WO2011060553A1 - Formulation and method for rapid preparation of isotonic and cytocompatible chitosan solutions without inducing chitosan precipitation - Google Patents

Abstract

The present disclosure is directed to a two-part cytocompatible system for preparing a cytocompatible composition for use in repairing tissue of a subject. The system comprises an admixture of a first-part liquid solution of a polysaccharide, such as chitosan and a second-part liquid solution of a salt, such as glycerol phosphate. When the chitosan and glycerol phosphate solutions are combined, a cytocompatible composition is produced which is free of polymer precipitation at room temperature. The composition also promotes non-covalent complex formation between chitosan and therapeutic anionic proteins with a pl < 6 78.

Description

FORMULATION AND METHOD FOR RAPID PREPARATION OF ISOTONIC AND CYTOCOMPATIBLE CHITOSAN SOLUTIONS WITHOUT INDUCING

CHITOSAN PRECIPITATION

TECHNICAL FIELD

[0001] The present disclosure relates to a two-part cytocompatible composition for use in repairing tissue of a patient comprising an admixture of a first-part liquid polymer solution with a second-part liquid salt solution, the composition being free of polymer precipitation at room temperature and the composition being admixed just prior to administration or use in the patient.

BACKGROUND ART

[0002] Lesions in the articular cartilage layer can be resurfaced with repair tissue via surgical treatments that induce bleeding from subchondral bone (Insall et al., 1967, J Bone Joint Surg Br, 49(2): 211-228; Steadman et al., 1997, Operative techniques in orthopaedics, 7(4): 300-304). These surgical techniques are part of a family of methods called marrow stimulation therapy, where the surgeon debrides the damaged cartilage to remove glycosaminoglycan-containing tissue (non-calcified and calcified cartilage) then perforates holes into the highly vascularized subchondral bone with a drill or microfracture awl, or abrades the surface of the bone until punctuate bleeding is observed throughout the bed of the lesion. The ensuing repair response leads to the formation of a fibrous repair tissue that can rapidly degrade under normal daily load-bearing (Nehrer and Minas, 1999, Clin Orthop, 365: 149-162; Knutsen et al., 2004, Journal of Bone and Joint Surgery-American, 86A(3): 455- 464). In a 5-year follow-up, 10 out of 40 patients treated with microfracture were considered failures in need of total knee arthoplasty (Knutsen et al., 2007, Journal of Bone and Joint Surgery-American, 89A(10): 2105-21 12).

[0003] To enhance the osteochondral repair response following marrow stimulation, a wound-stimulatory implant has been developed, consisting of an autologous, in situ solidifying scaffold-stabilized blood clot. The scaffold- stabilized clot is generated by mixing a cytocompatible polymer solution such as glycerol phosphate (GP)-buffered chitosan with unclotted whole blood (International application publication No. WO 02/000272, the content of which is enclosed herewith by reference).

[0004] Previous methods that describe how to prepare of isotonic chitosan solutions buffered with disodium glycerol phosphate were time-consuming, and required drop-wise addition of a concentrated GP into chitosan (Chenite et al., 2000, Biomaterials, 21 : 2155-2161). Furthermore, these thermogelling chitosan- GP solutions tended to gel or precipitate with extended storage at room temperature. Lowering the pH by reducing the level of disodium GP resulted in formulations that showed a slower time to gel during room temperature storage but these pH 6.0 chitosan solutions were hypotonic (~70 mOsm) and not cytocompatible, as evidenced herein below:

[0005] The initial chitosan-GP formulation that improved cartilage repair in animal models consisted in 1.5% to 1.7% chitosan, 70 mM HCI and 135 mM disodium β-glycerol phosphate, pH 6.7 to 6.8 (390 to 520 mOsm) (Hoemann et al., 2007, Osteoarthritis and Cartilage, 15: 78-89; Hoemann et al., 2005, Journal of Bone and Joint Surgery-American Volume, 87A(12): 2671-2686; Chevrier et al, 2009, Osteoarthritis and Cartilage, 15: 316-327). To prepare a sterile solution, the chitosan powder was dissolved in 77 mM HCI to achieve 90% to 100% chitosan protonation (pH 4.5 to 5.0), autoclave sterilized, and then combined drop-wise at 4°C with 1/10^th volume of filter-sterile 1.35 M disodium β- glycerol phosphate pH 9.3.

[0006] Rapid combination of 1.0 ml of 1.35 M disodium glycerol phosphate (GP) pH 9.3, into 9 ml of an acidic solution of 1.7% to 1.9% w/v chitosan-HCI pH 4.5 (100% chitosan protonation) resulted in chitosan precipitation. [0007] In summary, isotonic and cytocompatible chitosan-GP solutions may be prepared by drop-wise addition of concentrated GP, pH ~9.3, into acidic chitosan solutions pH 4.5 and that such solutions are unstable at room temperature because they solidify spontaneously.

[0008] For practical reasons, it would thus be desirable and advantageous to be provided with a method and formulation that permits room temperature storage, and rapid reconstitution of cytocompatible solutions of chitosan for clinical use.

[0009] Chitosan-GP can also serve as a delivery vehicle for biologically active therapeutics (Chenite et al., 2000, Biomaterials, 21 : 2155-2161 ; International application publication No. WO 02/000272). Therefore, for additional practical reasons, it would be desirable and advantageous to be provided with a method and formulation that favors non-covalent coupling of specific biological factors with chitosan, to immobilize the factor at the site of implantation. However it is currently unknown how to couple proteins non- covalently with chitosan particles

SUMMARY

[0010] In accordance with the present disclosure there is now provided a two-part cytocompatible system for providing a cytocompatible polymer composition for use in repairing tissue(s) of a subject in need thereof. The system comprises at least two containers, the first one comprising a first-part liquid solution of a polymer and the second one comprising a second-part liquid solution of a salt. This system enables the generation, just prior to administration, of a cytocompatible polymer composition that is substancially homogeneous and/or substantially free of polymer precipitation at room temperature.

[0011] According to a fist aspect, the present application provides a two-part cytocompatible system for preparing a cytocompatible polymer composition for use in repairing a tissue of a subject. Broadly, the system comprises a first part liquid solution of a polymer in a first container and a second part liquid solution of a salt in a second container. Advantageously, the first part and said second part are to be combined, prior to said use, to provide said cytocompatible polymer composition and wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature. In an embodiment, the polymer is a modified or natural polysaccharide. In another embodiment, the polymer is dissolved in a mineral acid or an organic acid to prepare the first part liquid solution, such as, for example, the acid is hydrochloric acid, lactic acid, citric acid, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid and/or hydrobromic acid. In yet another embodiment, the polysaccharide is chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, heparin and/or heparin sulfate. Still according to a further embodiment, the salt is an organic salt or an inorganic salt, such salts including, but not limited to sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt (for example NaCI, KCI, CsCI, CaCI₂, CsF, KCI0₄, NaN0₃ and/or CaS0₄). In an embodiment, the organic salt is glycerol-phosphate. In another embodiment, the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower about 7.8. In still another embodiment, the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower than about 6.8. In an embodiment, the polymer is chitosan. In still another embodiment, the chitosan has a degree of deacetylation equal to or higher than about 20% and equal to or lower than about 100% deacetylated. In yet another embodiment, the chitosan has an average molecular weight equal to or higher than about 1 kDa and equal to or lower than about 10 MDa. In another embodiment, the chitosan has an average molecular weight equal to or higher than about 10 kDa and equal to or lower than about 150 kDa. In still another embodiment, the concentration of chitosan in the first part liquid solution is equal to or higher than about 1.5% and equal to or lower than about 2.2% w/v. In yet a further embodiment, the system further comprises a third part blood component. In an embodiment, the third part blood component is whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood and/or placenta blood. In still another embodiment, the third part blood component is plasma, erythrocytes, leukocytes, monocytes, platelets, fibrinogen, stem cells and/or thrombin. In still a further embodiment, the third part blood component comprises a platelet-rich plasma substantially free of erythrocytes. In yet another embodiment, the mixing ratio of (i) the third part blood component and (ii) the first part liquid solution of polymer solution combined with the second part liquid solution of the salt is equal to or higher than about 1 :3 and equal to or lower than about 1 :6. In still another embodiment, the cytocompatible polymer composition is capable of activating complement through platelet activation and/or capable of stimulating release of C5a peptide in whole blood or blood fractions containing platelets. In a further embodiment, the concentration of glycerol-phosphate in the second part liquid solution is equal to or higher than about 400 and equal to or lower than about 1000 mM. In another embodiment, the system further comprises a buffer to achieve near-neutral pH and isotonicity of the cytocompatible polymer solution. In still another embodiment, the first and second biological containers are independently selected from a vial or a syringe.

[0012] According to a second aspect, the present application provides a cytocompatible polymer composition for use in repairing a tissue of a subject, wherein said cytocompatible polymer. Broadly, the composition is prepared by admixing a first part liquid solution of a polymer with a second art liquid solution of a salt prior to said use. Advantageously, the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature. Various embodiments of the polymer (type and method of preparing the solution), the acid (type, concentration), the salt (type, concentration) the cytocompatible polymer composition characterisitics (pH, biological properties) , the chitosan (degree of deacetylation, average molecular weight, concentration in first part liquid solution), the third part blood component (type), mixing ratio, the buffer and the first and second biological containers have been described and do apply herein.

[0013] According to a third aspect, the present application provides the use a cytocompatible polymer composition as defined herein or prepared from a two- part composition as defined herein for repairing a tissue in a subject. In an embodiment, the tissue is cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors and/or ulcers. In another embodiment, the cytocompatible polymer composition is capable of stimulating subchondral angiogenesis, bone remodeling and/or osteochondral repair.

[0014] According to a fourth aspect, the present application provides the use of a cytocompatible polymer composition (i) prepared with a two-part system as defined herein or (ii) defined herein for delivering a therapeutic substance to a subject. In an embodiment, the therapeutic substance is a polysaccharide, a polypeptide, a drug, a liposome, a DNA, DNA-polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, a colony stimulating factor, a cytokine, a complement factor, and/or an angiogenic factor. In another embodiment, therapeutic substance is an anionic protein with a pi less than the maximum pKa=6.78 of chitosan.

[0015] According to a fifth aspect, the present application provides a method for repairing and/or regenerating a tissue of a subject in need thereof. Broadly, the method comprises administering a cytocompatible polymer composition (i) prepared with a two-part system as defined herein or (ii) as defined herein into said tissue in need of repair and/or regeneration. Advantageously, the cytocompatible composition is capable of adhering to the tissue to be repaired and/or regenerated so as to repair and/or regenerate the tissue. In an embodiment, the tissue is cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors and/or ulcers. In a further embodiment, the cytocompatible polymer composition is capable of stimulating subchondral angiogenesis, bone remodeling and/or osteochondral repair.

[0016] According to a sixth aspect, the present application provides a method for delivering a therapeutic substance in a subject in need thereof. Broadly, the method comprises administering a cytocompatible polymer composition admixed with the therapeutic substance into said subject, wherein said cytocompatible polymer composition (i) is prepared with a two-part system as defined herein or (ii) is defined herein. In an embodiment, the therapeutic substance is a polysaccharide, a polypeptide, a drug, a liposome, a DNA, a DNA-polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, a colony stimulating factor, a complement factor, a cytokine and/or an angiogenic factor. In an embodiment.the therapeutic substance is an anionic protein with a pi less than the maximum pKa=6.78 of chitosan.

[0017] According to a seventh aspect, the present application provides a kit for preparing a cytocomptabile polymer composition for use in repairing and/or regenerating a tissue of a subject. Broadly, the kit comprises i) a first container comprising a liquid polymer solution and ii) a second container comprising a liquid salt solution. Advantageously, when the polymer solution and liquid salt solution are to be combined prior to said use, the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature. In an embodiment, the polymer is dissolved in an acid solution to provide the polymer solution, such as, for example, hydrochloric acid, acetic acid, lactic acid, citric acid and/or glycolic acid solution. In an embodiment, the kit can further comprise a concentrated buffer solution so as to adjust the cytocompatbile polymer solution to a cytocompatible pH and isotonicity. In an embodiment, the polymer is a modified or natural polysaccharide including, but not limited to chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, heparin, and/or heparin sulfate. In a further embodiment, the salt in said liquid salt solution is an organic or inorganic salt, such as, for example, sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt and/or carboxylate salt (e.g. NaCI, KCI, CsCI, CaCI₂, CsF, KCI0₄, NaN0₃ and/or CaS0₄). In another embodiment, the salt is an organic salt, for example, glycerol-phosphate. In still a further embodiment, the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower than about 7.8. In another embodiment, the cytocompatible polymer composition has a pH equal to or higher than about 5.8 and equal to or lower than 6.8. In a further embodiment, the polymer is chitosan. In still another embodiment, chitosan has a degree of deacetylation equal to or higher than about 20% and equal to or lower than about 100%. In yet another embodiment, the chitosan has an average molecular weight equal to or higher than about 1 kDa and equal to or lower than about 10 MDa. In still another embodiment, the chitosan has an average molecular weight equal or higher than about 10 kDa and equal to or lower than about 150 kDa. In yet another embodiment, the concentration of in the polymer solution equal or higher than about 1.3% and equal or lower than about 2.5% w/v. In yet another embodiment, the concentration of glycerol-phosphate in the salt solution is equal or higher than about 600 and equal or lower than 1000 mM. In still another embodiment, the cytocompatible polymer composition has a pH equal to or higher than about 6.9 and equal to or lower about 7.4. In still another embodiment, the kit further comprises instructions for use of said kit to repair and/or regenerate the tissue of the subject. In yet another embodiment, the first and second biological containers are independently selected from a vial and a syringe.

[0018] According to an eight aspect, the present application provides a method for preparing a cytocompatible polymer solution for use in repairing a tissue of a subject. Broadly, the method comprises combining a first part liquid solution of a polymer in a first container and a second part liquid solution of a salt in a second container. When said first part and said second part are combined, prior to said use, they provide a cytocompatible polymer composition wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature. Preferably, the cytocompatible polymer composition is administered rapidly to the subject in need thereof. The method can also comprise the combination of other solution such as a blood component or a buffer. Various embodiments of the polymer (type and method of preparing the solution), the acid (type, concentration), the salt (type, concentration) the cytocompatible polymer composition characterisitics (pH, biological properties), the chitosan (degree of deacetylation, average molecular weight, concentration in first part liquid solution), the third part blood component (type), mixing ratio, the buffer and the first and second biological containers have been described and do apply herein.

[0019] According to a ninth aspect, the present application provides a method for repairing a tissue of a subject. Broadly, the method comprises combining a first part liquid solution of a polymer in a first container and a second part liquid solution of a salt in a second container and administering the resulting composition to the subject. When said first part and said second part are combined, prior to said use, they provide a cytocompatible polymer composition wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature. Preferably, the cytocompatible polymer composition is administered rapidly to the subject in need thereof. The method can also comprise the combination of other solution such as a blood component or a buffer. Various embodiments of the polymer (type and method of preparing the solution), the acid (type, concentration), the salt (type, concentration) the cytocompatible polymer composition characterisitics (pH, biological properties), the chitosan (degree of deacetylation, average molecular weight, concentration in first part liquid solution), the third part blood component (type), mixing ratio, the buffer and the first and second biological containers have been described and do apply herein.

[0020] According to a tenth aspect, the present application provides a method for administering a therapeutic substance to a subject. Broadly, the method comprises combining a first part liquid solution of a polymer in a first container, a second part liquid solution of a salt in a second container and the therapeutic substance and administering the resulting composition to the subject. When said first part and said second part are combined, prior to said use, they provide a cytocompatible polymer composition wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature. Preferably, the cytocompatible polymer composition is administered rapidly to the subject in need thereof. The method can also comprise the combination of other solution such as a blood component or a buffer. Various embodiments of the polymer (type and method of preparing the solution), the acid (type, concentration), the salt (type, concentration) the cytocompatible polymer composition characterisitics (pH, biological properties) , the chitosan (degree of deacetylation, average molecular weight, concentration in first part liquid solution), the third part blood component (type), mixing ratio, the buffer and the first and second biological containers have been described and do apply herein.

[0021] It is also provided a two-part cytocompatible composition for use in repairing tissue of a patient prepared by admixing just before use a first-part liquid polymer solution with a second-part liquid salt solution, said composition being free of polymer precipitation at room temperature.

[0022] In an embodiment, the polymer is a modified or natural polysaccharide, such as polysaccharide selected from the group consisting of chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, decorin, heparin, and heparin sulfate.

[0023] In another embodiment, the acid comprises a mineral acid or an organic acid, such as, but not restricted to, hydrochloric acid, lactic acid, citric acid, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid or hydrobromic acid.

[0024] In another embodiment, the salt can be an inorganic or organic salt, such as sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt; at least one of NaCI, KCI, CsCI, CaCI₂, CsF, KCIO4, NaN03 or CaS0₄; or disodium sodium glycerol-phosphate.

[0025] In an embodiment, the final composition has a pH between 5.8 and 7.8, more preferably between 5.8 and 6.8.

[0026] In a further embodiment, the chitosan solution can have a pH 4.5 to 5.6 and the salt solution either with no fixed pH (NaCI) or pH range 6.4 to 7.4 (GP-HCI).

[0027] The chitosan can be 20% to 100% deacetylated with an average molecular weight ranging from 1 kDa to 10 MDa, or preferably having an average molecular weight ranging from 10 kDa to 150 kDa, and in a concentration between 1.5% to 2.2% w/v. In a preferred embodiment, chitosan- HCI preparations with 70% to 90% protonation could generate a room temperature-stable chitosan solution. In another embodiment, the HCI concentration does not exceed 79 mM of the re-constituted 1.6% w/v chitosan- HCI and glycerol phosphate solution.

[0028] In another embodiment, a 2-part chitosan/blood system is feasible when 1 ml chitosan-HCI is packaged in a mixing vial, and 0.11 ml of β-GP in 50 mM or 100 mM HCI is injected directly into the vial and shaken immediately before use. Another embodiment is when 1.2 ml chitosan-HCI can be packaged in a mixing vial, and 0.3 ml of 0.5M β-GP in 50 mM HCI is added directly into the vial immediately before use. The chitosan will be in non-aggregated form when prepared the day of use, and will have the same formulation as the chitosan-GP solution that generated excellent repair results. Another embodiment is when 1 ml chitosan-HCI is packaged in a mixing vial, and 0.11 ml of 750 mM NaCI is used to generate an isotonic and slightly acidic (pH 5.8 to pH 6.1) chitosan solution.

[0029] In a further embodiment, the composition further comprises a blood component which can be selected from the group consisting of whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood and placenta blood; and further comprises plasma or platelet rich plasma free of erythrocytes.

[0030] The blood component can also be selected from the group consisting of plasma erythrocytes, lymphocytes, polymorphonuclear cells, monocytes, platelets, fibrinogen, stem cells and thrombin; more preferably is plasma, erythrocytes, polymorphonuclear cells, monocytes, or platelets.

[0031] In another embodiment, the glycerol-phosphate or salt component is generated as a stock solution and mixed with the liquid polymer solution such that the final salt concentration is cytocompatible between 250 to 520 milliOsmolar. [0032] In another embodiment, the mixing ratio of blood component and admixture of the liquid polymer-salt solution is in between and including 1 :3 to 1 :12 v/v admixture:blood.

[0033] Further, composition permits propagation of the clotting cascade, and can activate complement through platelet activation, stimulating release of C5a peptide.

[0034] In another embodiment, the composition also comprises admixing a buffer to achieve neutral pH and isotonicity, the chitosan being dissolved in an acid solution, such as hydrochloric acid, acetic acid, lactic acid or glycolic acid.

[0035] In accordance with the present description, there is provided of the use of the two-part composition described herein for repairing tissue in a patient.

[0036] The tissue can be selected from the group consisting of cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, infarcted cardiac tissue, ischemic tissue, resected tumors and ulcers.

[0037] The composition can also stimulate subchondral angiogenesis, bone remodeling, osteoclast formation, or osteochondral repair.

[0038] In accordance with the present description, there is provided of the use of the two-part composition described herein for delivering a therapeutic substance in a patient. The therapeutic substance can be selected from the group consisting of a polysaccharide, a polypeptide, a drug, a liposome, a DNA, DNA-polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, osteoclast formation, a colony stimulating factor, an anionic factor, a cytokine, and an angiogenic factor, preferably an anionic protein with a pi less than the maximum p a=6.78 of chitosan.

[0039] In accordance with the present description, there is provided a method for repair and/or regeneration of a tissue of a patient comprising administering a composition as defined herein into the tissue in need of repair and/or regeneration, wherein composition when placed at the site in need of repair will adhere to the site in need of repair to effect repair and/or regeneration of the tissue.

[0040] Also provided is a method for delivering a therapeutic substance in a patient comprising administering a two-part composition as defined herein.

[0041] In accordance with the present description, there is provided a method of preparing a cytocompatible composition for use in repairing tissue of a patient comprising dissolving a polymer in order to obtain a polymer solution free of precipitate, mixing a liquid salt solution to the polymer solution obtaining the cytocompatible composition free of polymer precipitate at room temperature. In another embodiment, the composition can be also be admixed at 4°C, preferably in a temperature range between 4°C and 37°C.

[0042] The mixing of the liquid salt solution to the polymer solution obtaining the cytocompatible composition free of polymer precipitate at room temperature can be done 0.5 minutes to 1 day prior to mixing the composition with a therapeutic substance or blood component.

[0043] In accordance with the present description, there is provided a kit for repairing and/or regenerating a tissue of a patient, the kit comprising i) a soluble acidic polymer solution and ii) a liquid salt solution.

[0044] The kit can also comprise instruction(s) for use.

[0045] In an embodiment, the liquid polymer solution and the liquid salt solution are packaged prior two admixture in two separate biological containers. It is encompassed herein any biological container allowing storage of the polymer solution and the liquid salt solution prior to admixing them together, such as for example but not limited to, a vial or a syringe.

[0046] In another embodiment, anionic biologically active factors can be added to the polysaccharide solution and remain tethered to the polysaccharide, and immobilized within the implant following mixture with blood or blood factors. [0047] In one aspect, the present description provides for the preparation of a chitosan composition via the mixing of two compositions, where the first composition, optionally referred to as the mixing composition, is prepared from or contains water, chitosan and hydrochloric acid, while the second composition, optionally referred to as the additive composition, is prepared from or contains water, glycerolphosphate salt, and hydrochloric acid. It is a feature of this aspect of the disclosure that each of these two precursor compositions contains hydrochloric acid.

[0048] For example, the additive composition may comprise, or consist entirely of, water, hydrochloric acid at a concentration of 25 to 75 mM, preferably 35 to 65 mM, and more preferably 40 to 60 mM, and glycerolphosphate salt at a concentration of 250 to 750 mM, preferably 350 to 650 mM, and still more preferably 400 to 600mM. The mixing composition may comprise, or consist entirely of, water, chitosan at a %wt/wt concentration in the range of 1.8 to 2.3, preferably 1.9 to 2.2, and more preferably about 2.05, while the hydrochloric acid concentration in term of millimolar, is in the range of 60 to 80 mM, preferably 65 to 75 mM, and more preferably 69-70 mM.

[0049] As another example, the additive composition may comprise, or consist essentially entirely of, water, hydrochloric acid and glycerolphosphate salt such that the pH of the additive composition is within the range of 6.7 to 7.3, preferably 6.9 to 7.1 , and most preferably around 7.0, while the mixing composition comprises, or consists essentially entirely of, chitosan and hydrochloric acid with a pH in the range of 5.5 to 6.2, preferably 5.7 to 6.0 and most preferably around pH 5.9.

[0050] Thus, in one related aspect of the disclosure, the mixing composition comprises chitosan at a %wt/wt concentration in the range of 1.8 to 2.3, preferably 1.9 to 2.2, and more preferably about 2.05, while the pH of the mixing composition is in the range of about 5.5 to 6.2, preferably 5.7 to 6.0 and most preferably 5.9. In yet another related aspect, the additive composition comprises glycerol phosphate salt at a %wt/wt concentration in the range of 8 to 12, preferably 9 to 11 , and more preferably around 10.2, while the pH of the mixing composition is in the range of 6.7 to 7.3, preferably 6.9 to 7.1 , and more preferably around 7.0, where the pH of the additive composition is adjusted by use of hydrochloric acid.

[0051] In a preferred embodiment, the mixing composition has a dynamic viscosity as measured in mPa-s, in the range of 500 to 1500, preferably 1350 to 1450.

[0052] Upon combination of the two compositions, the chitosan concentration is preferably 1.4 to 1.8 wt%, more preferably 1.5 to 1.7 wt%, and still more preferably around 1.6wt%; the glycerol phosphate salt concentration is preferably 1.8 to 2.2 wt%, more preferably 1.9 to 2.1 , and more preferably about 2.0wt%; the pH of the combination is preferably 6.4 to 7.0, more preferably 6.6 to 6.8 and still more preferably about 6.7. The osmolarity of the resulting combination, as determined mOsm/kg, is preferably within the range of 200 to 400, more preferably 220 to 380, and still more preferably 250 to 350, and yet more preferably 280 to 320.

[0053] In another preferred embodiment, each of the mixing and additive compositions is sterile.

[0054] In this aspect of the disclosure, in one embodiment the glycerol phosphate salt is selected from glycerol-2-phosphate salts, syn-glycerol-3- phosphate salts, and L-glycerol-3-phosphate salts, where a preferred glycerol phosphate salt is a salt of beta-glycerolphosphate (BGP), where suitable counterions are the disodium or dipotassium salts of the glycerol phosphate.

[0055] The chitosan used in this aspect of the disclosure preferably has a degree of deacetylation in the range of 70 to 85%, more preferably 78 to 84%, and still more preferably around 81 %.

[0056] After formation from the mixing composition and the additive composition, the resulting chitosan composition is preferably mixed with blood or component(s) thereof, in a volumetric ratio of about 3 parts blood to 1 part chitosan solution. [0057] Based on the foregoing, one statement of this aspect of the disclosure is a kit comprising mixing and additive containers, the mixing container comprising chitosan at a concentration of 1.9 to 2.2 wt% and a pH of 5.7 to 6.0 adjusted with hydrochloric acid, while the additive container comprises beta- glycerol phosphate at a concentration of 8 to 12 wt% and a pH of 6.9 to 7.1 adjusted with hydrochloric acid. The container can be for example a vial and/or a syringe.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Reference will now be made to the accompanying drawings.

[0059] Fig. 1 corresponds to a graphic representation of the pH of a 1 % w/v chitosan solution in relation with the protonation level.

[0060] Fig. 2 corresponds to a graphic representation of the correlation between HCI concentration needed to obtain a particular protonation level for a given chitosan solution (82.6% DDA) at a given w/v percent concentration.

[0061] Fig. 3 corresponds to the effect of increasing HCI in 1 volume of 500 mM β-GP admixed into 4 volumes of 2% w/v chitosan-HCI pH 5.6, on the β-GP solution (diamond symbol) and on the final pH of the isotonic chitosan-HCI- β- GP solution (square symbol), and the formation of visible precipitate in the vial after adding the β-GP solution.

[0062] Fig. 4 illustrates the effect of substituting NaCI for β-GP on an isotonic chitosan solution pH.

[0063] Fig. 5 illustrates the coagulation of mixtures of whole human blood and rapidly reconstituted chitosan-HCI pH 5.5 and β-GP pH 7.2, and C5a generation during coagulation of whole blood and chitosan-GP/blood mixtures as a function of time. C5a appeared in parallel with platelet degranulation and thrombin activation in coagulating chitosan-GP/blood (A, C, E) and whole blood (B, D, F). Panels A and B show the time-dependent increase in clot tensile strength (Amplitude (A), mm, black trace) and thrombin generation (Thrombin/Anti-Thrombin (TAT) levels, dashed bars) for a representative donor (out of 4 donors). Panels C and D show time-dependent increase of serum C5a fragment detected by goat anti-C5a polyclonal antiserum, and a positive control zymosan-activated serum (ZAS) incubated 60 min. Panels E and F show time- dependent increase of serum platelet factor 4 (PF4) as a marker of platelet activation, and a positive control chitosan-GP/blood incubated 75 min with thrombin (10 U/ml) (lla).

[0064] Fig. 6 illustrates an assay for C5a generation in serum and citrated plasma. Panel A shows serum exposed to either 4 mg/ml zymosan particles dispersed in NaCI (ZAS-NaCI), isotonic glycerol phosphate buffer (GP), or 4 mg/ml 80% DDA chitosan in isotonic GP buffer (Chi80-GP). Other lanes in Panel A shows serum samples collected from whole blood clotted in glass tubes at room temperature (RT) or 37°C, then allowed to retract at RT or 4°C for 4 h (pooled from N=3 donors), as well as citrated plasma incubated for 60 min at 37°C (Plasma), citrated plasma never heated or boiled (Plasma - not boil), purified human C5 (C5) and recombinant C5a (rC5a). Panels B and C: C5a fragments in citrated plasma exposed to various test activators as indicated. Symbols: the black circle indicates 16 kDa C5a constitutively present in plasma and serum. The arrow indicates C5a produced by complement activation.

[0065] Fig. 7 illustrates an assay using citrated plasma for C3 binding to insoluble zymosan or chitosan particles. Zymosan-NaCI (ZAP) and 80% DDA chitosan-GP (Chi80-GP) were exposed to citrated plasma for 60 min at 37°C, then the insoluble pellets were rinsed in EDTA buffer, extracted with 8 M urea, and urea extracts loaded on a nonreducing gel (Panel A), or a reducing gel (Panel B). Migration is shown relative to 0.2-5 μΙ human plasma (P) that was incubated for 1 h at 37°C. Arrows indicate C3 and C3b, open arrowheads indicate iC3b, and brackets indicate C3 cross-linked products. Panel C illustrates the nonreduced molecular masses of C3 and various degradation products (in gray) as well as the 42 kDa reduced molecular mass of the iC3b subfragment recognized by the anti-C3b antibody used. The boxes indicate the protein detected, and antisera used for immunodetection. [0066] Fig. 8 illustrates an assay using citrated plasma to measure C5a/C3a generation and C5/C3 binding to insoluble zymosan or chitosan particles. Citrated plasma (lanes 1-6) or recalcified plasma (lane 7) was incubated for 1 h at 37°C with various test conditions as indicated, and cleared Supernatants, or the 8M urea Pellet extracts from the same samples were analyzed by Western blot for C5a and C5 proteins under nonreducing conditions (Panels A, B, respectively) and for C3a and C3/iC3b proteins under reducing conditions (Panels C-E, respectively). Lanes 8-10 were loaded with appropriate controls: rC5a (recombinant C5a), C5 (purified C5), C3 (purified C3), P (plasma incubated for 1 h at 37°C); P-not boil (plasma never heated or boiled in sample buffer). Panel E shows a Western blot of panel D that was stripped and re- probed with anti-C3a antibody. The boxes indicate the protein detected, and antisera used for immunodetection.

[0067] Fig. 9 illustrates native C5 and C3 bound to chitosan under complement-inhibiting conditions. Zymosan or 95% DDA chitosan-NaCI (Chi95) were incubated at 4 mg/mL with pooled serum (-), serum with 10 mM EDTA (E), or methylamine-treated plasma (M) for 1 h at 37°C. Resulting Supernatants were analyzed by Western blot for C5a (Panel A, nonreducing conditions) and 8M urea Pellet extracts from same samples were analyzed for C5 with an anti- C5a antibody (Panel B, nonreducing conditions), and C3 with an anti-C3b antibody (Panel C, reducing conditions). Panel D shows Western blot of panel C that was stripped and reprobed with anti-C3a antibody. The boxes indicate the protein detected, and antisera used for immunodetection.

[0068] Fig. 10 illustrates the binding of pure C3 to chitosan verified by surface plasmon resonance (SPR) biosensing. The top trace shows protein accumulation on biotinylated chitosan coupled to streptavidin (SA) sensorchip and the bottom negative control trace shows bare SA sensorchip exposed simultaneously to the same conditions. Serial injections were as follows: buffer (1-4), pure C3 protein at 1 , 10, 20, and 100 pg/ml concentrations (5-8, respectively), anti-C3a antibody at 0.3 pg/ml (9), and anti-C3b antibody at 0.05 and 0.13 pg/ml (10, 11). [0069] Fig. 11 illustrates an assay for association of factor B, factor B cleavage products, and antithrombin (AT) in citrated plasma to chitosan and zymosan particles. Zymosan and chitosan particles were incubated in plasma for 1 h incubation at 37°C, the pellets extracted with 8M urea, and pellet extracts analyzed by Western blot under nonreducing conditions using anti-factor B polyclonal antiserum (Panel A) and monoclonal anti-human AT (Panel B). Panel C shows a nonreducing Western blot analysis of factor B, using urea pellet extracts (lanes 1-4) and supernatants (lanes 5-7) from the same set of samples as those analyzed in Panel A. Appropriate quantities of purified protein standard (factor B, AT) and plasma were loaded onto each gel as a control.

[0070] Fig. 12 corresponds to a schematic representation of the vial/syringe used to mix the polymer and salt solution.

[0071] Fig. 13 corresponds in (A) to a photographic representation showing that the implant described herein was delivered to an acute rabbit femoral trochlear cartilage defect with 4,1 mm diameter microdrill holes pre-treated with 3 μΙ_ purified human thrombin. The contralateral drilled cartilage defect was treated with 3 μΙ_ of purified human thrombin. After 8 weeks of repair, the implant-treated defects had significantly greater subchondral bone repair (compare A1 and B1) and significantly greater basal integration (compare C1 and D1 where open arrows indicate detached repair tissue) compared to the control defects. In (B), a graphic representation of results obtained with the implant delivered to an acute rabbit femoral trochlear cartilage defect with 4,1 mm diameter microdrill holes pre-treated with 3 μΙ_ purified human thrombin. The contralateral drilled cartilage defect was treated with 3 μΐ_ of purified human thrombin. After 8 weeks of repair, the implant-treated defects had significantly greater subchondral bone repair (right panel) and significantly greater basal integration (left panel) compared to the control defects.

[0072] Fig. 14 corresponds to a graphic representation (A) and to photographic representations (B and C) of the liquid mixture solidified in situ, in a 3.5 x 4.5 mm cartilage defect with 0.5 mm and 1 mm diameter microdrill holes, using pre-application of thrombin to accelerate solidification (N=8 bilateral defects; see Fig. 14A). The repair period was 6.5 months. Treated repair tissue (see Fig. 14C) contained more glycosaminoglycan than controls throughout the defect (see Fig. 14B).

[0073] Fig. 15 corresponds to a graphic representation of the liquid mixture of chitosan-GP reconstituted in the clinic during the operative procedure. Blood was added and mixed with the solution, then the implant was solidified in situ, to treat microfracture lesions of human patients. In other patients, lesions were treated with microfracture-only. Repair tissue biopsies were obtained 13 months post-treatment in 21 patients from the estimated geometric center of the initial lesion (13 treated, 9 Microfracture-only). The cartilage repair tissue in biopsies from implant-treated defects was significantly thicker (A) and had a significantly higher over-all histology score (B) for quality of repair as judged by 3 independent blinded readers, for 14 different repair tissue features.

[0074] Fig. 16 corresponds to photographic representations of average control biopsies (A) and average treated biopsies (B) showing that the cartilage repair tissue in biopsies from implant-treated defects had a significantly higher quality of repair.

DETAILED DESCRIPTION

[0075] It is provided a system and related composition for rapid combination of glycerol phosphate or salt solutions with acid chitosan solutions, to produce a cytocompatible polymer composition substantially free of polymer precipitates. The resulting solution can be further combined with whole blood or blood derivatives to generate mixtures capable of in s/iiv-so I id if i cati o n over wounds or surgical defects, or extracorporeal devices. Alternatively or in combination, the rapidly generated solution can also be combined with biological factors, non- covalently immobilize anionic proteins with a pi less than the maximal pKa =6.78 of chitosan, and maintain their activity prior to mixing with blood. The resulting solid implants stimulate the repair and regeneration of articular cartilage, joint tissues and other tissues including but not limited to meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, abscesses, resected tumors, ulcers, aorta, and cardiac tissue. [0076] The advantage of using a system with two distinct containers for the polymer solution and the salt solution is that the resulting polymer composition can be generated shortly prior to its administration to the subject. Because the polymer composition is produced shortly before it is administered, the polymer in the composition is substantially dissolved and/or in an unprecipitated form. As used herein, a "polymer is substantially dissolved", when the majority of the polymer in the solution are dissolved in the solution. As used herein, a "polymer is substantially in an unprecipitated form" when the majority of the polymer in the solution does not form a precipitate with other components of the solution (for example, other polymers). The final product is thus considered to be more homogeneous and easier to handle than polymer compositions that have been generated and stored prior to use.

[0077] The system proposed for generating a room temperature-stable 2- part formulation consist in (1) a polymer composition (such as an acid chitosan solution) and (2) a compatible salt solution or buffer of appropriate pH that can be rapidly reconstituted to form a 1-part polymer solution (such as an isotonic chitosan solution) that is free of polymer precipitates. Upon rapid mixing, the resulting cytocompatible polymer solution can be further combined with or without bioactive factors, and with whole blood or blood fractions, to allow propagation of the enzymatic clotting cascade, coagulation, and the formation of a cytocompatible implant containing chitosan polymer, polymerized fibrin, and blood elements.

[0078] For example liquid chitosan in dilute HCI (around 70 mM), pH 4.5 - 5.6 is prepared (see Fig. 12). Viscosity of this chitosan-HCI solution may slowly decline over time due to spontaneous hydrolysis of the chitosan chains. The solution could be frozen, or heated, with no fear of gellation or precipitation. A syringe containing 5x to 10x solution β-GP/NaCI, for asceptic injection into the chitosan vial, is prepared. Passive diffusion can be allowed, or the solution may be vortex mixed or shaken (i.e. for 10 seconds) to reconstitute a chitosan-salt solution. The correct ratio of chitosan and β-GP or NaCI is determined by the final solution transparency (lack of chitosan precipitation), osmolality (needs to be close to isotonic) and pH (isotonic NaCI brings chitosan to pH 5.8-6.1 and GP can bring the pH to pH 6.4 to 6.8). To add fresh blood, a vented injection device could be use in order to relieve any pressure introduced by the injected volume of blood or β-GP/NaCI.

[0079] Chitosan-GP product performance and clinical ease-of-use is now improved by the method and formulation described herein that permit room temperature storage and rapid reconstitution to generate a formulation similar to those formulations showing efficacy in cartilage repair studies. The rapidly reconstituted formulation should generate a chitosan solution that is isotonic, near-neutral pH and free of precipitate. The following disclosure demonstrates the disclose method and formulation generate room temperature-stable formulations that can be rapidly combined to form a cytocompatible and sterile solution of chitosan-glycerol phosphate or chitosan-sodium chloride. In addition, the in vivo efficacy of the composition described herein is demonstrated in rabbits and in humans (see Fig. 13-16).

[0080] Further, the formulation and method described permits immobilization of anionic bioactive factors such as complement C3 and C5 in the chitosan- salt blood implant, and the release of cationic chemotactic factors C3a and C5a via specific non-covalent tethering of anionic proteins to the chitosan scaffold before and after liquid-solid phase transition (see Table 1).

Table 1

Theoretical pi of human complement, clotting, and platelet factors

Prothrombin, P00734; C2, P06681 ; C4, P0C0L4; D, P00746; I, P05156; FVII, P08709; PF4, P02776.

b theoretical Mw without post-translational modifications, leader sequence removed, ExPASy.

c Total number of positively charged (Lys + Arg) minus total number of negatively charged (Asp + Glu)

d pK₀=6.78, the upper limit of chitosan pK_a, 72%-98% DDA at 150 mM NaCI, cor^ 30

[0081] Chitosan is a polycationic and biocompatible polysaccharide that is thrombogenic, chemotactic for neutrophils, and stimulates angiogenesis and wound repair (Hoemann et al., 2005, Journal of Bone and Joint Surgery- American Volume, 87A(12): 2671-2686) through mechanisms that remain unclear. Chitosan is produced by chemical deacetylation of chitin, resulting in a polymer composed of β(1~4) O-linked -glucosamine (Glc) and N-acetyl-p-D- glucosamine (GlcNA) with variable degree of deacetylation (DDA), for example, 90% DDA chitosan has 90% Glc and 10% GlcNA monomer. Chitin and chitosan were previously shown to deplete or adsorb complement C3 and C5 proteins from serum and plasma, suggesting that chitosan may activate complement.

[0082] There are three known complement activation pathways: classical, lectin, and the alternative pathway which is triggered by pathogens and activating biomaterial surfaces. Complement activation is marked by the formation of a C3 convertase complex on the surface of target cells or activating surfaces. C3 convertase generates a local amplification of the complement cascade and C5 cleavage, culminating in the release a 70 amino acid cationic C5a anaphylatoxin peptide fragment. The release of C5a provides a potent stimulus for neutrophil chemotaxis, while C3b fragments adsorbed onto biomaterial surfaces serve as opsonins or coatings that mediate recognition and clearance by phagocytes (reviewed in Law and Dodds, 1997, Protein Sci, 6: 263-274). Activation of complement could potentially explain how neutrophils become attracted to chitosan particles, yet evidence of C5a generation by chitosan was lacking. Moreover, although partly acetylated chitosans may still bear resemblance to chitin, a yeast cell wall molecular pattern, highly deacetylated chitosans, (i.e., 90% Glc or higher) have little chemical similarity with chitin, and would not necessarily be predicted to interact with the complement system in the same manner.

[0083] Complement activation is propagated through a cascade of plasma serine proteases, and represents the liquid phase of the innate immune reaction to trauma and foreign molecular patterns. Human complement C3 and C5 are ~190 kDa proteins that circulate as heterodimers of 115 kDa alpha and 75 kDa beta chains linked by a disulfide bond. Cleavage of the small peptide C3a from C3 generates a metastable C3b fragment with a reactive internal thioester bond that is able to form covalent linkages with activating surfaces. Solid-phase C3b associates with the proenzyme factor B to form a C3bB complex that is cleaved once in the factor B subunit by factor D; the Ba cleavage product dissociates leaving an active C3bBb solid-phase convertase for C3 and C5 that locally amplifies the complement cascade. [0084] C3b is rapidly cleaved in the alpha chain to form iC3b, by plasma protease I and its cofactor H, and cell-derived serine proteases can further degrade the iC3b alpha chain to produce C3c and C3dg. As C3b and C3b break-down products remain associated with the activating surface as opsonins, complement activation can therefore be assayed by the appearance of fluid- phase C5a, and solid-phase associated C3b and break-down products.

[0085] It was previously showed that homogenous mixtures of liquid chitosan-glycerol phosphate (GP) and whole blood gel and clot to form an in s/^'fiv-solidifying implant with an interpenetrating polysaccharide-fibrin scaffold network (Marchand et al., 2009, Osteoarthritis Cartilage, 17: 950-957; lliescu et al., 2007, Microscopy Res Tech Hybrid, 70: 236-247). Chitosan clot implants attract neutrophils and stimulate angiogenesis during wound repair.

[0086] It was previously shown that chitosan depletes complement proteins from plasma, suggesting that chitosan activates complement. Complement activation leads to cleavage of C5 to produce C5a, a neutrophil chemotactic factor. Towards elucidating the mechanisms of chitosan-induced wound repair, it was determined, as described herein, whether chitosan induces complement activation in chitosan-GP/whole blood clots. Since platelets and thrombin have been shown to activate complement in whole blood, the timing of C5a generation, platelet degranulation, and thrombin generation was determined in coagulating mixtures of chitosan-GP/blood.

[0087] Using western blot analysis of complement proteins in nonreducing and reducing gels, as also described herein, the binding of complement proteins to chitosan was evaluated to test the hypothesis that isotonic solutions of chitosan activate complement in human plasma and serum resulting in the release of C5a chemotactic peptide.

[0088] Here, the hypothesis that chitosan generates C5a in human whole blood, citrated plasma, and serum was tested using the formulation described herein. C5a fragment appeared in coagulating whole blood, and mixtures of chitosan-glycerol phosphate/whole blood, in parallel with platelet and thrombin activation. However, in plasma and serum, thrombin and chitosan-GP failed to generate C5a, although native C3, C5, and factor B adsorbed noncovalently to insoluble chitosan particles incubated in citrated plasma, serum, EDTA-serum and methylamine-treated plasma. By surface plasmon resonance, pure C3 adsorbed to chitosan. The profile of serum factors associating with chitosan was consistent with a model in which anionic blood proteins with a pi lower than the pKa 6.78 of chitosan associate electrostatically with cationic chitosan particles.

[0089] Zymosan, a yeast ghost particle, activated complement in serum and citrated plasma, but not in EDTA-serum or methylamine plasma, to generate fluid-phase C5a, while C3b formed covalent cross-links with zymosan- associated proteins and became rapidly cleaved to iC3b, with factor Bb stably associated. These data demonstrate that chitosan contained in the formulation described herein is a nonreactive biomaterial that does not directly activate complement, and provide a novel basis for predicting anionic serum protein- chitosan interactions.

[0090] Chitosan forms a noncovalent and tight association with AT in a charge- and DDA-dependent manner (see Fig.8). Furthermore, pure C3 bound to chitosan biosensor surfaces in a manner that selectively exposed the cationic C3a epitope. Altogether, these collective data allow generating a new and predictive model of chitosan-blood protein interaction, whereby anionic proteins with a pi lower than the pK_a of chitosan can deposit onto positively charged chitosan nonspecifically, and without requiring a reactive internal thioester bond (Fig. 9B and C; lane Chi95-M).

[0091] In Western blot analyses disclosed herein, C3b became covalently tethered to zymosan-associated proteins and rapidly converted to iC3b in citrated plasma and in serum. The data disclosed herein further demonstrate that citrated plasma leaves sufficient free magnesium ions in solution (i.e., on the order of 1.6 X 10^"6M) to support alternative complement activation by zymosan. Interestingly, low levels of iC3b were found to associate with zymosan in EDTA-serum but not in methylamine plasma (Fig. 9C). These data suggest that EDTA only partly interferes with the chemical ester linkage of C3b to zymosan-associated proteins, and splitting of C3b to iC3b. The data disclosed support a model whereby factor Bb fragment remains stably associated on the zymosan particle surface, in a complex with iC3b which is tethered to zymosan- associated factor(s).

[0092] The study disclosed herein has also shed some more light on the relationship between coagulation and complement activation. Glass is a well- known activator of thrombin through the contact pathway in whole blood and plasma. Here, it is shown using purified thrombin, and glass beads in recalcified plasma, that thrombin activity sufficient to cleave fibrinogen and generate a chitosan-NaCI/plasma clot or chitosan-glycerol phosphate/plasma clot was nonetheless insufficient to generate detectable levels of C5a in plasma. If thrombin is a C5 convertase, the data indicates that it is a rather weak convertase in human plasma compared to zymosan, and suggest that platelet activation (Fig. 5) which is triggered by thrombin, is a more significant driver of C5a generation in normal whole blood.

[0093] The generation of C5a in chitosan-GP/blood mixtures can therefore be best explained by complement activation through platelet activation, which is promoted by chitosan. It is generally held that complement activation may have pathological consequences by promoting inflammatory processes, however the data disclosed herein point to the therapeutic possibilities of in situ complement activation during wound repair. C5a release from blood clots and from chitosan- GP/blood clots could be related to the increased neutrophil chemotaxis seen towards chitosan-GP/blood implants during guided cartilage repair (Chevrier et al., 2007, Osteoarthritis Cartilage, 15: 316-327). Transient neutrophil chemotaxis toward chitosan-GP/blood implants was followed by angiogenesis, bone remodeling, and a more structurally improved cartilage repair tissue, compared to blood clots induced by surgical intervention alone (Hoemann et al., 2005, Bone Joint Surg-AM, 87: 2671-2686). These data suggest that transient C5a release could be a therapeutic initiating event in wound repair.

[0094] Chitosan is a nonactivating biomaterial that is positively charged, and binds to anionic plasma and serum proteins C3, C5, factor B, Ba fragment, and AT without leading to complement activation or release of C5a anaphylatoxin. C5a fragment appeared in coagulating whole blood serum in parallel with platelet degranulation, with or without chitosan. Together, these data allow to conclude that a C5 convertase is not activated in coagulating blood directly at the chitosan particle surface. The present disclosure reveals that chitosan particles selectively adsorb serum proteins with a pi below the insoluble chitosan pK_a=6.78, suggesting that ionic interactions could be the leading force for the adsorption of a variety of serum factors, including complement factors, to chitosan.

[0095] The term "repair" when applied to cartilage and other tissues is intended to mean without limitation repair, regeneration, reconstruction, reconstitution or bulking of cartilage or tissues.

[0096] The term "blood" is intended to mean whole blood, processed blood, venous blood, arterial blood, blood from bone-marrow, umbilical cord blood and placenta blood. It may be enriched in platelets.

[0097] The term "blood component" is intended to mean erythrocytes, leukocytes, monocytes, platelets, fibrinogen, and thrombin. It may further comprise platelet rich plasma free of erythrocytes. In another embodiment, blood component is intended to mean any component of the blood retaining clotting properties.

[0098] The term "biocompatible polymer" is intended to mean a polymer that can be contacted with a tissue, without altering the tissue viability and that is tolerated or accepted by the tissue or the organism.

[0099] The term "patient" is intended to mean a human or an animal.

[00100] The term "solidification" or "presolidification" is intended to mean the loss of the liquid state to the benefit of the solid state.

[00101] The term "thermogelling" is intended to mean the characteristic of a polymer which becomes non-liquid at a certain temperature over a certain period of time. [00102] The term "clotting" is intended to mean a type of solidification involving formation of a blood clot or plasma clot.

[00103] The term "cytocompatible" is intended to define the property of a composition or solution to not be toxic to the living cells.

[00104] The expression "therapeutic substance" is intended to refer to the property of any substance to have beneficial or therapeutic effect on the patient administered with the substance. Such therapeutic substance can be, but not limited to, a polysaccharide, a polypeptide, a drug, a liposome, a DNA, DNA- polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, a colony stimulating factor, a cytokine and an angiogenic factor.

[00105] The present disclosure will be more readily understood by referring to the following examples which are given to illustrate embodiments rather than to limit its scope.

EXAMPLE I

Determining the relationship between chitosan protonation level and solution pH.

[00106] To be cytocompatible, the optimal chitosan-GP solution should be close to isotonic and the solution pH should be between 5.8 to 6.8. Therefore it is necessary to determine appropriate level of acid needed in the composition to attain this pH range.

[00107] All chitosan-GP formulations employed in proof-of-efficacy animal studies (Hoemann et al., 2007, Osteoarthritis and Cartilage, 15: 78-89; Hoemann et al., 2005, Journal of Bone and Joint Surgery-American Volume, 87A(12): 2671-2686; Chevrier et al., 2007, Osteoarthritis and Cartilage, 15(3): 316-327) used HCI concentrations that yielded 90% or 100% theoretical chitosan protonation in the chitosan-GP solution. To determine the effect of chitosan protonation on chitosan solution pH, chitosan (83%DDA) was dissolved at 1% wt/vol in 10 ml total volume with varying levels of HCI to give 70%, 80%, 90%, and 100% protonation. For precision, the Loss on Drying (LOD) according to the Certificate of Analysis (COA) was taken into account when calculating the amount of chitosan to add to 10 ml. The solution pH was measured at room temperature.

[00108] As seen in Fig. 1 , the pH is maintained close to pH 5.5 when the protonation is below 90%, and drops abruptly when protonation exceeds 90%. The HCI content could potentially influence the chitosan polymer conformation in solution, and the interaction of chitosan with blood components and cells. It is clear that chitosan solutions maintained around pH 5.5 could be room temperature stable. It is thus demonstrated herein that chitosan-HCI preparations with 70% to 90% protonation could generate a room temperature- stable chitosan solution.

EXAMPLE II

Concentration of HCI calculated that generates a 100% protonated

chitosan in solutions with 1.5% to 2.2% w/v of chitosan.

[00109] It was evaluated the concentration of HCI that is needed to generate a chitosan being 100% protonated in solutions with a final 1.5% to 2.2%w/v of chitosan.

Table 2

% of protonation of chitosan in view of the concentraiton of HCI used

[00110] As seen in Table 2 and Fig. 2, the HCI concentration should not exceed 79 mM of the re-constituted 1.6% w/v chitosan-HCI and glycerol phosphate solution. EXAMPLE 111

Determination of the feasibility of 2-part component room temperature stable chitosan-HCI mixed with room temperature-stable GP-HCI, GP-NaCI or NaCI.

[00111] It was tested whether chitosan-GP could be reconstituted at room temperature from separate chitosan-HCI and salt solutions before use. Each separate solution is predicted to be room-temperature stable. The chitosan solution was pH -5.6. The aim of this experiment was to determine feasibility of rapid recombination of a 2-part chitosan and salt solution that generates a sterile isotonic and near-neutral chitosan solution free of chitosan precipitates.

[00112] The following materials were used:

-Chitosan preparation, 82.6% DDA (73 mM HCI needed to give 100% protonation of a 1.5% w/v solution);

-Chitosan-HCI solution: 1.66% chitosan-58 mM HCI. 50 ml preparation autoclaved for 40 minutes;

-10x stock β-GP solutions: 1 M disodium β-GP was dissolved in water, or in HCI solutions between 50 and 500 mM HCI;

-10x stock NaCI solutions: 1.5M NaCI; (0.5M β-GP in water/750 mM NaCI); (0.5M β-GP in 500 mM HCI 750 mM NaCI).

Table 3

Calculations

CHITOSAN %DDA Molec. Final concentration of HCI needed to protonate

Mass (MDa) "x %" free NH2 groi jps in a 1.5% chitosan so ution

100% 95% 90% 80%

Preparation 82.6 0.367 73.5 mM 69.8mM 66.1 mM 58.7 mM (lot #CH 10064) Table 4

Solution preparation: Chitosan-HCI -with 80% protonation

Table 5

Results obtained with 1 10 μΙ of Additive rapidly combined with 1000 μ1 1.66% chitosan pH 5.6.

Table 6

Solution preparation

Additive : 750 mM GP was cor nbined with the below concentration of HCI, then the a idditive was mixed with chitosan-HCI ?H 5.6

A2 B2 C2 D2 E2 amount of HCI in bGP 0 mM 50 mM 100 mM 250 mM 500 mM pH of additive 9.306 7.35 6.994 6.43 JMD final composition of each condition tested

A2 B2 C2 D2 E2

% chitosan (wt vol) 1.50% 1.50% 1.50% 1.50% 1.50% mM bGP 100 mM 100 mM 100 mM 100 mM 100 mM mM HCI 53 mM 58 63

final pH 6.66 6.67 6.62

* over 100% chitosan protonation Table 7

Results obtained with 110 μΙ of Additive was rapidly combined with 1000 μΙ

.66% chitosan pH 5.6.

* Precipitation may happen within a few seconds.

** Delayed shaking may cause the precipitate to persist.

[00113] Direct mixture of 110 μΙ 1M β-GP (conditions A1 and A2) into 1000 μΙ 1.66% chitosan generated a slightly cloudy solution that went clear if agitated immediately. If the solution was agitated after waiting a few minutes, some particle precipitates were present. Regarding conditions B1 through E1 and B2 through E2, all solutions were clear upon mixing with 1.66% chitosan pH 5.6. The final pH of the chitosan-GP solution was maintained at pH 6.6 as long as the final HCI concentration gave less than 100% protonation of chitosan 82.6%DDA. The pH dropped proportionally to 6.4 and 6.1 , when the HCI concentration exceeded 100% chitosan protonation.

[00114] A 2-part chitosan/blood system is feasible when 1 ml chitosan-HCI is packaged in a mixing vial, and 0.11 ml of β-GP in 50 mM or 100 mM HCI is injected directly into the vial and shaken immediately before use. Another embodiment is when 1.2 ml chitosan-HCI can be packaged in a mixing vial, and 0.3 ml of 0.5M β-GP in 50 mM HCI is added directly into the vial immediately before use. The chitosan will be in non-aggregated form when prepared the day of use, and will have the same formulation as the chitosan-GP solution that generated excellent repair results. Another embodiment is when 1 ml chitosan- HCI is packaged in a mixing vial, and 0.11 ml of 750 mM NaCI is used to generate an isotonic and slightly acidic (pH 5.8) chitosan solution (see Figs. 3 and 4).

EXAMPLE IV

Complement activation during coagulation of whole blood and chitosan-

GP/blood mixtures

[00115] The hypothesis that complement is activated in chitosan-GP/blood mixtures was tested in a time-dependent manner, using 80% DDA (i.e., 80% Glc and 20% GlcNA) chitosan.

[00116] Chitosan was dissolved at 2.05% w/w, pH 5.6 in dilute HCI, and autoclave-sterilized to produce liquid solutions with dynamic viscosities of 1422 mPa s (80.2% DDA), 1849 mPa s (80.6% DDA), 2964 mPa s (94.6% DDA) at 25°C and stored as sterile aliquots at room temperature for up to 6 months or frozen at -80°C. Before vortex mixing with plasma or blood, liquid chitosan-HCI was combined at a 4:1 v/v ratio with sterile ddH20, filter-sterile 500 mM disodium β-glycerol phosphate/ 50 mM HCI pH 7.2 (GP) or autoclave-sterile 750 mM NaCI to generate solutions with 1.6% w/v chitosan at pH 5.6 (chitosan-HCI, hypotonic), pH 6.6 (chitosan-GP, isotonic) or pH 6.1 (chitosan-NaCI, isotonic).

[00117] The disodium glycerol phosphate (GP) levels were adjusted to be isotonic and cytocompatible.

[00118] Clot tensile strength was evaluated with 4 Thromboelastograph® (TEG) instruments (5000 series TEG analyzer Software Version 3, Haemoscope, Niles IL) which permits the simultaneous analysis of 8 samples. Fresh peripheral venous blood was obtained from N=4 healthy consented nonfasting male/female volunteers (use of drugs that interfere with clotting and pregnancy excluded) under institutional-approved ethics protocols. All subjects enrolled in this research responded to an Informed Consent which has been approved by the Institutional Committee on Human Research and this protocol was accepted by them. Unmodified whole blood was homogenously mixed with chitosan-GP at a 3:1 ratio blood:chitosan-GP, deposited in plastic sample TEG cups, and allowed to coagulate for up to 75 min at 37°C. Samples were removed at specific intervals, the blood volume equivalent diluted 10-fold in ice- cold quench buffer (20 mM HEPES, 50 mM EDTA, 10 mM benzamidine, 150 mM NaCI, pH 7.4 with 100 μΜ PMSF and 33 μΜ FPR-ck), vortexed, and the serum cleared by centrifugation and stored at -80°C, as previously described. Whole unmodified blood was analyzed in parallel as a control. In other experiments, whole blood was allowed to clot for 30 min in sterile glass borosilicate vials, plastic vials, or serum-separating vacutainer tubes at room temperature or 37°C, then clotted at room temperature or 4°C for 4 h (N=4 donors) before centrifuging at 2000g for 5 min at room temperature to obtain serum which was stored at -80°C.

[00119] Thromboelastography was used to monitor coagulation and the development of clot tensile strength and thrombin generation was monitored in serum by thrombin-antithrombin (TAT) levels as previously described (Marchand et al., 2009, Osteoarthristis Cartilage, 17: 950-957). In both whole blood and chitosan-GP/blood mixtures, abundant levels of C5a fragment appeared in serum at 30 min, around the onset of thrombin generation and coagulation (Fig. 5A-D), and in parallel with platelet factor 4 (PF4), a marker of platelet degranulation (Fig. 5E,F). C5a, TAT, and PF4 levels began to decay in whole blood serum but not in chitosan-GP/blood clot serum after 75 min of coagulation (Fig. 5). In chitosan-GP/blood mixtures, minor complement and platelet activation was seen before the onset of thrombin generation in experiments using four different blood donors. These data show that complement was activated both in whole blood, and chitosan-GP/blood mixtures, in parallel with platelet activation.

EXAMPLE V

C5a generation in human serum and plasma

[00120] The hypothesis that 80% DDA chitosan generates C5a fragment in human serum was tested. As a positive control, zymosan was used, a well- known activator of the alternative pathway. Zymosan is a yeast ghost structure that is composed of chitin, the GlcNA parent molecule of chitosan, along with mannan polysaccharide, and residual yeast proteins and lipids. After 1 h incubation at 37°C, zymosan-activate serum (ZAS) contained abundant C5a fragments that migrated around 13 kDa, slightly faster than the 16.1 kDa marker (Fig. 6A). Upon mixing chitosan-GP with serum, 80% DDA chitosan (pK_a ~6.78) precipitated into insoluble particles that were cleared from the liquid phase by centrifugation. No C5a fragments were detected in serum exposed to GP buffer alone, or to 80% DDA chitosan-GP (Fig. 6A; lanes Serum-GP & Chi80-GP, respectively). These data showed that incubation of chitosan-GP with serum did not lead to generation of liquid phase C5a.

[00121] Complement activation is frequently tested using fresh serum, however, results in Fig. 5 suggested that residual levels of C5a could be present in the serum before adding zymosan. Indeed, high sensitivity chemiluminesence reagent (ECL+) revealed that serum generated in glass vials at various temperatures contained trace levels of two principle C5a fragments. One fragment migrated with the 16.1 kDa molecular weight marker, and a ~13 kDa fragment comigrated with C5a produced by zymosan (Fig. 6A), middle panel). These 16 and 3 kDa C5a proteins were both detected by goat polyclonal anti- C5a (Fig. 6) and by mouse monoclonal anti-C5a. The nonglycosylated recombinant C5a (rC5a) peptide standard migrated faster in the gel than the glycosylated 13 kDa zymosan-activated C5a. By contrast, citrated plasma in high sensitivity Western blots only contained the 16 kDa fragment (Fig. 6A, ECL+). At high-sensitivity, serum exposed to GP or chitosan-GP contained the 16 kDa and not the 3 kDa C5a fragments (Fig. 6A, ECL+). These data further supported the conclusion that chitosan did not generate activated C5a fragment in serum.

[00122] Given that serum already contained variable levels of a 13 kDa activated C5a fragment, and citrated plasma did not, citrated human plasma was used, which can still undergo complement activation by zymosan, to analyze the effect of chitosan on C5a generation. Zymosan particles dispersed in NaCI or in isotonic GP buffer were combined for 1 h with plasma at 4 mg/ml. The cleared supernatant contained an abundant 13 kDa C5a fragment (Fig. 6B and C; ZAP). These data confirmed that alternative complement activation could proceed in citrated plasma, and in the presence of GP buffer. However, at 4 mg/ml, chitosan-GP (80% or 95% DDA), chitosan-NaCI, chitosan with LPS, and solid chitosan particles all failed to generate detectable levels of the 13 kDa C5a fragment in plasma (Fig. 6B and C; Chi80, Chi95). Purified thrombin also failed to generate C5a fragments in citrated human plasma (Fig. 6C; lla), even though a fibrin clot was produced. These data showed that chitosan did not generate C5a either as solid particles, or as liquid chitosan solutions mixed into plasma that subsequently precipitated.

EXAMPLE VI

C3 binds to chitosan noncovalently while C3b and iC3b become cross- linked to zymosan-associated proteins

[00123] Zymosan and chitosan particles were incubated in citrated plasma, pelleted, rinsed in EDTA buffer and extracted with 8M urea, a chaotropic agent that denatures C3 without hydrolyzing thioester bonds. Western blot analysis of pellet extracts using an anti-C3b alpha chain antibody demonstrated that C3 proteins could be eluted from zymosan by urea (Fig. 7A and B). Since chitin is F3 insoluble in urea, these data also indicated that the C3 proteins eluted from zymosan were not covalently bound to chitin. In nonreducing conditions, zymosan-associated C3 proteins migrated at 190 kDa and as cross-linked higher molecular products (Fig. 7A; ZAP), and in reducing conditions C3 migrated as a strong ~42 kDa fragment along with a faint C3b band (Fig. 7B; ZAP). The 42 kDa band could arise from iC3b or C3c, although the absence of a faster-migrating 35 kDa C3c product in the nonreducing gel, due to cleavage of the 40 kDa C3dg fragment (Fig. 7C), indicated that the 42 kDa fragment was a subfragment of iC3b. Altogether, these data were consistent with C3b becoming covalently cross-linked to zymosan-associated proteins, and then being rapidly cleaved to iC3b.

[00124] C3 proteins were also eluted from the chitosan pellet. In nonreducing conditions, C3 proteins migrated as intact C3 and discrete high molecular weight products (Chi80-GP; Fig. 7A). Under reducing conditions, C3 protein migrated as an intact C3 alpha chain (-115 kDa), and discrete high molecular weight products above 190 kDa, that comigrated with cross-linked C3 proteins present in plasma (C3-X; Fig. 7B). These data showed that chitosan particles adsorbed abundant levels of intact C3 protein from citrated plasma.

EXAMPLE VII

C3 and C5 bind to chitosans with 80% DDA and 95% DDA as intact proteins.

[00125] Using citrated plasma, it was evaluated the effect of chitosan deacetylation level on complement protein binding, using 95% DDA chitosan (95% Glc and 5% GlcNA) and 80% DDA chitosan (80% Glc and 20% GlcNA). Fluid phase samples were analyzed for C5a and C3a, the urea pellet extracts were analyzed for bound C5 and C3, and zymosan served as a control. Zymosan is a strong complement activator and data are consistent with this, because C5a was present in the supernatant, and no native C5 was associated with the zymosan pellet (Fig. 8A and B; ZAP). Plasma C3a was much more F4 abundant than C5a, consistent with previous assays of C5a and C3a in human plasma and serum (Fig. 8C; Plasma). Zymosan generated slightly more C3a in the fluid phase (Fig. 8C; ZAP). In zymosan pellet extracts, iC3b was the main product eluted, which was detected by anti-C3b antisera and not anti-C3a antisera (Fig. 8D vs. E, respectively; ZAP).

[00126] When the chitosans were incubated with plasma, no C5a was produced in the fluid phase, and no increase in C3a was observed (Fig. 8A,C). The failure to detect C5a in chitosan supernatant was not an artifact of C5a associating with the chitosan pellet since 8M urea failed to elute C5a from the pellet. By contrast, chitosan pellet extracts contained intact C5, which was detected by anti-C5a and comigrated with pure C5 standard (Fig. 8B). Moreover, intact C3 alpha chain, detected by both anti-C3b and anti-C3a antisera, was eluted from chitosan pellets (Fig. 8D and E, respectively). Chitosan also captured 2 higher molecular weight C3 proteins from plasma that migrated above the intact 115 kDa C3 alpha chain. These proteins were not activation-fragments of C3, because they were recognized by both anti-C3b and anti-C3a antisera (8D, E). These species comigrated with or close to 2 cross- linked proteins in the purified C3 standard (C3-X; Fig. 8D and E). Although the nature of these 2 proteins is not completely clear, the apparent molecular mass and retention of C3a and C3b epitopes suggested that one of the species represented an intact 230 kDa alpha chain dimer (C3-X; Figs. 7B and 8D and E).

[00127] More C5 and C3 were associated with 95% DDA chitosan than 80% DDA chitosan (Fig. 8B, D and E; Chi95 vs. Chi80). Low levels of iC3b, proportional to the iC3b levels detected in plasma, were detected in C3 proteins eluted from chitosan (Fig. 8D). Note that the level of iC3b in plasma was diminished by protecting the sample from heat (Fig. 8D; P-not boil vs Plasma). To summarize, these data demonstrate that nonactivated C5 and C3 proteins adsorbed to insoluble chitosan particles. More C5 and C3 adsorbed to chitosan with higher glucosamine content.

EXAMPLE VIII

Complement C3 and C5 still bind to chitosan in EDTA and methylamine inhibiting conditions.

[00128] Complement protein-chitosan binding with human serum containing 10 mM EDTA which chelates divalent metal ions essential for complement activation was tested, and human plasma previously treated with 100 mM methylamine which reacts with and inactivates the C3 thioester internal bond was also tested. Zymosan incubated in serum, but neither serum-EDTA nor plasma-methylamine, generated fluid-phase C5a F5 (Zymosan; Fig. 9A). Solid- phase iC3b was associated with zymosan particles in serum, in EDTA-serum at trace levels, but not in methylamine-plasma (Zymosan; Fig. 9C and D). Interestingly, a novel and faster migrating "short" form of the C5 protein, sC5, was present in serum, eluted from zymosan particles, and detected by anti-C5a antisera (Fig. 9B).

[00129] When 95% DDA chitosan was incubated with the same three conditions, no fluid-phase C5a was generated (Chi95; Fig. 9A). Intact C5 and C3 proteins were eluted by urea from chitosan particles under all three conditions (Fig. 9B, C, and D). Chitosan also captured high molecular weight alpha chain products from serum and EDTA-serum, but not from methylamine plasma (Fig. 9C and D). These data supported the notion that the high molecular weight C3 products captured from plasma and serum by chitosan were thioester-dependent, covalently linked alpha chain multimers.

EXAMPLE IX

Specific binding of C3 protein to chitosan detected by SPR.

[00130] To further investigate pure C3-chitosan interactions, an SPR biosensor assay using biotinylated chitosan immobilized at the surface of a strepatavidin-coated biosensor surface compared to a streptavidin-only surface was conducted.

[00131] Interactions between purified human C3 and chitosan were monitored using a surface plasmon resonance (SPR)-based optical biosensor (Biacore 3000, GE Healthcare, Piscataway, NJ), and a streptavidin-coated (SA) sensorchip flowcell coupled with biotinylated chitosan at 25°C. Chitosan (98.4% DDA) was biotinylated to 1% mol/mol with succinimidyl ester (SE)-biotin (Invitrogen, Missisauga, ON) to produce B-chitosan. B-chitosan was dissolved in acetic acid pH 6.1 , 0,2 pm filtered, and further diluted to 50 pg/ml in Coupling Buffer (50 mM HEPES, 150 mM NaCI pH 6.1). Running buffer (10 mM HEPES, pH 7.4, 150 mM NaCI, 3.0 mM EDTA, 0.005% Tween™ 20) was used to equilibrate the chip and to dilute C3 and anti-C3 antibodies before injection. After buffer injection and baseline stabilization, three 180s pulse injections of 50 pg/ml B-chitosan (in HEPES buffer, pH 6.1, flow rate of 20 μΙ/min) resulted in a mass accumulation of -1300 Resonance Units (corresponding to 1.3 ng/mm² of material bound to the surface). C3 protein was serially injected (180 s, flow rate 5 μΙ/min) of buffer (4 times), at 1 , 10, 20, and 100 pg/rnl concentration followed by anti-C3a antibody (1/200 dilutions of a 60 pg/ml stock) and injections of anti- C3b antibody (1/500 and 1/200 dilution of a 25 pg/ml stock with 50% v/v glycerol). Control surface corresponded to bare streptavidin-coated sensorchip flowcell for parallel monitoring of nonspecific protein adsorption.

[00132] Injections of buffer or low concentrations of C3 (≤50 pg/ml), did not result in significant accumulation of C3 at the surface of the chitosan-coated biosensor (Fig. 10; compare injections 1-7 on chitosan surface, top line, and control surface, bottom line). In contrast, C3 injection at 100 g/mL, a concentration that is 10 times lower than that found in plasma resulted in a net and stable accumulation of 350 RUs on the chitosan surface while no accumulation could be detected on control surface (Fig. 10; injection 8). Injection of anti-C3a antibody led to an additional accumulation of 350 RUs on the chitosan surface on which C3 had been pre-bound while no accumulation was detected on the mock surface (Fig. 10; injection 9). By comparison, injection of anti-C3b antibody (Fig. 10; injections 10 and 11) only generated a nonspecific signal on both chitosan-C3 and the mock surface due to glycerol present in the antibody preparation. This latter result was consistent with C3 binding with chitosan via its anionic C3b moiety, while the cationic C3a peptide moiety was free to interact with other binding partners (e.g., anti-C3a antibody).

EXAMPLE X

Novel model of chitosan-anionic serum protein interaction.

[00133] Factor B is cleaved during complement activation by factor D to form factor Bb, the enzymatically active subunit of the C3/C5 convertase complex. When zymosan particles were incubated for 1 h incubation at 37°C in citrated plasma, rinsed with EDTA buffer and extracted with 8M urea, solid-phase Bb was abundantly present in the pellet extract (ZAP; Fig. 1 A and C). By contrast chitosan pellet F7 extracts under identical conditions contained intact factor B and no fragment Bb (Chi95; Fig. 11A and C). These data demonstrated unequivocally that chitosan did not trigger the formation of solid-phase complement C3/C5 convertase. Factor B associated more tightly with 95% DDA than 80% DDA chitosan (Fig. 1 1A), as was the case for C5 and C3 (Fig. 8B, D and E respectively). Most strikingly, fragment Ba was eluted from chitosan particles after incubation in plasma (Fig. 1 1A). Further analysis of the chitosan treated plasma supernatants revealed that chitosan adsorbed out low-level Ba fragment (and not Bb) already present in plasma (Fig. 11 C). [00134] Antithrombin (AT), an anionic serum protease inhibitor, also adsorbed to chitosan particles, with stronger affinity for 95% DDA compared to 80% DDA chitosan (Fig. 11 B). Altogether these data showed that a variety of anionic plasma and serum proteins, including C5, C3, factor B, Ba, and AT selectively adsorbed to chitosan, and all of these proteins had a theoretical pi lower than the pK_a 6.78 of chitosan.

EXAMPLE XI

Conditions that activated coagulation of plasma did not activate

complement.

[00135] To further elucidate the role of thrombin generation in complement activation, C5a generation was analyzed in clot-activated plasma. Negatively charged glass surfaces activate the contact pathway in plasma, but not in citrated plasma which is chelated of calcium ions necessary for propagation of the clotting cascade. Glass beads incubated in citrated plasma for 1 h at 37°C failed to induce coagulation, or to bind to C5, C3, factor B, AT, or to activate complement (Fig. 8A-D and Fig. 11A and B; lane Glass). Glass beads incubated in recalcified plasma induced fibrin clot formation. The resulting glass bead- fibrin pellet contained trace levels of urea-extractable AT (Fig. 11 B; lane Glass+Ca++) but the glass beads and fibrin clot failed to generate C5a or to bind C3, C3b, C5, or factor B (Figs. 8A-D and 1 1 B; Glass+Ca++]. When purified thrombin was added to citrated plasma, or to chitosan-GP/plasma samples, a fibrin clot was formed without generating activated C5a fragment (Fig. 6C; lane Ma). Since chitosan-GP/plasma mixtures permitted fibrinogen cleavage by thrombin, a serine protease, this indicated that the lack of complement activation by chitosan-GP and chitosan-NaCI solutions was not due to complement enzyme denaturation or nonspecific inhibition of serine protease activity. These results also demonstrate that chitosan-GP/citrated plasma mixtures when exposed to thrombin can solidify to form a polysaccharide-fibrin implant. EXAMPLE XII

In vivo efficacy of the 2-part formulation in rabbits and humans

Example of use of the 2-part formulation in an in vivo rabbit model

[00136] An implant prepared from the mixture of 400 μΙ_ 2.05% w/w Chitosan solution (pH 5 to 6) (without N=4, and with fluorescent rhodamine isothiocyanate-chitosan tracer N=3), 100 μΙ_ 10.05% w/w beta-Glycerol Phosphate solution pH 7.2, and 1.5 mL fresh rabbit unclotted whole blood was delivered to an acute rabbit femoral trochlear cartilage defect with 4,1 mm diameter microdrill holes pre-treated with 3 pL purified human thrombin. The contralateral drilled cartilage defect was treated with 3 pL of purified human thrombin. After 8 weeks of repair, the implant-treated defects had significantly greater subchondral bone repair (compare A1 and B2 in Fig. 13A; and right panel in Fig. 13B) and significantly greater basal integration (compare C1 and D1 in Fig. 13A; and left panel in Fig. 13B) compared to the control defects.

Autologous implant preparation: in vivo rabbit cartilage repair model

[00137] A liquid mixture containing 1200 pL 2.05% w/w Chitosan solution (pH 5 to 6), 300 pL 10.05% w/w beta-Glycerol Phosphate solution pH 7.2, and 4.5 mL fresh rabbit unclotted whole blood was solidified in situ, in a 3.5 x 4.5 mm cartilage defect with 0.5 mm and 1 mm diameter microdrill holes, using pre- application of thrombin to accelerate solidification (N=8 bilateral defects; see Fig. 14A). The repair period was 6.5 months. Treated repair tissue (Fig. 14C) contained more glycosaminoglycan than controls (Fig. 14B) throughout the defect .

Autologous implant preparation in human patients

[00138] A liquid mixture containing 1200 pL 2.05% w/w Chitosan solution (pH 5 to 6), 300 pL 10.05% w/w beta-Glycerol Phosphate solution pH 7.2, and 4.5 mL fresh human unclotted whole blood of chitosan-GP was reconstituted in the clinic during the operative procedure. The 2-part formulation described herein was used as a two vial product comprised of a mixing vial containing the chitosan solution and an additive vial containing the beta-glycerol phosphate buffer. Blood was added and mixed with the solution, then the implant was solidified in situ, to treat microfracture lesions of human patients. In other patients, lesions were treated with microfracture-only. Repair tissue biopsies were obtained 13 months post-treatment in 21 patients from the estimated geometric center of the initial lesion (13 treated, 9 Microfracture-only). The cartilage repair tissue in biopsies from implant-treated defects was significantly thicker (see Fig. 15A) and had a significantly higher over-all histology score for quality of repair (see Fig. 15B and 16A, B) as judged by 3 independent blinded readers, for 14 different repair tissue features. Statistically significant improvements were identified for critical tissue characteristics following treatment with the 2-part formulation described herein compared to microfracture alone. In particular, after applying the International Cartilage Repair Society's (ICRS) histological scoring systems I and II to the biopsies in a blinded manner, several parameters were found to be statistically significant between treatment groups at p<0.05. First, the Overall ICRS II score was significantly better for the group treated with the 2-part formulation than for the microfracture group (p=0.045), with scores of 64.5 and 36.9 respectively (see Figs. 15A and B). Considered to be the most important parameter within the ICRS II scoring system, the ICRS II Overall score assimilates all the parameters listed in the grading system to generate an overall assessment of tissue repair. Other statistically significant improvements were found in the assessments of cell morphology, cell viability and superficial zone morphology in the biopsies. Furthermore, surface architecture, collagen structure as observed by Polarized Light Microscopy, and repair tissue thickness measurements demonstrated improvement in favour of the 2-part formulation. Macroscopic grading of the cartilage repair by the surgeon at the time of biopsy, which included the extent of lesion filling, tissue surface characteristics and integration with surrounding tissue, was also significant (p=0.016).

[00139] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

- 46 -

WHAT IS CLAIMED IS:

1- A two-part cytocompatible system for preparing a cytocompatible polymer composition for use in repairing a tissue of a subject, said system comprising:

• a first part liquid solution of a polymer in a first container; and

• a second part liquid solution of a salt in a second container, wherein said first part and said second part are to be combined, prior to said use, to provide said cytocompatible polymer composition and wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.

2- The two-part cytocompatible system of claim 1 , wherein the polymer is a modified or natural polysaccharide.

3- The two-part cytocompatible system of claim 1 or 2, where the polymer is dissolved in a mineral acid or an organic acid to prepare the first part liquid solution.

4- The two-part cytocompatible system of claim 3, wherein the acid is hydrochloric acid, lactic acid, citric acid, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid or hydrobromic acid.

5- The two-part cytocompatible system of claim 2, wherein the polysaccharide is chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, decorin, keratan sulfate, dermatan sulfate, heparin or heparin sulfate.

6- The two-part cytocompatible system of any one of claims 1-5, wherein the salt is an organic salt or an inorganic salt. - 47 -

7- The two-part cytocompatible system of claim 6, wherein the inorganic salt is sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt.

8- The two-part cytocompatible system of claim 6, wherein the inorganic salt is NaCI, KCI, CsCI, CaCI₂, CsF, KCI0₄, NaN0₃ or CaS0₄.

9- The two-part cytocompatible system of claim 6, wherein the organic salt is glycerol-phosphate.

10- The two-part cytocompatible system of any one of claims 1-9, wherein the cytocompatible polymer composition has a pH between about 5.8 and about 7.8.

11 - The two-part cytocompatible system of any one of claims 1-9, wherein the cytocompatible polymer composition has a pH between about 5.8 and about 6.8.

12- The two-part cytocompatible system of claim 5, wherein the polymer is chitosan.

13- The two-part cytocompatible system of claim 12, wherein the chitosan has a degree of deacetylation between about 20% to about 100% deacetylated.

14- The two-part cytocompatible system of claim 12 or 13, wherein the concentration of chitosan in the first part liquid solution is between about 1.5% to about 2.2% w/v.

15- The two-part cytocompatible system of any one of claims 1-14, further comprising a third part blood component.

16- The two-part cytocompatible system of claim 15, wherein the third part blood component is whole blood, processed blood, venous blood, arterial - 48 - blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood or placenta blood.

17- The two-part cytocompatible system of claim 15, wherein the third part blood component is plasma, erythrocytes, leukocytes, monocytes, platelets, fibrinogen, stem cells or thrombin.

18- The two-part cytocompatible system of claim 15, wherein the third part blood component comprises a platelet-rich plasma substantially free of erythrocytes.

19- The two-part cytocompatible system of any one of claims 15-18, wherein the mixing ratio of (i) the third part blood component and (ii) the first part liquid solution of polymer solution combined with the second part liquid solution of the salt is between about 1 :3 to about 1 :12.

20- The two-part cytocompatible system of any one of claims 15-19, wherein said cytocompatible polymer composition is capable of activating complement through platelet activation.

21 - The two-part cytocompatible system of claim 20, wherein said cytocompatblie polymer composition is capable of stimulating release of C5a peptide in whole blood or blood fractions containing platelets.

22- The two-part cytocompatible system of claim 9, wherein the concentration of glycerol-phosphate in the second part liquid solution is between about 400 to about 1000 mM.

23- The two-part cytocompatible system of any one of claims 1-22, further comprising a buffer to achieve near-neutral pH and isotonicity of the cytocompatible polymer solution. - 49 -

24- The two-part cytocompatible system of any one of claims 1-23, wherein the first and second biological containers are independently selected from a vial or a syringe.

25- A cytocompatible polymer composition for use in repairing a tissue of a subject, wherein said cytocompatible polymer is prepared by admixing a first part liquid solution of a polymer with a second art liquid solution of a salt prior to said use, wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature.

26- The cytocompatible polymer composition of claim 25, wherein the polymer is a modified or natural polysaccharide.

27- The cytocompatible polymer composition of claim 25 or 26, where the polymer is dissolved in a mineral acid or an organic acid to prepare the first part liquid solution.

28- The cytocompatible polymer composition of claim 27, wherein the acid is hydrochloric acid, lactic acid, citric acid, acetic acid, nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid or hydrobromic acid.

29- The cytocompatible polymer composition of claim 26, wherein the polysaccharide is chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, dermatan sulfate, heparin or heparin sulfate.

30- The cytocompatible polymer composition of any one of claims 25-29, wherein the salt is an organic salt or an inorganic salt.

31 - The cytocompatible polymer composition of claim 30, wherein the inorganic salt is sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt. - 50 -

32- The cytocompatible polymer composition of claim 30, wherein the inorganic salt is NaCI, KCI, CsCI, CaCI₂, CsF, KCI0₄, NaN0₃ or CaS0₄.

33- The cytocompatible polymer composition of claim 30, wherein the organic salt is glycerol-phosphate.

34- The cytocompatible polymer composition of any one of claims 25-33, wherein the cytocompatible polymer composition has a pH between about 5.8 and about 7.8.

35- The cytocompatible polymer composition of any one of claims 25-33, wherein the cytocompatible polymer composition has a pH between about 5.8 and about 6.8.

36- The cytocompatible polymer composition of claim 29, wherein the polymer is chitosan.

37- The cytocompatible polymer composition of claim 36, wherein the chitosan has a degree of deacetylation between about 20% to about 100% deacetylated.

38- The cytocompatible polymer composition of claim 36 or 37, wherein the concentration of chitosan in the first part liquid solution is between about 1.5% to about 2.2% w/v.

39- The cytocompatible polymer composition of any one of claims 25-38, further comprising a third part blood component combined to the first part liquid and the second part liquid.

40- The cytocompatible polymer composition of claim 39, wherein the third part blood component is whole blood, processed blood, venous blood, arterial blood, blood from bone, blood from bone-marrow, bone marrow, umbilical cord blood or placenta blood. - 51 -

41 - The cytocompatibie polymer composition of claim 39, wherein the third part blood component is plasma, erythrocytes, leukocytes, monocytes, platelets, fibrinogen, stem cells or thrombin.

42- The cytocompatibie polymer composition of claim 39, wherein the third part blood component comprises a platelet-rich plasma substantially free of erythrocytes.

43- The cytocompatibie polymer composition of any one of claims 39-42, wherein the mixing ratio of (i) the third part blood component and (ii) the first part liquid solution of polymer solution combined with the second part liquid solution of the salt is between about 1 :3 to about 1 :12.

44- The cytocompatibie polymer composition of any one of claims 39-43, wherein said cytocompatibie polymer composition is capable of activating complement through platelet activation.

45- The cytocompatibie polymer composition of claim 44, wherein said cytocompatibie polymer composition is capable of stimulating release of C5a peptide in whole blood or blood fractions containing platelets.

46- The cytocompatibie polymer composition of claim 33, wherein the concentration of glycerol-phosphate in the second part liquid solution is between about 400 to about 1000 mM.

47- The two-part cytocompatibie system of any one of claims 1-24, further comprising a buffer to achieve near-neutral pH and isotonicity of the cytocompatibie polymer solution.

48- The two-part cytocompatibie system of any one of claims 25-47, wherein the first and second biological containers are independently selected from a vial or a syringe. - 52 -

49- Use of a cytocompatible polymer composition (i) prepared with a two-part system as defined in any one of claims 1-24 or (ii) as defined in any one of claims 25-48 for repairing a tissue in a subject.

50- The use of claim 49, wherein the tissue is selected from the group consisting of cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, infarcted cardiac tissues, ischemic tissues abscesses, resected tumors and ulcers.

51 - The use of claim 49 or 50, wherein said cytocompatible polymer composition is capable of stimulating subchondral angiogenesis, bone remodeling and/or osteochondral repair.

52- Use of a cytocompatible polymer composition (i) prepared with a two-part system as defined in any one of claims 1-24 or (ii) as defined in any one of claims 25 to 48 for delivering a therapeutic substance to a subject.

53- The use of claim 52, wherein said therapeutic substance is selected from the group consisting of a polysaccharide, a polypeptide, a drug, a liposome, a DNA, DNA-polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, an osteoclast-forming factor, a chemotactic factor, a colony stimulating factor, a cytokine, a complement factor, and an angiogenic factor.

54- The use of claim 53, wherein therapeutic substance is an anionic protein with a pi less than the maximum pKa=6.78 of chitosan.

55- A method for repairing and/or regenerating a tissue of a subject in need thereof, said method comprising administering a cytocompatible polymer composition (i) prepared with a two-part system as defined in any one of claims 1-24 or (ii) as defined in any one of claims 25-48 into said tissue in need of repair and/or regeneration, wherein said cytocompatible composition is capable of adhering to the tissue to be repaired and/or regenerated so as to repair and/or regenerate the tissue. - 53 -

56- The method of claim 55, wherein the tissue is selected from the group consisting of cartilage, meniscus, ligament, tendon, bone, skin, cornea, periodontal tissues, infarcted cardiac tissues, ischemic tissues, abscesses, resected tumors and ulcers.

57- The method of claim 55 or 56, wherein said cytocompatible polymer composition is capable of stimulating subchondral angiogenesis, osteoclast formation, bone remodeling and/or osteochondral repair.

58- A method for delivering a therapeutic substance in a subject in need thereof, said method comprising administering a cytocompatible polymer composition admixed with the therapeutic substance into said subject, wherein said cytocompatible polymer composition (i) prepared with a two-part system as defined in any one of claims 1-24 or (ii) as defined in any one of claims 25-48.

59- The method of claim 58, wherein said therapeutic substance is selected from the group consisting of a polysaccharide, a polypeptide, a drug, a liposome, a DNA, a DNA-polymer complex, an antibody, a siRNA, an extracellular matrix fragment, a growth factor, a chemotactic factor, an osteoclast-promoting factor, a colony stimulating factor, a complement factor, a cytokine and an angiogenic factor.

60- The method of claim 59, wherein said therapeutic substance is an anionic protein with a pi less than the maximum pK₀=6.78 of chitosan.

61 - A kit for preparing a cytocomptabile polymer composition for use in repairing and/or regenerating a tissue of a subject, said kit comprising i) a first container comprising a liquid polymer solution and ii) a second container comprising a liquid salt solution, wherein said polymer solution and liquid salt solution are to be combined prior to said use and wherein said wherein the polymer in said cytocompatible polymer composition is substantially in a dissolved, unprecipitated form at room temperature. - 54 -

62- The kit of claim 61 , wherein the polymer is dissolved in an acid solution to provide the polymer solution.

63- The kit of claim 62, wherein said acid solution is hydrochloric acid, acetic acid, lactic acid, citric acid or glycolic acid.

64- The kit of claim 61 or 62, further comprising a concentrated buffer solution so as to adjust the cytocompatbile polymer solution to a cytocompatible pH and isotonicity.

65- The kit of any one of claims 61-64, wherein the polymer is a modified or natural polysaccharide.

66- The kit of claim 65, wherein the polysaccharide is selected from the group consisting of chitosan, chitin, hyaluronan, glycosaminoglycan, chondroitin sulfate, hydroxyethyl cellulose, keratan sulfate, decorin, dermatan sulfate, heparin, and heparin sulfate.

67- The kit of any one of claims 60-66, wherein the salt in said liquid salt solution is an organic or inorganic salt.

68- The kit of claim 67, wherein the inorganic salt is sodium salt, chloride salt, potassium salt, calcium salt, magnesium salt, phosphate salt, sulfate salt or carboxylate salt.

69- The kit of claim 67, wherein the inorganic salt is NaCI, KCI, CsCI, CaC^, CsF, KCI0₄ NaN0₃ or CaS0₄.

70- The kit of claim 67, wherein the organic salt is glycerol-phosphate.

71 - The kit of any one of claims 61-70, wherein the cytocompatible polymer composition has a pH between about 5.8 and about 7.8. - 55 -

72- The kit of any one of claims 61-70, wherein the cytocompatible polymer composition has a pH between about 5.8 and about 6.8.

73- The kit of claim 65, wherein the polymer is chitosan.

74- The kit of claim 73, wherein chitosan has a degree of deacetylation between about 20% to about 100%.

75- The kit of any one of claims 61 to 74, wherein the concentration of in the polymer solution between about 1.3% to about 2.5% w/v.

76- The kit of claim 70, wherein the concentration of glycerol-phosphate in the salt solution is between about 600 to about 1000 mM.

77- The kit of any one of claims 61-76, wherein the cytocompatible polymer composition has a pH between about 6.9 to about 7.4.

78- The kit of any one of claims 61-77, further comprising instruction for use of said kit to repair and/or regenerate the tissue of the subject.

79- The kit of any one of claims 61-78, wherein the first and second biological containers are independently selected from a vial and a syringe.