NZ618430B2 - Hyaluronic acid-binding synthetic peptidoglycans, preparation, and methods of use - Google Patents

Abstract

Discloses a hyaluronic acid-binding synthetic peptidoglycan comprising a glycan and 1 to 20 synthetic peptides conjugated to the backbone of the glycan, wherein each synthetic peptide is 5 to 40 amino acids in length and can bind to a hyaluronic acid. Further discloses an engineered collagen matrix comprising the same, and use of such compositions to treat arthritis. comprising the same, and use of such compositions to treat arthritis.

Description

HYALURONIC ACID-BINDING SYNTHETIC PEPTIDOGLYCANS, PREPARATION, AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to US. Provisional Application Serial No. 61/489,602 filed May 24, 2011 and US. Provisional ation Serial No. 61/550,621 filed October 24, 2011. The disclosures of both of which are incorporated herein by reference.

CAL FIELD This invention pertains to the field of hyaluronic acid-binding synthetic peptidoglycans and methods of forming and using the same.

OUND AND SUMMARY OF THE INVENTION Articular cartilage is an important component for the protection ofbones in the body. In ular, lar cartilage functions to protect articulating bones from damage by providing a near-frictionless surface for bone movement and also providing compressive strength to joints. Articular cartilage broadly includes an extracellular matrix (ECM) derived from three main ents: a collagen scaffold, hyaluronic acid (HA), and aggrecan. The material composition of articular cartilage dictates the tissue’s biological, chemical and mechanical properties. The extracellular matrix (ECM) of healthy cartilage is primarily composed of a network of collagen fibrils (15-22% wet weight type II collagen), proteoglycans (4-7% wet weight), glycoproteins, water (60-85%) and electrolytes, giving rise to a viscoelastic tissue with dependent structural and mechanical anisotropy.

Cartilage degradation and wear is a hallmark of osteoarthritis (OA). During the initial stages of OA, aggrecan, a major proteoglycan in cartilage, is an early ent to be degraded. The aggrecan monomer is a protein core with covalently attached glycosaminoglycan (GAG) side chains that bind to filamentous hyaluronic acid via globular s and a link protein. ses, such as aggrecanases, cleave an at specific sites creating protein fragments and free GAG chains that are unable to rebind to HA. Instead, these free GAG chains are extruded from the matrix, which not only reduces compressive th, but also initiates an increase in pro—inflarnrnatory cytokines and matrix metalloproteases. The presence of aggrecan has been shown to reduce ion of proteases protecting underlying collagen fibers from proteolytic cleavage. Loss of an occurs even in normal cartilage and According to a first aspect of the present invention, there is provided a hyaluronic acid-binding synthetic peptidoglycan comprising a glycan and 1 to 20 synthetic peptides conjugated to the backbone of the , wherein each synthetic peptide is 5 to 40 amino acids in length and can bind to a hyaluronic acid.

According to a second aspect of the t invention, there is provided an engineered collagen matrix sing polymerized collagen, hyaluronic acid, and a hyaluronic acid-binding tic peptidoglycan comprising a glycan and 1 to 20 synthetic peptides conjugated to the backbone of the glycan, wherein each synthetic peptide is 5 to 40 amino acids in length and can bind to a hyaluronic acid.

AH26(11211529_2):GCC According to a third aspect of the present invention, there is provided the use of a hyaluronic acid-binding synthetic peptidoglycan in the manufacture of a medicament for the treatment of arthritis, n the treatment reduces a symptom associated with the arthritis, and wherein the synthetic peptidoglycan comprises a glycan and 1 to 20 synthetic peptides conjugated to the backbone of the glycan, wherein each synthetic peptide is 5 to 40 amino acids in length and can bind to a hyaluronic acid.

According to a fourth aspect of the present invention, there is provided a hyaluronic acidbinding synthetic oglycan comprising a glycan and 1 to 20 synthetic peptides conjugated to the ne of the glycan, wherein each synthetic peptide is 5 to 40 amino acids in length and comprises NALTVRGG or an amino acid sequence having at least 90% sequence identity to GAHWQFNALTVRGG.

AH26(11211529_2):GCC GAHWQFNALTVRGG; GDRRRRRMWHRQ; KHRRSR; QKRRS; RRHKSGHIQGSK; VRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; HRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; RGRRNVGPVSRSTLRDPIRR; VGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C—terminus of the peptide, or a glycine-cysteine- glycine (GCG) attached to the N—terminus. 4. The synthetic peptidoglycan of any one of clauses l to 3 wherein the glycan is selected from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid.

. The synthetic peptidoglycan of any one of clauses l to 4 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 6. The tic peptidoglycan of any one of clauses l to 5 wherein the synthetic peptidoglycan is ant to aggrecanase. 7. The synthetic peptidoglycan of any one of clauses l to 6 wherein the synthetic peptidoglycan is lyophilized. 8. A compound of formula PnGX n n is l to 20; wherein X is l to 20; wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a hyaluronic acid binding sequence; and wherein G is a glycan. 9. A compound of formula (PnL)xG n n is 1 to 20; wherein x is 1 to 20; n P is a synthetic peptide of about 5 to about 40 amino acids sing a hyaluronic acid binding sequence; wherein L is a linker; and wherein G is a glycan.

. A compound of formula P(LGn)X n n is l to 20; wherein x is l to 20; wherein P is a tic peptide of about 5 to about 40 amino acids comprising a hyaluronic acid binding sequence; wherein L is a linker; and wherein G is a glycan. ll. A compound of formula PnGx wherein n is MWG/1000; wherein MWG is the molecular weight of G rounded to the nearest 1 kDa; n X is l to 20; wherein P is a synthetic e of about 5 to about 40 amino acids comprising a hyaluronic acid binding sequence; and wherein G is a glycan. 12. A compound of formula (PnL)xG wherein n is MWG/1000; wherein MWG is the molecular weight of G rounded to the nearest 1 kDa; wherein x is l to 20; wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a hyaluronic acid binding sequence; n L is a linker; and wherein G is a glycan. 13. The compound of any one of clauses 8 to 12 wherein the synthetic peptide comprises an amino acid sequence of the formula Bl-Xl-XZ-X3-X4-X5-X6- X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and n Xl-X8 are idic amino acids. 14. The compound of any one of clauses 8 to 13 wherein the synthetic peptide comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; VGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; RRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; ISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the e may have a glycine-cysteine (GC) attached to the inus of the peptide, or a glycine-cysteineglycine (GCG) attached to the N—terminus.

. The compound of any one of clauses 8 to 14 wherein the glycan is selected from the group ting of n, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan e, and hyaluronic acid. 16. The compound of any one of clauses 8 to 15 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 17. The compound of any one of clauses 8 to 16 wherein the synthetic peptidoglycan is ant to aggrecanase. 18. An engineered collagen matrix comprising polymerized collagen, onic acid, and a hyaluronic inding synthetic peptidoglycan. 19. The engineered collagen matrix of clause 18 wherein the collagen is selected from the group ting of type I collagen, type II collagen, type III collagen, type IV collagen, type IX collagen, type XI collagen, and combinations thereof.

. The engineered collagen matrix of clause 18 or 19 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid ce of the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein Xl-X8 are non-acidic amino acids. 21. The engineered collagen matrix of any one of clauses 18 to 20 wherein the peptide component ofthe synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; QGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and TRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C—terminus of the peptide, or a glycine-cysteine- glycine (GCG) attached to the inus. 22. The engineered collagen matrix of any one of clauses 18 to 21 wherein the glycan ent of the synthetic peptidoglycan is selected from the group consisting of dextran, chondroitin, chondroitin sulfate, an, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. 23. The engineered collagen matrix of any one of clauses 18 to 22 n the glycan ent of the synthetic peptidoglycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 24. The engineered collagen matrix of any one of clauses 18 to 23 wherein the synthetic peptidoglycan is resistant to aggrecanase.

. The ered collagen matrix of any one of clauses 18 to 24 wherein the matrix is effective as a tissue graft. 26. The ered collagen matrix of clause 25 wherein the tissue graft is implanted into a patient. 27. The engineered collagen matrix of any one of clauses 18 to 24 wherein the matrix is in the form of a gel. 28. The engineered en matrix of clause 27 wherein the gel is stered to a patient by injection. 29. The engineered collagen matrix of any one of s 18 to 24 wherein the matrix is effective as a composition for in Vitro culture of cells.

. The engineered en matrix of clause 29 wherein the matrix further comprises an exogenous population of cells. 31. The engineered collagen matrix of clause 30 n the cells are selected from the group consisting of chondrocytes and stem cells. 32. The engineered collagen matrix of clause 31 wherein the stem cells are selected from the group consisting of lasts, osteogenic cells, and mesenchymal stem cells. 33. The engineered collagen matrix of any one of clauses 18 to 32 further comprising one or more nutrients. 34. The engineered collagen matrix of any one of clauses 18 to 33 filrther sing one or more growth factors.

. The engineered collagen matrix of any one of clauses 18 to 34 wherein the matrix is sterilized. 36. A composition for in Vitro culture of chondrocytes or stem cells comprising a hyaluronic acid-binding synthetic peptidoglycan. 37. The ition of clause 36 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence of the formula B1-X1-X2- X3-X4-X5-X6-X7-X8-B2, wherein X8 is t or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein Xl-X8 are non-acidic amino acids. 38. The composition of clause 36 or clause 37 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; RVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; LGRRGH; VSKRGHRRTAHE; STR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; KKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C-terminus of the peptide, or a glycine-cysteineglycine (GCG) attached to the N—terminus. 39. The composition of any one of clauses 36 to 38 wherein the glycan component of the synthetic peptidoglycan is selected from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. 40. The composition of any one of clauses 36 to 39 wherein the glycan component of the tic peptidoglycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 41. The composition of any one ofclauses 36 to 40 wherein the synthetic peptidoglycan is resistant to aggrecanase. 42. The composition of any one of clauses 36 to 41 wherein the stem cells are selected from the group consisting of lasts, osteogenic cells, and mesenchymal stem cells. 43. The composition of any one of clauses 36 to 42 further sing one or more nutrients. 44. The composition of any one of clauses 36 to 43 fithher comprising one or more growth factors. 45. The composition of any one of clauses 36 to 44 wherein the composition is sterilized. 46. An additive for a biomaterial cartilage or bone replacement ition comprising a hyaluronic acid-binding synthetic peptidoglycan for on to an existing biomaterial age or bone replacement material. 47. The additive of clause 46 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence of the formula B1-X1-X2-X3-X4-X5- X6-X7-X8-B2, n X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein Xl—X8 are non—acidic amino acids. 48. The additive of clause 46 or clause 47 wherein the peptide component of the synthetic oglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; TAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKJKGRR; RMRRKGRVKHWG; RHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; VKRK; PRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C—terminus of the peptide, or a glycine-cysteine- glycine (GCG) attached to the N-terminus. 49. The additive of any one of s 46 to 48 wherein the glycan component of the synthetic peptidoglycan is selected from the group consisting of dextran, chondroitin, chondroitin e, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. 50. The additive of any one of clauses 46 to 49 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 51. The additive of any one of clauses 46 to 50 wherein the synthetic oglycan is ant to aggrecanase. 52. A method of treatment for tis in a patient, said method comprising the step of administering to the patient a hyaluronic acid-binding synthetic peptidoglycan, wherein the synthetic peptidoglycan reduces a m associated with the arthritis. 53. The method of clause 52 wherein the peptide ent of the synthetic oglycan comprises an amino acid sequence of the formula Bl-Xl-X2-X3-X4-X5- X6-X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein X1-X8 are non-acidic amino acids. 54. The method of clause 52 or clause 53 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; HRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above e embodiments, the peptide may have a glycine-cysteine (GC) attached to the C—terminus of the peptide, or a glycine-cysteine- glycine (GCG) ed to the N—terminus. 55. The method of any one of clauses 52 to 54 wherein the glycan component of the synthetic peptidoglycan is selected from the group consisting of n, chondroitin, oitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. 56. The method of any one of clauses 52 to 55 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan e. 57. The method of any one of clauses 52 to 56 wherein the synthetic peptidoglycan is resistant to aggrecanase. 58. The method of any one ofclauses 52 to 57 wherein the arthritis is osteoarthritis. 59. The method of any one of clauses 52 to 57 wherein the arthritis is rheumatoid arthritis. 60. The method of any one of clauses 52 to 59 wherein the synthetic peptidoglycan is administered to the patient by injection. 61. The method of clause 60 wherein the injection is an intraarticular injection. 62. The method of clause 60 wherein the injection is into a joint capsule of the patient. 63. The method of any one of clauses 52 to 62 wherein the synthetic peptidoglycan is administered using a needle or a device for infusion. 64. The method of any one of clauses 52 to 63 wherein the synthetic peptidoglycan acts as a lubricant. 65. The method of any one of clauses 52 to 64 wherein the synthetic peptidoglycan prevents bone on bone articulation or prevents cartilage loss. 66. A method aring a biomaterial or bone cartilage replacement, said method comprising the step of combining the synthetic peptidoglycan and an existing biomaterial or bone cartilage replacement material. 67. The method of clause 66 n the peptide component of the synthetic peptidoglycan ses an amino acid sequence of the a Bl-Xl-X2-X3-X4-X5- X6-X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein Xl-X8 are idic amino acids. 68. The method of clause 66 or clause 67 wherein the e component of the synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; RLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; GLQGGWGPRHLRGKDQPPGR; LTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; ISR; RRRCGQKKK; VVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C—terminus of the peptide, or a glycine-cysteine- glycine (GCG) attached to the N—terminus. 69. The method of any one of clauses 66 to 68 wherein the glycan component of the synthetic peptidoglycan is ed from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. 70. The method of any one of clauses 66 to 69 wherein the glycan is selected from the group consisting of oitin sulfate and keratan sulfate. 71. The method of any one of clauses 66 to 70 wherein the synthetic peptidoglycan is resistant to aggrecanase. 72. A method ofreducing or preventing hyaluronic acid degradation in a patient, said method comprising administering to the patient a hyaluronic acid-binding synthetic oglycan. 73. The method of clause 72 wherein the peptide component of the synthetic peptidoglycan ses an amino acid sequence of the formula Bl-Xl-XZ-X3-X4-X5- X6-X7-X8-B2, wherein X8 is present or is not present, n B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein Xl-X8 are non—acidic amino acids. 74. The method of clause 72 or clause 73 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; VGGRRN; AGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C-terminus of the e, or a glycine-cysteineglycine (GCG) attached to the inus 75. The method of any one of s 72 to 74 wherein the glycan component of the synthetic peptidoglycan is selected from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, n sulfate, and hyaluronic acid. 76. The method of any one of clauses 72 to 75 n the glycan is ed from the group consisting of chondroitin sulfate and keratan sulfate. 77. The method of any one of clauses 72 to 76 n the synthetic peptidoglycan is resistant to anase. 78. The method of any one of clauses 72 to 77 n the synthetic peptidoglycan is administered to the patient by injection. 79. The method of clause 78 wherein the injection is an intraarticular injection. 80. The method of clause 78 wherein the injection is into a joint capsule of the patient. 8 l. The method of any one ofclauses 72 to 80 wherein the rate of hyaluronic acid degradation is reduced. 82. A method for correcting or modifying a tissue defect in a patient sing administering into the tissue defect a hyaluronic acid-binding synthetic peptidoglycan wherein the defect is corrected or modified. 83. The method of clause 82 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence of the formula Bl-Xl-X2-X3-X4-X5- X6-X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein Xl-X8 are non-acidic amino acids. 84. The method of clause 82 or clause 83 wherein the e component of the synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; VRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; GLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; LGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; ISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C—terminus of the peptide, or a glycine-cysteine- glycine (GCG) attached to the N—terminus. 85. The method of any one of clauses 82 to 84 wherein the glycan ent of the synthetic peptidoglycan is ed from the group consisting of dextran, oitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, n, keratin, keratan sulfate, and hyaluronic acid. 86. The method of any one of clauses 82 to 85 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 87. The method of any one of clauses 82 to 86 wherein the synthetic peptidoglycan is resistant to aggrecanase. 88. The method of any one of clauses 82 to 87 wherein the synthetic oglycan is administered to the patient by inj ection. 89. The method of clause 88 wherein the injection is subcutaneous. 90. The method of any one of clauses 82 to 89 wherein the defect is a cosmetic defect. 91. A dermal filler comprising a hyaluronic acid-binding synthetic peptidoglycan. 92. The dermal filler of clause 91 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid ce of the formula B1-X1-X2- X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not t, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein X1-X8 are non-acidic amino acids. 93. The dermal filler of clause 91 or clause 92 wherein the e component ofthe synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; VVKLK; KLKSQLVKRK; RYPISRPRKR; PVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine (GC) attached to the C-terminus of the peptide, or a glycine-cysteineglycine (GCG) attached to the N—terminus. 94. The dermal filler of any one of clauses 91 to 93 further comprising hyaluronic acid. 95. A method ofreducing or preventing collagen degradation, said method sing the steps of contacting a hyaluronic inding synthetic peptidoglycan with hyaluronic acid in the presence of collagen, and ng or preventing collagen degradation. 96. The method of clause 95 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence ofthe formula Bl-Xl-XZ- X3-X4-X5-X6-X7-X8—B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein Xl-X8 are non-acidic amino acids. 97. The method of clause 95 or clause 96 wherein the e component of the synthetic peptidoglycan comprises an amino acid ce selected from the group consisting of: GAHWQFNALTVRGG; RMWHRQ; KHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; QGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR. 98. The method of any one of clauses 95 to 97 wherein the glycan component of the synthetic peptidoglycan is ed from the group consisting of dextran, oitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, n sulfate, and hyaluronic acid. 99. The method of any one of clauses 95 to 98 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 100. The method of any one of clauses 95 to 99 wherein the synthetic peptidoglycan is resistant to aggrecanase. 101. The method of any one of clauses 95 to 100 wherein the rate of hyaluronic acid degradation is reduced. 102. A method of increasing pore size in an engineered collagen matrix, said method comprising the steps of combining collagen, onic acid, and a hyaluronic acid-binding synthetic peptidoglycan, and increasing the pore size in the matrix. 103. The method of clause 102 wherein the e component of the tic peptidoglycan comprises an amino acid sequence of the formula B1-X1-X2- X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and n X1—X8 are non—acidic amino acids. 104. The method of clause 102 or clause 103 wherein the peptide component of the tic peptidoglycan comprises an amino acid sequence ed from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; HIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; TSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; VVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR. 105. The method of any one of clauses 102 to 104 wherein the glycan component of the synthetic oglycan is selected from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. 106. The method of any one of clauses 102 to 105 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 107. The method of any one of clauses 102 to 106 wherein the synthetic peptidoglycan is resistant to aggrecanase. 108. The method of any one ofclauses 102 to 107 wherein the matrix is sterilized. 109. The method of any one of clauses 102 to 108 wherein the matrix further ses chondrocytes or stem cells. 110. The method of clause 109 wherein the stem cells are ed from the group consisting of osteoblasts, osteogenic cells, and mesenchymal stem cells. 111. The method of any one of clauses 102 to 110 wherein the matrix further comprises one or more nutrients. 112. The method of any one of clauses 102 to 111 wherein the matrix further comprises one or more growth factors. 113. A method ofreducing or preventing oitin sulfate ation, said method comprising the steps of contacting a hyaluronic acid—binding synthetic peptidoglycan with hyaluronic acid in the presence of collagen, and reducing or preventing chondroitin sulfate degradation. 114. The method of clause 113 wherein the peptide ent of the synthetic peptidoglycan comprises an amino acid sequence of the formula B1-X1-X2- X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, n B2 is a basic amino acid, and wherein X1-X8 are non—acidic amino acids. 115. The method of clause 113 or clause 114 wherein the peptide component of the tic peptidoglycan comprises an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; QKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR. 116. The method of any one of clauses 113 to 115 wherein the glycan component of the tic peptidoglycan is selected from the group consisting of n, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan e, and hyaluronic acid. 117. The method of any one of clauses 113 to 116 wherein the glycan is selected from the group consisting of chondroitin sulfate and keratan sulfate. 118. The method of any one of clauses 113 to 117 wherein the synthetic peptidoglycan is resistant to aggrecanase. 119. The method of any one of clauses 113 to 118 n the rate of chondroitin sulfate degradation is reduced. 120. The tic peptidoglycan, compound, engineered collagen matrix, composition, additive, method, or dermal filler of any of the preceding clauses wherein the e component of the tic peptidoglycan has a glycine-cysteine (GC) attached to the C-terminus of the peptide. 121. The synthetic peptidoglycan, compound, engineered en , composition, additive, method, or dermal filler of any of the preceding s wherein the peptide component ofthe synthetic peptidoglycan has a glycine-cysteine-glycine (GCG) attached to the N-terminus of the peptide. 122. The synthetic peptidoglycan, compound, engineered en matrix, composition, additive, method, or dermal filler of any of the preceding clauses wherein the synthetic peptidoglycan is resistant to matrix metallo proteases. 123. The synthetic peptidoglycan, compound, engineered collagen matrix, composition, ve, method, or dermal filler of clause 122 wherein the matrix metallo se is aggrecanase. 124. The synthetic peptidoglycan, nd, engineered collagen matrix, ition, ve, method, or dermal filler of any of the ing clauses wherein the dosage of the synthetic peptidoglycan is in a concentration ranging from about 0.01 uM to about 100 uM. 125. The synthetic peptidoglycan, compound, engineered collagen matrix, composition, ve, method, or dermal filler of any of the preceding s wherein the dosage of the synthetic peptidoglycan is in a concentration ranging from about 0.1 uM to about 10 uM.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows a reaction schematic for the production of an embodiment of the hyaluronic acid-binding synthetic peptidoglycan. Reaction steps are detailed in bold font.

FIGURE 2 shows a standard curve of N—[B-Maleimidopropionic acid]hydrazide,trifluoroacetic acid salt (hereinafter “BMPH”) absorbance (215 nm) based on amount (mg) ofBMPH injected. The standard curve was used to determine the amount of BMPH ed during the coupling reaction.

FIGURE 3 shows binding of the hyaluronic acid-binding tic peptidoglycan to the immobilized hyaluronic acid (HA). Nine HA-binding peptides (e.g., GAHWQFNALTVRGGGC; hereinafter “GAH” or “mAGC”) were ed to the fimctionalized glycosaminoglycan (e.g., chondroitin sulfate, hereinafter “CS”) backbone.

Concentrations of synthetic peptidoglycans were increased from 0.01 uM to 100 uM.

FIGURE 4 shows HA binding ofthe synthetic peptidoglycan as determined by gical frequency sweep (Panel A). The storage modulus of the HA mixtures was analyzed at an oscillatory frequency of 5.012 H2. At this frequency, a noticeable load was provided while the integrity of the HA chains was maintained. Statistical analysis (0t=0.05) showed that HA+CS and HA were significantly different (denoted *), and that HA+10.5GAH-CS and HA+CS were significantly different (denoted **). Panel B is an alternative representation of the same data shown in Panel A.

FIGURE 5 shows quatification of the turbidity of the collagen type I plus treatment groups during collagen fibril formation. The absorbance at 3 13 nm was measured every 3 minutes. After one hour (i.e., timepont number 20), all ent groups had completely formed networks. No cant differences 5) existed n treatment groups with t to the maximum absorbance or the time to halfmaximum absorbance.

FIGURE 6 shows the compressive engineering stress withstood by the collagen gels based on an applied engineering Strain of 1% per second. Statistical is (u=0.05) trated that the addition of lO.5GAH-CS ed in a significant difference in peak engineering stress, in addition to the engineering stress analyzed at engineering s of 5%, 7.5%, and 10%.

FIGURE 7 shows the storage modulus of collagen es measured at an oscillatory frequency of 0.5012 Hz. Statistical analysis (0L=0.05) demonstrated that the addition of lO.5GAH-CS resulted in a significant increase in the storage modulus of the collagen gel (denoted *).

FIGURE 8 shows the percent degradation ofHA mixtures due to the addition of onidase to the mixtures (Panel A). The percent degradation was determined by the changes in the dynamic viscosity ofthe HA mixtures. Dynamic ity measurements were initially taken of the mixtures, and served as the ne from which the percent degradations were calculated. The 0 hour timepoint was taken after the addition of hyaluronidase, the sufficient mixing of the samples, and the pipetting onto the rheometer stage, and approximately 2 minutes passed between the addition of the hyaluronidase and the measurement of the dynamic ity. Statistical analysis trated significant differences (0L=O.05) in the percent degradation for the lO.5GAH—CS sample at both the 0 hour and 2 hour timepoints.

Panel B shows the same data represented as normalized dynamic viscosity (mean :: SE, n=3) of HA mixtures due to the addition ofhyaluronidase. Dynamic viscosity measurements were initially taken of the mixtures before hyaluronidase was added, and these values served as the baseline from which the normalized dynamic viscosities were calculated. The normalized dynamic viscosities were determined by taking each measured dynamic viscosity after the addition of hyaluronidase and dividing this value by the initial dynamic viscosity of that sample. Statistical analysis (u=0.05) was ted, and significant differences were seen in the normalized degradation for the lO.5GAH—CS sample at both the 0 hr and 2 hr timepoints.

FIGURE 9 shows representative cryo-SEM images (10,000x magnification with um scale bar) of the CI scaffold associated with each cartilage ECM replicate. Panel A represents the CI control. Panel B represents CI+HA+CS. Panel C represents CI+HA+10.5GAH-CS.

FIGURE 10 shows the percent degradation (mean :: SE, n=3) of CI in ECM replicates exposed to MMP-I throughout a 50 hr duration. Statistical analysis (p<0.05) of the different ents revealed that all three treatments (CI control, CS, and CI+HA+lO.5GAH-CS) were significantly different from each other.

FIGURE 11 shows the cumulative chondroitin sulfate (CS) loss over an eight- day culture period in media stimulated with and without IL-lB. CS loss was measured by a DMMB assay. The addition ofmAGC had a significant effect on loss of CS from the scaffolds (p<0.001). ** denotes statistical significance between scaffold prepared without aggrecan mimic and those prepared with mAGC. + denotes statistical significance between scaffold treated with and t IL- 1[3 (p<0.05). Bars represent average i SEM (n=3).

FIGURE 12 shows the cumulative collagen breakdown over an eight-day culture period in media stimulated with and without IL—lB. Collagen breakdown was measured by a Sircol assay. The addition of aggrecan mimic had a significant effect on loss of collagen from the scaffolds (p<0.02). ** denotes statistical significance between ld prepared t aggrecan mimic and those prepared with mAGC. + denotes statistical significance between scaffold treated with and t IL-IB (p<0.05). Bars represent e :: SEM (n=3).

FIGURE 13 represents a platform to study the efficacy ofthe peptidoglycan ex vivo. 0.5% trypsin was used to remove native aggrecan from bovine cartilage explants.

Removal of aggrecan was confirmed by DMMB assay. Graphs ent the amount of an removed compared to positive control.

FIGURE 14 shows an assay to monitor peptidoglycan diffusion through the cartilage matrix. The Y-axis ents the difference in DMMB assay ance values read from aggrecan-depleted cartilage plugs treated with/without peptidoglycan. The X-axis represents the distance from the articular e of cartilage to subchondral bone. Bars ent average difference i SEM (n=3).

FIGURE 15 shows Safranin O and Avidin-Biotin stains ofbovine cartilage explants. A midsagittal cut was made through the matrix and probed for residual aggrecan (top panel, dark staining) and biotin m panel, dark ng) respectively. en type II binding peptidoglycan [WYRGRLGC; “mAG(II)C”] was diffused through the explant. Higher magnification (20X) of this tissue slice indicated that mAG(II)C penetrates approximately 200 um into tissue.

FIGURE 16 shows Avidin—Biotin stains of cartilage explants. Peptidoglycans (mAG(II)C and mAGC) were diffused through the cartilage explant. Images indicate depth of penetration of each (dark staining).

FIGURE 17 shows that the addition ofpeptidoglycans in an depleted (AD) explants increased compressive stiffness. Addition of the HA binding peptidoglycan (mAGC) significantly restored stiffness of cartilage explants to a higher extent as compared to the collagen type II binding peptidoglycan (mAG(II)C). Significance, denoted as *, specified an increase in compressive stiffiless between AD and AD+mACG augmented explants (p<0.005). Data is presented as meani SEM (n=5).

FIGURE 18 (A) shows a schematic representation of probe bound to MMP-13.

BHQ-3 black hole quencher 3 and CY5.5 absorbed and emitted at 695 nm respectively. Arrow and italics indicate the cleavage site. (B) shows the concentration profile e activity with and without MMP-13: Left, fluorescence imaging sections of 96-well microplate; Right, recovery of fluorescence emission ity (695 nm).

FIGURE 19 shows the extent of inflammation indicated by the MMP-l3 probe in Sprague-Dawley rats treated with and t peptidoglycan at four, six and eight weeks post FIGURE 20 shows a x-ray images of e-Dawley rat knee joints showing injured knee 6 weeks and 8 weeks following OA induction (Panels A and D, respectively), injured knee with peptidoglycan treatment s B and E, respectively), and normal knee (Panel C) six weeks after osteoarthritis induction surgery.

FIGURE 21 shows microCT of Sprague-Dawley rats indicating re-growth of new cartilage six and eight weeks after OA induction surgery. Injured knees 6 weeks and 8 weeks ing OA induction are shown in Panels A and D, respectively. Injured knees ing peptidoglycan treatment are shown in Panels B and E, respectively. Normal knee is shown in panel C.

FIGURE 22 shows that the on ofmAGC to collagen scaffolds increased the storage modulus and compressive stiffness. Frequency sweeps (A) on collagen scaffolds indicated an increase in storage modulus over a range of 0. 1 —2.0 Hz. Similarly, compressive stiffness (B) showed an increase in values when the scaffold was prepared with the addition of mAGC. Significance is denoted as * (p<0.0001). Data is presented as mean i SEM (n=5).

FIGURE 23 shows cumulative chondroitin sulfate (CS) loss over an eight-day e period in media stimulated with and without IL—IB. CS loss was measured by a DMMB assay. Scaffold compositions (A—H) are described in Table 3. The addition ofmAGC had a significant effect on loss of CS from the scaffolds (p<0.001). * denotes statistical significance between ld A and C, and ld E and G. (p<0.05). Bars represent average i SEM (n=3).

FIGURE 24 shows cumulative collagen breakdown over an day culture period in media stimulated with and without IL—1 [3. Collagen breakdown was measured by a Sircol assay. Scaffold itions (A—H) are bed in Table 3. The addition of aggrecan mimic had a significant effect on loss of collagen from the scaffolds 2). * denotes statistical significance between scaffold A and C, and scaffold E and G. (p<0.05). Bars represent average i SEM (n=3).

FIGURE 25 shows real—time PCR analysis for aggrecan and collagen type II expressed by bovine chondrocytes cultured in unaligned (A) and aligned (B) collagen scaffolds.

Values were normalized to endogenous GAPDH expression. on ofmAGC statistically changed aggrecan and collagen type II expression (paggrecan<0.02 and pconagen<0.001) respectively. There was also a statistical difference in aggrecan and collagen type II sion between unaligned and d scaffolds 01). Similarly, the aggrecan and collagen type II expression differed between scaffolds treated with and t IL-lB (p<0.01). Scaffold itions (A-H) are described in Table 3. Bars represent average i SEM (n=4).

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS As used herein, a “hyaluronic acid—binding synthetic peptidoglycan” means a synthetic peptide conjugated to a glycan where the peptide comprises a hyaluronic acid-binding sequence.

Various embodiments of the invention are described herein as follows. In one ment described herein, a hyaluronic acid-binding synthetic oglycan is provided.

The hyaluronic inding synthetic peptidoglycan comprises a synthetic peptide ated to a glycan wherein the synthetic peptide comprises a hyaluronic acid-binding sequence.

In another embodiment, a compound of the formula PnGX is described wherein n is l to 20; wherein x is l to 20; wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a onic acid binding sequence; and wherein G is a glycan.

In yet another embodiment, a compound of the formula (PnL) XG is described wherein n is l to 20; wherein x is l to 20; wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a hyaluronic acid binding sequence; n L is a linker; and wherein G is a glycan.

In another embodiment, a compound of the formula P(LGn)X is described wherein n is l to 20; wherein X is l to 20; wherein P is a synthetic peptide of about 5 to about 40 amino acids sing a hyaluronic acid binding sequence; n L is a linker; and wherein G is a glycan.

In yet r embodiment, a compound of the formula PnGX is described wherein n is MWG/1000; n MWG is the molecular weight of G rounded to the nearest 1 kDa; wherein x is 1 to 20; wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a hyaluronic acid binding ce; and wherein G is a glycan.

In r embodiment, a compound of the formula (PnL)xG is described wherein n is 00; wherein MWG is the molecular weight of G rounded to the nearest 1 kDa; wherein x is l to 20; wherein P is a tic peptide of about 5 to about 40 amino acids comprising a hyaluronic acid binding sequence; wherein L is a linker; and wherein G is a glycan.

For purposes of this disclosure, the hyaluronic acid-binding synthetic peptidoglycans and compounds described in the preceding paragraphs are collectively referred to as “hyaluronic acid-binding synthetic peptidoglycans” or “synthetic peptidoglycans.” In each of the above peptide embodiments, the synthetic peptidoglycan may se 5-15 peptide molecules (11 is 5-15), 5-20 peptide molecules (11 is 5-20), 1-20 peptide molecules (11 is 1-20), or 1-25 e molecules (11 is 1—25). In one embodiment, n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19,20,21, 22, 23, 24, and 25 peptide les.

In another illustrative embodiment described herein, an engineered collagen matrix is provided. The matrix comprises rized collagen, hyaluronic acid, and a hyaluronic acid-binding synthetic peptidoglycan. In another embodiment, a composition for in vitro culture of chondrocytes or stem cells is provided. The composition comprises any of the hyaluronic acid-binding synthetic peptidoglycans described in this disclosure.

In another embodiment described herein, a method of increasing pore size in an engineered collagen matrix is provided. The method comprises the steps of combining collagen, hyaluronic acid, and a hyaluronic acid-binding synthetic peptidoglycan and increasing the pore size in the matrix.

In yet another rative embodiment, a method of decreasing cartilage wear or erosion in a t is provided. The method comprises the step of administering to the patient a hyaluronic acid-binding synthetic peptidoglycan, wherein the synthetic peptidoglycan decreases wear or erosion of the cartilage. In one embodiment, the cartilage erosion or wear may be caused by arthritis. In one embodiment, the cartilage erosion or wear may be caused by aging, obesity, trauma or injury, an anatomic abnormality, genetic diseases, metabolic imbalances, inflammation, or the like.

In yet another illustrative embodiment, a method of ent for arthritis in a patient is provided. The method comprises the step of administering to the patient a hyaluronic acid-binding synthetic peptidoglycan, n the synthetic oglycan reduces a symptom associated with the arthritis.

In another illustrative embodiment, a method of reducing or preventing hyaluronic acid degradation in a patient is provided. The method comprises administering to the patient a hyaluronic acid-binding synthetic peptidoglycan.

In another illustrative ment, a method of ng or preventing collagen degradation is provided. The method comprises the steps of contacting a hyaluronic acidbinding synthetic peptidoglycan with hyaluronic acid in the presence of en, and ng or preventing collagen degradation.

In yet another illustrative ment, a method for correcting or modifying a tissue defect in a patient is provided. The method comprises administering into the tissue defect a onic acid-binding synthetic oglycan wherein the defect is corrected or d.

In another illustrative embodiment described herein, a dermal filler is provided. The filler comprises a hyaluronic acid-binding synthetic peptidoglycan. In one embodiment, the filler further comprises hyaluronic acid.

In yet another embodiment, an additive for a biomaterial cartilage or bone replacement composition is provided. The additive comprises a hyaluronic acid-binding synthetic peptidoglycan for addition to an ng biomaterial cartilage or bone ement material. In r ment described herein, a method of ing a biomaterial or bone cartilage replacement is provided. The method comprises the step of combining the synthetic peptidoglycan and an existing biomaterial or bone cartilage replacement material.

In the various embodiments, the peptide component of the synthetic peptidoglycan comprises an amino acid sequence of the formula Bl-Xl-X2-X3-X4-X5-X6-X7- X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and n X1-X8 are non-acidic amino acids.

In another embodiment, the peptide component of the synthetic oglycan can comprise or can be an amino acid sequence of the a B1-X1-B2-X2-X3-X4-X5-X6- X7-X8-X9-B3, wherein X9 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, wherein B3 is a basic amino acid, and wherein X1 -X9 are non—acidic amino acids.

In another embodiment, the tic peptide can comprise or can be an amino acid sequence of the a Bl-Xl-X2-X3-X4—X5—X6-X7-X8-B2-X9-B3, wherein X8 is present or is not present, wherein Bl is a basic amino acid, wherein B2 is a basic amino acid, wherein B3 is a basic amino acid, and wherein Xl-X9 are non—acidic amino acids.

As used herein, a “basic amino acid” is selected from the group consisting of lysine, arginine, or histidine. As used herein, a “non—acidic amino acid” is selected from the group consisting of alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, ine, tryptophan, tyrosine, and valine.

In the various illustrative ments described herein, the peptide component ofthe synthetic peptidoglycan can se an amino acid sequence selected from the group consisting of: GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RVKHWG; RHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and TRR.

In each of the above peptide embodiments, the peptide may have a glycine-cysteine attached to the C-terminus of the peptide, and/or a glycine-cysteine-glycine (GCG) attached to the N- terminus of the peptide. In various embodiments described herein, the peptide component of the synthetic peptidoglycan comprises any amino acid sequence described in the preceding paragraph or an amino acid sequence with 80%, 85%, 90%, 95%, 98%, or 100% homology to any of these amino acid sequences.

Additional peptides that can be included as the peptide component of the hyaluronic acid-binding synthetic peptidoglycans include es described in Amemiya et a1., Biochem. s. Acta, vol. 1724, pp. 94-99 , incorporated herein by reference. These peptides have an Arg-Arg motif and include peptides selected from the group consisting of: RRASRSRGQVGL; GRGTHHAQKRRS; QPVRRLGTPVVG; ARRAEGKTRMLQ; PKVRGRRHQASG; SDRHRRRREADG; VKHPPG; RERRERHAVARHGPGLERDARNLARR; TVRPGGKRGGQVGPPAGVLHGRRARS; NVRSRRGHRMNS; DRRRGRTRNIGN; KTAGHGRRWSRN; AKRGEGRREWPR; GGDRRKAHKLQA; RRGGRKWGSFEG; and LTRVEG.

In each of the above peptide embodiments, the peptide may have a e-cysteine attached to the C-terminus of the peptide. In each of the above peptide embodiments, the peptide may have a glycine-cysteine-glycine (GCG) ed to the N-terminus of the peptide. In various embodiments described herein, the e component of the tic peptidoglycan comprises any amino acid sequence described in the preceding paragraph or an amino acid ce with 80%, 85%, 90%, 95%, 98%, or 100% homology to any ofthese amino acid sequences.

In other embodiments, peptides described in Yang et a1., EMBO Journal, vol. 13, pp. 286-296 (1994), incorporated herein by reference, and Goetinck et al., J. Cell. Biol, vol. 105, pp. 408 (1987), incorporated herein by reference, can be used in the hyaluronic acid-binding synthetic peptidoglycans bed herein including peptides selected from the group consisting of RDGTRYVQKGEYR, HREARSGKYK, PDKKHKLYGV, and WDKERSRYDV. In each of these embodiments, the peptide may have a glycine-cysteine attached to the C-terminus of the peptide. In each of these ments, the peptide may have a glycine-cysteine-glycine (GCG) attached to the N-terminus of the peptide. In other embodiments, the peptide component ofthe synthetic peptidoglycan comprises an amino acid sequence with 80%, 85%, 90%, 95%, 98%, or 100% gy to any ofthese amino acid sequences.

In various embodiments, the e component of the synthetic peptidoglycan described herein can be modified by the inclusion ofone or more conservative amino acid substitutions. As is well-known to those skilled in the art, altering any non-critical amino acid of a peptide by conservative substitution should not significantly alter the activity of that peptide because the side-chain of the replacement amino acid should be able to form similar bonds and contacts to the side chain of the amino acid which has been replaced. Non- conservative substitutions are possible provided that these do not excessively affect the hyaluronic acid binding activity of the peptide.

As is well-known in the art, a “conservative tution” of an amino acid or a “conservative substitution variant” of a peptide refers to an amino acid substitution which maintains: 1) the secondary structure of the peptide; 2) the charge or hydrophobicity of the amino acid; and 3) the bulkiness of the side chain or any one or more of these characteristics.

Illustratively, the well-known terminologies “hydrophilic residues” relate to serine or threonine.

“Hydrophobic residues” refer to leucine, isoleucine, phenylalanine, valine or alanine, or the like. “Positively charged residues” relate to lysine, arginine, ornithine, or histidine.

“Negatively charged residues” refer to aspartic acid or glutamic acid. Residues having “bulky side chains” refer to alanine, tryptophan or ne, or the like. A list of illustrative conservative amino acid substitutions is given in TABLE 1.

TABLE 1 For Amino Acid Replace With Alanine D-Ala, Gly, Aib, B-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Om D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D- Aspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D- Cysteine D—Cys, S—Me-Cys, Met, D-Met, Thr, D- Glutamine D—Gln, Asn, D—Asn, Glu, D-Glu, Asp, D- Glutamic Acid D—Glu, D—Asp, Asp, Asn, D-Asn, Gln, D- _lycineA—b,B-A_aAla, D—Ala, Pro, D-Pro, e, Val, D-,Val Leu DLeu, Met, D- Valt, D-Val, Met DMet, DIle, DLeu, Ile D——Lys, Arg, D-Arg, 0m, D-Orn Methionine D—,Met S--Me-Cys, Ile, D-Ile, Leu, D-Leu, Phenylalanine D—Phe, Tyr, D--Tyr, His, D-His, Trp, D- D—Ser, Thr, D-Thr, allo-Thr, L-Cys, DCys Val, D-Val , Phe, D-Phe, His, D-His, Trp, D- In one embodiment, the vative amino acid substitutions appicable to the molecules lO described herein do not alter the motifs that consist of the Bl-Xl-XZ-X3-X4-X5-X6-X7-X8-B2 formula, the B 1 -X1 -B2-X2—X3—X4—X5—X6—X7—X8—X9—B3 formula, the B 1 -X1 -X2-X3-X4-X5 - X6-X7-X8-B2-X9-B3 formula, or the Arg—Arg motif.

In various embodiments bed herein, the glycan (e.g. glycosaminoglycan, abbreviated GAG, or polysaccharide) component of the synthetic peptidoglycan described herein can be selected from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan e, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. In one embodiment, the glycan is selected from the group ting of chondroitin sulfate and n sulfate. In another rative embodiment, the glycan is chondroitin sulfate.

In one embodiment described herein, the hyaluronic inding synthetic peptidoglycan comprises (GAHWQFNALTVRGG)10 conjugated to chondroitin sulfate wherein each peptide in the peptidoglycan molecule is linked separately to chondroitin sulfate. In r ment described herein, the hyaluronic acid-binding synthetic peptidoglycan comprises (GAHWQFNALTVRGGGC)11 conjugated to chondroitin sulfate wherein each peptide in the peptidoglycan molecule is linked separately to chondroitin sulfate. In each of the above peptide embodiments, the peptide number may be selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 peptide les.

In s embodiments described herein, the synthetic peptidoglycan is resistant to aggrecanase. An aggrecanase is characterized in the art as any enzyme known to cleave aggrecan.

In one illustrative aspect, the hyaluronic acid-binding synthetic peptidoglycan may be sterilized. As used herein “sterilization” or “sterilize” or “sterilized” means disinfecting the hyaluronic acid-binding synthetic peptidoglycans by removing unwanted contaminants including, but not d to, endotoxins and infectious agents.

In various illustrative embodiments, the hyaluronic acid-binding synthetic peptidoglycan can be disinfected and/or sterilized using conventional sterilization techniques including ene oxide or ethylene oxide treatment, gas plasma sterilization, gamma radiation (e.g., 1-4 Mrads gamma ation or 1—2.5 Mrads of gamma irradiation), electron beam, and/or sterilization with a peracid, such as peracetic acid. Sterilization techniques which do not adversely affect the structure and biotropic properties of the hyaluronic acid-binding synthetic peptidoglycan can be used. In one embodiment, the hyaluronic acid-binding tic peptidoglycan can be subjected to one or more sterilization processes. In r illustrative ment, the hyaluronic inding synthetic peptidoglycan is ted to sterile filtration. The hyaluronic acid—binding synthetic peptidoglycan may be wrapped in any type of container including a plastic wrap or a foil wrap, and may be further sterilized. The hyaluronic acid-binding synthetic peptidoglycan may be prepared under sterile conditions, for example, by lisation, which may readily be accomplished using standard techniques well-known to those skilled in the art.

In various embodiments described herein, the hyaluronic acid-binding synthetic peptidoglycans can be combined with minerals, amino acids, sugars, peptides, proteins, vitamins (such as ascorbic acid), or laminin, collagen, fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, or growth factors such as epidermal growth , et-derived growth factor, transforming growth factor beta, or fibroblast growth , and orticoids such as dexamethasone or viscoelastic altering agents, such as ionic and non-ionic water soluble polymers; acrylic acid polymers; hydrophilic rs such as hylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers and cellulosic polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, ypropyl methylcellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acids, or other polymeric agents both natural and synthetic.

In various embodiments described herein, the peptide component of the synthetic peptidoglycan is synthesized according to solid phase peptide synthesis protocols that are well- known by persons of skill in the art. In one embodiment a peptide precursor is synthesized on a solid t according to the well-known Fmoc protocol, cleaved from the support with trifluoroacetic acid and d by chromatography according to methods known to persons skilled in the art.

In various embodiments described herein, the peptide component of the synthetic peptidoglycan is synthesized utilizing the methods of biotechnology that are well-known to persons skilled in the art. In one embodiment a DNA sequence that encodes the amino acid ce information for the d peptide is ligated by recombinant DNA techniques known to persons skilled in the art into an sion plasmid (for example, a plasmid that incorporates an y tag for affinity purification of the peptide), the plasmid is transfected into a host organism for expression, and the peptide is then isolated from the host organism or the growth medium ing to methods known by persons skilled in the art (e.g., by affinity purification). Recombinant DNA technology methods are described in Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 3rd n, Cold Spring Harbor Laboratory Press, (2001), incorporated herein by reference, and are nown to the d artisan.

In various embodiments described herein, the e component of the hyaluronic acid-binding synthetic peptidoglycan is conjugated to a glycan by reacting a free amino group of the peptide with an aldehyde function of the glycan in the presence of a reducing agent, utilizing methods known to persons skilled in the art, to yield the peptide glycan conjugate. In one embodiment an aldehyde on of the glycan (e.g. polysaccharide or glycosaminoglycan) is formed by reacting the glycan with sodium metaperiodate according to methods known to persons skilled in the art.

In one embodiment, the peptide component of the synthetic oglycan is conjugated to a glycan by reacting an aldehyde fimction of the glycan with 3-(2- pyridyldithio)propionyl hydrazide (PDPH) to form an ediate glycan and further reacting the intermediate glycan with a peptide containing a free thiol group to yield the peptide glycan conjugate. In yet another embodiment, the sequence of the peptide component of the synthetic peptidoglycan may be modified to include a glycine-cysteine segment to provide an attachment point for a glycan or a glycan-linker conjugate. In any of the embodiments described herein, the crosslinker can be aleimidopropionic acid]hydrazide (BMPH). gh specific ments have been described in the preceding paragraphs, the hyaluronic acid—binding synthetic peptidoglycans described herein can be made by using any art-recognized method for conjugation of the peptide to the glycan (e.g. polysaccharide or glycosaminoglycan). This can include covalent, ionic, or en bonding, either directly or indirectly via a linking group such as a divalent linker. The conjugate is typically formed by covalent bonding ofthe peptide to the glycan through the formation of amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or o groups on the respective components of the conjugate. All ofthese methods are known in the art or are further described in the es section of this application or in Hermanson G.T., Bioconjugate Techniques, ic Press, pp.169-186 (1996), incorporated herein by reference. The linker typically comprises about 1 to about 30 carbon atoms, more lly about 2 to about 20 carbon atoms. Lower molecular weight linkers (z'.e., those having an approximate molecular weight of about 20 to about 500) are lly employed.

In addition, structural modifications of the linker portion of the ates are contemplated herein. For e, amino acids may be included in the linker and a number of amino acid substitutions may be made to the linker portion of the conjugate, including but not limited to naturally occurring amino acids, as well as those available from conventional synthetic methods. In another aspect, beta, gamma, and longer chain amino acids may be used in place of one or more alpha amino acids. In another aspect, the linker may be shortened or lengthened, either by changing the number of amino acids included therein, or by including more or fewer beta, gamma, or longer chain amino acids. Similarly, the length and shape of other chemical fragments of the linkers described herein may be modified.

In various ments described herein, the linker may include one or more bivalent fragments selected independently in each instance from the group consisting of alkylene, heteroalkylene, cycloalkylene, eteroalkylene, e, and heteroarylene each ofwhich is ally substituted. As used herein alkylene represents a group resulting from the replacement of one or more carbon atoms in a linear or branched ne group with an atom independently selected in each instance from the group consisting of oxygen, nitrogen, phosphorus and sulfur. In an alternative embodiment, a linker is not present.

In one embodiment described herein, an engineered collagen matrix is ed.

The previously described embodiments of the hyaluronic acid-binding synthetic peptidoglycan are applicable to the engineered collagen matrix described . In one embodiment, the engineered collagen matrix comprises polymerized collagen, hyaluronic acid, and a hyaluronic acid-binding synthetic peptidoglycan. In one embodiment, the engineered collagen matrix comprises polymerized collagen and a hyaluronic—binding synthetic peptidoglycan. In various illustrative embodiments, crosslinking agents, such as carbodiimides, aldehydes, lysl-oxidase, N—hydroxysuccinimide esters, sters, ides, and maleimides, as well as various natural crosslinking agents, including genipin, and the like, can be added before, during, or after polymerization of the collagen in solution.

In various illustrative embodiments, the collagen used herein to prepare an engineered collagen matrix may be any type of collagen, including collagen types I to XXVIII, alone or in any combination, for example, collagen types I, II, III, and/or IV may be used. In some ments, the collagen used to prepare an engineered collagen matrix is selected from the group consisting oftype I collagen, type II collagen, type III collagen, type IV en, type IX collagen, type XI collagen, and combinations thereof. In one embodiment, the ered collagen matrix is formed using commercially ble collagen (e.g., Sigma, St.

Louis, MO). In an alternative embodiment, the collagen can be purified from submucosa- containing tissue material such as intestinal, urinary bladder, or stomach tissue. In a r embodiment, the collagen can be purified from tail tendon. In an additional embodiment, the collagen can be purified from skin. In various aspects, the en can also contain endogenous or exogenously added non-collagenous proteins in addition to the collagen-binding synthetic peptidoglycans, such as fibronectin or silk ns, glycoproteins, and polysaccharides, or the like. The engineered collagen matrices prepared by the methods described herein can be in the form of a tissue graft (e.g., in the form of a gel) which can assume the characterizing features of the tissue(s) with which they are associated at the site of implantation or injection. In one embodiment, the engineered collagen matrix is a tissue graft that can be implanted into a patient. In another embodiment, the ered collagen matrix can be administered to a patient by injection. In either embodiment, the matrix can be in the form of a gel or a powder, for example.

In one embodiment, the collagen in the engineered collagen matrix ses about 40 to about 90 dry weight (wt) % of the matrix, about 40 to about 80 dry wt % of the matrix, about 40 to about 70 dry wt % of the matrix, about 40 to about 60 dry wt % of the matrix, about 50 to about 90 dry wt % of the matrix, about 50 to about 80 dry wt % of the matrix, about 50 to about 75 dry wt % of the matrix, about 50 to about 70 dry wt % of the matrix, or about 60 to about 75 dry wt % of the matrix. In another embodiment, the collagen in the engineered collagen matrix comprises about 90 dry wt %, about 85 dry wt %, about 80 dry wt %, about 75 dry wt %, about 70 dry wt %, about 65 dry wt %, about 60 dry wt %, about 50 dry wt %, about 45 dry wt %, about 40 dry wt %, or about 30 dry wt % of the .

In one embodiment, the final collagen concentration of the matrix in gel form is about 0.5 to about 6 mg per mL, about 0.5 to about 5 mg per mL, about 0.5 to about 4 mg per mL, about 1 to about 6 mg per mL, about 1 to about 5 mg per mL, or about 1 to about 4 mg per mL. In one embodiment, the final collagen concentration ofthe matrix is about 0.5 mg per mL, about 1 mg per mL, about 2 mg per mL, about 3 mg per mL, about 4 mg per mL, or about 5 mg per mL.

In one ment, the hyaluronic acid—binding synthetic peptidoglycan in the engineered collagen matrix comprises about 2 to about 60 dry weight (wt) % of the matrix, about 2 to about 50 dry wt % of the matrix, about 5 to about 50 dry wt % of the matrix, about to about 50 dry wt % of the matrix, about 10 to about 20 dry wt % of the matrix, about 10 to about 30 dry wt % of the matrix, about 10 to about 25 dry wt % of the matrix, about 15 to about dry wt % of the matrix, or about 15 to about 45 dry wt % of the matrix. In another ment, the hyaluronic inding synthetic peptidoglycan in the engineered en matrix comprises about 2 dry wt %, about 5 dry wt %, about 10 dry wt %, about 15 dry wt %, about 20 dry wt %, about 25 dry wt %, about 30 dry wt %, about 35 dry wt %, about 40 dry wt %, about 45 dry wt %, or about 50 dry wt % of the matrix In another embodiment, the engineered collagen matrix comprises hyaluronic acid and the hyaluronic acid in the engineered collagen matrix comprises about 2 to about 60 dry weight (wt) % of the matrix, about 2 to about 50 dry wt % of the matrix, about 5 to about 50 dry wt % of the matrix, about 10 to about 50 dry wt % of the matrix, about 10 to about 20 dry wt % of the matrix, about 10 to about 30 dry wt % of the matrix, about 10 to about 25 dry wt % ofthe matrix, about 15 to about 30 dry wt % of the matrix, or about 15 to about 45 dry wt % of the matrix. In another embodiment, the hyaluronic acid in the engineered en matrix comprises about 2 dry wt %, about 5 dry wt %, about 10 dry wt %, about 15 dry wt %, about 20 dry wt %, about 25 dry wt %, about 30 dry wt %, about 35 dry wt %, about 40 dry wt %, about 45 dry wt %, or about 50 dry wt % ofthe matrix.

In one embodiment, the engineered collagen matrix comprises hyaluronic acid and a hyaluronic acid-binding synthetic peptidoglycan. The hyaluronic acid and onic acid-binding tic peptidoglycan in the engineered collagen matrix comprise about 10 to about 60 dry weight (wt) % of the , about 20 to about 60 dry wt % of the matrix, about 30 to about 60 dry wt % of the matrix, about 40 to about 60 dry wt % of the matrix, about 10 to about 50 dry wt % of the matrix, about 20 to about 50 dry wt % of the matrix, about 25 to about 50 dry wt % of the , about 30 to about 50 dry wt % ofthe matrix, or about 25 to about 40 dry wt % of the matrix. In another embodiment, the hyaluronic acid and hyaluronic acid- g synthetic peptidoglycan in the ered collagen matrix comprises about 10 dry wt %, about 15 dry wt %, about 20 dry wt %, about 25 dry wt %, about 30 dry wt %, about 35 dry wt %, about 40 dry wt %, about 50 dry wt %, about 55 dry wt %, about 60 dry wt %, or about 70 dry wt % of the matrix.

In one illustrative , the engineered collagen matrix may be sterilized. As used herein lization” or “sterilize” or “sterilized” means disinfecting the matrix by removing unwanted contaminants including, but not limited to, endotoxins, nucleic acid contaminants, and infectious agents.

In s illustrative embodiments, the engineered collagen matrix can be disinfected and/or sterilized using conventional sterilization techniques including glutaraldehyde tanning, formaldehyde tanning at acidic pH, propylene oxide or ethylene oxide treatment, gas plasma sterilization, gamma radiation (e.g., 1-4 Mrads gamma irradiation or 1- 2.5 Mrads of gamma irradiation), electron beam, and/or sterilization with a peracid, such as peracetic acid. Sterilization techniques which do not adversely affect the ure and biotropic properties of the matrix can be used. In one embodiment, the engineered collagen matrix can be subjected to one or more sterilization processes. In illustrative ments, the collagen in solution, prior to polymerization, can also be sterilized or ected. The engineered collagen matrix may be wrapped in any type of container including a plastic wrap or a foil wrap, and may be further sterilized.

In any of these embodiments the engineered collagen matrix may fiirther comprise an exogenous population of cells. The added population of cells may comprise one or more cell tions. In s embodiments, the cell populations comprise a population of non-keratinized or keratinized epithelial cells or a population of cells selected from the group consisting of endothelial cells, mesodermally derived cells, mesothelial cells, synoviocytes, neural cells, glial cells, osteoblasts, fibroblasts, chondrocytes, tenocytes, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, multi-potential progenitor cells (e. g., stem cells, including bone marrow progenitor cells), and osteogenic cells. In some embodiments, the population of cells is selected from the group consisting of chondrocytes and stem cells. In some embodiments, the stem cells are selected from the group ting of osteoblasts, osteogenic cells, and mesenchymal stem cells. In various embodiments, the ered collagen matrix can be seeded with one or more cell types in combination.

In various aspects, the ered collagen matrices or engineered graft constructs of the present invention can be ed with nutrients, including minerals, amino acids, sugars, es, proteins, vitamins (such as ascorbic acid), or laminin, fibronectin, hyaluronic acid, fibrin, elastin, or an, or growth factors such as epidermal growth factor, platelet-derived growth factor, transforming growth factor beta, or fibroblast growth factor, and glucocorticoids such as dexamethasone or viscoelastic ng agents, such as ionic and non- ionic water soluble polymers; c acid rs; hydrophilic polymers such as polyethylene oxides, polyoxyethylene—polyoxypropylene copolymers, and polyvinylalcohol; osic polymers and cellulo sic polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acids, or other polymeric agents both natural and synthetic. In other illustrative ments, cross—linking agents, such as carbodiimides, aldehydes, lysl-oxidase, N—hydroxysuccinimide esters, imidoesters, hydrazides, and maleimides, as well as natural crosslinking agents, including genipin, and the like can be added before, concurrent with, or after the addition of cells.

As discussed above, in accordance with one embodiment, cells can be added to the engineered collagen matrices or the engineered graft constructs after polymerization of the collagen or during collagen polymerization. The engineered collagen matrices comprising the cells can be subsequently injected or implanted in a host for use as engineered graft constructs.

In another embodiment, the cells on or within the engineered collagen matrices can be cultured in vitro, for a ermined length of time, to increase the cell number or to induce d remodeling prior to implantation or injection into a patient.

In one embodiment described herein, a composition for in vitro culture of chondrocytes or stem cells is provided (i.e., for in vitro culture of cells without subsequent implantation or injection into a patient). The composition for in vitro culture ses a hyaluronic acid-binding tic peptidoglycan. The previously described embodiments of the hyaluronic acid-binding synthetic oglycan are applicable to the composition for in vitro culture described herein.

In various aspects, the composition for in vitro culture of the present invention can be combined with nutrients, including ls, amino acids, sugars, peptides, proteins, vitamins (such as ascorbic acid), or laminin, fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, or growth factors such as epidermal growth factor, et-derived growth factor, transforming growth factor beta, or fibroblast growth factor, and glucocorticoids such as dexamethasone.

In some embodiments, the composition for in vitro culture es stem cells selected from the group consisting of osteoblasts, osteogenic cells, and mesenchymal stem cells.

In various embodiments, the ition for in vitro culture can be seeded with one or more cell types in ation.

In one illustrative aspect, the composition for in vitro culture may be sterilized.

As used herein “sterilization” or “sterilize” or “sterilized” means disinfecting the composition by removing unwanted contaminants including, but not limited to, endotoxins, nucleic acid contaminants, and infectious agents. The sterilization procedures, methods and embodiments provided in the preceding paragraphs are also able to the ition for in vitro culture bed herein. The in vitro e composition may be used to expand populations of cells for implantation or injection into a patient.

In one ment described herein, an additive for a biomaterial age replacement composition is provided. The additive comprises a hyaluronic acid-binding synthetic peptidoglycan for addition to an existing biomaterial cartilage replacement al.

The previously described embodiments ofthe hyaluronic acid-binding synthetic oglycan are able to the additive described herein.

As used herein, the phrase “existing biomaterial cartilage replacement material” means a biologically compatible composition that can be utilized for replacement of d, defective, or missing cartilage in the body. Various types of existing biomaterial cartilage replacement compositions are well-known in the art and are contemplated. For example, existing biomaterial cartilage or bone replacement compositions include the DeNovo® NT Natural Tissue Graft r), MaioRegenTM (JRI Limited), or the collection of cryopreserved osteoarticular tissues produced by Biomet.

In one embodiment, a method of preparing a biomaterial or bone cartilage replacement is ed. The method comprises the step of combining the synthetic peptidoglycan and an existing biomaterial or bone cartilage replacement material. The previously described embodiments of the hyaluronic acid-binding synthetic peptidoglycan are applicable to the method described herein.

In one ment, a method of treatment for arthritis in a patient is provided.

The method comprises the step of administering to the patient a hyaluronic acid-binding synthetic peptidoglycan, wherein the synthetic peptidoglycan reduces one or more symptoms associated with arthritis. The previously described embodiments of the hyaluronic acid-binding tic peptidoglycan are applicable to the method described herein.

In various embodiments, the synthetic peptidoglycan used in the method of treatment for arthritis s one or more symptoms associated with arthritis. Various symptoms are known in the art to be associated with arthritis, including but not limited to pain, ess, tenderness, inflammation, swelling, redness, warmth, and decreased mobility. The symptoms of arthritis may be present in a joint, a tendon, or other parts of the body. As used herein, “reducing” means preventing or completely or lly alleviating a symptom of arthritis.

In various ments, the arthritis is osteoarthritis or toid arthritis.

The pathogenesis and clinical symptoms of osteoarthritis and rheumatoid arthritis are well- known in the art. In one embodiment of this method, the synthetic peptidoglycan acts as a lubricant following administration or prevents loss of cartilage. In another embodiment, the synthetic peptidoglycan prevents articulation of bones in the patient. For example, the synthetic peptidoglycan inhibits bone on bone articulation in a patient with reduced or damaged cartilage.

In one embodiment, a method ofreducing or preventing degradation ofECM components in a patient is provided. For example, a method of reducing or ting degradation ofECM components in the cartilage of a patient is provided. The method comprises administering to the patient a hyaluronic acid-binding synthetic peptidoglycan. The usly described embodiments of the onic acid-binding synthetic oglycan are applicable to the method described herein. In one embodiment, the synthetic peptidoglycan is resistant to matrix metallo proteases, e.g., an aggrecanase.

In r embodiment, a method ofreducing or preventing hyaluronic acid degradation in a patient is provided. The method comprises administering to the patient a onic acid-binding synthetic peptidoglycan. The previously described ments of the hyaluronic acid-binding synthetic peptidoglycan are applicable to the method described herein.

In another embodiment, a method of reducing or preventing collagen degradation is provided. The method comprises the steps of contacting a hyaluronic acid- binding synthetic peptidoglycan with hyaluronic acid in the presence of collagen, and reducing or preventing collagen degradation. The previously described embodiments of the onic acid-binding synthetic peptidoglycan are applicable to the method bed herein.

In another ment, a method of reducing or preventing chondroitin sulfate degradation is provided. The method comprises the steps of contacting a hyaluronic acid- g synthetic peptidoglycan with hyaluronic acid in the presence of collagen, and reducing or preventing oitin sulfate degradation. The previously bed embodiments of the hyaluronic acid-binding synthetic peptidoglycan are applicable to the method described herein.

“Reducing” ECM component degradation, e. g., onic acid, collagen, or chondroitin sulfate degradation, means completely or lly reducing degradation of hyaluronic acid, collagen, or chondroitin sulfate, respectively.

In one embodiment, reducing hyaluronic acid degradation in a t means reducing the rate of hyaluronic acid degradation. For example, Figure 8 described in the Examples section of the application shows that the rate of hyaluronic acid degradation in a mixture of hyaluronic acid and a hyaluronic inding synthetic peptidoglycan is significantly reduced upon addition ofthe synthetic peptidoglycan.

In one embodiment, reducing en degradation means reducing the rate of collagen degradation. For example, Figure 10 described in the Examples section of the application shows that the rate of collagen degradation in the presence of hyaluronic acid and a hyaluronic acid-binding synthetic peptidoglycan is significantly reduced upon addition of the synthetic peptidoglycan.

In one embodiment, reducing chondroitin sulfate degradation means reducing the rate of chondroitin sulfate degradation. For example, Figure ll described in the Examples section of the application shows that the rate of chondroitin sulfate degradation in the presence of a onic acid-binding synthetic peptidoglycan is significantly reduced upon on of the synthetic peptidoglycan.

In one embodiment described herein, a method for correcting or modifying a tissue defect in a patient is provided. The method comprises administering into the tissue defect hyaluronic acid and a hyaluronic inding synthetic peptidoglycan wherein the defect is corrected or d. The previously described embodiments of the hyaluronic acid-binding synthetic oglycan are applicable to the method described herein. In one embodiment, the tissue defect is a cosmetic defect.

The following embodiments are applicable to methods described herein where the hyaluronic acid-binding synthetic peptidoglycan is administered to a patient. In various embodiments, the hyaluronic inding synthetic peptidoglycan can be injected or ted (e.g., incorporated in a age repair composition or ). In some embodiments described herein, the injection is an intraarticular injection. In r embodiment described herein, the injection is into a joint capsule of the patient. In other embodiments, the injection is a subcutaneous injection, as in the case of dermal fillers. Suitable means for injection e a needle (including microneedle) injector or a device for infusion.

In an illustrative embodiment, pharmaceutical formulations for use with hyaluronic acid-binding synthetic peptidoglycans for administration to a patient comprise: a) a pharmaceutically active amount of the hyaluronic acid-binding synthetic oglycan; b) a pharmaceutically acceptable pH buffering agent to provide a pH in the range of about pH 4.5 to about pH 9; c) an ionic strength ing agent in the concentration range of about 0 to about 300 millimolar; and d) water soluble Viscosity modifying agent in the concentration range of about 0.25% to about 10% total formula weight or any individual component a), b), c), or d) or any combinations of a), b), c) and d).

In various ments described , the pH buffering agents are those agents known to the skilled artisan and include, for example, e, borate, carbonate, citrate, and ate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic acid, hloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethyl barbituric acid, and proteins, as well as s biological buffers, for example, TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate, or MES.

In various embodiments described , the ionic strength modifying agents include those agents known in the art, for example, in, propylene glycol, mannitol, e, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

Useful viscosity modulating agents include but are not d to, ionic and non- ionic water soluble polymers; crosslinked acrylic acid polymers such as the “carbomer” family ofpolymers, e.g., carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark; hydrophilic polymers such as polyethylene oxides, polyoxyethylene- ypropylene copolymers, and nylalcohol; cellulosic polymers and cellulosic polymer tives such as ypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; gums such as tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acid and salts thereof, chitosans, gellans or any combination thereof.

Typically, non-acidic viscosity enhancing , such as a neutral or basic agent are employed in order to facilitate achieving the desired pH of the formulation.

In various embodiments described herein, formulations for injection may be suitably formulated as a sterile non—aqueous solution or as a dried form (e.g., lyophilized) to be used in conjunction with a suitable vehicle such as sterile, pyrogen—free water. The preparation of formulations for injection under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical ques well-known to those skilled in the art. In one embodiment, the viscosity of a solution containing hyaluronic acid is increased by addition of a hyaluronic acid—binding synthetic peptidoglycan.

In various embodiments described herein, the solubility of a hyaluronic acid- binding synthetic peptidoglycan used in the preparation of ations for administration via injection may be sed by the use of appropriate ation ques, such as the incorporation of so lubility-enhancing compositions such as mannitol, ethanol, glycerin, hylene glycols, ene glycol, poloxomers, and others known to those of skill in the art.

In various embodiments described herein, formulations for administration via injection may be formulated to be for immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations. Thus, a hyaluronic acid-binding synthetic oglycan may be formulated as a solid, semi-so lid, or thixotropic liquid for administration as an implanted depot providing modified e of the active compound. Illustrative examples of such formulations e drug-coated stents and copolymeric(dl-lactic, glycolic)acid (PGLA) microspheres. In another embodiment, hyaluronic acid-binding synthetic peptidoglycans or compositions comprising hyaluronic acid-binding synthetic peptidoglycan may be continuously administered, Where appropriate.

In any of the embodiments described herein, the hyaluronic acid-binding synthetic peptidoglycan can be administered alone or in combination with le pharmaceutical carriers or diluents. Diluent or carrier ingredients used in the hyaluronic acid- binding synthetic peptidoglycan formulation can be selected so that they do not sh the desired effects of the hyaluronic acid-binding synthetic peptidoglycan. The hyaluronic acid- binding synthetic oglycan formulation may be in any suitable form. es of suitable dosage forms include aqueous solutions of the hyaluronic acid-binding tic peptidoglycan, for example, a solution in isotonic saline, 5% e or other well-known pharmaceutically acceptable liquid carriers such as alcohols, glycols, esters and amides.

Suitable dosages of the hyaluronic inding tic peptidoglycan can be determined by standard methods, for example by establishing dose-response curves in laboratory animal models or in clinical trials. In various embodiments described herein, the dosage of the hyaluronic acid—binding synthetic peptidoglycan, can vary significantly depending on the patient condition, the disease state being treated, the route of administration and tissue distribution, and the possibility of co—usage of other therapeutic treatments. Illustratively, suitable dosages of onic acid-binding synthetic peptidoglycan (administered in a single bolus or over time) include from about 1 ng/kg to about 10 mg/kg, from about 100 ng/kg to about 1 mg/kg, from about 1 ug/kg to about 500 ug/kg, or from about 100 ug/kg to about 400 ug/kg. In each of these embodiments, dose/kg refers to the dose per kilogram of patient mass or body weight. In other illustrative aspects, ive doses can range from about 0.01 ug to about 1000 mg per dose, from about 1 ug to about 100 mg per dose, or from about 100 ug to about 50 mg per dose, or from about 500 ug to about 10 mg per dose, or from about 1 mg to 10 mg per dose, or from about 1 to about 100 mg per dose, or from about 1 mg to 5000 mg per dose, or from about 1 mg to 3000 mg per dose, or from about 100 mg to 3000 mg per dose, or from about 1000 mg to 3000 mg per dose. In one embodiment, suitable dosages of a hyaluronic acid-binding synthetic oglycan include trations ranging from about 0.01 uM to about 100 uM, about 0.05 to about 100 uM, about 0.1 uM to about 100 uM, about 0.1 uM to about 50 uM, about 0.1 uM to about 20 uM, about 0.1 uM to about 10 uM, about 0.5 uM to about 10 uM, about 0.5 uM to about 50 uM, and about 0.5 uM to about 100 uM. In another embodiment, suitable dosages of a hyaluronic inding synthetic oglycan include concentrations of about 0.01 uM, 0.1 uM, 0.2 uM, 0.5 uM, 1 uM, 2 uM, 5 uM, 10 uM, 20 uM, 50 uM, and 100 uM.

The hyaluronic acid-binding synthetic peptidoglycan can be formulated in an excipient. In any of the ments described herein, the excipient can have a concentration ranging from about 0.4 mg/ml to about 6 mg/ml. In various embodiments, the concentration of the excipient may range from about 0.5 mg/ml to about 10 mg/ml, from about 0.1 mg/ml to about 6 mg/ml, from about 0.5 mg/ml to about 3 mg/ml, from about 1 mg/ml to about 3 mg/ml, from about 0.01 mg/ml to about 10 mg/ml, and from about 2 mg/ml to about 4 mg/ml.

In embodiments where the hyaluronic acid-binding synthetic peptidoglycan is implanted as part of a cartilage repair composition or device (e.g., a gel for implantation), any suitable formulation described above may be used.

Any effective regimen for administering the hyaluronic acid-binding synthetic oglycan can be used. For example, the hyaluronic acid-binding synthetic oglycan can be administered as a single dose, or as a multiple—dose daily regimen. Further, a staggered regimen, for example, one to five days per week can be used as an alternative to daily treatment.

In various embodiments bed herein, the patient is treated with multiple injections of the hyaluronic acid—binding synthetic peptidoglycan. In one embodiment, the patient is injected multiple times (e.g, about 2 up to about 50 times) with the hyaluronic acid- binding synthetic peptidoglycan, for e, at 12—72 hour intervals or at 48-72 hour intervals.

Additional injections of the hyaluronic acid—binding synthetic peptidoglycan can be administered to the patient at an interval of days or months after the initial inj ections(s).

In any of the embodiments herein described, it is to be understood that a combination of two or more hyaluronic acid—binding synthetic peptidoglycans, differing in the peptide portion, the glycan n, or both, can be used in place of a single hyaluronic acidbinding synthetic peptidoglycan.

It is also appreciated that in the foregoing embodiments, certain aspects of the compounds, compositions and s are presented in the alternative in lists, such as, ratively, selections for any one or more of G and P. It is therefore to be understood that various alternate embodiments of the ion include individual members of those lists, as well as the various subsets of those lists. Each of those combinations is to be understood to be described herein by way of the lists.

In the following illustrative examples, the terms “aggrecan c” and “mimetic” are used synonymously with the term “hyaluronic acid-binding synthetic peptidoglycan.” EXAMPLE 1 Peptide Synthesis All peptides were synthesized using a Symphony peptide synthesizer (Protein Technologies, Tucson, AZ), utilizing an FMOC protocol on a Knorr resin. The crude peptide was released from the resin with TFA and purified by reverse phase chromatography on an AKTAexplorer (GE Healthcare, Piscataway, NJ) utilizing a Grace-Vydac 218TP C-18 reverse phase column and a gradient of water/acetonitrile 0.1%TFA. Dansyl—modified peptides were prepared by adding an additional coupling step with dansyl-Gly (Sigma) before release from the resin. Peptide structures were ed by mass spectrometry. The following peptides were prepared as described above: NALTVRGGGC, KQKIKHVVKLKGC, and KLKSQLVKRKGC.

EXAMPLE 2 Chondroitin Sulfate Functionalization and Synthetic Peptidoglycan Formation The reaction schematic for the creation of the an mimic (i.e., GAH) can be seen in Figure 1. onalization of the chondroitin sulfate (CS) (Sigma, St. Louis, MO) was accomplished using sodium periodate (Thermo Scientific, m, MA) to oxidize the CS.

By varying the reaction duration and sodium ate tration, the number of de groups produced by the ion reaction was controlled, values presented in Table 2. Table 2 details the sodium periodate concentration and the reaction duration needed to obtain the desired number of aldehydes per CS chain. Through progressive chemical reactions, schematic shown in Figure l, the number ofBMPH attached per CS chain is assumed to equal the number of aldehydes produced and the number of hyaluronic acid (HA) binding peptides attached.

Based on the reaction on and the concentration of sodium periodate, the number of peptides (average) per CS chain is shown in Table 2.

TABLE 2.

Sodium Periodate Concentration (mM) Reaction Duration (hr) # Aldehydes/ CS Chain —20 —— The tration of CS was kept constant at 20 mg per mL for all oxidation ons. The measured amounts of CS and sodium periodate were reacted and protected from light in 0.1 M sodium e buffer (pH 5.5) for the durations specified. tion of the reaction was obtained by removing sodium periodate by performing gel filtration chromatography with a Bio-Scale Mini Bio—Gel column packed with polyacrylamide beads (Bio-Rad tories, Hercules, CA) using an AKTA Purifier FPLC (GE Healthcare, Piscataway, NJ). The running buffer used for the desalting process was lx Phosphate Buffered Saline (PBS, pH 7.4, Invitrogen, Carlsbad, CA).

N—[B-Maleimidopropionic acid]hydrazide,trifluoroacetic acid salt (BMPH, Pierce, Rockford, IL) was reacted in a 50 M excess with the desalted, oxidized CS in lx PBS.

The hydrazide end ofBMPH reacts to covalently attach to the fiinctionalized CS, via the newly created aldehydes, to form a Schiff base intermediate. Sodium cyanoborohydride (5 M, Pierce) was added to the reaction to reduce the Schiff base intermediate imine to a more stable amine.

Excess BMPH was removed from the solution by FPLC ing in deionized water. Due to the absorbance ion capabilities on the AKTA Purifier FPLC, the amount of excess BMPH was measured. The small size and low lar weight ofBMPH (297.19 g/mol) resulted in its elution from the column at a separate, much later timepoint. With the presence of its numerous single bonds and occasional double bonds, BMPH produced a strong absorbance spectrum at both the 215 nm wavelength (characteristic of single bonds) and 254 nm wavelength (characteristic of double bonds). Therefore, a standard curve was produced, correlating known BMPH masses to the integrated area of the 215 nm absorbance spectra, Figure 2. With this standard curve, the mass of excess BMPH was ined. Subtracting the excess BMPH mass from the original reaction mass allows the determination of the mass of BMPH consumed in the reaction. Using the consumed mass, the number ofBMPH bound to the oxidized CS was calculated. The collected CS-BMPH t was frozen, lyophilized, and stored at -80° Celsius.

The HA binding peptide sequence was identified by Mummert. Slight modifications to the identified sequence produced the specific HA binding sequence, GAHWQFNALTVRGGGC (noted as GAH), that was used in this research. The peptide was ed by and purchased from Genscript (Piscataway, NJ). The cysteine amino acid was ed to allow coupling, by way of thioether bond formation, to the maleimide group of BMPH. This on occurs at a 1:1 ratio, allowing the assumption that the number ofBMPH bound to the functionalized CS will equal the number ofGAH peptides attached. GAH peptide, at one molar excess to the number ofBMPH coupled per chain, was dissolved in dimethyl sulfoxide (DMSO, Sigma) and was added to the CS-BMPH solution in 15 minute intervals, a quarter of the volume at a time. After the last addition of GAH peptide, the reaction was allowed to progress for two hours. During this time, the excess GAH peptide formed particulates. Before purifying the on to obtain GAH functionalized CS, the on was passed through an Acrodisc 0.8 um pore er filter (Pall, Port Washington, NY) to remove the excess peptide particulates. The solution was then passed, with deionized water, through the AKTA Purifier FPLC to purify the GAH-CS compound. The collected compounds were then frozen at -80° Celsius and lyophilized to produce the desired aggrecan-mimics. By laboratory convention, the aggrecan mimic was named by (# ofpeptides attached) (first three letters of e sequence) — (GAG abbreviation that was fianctionalized) i.e. for the an mimic, 3GAH-CS for 3 GAH HA binding peptides functionalized to a chondroitin sulfate GAG backbone.

EXAMPLE 3 Binding of Synthetic Peptidoglycan to Hyaluronic Acid Synthetic Peptidoglycan Binding to Immobilized onic Acid Hyaluronic Acid (HA, from Streptococcus equi, Sigma) at a concentration of 4 mg per mL, was immobilized to a 96-well plate (Costar, blk/clr, Corning, Corning, New York) ght at 4° Celsius. Biotin labeled GAH peptides were bound, by way ofBMPH, to functionalized CS at a concentration of 1 biotin-GAH per CS chain. Unlabeled GAH peptides bound to the remaining unreacted aldehydes of CS. Standard biotin-streptavidin ion methods were ed to determine the degree of aggrecan mimic binding to the immobilized HA. ng of the HA surface was done for one hour with 1% Bovine Serum Album (BSA, Sera Care Life Sciences, Milford, MA) in 1x PBS solution. After washing with lx PBS, the biotin-labeled aggrecan mimic was incubated in the well for 30 minutes and then washed with 1X PBS. Streptavidin-horseradish peroxidase (R&D s, Minneapolis, MN) solution was added to each well, and allowed to react for 20 minutes. After reaction completion and washing, chromogen solution was added (Substrate Reagent Pack, R&D Systems) and developed for 15 min. At 15 min, sulfuric acid ) was added directly to each well to stop the reaction. The well plate was then read on the M5 SpectraMax Plate Reader (Molecular s, Sunnyvale, CA) at 450 and 540 nm wavelengths. By subtracting the two absorbance readings produced, the absorbance due to the bound biotin-labeled aggrecan-mimic was determined.

One GAH e per aggrecan mimic was replaced by a biotin-labeled GAH peptide and the now-labeled aggrecan mimic was ted with immobilized HA.

Commercially available biotin detection products (through streptavidin and HRP) demonstrated the degree of mimic g to the immobilized HA (see Figure 3). Starting at a concentration of 1 uM, the aggrecan mimic had a dose dependent increase in ce on the immobilized HA, proving that the mimic was binding to the HA. However, the ination of the mimic’s binding affinity was not pursued due to the uncertainty of the amount ofHA immobilized.

Rheometer Derived Synthetic Peptidoglycan Binding to Hyaluronic Acid HA solutions were created to test the aggrecan-mimic’s ability to bind to HA in a more physiologically relevant situation. The ability of the an-mimic to bind to HA was deduced by the improvement in storage modulus of the solution, indicating HA crosslinking by the mimic. Multiple treatments were created in 1x PBS pH 7.4 to test the aggrecan mimic’s ability to bind HA: 2.5 wt% HA control, HA+CS at a 25:1 molar ratio of CS:HA, HA+3GAH- CS at 25:1, HA+7.2GAH-CS at 25:1, HA+10.5GAH-CS at 25:1.

Using the AR—G2 Rheometer (TA Instruments, New Castle, DE), frequency (0.1 — 100 Hz, 2.512 Pa) and stress (0.1 — 100 Pa, 1.0 Hz) sweeps were conducted to measure the storage s of each solution.

Rheology studies the flow of a nce in response to d forces and is often used when measuring viscoelastic materials. In particular, the rheometer determines the storage modulus and the loss modulus based on the substance feedback to the d force.

The storage modulus is a measure of the amount of energy that is elastically absorbed by the substance and the loss modulus s the amount of energy lost through heat. A large storage modulus is indicative of a gel—like nce with a more rigid, elastic structure; whereas, a small storage modulus and a large loss modulus te a viscous material that does not elastically retain the applied load. The high molecular weight HA (~1.5MDa) is a very viscous material which elastically retains a portion of the applied load due to a pseudo-gel formed by HA chain entanglement. The d aggrecan mimic contains le HA binding peptides which can act as a type ofHA chain crosslinker assuming adequate mimic binding to the HA.

In solution with the high molecular weight HA, it is esized that the aggrecan mimic could increase the rigidity of the solution, creating a larger storage modulus. A larger storage modulus would be indicative of ive HA crosslinking, proving a strong binding y n the an mimic and the HA chains present in the mixture. Multiple versions of the aggrecan mimic were tested, entiated by the number of GAH peptides (on average either 3, 7.2, or 10.5) attached per functionalized CS chain.

The s of the experiment, shown in Figure 4, showed that the addition of CS significantly (0t=0.05) lowered the storage modulus of the HA solution. The addition of the dense ve charges associated with the CS helped spread the HA chains, easing the degree ofHA entanglement and removing the pseudo—gel that stored the applied energy. Confirming the hypothesis, as the number of GAH peptides per CS increased from 3 to 10.5, the storage modulus of the mixture sed as well. This increase can be attributed to two beneficial attributes of having a higher number ofGAH peptides per aggrecan mimic. First, the more GAH peptides attached per CS, the higher the avidity of the mimic, resulting in a stronger mimic binding to the HA molecule. Second, the more GAH peptides attached per CS, the greater the likelihood of the mimic acting as a crosslinker between the HA molecules. Both effects contributed to a more gel-like mixture, resulting in a larger measured storage modulus.

Weaker binding between the mimic and HA would not restore the pseudo-gel and would be unable to store the applied energy from the rheometer. The increase in storage modulus confirms the strong mimic binding to the immobilized HA shown in Figure 3. Specifically at .5 GAH peptides per CS chain, the storage modulus was significantly (u=0.05) higher than the HA+CS control, reaching an average storage modulus similar to the HA control.

EXAMPLE 4 Sflthetic Peptidoglycan Compression Studies Collagen Gel Formation and Turbidity To mimic the native cartilage extracellular matrix, en was utilized to entrap the HA and aggrecan-mimic ates within a natural scaffold. en type 11 (CH) was obtained from two different commercial sources (Affymetrix, Santa Clara, CA and Sigma).

Mixtures of the cartilage ECM components were ed in TBS Buffer (60 mM TES, 20 mM NazHPO4, 0.56 M NaCl, chemicals from Sigma) pH 7.6 according to the native component breakdown, where CII comprised 70 dry wt% and the combination ofHA and the aggrecan mimic/CS control formed the remaining 30 dry wt% ofthe mixture. The final concentration of C11 in the gel was 2 mg per ml. s consisted of a C11 control, CII+HA+CS control, and CII+HA+aggrecan mimic AH-CS). To prevent premature fibrillogenesis and gel ion, the solutions were kept on ice at an acidic pH. Solution mixtures of the components were placed in a 384 well plate (Greinier blk/clr, Monroe, NC), placed at 37° C and physiological pH to initiate fibrillogenesis, and were monitored at 3 13 nm on the M5 aMax to determine gel formation. CII was unable to form gels when included with the varying treatments (See Supplementary Information). ore, collagen type I (CI, High Concentration Rat Tail Collagen Type 1, BD Biosciences, Bedford, MA) was ed for the gel formation. The same treatments and procedure were used with the CI, except that the component masses were d for a CI final concentration of 4 mg per mL. CI was used for all ing experiments.

Turbidity with C1 was performed to measure the formation of the cartilage replicate, results shown in Figure 5. As demonstrated, the addition ofHA+lO.5GAH-CS did not affect the fibrillogenesis of the collagen fibers. All treatments followed a similar curve and reached similar absorbance peaks at about the same time. HA+lO.5GAH-CS treatment had a higher initial absorbance due to the an mimics tendency to form self-aggregates in lx PBS solution, not due to premature CI fibril ion. The aggregation of lO.5GAH-CS was recognized during the initial HA rheometer tests, but the ation did not inhibit the an mimic’s ability to bind to HA.

Collagen Gel Property Testing Collagen-based gel compression tests and frequency sweeps were conducted using an AR-G2 Rheometer using a 20-millimeter parallel plate geometry (TA Instruments).

The 375 uL gel mixtures were prepared on ice and pipetted onto the rheometer base plate. The geometry was lowered to a gap distance of 1 mm and the solution was heated to 37° Celsius. A humidity trap was utilized to t gel dehydration while the mixture was allowed to gel over two hours. This two hour value was determined by the demonstrated time to gelation data from the turbidity data. After the two hour time period, the gels were compressed or oscillated depending on the test. Compression tests occurred at an engineering strain rate of 1% (10 um) per second. The gap distance and the normal force on the geometry head were measured. The frequency sweeps ed the storage modulus of the created gels during a logarithmic base ten increase in frequency from 0.1 to 1 Hz.

The simultaneous normal force and displacement were measured, and the engineering stress and strain were calculated for the treatments. As shown in Figure 6, the inclusion of the aggrecan mimic significantly (0t=0.05) increased the compressive strength of the gel complex. The peak engineering stress of the collagen+HA+AGG mimic reached 7.5 kPa at an engineering strain of 9%, whereas the collagen+HA+CS control reached a peak of 4.8 kPa at 4%, and the collagen control reached a peak of 4.2 kPa at 15% strain.

Two factors contributed to the se in compressive strength of the CI+HA+lO.5 gel, the first being the mimic’s ability to t water and the second being the HA crosslinking ability of the aggrecan mimic. In native age, the predominance of the entrapped negative charges ed by the HA and CS attract water and retard its diffiasion from the cartilage. When a compressive force is applied to the cartilage, the water is not able to diffuse out into the synovial e. Retaining this incompressible water increases the compressive strength of the ure. Similarly in the tested gel complexes, the inclusion of the negative charges associated with CS in the gel provides the same attraction. As can be seen in Figure 6, both the CS and 10.5GAH-CS treatments have an increased compressive strength.

The CS treatment is not fixed within the CI complex (it is not bound to HA) and ore after a small compressive ation, the CS and its attracted water diffuse out of the complex into the surrounding fluid. The diffusion of the CS and water from the complex shes the compressive strength of the complex, causing the resulting gel’s compressive profile to resemble that of the collagen scaffold control. In contrast, 10.5GAH-CS is bound to the interwoven HA. Therefore, a much higher compressive stress is required to overcome the binding of the mimic to HA and cause the diffusion of CS and attracted water from the complex. ly, the ability of the aggrecan mimic to act as a HA crosslinker results in a higher degree of entrapment for the HA and mimic. Effectively, the HA crosslinking nature creates large aggregates within the collagen x, similar to the native aggrecan/HA aggregates. The main difference between the aggrecan mimic and native an is the size of the molecule. The protein backbone of aggrecan alone weighs ~220 kDa, whereas the aggrecan mimic, in entirety, only weighs around 30 kDa. Therefore, the native aggregate complex, with over 100 aggrecan molecules bound to the HA, produces much larger aggregates than the aggrecan mimic could produce. However, by acting as a crosslinker between HA chains, the an mimic can produce its own form of an aggregate that also portrays the main characteristics of native aggregates; voluminous, negatively-charged structures. The role of the aggrecan mimic as an HA crosslinker was further igated by applying shear loads through rheological tests on the CI gels described above. The results of these experiments can be seen in Figure 7.

The ion of 10.5GAH-CS cantly (0t=0.05) increased the storage modulus of the formed gel. The network created by the binding of the mimic to the HA supplemented the existing rigidity ofthe CI matrix, ng an increased elastic absorbance of the energy applied by shear loading. This study was important as it verified the crosslinking y of the 10.5GAH-CS and the creation of an alternate aggregate form.

EXAMPLE 5 Sflthetic oglycan Protection of onic Acid Degradation Dynamic viscosity values ofHA solutions were determined using the AR—GZ.

High molecular weight HA solutions have a large viscosity due to the extensive chain entanglement caused by the long chain length. Hyaluronidase (Type II from Sheep Testes, Sigma) cleaves the HA chain, creating shorter chains with less entanglement. The shorter HA chains will have a ably lower viscosity. HA solutions were incubated with 100 units/ mL hyaluronidase. Dynamic viscosities were determined using a time sweep with constant angular frequency and oscillatory stress initially and at 2 and 4-hour timepoints. Samples (at 0.5 wt% HA) consisted of HA, HA+CS, and HA+10.5GAH-CS. The treatment values were added at a 75:1 treatment to HA molar ratio. The percent degradation was calculated for each measurement by dividing the initial viscosity from the ence of the measured viscosity minus the initial viscosity.

Work by Pratta et al. and Little et al. has shown the importance of aggrecan in preventing cartilage component ation. The demolition of the cartilage matrix in rthritis is started with the cleavage of the aggrecan proteoglycans. The removal of the GAG-rich region ofthe proteoglycan exposes the remaining components, C11 and HA, to degrading enzymes. With the knowledge of the importance of aggrecan in preventing degradation, studies were conducted to determine the ability of the aggrecan-mimic in preventing HA degradation.

The viscosity of a HA solution is dependent on the size of the HA chains. Due to entanglement, larger HA chains will produce a higher viscosity. When exposed to hyaluronidase, the HA chain is cleaved into smaller units. Therefore, the size of the HA and the amount of HA entanglement decreases. This decrease prompts a similar decrease in the ed ity. The percent change in viscosity ofHA solutions in the ce of hyaluronidase will provide key information into the amount of degradation the HA has undergone. Figure 8 presents the percent degradation of HA control versus the ated ents. As can be seen, the AGG mimic, GAH, significantly reduced the rate of degradation of HA, indiating that it behaves similarly to native AGG in its protection ofECM components.

Viscosities of each treatment without hyaluronidase (TES Buffer replaced the hyaluronidase volume) were initially measured and served as the baseline for the percent degradation calculations. The 0 hr timepoint involved the addition of the hyaluronidase, mixing ofthe solution, pipetting onto the rheometer, and the beginning equilibration operation of the machine. Therefore, the 0 hr timepoint occurred approximately two minutes after the addition of hyaluronidase. A high concentration of hyaluronidase (25 units per mL) was utilized to replicate the worst possible scenario. In addition, the HA molecules were dispersed in on, rather than tightly interwoven into a en network. As can be seen from Figure 8, both the HA Control and the HA+CS ent had almost complete ation ofthe HA on at the 0 hr timepoint. In contrast, the addition of 10.5GAH-CS significantly (0t=0.05) d the amount of HA degradation. In fact, the presence of 10.5GAH-CS increased the Viscosity above the baseline values. It is believed that the addition of hyaluronidase cleaves some of the excess HA. This allows lO.5GAH-CS to better crosslink the remaining, intact chains, creating a denser gel which produced the larger Viscosity.

At the 2 hr timepoint, both the HA control and HA+CS had completely degraded with percent degradations above 90%, but the HA solution with 10.5GAH-CS had a significantly (0L=O.05) lower percent degradation. Lastly, at the 4 hr timepoint, all treatments had been degraded, with their percent degradations all above 90%. Amongst the three timepoints, lO.5GAH-CS was not able to completely prevent HA degradation, but it cally reduced the rate of degradation compared to the degradations of the HA Control and HA+CS.

This reduced rate demonstrates that the 10.5GAH-CS prevents the degradation of the HA chains. It is ed that this prevention is being accomplished through competitive inhibition ofthe hyaluronidase ge point on the HA chain. The non-covalent binding of the mimic to the HA chain coupled with the gradual degradation rate of the HA chains appear to validate this belief. In addition, the degradation rate of the 10.5GAH-CS solution is still believed to be artificially high. Upon incubation ofthe mimic within the HA solution, HA+lO.5GAH-CS ates were . However, these aggregates did not spread uniformly throughout the solution volume. Therefore, the solutions were mixed, similarly to the other samples, before a measurement was taken. The mixing of the solution disrupted the aggregates, dislodging lO.5GAH-CS and exposing the hyaluronidase ge point. Even after the 4 hr timepoint, when supposedly complete degradation had ed, ntial aggregation of HA+lO.5GAH-CS still occurred. In a t matrix like the ECM of age, it is possible that lO.5GAH-CS could not only significantly reduce the degradation rate, but suppress HA ation.

EXAMPLE 6 C oScannin Electron Microsco SEM The sed constructs, as described for turbidity measurements, were formed on an SEM plate at 37°C overnight. The SEM plates were d into a holder, and were plunged into a liquid nitrogen slush. A vacuum was pulled on the sample as it was transferred to the Gatan Alto 2500 pre—chamber. Within the chamber, cooled to -l70°C, a cooled scalpel was used to create a free break surface on the sample. The sample was subjugated to sublimation at -85°C for 15 minutes followed by a sputter-coating ofplatinum for 120 seconds. After sputter-coating, the sample was transferred to the microscope stage and images were taken at -l30°C.

Representative images were ed at a magnification of 10,000x, as shown in Figure 9. Panel A shows the CI control, and is characterized by extensive crosslinking between major fibrils, and relatively small matrix pore size. Panel B shows CI+HA+CS, and contains extensive crosslinking, but larger pore size, due to the presence of the large HA chains. Panel C shows CI+HA+lO.5GAH-CS and illustrates a noticeably smaller degree of crosslinks in on to a very large pore size. The AGG mimic can bind to the HA creating a relatively large, cumbersome complex that s the CI crosslinking.

As can be qualified in the representative images, the addition ofHA+CS did not have an effect on the variation of collagen fibril diameters, but the HA+CS sample did have a larger representative void space. In comparison to the control groups, the addition of the AGG mimic with the HA resulted in a smaller variation ofcollagen fibril diameters due to the limited number of small fibril diameters, and an overall increase in the void space of the sample. The binding of the AGG mimic to the HA molecule created an aggregate complex that was trapped within the collagen scaffold and excluded smaller fibril formation between the larger fibrils due to steric nce.

EXAMPLE 7 Collagen Protection sed constructs containing en alone, collagen+HA+CS, or collagen+HA+lO.5GAH—CS were created in 8-well chambered slides as described previously.

The final sample volume was 200 uL consisting of 0.8 mg of collagen type 1. Matrix metalloprotease-I (MMP-I, R&D Systems, Minneapolis, MN) at a tration of0. 133 mg/mL, was activated following the ol ed in the manufacturer’s instructions.

Briefly, MMP-l, already dissolved in manufacturer’s buffer (50 mM Tris, 10 mM CaClz, 150 mM NaCl, 0.05% Brij-35, pH 7.5), was combined with an equal volume of 25 mM APMA ) in DMSO at 37° C for 2 hrs to activate the enzyme. Upon activation, the MMP-l solution was d two fold in water and was added to the sample as a 100 uL supernatant.

The samples were incubated at 37°C with gentle shaking. Twenty-five hrs after the addition of the initial enzyme solution, the supernatant was removed and replaced with a fresh batch of enzyme. After 50 total hr of incubation with the enzyme, the remaining gels were removed from the red slides, washed with deionized water to remove any enzyme solution or degradation products, and resolubilized in 12 M HCl. The samples were diluted in water to reach a final concentration of 6 M HCl, and were hydrolyzed overnight at 110°C. ing hydrolysis, the amount of hydroxyproline (hyp) was analyzed according to the protocol developed by Reddy, et al. (Clin Biochem, 1996, 29: . Briefly, the hydrolyzed samples were ted with Cholramine T solution (0.56 M) for 25 minutes at room temperature before the addition of ’s reagent and uent chlorophore development for 20 minutes at 65°C. After the development of the chlorophore, the s were read on a spectrophotometer at a wavelength of 550 nm. Absorbance readings were compared to those obtained from known concentrations of collagen to determine the amount of collagen remaining in each sample.

Each replicate sample was constructed with 0.8 mg of CI, and after degradation, the ing CI amount was determined by the protocol ped by Reddy et al. and converting that to CI amount by a set of CI standards. The percent degradation was determined by subtracting the ing CI from the initial CI, ng by the l CI, and multiplying by 100. The percent degradation of the three treatments is shown in Figure 10. All the treatments were significantly different from each other (p<0.05). In particular, the percent degradation of the AGG mimic sample (CI+HA+10.5GAH-CS = 41.0%) was significantly less (p<0.05) than the other two treatments (CI=64.5% and CI+HA+CS=74.7%). The presence of the AGG mimic significantly reduced the CI degradation. The presence of the AGG mimic can act as a hindrance to the cleavage sites of the degrading enzymes. By creating the large aggregates with HA that are tightly trapped within the collagen scaffold, the AGG mimic can occupy the space proximal to the collagen, preventing enzyme access to degradation locations.

EXAMPLE 8 Diffusion of Peptidoglycans Through Cartilage Matrix Cartilage explants were obtained from the load bearing region of three month old bovine knee joints. Native aggrecan was removed from ted cartilage explants leaving a matrix consisting primarily of type II collagen and residual GAG. This was achieved by treating explants with 0.5% (w/v) trypsin in HBSS for 3 hours at 37 °C (Figure 13). After trypsin treatment explants were washed three times in HBSS and incubated with 20 % FBS to inactivate residual trypsin activity. Peptidoglycan was dissolved in led water at 10 uM concentration and diffiised through the lar surface of cartilage explants by g lO uL ofthe solution on the surface every ten minutes for one hour at room temperature (Figure 14).

Normal cartilage and aggrecan depleted cartilage were treated with 1X PBS as positive and negative controls respectively. After diffusion, explants were washed three times with lX PBS and stored at -20°C until further testing. ion of peptidoglycan was confirmed by staining a midsagittal section of the tissue with streptavidin-horseradish peroxidase stain. The streptavidin stain binds to the biotin labeled molecule and is depicted as a brown color (Figures and 16).

EXAMPLE 9 Bulk Compression g Displacement-controlled unconfined compression was performed on an AR G2 rheometer with force transducers capable of ing normal forces in the range of 001-50 N (TA Instruments). The explants were glued to the bottom of a hydrophobic printed slide (Tekdon) and covered in a 1X PBS bath. A 20mm diameter stainless steel el plate geometry head was lowered until initial contact was made. Explant height was measured using a digital micrometer (Duratool). Compressive loads from 0-30% nominal strain (at 5% intervals) were applied to the explants through a stepwise g that involved a ramp duration of 5 sec (i.e. a strain rate of 1.0 %/sec) and hold time of 30 sec. Compressive stiffness values were obtained by using the slope of equilibrium stress , computed during each hold section, versus respective strain values, based on a linear fit model. Scaffolds tested for bulk compression included: 1) Normal cartilage, 2) Aggrecan depleted cartilage (AD), and 3) AD+mAGC (Figure 17). Addition of the HA binding peptidoglycan (mAGC) significantly restored stiffness of cartilage explants to a higher extent as compared to the collagen type II g peptidoglycan (mAG(II)C).

EXAMPLE 10 Animal Model Sprague—Dawley rats (250~300g) were used for surgery. The patellar tendon, the or and posterior te ligaments and the medial, lateral collateral ligaments were transected. The medial and lateral meniscuses were totally menisectomized. The knee joint e was ed with an absorbable suture and the skin was closed with a 4-0 monofilament nylon. Starting at week 4, 10 ul of a 1 um mAGC was administered weekly.

The extent of inflammation was indicated by the MMP-13 probe e 18) in Sprague-Dawley rats treated with and without peptidoglycan at four, six and eight weeks post surgery (Figure 19). X-ray images of Sprague—Dawley rat knee joints showed injured knee 6 weeks and 8 weeks following OA induction (Figure 20, Panels A and D, respectively), injured knee with peptidoglycan treatment (Figure 20, Panels B and E, respectively), and normal knee (Figure 20, Panel C) six weeks after osteoarthritis induction surgery. MicroCT of Sprague- Dawley rats indicated re-growth ofnew cartilage six and eight weeks after OA induction surgery. Injured knees 6 weeks and 8 weeks following OA ion, e 21, Panels A and D, respectively), injured knees following peptidoglycan treatment (Figure 21, Panels B and E, respectively), and Normal knee (Figure 21, Panel C), are shown.

EXAMPLE 11 Reagents Peptide GAHWQFNALTVRGGGC (GAH) was purchased from Genscript (Piscataway, NJ). N—[B-maleimidopropionic acid] hydrazide, trifluoroacetic acid salt (BMPH) was purchased from Pierce (Rockford, IL). Rat tail type I collagen was sed from BD Biosciences (Bedford, MA). Human recombinant interlukin-lB was purchased from Peprotech (Rocky Hill, NJ). All other supplies were sed from VWR (West Chester, PA) or Sigma- Aldrich (St. Louis, MO) unless otherwise noted.

E 12 en ld Synthesis Collagen scaffolds were prepared in TBS buffer (60 mM TES, 20 mM NazPO4, 0.56 M NaCl) at a pH of 7.6. Scaffold composition for mechanical testing and in vitro atory model studies are described in their respective sections. All solutions were maintained on ice until fibrillogenesis was initiated at 37 OC. Aligned collagen lds were created by placing the collagen on at the isocenter of a 9.4 Tesla magnet (Chemagnetics CMX400) at 37 °C for one hour, whereas unaligned gels were prepared similarly but without magnetic exposure.

The slide containing the collagen solution was placed parallel to the magnetic field, orienting the collagen fibers in a ion perpendicular to the bottom of the slide. The gels were then maintained at 37 °C for 24 hours in a humidity—controlled chamber to prevent evaporation.

EXAMPLE l3 Rheological Mechanical Testing Shear and compression testing was performed on a stress-controlled AR G2 rheometer (TA Instruments) using a 20 mm er stainless steel parallel plate geometry head. Collagen scaffolds were prepared on 20 mm diameter hydrophobic printed slides (Tekdon). For shear tests, the geometry head was lowered until contact was made at a gap height of 950 um. inary frequency and stress sweeps were performed to determine a linear and stress-independent storage modulus range. Frequency sweeps were then performed on all gels with an oscillatory stress of 0.2 Pa over a frequency range of 0.1 to 2 Hz. For compression tests, the ry head was lowered until contact was made with the scaffold at a gap height of 1000 um. Compressive loads from 0-30% nominal strain (at 5% intervals) were applied to the collagen scaffold through a stepwise loading that involved a ramp duration of 5 sec (i.e. a strain rate of 1.0 %/sec) and hold time of 30 sec. Compressive stiffness values were obtained by using the slope of equilibrium stress , computed during each hold section, versus respective strain values, based on a linear fit model. Collagen scaffold composition for mechanical tests were: 1) ned collagen, 2) Aligned collagen, 3) Unaligned collagen+mAGC and 4) Aligned collagen+mAGC.

Balk Mechanical Analysis: The aggrecan mimic, mAGC, enhanced bulk mechanical properties of scaffolds, irrespective of fiber ent e 22). For shear testing, the storage moduli values at 0.5 Hz for unaligned and aligned collagen gels were 104. 1:36 Pa and 49.9::5.4 Pa respectively. The addition ofmAGC to the collagen scaffold showed a significant increase in the storage moduli of the unaligned and aligned gels to 113.9i4.6 Pa and 76.6::3.6 Pa respectively 01). Unaligned gels showed a higher storage modulus as compared to d gels (p<0.0001). For compression testing, the compressive stiffness for aligned scaffolds (2478i250 Pa) was lower than unaligned scaffolds (3564i315 Pa) 01).

Addition ofmAGC to these scaffold s increased ssive stiffness of the aligned and unaligned scaffolds to 4626i385 Pa and 57472806 Pa, respectively (p<0.0001).

E 14 In Vitro Inflammation Model Collagen scaffolds seeded with chondrocytes were stimulated with IL-lB and assessed for degradation products.

Chondrocyte Isolation: Primary ocytes were harvested from three-month-old bovine knee joints ed from an abattoir within 24 hours of slaughter (Dutch Valley Veal).

Cartilage slices, 150-200 um thick, were shaved from the lateral l condyle and washed three times in serum-free DMEM/F-l2 medium (50 ug/mL ascorbic acid 2-phosphate, 100 ug/mL sodium pyruvate, 0.1% bovine serum albumin, 100 units/mL penicillin, 100ug/mL streptomycin and 25 mM HEPES) prior to digestion with 3% fetal bovine serum (FBS) and 0.2% collagenase-P (Roche Pharmaceuticals) at 37°C for six hours. Released chondrocytes were filtered through 70 um cell strainer and centrifiiged at 1000 rpm three times for five minutes each in medium listed above supplemented with 10% PBS. The cell pellet was resuspended in 10% FBS mented media and plated on 10 cm dishes at 10,000 cells/mL in a 37 °C, 5% C02 humidified incubator until confluent.

Scafi’ola’ Fabrication: Upon reaching confluency, cells were trypsinized and encapsulated at ,000 cells/mL within collagen lds (Table 3) and allowed to equilibrate for 3 days prior to treatment.

TABLE 3: Scaffold composition for in vitro testing Unalignea’ Collagen Experimental Setup Aligned Collagen Experimental Setup A: en CS HA : IL— 18 E: Collagen+CS+HA+IL-1 B B: Collagen CS HA F: Collagen+CS+HA C: Collagen mAGC HA i IL—lB G: Collagen+mAGC+HA+IL—IB mAGC HA H: Collaen+mAGC+HA Inflammation Model: Constructs were incubated with or without 20 ng/mL IL-lB in chemically-defined media supplemented with 5% FBS and otics (100 units/mL penicillin and 100 ug/mL streptomycin). Culture medium was replaced every two days. Removed media extracts were stored at -80°C until further testing.

Degradation Assay: GAG degradation was monitored by measuring CS released in cell culture media using the ylmethylene blue (DMMB) dye assay and computed with a oitin— 6-sulfate standard curve. Similarly, type I collagen degradation in cell culture media was monitored using the Sircol en Assay using manufacturer ed protocols (Bio-Color).

GAG and collagen degradation were reported as cumulative release over an eight-day culture period.

Proteolytic Degradation Analysis: The amount of CS and collagen ed into cell culture media was significantly decreased when lds that ned mAGC (Figures ll, 12, 23 and 24) (pcs<0.001 and pcouagen<0.02, respectively). Aligned collagen gels showed a statistically higher CS and collagen release into the media as compared to unaligned collagen fibers (p<0.00 l ).

As bed herein, the hyaluronic-binding synthetic peptidoglycan is able to t HA and the underlying collagen fibers in the scaffold from proteolytic cleavage. The synthesis of the hyaluronic-binding synthetic peptidoglycan utilized the chondroprotective benefits of CS. CS has been shown to down-regulate matrix metalloproteases tion. Our synthetic peptidoglycan design herein bed allowed CS chains to be attached to HA, ting degradation of both molecules. By placing the synthetic peptidoglycan in an environment rich in proteolytic enzymes, its y to prevent excessive loss ofECM components has been demonstrated.

E 15 Real—time PCR Following the cell e study, constructs were stored in RNAlater solution (Ambion) at 4 CC for less than one week. Total mRNA was extracted using Nucleospin RNA 11 (Clontech) according to manufacturer’s protocols. Extracted mRNA from all samples was quantified using Nanodrop 2000 ophotometer (Thermo Scientific) and reverse transcribed into cDNA using High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). Real- time PCR was performed using Taqman Gene Expression Assays (Applied Biosystems) with the following primers: GAPDH (Bt03210913_g1), aggrecan (Bt032l2l86_ml) and collagen type II 5186l_ml). 60 ng of cDNA template was prepared per 20 uL reaction for the two genes of interest and the endogenous gene. Real-time PCR analysis was carried out using a Taqman PCR Master Mix and 7500 ime PCR System (Applied Biosystems). Data reported was normalized to GAPDH gene expression. mRNA Expression Analysis: Collagen alignment, presence of aggrecan mimic and stimulation with IL-lB significantly effected aggrecan (pangnment , ppeptidoglycan <0.02 and p1L_1B<0.001) and collagen type II expression (palignmem <0.01, ppeptidoglycan <0.001 and p1L_1[3<0.015). The presence ofmAGC limited excessive loss ofCS from the scaffold, which results in a lower aggrecan expression (p<0.02) (Figure 25). The presence ofmAGC also limited collagen degradation. However, collagen type II expression depended on the extent of collagen lost during degradation (Figure 25). In unaligned scaffolds, the level of collagen type II expression was higher in scaffolds prepared without mAGC, whereas in aligned en scaffolds, the level of en type II was higher in scaffolds prepared with mAGC (p<0.05).

EXAMPLE 16 Statistical Analysis Each experiment was repeated twice, with at least n=3 in each data set.

Statistical significance for mechanical test data was analyzed with a y ANOVA with alignment and addition of peptidoglycan as factors. The cell culture data was analyzed using a three-way ANOVA with alignment, addition ofpeptidoglycan, and treatment with IL-lB as factors. A oe Tukey pairwise comparison (or: 0.05) was used to directly compare scaffolds prepared with and without the aggrecan mimic in each system.

I

Claims (29)

/WE CLAIM:

1. A hyaluronic inding tic peptidoglycan comprising a glycan and 1 to 20 synthetic es conjugated to the ne of the glycan, wherein each synthetic peptide is 5 to 40 amino acids in length and can bind to a hyaluronic acid. 5

2. The synthetic peptidoglycan of claim 1 wherein the synthetic peptide comprises an amino acid sequence of the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and 10 wherein X1-X8 are non-acidic amino acids.

3. The synthetic peptidoglycan of claim 1 or claim 2 wherein the synthetic peptide comprises an amino acid sequence selected from the group consisting of: (i) GAHWQFNALTVRGG; GDRRRRRMWHRQ; 15 GKHLGGKHRRSR; RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; 20 RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; RLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; 25 RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; 30 RRIGHQVGGRRN; RLESRAAGQRRA; AH26(11211529_2):GCC GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; 5 YQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; 10 RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR or 15 (ii) an amino acid sequence having at least 90% sequence identity to an amino acid sequence of (i).

4. The synthetic peptidoglycan of any one of claims 1 to 3 wherein the glycan is selected from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid. 20

5. The tic peptidoglycan of any one of claims 1 to 4 wherein the synthetic peptidoglycan is resistant to anase.

6. The tic peptidoglycan of any one of claims 1 to 5 wherein the peptide component of the synthetic peptidoglycan has a glycine-cysteine attached to the C-terminus of the peptide.

7. An engineered collagen matrix comprising polymerized en, hyaluronic acid, and a 25 hyaluronic acid-binding synthetic peptidoglycan comprising a glycan and 1 to 20 synthetic peptides conjugated to the backbone of the glycan, n each tic peptide is 5 to 40 amino acids in length and can bind to a hyaluronic acid.

8. The ered collagen matrix of claim 7 wherein the peptide component of the synthetic peptidoglycan comprises an amino acid sequence of the formula B1-X1-X2-X3-X4- 30 X5-X6-X7-X8-B2, AH26(11211529_2):GCC n X8 is present or is not present, wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein X1-X8 are non-acidic amino acids. 5

9. The ered collagen matrix of claim 7 or 8 wherein the peptide component of the synthetic oglycan comprises an amino acid sequence selected from the group consisting (i) GAHWQFNALTVRGG; GDRRRRRMWHRQ; 10 GKHLGGKHRRSR; RGTHHAQKRRS; HIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR; 15 RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; 20 RMRRKGRVKHWG; RGGARGRHKTGR; TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; 25 RRIGHQVGGRRN; RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; 30 RRRKKIQGRSKR; RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; AH26(11211529_2):GCC VVKLK; KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; 5 KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR or (ii) an amino acid sequence having at least 90% sequence identity to an amino acid sequence of (i).

10 10. The engineered collagen matrix of any one of claims 7 to 9 wherein the glycan component of the tic peptidoglycan is selected from the group consisting of dextran, chondroitin, oitin sulfate, dermatan, dermatan sulfate, heparan, n, keratin, keratan sulfate, and hyaluronic acid.

11. The engineered collagen matrix of any one of claims 7 to 10 wherein the synthetic 15 peptidoglycan is resistant to aggrecanase.

12. The engineered collagen matrix of any one of claims 7 to 11 wherein the peptide component of the synthetic peptidoglycan has a glycine-cysteine attached to the inus of the peptide.

13. The engineered collagen matrix of any one of claims 7 to 12 wherein the matrix further 20 comprises an exogenous population of cells.

14. Use of a hyaluronic acid-binding synthetic peptidoglycan in the cture of a medicament for the treatment of tis, wherein the treatment reduces a symptom associated with the arthritis, and wherein the synthetic peptidoglycan comprises a glycan and 1 to 20 synthetic peptides ated to the backbone of the glycan, wherein each synthetic peptide is 5 25 to 40 amino acids in length and can bind to a hyaluronic acid.

15. The use of the onic acid-binding synthetic peptidoglycan of claim 14 wherein the peptide component of the tic peptidoglycan comprises an amino acid sequence of the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not present, AH26(11211529_2):GCC wherein B1 is a basic amino acid, wherein B2 is a basic amino acid, and wherein X1-X8 are non-acidic amino acids.

16. The use of the hyaluronic acid-binding synthetic peptidoglycan of claim 14 or 15 wherein 5 the peptide component of the synthetic peptidoglycan comprises an amino acid sequence selected from the group consisting of: (i) GAHWQFNALTVRGG; RMWHRQ; GKHLGGKHRRSR; 10 RGTHHAQKRRS; RRHKSGHIQGSK; VRGRHE; TAGRPR; RYGGHRTSRKWV; 15 RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR; DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; 20 RGGARGRHKTGR; GLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG; STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; 25 RLESRAAGQRRA; GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; 30 RKSYGKYQGR; KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; AH26(11211529_2):GCC KLKSQLVKRK; RYPISRPRKR; KVGKSPPVR; KTFGKMKPR; 5 RIKWSRVSK; and TRR or (ii) an amino acid sequence having at least 90% sequence ty to an amino acid sequence of (i).

17. The use of the hyaluronic inding synthetic peptidoglycan of any one of claims 14 10 to 16 wherein the glycan component of the synthetic peptidoglycan is selected from the group consisting of dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and onic acid.

18. The use of the hyaluronic acid-binding synthetic peptidoglycan of any one of claims 14 to 17 wherein the synthetic peptidoglycan is resistant to aggrecanase. 15

19. The use of the hyaluronic acid-binding synthetic peptidoglycan of any one of claims 14 to 18 wherein the peptide component of the synthetic peptidoglycan has a glycine-cysteine ed to the C-terminus of the peptide.

20. The use of the hyaluronic acid-binding synthetic peptidoglycan of any one of claims 14 to 19 wherein the arthritis is selected from the group consisting of osteoarthritis and rheumatoid 20 arthritis.

21. The use of the hyaluronic acid-binding synthetic oglycan of any one of claims 14 to 20 wherein the dosage of the synthetic peptidoglycan is in a concentration ranging from about 0.1 µM to about 10 µM.

22. The synthetic peptidoglycan of any one of claims 1 to 6 wherein the synthetic 25 oglycan is ant to matrix metallo proteases.

23. The engineered en matrix of any one of claims 7 to 13 wherein the synthetic peptidoglycan is resistant to matrix metallo proteases. AH26(11211529_2):GCC

24. The use of the hyaluronic acid-binding synthetic peptidoglycan of any one of claims 14 to 21 wherein the synthetic peptidoglycan is resistant to matrix metallo proteases.

25. The synthetic oglycan of any one of claims 1-6 comprising 2 to 20 synthetic es conjugated to the backbone of the glycan. 5

26. The synthetic peptidoglycan of any one of claims 1-6 comprising 5 to 15 synthetic peptides conjugated to the backbone of the .

27. The use of the hyaluronic acid-binding synthetic peptidoglycan of any one of claims 14 to 21, wherein the synthetic peptidoglycan comprises 2 to 20 synthetic peptides conjugated to the backbone of the glycan. 10

28. The use of the hyaluronic acid-binding synthetic oglycan of any one of claims 14 to 21, wherein the synthetic peptidoglycan comprises 5 to 15 synthetic peptides conjugated to the backbone of the glycan.

29. A hyaluronic acid-binding synthetic peptidoglycan comprising a glycan and 1 to 20 synthetic es conjugated to the backbone of the glycan, wherein each synthetic peptide is 5 15 to 40 amino acids in length and comprises GAHWQFNALTVRGG or an amino acid sequence having at least 90% sequence identity to GAHWQFNALTVRGG.