WO2014065863A1 - Compositions and methods for promoting collagen-crosslinking - Google Patents

Abstract

The present disclosure generally relates to the field of tissue engineering. The collagenous tissue produced using methods of the present disclosure is extensively cross- linked and has high mechanical properties. Specifically, the present disclosure involves an enzyme-mediated collagen-crosslinking process to produce tissue such as cartilage, and to enhance its maturation and integration.

Description

COMPOSITIONS AND METHODS FOR PROMOTING COLLAGEN- CROSSLINKING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/717,069, filed October 22, 2012, which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure generally relates to the field of tissue engineering. The collagenous tissue produced using methods of the present disclosure is extensively cross- linked and has high mechanical properties. Specifically, the present disclosure involves an enzyme-mediated collagen-crosslinking process to produce tissue such as cartilage, and to enhance its maturation and integration.

BACKGROUND

[0003] Repair of musculoskeletal tissues such as cartilage following damage or degeneration as a result of many diverse factors, including direct impact or disease, is a major health concern. For example, articular cartilage lacks inherent healing potential. As a result, cartilage lesions tend to degenerate into osteoarthritis, a significant problem affecting over a third of adults aged 65 (Athanasiou et al., Electronic Reference, 2009). Currently, there are no cartilage treatments that offer long-term functionality. Mosaicplasty and microfracture require defect site preparation via cartilage removal. Subsequently, either cartilage plugs or a "super clot" fills the defect (Smith et al., Journal of Bone and Joint Surgery, 87:445-449, 2005). Autografts and allografts are also options. For these and other procedures, success is predicated upon the integration of the fill tissue with native cartilage. Various strategies and materials have been proposed to integrate cartilage and bone, however, cartilage-to-cartilage integration has proven to be notoriously difficult (Jiang et al., Annals of Biomedical

Engineering, 38: 2183-2196, 2010; Kon et al., Journal of Orthopaedic Research, 28: 116- 124, 2010; St-Pierre et al., Acta Biomaterialia, 8: 1603-1615, 2012; Augst et al., Journal of the Royal Society, 5: 929-939, 2008).

[0004] Recent efforts have focused on developing de novo musculoskeletal tissues such as cartilage, bone, tendons, and ligaments for reparative and regenerative medicine strategies. Tissue engineering has the potential to substantially improve treatment options for musculoskeletal disease and injury by generating neotissue that mimics the complex structure of native tissue (Hu et al., Tissue Eng 12:969-979, 2006). Despite recent advances, current problems associated with engineered tissues limit their potential for clinical application. In particular, tensile properties of engineered tissues are still not on par with native tissue values, potentially due to the lack of interfibrillar collagen crosslinks (Bastiaansen-Jenniskens et al, Osteoarthritis Cartilage 16:359-366, 2008). For example, the tensile properties of articular cartilage are attributed primarily to the fibrillar collagen network.

[0005] Previous work has shown that immature native tissue and neotissue, which has applications for tissue engineering and drug development, generally have fewer crosslinks than mature native tissue (Riesle et al, J Cell Biochem, 71:313-327, 1998; Beekman et al., Exp Cell Res, 237: 135-141, 1997; Bank et al., Biochem J, 330:345-351, 1998). For example, a 6 week culture of bovine chondrocytes resulted in crosslink abundance that was only 30% of native values (Riesle et al., J Cell Biochem, 71:313-327, 1998). Likewise, culturing chondrocytes for 4 weeks in alginate bead culture produced only 22% of native pyridinoline values (Beekman et al., Exp Cell Res, 237: 135-141, 1997). In another study, three- dimensional cultivation of human osteoblast-like cells on highly porous natural bone mineral resulted in increased amount of collagen crosslinks in a time-dependent manner, although the reported values where still far from native bone tissue (Acil et al., J Biomed Mater Res, 51:703-710, 2000). The general lack of pyridinoline and the functional role of crosslinking in collagen-rich tissues highlight the necessity of developing new strategies for recapitulating native levels of crosslinking.

[0006] To achieve long-term and durable repair, tissue grafts and engineered articular cartilage alike need to be efficiently integrated with native cartilage. There is a general consensus regarding the main factors that hinder integration. First, cell death at the wound edge (Hunziker, et al., The Journal of Bone and Joint Surgery, American volume 85-A Suppl 2:85-92, 2003) in surgically prepared defects leads to metabolically inactive tissue, which prevents cell adhesion and migration to the injury site (van de Breevaart Bravenboer et al, Arthritis Res Ther, 6:R469-476, 2004; Peretti et al, J Biomed Mater Res, A64:517-524, 2003; Bos et al, Arthritis Rheum, 46: 976-985, 2002; Hunziker et al, Clinical Orthopaedics and Related Research: S182-189, 2001; Tew et al, Arthritis Rheum, 43:215-225, 2000). Second, cell migration to the wound edge is hindered by the dense collagen network. In native cartilage, cells are locked into lacunae and are not observed to migrate (Minns et al, Journal of Anatomy, 123:437-457, 1977). Third, there is a lLack of cross-links between matrices of native and implant tissues (Ahsan et al, Journal of Orthopaedic Research, 17:850-857, 1999; McGowan et al, Journal of Orthopaedic Research, 23:594-601, 2005). [0007] In short, insufficient viable cells at the wound edge prevents synthesis of integrative matrix between the two surfaces to be joined, in part by lack of matrix synthesis (Bos et al., Arthritis Rheum, 46: 976-985, 2002; Hunziker et al., Clinical Orthopaedics and Related Research: S 182- 189, 2001 ; Tew et al., Arthritis Rheum, 43:215-225, 2000;

Obradovic et al., Journal of Orthopaedic Research, 19: 1089-1097, 2001 ; Reindel et al., Journal of Orthopaedic Research, 13:751-760, 1995). Even when viable cells are present, the newly synthesized matrix may not be sufficiently cross-linked to the native tissue.

Without proper integration, tissue implants fall out of place or degrade rapidly (Hunziker et al., Clinical Orthopaedics and Related Research: S 182-189, 2001), likely due to the high stress concentrations that occur at cartilage interfaces in vivo. Further, improperly integrated transplanted tissue does not possess the same degree of tensile strength as the native tissue even if the transplanted tissue remains in place.

[0008] Efforts to resolve cartilage integration problems have not yielded adequate results. Accordingly, there exists a need for tools to produce neotissue that can be efficiently integrated with native tissue for use in treating musculoskeletal damage. There also exists a need for engineered tissue possessing biochemical and biomechanical properties on par with that of native tissue.

SUMMARY

[0009] The present disclosure generally relates to the field of tissue engineering. The collagenous tissue produced using methods of the present disclosure is extensively cross- linked and has high mechanical properties. Specifically, the present disclosure involves an enzyme-mediated collagen-crosslinking process to produce tissue such as cartilage, and to enhance its maturation and integration.

[0010] In particular, the present disclosure provides methods of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength. In some embodiments, the conditions comprise culturing the connective tissue cells in the presence of 0.015 μg/ml to 1.5 mg/ml lysyl oxidase (LOX). In a subset of these embodiments, LOX is present at a concentration of 0.075 μg/ml to 0.75 μg/ml or about 0.15 μg/ml. In some embodiments, the conditions comprise culturing the connective tissue cells under hypoxic conditions of 0.5% to 11% oxygen. In a subset of these embodiments, oxygen is present at 1% to 10%, 2% to 9%, 3% to 8%, 4% to 7% or about 5%. In some embodiments, the conditions comprise culturing the connective tissue cells in the presence of glucose oxidase (GOX) and catalase (CAT), In a subset of these embodiments, GOX:CAT ratio is 25:1 to 1:25 or about 10:1. In some embodiments, the level of enzyme-mediated collagen-crosslinks is directly proportional to the concentration of exogenously- supplied or endogenously-expressed LOX present during the treating step. In some embodiments, the connective tissue is selected from the group consisting of cardiovascular tissue, musculoskeletal tissue, and skin. In some embodiments, the connective tissue is an engineered scaffold-based tissue or a scaffoldless tissue.

[0011] Also provided by the present disclosure are methods of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength, wherein the conditions comprise culturing the connective tissue cells in the presence of 0.015 μg/ml to 1.5 mg/ml lysyl oxidase (LOX). In a subset of these embodiments, LOX is present at a concentration of 0.075 μg/ml to 0.75 μg/ml or about 0.15 μg/ml. Additionally, the present disclosure provides methods of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength, wherein the conditions comprise culturing the connective tissue cells under hypoxic conditions of 0.5% to 11% oxygen. In a subset of these embodiments, oxygen is present at 1% to 10%, 2% to 9%, 3% to 8%, 4% to 7% or about 5%. Moreover, the present disclosure provides methods of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength, wherein the conditions comprise culturing the connective tissue cells in the presence of glucose oxidase (GOX) and catalase (CAT), In a subset of these embodiments, GOX:C AT ratio is 25: 1 to 1:25 or about 10: 1. In some preferred embodiments, the connective tissue cells comprise chondrogenic cells. In some embodiments, the chondrogenic cells are primary cells isolated from one or more of the group consisting of articular cartilage, fibrocartilage, elastic cartilage, and hyaline cartilage. In a preferred embodiment, the chondrogenic cells are primary chondrocytes isolated from articular cartilage. In some embodiments, the

chondrogenic are cultured cells (e.g., passaged in vitro) expanded from primary cells isolated from one or more of the group consisting of articular cartilage, fibrocartilage, elastic cartilage, and hyaline cartilage. In a preferred embodiment, the chondrogenic cells are cultured chondrocytes expanded from primary cells isolated from articular cartilage. In some embodiments, the chondrogenic cells comprise mesenchymal stem cells isolated from a source selected from the group consisting of bone marrow, adipose tissue, synovium, periosteum, dermis, umbilical cord blood, synovial fluid, muscle, and tendon. In some embodiments, the chondrogenic cells comprise pluripotent stem cells, which in a preferred embodiment comprise induced-pluripotent stem cells. In some embodiments, the

chondrogenic cells are cultured during the contacting step using a construct formation technique selected from the group consisting of self-assembly, centrifugation, scaffold seeding, hydrogel encapsulation, and in vivo cell-seeding (e.g., autologous chondrocyte implantation). In a preferred embodiment, the chondrogenic cells are cultured during the contacting step using a self-assembly construct formation technique. In some preferred embodiments, the collagenous tissue comprises cartilage. In some embodiments, the cartilage is engineered tissue. In some embodiments, the cartilage is native tissue. In some embodiments, the collagenous tissue comprises one or both of a tendon and a ligament. In some preferred embodiment, the collagenous tissue has a higher Young's modulus value than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression (e.g., under conditions ineffective for formation of enzyme-mediated collagen-crosslinks). In some embodiment, the Young's modulus value is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more fold (MPa) over the control; or up to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 fold (MPa) over the control. In some preferred embodiments, the collagenous tissue has a higher ultimate tensile strength (UTS) than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression (e.g., under conditions ineffective for formation of enzyme-mediated collagen-crosslinks). In some embodiments, the UTS is at least 2, 3, 4, 5 , 6, 7, 8, 9, or 10 or more fold (MPa) over the control; or up to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 fold (MPa) over the control. In some preferred embodiments, the collagenous tissue has a higher pyridinoline content than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression (e.g., under conditions ineffective for formation of enzyme-mediated collagen-crosslinks). In some embodiments, the pyridinoline content is at least 2, 3, 4, 5 , 6, 7, 8, 9, or 10 or more fold (pyr/ww or pyr/col) over the control; or up to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 fold (pyr/ww or pyr/col) over the control. In some preferred embodiments, the collagenous tissue has a higher pyrrole content than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression (e.g., under conditions ineffective for formation of enzyme-mediated collagen-crosslinks). In some embodiments, the pyrrole content is at least 2, 3, 4, 5 , 6, 7, 8, 9, or 10 or more fold (pyrrole/ww or pyrrole/col) over the control; or up to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 fold (pyrrole/ww or pyrrole/col) over the control. In some embodiments, the collagenous tissue comprises cornea. In other embodiments, the collagenous tissue comprises a synthetic collagen membrane. In some preferred embodiments, the collagenous tissue comprises a tendon, a ligament, a bone, cartilage and decellularized compositions thereof. In some embodiments, the collagenous tissue is native tissue. In some embodiments, the collagenous tissue is engineered tissue. The present disclosure further provides methods of producing collagenous tissue possessing a high tensile property, comprising: contacting connective tissue cells with a composition comprising an effective amount of copper to produce collagenous tissue possessing a high tensile property. In some embodiments, the copper is in the form of a salt (e.g., cupric sulfate) or bound to a carrier (e.g., ceruloplasmin, also known as ferroxidase). In some embodiments, the composition further comprises hydroxylysine. In some embodiments, the high tensile property comprises a high Young's modulus value. In some embodiments, the high tensile property comprises a high ultimate tensile strength (UTS). In some

embodiments, the high tensile property comprises both a high Young's modulus value and a high ultimate tensile strength (UTS). In some embodiments, the collagenous tissue is selected from the group consisting of cardiovascular tissue, musculoskeletal tissue, and skin. In some embodiments, the connective tissue is an engineered scaffold-based tissue or a scaffoldless tissue.

[0012] Additionally, the present disclosure provides compositions comprising the collagenous tissue prepared by a method of the two preceding paragraphs. In some embodiments, the present disclosure provides compositions comprising an isolated collagenous tissue and a soluble oxygen-depleting compound. In some embodiments, the present disclosure provides compositions comprising isolated collagenous tissue, and exogenous lysyl oxidase. In some embodiments, the present disclosure provides

compositions comprising isolated collagenous tissue, glucose oxidase (GOX), and caialase (CAT). In some embodiments, the present disclosure provides compositions comprising isolated collagenous tissue, and copper. In some embodiments, the copper is in the form of a salt (e.g., cupric sulfate) or bound to a carrier (e.g., ceruloplasmin, also known as

ferroxidase).. In some embodiments, the compositions further comprise hydroxylysine.

[0013] Moreover, the present disclosure provides methods of producing an integrated collagenous tissue possessing a high tensile strength, comprising: contacting a first collagenous tissue with a second collagenous tissue under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce an integrated collagenous tissue possessing a high tensile strength. In some embodiments, the conditions comprise treating the first and/or the second collagenous tissues with a composition comprising of 0.015 μg/ml to 1.5 mg/ml lysyl oxidase (LOX). In a subset of these embodiments, LOX is present at a concentration of 0.075 μg/ml to 0.75 μg/ml or about 0.15 μg/ml. In some embodiments, the conditions comprise subjecting the first and/or the second collagenous tissues to hypoxic conditions of 0.5% to 11% oxygen. In a subset of these embodiments, oxygen is present at 1% to 10%, 2% to 9%, 3% to 8%, 4% to 7% or about 5%. In some embodiments, the conditions comprise treating the first and/or the second collagenous tissues with a composition comprising glucose oxidase (GOX) and catalase (CAT). In a subset of these embodiments, GQX:CAT ratio is 25: 1 to 1 :25 or about 10: 1. In some preferred

embodiments, the level of enzyme-mediated collagen-crosslinks is directly proportional to the concentration of exogenously- supplied or endogenously-expressed lysyl oxidase present during the treating step. In some embodiments, the collagen-crosslinks comprise one or both of pyridinoline cross-links and pyrrole cross-links. In some embodiments, the first collagenous tissue is native cartilage and the second collagenous tissue is engineered cartilage. In some embodiments, both the first and second collagenous tissues are native cartilage. In some embodiments, the first collagenous tissue is a native tendon or ligament, and the second collagenous tissue is an engineered tendon or ligament. In some

embodiments, both the first and the second collagenous tissues are native tendons or ligaments. In some embodiments, both the first and the second collagenous tissues are independently selected from the group consisting of tendons, ligaments, bones, cartilage and decellularized compositions thereof. In some embodiments, both the first and second collagenous tissues are independently selected from the group consisting of cardiovascular tissue, musculoskeletal tissue, and skin. In some embodiments, both the first and second collagenous tissues are independently selected from an engineered scaffold-based tissue or a scaffoldless tissue. In some embodiments, the collagenous tissues comprises a synthetic collagen membrane. In some embodiments, both of the collagenous tissues are native tissues. In other embodiments, one of the collagenous tissues is an engineered tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a hierarchical depiction of a heterotypic collagen fibril, emphasizing the internal axial relationships required for mature crosslink formation. Upper: Three- dimensional concept of the type II/IX/XI heterotypic fibril of developing musculoskeletal tissues extracellular matrix. Middle: Detail illustrating required nearest neighbor axial relationships for trifunctional intermolecular crosslinks to form collagens of cartilage, bone, and other high-tensile strength tissue matrices. The exact 3D spatial pattern of crosslinking bonds is still unclear for any tissue. Lower: Detail of the axial stagger of individual collagen molecules required for pyridinoline crosslinking (adapted from Eyre et al., Methods 45, 65- 74, 2008).

[0015] FIG. 2 illustrates the molecular pathway of collagen crosslinks in musculoskeletal tissues initiated by the extracellular enzyme lysyl oxidase (LOX). Triple helix and teloppetide lysines are tracked by color to the initial and mature crosslinks. Bone contains both pyridinoline and pyrrole collagen crosslinks while articular cartilage features only pyridinoline crosslinks (adapted from Eyre et al., Methods 45, 65-74, 2008).

[0016] FIG. 3 illustrates the biomechanical and biochemical properties of tissue constructs treated with different concentrations of LOX. FIG. 3A shows an assessment of biomechanical properties of the constructs through quantification of the Young's modulus (MPa), a measure of tensile stiffness. FIG. 3B shows an assessment of biochemical properties of the constructs through quantification of pyridinoline content (PYR/WW).

Groups not connected by the same letter are statistically different.

[0017] FIG. 4 illustrates the biomechanical properties and biochemical content of tissue constructs treated with high LOX (0.15μg/ml) at different times during tissue culture. FIG. 4A shows an Assessment of biomechanical properties of the constructs through quantification of the Young's modulus (MPa), a measure of tensile stiffness. FIG. 4B shows an assessment of biochemical properties of the constructs through quantification of pyridinoline content (PYR/WW). FIG. 4C shows an assessment of the aggregate modulus (kPa), a measure of compressive stiffness. Groups not connected by the same letter are statistically different. [0018] FIG. 5 provides a schematic of the experiment examining integration of tissue engineered cartilage to native cartilage. For Group B, LOX was applied during construct formation, t=15-28d. For Group C, LOX was applied after forming the construct-to-native assemblies, t=29-35d. For Group D, LOX was applied both before and after the formation of the construct- to-native assemblies, t=15-35d.

[0019] FIG. 6 illustrates how LOX application effects integration of engineered constructs with native articular cartilage. LOX-treated constructs received 0.15 μg/ml LOX both before and after the formation of the construct-to-native assemblies, t=15-35d (Group D). Gaps were seen histologically one third of the controls (left panel), but not in the LOX- treated samples (right panel). The presence of gaps between the two construct types is indicative of lack of tissue integration.

[0020] FIG. 7 illustrates how LOX application impacts the biomechanical properties of integrated engineered tissues. FIG. 7A shows the assessment of biomechanical properties of the constructs through quantification of the Young's modulus (MPa), a measure of tensile stiffness. FIG. 7B shows the ultimate tensile strength (UTS) of the constructs as a function of time of LOX application during tissue integration. Bars with different letters are significantly different (p<0.05).

[0021] FIG. 8 illustrates how LOX effects native-to-native tissue integration. Native-to- native tissue assemblies consisted of tissue explants instead of constructs. The control group was incubated for 14 days in culture medium, while the LOX-treated group was incubated in a LOX-containing medium (0.15 μg/ml) during the same time period. Gaps were observed histologically in the control group (left panel), but not the LOX-treated group (right panel).

[0022] FIG. 9 illustrates how LOX impacts the mechanical properties of native-to-native integrated tissues. FIG. 9A shows the assessment of biomechanical properties of the control and LOX-treated tissues through quantification of the Young's modulus (MPa), a measure of tensile stiffness. FIG. 9B shows the ultimate tensile strength (UTS) of the control and LOX- treated tissues.

[0023] FIG. 10 illustrates how generation of a hypoxic environment in articular cartilage tissue can impact various properties of the tissue explants. β-aminopropionitrile (BAPN) is an inhibitor of LOX activity. FIG. 10A shows the quantification of LOX mRNA expression in various treatment groups (x-axis) as determined by real-time PCR. FIG. 10B shows an assessment of the pyridinoline per collagen (PYR/COL) content of the tissue explants in various treatment groups. FIG. IOC shows an assessment of the tensile strength of the tissue explants in various treatment groups. Groups not connected by the same letter are statistically different.

[0024] FIG. 11 provides a graph showing the biomechanical properties of anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), patella tendon (Pat), and medial meniscus (Men) explants after 4 weeks incubation under normoxic or hypoxic conditions in the presence or absence of β-aminopropionitrile (BAPN). Bars delineate means+SD and groups not sharing a letter are statistically different from one another.

[0025] FIG. 12 provides graphs showing the effect of copper (Cu) and hydroxylysine (AA) on tensile properties of neocartilage. FIG. 12A shows the Young's modulus values, while FIG. 12B shows the ultimate tensile strength (UTS) of self-assembled neocartilage.

DETAILED DESCRIPTION

[0026] The present disclosure generally relates to the field of tissue engineering. The collagenous tissue produced using methods of the present disclosure is extensively cross- linked and has high mechanical properties. Specifically, the present disclosure involves an enzyme-mediated collagen-crosslinking process to produce tissue such as cartilage, and to enhance its maturation and integration.

[0027] In some embodiments, the present disclosure provides methods for improving engineered tissues. An example of a tissue suitable for the methods of the present disclosure is a collagen-containing, or collagenous, tissue, such as cartilage. Collagenous tissue contains an intricate architecture of collagen crosslinks, as seen in FIG. 1, as well as a variety of other components. As this collagen network structure and associated properties is inherent to native collagenous tissues, a goal of tissue engineering is to produce neotissues that mimic or are superior to native tissues.

[0028] Current information on the biochemistry of engineered musculoskeletal tissues such as tendons, ligaments, or fibrocartilage is limited (Kelly et al., J Mater Sci Mater Med, 18:273-281, 2007; Doroski et al, Biomate rials, 28: 187-202, 2007). However, it is thought that tissue mechanical properties are dependent on more than the quantity of any single biochemical component (Tran-Khanh et al., J Orthop Res, 23: 1354-1362, 2005). The unique structure and architecture of the collagen network plays as important a role as collagen quantity in determining the load-bearing function of the tissue. [0029] Collagen, comprising the major fraction of the ECM of musculoskeletal tissues, accounts for approximately 65-80% of the tissue's dry weight (Lewis et al., J Orthop Sports Phys Ther, 36:717-727, 2006). In tissues, collagen fibrils noncovalently stabilize the highly hydrated, negatively charged proteoglycans (Buckwalter et al., Instr Course Lect, 47:477- 486, 1998). After collagen biosynthesis and triple -helix formation, extracellular modification of the collagen molecules by endopeptidases results in covalent crosslinks between individual collagen molecules (FIG. 1). Such intermolecular collagen and elastin crosslinks are pyrrole and pyridinoline (FIG. 2), which are present in a wide variety of tissues such as bone, cartilage, ligaments, and tendons. The precursor of crosslinks is formed by the extracellular enzyme lysyl oxidase (LOX)( Siegel et al., J Biol Chem 251:5786-5792, 1976). The major collagen crosslinks in articular cartilage are the difunctional (initial/immature) crosslink, dehydrodihydroxylysinonorleucine (DDHLNL) and the trifunctional (final/mature) crosslink, hydroxylysylpyridinoline (HP). Bone features both pyridinoline and pyrrole collagen crosslinks in about equal amount with the major ones being dehydrohydroxylysinonorleucine (deH-HLNL), and the mature forms, pyridinium and pyrrole cross-links (Robins et al., Biochem J, 163:339-346, 1977). In ligaments the most predominant collagen crosslink is dihydroxylysinonorleucin (Fujii et al, Knee Surg Sports Traumatol Arthrosc, 2:229-233, 1994) that turns into the mature pyridinoline form. Several studies have highlighted how biomechanical properties correlate with collagen crosslinking in native tissues (Bastiaansen- Jenniskens et al., Osteoarthritis Cartilage, 16:359-366, 2008; Ahsan et al., Osteoarthritis Cartilage, 13:709-715, 2005; Williams et al., Tissue Eng Part A, 17: 17-23, 2011).

The methods and compositions described herein may find use as in treating diseases and syndromes related to collagen crosslinking deficiency {e.g., osteolathyrism, Ehlers Danlos syndrome Type IV, etc.).

Processes for Producing Collagenous Tissue

[0030] The present disclosure relates to methods of producing collagenous tissue possessing a high tensile strength involving treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen cross-links to produce collagenous tissue possessing a high tensile strength. Conditions effective for formation of enzyme- mediated collagen cross-links to produce a collagenous tissue having a high tensile strength may comprise exogenous application of lysyl oxidase (LOX). In some embodiments, LOX is in its active form, while in others LOX is in an inactive form (e.g., prodrug activatable by hypoxia). Conditions effective for formation of enzyme-mediated collagen cross-links to produce a collagenous tissue having a high tensile strength may comprise exogenous application of both glucose oxidase (GOX) and catalase (CAT), GOX-CAT enzymatic system. Conditions effective for the formation of enzyme-mediated collagen cross-links to produce a collagenous tissue having a high tensile strength may comprise culturing connective tissue cells under hypoxic conditions. In some embodiments, the level of enzyme-mediated collagen-crosslinks is directly proportional to the concentration of exogenously- supplied or endogenously-expressed LOX present during the treating step.

[0031] In some embodiments of the present disclosure, the collagenous tissue possessing a high tensile strength has a higher Young's modulus than does a control collagenous tissue. Control collagenous tissue may be produced under conditions ineffective for the formation of enzyme-mediated collagen-crosslinks. Control collagenous tissue may be produced essentially in the absence of exogenously supplied lysyl oxidase. Control collagenous tissue may be produced essentially in the absence of induction of endogenous expression of lysyl oxidase. Control collagenous tissue may be produced essentially by providing adequate oxygen to the connective tissue cells in the growth media (i.e. not under hypoxic conditions).

[0032] In some embodiments, collagenous tissue possessing a high tensile strength has a Young's modulus value at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more over the Young's modulus value of a control collagenous tissue. In some embodiments, collagenous tissue possessing a high tensile strength has a Young's modulus value of up to 6-fold, up to 7- fold, up to 8-fold, up to 9-fold, up to 10-fold, up to 11-fold, up to 12-fold, up to 13-fold, up to 14-fold, up to 15-fold, or up to 16-fold over the Young's modulus value of a control collagenous tissue.

[0033] In some embodiments of the present disclosure, the collagenous tissue possessing a high tensile strength has a higher ultimate tensile stress value than does a control collagenous tissue. In some embodiments, collagenous tissue possessing a high tensile strength has an ultimate tensile stress value at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more over the ultimate tensile stress value of a control collagenous tissue. In some embodiments, collagenous tissue possessing a high tensile strength has an ultimate tensile stress value of up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, up to 10-fold, up to 11- fold, up to 12-fold, up to 13-fold, up to 14-fold, up to 15-fold, or up to 16-fold over the ultimate tensile stress value of a control collagenous tissue.

[0034] In some embodiments of the present disclosure, the collagenous tissue possessing a high tensile strength has a higher pyridinoline content than does a control collagenous tissue. In some embodiments, collagenous tissue possessing a high tensile strength has a pyridinoline content at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more over the pyridinoline content of a control collagenous tissue. In some embodiments, collagenous tissue possessing a high tensile strength may has a pyridinoline content of up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, up to 10-fold, up to 11-fold, up to 12-fold, up to 13-fold, up to 14-fold, up to 15-fold, or up to 16-fold over the pyridinoline content of a control collagenous tissue.

[0035] In some embodiments of the disclosure, a collagenous tissue possessing a high tensile strength is provided in the form of a composition prepared according to the methods provided herein. In some embodiments, a composition is provided comprising an isolated collagenous tissue, and exogenous lysyl oxidase. In some embodiments, a composition is provided comprising an isolated collagenous tissue, a glucose oxidase, and a catalase. In some embodiments, the isolated collagenous tissue is engineered tissue. In other embodiments, the isolated collagenous tissue is native tissue. In some embodiments, the composition is an isotonic solution.

Connective Tissue Cells

[0036] The four types of tissues that make up mammalian bodies include connective, muscle, nervous and epithelial tissues. Connective tissue is fibrous tissue made up of cells, fibers, and an extracellular matrix. Of particular relevance to the present disclosure is connective tissue, which comprises collagen fibers (e.g., collagenous tissue such as tendons, ligaments, cartilage, bone, etc.). The connective tissue may be cardiovascular tissue, such as heart valves and the parietal pericardium. The connective tissue may be musculoskeletal tissue, such as fibrocartilage, attachments, enthuses, and muscle. The connective tissue may be derived from skin.

[0037] The connective tissue cells of the present disclosure, from which collagenous tissue is produced, may comprise chondrogenic cells. As used herein, the term

"chondrogenic cell" refers to a cell capable of forming cartilage. The term "chondrogenic cell" encompasses chondrocytes and bone marrow. Other chondrogenic cells include but are not limited to adipose-derived, skin-derived (see, e.g., US 2009/015533), synovium-derived, periosteum-derived, induced pluripotent, and embryonic stem cells (see, e.g., US

2009/0136559). All cell types that are capable of producing matrix specific to cartilage (e.g., collagen type II, aggrecan, glycosaminoglycans, cartilage oligomeric protein, and superficial zone protein) are chondrogenic cells suitable for use in the compositions and methods of the present disclosure. Thus the cells and cell samples used in conjunction with the methods of the present disclosure may comprise chondrocytes, chondro-differentiated cells,

fibrochondrocytes, fibrochondro-differentiated cells, and combinations thereof (referred to herein as chondrocytes).

[0038] The chondrocytes of the present disclosure may be articular chondrocytes or meniscal fibrochondrocytes. Generally, chondrocytes may be from a bovine or porcine source, or another animal source. Alternatively, if the constructs are to be used for in vivo tissue replacement, the source of chondrocytes may be autologous cartilage from a small biopsy of the patient's own tissue, provided that the patient has healthy cartilage that may be used as the start of in vitro expansion. Another suitable source of chondrocytes is allogeneic chondrocytes, such as those from histocompatible cartilage tissue obtained from a donor or cell line.

[0039] In some embodiments, the connective tissue cells used in conjunction with the methods of the present disclosure may be derived from mesenchymal, embryonic, induced pluripotent stem cells, skin cells, or other stem cells. The connective tissue cells may be derived from any source and site for obtaining a cell sample comprising a sufficient number of cells to produce a collagenous tissue. Such cells and cell samples may be obtained by any means suitable for obtaining a cell sample comprising a sufficient number of connective tissue cells. In certain embodiments, such a means may comprise enzymatic digestion of native tissue. Suitable enzymes for such an enzymatic digestion include, but are not limited to, one or more collagenases.

[0040] In some embodiments of the present disclosure, methods are provided for producing collagenous tissue possessing a high tensile strength from native connective tissue cells in situ (e.g., have not been isolated from an animal). Generally, these cells remain substantially associated with the animal (e.g., are not removed for in vitro use). Conditions Effective for Formation of Enzyme-Mediated Collagen- Crosslinks

[0041] Enzymatic reactions can be classified according to their Enzyme Commission (EC) number. The EC number associated with a given enzyme specifies the classification of the type of enzymatic reaction that a given enzyme is capable of catalyzing. EC numbers do not specify identities of enzymes, but instead specify the identity of the chemical reaction that a given enzyme catalyzes. EC classifications are helpful to those skilled in the art in identifying the molecular function and/or activity of a given protein outside of knowing its unique identifying classification with regard to the organism it came from, such as its NCBI (National Council for Biotechnology) identifier.

Exogenous Lysyl Oxidase (LOX)

[0042] The present disclosure relates to methods involving treating connective tissue cells under conditions effective for formation of enzyme-mediate collagen cross-links to produce collagenous tissue possessing a high tensile strength. In some embodiments, the enzyme comprises exogenous lysyl oxidase (LOX).

[0043] Lysyl oxidase catalyzes the following reaction: Peptidyl-L-lysyl-peptide + O₂ + H₂O pep tidyl- ally syl-pep tide + NH₃ + H₂O₂. Lysyl oxidase enzymes are characterized has having EC 1.4.3.13 (lysyl oxidase) activity. Examples of lysyl oxidase enzymes may include, without limitation, gil257065lgblAAB23549.1l [Homo sapiens], gil244146lgblAAB21243.1l [Homo sapiens], gil726455lgblAAC52176.1l [Rattus norvegicus], gil205227lgbl AAA41537. il [Rattus norvegicus], gill98953lgblAAA19032.1l [Mus musculus],

gil326778284lreflZP_08237549.ll [Streptomyces griseus XylebKG-1],

gil326658617lgblEGE43463.ll [Streptomyces griseus XylebKG-1],

gill98958lgblAAA20185. ll [Mus musculus], gil302136986lgblADK94439.1l [Gadus morhua], gill87189lgblAAA59525.1l [Homo sapiens], gil212269lgblAAA48942.1l [Gallus gallus], gil386620lgblAAB27430.1l [swine, Peptide Partial, 36 aa],

gill6033746lgblAAL13313.1IAF421186_ll [Bos taurus], gill55733227lgblABU39845.1l [Salmo salar], gill55733223lgblABU39843.1l [Salmo salar], gil 155733219lgbl ABU39841.11 [Salmo salar], gill55733215lgblABU39839.1l [Salmo salar], gill55733211lgblABU39837.1l [Oncorhynchus mykiss], gil l55733207lgblABU39835.1l [Oncorhynchus mykiss], and gill55733203lgblABU39833. ll [Oncorhynchus mykiss]. Each sequence associated with the foregoing accession numbers is incorporated herein by reference. [0044] Various forms of lysyl oxidase and lysyl oxidase-like enzymes may be used in the methods and compositions of the present disclosure such as, for example, lysyl hydroxylase and other lysyl oxidase variants with similar enzymatic function. The lysyl oxidase may be, for example, lysyl oxidase 1, 2, 2a, 2b, and 4 and homologs thereof. The lysyl oxidase may be, for example, an enzyme belonging to the family of lysyl oxidases. The lysine oxidase may be, for example, an enzyme that functions as a protein-lysine 6-oxidase.

[0045] Methods of obtaining lysyl oxidase are well-known in the art. Lysyl oxidase may be obtained from a commercial supplier of purified catalase enzyme. Examples of commercially available lysyl oxidase include, without limitation recombinant human proteins such as Lysyl Oxidase-Like 4 (LOXL4) and Lysyl Oxidase-Like 2 (LOXL2) , both obtainable from Genway Biotech, Inc. (Genway ID: GWB-931D01, and Genway ID: GWB- A8612E, respectively). Lysyl oxidase may be recombinantly expressed and purified.

Additional methods of obtaining purified lysyl oxidase will be apparent to those skilled in the art.

[0046] In some embodiments, lysyl oxidase may contact connective tissue cells in accordance with the methods of the present disclosure. In some embodiments, contacting connective tissue cells with exogenous lysyl oxidase may produce a collagenous tissue possessing a high tensile strength. Lysyl oxidase may be present in the culture media of connective tissue cells. Lysyl oxidase may be applied exogenously to connective tissue cells by spraying or other exogenous application method. One of skill in the art will recognize various acceptable methods for contacting exogenous lysyl oxidase to connective tissue cells and such methods may be used in accordance with the tensile strength enhancement methods of the present disclosure.

[0047] In some embodiments, exogenous lysyl oxidase is present at a concentration of 0.015 μg/ml to 1.5 mg/ml lysyl oxidase. In some embodiments, lysyl oxidase is present at a concentration of more than 0.025 μg/ml, 0.05 μg/ml, 0.075 μg/ml, 0.1 μg/ml, 0.125 μg/ml, 0.15 μ^πύ, 0.175 μ^πύ, 0.2 μ^πύ, 0.25 μ^πύ, 0.3 μ^πύ, 0.35 μ^πύ, 0.4 μ^πύ, 0.45 μg/ml, 0.5 μg/ml, 0.55 μ^πύ, 0.6 μg/ml, 0.65 μ^πύ, 0.7 μ^πύ, 0.75 μg/ml, 0.8 μ^πύ, 0.85 μ^πύ, 0.9 μg/ml, or 0.95 μ^πύ. In some embodiments, the lysyl oxidase is present at a concentration of less than 1.5 mg/ml, 1.4 mg/ml, 1.3 mg/ml, 1.2 mg/ml, 1.1 mg/ml, 1.0 mg/ml, 0.9 μg/ml, 0.8 μg/ml, 0.7 μ^πύ, 0.6 μg/ml, 0.5 μg/ml, 0.4 μg/ml, 0.3 μg/ml or 0.2 μg/ml. In some embodiments, exogenous LOX is provided at a concentration of between 0.075 to 0.75 μ^πύ.

[0048] Lysyl oxidases (or other lysyl oxidase-like enzyme such as lysyl hydroxylase) may be provided in either an active or inactive form. An active lysyl oxidase may have, for example, enzymatic activity directly upon application to connective tissue cells. An inactive lysyl oxidase may be inactive upon direct application, but become activated following exposure to an activate condition such as, for example, hypoxia.

Hypoxic Conditions

[0049] The present disclosure relates to methods involving treating connective tissue cells under conditions effective for formation of enzyme-mediate collagen cross-links to produce collagenous tissue possessing a high tensile strength. In some embodiments, the methods involve culturing connective tissue cells under hypoxic conditions to induce endogenous lysyl oxidase expression.

[0050] Hypoxic conditions refer to conditions in which hypoxia is present. Hypoxia refers to a state in which the surrounding environment is substantially deprived of adequate oxygen supply. In some embodiments, hypoxic conditions are those in which oxygen is present at fromO.5% to 11%. In some embodiments, the hypoxic environment has no more than 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9% or 10%, but less than 11% oxygen. In some embodiments, the hypoxic environment has less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% oxygen. In some embodiments, the hypoxic conditions are an environment having from 1.0% to 10% oxygen.

Exogenous Glucose Oxidase (GOX) and Catalase (CAT)

[0051] The present disclosure relates to methods involving treating connective tissue cells under conditions effective for formation of enzyme-mediate collagen cross-links to produce collagenous tissue possessing a high tensile strength. In some embodiments, the enzyme comprises exogenous glucose oxidase (GOX) and catalase (CAT).

[0052] Glucose oxidase is an oxido-reductase enzyme that catalyzes the oxidation of glucose, resulting in the formation of D-glucono-l,5-lactone and hydrogen peroxide (H₂O₂). Glucose oxidase enzymes are characterized as having EC 1.1.3.4 (glucose oxidase) activity. Examples of glucose oxidase enzymes may include, without limitation,

gil2355lemblCAA39826. ll [Aspergillus niger], gil58585090lreflNP_001011574.11 [Apis mellifera], gill575046lgblAAB09442.1l [Talaromyces flavus], gil300100567lgblEFI91979.1l, [Schizophyllum commune H4-8], gil393716500lgblAFN20671.1l [Aspergillus niger], gil310687275lgblADP03053.ll [Aspergillus niger], gil4102458lgblAAD01493.1l [Penicillium amagasakiense], gil302403429lgblADL38963.1l [Spodoptera exigua],

gil302674395lreflXP_003026882. ll [Schizophyllum commune H4-8],

gill3236685lgblAAF59929.2IAF234246 [Aspergillus niger], gil238801174lgblACR56326.1l [Aspergillus niger], gill21592054lgblABM63225.1l g [Penicillium adametzii],

gil215982092lgblACJ71598.1l[He/icoverpa zea], gil 126140618 Igb I ABN79922.11 [Penicillium expansum], gil l l0294440lgblABG66642.1l [Aspergillus niger],

gil 110180404lgb I ABG54443.11 [Aspergillus niger],

gill0798449lemblCAC12802.1l[A¾?ergi7/ws niger],

gil34850706lemblCAE47418.1l[ra/flram c^ variabilis], gil40642966lemblCAD88590.ll [Botryotinia fuckeliana], gil6448461ldbj IB AA86908. il [Apis mellifera]. Each sequence associated with the foregoing accession numbers is incorporated herein by reference.

[0053] Methods of obtaining glucose oxidase are well4inown in the art. Glucose oxidase may be obtained from a commercial supplier of purified glucose oxidase enzyme. Glucose oxidase may be recombinantly expressed and purified. Additional methods of obtaining purified glucose oxidase will be apparent to those skilled in the art.

[0054] Catalase is an enzyme that catalyzes the hydrolysis of hydrogen peroxide to water (H₂0) and oxygen (0₂). Catalase enzymes are characterized as having EC 1.11.1.6 (catalase) activity. Examples of catalase enzymes include, without limitation,

gil22234lemblCAA38588. ll [Zea mays], gil6006609lemblCAB56850.1l [Prunus persica], gil203335lgblAAB42378.1l[Raftto norvegicus], gil387907809lreflYP_006338143. II

[Helicobacter pylori XZ274], gil387572744lgbl AFJ81452.11 [Helicobacter pylori XZ274], gil211638131lemblCAR66757.ll; (ec 1.11.1.6) [Photorhabdus asymbiotica subsp.

asymbiotica ATCC 43949], gil78369302lreflNP_001030463.1l [Bos taurus],

gil 157951741 lreflNP_033934.2l [Mus musculus], gil328952878lreflYP_004370212.1l

[Desulfobacca acetoxidans DSM 11109], gil320333277lref I YP_004169988.11 [Deinococcus maricopensis DSM 21211], gil319754566lgblADV66323.11 [Deinococcus maricopensis DSM 21211], gil4557014lreflNP_001743.11 [Homo sapiens], gil6978607lreflNP_036652.1l [Rattus norvegicus], gil340622096lreflYP_004740548.1l [Capnocytophaga canimorsus Cc5], gil319954516lreflYP_004165783.1l[Ce//«to/?^fl algicola DSM 14237],

gil339902362lgblAEK23441. ll [Capnocytophaga canimorsus Cc5],

gil319423176lgblADV50285. ll [Cellulophaga algicola OSM 14237], gil297563504lreflYP_003682478. ll [Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111], gil297559004lreflYP_003677978.1l [Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111], and gil297561653lreflYP_003680627.1l [Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111]. Each sequence associated with the foregoing accession numbers is incorporated herein by reference.

[0055] Enzymes with similar enzymatic function as catalase, such as those enzymes belonging to the family of hyperoxidases, may also be used in the methods and compositions of the present disclosure. Examples of hyperoxidases may include, for example, glutathione peroxidase, haloperoxidase, and myeloperoxidase. One of skill in the art will readily recognize additional enzymes with similar enzymatic function as catalase and that may be used in the methods and compositions described herein.

[0056] Methods of obtaining catalase or other hyperoxidase enzymes are well-known in the art. Catalase may be obtained from a commercial supplier of purified catalase enzyme. Catalase may be recombinantly expressed and purified. Additional methods of obtaining purified catalase will be apparent to those skilled in the art.

[0057] In some embodiments, glucose oxidase and catalase may contact connective tissue cells in accordance with the methods of the present disclosure. In some embodiments, contacting connective tissue cells with exogenous glucose oxidase and catalase may produce a collagenous tissue possessing a high tensile strength. The glucose oxidase and catalase may be present in the culture media of connective tissue cells. The glucose oxidase and catalase may be applied exogenously to connective tissue cells by spraying or other exogenous application method. One of skill in the art will recognize various acceptable methods for contacting exogenous glucose oxidase and catalase to connective tissue cells and such methods may be used in accordance with the tensile strength enhancement methods of the present disclosure.

[0058] In some embodiments, connective tissue cells are cultured in the presence of glucose oxidase and catalase. In some embodiments, glucose oxidase and catalase are present in the growth media of the cultured connective tissue cells. In some embodiments, connective tissue cells are cultured in the presence of glucose oxidase and catalase present in a 10: 1 (glucose oxidase:catalase) ratio in the culture media. In some embodiments, the glucose oxidase and catalase are present in a glucose oxidase: catalase ratio of between 25: 1 to 1:25. Exogenous Copper and Hydroxylysine

[0059] The present disclosure relates to methods of producing collagenous tissue possessing a high tensile property involving contacting connective tissue cells with copper, contacting connective tissue cells with hydroxylysine, or contacting connective tissue cells with both copper and hydroxylysine to produce collagenous tissues possessing a high tensile property. In some embodiments, the copper is in the form of a salt (e.g., cupric sulfate) or bound to a carrier (e.g., ceruloplasmin, also known as ferroxidase)

[0060] Connective tissue cells contacted with copper, hydroxylysine, or both copper and hydroxylysine exhibit high tensile properties. The methods described herein involving contacting connective tissue cells with copper, hydroxylysine, or both copper and

hydroxylysine result in a neocartilage possessing an increased Young's modulus, an increased ultimate tensile strength, an increased aggregate modulus, an increase in PYR crosslinks, and an increase in collagen crosslinks when compared to a neocartilage not contacted with copper, hydroxylysine, or both copper and hydroxylysine.

Cartilage Construct Formation

[0061] Cartilage constructs can be formed using chondrogenic cells via a number of methods including but not limited to self-assembly, centrifugation, scaffold- seeding, hydrogel encapsulation, and in vivo cell-seeding.

[0062] In some embodiments, cartilage constructs may be formed using chondrogenic cells via self-assembly (see, e.g., US 2009/0142307 and US 2010/0303765). Briefly, the self- assembly process is a process of producing a cartilage construct which comprises culturing an aggregate of chondrogenic cells on a material that is not conducive to cell attachment (e.g., hydrogel such as agarose, alginate, 2-hydroxyethyl methacrylate polymer, etc.).

[0063] In some embodiments, cartilage constructs may be formed using chondrogenic cells via centrifugation. The centrifugation method involves the use of "pellet culture," in which isolated chondrocytes are first centrifuged into pellets inside centrifuge tubes. After several days of culture in the centrifuge tubes (to allow the mass of cells to aggregate), the pellets are then transferred onto various surfaces, including hydrogels, or left in the centrifuge tubes, either of which allows for the formation of constructs.

[0064] In some embodiments, cartilage constructs may be formed using chondrogenic cells via scaffold- seeding. The scaffold- seeding process involves forming cartilage constructs in which chondrocytes are attached to a scaffold or other surface to promote cell attachment. Methods of achieving cell attachment to a scaffold or other surface include but are not limited to employing a cross-linking mechanism, such as polymerization activators and ultraviolet (UV) radiation.

[0065] In some embodiments, cartilage constructs may be formed using chondrogenic cells via hydrogel encapsulation. Hydrogel encapsulation involves mixing chondrocytes with molten hydrogel to form aggregated chondrocytes surrounded by a hydrogel coat.

Alternative biomaterials to hydrogel may also be used to form the aggregation coat, including but not limited to biopolymers such as agarose or alginate.

[0066] In some embodiments, cartilage constructs may be formed using chondrogenic cells via a process involving sedimentation of the chondrocytes onto a hydrogel coated culture vessel, onto a shaped hydrogel negative mold.

[0067] In some embodiments, cartilage constructs may be formed using chondrogenic cells via in vivo cell-seeding. The in vivo cell-seeding process involves forming cartilage constructs in which chondrocytes are deposited into a joint of a subject by intra-articular injection.

Analysis of Cartilage Constructs

[0068] The properties of cartilage constructs may be tested using any number of criteria including, but not limited to, morphological, biochemical, and biomechanical properties, which also may be compared to native tissue levels. Morphological examination includes but is not limited to histology analysis using safranin- 0 and fast green staining for proteoglycan and GAG content, as well as picro-sirius red staining for total collagen,

immunohistochemistry for collagens I and II, and confocal and scanning electron

microscopies for assessing cell-matrix interactions. Biochemical assessments includes picogreen for quantifying DNA content, DMMB for quantifying GAG content,

hydroxyproline assay for quantifying total collagen content, ELISA for quantifying amounts of specific collagens (I and II), and RT-PCR for analysis of mRNA expression of proteins associated with the extracellular matrix (e.g. collagen and aggrecan).

Constructs also may be evaluated using one or more of incremental tensile stress relaxation, incremental compressive stress relaxation, and biphasic creep indentation testing to obtain moduli, strengths, and viscoelastic properties of the constructs. Incremental compressive testing under stress relaxation conditions may be used to measure a constructs compressive strength and stiffness. Incremental tensile stress relaxation testing may be used to measure a constructs tensile strength and stiffness. Additionally, indentation testing under creep conditions may be used to measure a constructs modulus, Poisson's ratio, and permeability. Analysis of the Young's modulus value and Aggregate modulus value of a construct are examples of parameters that may be used to assess properties of the construct relating to tensile strength.

Processes for Producing Integrated Collagenous Tissue

[0069] The present disclosure further relates to methods of producing an integrated collagenous tissue possessing a high tensile strength. In some embodiments, the methods comprise contacting a first collagenous tissue with a second collagenous tissue under conditions effective for the formation of enzyme-mediated collagen-crosslinks. In some embodiments, the methods may produce an integrated collagenous tissue possessing a high tensile strength. Conditions effective for formation of enzyme-mediated collagen cross-links to produce an integrated collagenous tissue having a high tensile strength may involve exogenous application of lysyl oxidase (LOX). Conditions effective for formation of enzyme-mediated collagen cross-links to produce an integrated collagenous tissue having a high tensile strength may involve exogenous application of both glucose oxidase (GOX) and catalase (CAT). Conditions effective for the formation of enzyme-mediated collagen crosslinks to produce an integrated collagenous tissue having a high tensile strength may involve culturing connective tissue cells under hypoxic conditions. In some embodiments, the level of enzyme-mediated collagen-crosslinks is directly proportional to the concentration of exogenously- supplied or endogenously-expressed lysyl oxidase present during the treating step. Conditions effective for formation of collagen cross-links to produce an integrated collagenous tissue having a high tensile strength may involve exogenous application of copper, exogenous application of hydro ylysine, or exogenous application of both copper and hydroxylysine. In some embodiments, the copper is in the form of a salt (e.g., cupric sulfate) or bound to a carrier (e.g., ceruloplasmin, also known as ferroxidase)

[0070] In some embodiments, the first collagenous tissue is a native cartilage. In some embodiments, the second collagenous tissue is engineered cartilage. In some embodiments, the first and second collagenous tissues are both native cartilage. In some embodiments, the first and second collagenous tissues are both engineered cartilage. [0071] In some embodiments, the collagenous tissues are contacted with lysyl oxidase. In some embodiments, lysyl oxidase is present in the growth media of the cultured

collagenous tissues. In some embodiments, collagenous tissues are contacted with lysyl oxidase via spraying or other exogenous application method. In some embodiments, collagenous tissues are contacted with lysyl oxidase in vivo on native tissue. In some embodiments, collagenous tissues are contacted with 0.015 μg/ml to 1.5 mg/ml lysyl oxidase. In some embodiments, collagenous tissues are contacted with lysyl oxidase at a concentration of more than 0.025 μ^πύ, 0.05 μ^πύ, 0.075 μ^πύ, 0.1 μ^πύ, 0.125 μ^πύ, 0.15 μ^πύ, 0.175 μ^πύ, 0.2 μ§/πύ, 0.25 μ^πύ, 0.3 μ^πύ, 0.35 μ^πύ, 0.4 μ^πύ, 0.45 μ^πύ, 0.5 μg/ml, 0.55 μ^πύ, 0.6 μ^πύ, 0.65 μ^πύ, 0.7 μ^πύ, 0.75 μ^πύ, 0.8 μ^πύ, 0.85 μ^πύ, 0.9 μg/ml, or 0.95 μg/ml. In some embodiments, the collagenous tissues are contacted with lysyl oxidase at a concentration of less than 1.5 mg/ml, 1.4 mg/ml, 1.3 mg/ml, 1.2 mg/ml, 1.1 mg/ml, 1.0 mg/ml, 0.9 μ^πύ, 0.8 μg/ml, 0.7 μ^πύ, 0.6 μ^πύ, 0.5 μg/ml, 0.4 μ^πύ, 0.3 μg/ml or 0.2 μ^ηύ. In some embodiments, exogenous LOX is provided at a concentration of between 0.075 to 0.75 μ^πύ.

[0072] In some embodiments, collagenous tissues are placed under hypoxic conditions. In some embodiments, the collagenous tissues are placed in an environment of from 0.5% to 11% oxygen. In some embodiments, the collagenous tissues are placed in a hypoxic environment of more than 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9% or 10% but less than 11% oxygen. In some embodiments, the collagenous tissues are placed in a hypoxic environment of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% oxygen. In some embodiments, the collagenous tissues are placed in an environment of from 1.0% to 10% oxygen.

[0073] In some embodiments, the collagenous tissues are contacted with glucose oxidase and catalase. In some embodiments, glucose oxidase and catalase are present in the growth media of the collagenous tissues. In some embodiments, collagenous tissues are contacted with glucose oxidase and catalase via spraying or other exogenous application method. In some embodiments, collagenous tissues are contacted with glucose oxidase and catalase in vivo on native tissue. In some embodiments, collagenous tissues are contacted with glucose oxidase and catalase present in a 10: 1 ratio (10 glucose oxidase: 1 catalase) in the culture media,. In some embodiments, collagenous tissues are contacted with glucose oxidase and catalase in a glucose oxidase:catalase ratio of between 25: 1 to 1:25.

[0074] The compositions of the present disclosure may be applied in a variety of ways known in the art. In some embodiments, the methods may involve direct, in vivo application of the compositions, such as compositions comprising one or more of lysyl oxidase, glucose oxidase, catalase, copper, and/or hydroxylysine, to connective tissue cells. The compositions may be administered either in their active form (e.g. intra- articular, intrasheath, and oral) or as pro-drugs. When the compositions are delivered as pro-drugs, the composition may remain inactive under certain conditions (e.g. normoxia), but are capable of being activated by other conditions (e.g. hypoxia). Methods of designing and using pro-drugs are well- known in the art. For example, an exemplary pro-drug delivery method may involve a one- electron reduction mediated by ubiquitous cellular reductases, such as the NADPH cytochrome P450, to generate a radical anion pro-drug (RP). In the presence of oxygen (normoxia), the RP reacts rapidly with oxygen to generate the original prodrug and superoxide (SO). Under the low oxygen conditions of the hypoxic zones, the radical anion pro-drug undergoes further irreversible reductions to the hydroxylamine (HA) followed by elimination, releasing the active drug and an azole derivative (AZ).

[0075] A variety of additional application methods are known in the art and may be used to apply compositions of the present disclosure to connective tissue cells so as to effect the formation of enzyme-mediated collagen-crosslinks. For example, the compositions may be applied as a liquid solution for intra- articular administration, as a cream solution for application to the skin, or as an orally administered drug.

Definitions

[0076] Unless defined otherwise, all scientific and technical terms are understood to have the same meaning as commonly used in the art to which they pertain. For the purpose of the present disclosure, the following terms are defined.

[0077] The term "construct" or "cartilage construct" as used herein refers to a three- dimensional mass having length, width, and thickness, and which comprises living mammalian tissue produced in vitro.

[0078] The terms "self-assemble" or "self-assembly" as used herein refer to a process in which specific local interactions and constraints between a set of components cause the components to autonomously assemble, without external assistance, into the final desired structure.

[0079] As used herein, the term "cell aggregate" refers to a cluster of cells. In contrast, the term "cell suspension" refers to a dispersion of cells in a liquid.

[0080] As used herein, the term "cartilage" refers to an avascular protective tissue in the form of a matrix comprising collagen, proteoglycans and elastin.

[0081] The term "about" as used herein in reference to a numerical value refers to a value that is from 10% below to 10% above the numerical value. For instance "about 5% oxygen" refers to an oxygen content of 4.5% to 5.5%.

EXAMPLES

[0082] Abbreviations: ACs (articular chondrocytes); ACL (anterior cruciate ligament); BAPN (beta-aminoproprionitrile); CAT (catalase); CM (culture medium); col I (collagen type I); col II (collagen type II); DMEM (Dulbecco's modified Eagle's medium); ECM

(extracellular matrix); FBS (fetal bovine serum); GAG (glycosaminoglycan); GOX (glucose oxidase); IHC (immunohistochemistry); ITS (insulin, transferrin, selenium); LOX (lysyl oxidase); MCs (meniscus cells); Men (medial meniscus); NEAA (non-essential amino acids); Pat (patellar tendon); PCL (posterior cruciate ligament); PSF

(penicillin/streptomycin/fungizone); PYR (pyridinoline); SD (standard deviation); UTS (ultimate tensile strength); and WW (wet weight).

EXAMPLE 1

Use of Lysyl Oxidase to Promote Collagen- Crosslinking in Engineered Tissues

[0083] This example describes how application of exogenous lysyl oxidase (LOX) alters the biochemical and biomechanical properties of engineered tissue in both a concentration- dependent and time of application-dependent manner.

Materials and Methods

[0084] Chondrocyte Isolation. Articular cartilage were harvested from the distal femur of 1 week old calves, diced into 1 mm pieces and digested in 0.2% collagen 's type II

(Worthington) in cell medium culture. The medium formulation follows: Dulbecco's modified Eagle's medium (DMEM) (Invitrogen), 10% fetal bovine serum (FBS)

(Benchmark), 1 % non-essential amino acids (NEAA) (Invitrogen), 25 p of 1- ascorbic acid (Sigma) and 1% penicillin/stre tomycin/fungizone (PSF) (Biowhittaker/Cambrex).

Following an 18 hour digestion in collagenase, articular chondrocytes were isolated, frozen at -80°C for 24 hours, and stored in liquid nitrogen until needed. [0085] Self-assembly and Culture ofNeocartilage. Two days after freezing cells were thawed and counted. Viability using trypan blue was over 90%. Articular chondrocytes were seeded into cylindrical, non-adherent agarose wells at a concentration of 5.5 million in 100 μΐ of medium, as previously described (Hu et al., Tissue Eng 12:969-979, 2006; Ofek., et al., PLoS One, 3:e2795, 2008), and fed with chondrogenic medium (Elder et al., Tissue Eng Part A, 15: 1151-1158, 2009; Natoli et al., Arthritis Rheum 62: 1097-1107, 2010; Natoli et al., Tissue Eng Part A, 15:3119-3128, 2009): DMEM (Invitrogen), 1% NEAA (Invitrogen), 100 nm dexamethasone (Sigma), 1% ITS + premix (BD Biosciences), 40 mg mL L-proline (Sigma), 50 mg/mL ascorbate-2-phosphate (Sigma), 100 mg/mL sodium pyruvate (Fisher Scientific), 0.146 mg/ml hydroxylysine (Sigma), 0.0016 mg/ml copper (Sigma), and 1% PSF (Biowhittaker/Cambrex)). At 4 hours post-seeding, 400 μΐ of medium was added. Through self-assembly, cells coalesced into disc-shaped constructs. At t=10 days constructs were unconfined from the agarose wells and transferred into 48-well plates as previously described (Hu et al., Tissue Eng 12:969-979, 2006). Constructs were incubated (37°C and 10% C0₂) until t=28 days receiving 500 μΐ medium, changed every 24 hours.

[0086] LOX Application. This study includes two phases. Two different types of culture medium were employed in both phases: control medium and medium supplemented with LOX. Phase I investigated the effects of LOX concentration. Three different concentrations of LOX were investigated: Low (0.0015μ /πι1), Medium (0.015 μ^πύ), and High

(0.15μ /Γπ1) during a 4 week culture period. LOX-treated groups received LOX medium during t=8-22 days. In phase II, LOX was applied at two different time points at the concentration identified in Phase I (i.e., 0.15μ /πι1). This study examined three different groups: Control, Early LOX (applied t=8-14 days), and Late LOX (applied t=15-21 days) during a 6 week culture period.

[0087] Gross Morphology. After the end of culture period (t=28 days and t=42 days for phase I and II, respectively), constructs were removed from the incubator and photographs were taken to measure construct dimensions (diameter and thickness), using ImageJ. After recording the wet weight, constructs were portioned for biochemical and biomechanical evaluation. A 3 mm diameter cylindrical punch, taken from the center of each construct, was used for creep indentation testing¹ while the outer ring was divided for tensile testing, histology, and quantitative biochemistry as previously described (Natoli et al., J Orthop Res, 27:949-956, 2009). [0088] Histology/Immunohistochemistry (IHC). For histology, constructs were cryo- embedded at -20 °C in HISTOPREP™ (Fisher Scientific) and were sectioned at 14 μηι, placed on histological glass slides and warmed at 37 °C overnight. The sections were then fixed in 10% formalin and stained using Safranin-O/fast green and Picrosirius red for GAG and collagen respectively. Phenotype maintenance of articular chondrocytes was evaluated with IHC. Samples sections were fixed in 4°C acetone and stained for collagen type I and II as previously described (Hennerbichler et al., Am J Sports Med, 35:754-762, 2007).

[0089] Quantitative Biochemistry. Samples collected for biochemical evaluation were weighed, frozen, lyophilized, and the dry weight was recorded. Samples were then digested in phosphate buffer with 5 mm EDTA, 5 mm N-acetyl-cysteine, and 125 ^ig/mL papain (Sigma) for 18 h at 65 °C. Following digestion of the samples total collagen was quantified using chloramine-T hydroxyproline assay (Woessner et al., J.F., Arch Biochem Biophys, 93:440-447, 1961). Sulfated GAG was quantified using the Blyscan Glycosaminoglycan Assay (Biocolor) and total number of cells was estimated with a Picogreen DNA Assay (Invitrogen) assuming 7.7 pg DNA per cell as previously described (Natoli et al., J Orthop Res, 27:949-956, 2009).

[0090] High Performance Liquid Chromatography (HPLC). HPLC samples were digested in 800 μΐ_^ of 6 N HC1 at 100°C for 18 hours. Following digestion, samples were dried using a vacuum concentrator and re-suspended in 50 μΐ_^ a solution of 10 nmol pyridoxine/ml and 2.4 μιηοΐ homoarginine/ml. Samples were subsequently diluted fivefold with 0.5% HFBA in 10% acetonitrile. 10 μΐ of each sample was analyzed as described previously (Bank et al., J Chromatogr B Biomed Sci Appl 703:37-44, 1997). Pyridinoline standards (Quidel, San Diego, CA) were used to quantify crosslink content.

[0091] Creep Indentation Testing. The compressive properties of each construct were evaluated using a creep indentation apparatus as previously described (Athanasiou et al., Clin Orthop Relat Res, 254-266, 1995). Sample thickness was measured using a micrometer. Specimens were glued to a stainless steel surface and kept until equilibrium in PBS for 15 minutes. A 0.7g mass applied to specimens through a flat, porous indenter tip (0.8 mm diameter). Samples crept until equilibrium. The compression properties of the samples approximate by calculating the aggregate modulus using as a semi-analytical, semi-numeric, linear biphasic model (Athanasiou et al., Clin Orthop Relat Res, 254-266, 1995). [0092] Tensile Testing. The tensile properties of the samples were determined using an uniaxial materials testing apparatus (Instron Model 5565). Specimens were prepared into dog-bone shapes and their dimensions (thickness and width) measured from photographs using ImageJ software. Samples then tested as previously described (Elder et al., Tissue Eng Part A, 15: 1151-1158, 2009; Natoli et al., Arthritis Rheum 62: 1097-1107, 2010). Applying a strain rate of 1% of the gauge length per second the samples were tested until failure. Tensile stiffness of the samples was represented by Young's modulus, which was calculated by least squares fitting of the linear region of the stress-strain curve. The ultimate tensile strength (UTS) was represented the maximum stress reached during sample testing.

[0093] Statistical Analyses. All quantitative assessments in this study were performed using n = 5-7 samples per group. Numerical data are represented as means + standard deviations. To compare among treatment groups, one-way ANOVA analysis of variance was performed. Fisher LSD post hoc testing was applied, if significance (p < 0.05) was identified. Statistical significance in figures illustrating biornechanical and biochemical data among individual groups or levels of a given factor is shown with non-shared letters.

Results

Phase 1 - Effects ofLOX Concentration on Properties of^'Neocartilage

[0094] At the end of the 4 week self-assembly process, tissue engineered cartilage constructs treated with Low, Medium, or High LOX were evaluated, along with controls (no LOX), using gross morphology and histology to assess the composition and gross properties of the tissue. Neocartilage constructs from all groups exhibited no significant differences in their morphology. All presented similar flat surfaces without abnormalities. No contraction was detected in any of the constructs, which exhibited the following diameters: 5.03+0.05, 5.05+0.03, 5.06+0.06, and 5.08+0.06 mm for control, Low, Medium, and High LOX treatments, respectively. No significant differences were detected in terms of the thickness of the constructs, having values of 0.53+0.01, 0.53+0.02, 0.52+0.02, and 0.51+0.01 mm for control, Low, Medium, and High LOX treatments, respectively. Histology showed that all constructs stained positive for both GAG and collagen. Additionally, immunohistochemistry (IHC) showed that all neotissue from all groups stained positively for collagen type II and negatively for collagen type I. In general, the presence of total collagen and GAG, and specifically of collagen type II but not type I, shows normal cartilage phenotype maintenance in all groups. [0095] Maturational growth of neotissue was evaluated by assessing total collagen, GAG, and pyridinoline (PYR) at the end of the culture period. No significant differences were detected concerning the percentage of GAG normalized to construct wet weight (GAGAVW). The mean+SD percentages of GAG per construct wet weight (WW) were 1.50+0.21, 1.60+0.22, 1.23+0.03 and 1.46+0.41% for control, Low, Medium, and High LOX, respectively. No significant differences among groups were detected for total collagen per construct wet weight (collagenAVW); mean+SD values were 1.87+0.25, 1.64+0.21,

1.83+0.18, and 1.95+0.33% for control, Low, Medium, and High LOX, respectively. The results for PYR normalized to construct wet weight are presented in FIG. 3B. High LOX treatment resulted in constructs with significantly increased PYR concentration compared with controls or other LOX treatments. There was a 46.4% increase over control in the percentage of PYR normalized to wet weight for the High LOX treated group.

[0096] No significant differences in compressive properties were measured among groups. The mean+SD construct aggregate modulus values measured were 130+30, 111+19, 108+15, and 124+23 kPa for control, Low, Medium, and High LOX treatment, respectively. Permeability values were 16.7+13.4, 19.2+10.5, 17.8+12.8 and 16.8+11.2 x 10^"15 m⁴/N s, and the compressive Poisson's ratio values were 0.13+0.08, 0.19+0.13, 0.17+0.11, and 0.13+0.08 for control, Low, Medium, and High, respectively. FIG. 3A shows the construct tensile stiffness. High LOX treatment significantly increased Young's moduli compared with control and other LOX treated groups. There was a 164% increase in tensile stiffness compared with control. Other than the High LOX group, no other LOX treated groups were shown to have significant differences in tensile stiffness. Only the High LOX treated constructs had significantly greater ultimate tensile strength (UTS) than control (an 78% increase); values for the UTS were 259+42, 292+58, 296+66, and 461+100 kPa for control, Low, Medium, and High LOX treatment, respectively.

Phase II - Effect of Time of LOX Application on Properties of Neocartilage

[0097] Phase II examined the effects of Early and Late LOX application on self- assembled articular cartilage. At the end of the 6 week culture period, the biochemical composition, biomechanical properties, gross morphology, and histology of constructs were evaluated.

[0098] As with Phase I, constructs from all groups had no significant differences in their morphology and presented similar flat surfaces without abnormalities. No contraction was detected in any of the constructs, which exhibited the following diameters: 5.52+0.20, 5.83+0.38, and 5.47+0.12 mm for control, Early, and Late LOX treatment, respectively. No significant differences were detected in terms of the thickness of the constructs, having values of 0.53+0.01, 0.53+0.02, 0.52+0.02, and 0.51+0.01 mm for control, Early, and Late LOX treatment, respectively. Histology results showed that all constructs were positively stained for both collagen and GAG. IHC showed positive staining for collagen type II and negative staining for collagen type I, for all samples, demonstrating normal cartilage phenotype maintenance.

[0099] Assessment of total collagen, GAG, and PYR evaluated maturational growth of self-assembled articular cartilage at the end of the 6 week culture period. No significant differences were detected among groups for the percentage of GAGAVW or total collagen per construct- wet weight. The mean+SD percentages of GAGAVW were 1.83+0.23, 1.62+0.08, and 1.73+0.11% for control, early, and late LOX groups, respectively. CollagenAVW values werel+0.18, 1.21+0.20, and 1+0.12% for control, early, and late LOX respectively. FIG. 4B shows the results for PYR normalized to construct wet weight (PYRAVW). Both Early and Late LOX treatments resulted in constructs with significantly increased PYR contents compared to control. There was a -100% and -150% increase over control in the percentage of PYR normalized to wet weight for the Early and Late LOX treated groups, respectively.

[00100] Regarding the neotissue's mechanical properties, early LOX application significantly increased the compressive properties of the constructs as shown in FIG. 4C. Construct aggregate modulus values were 153+30, 220+47, and 175+65 kPa for control, Early and Late LOX groups, respectively. Permeability values were 17.8+13.1, 18.2+17.1, and 16.1+13.9X 10^"15 m⁴/N s, while the compressive Poisson's ratio values were 0.14+0.08, 0.09+0.05, and 0.10+0.09 for control, Early, and Late LOX groups, respectively. As shown in FIG. 4A, Early LOX application increased the Young's modulus value by 106% over control, while Late LOX treatment had no significant effect on the tensile stiffness of the constructs. Similarly, only the Early LOX treated constructs had significantly greater UTS values than control, resulting in a 101% increase. UTS values were 436+144, 982+379, and 463+243 kPa for control, Early, and Late LOX groups, respectively.

Conclusions

[00101] As described herein, the present disclosure provides methods to enhance the biomechanical properties of engineered musculoskeletal tissue by temporal modulation of collagen crosslinking via exogenous lysyl oxidase (LOX). In short, LOX application was demonstrated to enhance pyridinoline abundance and concomitantly increase neocartilage tensile properties. Specifically exogenous LOX: 1) enhances the tensile stiffness and strength of self- assembled neocartilage; 2) increases pyridinoline abundance; 3) enhances biomechanical properties in a concentration-dependent manner; and 4) enhances

biomechanical properties in a time-dependent manner.

[00102] Phase I of this study demonstrated that a modest LOX concentration was necessary to enhance the biomechanical properties of neocartilage. A LOX concentration range of 0.075 μg/ml to 0.75 μg/ml or 7.5 μg/ml is contemplated to be effective in enhancing cross-linking of collagenous tissue. In fact, the exemplary LOX concentration of 0.15 μg/ml was orders of magnitude lower than a previous study that used a concentration of 2.5 ng/cell for treating smooth muscle cells (Kothapalli et al., J Tissue Eng Regen Med, 3:655-661, 2009). The measurable efficacy of the minute LOX doses used in this example is very encouraging since it is known that articular cartilage forms from a condensate of cells, and the use of LOX was heretofore considered to be impractical for treating highly cellular neotissue.

[00103] Phase II of this study demonstrated that earlier LOX administration had a more pronounced influence on both pyridinoline content and tensile properties. Although LOX forms the precursor for crosslink production, a series of intermediates must be produced prior to pyridinoline formation (Eyre et al., Biochem J 252: 495-500, 1988). The results of this study also show that even one additional week of culture significantly enhances crosslink formation, highlighting the importance of early LOX treatment, and that early LOX treatment also increases compressive stiffness without affecting the amount of GAG normalized to wet weight.

[00104] The Young's moduli of native and engineered articular cartilage demonstrated correlations with cartilage- specific macromolecules such as total collagen, collagen type II and IX, sulfated GAGs, and pyridinoline crosslinks (Yan et al., Biomaterials, 30:814-821, 2009). Importantly, the present disclosure highlights the strong correlation among the mechanical properties of engineered tissues and collagen crosslinking. Exogenous LOX application provides an exciting new technology for promoting collagen network

development in neocartilage. LOX has the potential to promote tissue maturation in a spectrum of collagen-rich tissues including bone, tendon, meniscus, and ligaments. EXAMPLE 2

Use of Lysyl Oxidase to Enhance Cartilage-to- Cartilage Integration

[00105] This example describes how application of exogenous lysyl oxidase (LOX) to cartilage-to-cartilage interfaces enhances integration of the two cartilage types and increases the tensile strength of the cartilage.

Materials and Methods

[00106] Chondrocyte Isolation. Articular cartilage was harvested from distal femurs of one- week old male calves (Research 87 Inc., Boston, MA) less than 36hr after sacrifice. Following harvest, the tissue was digested in 0.2% collagenase type II (Worthington, Lakewood, NJ) in culture medium for 24hr as previously described (Natoli et al., J Orthop Res, 27:949-956, 2009). The culture medium formulation used was follows: DMEM with 4.5 mg/mL glucose and L-glutamine, 100 nM dexamethasone, 1% fungizone, 1%

penicillin/streptomycin, 1% ITS+, 50 mg/mL ascorbate-2-phosphate, 40 mg/mL L-proline, and 100 mg/mL sodium pyruvate. Cell viability was assessed using trypan blue exclusion, and cells were frozen at -80°C using DMEM containing 20% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 10% dimethyl sulfoxide. Cells were maintained at - 80°C until use. To reduce variability, cells from four animals were pooled together for cell seeding.

[00107] Construct self-assembly. Cylindrical, non-adherent, agarose wells were prepared by placing 5mm diameter stainless steel posts in 48 well plates filled with 1ml of 2%, molten agarose, as previously described (Responte et al., Biomaterials, 33:3187-3194, 2012). After the agarose gelled, posts were removed. The resultant wells were saturated with two exchanges of medium. After thawing, cells were counted, viability was assessed using trypan blue exclusion, and cells were seeded into the agarose wells at a concentration of 5.5 million/100 ml medium. After 4hr, an additional 400ml of medium was added per well. Constructs were cultured at 10% CO2, 37°C, in a humidified incubator for a total of t=28d (t=ld defined as 24hr post seeding). Medium was changed daily (500 ml).

[00108] Tissue Integration. Two separate, but concurrent, experiments were conducted. First, the use of LOX was examined for the construct-to-native interface. At t=28d, engineered constructs were removed from culture and prepared for integration with native articular cartilage. Bovine articular cartilage explants, measuring 6mm x 1mm, were harvested using biopsy punches. A concentric, 4 mm diameter defect was punched from the explant. From the engineered constructs, 4 mm diameter biopsies were obtained and press- fitted into the defect in the explant, using three, 5 μΐ drops of cyanoacrylate at equally- spaced locations on top of the construct/explant assembly (FIG. 5). These constructs/explant assemblies were cultured for an additional 14d, at which point they were removed for assessments. The second experiment consisted of explants instead of constructs. Native-to- native tissue assemblies were formed using the same geometry as described above.

[00109] Collagen Cross-Linking Via Lysyl Oxidase. The LOX medium contained a concentration of 0.15 μg/ml LOX (GenWay Biotech, Inc., San Diego, CA). For the construct-to-native study, four groups were examined. The control (Group A) consisted of construct/explant assemblies maintained in culture medium only. Group B was treated with the LOX medium during t=15-28d; Group C was treated during t=29-35d; Group D was treated during t=15-35d. Assemblies were assessed at t=42d to allow for a total of 14d of integration time. For the native-to-native study, two groups were examined. The control group was allowed to integrate for 14d in culture medium, while the LOX Group was maintained in a LOX medium during the same time.

[00110] Histology: Frozen sections were fixed in 10% neutral buffered formalin and stained with picrosirius red to demonstrate collagen distribution, as previously described (Hu et al., Tissue Eng 12:969-979, 2006).

[00111] Biochemistry. Total collagen was assessed using a hydroxyproline assay, and glycosaminoglycan content was measured using a Blyscan kit, both as previously described (Hu et al., Tissue Eng 12:969-979, 2006).

[00112] Tensile Testing. Assemblies were cut into strips 1mm wide. Thickness and width were verified photographically using ImageJ (National Institutes of Health, Bethesda, MD). Specimens were clamped and exposed to constant uniaxial strain of 1% gauge length per second until failure using a uniaxial materials testing machine (Instron 5565). Force- deformation data were collected to yield the stress-strain response. The Young's modulus was the slope of the linear region of the graph. The ultimate tensile strength (UTS) was the maximum stress attained by the specimen before failure. The maximum strain was the strain corresponding to the maximum stress. Statistical analysis: Using n=6 samples, data were compiled as mean+standard deviation and analyzed using a single factor ANOVA. If the F- test was statistically significant, a Tukey' s post hoc test was employed to identify significant groups. Significance was defined as p<0.05.

Results

Integration of engineered constructs to native articular cartilage

[00113] For all treatment durations, LOX-treated construct-to-native assemblies displayed better integration as compared to controls using gross morphology, histology, and

biomechanical evaluations. Grossly, gaps were seen between the construct and native tissue in 33% of the controls, showing that the cyanoacrylate used to initially bond the assembly together did not persist throughout the culture period (FIG. 6). Gaps were not seen in any of the LOX-treated specimens. Similarly, histological evaluation showed gaps in the controls, while LOX-treated samples showed construct adherence to the native tissue. Tensile testing across the integration interface showed significantly higher Young's modulus values when LOX was applied during t=15-35d (Group D), 1.6+0.6 MPa, versus control values of 0.7+0.2 MPa (FIG. 7). Significantly higher UTS values were observed when LOX was applied before formation of the construct-to-native assembly (Groups B and D), 0.42+0.07 MPa and 0.39+0.06 MPa respectively, versus control and post-formation LOX application values of 0.23+0.08 MPa and 0.28+0.1 MPa respectively (FIG. 7). No significant differences were detected in the GAG/ww or collagen/ww among the construct or the explant portions of the assemblies. Specifically, no significant differences were detected in the GAG/ww content among the constructs (4.5+1.4%, 2.9+1.4%, 3.1+0.5%, and 4.5+0.6% for Groups A-D, respectively) or among the explant rings (8.6+1.1%, 10.9+1.9%, 9.6+01.2%, and 10.5+2.8% for Groups A-D, respectively). Additionally, no significant differences were detected in the collagen/ww content among the constructs (5.4+2.2%, 5.7+1.7%, 5.7+0.7%, and 7.2+3.1% for Groups A-D, respectively) or among the explant rings (5.6+3%, 10.6+7.5%, 6.2+3%, and 10.5+3.2% for Groups A-D, respectively).

Integration between native cartilage tissues

[00114] Qualitatively, the only difference between control and LOX-treated native-to- native assemblies was seen by histology (FIG. 8). Additionally, the Young's modulus values of the LOX-treated group was more than twice that of the control (1.5+1.1 MPa versus 0.7+0.4 MPa), though neither this property nor the UTS were statistically significant (FIG. 9). GAG and collagen content were not different between the two groups. Conclusions

[00115] As evidenced by the biomechanical and histological data, the present disclosure provides methods employing LOX to enhance cartilage-to-cartilage integration. Engineered tissues, formed using a self-assembling process, were integrated to native tissue explants by applying LOX to a ring-and implant assembly. Additionally, LOX application to native-to- native cartilage interfaces was found to enhanced integration. In short, cartilage integration can be enhanced when the interface is stocked with metabolically active cells and cross-links simultaneously. This was confirmed to be true because enhanced interfacial properties were observed for construct-to-native but not for the native-to-native case. Group D, which was treated with LOX for the longest period of time, had statistically higher tensile properties than did the other three groups. Group D had approximately 2.2 times the tensile strength of controls. This was confirmed with morphological and histological data. The results of this study are significant for cartilage regeneration and repair methods.

[00116] It is worth noting that, despite the lack of any significant differences in the collagen and GAG content in either the construct or explant groups, there were significant LOX-induced increases in interface biomechanics. The fact that interfacial strength increased manifold, in the absence of increases in the main ECM components, indicates that cross-links play a central role in integration.

[00117] The study demonstrates that LOX is a potent agent for enhancing integration between native and tissue engineered cartilage. It also paves the way for the use of LOX in improving native cartilage integration. Thus the present disclosure provides methods to solve the problem of repairing cartilage defects.

EXAMPLE 3

Use of Hypoxia to Enhance the Mechanical Properties of Articular Cartilage

through Collagen Crosslinking

[00118] This example describes how culturing articular cartilage under low oxygen conditions promotes collagen crosslinking and increases tensile strength of neocartilage.

Materials and Methods

[00119] A two-factor experimental design was employed to investigate the effect of hypoxia and beta-aminoproprionitrile (BAPN) on articular cartilage explants. Since hypoxia is hypothesized to act through LOX activity, BAPN was chosen as an inhibitor with demonstrated activity in preventing LOX-mediated collagen crosslinking (Ahsan et al., Osteoarthritis Cartilage, 13:709-715, 2005). Explants harvested from the patellofemoral groove of 6 juvenile calves were cultured in serum containing medium under either normoxic (21% 0₂) or hypoxic (4% 0₂) conditions with or without 0.25 mM BAPN. At t=3 days, LOX gene expression was evaluated. At t=2 weeks, tissue size, volume, tensile and compressive mechanical properties, and biochemical content (collagen, glycosaminoglycans, DNA, and pyridinoline) were assessed.

Results

[00120] Incubation of cartilage explants for 2 weeks resulted in expansive tissue growth with significant increase in thickness, wet weight, and water content in all groups. Real time PCR results showed elevated LOX gene expression in all groups treated with hypoxia, with the highest seen in the hypoxia only group, at 43% over controls (p<0.05). BAPN treatment also increased LOX expression over controls, though it was below that of groups treated with hypoxia (FIG. 10A).

[00121] Indicative of increased crosslinking, significantly higher pyridinoline per collagen (PYR/COL) was detected in the hypoxia treated group without BAPN inhibition (36% increase over controls, p<0.05). BAPN abolished hypoxia-induced increases in pyridinoline (FIG. 10B).

[00122] Concomitant with the high amount of PYR/COL, significantly higher tensile properties were measured for the hypoxia treated group without BAPN inhibition (138% over controls, p<0.05). BAPN treatment abolished hypoxia-induced gains in tensile properties (FIG. IOC). No significant differences in compressive properties, collagen, and GAG content were detected among groups.

[00123] Tensile stiffness represented by Young's modulus was significantly enhanced in explants treated with hypoxia over the normoxia treated explants as shown in FIG. 11. The hypoxia-induced gains in the Young' s modulus values of anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), patellar tendon (Pat) and medial meniscus (Men) explants were partially reduced by BAPN. Conclusions

[00124] Hypoxia has been successfully employed herein to specifically target and increase the tensile properties of articular cartilage. This has been shown to occur through hypoxia- induced LOX up-regulation and resultant increases in pyridinoline crosslinks per collagen fiber. These results were confirmed using the LOX inhibitor BAPN, since measured differences resulting from the application of hypoxia (Young's moduli, PYR/WW) were reduced with the addition of BAPN. These results are clinically relevant for articular cartilage repair therapies, since hypoxia positively influences tensile properties without diminishing compressive properties.

EXAMPLE 4

Use of Glucose Oxidase and Catalase to Enhance the Mechanical Properties of Articular

Cartilage through Collagen Crosslinking

[00125] This example describes how use of glucose oxidase (GOX) and catalase (CAT) can trigger hypoxic conditions in tissues and that the degree of hypoxia can be controlled as a function of enzyme concentration. Use of the GOX/CAT system to generate hypoxic conditions is an effective way to increase collagen crosslinking and increases tensile strength of neocartilage.

The GOX/CAT System

[00126] The GOX/CAT system is established by the addition of glucose oxidase (GOX) and catalase (CAT) to buffered solutions containing at least 5 mraol/L D-glucose. GOX generates hydrogen peroxide (H₂O₂) by consuming oxygen, while catalase degrades H₂O₂ back to water and half a molecule of oxygen. Thus, the overall reaction consumes oxygen, which is the prerequisite for generating hypoxia. Due to the special kinetic properties of GOX and CAT, the system generates stable H₂O₂ concentrations that depend on the ratio of the enzyme activities. In contrast, hypoxia is mainly controlled by the GOX activity and the medium volume since it defines the diffusion distance for gaseous oxygen to reach the adherent cells on the tissue culture vessel bottom. The GOX/CAT system offers the unique opportunity to independently control both hypoxia and I ! _.·(); in cell culture. All other molecules involved in the system are either physiological (water, glucose) or physiologically inert (gluconolactone) under cell culture conditions. Materials and Methods

[00127] Enzymatic hypoxia medium was prepared by diluting glucose oxidase (Sigma Catalog No. G0543, CAS No. 9001-05-2, EC No. 1.1.3.4) and catalase (Sigma Catalog No. C3155, CAS No. 9001-37-0, EC No. 1.11.1.6) at a constant 1: 10 ratio in cell culture medium. Enzyme activities of stock solutions were 3 mM/s for GOX and 998 mM/s for CAT, To obtain a defined, stable oxygen concentration of 2% at the cell surface, stock solutions were diluted by 1:10,000 for GOX and 1:1,000 for CAT, The medium volumes used were 2.5 ml for 6-well plates and the tissues were incubated at 37°C, Previous experiments using a computer-driven oxygen electrode Oxi 325-B (WTW, Weilheim, Germany) for oxygen measurement have revealed that at those conditions 2% hypoxia was rapidly induced within 15 min and maintained over 24 hours.

Results

[00128] The studies investigating the effects of the GOX/C AT enzymatic system both in native and engineered tissue cultures revealed the effectiveness of the system to replicate hypoxic conditions in tissue culture medium and maintain stable hypoxic conditions for over 24 hours. Mechanical testing and biochemical evaluation of the cultured tissues showed enhanced mechanical properties of the tissues and increased formation of pyridinoline collagen crosslinks. RT-PCR data showed increased gene expression of lysyl oxidase in the enzymatic hypoxia- treated groups.

EXAMPLE 5

Use of Copper and Hydroxylysine to Enhance the Mechanical Properties of

Neocartilage

[00129] In this example the effect of both copper (Cu) and hydroxylysine (AA) on tensile properties of neocartilage was examined. Briefly, neocartilage constructs were treated with 0.0016 mg/mL copper (Cu), provided as copper sulfate, and 0.146mg/ml hydroxylysine (AA), either alone or in combination, or left untreated as controls. Treatments were carried out for the entire culture duration, after which time properties of the neocartilage were assayed. As shown in FIG. 12A and FIG. 12B, copper significantly increases the Young's modulus and Ultimate Tensile Strength of neocartilage as compared to values determined for controls. [00130] A similar study was conducted to assess the impact of Cu and AA on PYR crosslinks and further parameters related to collagen tensile strength. Native and engineered tissues were either generated using a scaffoldless, self-assembly process or harvested directly and supplemented with Cu and AA, either alone or in combination, following a two-factor, full factorial study design. At the end of a 6-week culture period, the compressive and tensile properties of self-assembled neocartilage constructs along with collagen, glycosaminoglycan (GAG), DNA, and pyridinoline (PYR) content were measured for the following groups: control, AA, Cu, and Cu + AA. It was observed that Cu significantly increased PYR crosslinks in all applied groups over controls. When Cu and AA were combined, the result was synergistic, with a 1016% increase in PYR content over controls; this increase in the PYR crosslinks manifested in a synergistic 3.3-fold increase in the tensile properties in the Cu + AA group. Additionally, while all treatments significantly increased the compressive properties over controls, a 143% increase over control values was detected in the Cu group in terms of the aggregate modulus. These data further suggest a role for copper and

hydroxylysine in improving the mechanical properties of engineered neocartilage through collagen crosslinking enhancement.

Claims

CLAIMS We claim:

1. A method of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength.

2. The method of claim 1, wherein the conditions comprise culturing the connective tissue cells in the presence of 0.015 μg/ml to 1.5 mg/ml lysyl oxidase (LOX).

3. The method of claim 1, wherein the conditions comprise culturing the connective tissue cells under hypoxic conditions of 0.5% to 11% oxygen.

4. The method of claim 1, wherein the conditions comprise culturing the connective tissue cells in the presence of glucose oxidase (GOX) and catalase (CAT).

5. The method of any one of claims 1-4, wherein the level of enzyme-mediated collagen-crosslinks is directly proportional to the concentration of exogenously- supplied or endogenously-expressed lysyl oxidase present during the treating step.

6. A method of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength, wherein the conditions comprise culturing the connective tissue cells in the presence of 0.015 μg/ml to 1.5 mg/ml lysyl oxidase (LOX).

7. A method of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength, wherein the conditions comprise culturing the connective tissue cells under hypoxic conditions of 0.5% to 11% oxygen.

8. A method of producing collagenous tissue possessing a high tensile strength, comprising: treating connective tissue cells under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce collagenous tissue possessing a high tensile strength, wherein the conditions comprise culturing the connective tissue cells in the presence of glucose oxidase (GOX) and catalase (CAT),

9. The method of any one of claims 6-8, wherein the connective tissue cells comprise chondrogenic cells.

10. The method of claim 9, wherein the chondrogenic cells are primary cells isolated from one or more of the group consisting of articular cartilage, fibrocartilage, elastic cartilage, and hyaline cartilage.

11. The method of claim 9, wherein the chondrogenic cells are primary chondrocytes isolated from articular cartilage.

12. The method of claim 9, wherein the chondrogenic are cultured cells expanded from primary cells isolated from one or more of the group consisting of articular cartilage, fibrocartilage, elastic cartilage, and hyaline cartilage.

13. The method of claim 9, wherein the chondrogenic cells are cultured chondrocytes expanded from primary cells isolated from articular cartilage.

14. The method of claim 9, wherein the chondrogenic cells comprise

mesenchymal stem cells isolated from a source selected from the group consisting of bone marrow, adipose tissue, synovium, periosteum, dermis, umbilical cord blood, synovial fluid, muscle, and tendon.

15. The method of claim 9, wherein the chondrogenic cells comprise pluripotent stem cells.

16. The method of claim 9, wherein the chondrogenic cells are cultured during the contacting step using a construct formation technique selected from the group consisting of self-assembly, centrifugation, scaffold seeding, hydrogel encapsulation, and in vivo cell- seeding.

17. The method of claim 9, wherein the chondrogenic cells are cultured during the contacting step using a self-assembly construct formation technique.

18. The method of claim 9, wherein the collagenous tissue comprises cartilage.

19. The method of claim 18, wherein the cartilage is engineered tissue.

20. The method of claim 18, wherein the cartilage is native tissue.

21. The method of claim 9, wherein the collagenous tissue comprises one or both of a tendon and a ligament.

22. The method of claim 9, wherein the collagenous tissue has a higher Young's modulus than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression.

23. The method of claim 9, wherein the collagenous tissue has a higher ultimate tensile strength value than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression.

24. The method of claim 9, wherein the collagenous tissue has a higher pyridinoline content than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression.

25. The method of claim 9, wherein the collagenous tissue has a higher pyrrole content than does a control collagenous tissue produced essentially in the absence of exogenously- supplied lysyl oxidase or in the absence of induction of endogenous lysyl oxidase expression.

26. A method of producing collagenous tissue possessing a high tensile property, comprising: contacting connective tissue cells with a composition comprising an effective amount of copper to produce collagenous tissue possessing a high tensile property.

27. The method of claim 26, wherein the composition further comprises hydroxylysine.

28. A composition comprising isolated collagenous tissue, and exogenous lysyl oxidase.

29. A composition comprising isolated collagenous tissue, glucose oxidase (GOX), and catalase (CAT).

30. A composition comprising isolated collagenous tissue, and copper, wherein the copper is in the form of a salt or bound to a carrier.

31. A method of producing an integrated collagenous tissue possessing a high tensile strength, comprising: contacting a first collagenous tissue with a second collagenous tissue under conditions effective for formation of enzyme-mediated collagen-crosslinks to produce an integrated collagenous tissue possessing a high tensile strength.

32. The method of claim 31, wherein the conditions comprise treating the first and/or the second collagenous tissues with a composition comprising of 0.015 μg/ml to 1.5 mg/ml lysyl oxidase (LOX).

33. The method of claim 31, wherein the conditions comprise subjecting the first and/or the second collagenous tissues to hypoxic conditions of 0.5% to 11% oxygen.

34. The method of claim 31, wherein the conditions comprise treating the first and/or the second collagenous tissues with a composition comprising glucose oxidase (GOX) and caialase (CAT).

35. The method of any one of claims 31-34, wherein the level of enzyme- mediated collagen-crosslinks is directly proportional to the concentration of exogenously- supplied or endogenously-expressed lysyl oxidase present during the treating step.

36. The method of any one of claims 31-35, wherein the first collagenous tissue is native cartilage and the second collagenous tissue is engineered cartilage.

37. The method of any one of claims 31-35, wherein both the first and second collagenous tissues are native cartilage.

38. The method of any one of claims 31-35, wherein the first collagenous tissue is a native tendon or ligament, and the second collagenous tissue is an engineered tendon or ligament.

39. The method of any one of claims 31-35, wherein both the first and the second collagenous tissues are native tendons or ligaments.

40. The method of any one of claims 31-35, wherein both the first and the second collagenous tissues are independently selected from the group consisting of tendons, ligaments, bones, cartilage and decellularized compositions thereof.

41. The method of any one of claims 31-35, wherein both of the collagenous tissues are native tissues

42. The method of any one of claims 31-35, wherein one of the collagenous tissues is an engineered tissue.