Atraumatically formed chondrocyte compositions, methods of preparation and methods of treatment therewith

Abstract

The present application relates to chondrocyte compositions and kits comprising morselized cartilage tissue particles and a biodegradable matrix. Applications and methods of using the chondrocyte composition for cartilage grafts, repairing cartilage injuries and defects, and joint repair are provided.

Description

[0001] ATRAUMATICALLY FORMED CHONDROCYTE COMPOSITIONS, METHODS OF

[0002] PREPARATION AND METHODS OF TREATMENT THEREWITH

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004]

[0001] This application claims the benefit of the filing date of U.S. Provisional application Serial NO. 63 / 649,321, filed on May 17, 2024, the entire contents of which is incorporated herein by reference in its entirety.

[0005] SEQUENCE LISTING

[0006]

[0002] The instant application contains a Sequence Listing encoded in XML format which was filed electronically by EFS-web and is hereby incorporated by reference in its entirety. Said XML format Sequence Listing, created on April 8, 2024, is named 2709-9_P2_sequence_listing.xml and is 15,012 bytes in size.

[0007] FIELD

[0008]

[0003] The present disclosure relates to chondrocyte compositions and kits comprising morselized cartilage tissue particles and a biodegradable matrix. Applications and methods of using the chondrocyte composition for cartilage grafts, repairing cartilage injuries and defects, and joint repair are provided.

[0009] BACKGROUND OF THE INVENTION

[0010]

[0004] Articular cartilage is comprised of chondrons - chondrocytes that are surrounded by a pericellular matrix (PCM) that is rich in type VI collagen. Chondrons are surrounded by an organized extracellular matrix (ECM) that contains proteins such as collagen fibrils, proteoglycans, and non-collagenous proteins (Davies et al (2019) Bioengineering, 6(1), Article 1, doi.org / 10.3390 / bioengineering6010022). Although the structure of cartilage is relatively simple, injuries to cartilaginous structures from sports injuries, repetitive micro trauma, or biomechanical aberrations can lead to pain and’ disability, significantly impairing quality of life (Grande et al (2013) Cartilage, 4(4), 281-285 (“Grande et al. 2013”) doi.org / 10.1177 / 1947603513494402).

[0011]

[0005] Musculoskeletal conditions are among the most prevalent medical issues in the US, ranging from acute conditions like cartilage and bone trauma to chronic conditions like osteoarthritis (OA) and rheumatoid arthritis (RA) (United States Bone and Joint Initiative, The Burden of Musculoskeletal Diseases in the United States, 2nd ed (American Academy of Orthopaedic Surgeons, Rosemont, 2011)). Lesions in articular cartilage are typically due to trauma, predisposing patients to degenerative joint disease like post-traumatic osteoarthritis (PTOA) (Grande et al. 2013). The progression of acute to chronic inflammation in osteoarthritis can significantly impact human health and necessitates early efficacious intervention in cartilage repair (Grande et al. 2013). Therefore, there is a need to address these conditions and provide therapeutic remedies in patients with acute articular cartilage lesions to limit, reduce or control disease progression and to improve quality of life.

[0012]

[0006] Currently, therapeutic remedies to treat articular cartilage lesions include microfracture, mosaicplasty, autologous chondrocyte implantation (ACI), matrix-induced ACI (MACI), osteochondral autograft transfer system (OATS), and others (Grande et al, 2013). These methods have their intrinsic advantages and all have limitations. For example, osteochondral allografting attempts to integrate a healthy bone-cartilage unit into the damaged region, but limitations include insufficient integration or lack of healthy donor tissue. Additionally, microfracture has waned in popularity since it promotes the formation of cartilage that is lower quality than hyaline (Erggelet et al Journal of Clinical Orthopaedics and Trauma 7(3): 145-152 (2016)). Also, patients with acute articular cartilage lesions are often too young for total joint arthroplasty (TJA), necessitating a novel effective therapy to improve quality of life.

[0013]

[0007] Chondrons isolated from both healthy and damaged joints yield higher quality hyaline compared to isolated chondrocytes without their native pericellular matrix(Vonk, LA et al (2014) Osteoarthritis and Cartilage, 22(11), 1910-1917, doi.org / 10.1016 / j.joca.2014.08.005 ). This suggests it may be more efficacious to use chondrons with their native pericellular matrix (such as in minced cartilage morsels) to treat articularcartilagelesions as opposed to isolated chondrocytes (Vonk et al., (2014)). The challenge is expanding these chondrons without losing their pericellular matrix. Vonk et al. teach chondron isolation using enzymatic digestion of minced cartilage. However, themethods of Vonk et al. involve chondron isolation through enzymatic digestion and culture to obtain materials, thereby introducing significant and multiple steps and variability. The degree of ECM production and integration into native tissue affects the efficacy of these implants, which increases using a 3D matrix.

[0014]

[0008] Previous studies report the use of Autologous Chondrocyte Implantation (ACI) of minced cartilage morsels in point-of-care treatment for focal articular cartilage defects. In most studies, cultures of isolated cells or tissues are employed. Lu et al., report ACI without ex vivo cell expansion (Lu et al. Journal of Orthopaedic Research, 24(6): 1261—1270 (2006)). In these studies, minced cartilage culture was prepared on non-woven felts or meshes coated with fibrin and cultured. When used to treat full-thickness chondral defects in goats, the cartilage fragments on a resorbable scaffold produced some hyaline-like repair tissue at 6 months. While the methods of Lu et al. suggest that minced cartilage morsels and increased tissue surface area allow for some chondrocyte migration, growth, and redistribution, the methods result in reduced cell viability. Further, mincing resulted in reduced expression of Collagen Type II alpha 1 chain (COL2A1 or COL2) and Aggrecan (AC AN), and increased expression of inflammatory peptidases including Matrix Metallopeptidase -1 (MMP-1 or MMP1; Gene ID: 281308), and Matrix Metallopeptidase- 13 (MMP-13 or MMP13, Gene ID: 281914) expression (Moser et al, J. of Experimental Orthopaedics 10:97 (2023)). Cartilage Autograft Implantation System (CAIS, DePuy / Mitek, Raynham, MA) and DeNovo Natural Tissue (NT, Zimmer) use either autograft or juvenile allograft donor articular cartilage, respectively. These approaches allow the transplanted cartilage to form a new hyaline-like cartilage matrix in vivo, and fill a defect (Farr et al, The Journal of Knee Surgery, 25(1), 23-29 (2012)). The Instant MSC Product Accompanying Autologous Chondron Transplantation (IMPACT) study utilizes both allogenic mesenchymal stem cells (MSCs) and autologous chondrons isolated by enzymatic digestion of cartilage to completely fill focal articular cartilage defects with high regenerative and chondrogenic potential (de Windt et al, Stem Cells 35(1), 256-264 (2017)). However, all of these studies use inconsistent minced cartilage morsel sizes having low viability or inconsistent isolated chondron sizes. Further, as reported by Moser, chondrogenic gene expression is reduced. No study examines the effect of consistent morsel sizes and the impact of consistent morsel sizes on chondrogenesis and in vivo focal articular cartilage defect repair.

[0009] To repair and regenerate damaged tissue, scaffolds, such as porous meshes and hydrogels, are commonly used to drive tissue formation (Smith and Grande, Nature Reviews. Rheumatology, 11 (2015), doi.org / 10.1038 / nrrheum.2015.27). Factors such as biomechanics, pore size and porosity, degradation time, biocompatibility, effects of degradation products, and surface characteristics are important when considering scaffolds for applicability and success in clinical practice.

[0015]

[0010] Starting with cultured chondrocytes, in vitro chondrocyte differentiation has been shown to be greater in scaffolds of porous poly (1-lactic) acid with smaller pore sizes (100 um) (Conoscenti et al, (2017) Materials Science and Engineering: C, 80, 449^159, doi.org / 10.1016 / j.msec.2017.06.011). Additionally, a recently characterized alginate- Nanocellulose hydrogel scaffold has been shown to potently support the chondrocyte phenotype (Muller et al, (2017) Annals of Biomedical Engineering, 45(1), 210-223, doi.org / 10.1007 / s 10439-016- 1704-5).

[0016] [Oi l] A process, method, device, and system for the preparation of morselized tissues particles having high viability (“the TissueMill® system”) is described in U.S. Patent Application No. 16 / 584,755, now U.S. Patent No. 11,033,295, issued June 15, 2021, to Davenport et al (“Davenport et al.”) . The process and system atraumatically prepare morselized tissue particles, including cartilage, in a liquid medium. Related applications and patents include: US Patent Application No. 17 / 236, 130, now U.S. Patent 11,963,695, issued April 23, 2024; US Patent Application No. 17 / 236, 166, now U.S. Patent 11,918,248, issued March 5, 2024; U.S. Patent Application No. 17 / 093,803, now U.S. Patent 12,247,902, issued March 11, 2025; , International Patent Publication No. WO 2020 / 227196, published November 12, 2020; International Patent Publication No. WO 2021 / 225763, published November 11, 2021; US Patent Application No. 17 / 946,492, published as US Patent Publication No. 2023 / 0018416; US Patent Application No. 18 / 418,768, published July 12, 2024 as US Patent Publication No. 2024 / 0197361; US Patent Application No. 18 / 614,962, published July 11, 2024 as US Patent Publication No.

[0017] 2024 / 0225688; and US Patent Application No. 19 / 045,709 filed February 5, 2025. . Each of the foregoing applications are hereby incorporated in their entireties by reference (“the Davenport applications”). The Davenport applications describe a device and the use of the device thereof to prepare morselized full thickness skin and cartilage having an average size of between 0.2 mm to 1.5 mm and to produce morselized cartilage having a viability of between 87 to 98% using standard trypan blue exclusion, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and calcein AM assays. The Davenport applications do not describe formulations, methods of treatment using morselized cartilage particle (MCP) formulations, or provide guidance regarding suitable compositions for clinical application.

[0018]

[0012] In this present application we describe and provide methods and compositions for treating cartilage lesions using MCPs. As reported below, these studies identify and evaluate, both qualitatively and quantitatively, the optimal formed cartilage morsel size for use in compositions comprising MCPs and a biodegradable matrix. Notably, the MCPs are quantitatively and qualitatively different from previous cartilage preparations prepared by alternative approaches such as shaving and mincing. More specifically, the chondrocytes in the MCPs upregulate chondrogenic genes, and in a size dependent manner. In contrast, minced or shaved cartilage preparations have significantly lower cell viability, reduced metabolic activity, and higher expression of catabolic genes (Moser et al., Journal of Experimental Orthopaedics 10:97 (2023)).

[0019] SUMMARY OF THE INVENTION

[0020]

[0013] In a general aspect, the present invention provides chondrocyte composition comprising morselized cartilage tissue particles having an average length 1.0 mm or less and a chondrocyte viability of at least 85%, and a biodegradable matrix composition. Methods of repairing cartilage or treating a cartilage defect in a subject based on application of the composition are provided. In accordance with the compositions and methods, increased expression of one or more chondrogenic gene selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4 is achieved and is associated with the chondrocyte composition.

[0021]

[0014] In aspects, the invention provides a chondrocyte composition comprising: a. morselized cartilage tissue particles having an average length of about 0.1 to about

[0022] 1.0 mm and a chondrocyte viability of at least 85%, and b. a biodegradable matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from 1:10 and 5:1.

[0015] In an aspect, the volume ratio of morselized cartilage tissue particles to matrix is from 1 :10 to 5:1, from 1 :5 to 5:1, from 1:5 to 3:1, from 1 :5 to 2:1, from 1 :10 to 1:1, from 1 :5 to 1 :1, about 1:5, about 1 :3, at least 1 :2.

[0023]

[0016] In an aspect, said biodegradable matrix composition is a hydrogel, a dispersion, or a cream. In an aspect, said hydrogel is a synthetic polymer hydrogel selected from the group consisting of polylactide (PEA), poly-lactide- co-glycolide (PLGA), polyglycolide (PGA), poly- (D,L-lactic acid) (PDLLA), polycaprolactone (PCL), polyethylene-glycol (PEG), poly(vinyl alcohol) (PVA), poly (Nisopropylacrylamide)(PNIPAM), polyacrylamide (PAM), and combinations thereof.

[0024]

[0017] In another aspect, said biodegradable matrix composition is a natural polymer hydrogel selected from the group consisting of alginate-nanocellulose, chitosan, collagen, alginate, hyaluronic acid, gelatin, platelet-rich fibrin and combinations thereof.

[0025]

[0018] In an embodiment, the morselized cartilage tissue particles are selected from the group consisting of morselized articular cartilage tissue particles, morselized non-articular cartilage tissue particles, and combinations thereof. In one embodiment, the morselized tissue particles are morselized articular cartilage tissue particles.

[0026]

[0019] In some aspects, the morselized cartilage tissue particles have an average length is about 0.3 mm to about 0.7 mm. Thus, in some aspects, the cartilage tissue is morselized to obtain particles of size or length about 0.3 mm to about 0.7mm. In an aspect, the cartilage tissue is morselized such that the particles have a size or length of 1mm or less, or of less than lmm. In some aspects, the average length is about 0.5 mm. In aspects, the morselized particles are of a consistent or uniform size whereby at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% are less than 1 mm length. In aspects, the morselized particles are of a consistent or uniform size whereby at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% are about 0.5 mm length.

[0027]

[0020] In an embodiment, the biodegradable matrix composition comprises an autologous preparation comprising a platelet-rich fibrin. In one embodiment, the biodegradable matrix composition comprises a combination of: an autologous biodegradable matrix material derived from one or more of a platelet-rich fibrin, a lipoaspirate, a bone-marrow aspirate concentration, autologous mesenchymal derived stromal cells and combinations thereof; and an allogenic biodegradable matrix material derived from one or more of umbilical cord; umbilical cord blood, placental chorion, amnion, allogenic platelet-rich fibrin, allogenic lipoaspirate, allogenic bone-marrow aspirate concentration, allogenic mesenchymal derived stromal cells, and combinations thereof.

[0028]

[0021] In another aspect, the biodegradable matrix composition is an alginate-nanocellulose hydrogel. In one aspect, the alginate-nanocellulose hydrogel has a weight ratio of nanocellulose to alginate in the alginate-nanocellulose hydrogel from 0.01 to 10. In some embodiments, the alginate-nanocellulose hydrogel further comprises cellulose nanociystais, TEMPO-oxidized cellulose nanocry stals (CNCTs), cellulose nanofibers, TEMPO-oxidized cellulose nanofibers (CNFTs), and combinations thereof.

[0029]

[0022] In some aspects, the chondrocytes exhibit increased chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, compared to chondrocytes in a non-morselized cartilage tissue. In aspects, the expression of one or more inflammatory peptidase is not increased. In an aspect, the expression of MMP13 is not increased. In some aspects, the chondrocytes exhibit increased chondrogenic gene expression in two or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, compared to chondrocytes in a non-morselized cartilage tissue. In an aspect, the chondrocytes exhibit increased chondrogenic gene expression in three or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. In one aspect, the chondrocytes exhibit increased chondrogenic gene expression in the genes ACAN, COL2, COMP, PCNA, SOX9, and PRG4. In one aspect, the chondrocytes exhibit increased chondrogenic gene expression in the genes COMP, PCNA, SOX9, and PRG4. In one aspect, the chondrocytes exhibit increased chondrogenic gene expression in the genes ACAN and COL2. In one aspect, the chondrocytes exhibit increased chondrogenic gene expression in the genes ACAN and COL2 and the expression of one or more inflammatory peptidase is not increased. In one aspect, the chondrocytes exhibit increased chondrogenic gene expression in the genes AC AN and COL2 and the expression of MMP13 is not increased.

[0030]

[0023] In some embodiments, the chondrocyte composition further comprises at least one component selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin and salts thereof, glucosamine and salts thereof, methylsulfonylmethane (MSM), vitamins and combinations thereof. In an aspect, the fibrin glue and platelet rich plasma are autologous.

[0031]

[0024] In an aspect, the at least one component is at least one growth factor. In one aspect, the at least one growth factor is selected from the group consisting of TGF-β1, BMP-2, BMP-7, IGF-I, FGF-2, FGF-18, PDGF, and combinations thereof. In another aspect, the at least one growth factor increases or upregulates chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4.

[0032]

[0025] In one embodiment, the liquid medium is a hydrophilic medium, an oleophilic medium, or an emulsion of hydrophilic and oleophilic mediums.

[0033]

[0026] In some embodiments, the chondrocyte composition is a paste or a suspension. In one embodiment, the chondrocyte composition is a paste. In one embodiment, the chondrocyte composition is a suspension. In an embodiment, the suspension is a homogeneous suspension. In an embodiment, the paste or suspension is injectable.

[0034]

[0027] In an aspect, the at least one component is an active pharmaceutical ingredient and said active pharmaceutical ingredient is selected from the group consisting of non-steroidal antiinflammatory drugs (NSAIDs), steroids and corticosteroids; analgesics; anthraquinones and combinations thereof. In some embodiments, the NS AID is selected from ibuprofen, naproxen sodium, aspirin, diclofenac (and salts thereof), celecoxib, coxib, sulindac, oxaprozin, piroxicam, indomethacin, meloxicam, fenoprofen, diflunisal, etodolac, ketorolac tromethamine, meclofenamate, nabumetone, salsalate, or salts thereof. In some embodiments, the steroids and corticosteroids are selected from triamcinolone, cortisone, prednisone, methylprednisolone, hydrocortisone, or combinations thereof. In an aspect, the analgesic is selected from acetaminophen, capsaicin, camphor, menthol, lidocaine, or combinations thereof. In an aspect, the anthraquinone is diacerein.

[0035]

[0028] The invention provides a composition comprising: a. morselized articular cartilage tissue particles having an average length of between 0.1 and 1.0 mm and a chondrocyte viability of at least 85%; b. a biodegradable matrix composition comprising an alginate-nanocellulose hydrogel, wherein the volume ratio of morselized tissue particles to biodegradable matrix composition is about 1 :3; and c. at least one growth factor selected from the group consisting of TGF-β1, BMP -2, BMP-7, IGF-I, FGF-2, FGF-18, and PDGF.

[0036]

[0029] In an embodiment, the at least one growth factor comprises TGF-β1, FGF-18, or both.

[0037]

[0030] In another embodiment, the composition further comprises an active pharmaceutical ingredient selected from the group consisting of NSAIDs, steroids and corticosteroids; analgesics; anthraquinones and combinations thereof.

[0038]

[0031] In an aspect, the composition is a paste.

[0039]

[0032] In another aspect, the weight ratio of nanocellulose to alginate in the alginatenanocellulose hydrogel is in the range of greater than 0.01 to about 10.0. In one such aspect, the alginate-nanocellulose hydrogel further comprises cellulose nanocrystals, TEMPO-oxidized cellulose nanocrystals (CNCTs), cellulose nanofibers, and / or TEMPO-oxidized cellulose nanofibers (CNFTs).

[0040]

[0033] In an embodiment, the at least one growth factor increases or upregulates chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, compared to a composition lacking said at least one growth factor.

[0041]

[0034] In further aspects, the invention provides a cartilage graft composition comprising: a. morselized articular cartilage tissue particles having a length of between 0.3 mm or about 0.7 mm, a chondrocyte viability of at least about 90%; b. a biodegradable matrix; and c. an increased or upregulated chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, relative to a non-morselized articular cartilage tissue.

[0042]

[0035] In one such aspect, the biodegradable matrix composition is a hydrogel. In an aspect, the hydrogel is a natural polymer hydrogel selected from the group consisting of alginatenanocellulose, chitosan, collagen, alginate, hyaluronic acid, gelatin, platelet-rich fibrin and combinations thereof. In a particular aspect, the hydrogel is an alginate-nanocellulose hydrogel.

[0043]

[0036] The invention further provides methods of repairing cartilage or treating a cartilage defect in a subject in need thereof, comprising administering to the subject the chondrocyte composition hereof. The invention provides methods of repairing cartilage or treating a cartilage defect in a subject in need thereof, comprising administering to the subject a chondrocyte composition comprising morselized cartilage tissue particles having an average length of between about 0.1 and about 1.0 mm, a chondrocyte viability of at least 85%, and a biodegradable matrix composition (BMC).

[0044]

[0037] In one aspect, said administering comprises applying said chondrocyte composition using a spatula or syringe.

[0045]

[0038] In an aspect, the method further comprises introducing a divalent cation solution in situ to the chondrocyte composition applied to said subject thereby physically cross-linking the BMC. In one aspect, the divalent cation solution comprises CaCl2. In an aspect the solution of CaCl2comprises a 100 nM isotonic buffered solution of CaCl2. In an embodiment, the solution of CaCl2is applied for about 5 to about 15 seconds.

[0046]

[0039] In some aspects of the method(s), said chondrocyte composition is prepared from autologous tissues or cells. In an aspect, the chondrocyte composition is prepared from allogenic tissues or cells. In another aspect, the chondrocyte composition is prepared from a combination of allogenic and autologous tissues or cells.

[0040] In some embodiments of the method(s), the morselized tissue particles or the composition is administered to a joint of the subject. In one embodiment, the joint is a knee joint.

[0047]

[0041] In some embodiments the method(s) further comprise comprising covering the chondrocyte composition with a fibrin glue, a chitosan-based dressing, or combination thereof. In an embodiment, the fibrin glue is prepared from autologous fibrin.

[0048]

[0042] A method is provided for treating a subject in need of joint repair, comprising: a. harvesting at least one cartilage tissue portion from a subject; b. morselizing the harvested cartilage tissue portion for less than about 30 minutes to prepare morselized cartilage tissue particles having an average length of about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%; c. collecting the morselized cartilage tissue particles; d. mixing the morselized cartilage tissue particles with a biodegradable matrix composition (“BMC”) to produce an autologous chondrocyte composition; and e. applying and shaping said autologous chondrocyte composition in a joint of the subject.

[0049]

[0043] In one aspect, said morselizing comprises morselizing for about 4 to about 12 minutes. In one aspect, said morselizing comprises morselizing for about 5-10 minutes. In one aspect, said morselizing comprises morselizing for about 5 minutes. In one aspect, said morselizing comprises morselizing for less than 10 minutes. In one aspect, said morselizing comprises morselizing for about 5 to about 7 minutes. In one aspect, said morselizing comprises morselizing for about 5 to about 10 minutes.

[0050]

[0044] In an aspect, the method further comprises introducing a divalent cation solution in situ to the subject applied chondrocyte composition thereby physically cross-linking the BMC via ionic bonds. In one aspect, the divalent cation solution is a CaCl2solution.

[0051]

[0045] In some embodiments of the method of treating a subject in need of joint repair, the joint of the subject is a knee joint, an elbow joint, a hip joint, an ankle joint, a wrist joint, a finger joint, or a toe joint.

[0052]

[0046] In some embodiments, the composition is implanted in a knee joint of the subject.

[0047] In one embodiment, the joint has a defect and the method results in complete or almost complete defect closure 8-16 weeks post-surgery. In an embodiment, the joint has a defect and the method results in complete or almost complete defect closure about 12 weeks post-surgery.

[0053]

[0048] A further method is provided for repairing cartilage or treating a cartilage defect in a knee joint of a subject in need thereof, comprising: a. administering to the knee joint of the subject via syringe or spatula, autologous morselized cartilage tissue particles (MCP) having an average length of about 0. Ito about 1.0 mm and a chondrocyte viability of at least 85%, and a biodegradable matrix composition; b. physically cross-linking the MCP composition in situ with 100 nM CaCl2for about 10 seconds; and c. covering the morselized articular cartilage tissue particles or composition with protective layer.

[0054]

[0049] In an aspect, said protective layer is fibrin glue, chitosan, collagen, or a combination thereof. In an aspect, the fibrin glue is autologous.

[0055]

[0050] The invention provides a kit for treating cartilage degeneration, the kit comprising at least one of: a. an applicator dispensing device for receiving morselized cartilage tissue particles; b. one or more syringes for mixing and / or dispensing cartilage; and c. a biodegradable matrix;

[0056]

[0051] In an aspect, the syringes are selected from a mixing syringe, a two- (or multi) stage syringe, a dual- (or multi) chamber syringe, a dual (or multi) syringe, a two- (or multi) component syringe, or a spatula.

[0057]

[0052] In one aspect, the kit further comprises a disposable tissue harvesting device. In one aspect, the kit further comprises a disposable morselization chamber.

[0058]

[0053] In an embodiment, the kit further comprises reagents selected from a saline-containing solution or an isotonic compatible medium capable of buffering pH.

[0054] In an embodiment, the kit further comprises at least one element selected from an active pharmaceutical ingredient, a liquid medium, fibrin glue, growth factors, binders, saline, buffer solution, cell nurturing / preservation solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In aspects, the at least one element is at least one growth factor selected from the group consisting of TGF-β1, BMP-2, BMP-7, IGF-I, FGF-2, FGF-18, PDGF, and combinations thereof.

[0059]

[0055] In another aspect, the active pharmaceutical ingredient is selected from the group consisting of NSAIDs, steroids and corticosteroids; analgesics; anthraquinones, and combinations thereof.

[0060]

[0056] While there have been described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that other and further changes and modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such modifications and changes as come within the true scope of the invention.

[0061]

[0057] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

[0062] BRIEF DESCRIPTION OF DRA WINGS

[0063]

[0058] Figure 1 A and B presents exemplary results of in vitro testing. A) Confocal micrographs of morsel size (5.0mm, 1.0mm and 0.5 mm). Cell viability was maintained at >90% regardless of morsel size. Green living, red dead cells 100X (original magnification). B) Cartilage regenerative gene expression (upper panel) and MMP-13 expression (lower panel) are graphed. Morsels >5mm, 1mm, and 0.5mm sizes at 3 wks are graphed. A baseline of gene expression level in cartilage explants larger than 3 cm2is shown as a dashed line across. In the cartilage regenerative gene expression (left panel), relative expression of genes ACAN, COL2, PCNA, COMP, SOX9 and PRG4 is depicted. 0.5mm length morsels consistently demonstrated the highest chondrogenic potential based on gene expression. * p < 0.05 ** p < 0.01 *** P p < 0.001 **** p < 0.0001.

[0059] Figure 2 presents standard light microscopy images demonstrating in vitro chondrocyte migration patterns post cartilage tissue processing from weeks 1, 2 and 3, 2X (original magnification).

[0064]

[0060] Figure 3 presents results of an exemplary treatment in a macroscopic injury model. A) Defect treated with the minced cartilage morsel / nitrocellulose (NC) paste demonstrates no border between new and native cartilage. B) Marrow stimulation only leaves the defect largely unfilled.

[0065]

[0061] Figure 4 presents safranin-0 staining of cartilage treated in a knee according to the present methods. A) High quality cartilage stained in knee treated with the minced cartilage morsel / nitrocellulose (NC) paste. B) Marrow stimulation only demonstrates lack of cartilage formation.

[0066] DETAILED DESCRIPTION

[0067]

[0062] Numerous treatment modalities for focal articular cartilage defects exist, including the use of chondrons from minced cartilage morsels. It is more efficacious to use chondrons in minced cartilage morsels for cartilage implantation as opposed to isolated chondrocytes, since the interaction between chondrocytes and their native pericellular matrix yields higher quality hyaline. The degree of ECM production and integration into native tissue affects the efficacy of these implants. Notably, the efficacy of chondrocyte proliferation to a differentiated state increases under 3D surroundings, promoting ECM production.

[0068]

[0063] Previous minced cartilage approaches, such as CAIS, Zimmer DeNovo NT, and IMPACT, utilized minced cartilage morsels of inconsistent sizes. Each of their approaches, using either autologous or allogenic minced cartilage fragments, do validate point-of-care treatment models for focal articular cartilage defects. However, the present application demonstrates that controlling morsel size using the methods and devices such as described in the Davenport applications results in increased regenerative potential, with a matrix that supports chondrogenesis. Compared to prior art therapeutic remedies, and other remedies such as microfracture, ACI, MACI, or mosaicplasty, the present methods and compositions have numerous advantages and differences, including: 1) can particularly use autologous tissue, avoiding any allograft concerns, 2) are quick, cost effective, and readily available in the operating room (OR) or clinical setting, 3) can be performed peri-operatively, 4) control morsel size to provide a homogeneous morsel size population and maintain cell viability in a non- traumatic fashion, 5) provide rapid cartilage repair in vivo due to chondrocyte growth and migration, and provide complete healing of a focal deficit in a small animal model; and, 6) are readily adaptable to arthroscopic delivery, by syringe or arthroscopic portal, and can be used with fibrin glue or chitosan.

[0069]

[0064] The present invention provides chondrocyte compositions comprising morselized cartilage tissue particles having an average length 1.0 mm or less and a chondrocyte viability of at least 85%, and a biodegradable matrix composition. Methods of repairing cartilage or treating a cartilage defect in a subject based on application of the composition are provided. In accordance with the compositions and methods, increased expression of one or more chondrogenic gene selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4 is achieved and is associated with the chondrocyte composition.

[0070] Morselized cartilage particles

[0071]

[0065] The process, method, device, and system for the preparation of morselized tissues particles having high viability (“the TissueMill® system”) is described in U.S. Patent Application No. 16 / 584,755, now U.S. Patent No. 11,033,295, issued June 15, 2021, to Davenport et al (“Davenport et al.”) . Related applications and patents include: US Patent Application No. 17 / 236, 130, now U.S. Patent 11,963,695, issued April 23, 2024; US Patent Application No. 17 / 236, 166, now U.S. Patent 11,918,248, issued March 5, 2024; U.S. Patent Application No. 17 / 093,803, now U.S. Patent 12,247,902, issued March 11, 2025; , International Patent Publication No. WO 2020 / 227196, published November 12, 2020; International Patent Publication No. WO 2021 / 225763, published November 11, 2021; US Patent Application No. 17 / 946,492, published as US Patent Publication No. 2023 / 0018416; US Patent Application No. 18 / 418,768, published July 12, 2024 as US Patent Publication No. 2024 / 0197361; US Patent Application No. 18 / 614,962, published July 11, 2024 as US Patent Publication No.

[0072] 2024 / 0225688; and US Patent Application No. 19 / 045,709 filed February 5, 2025. Each of the foregoing applications are hereby incorporated in their entireties by reference (“the Davenport applications”).

[0066] The TissueMill® process and system atraumatically prepares and provides morselized tissue particles, including cartilage, in a liquid medium. The device and process can prepare morselized full thickness skin and cartilage to produce morselized cartilage having a viability of between 87 to 98%.

[0073]

[0067] TissueMill® cartilage processing leads to the production of precise and reproducible morsel size, controlled in part by processing time. Importantly, the processing occurs in a fluid environment where the pH, tonicity, and other critical parameters can be controlled. After the desired morsel size is obtained, the processing fluid and morselized cartilage tissue particles (MCPs) are collected. The MCPs and processing fluid are collected in a volume which is typically a volume of about 25 ml. The volume can be about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml. The MCPs are allowed to settle under gravity and the excess processing fluid is dispelled to produce a volume of morselized cartilage tissue particles and a volume of void buffer (e.g., the fluid remaining in the voids between particles). The MCP void ratio (e) can be calculated where e = Vv / Vs, where Vvis the volume of the voids (e.g., void buffer volume) and Vsis the volume of solids (e.g., MCPs). The void ratio (e) is related to the porosity (n), where e = n / (l-n) and n = e / (l+e). The MCPs together with the processing fluid remaining in the pores and interparticle spaces (“void volume”) can be used directly in the preparation of the chondrocyte compositions described herein. In some aspects, the MCPs can be washed with appropriate buffers (e.g., processing fluid, phosphate buffered saline (PBS), or other isotonic buffered solutions) to remove fines and other small particles or to exchange the void buffer.

[0074]

[0068] The TissueMill® process and system provides a device for processing organ tissue so as to maintain tissue viability comprising: a) an aseptic container for accommodating fluid and for receiving said organ tissue; b) a stationary cutting device and a movable cutting device supported in compressive engagement within said aseptic container, said movable cutting device being movable with respect to said stationary cutting device to cut organ tissue therebetween, said stationary cutting device including breaches therethrough configured to atraumatically cut said tissue in combination with said movable cutting device, in a slicing action; c) an agitation device supported within said aseptic container which causes repeated continuous recirculating flow of said fluid and organ tissue through said cutting devices within said aseptic container, said agitation device capable of moving in concert with said movable cutting device and repeatedly recirculating said fluid and organ tissue through said cutting devices to atraumatically cut said organ tissues repeatedly, while main-taining said tissue viability; and d) wherein said agitation device includes an impeller movable with said movable cutting device in a rota-tional direction about an axis, said impeller shaped to include a leading portion adjacent said cutting devices for imparting an axial thrust and a terminal portion for imparting a radial thrust for causing fluid repeated recirculating flow through said cutting devices.

[0075]

[0069] The TissueMill® process and system provides a method for processing organ tissue into particulate form comprising: providing a stationary cutting device and movable cutting device in mutual compressive engagement supported within an aseptic container; placing fluid and organ tissue into said container; moving said movable cutting device with respect to said stationary cutting device to cut said organ tissue through breaches formed in the stationary cutting device to atraumatically cut said tissue in combination with said movable cutting device, in a slicing action; providing an impeller to continuously recirculate said fluid and said organ tissue through said cutting devices to repeatedly atraumatically cut said organ tissue into progressively smaller particulates, said impeller shaped to include a leading portion proximal to said cutting devices for imparting an axial thrust and a terminal portion distal to said cutting devices for imparting a radial thrust for causing said continuous recirculation through said devices for repeated cutting of said organ tissue.

[0076]

[0070] Alternative approaches and methods can be utilized in line with the TissueMill® system and as described by Davenport, provided they achieve atraumatic morselization with a uniform or homogeneous morsel size in the range noted and provided herein, provided the noted viability of at least 87% is achieved, provided they retain the above-noted numerous advantages and differences versus alternative methods, and provided the derived morselized cartilage tissue particles or MCPs increase expression of one or more chondrogenic gene selected from the group of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, particularly when combined in composition with a matrix, particularly a biodegradable matrix..

[0077]

[0071] As used herein, “morselized cartilage particles” or “morselized cartilage tissue particles”, also denoted MCPs, refers to the settled MCPs including the void buffer. To prepare a chondrocyte composition as described herein, a volume of MCPs are mixed with a biodegradable matrix composition (“BMC”) at a ratio of from 1 : 10 to 5 : 1. In aspects, the ratio is determined by the volume of material required for the therapeutic use, particularly when the amount of cartilage material is limiting. In aspects, the ratio is selected to maximize the ratio of MCPs to biodegradable matrix.

[0078]

[0072] As used herein, the morselized cartilage tissue particles are selected from the group consisting of morselized articular cartilage tissue particles, morselized non-articular cartilage tissue particles, and combinations thereof. Thus, the morselized cartilage may derived from articular cartilage - cartilage from the joint or joints, or non-articular cartilage - cartilage not from the joint(s), or a combination of articular and non-articular cartilage. In an aspect, the morselized cartilage tissue particles comprise morselized articular cartilage tissue particles. In aspects, the morselized cartilage tissue particles are autonomously derived morselized cartilage tissue particles.

[0079]

[0073] In accordance with the present application and methods, gene expression analysis reveals that smaller formed cartilage morsel size and increased surface area are linked to increased chondrogenic potential. Not to be limited to theory, it is thought that increased surface area exposure to the surroundings promotes chondrogenesis. Regardless of processing time, cell viability is highly conserved and trends in gene expression are maintained for three weeks post tissue processing. These trends remain consistent for increased cartilage structure, function, and maintenance of the cartilage phenotype. Relevant genes including Collagen Type II alpha 1 chain (COL2A1 or COL2), Proliferating Cell Nuclear Antigen (PCNA), SRY-Box Transcription Factor 9 (SOX9 or SOX-9), and Cartilage and Proteoglycan 4 (PRG4) are significantly up- regulated early post tissue processing, such as up to three weeks. Relevant genes are upregulated in smaller morsel size, such as compared to minced cartilage or minced cartilage morsel size larger than 3 mm. In contrast, prior art methods and approaches, such as those described in Moser et al. (Moser et al, J. of Experimental Orthopaedics 10:97 (2023)) using mincing with commercially available shavers, report decreases in COL2 and ACAN expression and increased expression of MMP13, an inflammatory peptidase, in shaved cartilage.

[0080]

[0074] In aspects of the methods and compositions provided herein, smaller formed cartilage morsel size is linked to increased chondrogenic potential, including increased expression of chondrogenic or chondrogenesis relevant genes. In an aspect, 0.5mm morsels appear to produce the highest chondrogenic potential. In aspects, relevant gene expression is increased in smaller morsel sizes, particularly morsels smaller than 5mm, 1mm or less, about 0.5mm in size. As shown herein, COL2 and ACAN gene expression is higher in 0.5mm morsels than in 1mm morsels. Similarly, expression of PCNA, COMP and SOX9 genes is higher in 0.5mm morsels relative to PCNA, COMP and SOX9 gene expression in 1mm morsels. PRG4 expression is higher in 1mm morsels, when compared to 0.5mm morsels. Not to be limited by theory, these results reveal the importance of adjusting morsel size to optimize the expression of genes that are of interest in each clinical scenario. Notably, MMP-13 expression is lowest in 0.5mm morsels reflecting a synergistic relationship between cartilage anabolism and catabolism.

[0081]

[0075] In accordance with the present compositions and methods, chondrocyte compositions comprising morselized cartilage tissue particles and a biodegradable matrix provides for the formation of hyaline cartilage with complete defect closure. In particular, chondrocyte compositions comprising morselized cartilage tissue particles containing 0.5mm morsels and a biodegradable matrix demonstrates the formation of hyaline cartilage with complete defect closure. Chondrocyte compositions comprising morselized cartilage tissue particles and a matrix of nanocellulose-alginate (also “alginate-nanocellulose” or “NCA”) containing 0.5mm morsels (NCA implant paste) demonstrates the formation of hyaline cartilage with complete defect closure.

[0082]

[0076] Using cartilage morsels for cartilage implantation as opposed to isolated chondrocytes yields higher quality hyaline cartilage. Not to be limited by theory, it is believed that higher quality hyaline cartilage is due to the interaction between chondrocytes and their pericellular matrix. Additionally, implementing a biodegradable matrix provides significantly improved results. In an aspect, particularly using a nanocellulose-alginate hydrogel as a matrix base for the implant paste appears to further support the chondrocyte phenotype (Muller et al (2017) Annals of Biomedical Engineering 45(1 ):210-223). Safranin-0 staining reveals higher quality cartilage formation compared to marrow stimulation alone. Implant paste with 0.5mm morsels produces the greatest regenerative capacity in terms of quality and the ability to regain function.

[0083]

[0077] In accordance with the present application, methods and compositions, significant chondrocyte viability is maintained inthe morsels. Chondrocyte viability of at least 85% is maintained. Chondrogenic and proliferative genes such as Aggrecan (ACAN; Gene ID: 176), COL2, Cartilage Oligomeric Matrix Protein (COMP; Gene ID: 1311), PCNA, SOX9, and PRG4 all demonstrate increased expression, to a varying degree, as morsel size is decreased, particularly with morsel size less than 5mm, about 1mm, less than 1mm, about 0.5mm, 0.5mm. In particular, 0.5mm morsels exhibit the highest chondrogenic potential. As shown here, NCA implant paste containing 0.5mm morsels results in complete defect closure unlike marrow stimulation alone. Safranin-O staining demonstrates superior cartilage formation in the defect area.

[0084]

[0078] Smaller consistent morsel size is found to be linked to increased chondrogenic potential. Also, since PRG4 expression was highest in 1mm morsels, and MMP-13 gene expression was significantly decreased in only 0.5mm morsels, morsel sizes can be adjusted to optimize relevant gene expression of interest. Not to be limited by theory, it is thought that favorable gene expression will be exhibited in smaller morsels, however at morsel sizes below 0.1 mm in size, chondrocyte viability, favorable gene expression, or both may be reduced. Thus in an aspect, morsel size is 1mm or less, less than 1mm, about 0.5mm,.0.5mm, greater than 0.3mm, greater than 0.2mm, greater than 0.1mm, at least 0.1mm. A biodegradable matrix mixed with 0.5mm morsels produced the greatest regenerative capacity. In an aspect, a matrix comprising an NC hydrogel mixed with 0.5mm morsels produced significantregenerative capacity.

[0085]

[0079] The present application provides for, and includes, a chondrocyte composition comprising a morselized cartilage tissue having an average morsel size or length of about 0.1 to 1 ,0mm and a chondrocyte viability of at least 85%, and a matrix. The present application provides for, and includes, a chondrocyte composition comprising a morselized cartilage tissue having an average morsel size or length of about 0.1 to 1.0mm and a chondrocyte viability of at least 85%, and a biodegradable matrix. The present application provides for, and includes, a chondrocyte composition comprising a morselized cartilage tissue having an average morsel size or length of about 0.1 to 1.0mm and a chondrocyte viability of at least 85%, and a biodegradable matrix composition. The present application provides for, and includes, a chondrocyte composition comprising a morselized cartilage tissue having an average morsel size or length of about 0.1 to 1.0mm and a viability of at least 85%, and a biodegradable matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix is from 1 : 10 to 5 : 1 , from 1 :5 to 5:1, from 1 :5 to 3:1, from 1:5 to 2:1, from 1 :10 to 1:1, from 1:5 to 1 :1. The present application provides for, and includes, a chondrocyte composition comprising a morselized cartilage tissue having an average morsel size or length of about 0.1 to 1.0mm and a chondrocyte viability of at least 85%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from 1 : 10 to 5 : 1.

[0086]

[0080] As used herein, the average length refers to the average of a sample of at least ten (10) particles measured across the longest length. As used herein, the average size refers to the average of a sample of at least ten (10) particles measured across the longest length.

[0087]

[0081] As used herein, chondrocyte viability is determined according to methods known in the art. Known or available methods for determining cell viability or chondrocyte viability include 4 μM Calcein acetoxymethyl (AM) (ThermoFisher), trypan blue exclusion, MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). In an aspect, chondrocyte viability is determined using a method selected from4 μM Calcein acetoxymethyl (AM) (ThermoFisher), trypan blue exclusion, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and others.

[0088]

[0082] In aspects, the morselized cartilage tissue has an average length of from about 0.3 to about 0.7 mm and a chondrocyte viability of at least 85%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1. In an aspect, the average length is about 0.5 mm.

[0083] In aspects, the morselized cartilage tissue has an average length of between 0.2 and 0.9 mm and a chondrocyte viability of at least 85%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1.

[0089]

[0084] In aspects, the morselized cartilage tissue has an average length of from about 0.3 to about 0.8 mm and a chondrocyte viability of at least 85%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1. In another aspect, the morselized cartilage tissue has an average length of from about 0.4 to about 0.7 mm and a chondrocyte viability of at least 85%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1. In an aspect, the morselized cartilage tissue has an average length of from about 0.4 to about 0.7 mm and a chondrocyte viability of at least 85%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:5 to about 2:1.

[0090]

[0085] The present application provides for, and includes, a chondrocyte composition comprising a morselized cartilage tissue having an average length of from about 0.1 to about 1.0mm and a chondrocyte viability of at least 85%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1. In an aspect, the average length is about 0.5 mm.

[0091]

[0086] In aspects, the morselized cartilage tissue has an average length of from about 0.2 to about 0.9 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1.

[0092]

[0087] In aspects, the morselized cartilage tissue has an average length of from about 0.3 to about 0.8 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1. In another aspect, the morselized cartilage tissue has an average length of form about 0.4 to about 0.7 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1. In an aspect, the morselized cartilage tissue has an average length of from about 0.4 to about 0.7 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1. In an aspect, the morselized cartilage tissue has an average length of from about 0.4 to about 0.7 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :5 to about 2:1.

[0093]

[0088] The present application provides for, and includes, a chondrocyte composition comprising a morselized cartilage tissue having an average length of from about 0.1 to about 1.0mm and a chondrocyte viability of at least 95%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1. In an aspect, the average length is about 0.5 mm.

[0094]

[0089] In aspects, the morselized cartilage tissue has an average length of from about 0.2 to about 0.9 mm and a chondrocyte viability of at least 95%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 : 10 to about 5:1.

[0095]

[0090] In aspects, the morselized cartilage tissue has an average length of from about 0.3 to about 0.8 mm and a chondrocyte viability of at least 95%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1. In another aspect, the morselized cartilage tissue has an average length of from about 0.4 to about 0.7 mm and a chondrocyte viability of at least 95%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 : 10 to about 5:1. In an aspect, the morselized cartilage tissue has an average length of from about 0.4 to about 0.7 mm and a chondrocyte viability of at least 95%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1. In an aspect, the morselized cartilage tissue has an average length of from about 0.4 to about 0.7 mm and a chondrocyte viability of at least 95%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:5 to about 2:1.

[0091] In an aspect, the chondrocyte compositions hereof, particularly the chondrocyte and matrix, particularly biodegradable matrix, compositions hereof exhibit increased expression of one or more chondrogenic and proliferative genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. In an aspect, the chondrocyte compositions hereof, particularly the chondrocyte and matrix, particularly biodegradable matrix, compositions hereof exhibit increased expression of two or more chondrogenic and proliferative genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. In an aspect, the chondrocyte compositions hereof, particularly the chondrocyte and matrix, particularly biodegradable matrix, compositions hereof exhibit increased expression of three or more chondrogenic and proliferative genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. In an aspect, the chondrocyte compositions hereof, particularly the chondrocyte and matrix, particularly biodegradable matrix, compositions hereof exhibit increased expression of chondrogenic and proliferative genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. In an aspect, the chondrocyte compositions hereof, particularly the chondrocyte and matrix, particularly biodegradable matrix, compositions hereof exhibit increased expression of the chondrogenic and proliferative genes ACAN, COL2, COMP, PCNA, SOX9, and PRG4. In an aspect, the chondrocyte compositions hereof, particularly the chondrocyte and matrix, particularly biodegradable matrix, compositions hereof exhibit increased expression of the chondrogenic and proliferative genes COMP, PCNA, SOX9, and PRG4. In an aspect, the chondrocyte compositions hereof, particularly the chondrocyte and matrix, particularly biodegradable matrix, compositions hereof exhibit increased expression of at least two chondrogenic and proliferative genes selected from COMP, PCNA, SOX9, and PRG4.

[0096]

[0092] The present application further provides for, and includes, chondrocyte composition comprising morselized cartilage tissue has an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 : 10 to about 5:1, and the morselized cartilage tissue exhibits increased expression of chondrogenic and proliferative genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. The present application further provides for, and includes, chondrocyte composition comprising morselized cartilage tissue has an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1, and the morselized cartilage tissue exhibits increased expression of at least one chondrogenic and proliferative gene selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. The present application further provides for, and includes, chondrocyte composition comprising morselized cartilage tissue has an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1, and the morselized cartilage tissue exhibits increased expression of at least two chondrogenic and proliferative genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. The present application further provides for, and includes, chondrocyte composition comprising morselized cartilage tissue has an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1:10 to about 5:1, and the morselized cartilage tissue exhibits increased expression of at least three chondrogenic and proliferative genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4. The present application further provides for, and includes, chondrocyte composition comprising morselized cartilage tissue has an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%, and a matrix composition, wherein the volume ratio of morselized cartilage tissue particles to matrix composition is from about 1 :10 to about 5:1, and the morselized cartilage tissue exhibits increased expression of at least two chondrogenic and proliferative genes selected from the group consisting of COMP, PCNA, SOX9, and PRG4. In a further aspect, the morselized cartilage tissue exhibits decreased expression of MMP-13.

[0097]

[0093] As used herein, increased gene expression refers to a significantly increased expression of chondrogenic and proliferative genes ACAN, COL2, COMP, PCNA, SOX9, and PRG4 compared to cartilage explants larger than 3 mm at three weeks, wherein significance is determined using a standard method known and accepted in the art. In an aspect, increased gene expression refers to a significantly increased expression of chondrogenic and proliferative genes ACAN, COL2, COMP, PCNA, SOX9, and PRG4 compared to cartilage explants larger than 3 mm at three weeks, wherein significance is determined using Student’s T-Test. Similarly, as used herein, decreased gene expression refers to a significantly decreased expression of genes involved in the breakdown of cartilage and type II collage, compared to cartilage explants larger than 3 mm at three weeks, wherein significance is determined usinga standard method known and accepted in the art. In an aspect, decreased gene expression refers to a significantly decreased expression of genes involved in the breakdown of cartilage and type II collage, compared to cartilage explants larger than 3 mm at three weeks, wherein significance is determined using Student’s T-Test.

[0098]

[0094] In an aspect, the morselized cartilage tissues exhibit increased expression of chondrogenic and proliferative genes ACAN, COL2, COMP, PCNA, SOX9, and PRG4.

[0099] Matrices

[0100]

[0095] The present application provides for, and includes, biodegradable matrix compositions. The present application provides for, and includes, biodegradable matrix compositions that are hydrogels. Hydrogels are a soft, gel-like material that absorb and retain large amounts of water. As provided herein, the MCPs are mixed with, and embedded into, the matrix as opposed to applying the MCPs to a scaffold. In aspects, the MCPs are mixed with, and embedded into, the hydrogel as opposed to applying the MCPs to a scaffold, either of a hydrogel or other matrix. Not to be limited by theory, it is thought that an embedded MCP composition better mimics the natural state of the chondrocytes and encourages the development of natural cartilage structures.

[0101]

[0096] In aspects, the hydrogel is prepared from synthetic polymers selected from the group consisting of polylactide (PLA), poly-lactide-co-glycolide (PLGA), polyglycolide (PGA), poly- (D,L-lactic acid) (PDLLA), polycaprolactone (PCL), polyethylene-glycol (PEG), poly(vinyl alcohol) (PVA), poly (Nisopropylacrylamide)(PNIPAM), polyacrylamide (PAM), and combinations thereof. The preparation of suitable synthetic hydrogels is known in the art and hydrogels generally are reviewed in Bao et al., Frontiers in Chemistry 8:53 (2020), Zhu et al., Front. Bioeng. Biotechnol. 10:954501 (2022), and Zhao et al., Front. Bioeng. Biotechnol. 9:603444 (2021), hereby incorporated by reference. For use in the compositions and methods herein, suitable synthetic polymer hydrogels are selected for biocompatibility and biodegradability, and to reduce or eliminate immune responses and toxicity.

[0097] In aspects, the hydrogel is a hydrogel prepared from natural polymers. Natural polymer hydrogels exhibit high biocompatibility, biodegradability, and similar macromolecular structure to natural extracellular matrices found in cartilage. Natural polymer hydrogels include both protein based materials (e.g., gelatin, collagen, fibrin, and silk fibroin) and polysaccharide-based materials (hyaluronic acid, chondroitin sulfate, alginate, chitosan, cellulose, and others). Natural polymer hydrogels are non-toxic and non-immunogenic but can have poor stability, rapid degradation, and low mechanical strength. In aspects, natural polymer hydrogels are selected from, but are not limited to, alginate-nanocellulose hydrogels, chitosan, collagen, silk fibroin, alginate, hyaluronic acid, gelatin, platelet-rich fibrin, or combinations thereof.

[0102]

[0098] In aspects, the hydrogels are network hydrogels. Network hydrogels are stabilized by either physical or chemical cross-links. Chemical cross-links introduce covalent bonds, for example disulfide bonds or methacrylate chemistry, and are often incorporated into synthetic polymers. Physical cross links aggregate the hydrogel polymers by molecular entanglement (for example gelatin), hydrogen bonding, ionic bonding, or hydrophobic interactions. Notably, ionic bonding can be introduced after application of the therapeutic composition by the introduction of multivalent ions, typically Ca++, thereby allowing the repair material to be shaped, then stabilized. Physically crosslinked hydrogels generally exhibit increased biodegradability, but lower toxicity and immunogenicity compared to chemically cross-linked hydrogels.

[0103]

[0099] In aspects of the present chondrocyte compositions, the viscosity of the chondrocyte composition is suitable for injection. In aspect, the chondrocyte compositions are arthroscopically injectable compositions. In aspects, the injectable compositions can be physically cross-linked after injection through the application of divalent cations, for example Ca^, after injection or application to the target repair location.

[0104]

[0100] The present application provides for, and includes, chondrocyte compositions comprising morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm, a chondrocyte viability of at least 85%, and a biodegradable matrix composition (“BMC”), wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1. The present application provides for, and includes, chondrocyte compositions comprising morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm, a chondrocyte viability of at least 85%, and a biodegradable matrix composition (“BMC”), wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :5 to about 2:1. The present application provides for, and includes, chondrocyte compositions comprising morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm, a chondrocyte viability of at least 85%, and a biodegradable matrix composition (“BMC”), wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :5 to about 5:1. In aspects, the BMC is a natural polymer hydrogel. In an aspect, the hydrogel is alginate-nanocellulose hydrogel, chitosan hydrogel, collagen hydrogel, alginate hydrogel, hyaluronic acid hydrogel, gelatin hydrogel, platelet-rich fibrin, or combinations thereof. In aspects, the hydrogels further comprise a physical cross-link. In aspects, the physical cross-link is an ionic bond. In certain aspects, the ionic bond is formed by the addition of divalent cations, such as calcium. In aspects, the divalent cation is added after the chondrocyte compositions are applied and shaped at the site of cartilage repair thereby stabilizing the repair structure.

[0105]

[0101] In an aspect, the biodegradable matrix composition is a hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1 : 1 to about 1:5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1 :5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4

[0106] A Iginate-nanocellulose

[0107]

[0102] In an aspect, the biodegradable matrix composition is an alginate-nanocellulose hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1 :1 to about 1 :5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1:5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4. In aspects, the alginate-nanocellulose hydrogel has a weight ratio of nanocellulose to alginate in the alginate-nanocellulose hydrogel from about 0.01 to about 10.0. In further aspects, the alginate- nanocellulose hydrogel further comprises cellulose nanocrystals, TEMPO-oxidized cellulose nanocrystals (CNCTs), cellulose nanofibers, TEMPO-oxidized cellulose nanofibers (CNFTs), and combinations thereof. In aspects, the alginate-nanocellulose can further comprise one or more hydrogel forming polymers selected from chitosan, collagen, alginate, hyaluronic acid, gelatin, platelet-rich fibrin, or combinations thereof.

[0108] Gelatin

[0109]

[0103] In an aspect, the biodegradable matrix composition is a gelatin hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1 : 1 to about 1 :5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1 :5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4. In aspects, the gelatin hydrogel can further comprise one or more hydrogel forming polymers selected from chitosan, collagen, alginate, hyaluronic acid, alginate-nanocellulose, platelet-rich fibrin, or combinations thereof.

[0110] Chitosan

[0111]

[0104] In an aspect, the biodegradable matrix composition is a chitosan hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1 : 1 to about 1 :5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1 :5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4. In aspects, the chitosan is unmodified chitosan (e.g. without chemical modification to the free amines present in the chitosan backbone chain. In aspects, the chitosan comprises or may further include chemically modified chitosan via the free amine groups present in the chitosan backbone chain. In aspects, the chitosan hydrogel can further comprise one or more hydrogel forming polymers selected from collagen, gelatin, alginate, hyaluronic acid, alginate-nanocellulose, platelet-rich fibrin, or combinations thereof. In further aspects, chitosan (modified or unmodified) can be combined with poly (1-lactide) (PLLA) and pectin.

[0112] Collagen

[0113]

[0105] In an aspect, the biodegradable matrix composition is a collagen hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1:10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1:1 to about 1:5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1 :5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4. In aspects, the collagen hydrogel is prepared from Type I or Type II collagens. As provided herein, a collagen hydrogel may further comprise hyaluronic acid (HA). In aspects, the collagen hydrogel can further comprise one or more hydrogel forming polymers selected from chitosan, gelatin, alginate, hyaluronic acid, alginate-nanocellulose, platelet-rich fibrin, or combinations thereof.

[0114] Alginate

[0115]

[0106] In an aspect, the biodegradable matrix composition is a gelatin hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1 : 1 to about 1 :5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1 :5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4. In aspects, the alginate hydrogel can further comprise one or more hydrogel forming polymers selected from chitosan, collagen, gelatin, hyaluronic acid, alginate-nanocellulose, platelet-rich fibrin, or combinations thereof.

[0116] Hyaluronic acid

[0117]

[0107] In an aspect, the biodegradable matrix composition is a hyaluronic acid hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1 : 1 to about 1 :5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1 :5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4. In aspects, the hyaluronic acid hydrogel can further comprise one or more hydrogel forming polymers selected from chitosan, collagen, alginate, gelatin, alginate-nanocellulose, platelet-rich fibrin, or combinations thereof.

[0118] Platelet-rich fibrin

[0119]

[0108] In an aspect, the biodegradable matrix composition is a platelet-rich fibrin hydrogel provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. In aspects, the ratio of particles to matrix is from about 1 :1 to about 1 :5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about 1 :5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4. In aspects, the platelet-rich fibrin hydrogel can further comprise one or more hydrogel forming polymers selected from chitosan, collagen, alginate, hyaluronic acid, alginate-nanocellulose, gelatin, or combinations thereof.

[0120] Synthetic polymers

[0121]

[0109] In an aspect, the biodegradable matrix composition is a synthetic polymer hydrogel selected from the group consisting of polylactide (PLA), poly-lactide-co-glycolide (PLGA), polyglycolide (PGA), poly-(D,L-lactic acid) (PDLLA), polycaprolactone (PCL), polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), poly (Nisopropylacrylamide)(PNIPAM), polyacrylamide (PAM), and combinations thereof provided at a volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1. The preparation of synthetic polymer hydrogels are known in the art. In aspects, the ratio of particles to matrix is from about 1 : 1 to about 1 :5. In other aspects, the ratio of particle to matrix is from about 1 :2 to about to about 1:5. In another aspect, the ratio of particle to matrix is from about 1 :2 to about 1 :4.

[0122]

[0110] In aspects, the matrix composition is an autologous matrix composition. Suitable autologous matrix compositions comprise an autologous preparation of a platelet-rich fibrin and combinations of platelet-rich fibrin with the natural polymer hydrogels or a synthetic polymer hydrogels as provided herein. When combined with autologous morselized cartilage tissues, the biodegradable matrix compositions reduce or avoid immunogenic responses in subjects in need thereof. In aspects, the autologous morselized cartilage tissue and autologous matrix materials can be combined with one or more non-immunogenic materials, including but not limited to, alginate-nanocellulose hydrogels, poly (1-lactic) acid, chitosan, collagen, silk fibroin, alginate, hyaluronic acid, gelatin, cellulose, and poly(chitosan-g-lactic acid). .

[0123]

[0111] In aspects, the matrix composition is an allogenic matrix composition. Suitable allogenic matrix compositions comprise an allogenic preparations of platelet-rich fibrin, or other suitable allogenic materials. When combined with autologous morselized cartilage tissues, the allogenic matrix containing compositions have increased potential for immunogenic responses in subjects in need thereof compared to compositions prepared from autologous or non-immunogenic synthetic and natural hydrogels. In aspects, the morselized cartilage tissue and allogenic matrix materials may be combined with one or more non-immunogenic materials, including but not limited to, alginate-nanocellulose hydrogels, chitosan, collagen, silk fibroin, alginate, hyaluronic acid, gelatin, platelet-rich fibrin, or combinations thereof.

[0124]

[0112] In aspects, the present application provides for and includes, matrices comprising combinations of autologous and allogenic materials. The inclusion of allogenic materials provides for the capacity to supplement the autologous materials to provide sufficient therapeutic amounts.

[0125] Additional components

[0126]

[0113] The present application provides for, and includes, compositions and methods of use thereof, of chondrocyte compositions as provided herein, together with one or more additional components or elements. In aspects, the present application provides chondrocyte compositions comprising morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a biodegradable matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1:10 to about 5:1, and at least one component selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin and salts thereof, glucosamine and salts thereof, methylsulfonylmethane (MSM), vitamins and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5: 1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0127]

[0114] In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0128]

[0115] In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a nanocellulose-alginate hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1:10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a nanocellulose-alginate hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, binders, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0129]

[0116] In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a gelatin hydrogel matrix composition comprising, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a gelatin hydrogel matrix composition comprising, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1:10 to about 5: 1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0130]

[0117] In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a chitosan hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a chitosan hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0131]

[0118] In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, an alginate hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, an alginate hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1:10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0132]

[0119] In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a hyaluronic acid hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1:10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a hyaluronic acid hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0133]

[0120] In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a platelet-rich fibrin hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In an aspect, the chondrocyte composition comprises morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a plateletrich fibrin hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 :10 to about 5:1, and one or more components selected from the group consisting of an active pharmaceutical ingredient, fibrin glue, platelet rich plasma, growth factors, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), and combinations thereof. In aspects, the materials are autologously derived.

[0134] Active Pharmaceutical Ingredients

[0135]

[0121] In aspects, the chondrocyte compositions as provided herein include one or more active pharmaceutical ingredient. Suitable active pharmaceutical ingredients are provided at a therapeutically effective amount and can be selected from the group consisting of non-steroidal anti-inflammatory drugs (NSAID), steroids and corticosteroids; analgesics; anthraquinones, and combinations thereof. In an aspect, the active pharmaceutical ingredient is an NSAID selected from ibuprofen, naproxen sodium, aspirin, diclofenac (and salts thereof), celecoxib, coxib, sulindac, oxaprozin, piroxicam, indomethacin, meloxicam, fenoprofen, diflunisal, etodolac, ketorolac tromethamine, meclofenamate, nabumetone, salsalate, or salts thereof. In aspects, the steroids and corticosteroids are selected from triamcinolone, cortisone, prednisone, methylprednisolone, hydrocortisone, or combinations thereof. In aspects, the analgesic is selected from acetaminophen, capsaicin, camphor, menthol, lidocaine, or combinations thereof. In a further aspect, the active pharmaceutical ingredient is the anthraquinone diacerein.

[0136] Growth factors

[0137]

[0122] In aspects, chondrocyte compositions as provided herein can include one or more growth factors as at least one component. Suitable growth factors include growth factors known to stimulate cell growth, maintain potency (e.g., stem cell markers), or promote differentiation of chondrocytes. In aspects, the chondrocyte compositions include one or more growth factors selected from the group consisting of transforming growth factor β1 (TGF-β1), Bone morphogenetic protein 2 (BMP-2) (Homo sapiens Gene ID: 650), bone morphogenetic protein- 7 (BMP-7) (Homo sapiens Gene ID: 655), Insulin-like Growth Factor 1 (IGF-I) (Homo sapiens Gene ID: 3479), fibroblast growth factor 2 (FGF-2) (Homo sapiens Gene ID: 2247), fibroblast growth factor 18 (FGF-18) (Homo sapiens Gene ID: 8817), platelet derived growth factor (PDGF) (homo or hetero dimers of Homo sapiens Gene IDs: 5154, 5155, 56034, and 80310), and combinations thereof.

[0138]

[0123] In aspects, the present application provides for, and includes a chondrocyte composition comprising morselized cartilage tissue particles having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, a hydrogel matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1:10 to about 5:1, and transforming growth factor β1 (TGF-β1), fibroblast growth factor 18 (FGF-18), or a combination of both.

[0139] Liquid Media

[0140]

[0124] The present application provides for, and includes, liquid media as an additional component. As discussed above, the MCPs for use in the preparation of the chondrocyte compositions includes a void buffer. In aspects, this void buffer can be replaced by washing the settled MCPs with a liquid media. In other aspects, the liquid media can be added to MCPs without replacing the void buffer. As provided herein, suitable liquid media include hydrophilic mediums, oleophilic mediums, or emulsions of hydrophilic and oleophilic mediums. Suitable liquid media are buffered media and are isotonic.

[0141] Physical forms

[0142]

[0125] The chondrocyte compositions of the present application are generally prepared as either pastes or suspensions and can include additional components that are, dispersions, creams, fluids, and emulsions.

[0143]

[0126] In aspects, the paste or suspension can be prepared as an injectable form. Generally, the injectable form has a suitable viscosity to ensure that the composition can be delivered, desirably through arthroscopic means, and ensure it remains at the injection site. In aspects, the chondrocyte compositions are prepared for use as an arthroscopically injectable composition. In some aspects, the injectable chondrocyte composition can be treated with divalent cations after application to increase the viscosity, strength, and rigidity of the hydrogel to facilitate retention of the hydrogel in the application site.

[0144] Methods

[0145]

[0127] The present application provides for, and includes, methods to prepare chondrocyte compositions and methods of treating cartilage. In aspects, the methods are performed in a nonhospital clinical setting, or at the bedside of a patient. In aspects, the present methods include harvesting at least one cartilage portion from a donor and placing the harvested cartilage in a physiological buffer as a processing fluid, morselizing the harvested cartilage tissue portion in the physiological buffer for less than 30 minutes to prepare morselized cartilage tissue particles (MCPs) having an average length of from about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, collecting the morselized particles and removing the excess processing fluid, mixing the MCPs with a biodegradable matrix to produce a chondrocyte composition (“MCP composition”), and applying said MCP composition to a joint of subject in need of joint repair. In aspects, the harvested cartilage is morselized for from about 4 to about 12 minutes. In aspects, the harvested cartilage is morselized for from about 5 to about 15 minutes. In aspects, the harvested cartilage is morselized for from about 4 to about 20 minutes. In aspects, the harvested cartilage is morselized for from about 5 to about 20 minutes. In aspects, the harvested cartilage is morselized for from about 5 to about 25 minutes. In aspects, the harvested cartilage is morselized for from about 10 to about 25 minutes. In aspects, the harvested cartilage is morselized for less than 25 minutes. In aspects, the harvested cartilage is morselized for from less than 20 minutes. In aspects, the amount of time required is determined by monitoring the morselation process and determining the size of the particles.

[0146]

[0128] In aspects, the donor is a subject in need of joint repair and said MCP composition comprises an autologous MCP composition. In other aspects, the donor is an allogenic donor. In addition to said MCP composition, the present application provides for, and includes, obtaining other autologous materials for use in the methods, including fibrin glue, lipoaspirates, bone- marrow aspirate concentrations, or autologous mesenchymal derived stromal cells (MSCs). Similarly, an autologous MCP composition may include allogenic materials derived from umbilical cord; umbilical cord blood, placental chorion, amnion, allogenic platelet-rich fibrin, allogenic lipoaspirate, allogenic bone-marrow aspirate concentration, allogenic mesenchymal derived stromal cells, or combinations thereof. In aspects, the autologous or allogenic materials are used to prepare biodegradable matrix materials. In other aspects, the allogenic materials are provided as cell preparations to supplement the autologous cells of the MCPs.

[0147]

[0129] In aspects, the MCP composition can be treated to physically cross-link the biodegradable matrix composition to increase the viscosity and mechanical strength of the MCP composition. In aspects, the MCP composition is transferred to a form or mold to shape the MCP composition for therapeutic use and treated with a polyvalent cation in a physiological buffer to introduce ionic crosslinks. In other aspects, the MCP composition for therapeutic use is applied to the joint in need of repair and treated with polyvalent cations in situ to introduce ionic bonds as physical cross-links. In aspects, the MCP composition is applied and shaped for therapeutic use and treated with polyvalent cations in situ to introduce ionic bonds as physical cross-links. As provided herein, suitable polyvalent cations include Ca++Sr++, Fe++, Co++, and Ba++. In aspects, the polyvalent cation is a divalent cation. In certain aspects, the divalent cation is Ca++provided as a solution of CaCl2, CaCO3, or CaSO4. The selection of appropriate polyvalent cation is determined by the desired gelling rate where CaCO3, or CaSCU, exhibit slower gelling rates than CaCl2. In aspects, the concentration of the polyvalent cation is between 10 nM (1 x 10"9M) and 1 mM (lxlO"6M). In aspects, the polyvalent cation is a 100 nM isotonic buffered solution of CaCl2.

[0148]

[0130] The methods of treating provide for and include treatment of joints in subjects in need of joint repair. In aspects, the joint is selected from a knee joint, an elbow joint, a hip joint, an ankle joint, a wrist joint, a finger joint, or a toe joint. In aspects, the methods provide for implanting the MCP compositions described herein in the knee joint of a subject in need. In aspects, the treatment is performed using autologous MCPs. As provided herein, the treatment provides a complete, or almost complete defect closure after 8 to 16 weeks post-surgery. In aspects, the treatment results in complete or almost complete defect closure about 12 weeks post- surgery.

[0149]

[0131] In aspects of the present application, after treating the joint or cartilage defect, the MCP composition is covered with a protective layer. In aspects, the protective layer is fibrin glue, chitosan based dressing, collagen, or other biodegradable matrix. In aspects, the protective layer is prepared from an autologous tissue or a non-immunogenic material.

[0150]

[0132] The present methods provide for, and include, methods of repairing cartilage or treating a cartilage defect in subjects in need thereof. The methods comprise, preparing a MCP composition from autologous cartilage, wherein the MCP composition comprises morselized cartilage tissue particles having an average length of between 0.1 and 1.0 mm and a chondrocyte viability of at least 85%, and a biodegradable matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from about 1 : 10 to about 5:1, and applying the MCP composition to damaged cartilage or a cartilage defect. In aspects, the MCP composition is shaped during application and physically cross-linked using a polyvalent cation. In further aspects, the applied MCP composition is covered with a protective layer comprising fibrin glue, chitosan based dressing, collagen, or other biodegradable matrix. In aspects, the MCP composition comprises autologous materials. In other aspects, the MCP composition comprises autologous MCPs and allogenic materials. In yet other aspects, the MCP composition comprises allogenic materials.

[0151]

[0133] In aspects, the methods of repairing cartilage or treating a cartilage defect in subjects in need thereof comprises applying the MCP composition to a joint in need of repair. Suitable joints the joint are selected from a knee joint, an elbow joint, a hip joint, an ankle joint, a wrist joint, a finger joint, or a toe joint. As provided herein, the methods of repairing cartilage or treating a cartilage defect in subjects in need thereof further comprise applying a protective layer to the MCP compositions at the treatment site. In an aspect, the protective layer is a fibrin glue.

[0152]

[0134] Physical crosslinking of the MCP compositions with a polyvalent cation are also included and provided for in the methods of repairing cartilage or treating a cartilage defect in subjects in need thereof. In aspects, the MCP composition is applied to the treatment site, shaped to conform the repair according the therapeutic need, and then cross-linked using a polyvalent cation. In aspects, the polyvalent cation is Ca++, applied as a solution of CaCl2, CaCO2, or CaSO4at a concentration of between 10 nM (1 x 10"9M) and 1 mM (IxlO"6M). In aspects, a CaCl2solution at a concentration of 100 nM in an isotonic physiological buffer is applied for about 5 to 15 seconds. In other aspects, CaCO3or CaSO4solutions are applied as they provide for a slower rate of ionic cross-linking.

[0153]

[0135] The MCP compositions can be delivered and applied to the cartilage to be repaired or a joint having a cartilage defect by applying the MCP composition with a spatula or by injection using a large gauge needle. In some aspects, the repair of the cartilage or joint is performed arthroscopically. In some aspects, the MCP composition can be pre-formed into a suitable shape for therapeutic use and the shaped MCP composition transferred to the subject in need. In aspects, the methods provide for applying the MCP composition to a shaped form, applying a polyvalent cation to the shaped MCP composition to increase the viscosity, mechanical strength, and rigidity.

[0154] Kits

[0155]

[0136] The present disclosure further provides for, and includes, kits for treating cartilage degeneration, the kit comprising at least one of an applicator dispensing device for receiving morselized cartilage tissue particles; one or more syringes for mixing and / or dispensing cartilage; and a biodegradable matrix. In aspects, the syringes included in the kits are from a mixing syringe, a two- (or multi) stage syringe, a dual- (or multi) chamber syringe, a dual (or multi) syringe, a two- (or multi) component syringe. In other aspects, the kits include a spatula for applying the cartilage composition for cartilage repair.

[0137] The kits of the present application may further comprise a disposable tissue harvesting device. In aspects, kits include a disposable morselization chamber. In aspects, the disposable morselization chamber is a sterilized disposable morselization chamber.

[0156]

[0138] Further provided for, and included in, the kits of the present application are reagents selected from a saline-containing solution or an isotonic compatible medium capable of buffering pH. The kits may further comprise at least one element selected from an active pharmaceutical ingredient, a liquid medium, fibrin glue, growth factors, binders, saline, buffer solution, cell nurturing / preservation solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof. In aspects, the at least one element is at least one growth factor. In aspects, the growth factor is selected from the group consisting of TGF-β1, BMP -2, BMP-7, IGF-I, FGF-2, FGF-18, PDGF, and combinations thereof. In aspects, the kits include at least one active pharmaceutical ingredient selected from the group consisting of NSAIDs, steroids and corticosteroids; analgesics; anthraquinones, and combinations thereof.

[0157] Additional Definitions

[0158]

[0139] As used herein, a “physiological buffer” is an isotonic buffer having a buffered pH between 6 and 8. The buffer can involve one of many different organic buffers. In certain aspects, an organic buffer is selected from the group consisting of 2-(N- morpholino)ethanesulfonic acid (MBS), Tris (hydroxymethyl)aminomethane (TRIS) and (N-[2- hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES). Suitable physiological buffers are selected from the group consisting of Hanks' Balanced Salt Solution (HBSS), (N-[2- hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), Phosphate Buffered Saline (PBS), and Tris-Buffered Saline (TBS), and wherein the solution is maintained at a pH of 6-8. As used herein “isotonic solution” means an aqueous solution with a salt concentration approximately equal or equal to that found in normal cells of the body. In some aspects, isotonic solutions can include Lactated Ringer's solution, Normal Saline solution (0.9%), and Phosphate Buffered Saline (PBS).

[0140] An “effective amount” is that amount of a peptide or polypeptide provided herein, the administration of which to a subject, either in a single dose or as part of a series, is effective for treatment.

[0159]

[0141] The term “subject” is meant any subject, particularly a mammalian subject, in need of treatment with a peptide or polypeptide provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on. In one aspect, the subject is a human subject.

[0160]

[0142] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole.

[0161]

[0143] As used herein the term “about” refers to ± 10 %.

[0162]

[0144] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided.

[0163]

[0145] The term “consisting of’ means “including and limited to”.

[0164]

[0146] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0165]

[0147] As used herein, the singular form “a”, “ “aann”” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound" or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[0148] The term “biodegradable includes materials that are biocompatible and / or degrade in the body and can be safely disposed of through bodily functions and / or are bioabsorbable.

[0166] Embodiments

[0167]

[0149] Embodiment !. A chondrocyte composition comprising: a. morselized cartilage tissue particles having an average length of about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, and b. a biodegradable matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from 1:10 and 5:1.

[0168]

[0150] Embodiment 2. The chondrocyte composition of embodiment 1, wherein said biodegradable matrix composition is a hydrogel, a dispersion, or a cream.

[0169]

[0151] Embodiment 3. The chondrocyte composition of embodiment 2, wherein said hydrogel is a synthetic polymer hydrogel selected from the group consisting of polylactide (PEA), poly-lactide-co-glycolide (PLGA), polyglycolide (PGA), poly-(D,L-lactic acid) (PDLLA), poly caprolactone (PCL), polyethylene-glycol (PEG), poly(vinyl alcohol) (PVA), poly (Nisopropylacrylamide)(PNIPAM), polyacrylamide (PAM), and combinations thereof.

[0170]

[0152] Embodiment 4. The chondrocyte composition of any one of embodiments 1 to 3, wherein said biodegradable matrix composition is a natural polymer hydrogel selected from the group consisting of alginate-nanocellulose, chitosan, collagen, alginate, hyaluronic acid, gelatin, platelet-rich fibrin and combinations thereof.

[0171]

[0153] Embodiment 5. The chondrocyte composition of any one of embodiments 1 to 4, wherein said morselized cartilage tissue particles are selected from the group consisting of morselized articular cartilage tissue particles, morselized non-articular cartilage tissue particles, and combinations thereof.

[0172]

[0154] Embodiment 6. The chondrocyte composition of any one of embodiments 2 to 5, wherein the morselized tissue particles are morselized articular cartilage tissue particles.

[0173]

[0155] Embodiment 7. The chondrocyte composition of any one of embodiments 1 to 6, wherein the average length is about 0.3 mm to about 0.7 mm.

[0156] Embodiment 8. The chondrocyte composition of any one of embodiments 1 to 7, wherein the average length is about 0.5 mm.

[0174]

[0157] Embodiment 9. The chondrocyte composition of any one of embodiments 1 to 8, wherein said biodegradable matrix composition comprises an autologous preparation comprising a platelet-rich fibrin.

[0175]

[0158] Embodiment 10. The chondrocyte composition of any one of embodiments 1 to 9, wherein said biodegradable matrix composition comprises a combination of: an autologous biodegradable matrix material derived from one or more of a platelet-rich fibrin, a lipoaspirate, a bone-marrow aspirate concentration, autologous mesenchymal derived stromal cells and combinations thereof; and an allogenic biodegradable matrix material derived from one or more of umbilical cord; umbilical cord blood, placental chorion, amnion, allogenic platelet-rich fibrin, allogenic lipoaspirate, allogenic bone-marrow aspirate concentration, allogenic mesenchymal derived stromal cells, and combinations thereof.

[0176]

[0159] Embodiment 11. The chondrocyte composition of any one of embodiments 1 to 10, wherein said biodegradable matrix composition is an alginate-nanocellulose hydrogel.

[0177]

[0160] Embodiment 12. The chondrocyte composition of any one of embodiments 1 to 11, wherein said alginate-nanocellulose hydrogel has a weight ratio of nanocellulose to alginate in the alginate-nanocellulose hydrogel from 0.01 to 10.

[0178]

[0161] Embodiment 13. The chondrocyte composition of any one of embodiments 1 to 11, wherein said alginate-nanocellulose hydrogel further comprises cellulose nanocrystals, TEMPO- oxidized cellulose nanocrystals (CNCTs), cellulose nanofibers, TEMPO-oxidized cellulose nanofibers (CNFTs), and combinations thereof.

[0179]

[0162] Embodiment 14. The chondrocyte composition of any one of embodiments 1 to 13; wherein said chondrocytes exhibit increased chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, compared to chondrocytes in a non-morselized cartilage tissue.

[0163] Embodiment 15. The chondrocyte composition of any one of embodiments 1 to 14, further comprising at least one component selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin and salts thereof, glucosamine and salts thereof, methylsulfonyl methane (MSM), vitamins and combinations thereof.

[0180]

[0164] Embodiment 16. The chondrocyte composition of any one of embodiments 1 to 15, wherein the fibrin glue and platelet rich plasma are autologous.

[0181]

[0165] Embodiment 17. The chondrocyte composition of any one of embodiments 1 to 16, wherein the at least one component is at least one growth factor.

[0182]

[0166] Embodiment 18. The chondrocyte composition of embodiment 17, wherein the at least one growth factor is selected from the group consisting of TGF-β1, BMP -2, BMP-7, IGF-I, FGF-2, FGF-18, PDGF, and combinations thereof.

[0183]

[0167] Embodiment 19. The chondrocyte composition of any one of embodiments 17 or 18, wherein the at least one growth factor increases or upregulates chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4.

[0184]

[0168] Embodiment 20. The chondrocyte composition of any one of embodiments 1 to 19, wherein said liquid medium is a hydrophilic medium, an oleophilic medium, or an emulsion of hydrophilic and oleophilic mediums.

[0185]

[0169] Embodiment 21. The chondrocyte composition of any one of embodiments 1 to 20, wherein the chondrocyte composition is a paste or a suspension.

[0186]

[0170] Embodiment 22. The chondrocyte composition of any one of embodiments 1 to 21, wherein the paste or suspension is injectable.

[0187]

[0171] Embodiment 23. The chondrocyte composition of any one of embodiments 1 to 22, wherein the chondrocyte composition is a paste.

[0172] Embodiment 24. The chondrocyte composition of any one of embodiments 1 to 22, wherein said chondrocyte composition is a suspension.

[0188]

[0173] Embodiment 25. The chondrocyte composition of any one of embodiments 1 to 24, wherein said suspension is a homogeneous suspension.

[0189]

[0174] Embodiment 26. The chondrocyte composition of any one of embodiments 1 to 25, wherein said active pharmaceutical ingredient is selected from the group consisting of nonsteroidal anti-inflammatory drugs (NSAIDs), steroids and corticosteroids; analgesics; anthraquinones and combinations thereof.

[0190]

[0175] Embodiment 27. The chondrocyte composition of any one of embodiments 1 to 26, wherein said NSAID is selected from ibuprofen, naproxen sodium, aspirin, diclofenac (and salts thereof), celecoxib, coxib, sulindac, oxaprozin, piroxicam, indomethacin, meloxicam, fenoprofen, diflunisal, etodolac, ketorolac tromethamine, meclofenamate, nabumetone, salsalate, or salts thereof.

[0191]

[0176] Embodiment 28. The chondrocyte composition of any one of embodiments 1 to 27, wherein said steroids and corticosteroids are selected from triamcinolone, cortisone, prednisone, methylprednisolone, hydrocortisone, or combinations thereof.

[0192]

[0177] Embodiment 29. The chondrocyte composition of any one of embodiments 1 to 28, wherein said analgesic is selected from acetaminophen, capsaicin, camphor, menthol, lidocaine, or combinations thereof.

[0193]

[0178] Embodiment 30. The chondrocyte composition of any one of embodiments 1 to 29, wherein said anthraquinone is diacerein.

[0194]

[0179] Embodiment 31. A composition comprising : a. morselized articular cartilage tissue particles having an average length of between 0.1 and 1.0 mm and a chondrocyte viability of at least 85%; b. a biodegradable matrix composition comprising an alginate-nanocellulose hydrogel, wherein the volume ratio of morselized tissue particles to biodegradable matrix composition is about 1 :3; and c. at least one growth factor selected from the group consisting of TGF-J31, BMP-2, BMP-7, IGF-I, FGF-2, FGF-18, and PDGF.

[0195]

[0180] Embodiment 32. The composition of embodiment 31 , wherein said at least one growth factor comprises TGF-β1, FGF-18, or both.

[0196]

[0181] Embodiment 33. The composition of any one of embodiments 31 or 32, wherein said composition further comprises an active pharmaceutical ingredient selected from the group consisting of NSAIDs, steroids and corticosteroids; analgesics; anthraquinones and combinations thereof.

[0197]

[0182] Embodiment 34. The composition of any one of embodiments 31 to 33, wherein the composition is a paste.

[0198]

[0183] Embodiment 35. The composition of any one of embodiments 31 to 34, wherein the weight ratio of nanocellulose to alginate in the alginate-nanocellulose hydrogel is in the range of greater than 0.01 to about 10.0.

[0199]

[0184] Embodiment 36. The composition of any one of embodiments 31 to 35, wherein the alginate-nanocellulose hydrogel further comprises cellulose nanocrystals, TEMPO-oxidized cellulose nanocrystals (CNCTs), cellulose nanofibers, and / or TEMPO-oxidized cellulose nanofibers (CNFTs).

[0200]

[0185] Embodiment 37. The composition of any one of embodiments 31 to 36, wherein the at least one growth factor increases or upregulates chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, compared to a composition lacking said at least one growth factor.

[0201]

[0186] Embodiment 38. A cartilage graft composition comprising: a. morselized articular cartilage tissue particles having a length of between 0.3 mm or about 0.7 mm, a chondrocyte viability of at least about 90%; b. a biodegradable matrix; and c. an increased or upregulated chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, relative to a non-morselized articular cartilage tissue.

[0202]

[0187] Embodiment 39. The cartilage graft composition of embodiment 38, wherein said biodegradable matrix composition is a hydrogel.

[0203]

[0188] Embodiment 40. The cartilage graft composition of embodiment 39, wherein said hydrogel is a natural polymer hydrogel selected from the group consisting of alginatenanocellulose, chitosan, collagen, alginate, hyaluronic acid, gelatin, platelet-rich fibrin and combinations thereof.

[0204]

[0189] Embodiment 41. The cartilage graft composition of any one of embodiments 38 to 40, wherein said hydrogel is an alginate-nanocellulose hydrogel.

[0205]

[0190] Embodiment 42. A method of repairing cartilage or treating a cartilage defect in a subject in need thereof comprising, administering to the subject a chondrocyte composition comprising morselized cartilage tissue particles having an average length of between about 0.1 and about 1.0 mm, a chondrocyte viability of at least 85%, and a biodegradable matrix composition (BMC).

[0206]

[0191] Embodiment 43. The method of repairing cartilage or treating a cartilage defect of embodiment 42, wherein said administering comprises applying said chondrocyte composition using a spatula or syringe.

[0207]

[0192] Embodiment 44. The method of repairing cartilage or treating a cartilage defect of embodiments 42 or 43, further comprising introducing a divalent cation solution in situ to the chondrocyte composition applied to said subject thereby physically cross-linking the BMC.

[0208]

[0193] Embodiment 45. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 44, wherein divalent cation solution comprises CaCl2.

[0209]

[0194] Embodiment 46. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 45, where said solution of CaCl2comprises a 100 nM isotonic buffered solution of CaCl2.

[0195] Embodiment 47. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 46, wherein said solution of CaCl2is applied for about 5 to about 15 seconds.

[0210]

[0196] Embodiment 48. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 47, wherein said chondrocyte composition is prepared from autologous tissues or cells.

[0211]

[0197] Embodiment 49. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 48, wherein said chondrocyte composition is prepared from allogenic tissues or cells.

[0212]

[0198] Embodiment 50. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 49, wherein said chondrocyte composition is prepared from a combination of allogenic and autologous tissues or cells.

[0213]

[0199] Embodiment 51. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 50, wherein the morselized tissue particles or the composition is administered to a joint of the subject.

[0214]

[0200] Embodiment 52. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 451, wherein the joint is a knee joint.

[0215]

[0201] Embodiment 53. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 52, further comprising covering the chondrocyte composition with a fibrin glue, a chitosan-based dressing, or combination thereof.

[0216]

[0202] Embodiment 54. The method of repairing cartilage or treating a cartilage defect of any one of embodiments 42 to 53, wherein the fibrin glue is prepared from autologous fibrin.

[0217]

[0203] Embodiment 55. A method of treating a subject in need of joint repair, comprising: a. harvesting at least one cartilage tissue portion from a subject; b. morselizing the harvested cartilage tissue portion for less than about 30 minutes to prepare morselized cartilage tissue particles having an average length of about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%; c. collecting the morselized cartilage tissue particles; d. mixing the morselized cartilage tissue particles with a biodegradable matrix composition (“BMC”) to produce an autologous chondrocyte composition; and e. applying and shaping said autologous chondrocyte composition in a joint of the subject.

[0218]

[0204] Embodiment 56. The method of treating a subject in need of joint repair of embodiment 55, wherein said morselizing comprises morselizing is about 4 to aboutl2 minutes.

[0219]

[0205] Embodiment 57. The method of treating a subject in need of joint repair of embodiments 55 or 56, further comprising introducing a divalent cation solution in situ to the subject applied chondrocyte composition thereby physically cross-linking the BMC via ionic bonds.

[0220]

[0206] Embodiment 58. The method of any one of embodiments 55 to 57, wherein said divalent cation solution is a CaCl2solution.

[0221]

[0207] Embodiment 59. The method of treating a subject in need of joint repair of any one of embodiments 55 to 58, wherein the joint of the subject is a knee joint, an elbow joint, a hip joint, an ankle joint, a wrist joint, a finger joint, or a toe joint.

[0222]

[0208] Embodiment 60. The method of treating a subject in need of joint repair of any one of embodiments 55 to 59, wherein the composition is implanted in a knee joint of the subject.

[0223]

[0209] Embodiment 61. The method of treating a subject in need of joint repair of any one of embodiments 55 to 60, wherein the joint has a defect, and wherein the method results in complete or almost complete defect closure 8-16 weeks post-surgery.

[0224]

[0210] Embodiment 62. The method of treating a subject in need of joint repair of any one of embodiments 55 to 61, wherein the joint has a defect, and wherein the method results in complete or almost complete defect closure about 12 weeks post-surgeiy.

[0225]

[0211] Embodiment 63. A method of repairing cartilage or treating a cartilage defect in a knee joint of a subject in need thereof, comprising: a. administering to the knee joint of the subject via syringe or spatula, autologous morselized cartilage tissue particles (MCP) having an average length of about O.lto about 1.0 mm and a chondrocyte viability of at least 85%, and a biodegradable matrix composition; b. physically cross-linking the MCP composition in situ with 100 nM CaC12 for about 10 seconds; and c. covering the morselized articular cartilage tissue particles or composition with protective layer.

[0226]

[0212] Embodiment 64. The method of embodiment 63, wherein said protective layer is fibrin glue, chitosan, collagen, or a combination thereof.

[0227]

[0213] Embodiment 65. The method of embodiments 63 or 64, wherein said fibrin glue autologous.

[0228]

[0214] Embodiment 66. A kit for treating cartilage degeneration, the kit comprising at least one of: a. an applicator dispensing device for receiving morselized cartilage tissue particles; b. one or more syringes for mixing and / or dispensing cartilage; and c. a biodegradable matrix;

[0229]

[0215] Embodiment 67. The kit for treating cartilage degeneration of embodiment 66, wherein the syringes are selected from a mixing syringe, a two- (or multi) stage syringe, a dual- (or multi) chamber syringe, a dual (or multi) syringe, a two- (or multi) component syringe, or a spatula.

[0230]

[0216] Embodiment 68. The kit for treating cartilage degeneration of embodiments 66 or 67, further comprising a disposable tissue harvesting device.

[0231]

[0217] Embodiment 69. The kit for treating cartilage degeneration of any one of embodiments 66 to 68, further comprising disposable morselization chamber.

[0218] Embodiment 70. The kit for treating cartilage degeneration of any one of embodiments 66 to 69, further comprising reagents selected from a saline-containing solution or an isotonic compatible medium capable of buffering pH.

[0232]

[0219] Embodiment 71. The kit for treating cartilage degeneration of any one of embodiments 66 to 70, wherein the kit further comprises at least one element selected from an active pharmaceutical ingredient, a liquid medium, fibrin glue, growth factors, binders, saline, buffer solution, cell nurturing / preservation solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof.

[0233]

[0220] Embodiment 72. The kit for treating cartilage degeneration of any one of embodiments 66 to 71, wherein the at least one element is at least one growth factor selected from the group consisting of TGF-β1, BMP -2, BMP-7, IGF-I, FGF-2, FGF-18, PDGF, and combinations thereof.

[0234]

[0221] Embodiment 73. The kit for treating cartilage degeneration of any one of embodiments 66 to 72, wherein said active pharmaceutical ingredient is selected from the group consisting of NSAIDs, steroids and corticosteroids; analgesics; anthraquinones, and combinations thereof.

[0235] Examples:

[0236] TissueMill® Cartilage Processing

[0237]

[0222] A novel method to process cartilage tissue is explored using a TissueMill® reusable base unit and single use sterile dispensable unit. Two grams of cartilage is placed into the processor containing 25 to 50 milliliters of physiological buffer using forceps. Timed processing allows the production of precise and reproducible formed cartilage morsel-sizes. Cartilage is harvested and morselized into 0.5mm, 1.0mm, and 5.0mm lengths, and cultured or used in the production of chondrocyte compositions.

[0238]

[0223] Following morselization, the cartilage particles are collected from the sterile dispensable unit and allowed to settle, desirably under gravity. The excess fluid is expelled and the MCTPs collected for further processing and the preparation of chondrocyte compositions as described herein. Approximately two milliliters of collected cartilage morsels is obtained.

[0239] In Vitro Studies I

[0240]

[0224] Bovine cartilage is harvested from the tibial head of a young bovine knee joint from the slaughterhouse. This is morselized into 0.5mm, 1.0mm, and 5.0mm length morsels. These morsel sizes correspond to processing times of 4-12 minutes, respectively. The size of the cartilage fragments are checked through standard light microscopy. These morsels are cultured in DMEM / F12 (Coming) with 10% FBS and 1% antibiotic-antiymyotic (AA) for 3 weeks.

[0241]

[0225] Cell viability is assessed immediately after mincing using a live / dead viability / cytotoxicity kit for mammalian cells (ThermoFisher). To make the staining solution, a 4 μM Calcein acetoxymethyl (AM) solution is made in PBS (Coming), and 50 μM Ethidium Homodimer is added. Cells are stained for 15 minutes at room temperature in a dark environment. The unbound dye is washed with PBS. Cells are kept in PBS during imaging using a confocal fluorescence microscope with appropriate filters. The excitation wavelength for Calcein AM is 488 / 520 nm. The excitation / emission wavelength for ethidium homodimer is 528 / 617 nm.

[0242]

[0226] In vitro outgrowth from the minced cartilage morsels are captured at weeks, 1, 2, and 3.

[0243] Additionally, chondrogenic gene expression is conducted at week 3 through RNA isolation from varying morsels and subsequent qRT-PCR. Gene expression for Aggrecan (ACAN), Collagen Type II (COL2), Proliferating Cell Nuclear Antigen (PCNA), Cartilage Oligomeric Matrix Protein (COMP), SRY-Box Transcription Factor 9 (SOX-9), and Cartilage and Proteoglycan 4 (PRG4 / Lubricin) are analyzed. A brief description of these genes, along with their primer sequences is recorded in Table 1.

[0244] Table 1: Brief description of the genes analyzed in the bovine cartilage in vitro study with their associated primer sequences In Vitro Studies II

[0245]

[0227] In vitro viability studies of formed cartilage morsel-size based on fluorescent microscopy reveal that as processing time increased from 4-12 minutes, chondrocyte viability is highly conserved at >90% regardless of morsel size (Figure 1 A). Viability is measured immediately after mincing. Additionally, as processing time increased, morsel size uniformly decreased. Dead cells are localized to the periphery of the cartilage morsels only. While viability is maintained, gene expression varied across morsel sizes (Figure 1A). Several chondrogenic genes are significantly upregulated 3 weeks post-processing, including ACAN, COL2, PCNA, COMP, SOX-9, and PRG4 (Figure IB). The gene expression of these proteins is upregulated greatest in 0.5mm length morsels at 3 weeks post-processing, compared to 1mm and >5mm samples. As morsel size decreased, the expression of these chondrogenic genes increased. Of note, while PRG4 expression increases as morsel size decreases from >5mm, the 1mm morsels have greater expression than the 0.5mm morsels. Differences are significant among all genes between all morsel sizes, except for COL2 and ACAN, where the difference is only significant between 0.5mm and >5mm samples. Additionally, expression of Matrix Metalloprotease- 13 (MMP-13), an important enzyme involved in the breakdown of cartilage and type II collagen, is only significantly decreased in 0.5mm length morsels compared to cartilage explants greater than 3 cm2. Thus, the 0.5mm morsels show the highest chondrogenic potential with the highest gene expression.

[0246]

[0228] Furthermore, cellular outgrowth from the formed cartilage morsels is rapid following cartilage tissue processing. After 1 week, early chondrocyte migration from each cartilage morsel was visible (Figure 2). After 2 weeks, there was complete colonization of the culture plate. After 3 weeks, multilayer chondrocytes formed, with bridging between the cartilage morsels (Figure 2).

[0247] In Vivo Studies

[0248]

[0229] Hyaline cartilage is harvested from a skeletally mature 9-month-old donor rabbit on the day of surgery. The cartilage is morselized immediately to create morsels of 0.5mm length. The morsels are mixed with alginate-Nanocellulose gel at a ratio of 1:3 to produce a formed cartilage morsel / Nanocellulose (NC) paste. The alginate-Nanocellulose gel is prepared via methods cited in a previous study (Muller et al., Annals of Biomedical Engineering 45(1 ):210—223 (2017)). A rabbit articular cartilage injury modeling microfracture (MFX) is developed by drilling a 4mm diameter full thickness hole into the trochlear groove of each knee, reaching the marrow. In rabbits that receive repair, the NCA implant paste is placed into the defect with a spatula and ionically crosslinked in situ with 100 nM CaCl2for 10 seconds. Subsequently, after crosslinking, the filled-in defect is covered with fibrin glue (Baxter, Illinois). The knee joints with the articular cartilage injury, both those that did and did not receive repair, are fixed with 10% formalin, decalcified with 10% EDTA solution, and then embedded in paraffin. Safranin-0 staining is conducted to assess the quality of cartilage repair.

[0249]

[0230] All aspects of this study meet IACUC approval at the Feinstein Institutes of Medical Research. Descriptive statistics, ANOVA, and Student t-tests are used to compare groups.

[0250]

[0231] From the findings of the in vitro studies, 0.5 mm morsels are chosen to be tested in vivo. Macroscopically, NC implant paste containing 0.5mm morsels demonstrates the formation of hyaline cartilage from the implanted material with significant defect closure 12 weeks postoperation (Figure 3). Notably, there is no distinguishable border between the new and native cartilage. Marrow stimulation alone results in largely unfilled defects. Although a thin layer of cartilage-like tissue appears in some defects, the defects are not completely filled.

[0251]

[0232] Safranin-0 staining, useful for the detection of aggrecan, along with microscopic evaluation demonstrated superior quality cartilage formation in the defect area using the NCA implant paste compared to marrow stimulation alone (Figure 4). Staining revealed the regeneration of an aggrecan-rich matrix and regenerated articular surface. Overall, 0.5mm morsels are optimum for the NCA implant paste treatment.

[0252]

[0233] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[0234] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

Claims1. A chondrocyte composition comprising: a. morselized cartilage tissue particles having an average length of about 0.1 to about 1.0 mm and a chondrocyte viability of at least 85%, and b. a biodegradable matrix composition, wherein the volume ratio of morselized cartilage tissue particles to biodegradable matrix composition is from 1:10 and 5:1.

2. The chondrocyte composition of claim 1 , wherein said biodegradable matrix composition is a hydrogel, a dispersion, or a cream.

3. The chondrocyte composition of claim 2, wherein said hydrogel is a synthetic polymer hydrogel selected from the group consisting of polylactide (PLA), poly-lactide-co-glycolide (PLGA), polyglycolide (PGA), poly-(D,L-lactic acid) (PDLLA), polycaprolactone (PCL), poly ethylene-gly col (PEG), poly (vinyl alcohol) (PVA), poly (Nisopropylacrylamide)(PNIPAM), polyacrylamide (PAM), and combinations thereof.

4. The chondrocyte composition of claim 2, wherein said biodegradable matrix composition is a natural polymer hydrogel selected from the group consisting of alginate-nanocellulose, chitosan, collagen, alginate, hyaluronic acid, gelatin, platelet-rich fibrin and combinations thereof.

5. The chondrocyte composition of claim 1, wherein said morselized cartilage tissue particles are selected from the group consisting of morselized articular cartilage tissue particles, morselized non-articular cartilage tissue particles, and combinations thereof.

6. The chondrocyte composition of claim 2, wherein the morselized tissue particles are morselized articular cartilage tissue particles.

7. The chondrocyte composition of claim 1, wherein the average length is about 0.3 mm to about 0.7 mm.

8. The chondrocyte composition of claim 1, wherein the average length is about 0.5 mm.

9. The chondrocyte composition of claim 2, wherein said biodegradable matrix composition comprises an autologous preparation comprising a platelet-rich fibrin.

10. The chondrocyte composition of claim 2, wherein said biodegradable matrix composition comprises a combination of: an autologous biodegradable matrix material derived from one or more of a platelet-rich fibrin, a lipoaspirate, a bone-marrow aspirate concentration, autologous mesenchymal derived stromal cells and combinations thereof; and an allogenic biodegradable matrix material derived from one or more of umbilical cord; umbilical cord blood, placental chorion, amnion, allogenic platelet-rich fibrin, allogenic lipoaspirate, allogenic bone-marrow aspirate concentration, allogenic mesenchymal derived stromal cells, and combinations thereof.

11. The chondrocyte composition of claim 2, wherein said biodegradable matrix composition is an alginate-nanocellulose hydrogel.

12. The chondrocyte composition of claim 11, wherein said alginate-nanocellulose hydrogel has a weight ratio of nanocellulose to alginate in the alginate-nanocellulose hydrogel from 0.01 to 10.

13. The chondrocyte composition of claim 11, wherein said alginate-nanocellulose hydrogel further comprises cellulose nanocrystals, TEMPO-oxidized cellulose nanocrystals (CNCTs), cellulose nano fibers, TEMPO-oxidized cellulose nanofibers (CNFTs), and combinations thereof.

14. The chondrocyte composition of claim 1 ; wherein said chondrocytes exhibit increased chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, compared to chondrocytes in a non- morselized cartilage tissue.

15. The chondrocyte composition of claim 1, further comprising at least one component selected from the group consisting of an active pharmaceutical ingredient, liquid medium, fibrin glue, platelet rich plasma, growth factors, binders, saline, buffer solution, chondroitin and saltsthereof, glucosamine and salts thereof, methylsulfonylmethane (MSM), vitamins and combinations thereof.

16. The chondrocyte composition of claim 15, wherein the fibrin glue and platelet rich plasma are autologous.

17. The chondrocyte composition of claim 15, wherein the at least one component is at least one growth factor.

18. The chondrocyte composition of claim 17, wherein the at least one growth factor is selected from the group consisting of TGF-β1, BMP-2, BMP-7, IGF-I, FGF-2, FGF-18, PDGF, and combinations thereof.

19. The chondrocyte composition of claim 18, wherein the at least one growth factor increases or upregulates chondrogenic gene expression in one or more genes selected from the group consisting of AC AN, COL2, COMP, PCNA, SOX9, and PRG4.

20. The chondrocyte composition of claim 15, wherein said liquid medium is a hydrophilic medium, an oleophilic medium, or an emulsion of hydrophilic and oleophilic mediums.

21. The chondrocyte composition of claim 1 , wherein the chondrocyte composition is a paste or a suspension.

22. The chondrocyte composition of claim 21, wherein the paste or suspension is injectable.

23. The chondrocyte composition of claim 22, wherein the chondrocyte composition is a paste.

24. The chondrocyte composition of claim 22, wherein said chondrocyte composition is a suspension.

25. The chondrocyte composition of claim 24, wherein said suspension is a homogeneous suspension.

26. The chondrocyte composition of claim 15, wherein said active pharmaceutical ingredient is selected from the group consisting of non-steroidal anti-inflammatory drugs (NSAIDs), steroids and corticosteroids; analgesics; anthraquinones and combinations thereof.

27. The chondrocyte composition of claim 26, wherein said NSAID is selected from ibuprofen, naproxen sodium, aspirin, diclofenac (and salts thereof), celecoxib, coxib, sulindac, oxaprozin, piroxicam, indomethacin, meloxicam, fenoprofen, diflunisal, etodolac, ketorolac tromethamine, meclofenamate, nabumetone, salsalate, or salts thereof.

28. The chondrocyte composition of claim 26, wherein said steroids and corticosteroids are selected from triamcinolone, cortisone, prednisone, methylprednisolone, hydrocortisone, or combinations thereof.

29. The chondrocyte composition of claim 26, wherein said analgesic is selected from acetaminophen, capsaicin, camphor, menthol, lidocaine, or combinations thereof.

30. The chondrocyte composition of claim 26, wherein said anthraquinone is diacerein.

31. A composition comprising: a. morselized articular cartilage tissue particles having an average length of between 0.1 and 1.0 mm and a chondrocyte viability of at least 85%; b. a biodegradable matrix composition comprising an alginate-nanocellulose hydrogel, wherein the volume ratio of morselized tissue particles to biodegradable matrix composition is about 1 :3; and c. at least one growth factor selected from the group consisting of TGF-β1, BMP-2, BMP-7, IGF-I, FGF-2, FGF-18, and PDGF.

32. The composition of claim 31, wherein said at least one growth factor comprises TGF-β1, FGF-18, or both.

33. The composition of claim 31, wherein said composition further comprises an active pharmaceutical ingredient selected from the group consisting of NSAIDs, steroids and corticosteroids; analgesics; anthraquinones and combinations thereof.

34. The composition of claim 31, wherein the composition is a paste.

35. The composition of claim 31, wherein the weight ratio of nanocellulose to alginate in the alginate-nanocellulose hydrogel is in the range of greater than 0.01 to about 10.0.

36. The composition of claim 31, wherein the alginate-nanocellulose hydrogel further comprises cellulose nanocrystals, TEMPO-oxidized cellulose nanocrystals (CNCTs), cellulose nanofibers, and / or TEMPO-oxidized cellulose nanofibers (CNFTs).

37. The composition of claim 31, wherein the at least one growth factor increases or upregulates chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, compared to a composition lacking said at least one growth factor.

38. A cartilage graft composition comprising: a. morselized articular cartilage tissue particles having a length of between 0.3 mm or about 0.7 mm, a chondrocyte viability of at least about 90%; b. a biodegradable matrix; and c. an increased or upregulated chondrogenic gene expression in one or more genes selected from the group consisting of ACAN, COL2, COMP, PCNA, SOX9, and PRG4, relative to a non-morselized articular cartilage tissue.

39. The cartilage graft composition of claim 38, wherein said biodegradable matrix composition is a hydrogel.

40. The cartilage graft composition of claim 39, wherein said hydrogel is a natural polymer hydrogel selected from the group consisting of alginate-nanocellulose, chitosan, collagen, alginate, hyaluronic acid, gelatin, platelet-rich fibrin and combinations thereof.

41. The cartilage graft composition of claim 40, wherein said hydrogel is an alginatenanocellulose hydrogel.

42. A method of repairing cartilage or treating a cartilage defect in a subject in need thereof comprising, administering to the subject a chondrocyte composition comprising morselized cartilage tissue particles having an average length of between about 0.1 and about 1.0 mm, a chondrocyte viability of at least 85%, and a biodegradable matrix composition (BMC).

43. The method of repairing cartilage or treating a cartilage defect of claim 42, wherein said administering comprises applying said chondrocyte composition using a spatula or syringe.

44. The method of repairing cartilage or treating a cartilage defect of claim 42, further comprising introducing a divalent cation solution in situ to the chondrocyte composition applied to said subject thereby physically cross-linking the BMC.

45. The method of repairing cartilage or treating a cartilage defect of claim 42, wherein divalent cation solution comprises CaCl2.

46. The method of repairing cartilage or treating a cartilage defect of claim 45, where said solution of CaCl2comprises a 100 nM isotonic buffered solution of CaCl2.

47. The method of repairing cartilage or treating a cartilage defect of claim 45, wherein said solution of CaCl2is applied for about 5 to about 15 seconds.

48. The method of repairing cartilage or treating a cartilage defect of claim 42, wherein said chondrocyte composition is prepared from autologous tissues or cells.

49. The method of repairing cartilage or treating a cartilage defect of claim 42, wherein said chondrocyte composition is prepared from allogenic tissues or cells.

50. The method of repairing cartilage or treating a cartilage defect of claim 42, wherein said chondrocyte composition is prepared from a combination of allogenic and autologous tissues or cells.

51. The method of repairing cartilage or treating a cartilage defect of claim 42, wherein the morselized tissue particles or the composition is administered to a joint of the subject.

52. The method of repairing cartilage or treating a cartilage defect of claim 51, wherein the joint is a knee joint.

53. The method of repairing cartilage or treating a cartilage defect of claim 42, further comprising covering the chondrocyte composition with a fibrin glue, a chitosan-based dressing, or combination thereof.

54. The method of repairing cartilage or treating a cartilage defect of claim 53, wherein the fibrin glue is prepared from autologous fibrin.

55. A method of treating a subject in need of joint repair, comprising: a. harvesting at least one cartilage tissue portion from a subject; b. morselizing the harvested cartilage tissue portion for less than about 30 minutes to prepare morselized cartilage tissue particles having an average length of about 0.1 to about 1.0 mm and a chondrocyte viability of at least 90%; c. collecting the morselized cartilage tissue particles; d. mixing the morselized cartilage tissue particles with a biodegradable matrix composition (“BMC”) to produce an autologous chondrocyte composition; and e. applying and shaping said autologous chondrocyte composition in a joint of the subject.

56. The method of treating a subject in need of joint repair of claim 55, wherein said morselizing comprises morselizing is about 4 to about 12 minutes.

57. The method of treating a subject in need of joint repair of claim 55, further comprising introducing a divalent cation solution in situ to the subject applied chondrocyte composition thereby physically cross-linking the BMC via ionic bonds.

58. The method of claim 57, wherein said divalent cation solution is a CaCl2solution.

59. The method of treating a subject in need of joint repair of claim 55, wherein the joint of the subject is a knee joint, an elbow joint, a hip joint, an ankle joint, a wrist joint, a finger joint, or a toe joint.

60. The method of treating a subject in need of joint repair of claim 55, wherein the composition is implanted in a knee joint of the subject.

61. The method of treating a subject in need of joint repair of claim 55, wherein the joint has a defect, and wherein the method results in complete or almost complete defect closure 8-16 weeks post-surgery.

62. The method of treating a subject in need of joint repair of claim 61 , wherein the joint has a defect, and wherein the method results in complete or almost complete defect closure about 12 weeks post-surgery.

63. A method of repairing cartilage or treating a cartilage defect in a knee joint of a subject in need thereof, comprising: a. administering to the knee joint of the subject via syringe or spatula, autologous morselized cartilage tissue particles (MCP) having an average length of about 0. Ito about 1.0 mm and a chondrocyte viability of at least 85%, and a biodegradable matrix composition; b. physically cross-linking the MCP composition in situ with 100 nM CaCl2for about 10 seconds; and c. covering the morselized articular cartilage tissue particles or composition with protective layer.

64. The method of claim 63, wherein said protective layer is fibrin glue, chitosan, collagen, or a combination thereof.

65. The method of claim 64, wherein said fibrin glue autologous.

66. A kit for treating cartilage degeneration, the kit comprising at least one of: a. an applicator dispensing device for receiving morselized cartilage tissue particles; b. one or more syringes for mixing and / or dispensing cartilage; and c. a biodegradable matrix;67. The kit for treating cartilage degeneration of claim 66, wherein the syringes are selected from a mixing syringe, a two- (or multi) stage syringe, a dual- (or multi) chamber syringe, a dual (or multi) syringe, a two- (or multi) component syringe, or a spatula.

68. The kit for treating cartilage degeneration of claim 66, further comprising a disposable tissue harvesting device.

69. The kit for treating cartilage degeneration of claim 66, further comprising disposable morselization chamber.

70. The kit for treating cartilage degeneration of claim 66, further comprising reagents selected from a saline-containing solution or an isotonic compatible medium capable of buffering pH.

71. The kit for treating cartilage degeneration of claim 66, wherein the kit further comprises at least one element selected from an active pharmaceutical ingredient, a liquid medium, fibrin glue, growth factors, binders, saline, buffer solution, cell nurturing / preservation solution, chondroitin (and salts thereof), glucosamine (and salts thereof), methylsulfonylmethane (MSM), vitamins, nutrients, and combinations thereof.

72. The kit for treating cartilage degeneration of claim 71, wherein the at least one element is at least one growth factor selected from the group consisting of TGF-β1, BMP-2, BMP-7, IGF- I, FGF-2, FGF-18, PDGF, and combinations thereof.

73. The kit for treating cartilage degeneration of claim 71, wherein said active pharmaceutical ingredient is selected from the group consisting of NSAIDs, steroids and corticosteroids; analgesics; anthraquinones, and combinations thereof.

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