograft and a ligament augmentation device (LAD) . An experimental study in a goat model," Am. J. Sports Med. 15, 528-538 (1987) .

70. Jackson, D.W. et al . , Trans. Orhtop. Res. Soc. 16, 208 (Abstract) (1991) .

71. Jackson, D.W. et al . , "A comparison of patellar tendon autograft and allograft used for anterior cruciate ligament reconstruction in the goat model," Am. J. Sports Med. 21, 176-185 (1993).

72. Jiang, J., Nicoll, S. B. & Lu, H. H., "Effects of Osteoblast and Chondrocyte Co-Culture on Chondrogenic and Osteoblastic Phenotype In Vitro," Trans. Orhtop. Res. Soc. 49 (Abstract ) (2003 ).

73. Johnson, R.J., "The anterior cruciate: a dilemma in sports medicine," Int. J. Sports Med. 3, 71-79 (1982).

74. Kim et al . , Biomaterials (2003). 75. Kurosaka, M., Yoshiya, S. & Andrish, J. T., "A biomechanical comparison of different surgical techniques of graft fixation in anterior cruciate ligament reconstruction," Am. J Sports Med. 15, 225- 229 (1987).

76. Kurzweil, P. R., Frogameni , A. D. & Jackson, D. W., "Tibial interference screw removal following anterior cruciate ligament reconstruction," Arthroscopy 11, 289-291 (1995).

77. Larson, R. P., "The Crucial Ligaments: Diagnosis and Treatment of Ligamentous Injuries About the Knee, " John, A. Jr. & Feagin, J. A. (eds.), pp. 785-796 Churchill Livingstone, New York (1994) .

78. Liu, S. H. et al . , "Morphology and matrix composition during early tendon to bone healing, " Clinical Orthopaedics & Related. Research. 253-260 (1997).

79. Loh, J. C. et al . , "Knee stability and graft function following anterior cruciate ligament reconstruction: Comparison between 11 o'clock and 10 o'clock femoral tunnel placement," Arthroscopy 19, 297-304 (2003).

80. Lu, H.H. , Pollack, S. R. & Ducheyne, P., "Temporal zeta potential variations of 45S5 bioactive glass immersed in an electrolyte solution," J. Biomed. Matls . Res. 51, 80-87 (2000).

81. Lu, H. H., Pollack, S. R. & Ducheyne, P., "45S5 bioactive glass surface charge variations and the formation of a surface calcium phosphate layer in a solution containing fibronectin, " J. Biomed. Matls. Res. 54, 454-461 (2001).

82. Lu, H. H., Cooper, J.A., Ko, F. A., Attawia, M. A. & Laurencin, CT. , "Effect of polymer scaffold composition on the morphology and growth of anterior cruciate ligaments cells," Society for Biomaterials Proceedings (Abstract) (2001) .

83. Lu, H. H. , El Amin, S. F., Scott, K. D. & Laurencin, CT. , "Three dimensional, bioactive, biodegradable, polymer bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro," J. Biomed. Matls. Res. 64A, 465-474 (2003) .

84. Lu, H. H. et al . , "Evaluation of Optimal Parameters in the Co-Culture of Human Anterior Cruciate Ligament

Fibroblasts and Osteoblasts for Interface Tissue Engineering, " ASME 2003 Summer Bioengineering Conference (Abstract) (2003) .

85. Markolf, K. L. et al . , "Effects of femoral tunnel placement on knee laxity and forces in an anterior cruciate ligament graft," J. Orthop. Res. 20, 1016- 1024 (2002).

86. Matthews, L. S., Soffer, S. R., "Pitfalls in the use of interference screws for anterior cruciate ligament reconstruction: brief report," Arthroscopy 5, 225-226 (1989) .

87. Matyas, J. R., Anton, M. G., Shrive, N. G. & Frank, CB. , "Stress governs tissue phenotype at the femoral insertion of the rabbit MCL," J. Biomech. 28, 147-157 (1995) .

88. McCarthy, D.M. , Tolin, B. S., Schwendeman, L., Friedman, M.J. & Woo, S. L., "The Anterior Cruciate

Ligament: Current and Future Concepts," Douglas, W. & MD Jackson (eds.) Raven Press, New York (1993).

89. Messner, K., "Postnatal development of the cruciate ligament insertions in the rat knee. Morphological evaluation and immunohistochemical study of collagens types I and II," Acta Anatomica 160, 261-268 (1997).

90. Moore, P. B. & Dedman, J. R., "Calcium binding proteins and cellular regulation," Life Sci. 31, 2937-2946

(1982) .

91. Nicoll, S. B., Wedrychowska , A., Smith, N. R. & Bhatnagar, R. S., "Modulation of proteoglycan and collagen profiles in human dermal fibroblasts by high density micromass culture and treatment with lactic acid suggests change to a chondrogenic phenotype, " Connect. Tissue Res. 42, 59-69 (2001).

92. Niyibizi, C, Sagarrigo, V. C, Gibson, G. & Kavalkovich, K., "Identification and immunolocalization of type X collagen at the ligament- bone interface," Biochem. Biophys . Res. Coinmun. 222, 584-589 (1996).

93. Noyes, F. R. & Barber-Westin, S. D., "Revision anterior cruciate ligament surgery: experience from

Cincinnati," Clin. Orthop. 116-129 (1996).

94. Noyes, F. R., Mangine, R. E. & Barber, S., "Early knee motion after open and arthroscopic anterior cruciate ligament reconstruction," Am. J. Sports Med. 15, 149- 160 (1987) .

95. Panni, A. S., Milano, G., Lucania, L. & Fabbriciani, C, "Graft healing after anterior cruciate ligament reconstruction in rabbits," Clin. Orthop. 203-212 (1997) .

96. Pena, F . , Grontvedt , T . , Brown, G .A. , Aune , A. K . & Engebretsen, L. , "Comparison of failure strength between metallic and absorbable interference screws. Influence of insertion torque, tunnel-bone block gap, bone mineral density, and interference," Am. J. Sports Med. 24, 329-334 (1996) .

97. Petersen, W. & Tillmann, B., "Structure and vascularization of the cruciate ligaments of the human knee joint," Anat . Embryo1. (Berl) 200, 325-334 (1999) .

98. Robertson, D. B., Daniel, D. M. & Biden, E., "Soft tissue fixation to bone," Am. J. Sports Med. 14, 398- 403 (1986). 99. Rodeo, S.A., Suzuki, K., Deng, X. H., Wozney, J. & Warren, R. F., "Use of recombinant human bone morphogenetic protein-2 to enhance tendon healing in a bone tunnel," Am. J. Sports Med. 27, 476-488 (1999).

100. Rodeo, S. A., Arnoczky, S. P., Torzilli, P.A., Hidaka, C. Sc Warren, R. F., "Tendon-healing in a bone tunnel. A biomechanical and histological study in the dog, " J. Bone Joint Surg. Am. 75, 1795-1803 (1993).

101. Safran, M. R. & Harner, CD., "Technical considerations of revision anterior cruciate ligament surgery, " Clin. Orthop. 50-64 (1996) .

102. Sagarriga, V. C, Kavalkovich, K., Wu, J. & Niyibizi, C, "Biochemical analysis of collagens at the ligament-bone interface reveals presence of cartilage- specific collagens," Arch. Biochem. Biophys . 328, 135- 142 (1996) .

103. Scapinelli, R. & Little, K., "Observations on the mechanically induced differentiation of cartilage from fibrous connective tissue," J. Pathol. 101, 85-91 (1970).

104. Schafer, et al . , "In vitro generation of osteochondral composites," Biomaterials 21:2599-2606 (2000).

105. Schafer, et al . , "Tissue-engineered composites for the repair of large osteochondral defects," Arthritis Rheum. 46:2524-2534 (2002). 106. Shellock, F. G., Mink, J. H., Curtin, S. & Friedman, M.J., "MR imaging and metallic implants for anterior cruciate ligament reconstruction: assessment of ferromagnetism and artifact," J. Magn. Reson. Imaging 2, 225-228 (1992) .

107. Shin et al . , Polymer 42, (2001).

108. Sittinger, et al . , "Engineering of cartilage tissue using bieresorbable polymer carriers in perusion culture," Biomaterials 15 (6) : 451-456 (1994).

109. Spalazzi, J. P., Dionisio, K. L., Jiang, J. & Lu, H. H., "Chondrocyte and Osteoblast Interaction on a

Degradable Polymer Ceramic Scaffold, " ASME 2003 Summer Bioengineering Conference (Abstract ) (2003 ).

110. Steiner, M. E., Hecker, A. T. , Brown, CH. , Jr. & Hayes, W. C, "Anterior cruciate ligament graft fixation.

Comparison of hamstring and patellar tendon grafts," Am. J. Sports Med. 22, 240-246 (1994).

111. Thomas, N. P., Turner, I. G. & Jones, CB. , "Prosthetic anterior cruciate ligaments in the rabbit. A comparison of four types of replacement," J. Bone Joint Surg. Br. 69, 312-316 (1987).

112. Thomopoulos, S . et al . , "The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study," J. Orthop. Res. 20, 454-463 (2002). 113. Wei, X. & Messner, K., "The postnatal development of the insertions of the medial collateral ligament in the rat knee," Anat . Embryol. (Berl) 193, 53-59 (1996).

114. Weiler, A., Hoffmann, R. F., Bail, H.J., Rehm, 0. & Sudkamp, N. P., "Tendon healing in a bone tunnel. Part II: Histologic analysis after biodegradable interference fit fixation in a model of anterior cruciate ligament reconstruction in sheep, " Arthroscopy 18, 124-135 (2002) .

115. Weiler, A., Windhagen, H. J. , Raschke, M.J., Laumeyer, A. Sc Hoffmann, R. F., "Biodegradable interference screw fixation exhibits pull-out force and stiffness similar to titanium screws," Am. J. Sports Med. 26, 119-126 (1998) .

116. Woo, S. L., Gomez, M.A. , Seguchi, Y., Endo, CM. & Akeson, W. H., "Measurement of mechanical properties of ligament substance from a bone-ligament-bone preparation," J. Orthop. Res. 1, 22-29 (1983).

117. Woo, S. L., Newton, P.O., MacKenna, D. A. & Lyon, R. M. , "A comparative evaluation of the mechanical properties of the rabbit medial collateral and anterior cruciate ligaments," J. Biomech. 25, 377-386 (1992).

118. Wu H. et al . , J. Am. Chem. Soc. 2003 Jan 15; 125(2) :554-559. 119. Wuthier, R. E., "Involvement of cellular metabolism of calcium and phosphate in calcification of avian growth plate cartilage," J. Nutr. 123, 301-309 (1993).

120. Xu et al., Biomaterials 25, 877-886, (2004).

121. Yahia, L., "Ligaments and Ligamentoplasties, " Springer Verlag, Berlin Heidelberg (1997).

122. Yoshiya, S., Nagano, M., Kurosaka, M., Muratsu, H. & Mizuno, K. , "Graft healing in the bone tunnel in anterior cruciate ligament reconstruction, " Clin. Orthop. 278-286 (2000).

Claims

What is claimed is:

1. A scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising two portions, wherein each of the two portions comprising first through third phases, wherein (i) 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.

2. The scaffold apparatus of claim 1, wherein the two portions encase respective portions of a soft tissue graft .

3. The scaffold apparatus of claim 1, wherein the two portions, in combination, encase the entirety of a soft tissue graft on all sides.

4. The scaffold apparatus of claim 1, wherein a degradable cell barrier is inserted between two adjacent ones of said first through third phases.

5. The scaffold apparatus of claim 4, wherein the degradable cell barrier comprises a nanofiber mesh.

6. The scaffold apparatus of claim 5, wherein the nanofiber mesh comprises polylactide-co-glycolide (PLGA) .

7. The scaffold apparatus of claim 5, wherein the nanofiber mesh is electrospun.

8. An interference apparatus for affixing soft tissue to bone, comprising the scaffold apparatus of claim 1.

9. The interference apparatus of claim 8, wherein the interference apparatus is biomimetic.

10. The interference apparatus of claim 8, wherein the interference apparatus is biodegradable.

11. The interference apparatus of claim 8, wherein the interference apparatus is osteointegrative.

12. A scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a first phase comprising a material which promotes growth and proliferation of fibroblasts, (ii) a second phase adjacent to the first phase comprising a material which promotes growth and proliferation of chondroblasts , and (iii) a third phase adjacent to the second phase comprising a material which promotes the growth and proliferation of osteoblasts, wherein a degradable cell barrier is inserted between two adjacent ones of said first through third phases .

13. The scaffold apparatus of claim 12, wherein the degradable cell barrier is a nanofiber mesh.

14. The scaffold apparatus of claim 13, wherein the nanofiber mesh comprises polylactide-co-glycolide (PLGA) .

15. The scaffold apparatus of claim 3=13, wherein the nanofiber mesh is electrospun.

16. A scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a first phase comprising a material which promotes growth and proliferation of fibroblasts, (ii) a second phase adjacent to the first phase comprising a material which promotes growth and proliferation of chondroblasts , and (iii) a third phase adjacent to the second phase comprising a material which promotes the growth and proliferation of osteoblasts, wherein said first phase coupled to a soft tissue graft.

17. The scaffold apparatus of claim 16, wherein the soft tissue graft is synthetic.

18. The scaffold apparatus of claim 16, wherein the soft tissue graft is a graft for a ligament of the subject.

19. The scaffold apparatus of claim 18, wherein the ligament is an anterior cruciate ligament of the subject.

20. The scaffold apparatus of claim 16, wherein a portion of the scaffold apparatus is configured to be at least partially inserted into a femur of the subject and another portion of the scaffold apparatus is configured to be at least partially inserted into a tibia of the subject.

21. The scaffold apparatus of claim 16, wherein the scaffold apparatus is configured to be inserted in a femur of the subject through a tunnel.

22. The scaffold apparatus of claim 16, wherein the scaffold apparatus is configured to be inserted in a tibia of the subject through a tunnel.

23. The scaffold apparatus of claim 16, wherein the first phase is exposed to a joint cavity of the subject.

24. The scaffold apparatus of claim 16, wherein the second phase is positioned in proximate contact to articular cartilage of the subject.

25. The scaffold apparatus of claim 16, wherein the third phase is encased in bone tissue of the subject.

26. A scaffold apparatus for fixing musculoskeletal soft tissue to bone in a subject, said scaffold apparatus comprising (i) a graft collar and (ii) a polymer-fiber mesh coupled to the graft collar to apply compressive mechanical loading to the graft collar.

27. The scaffold apparatus of claim 26, wherein the polymer-fiber mesh wraps around the graft collar.

28. The scaffold apparatus of claim 26, wherein an outer surface of the graft collar is wrapped in its entirety by the polymer- fiber mesh.

29. The scaffold apparatus of claim 26, wherein the graft collar is bi-phasic.

30. The scaffold apparatus of claim 29, wherein the bi- phasic graft collar includes a first phase comprising a material which promotes growth and proliferation of chondrocytes, and a second phase adjacent to the first phase comprising a material which promotes the growth and proliferation of osteoblasts.

31. The scaffold apparatus of claim 26, wherein the polymer-fiber mesh comprises nanofibers.

32. The scaffold apparatus of claim 31, wherein the nanofiber mesh comprises polylactide-co-glycolide

(PLGA) .

33. The scaffold apparatus of claim 31, wherein the nanofiber mesh is electrospun.

34. The scaffold apparatus of claim 26, wherein the scaffold apparatus is coupled to a soft tissue graft.

35. The apparatus of claim 34, wherein the soft tissue graft is a graft for a ligament of the subject.

36. The apparatus of claim 35, wherein the ligament is an anterior cruciate ligament of the subject.

37. An graft-fixation apparatus comprising the scaffold apparatus of claim 26.

38. The apparatus of claim 37, wherein the graft fixation apparatus is an interference screw.

39. A scaffold apparatus for fixing musculoskeletal soft tissue to bone, said scaffold apparatus being configured to apply mechanical loading to a soft tissue graft to promote regeneration of a fibrocartilage interface between said soft tissue and said bone.

40. The scaffold apparatus of claim 39, wherein said scaffold apparatus comprises a nanofiber mesh configured to apply said mechanical loading to said soft tissue graft.

41. The scaffold apparatus of claim 40, wherein said mechanical loading is applied by said scaffold apparatus dynamically or intermittently to said soft tissue graft.

42. The scaffold apparatus of claim 40, wherein said mechanical loading is applied by said scaffold apparatus statically to promote regeneration of a fibrocartilage interface between said soft tissue and said bone in a subject.

43. The scaffold apparatus of claim 39, wherein said scaffold apparatus comprises a material that promotes growth and proliferation of chondroblasts .

44. The scaffold apparatus of claim 39, wherein said scaffold apparatus comprises first and second phases, wherein (i) the first phase comprises a material that promotes growth and proliferation of chondroblasts, (ii) the second phase adjacent to the first phase comprises a material that promotes growth and. proliferation of osteoblasts.

45. The scaffold apparatus of claim 39, wherein said scaffold apparatus comprises first, second and third phases, wherein (i) the first phase comprises a material that promotes growth and proliferation of fibroblasts, (ii) the second phase adjacent to the first phase comprises a material that promotes growth and proliferation of chondroblasts ,^■ and (iii) the third phase adjacent to the second phase comprises a material that promotes the growth and proliferation of osteoblasts .