Chitosan / thrombin formulations for tissue regeneration

Abstract

A tissue regeneration multi-component biomaterial composition, including: a) a chitosan component having a number average molar mass (Mn) of about 32-47 kilodalton (kDa) and a degree of deacetylation (DDA) of about 80- 90%; b) a thrombin component present in an amount effective to accelerate the solidification of the chitosan component with a blood-derived product mixture; and c) a trehalose component present in a lyoprotectant effective amount.

Description

CHITOSAN / THROMBIN FORMULATIONS FOR TISSUE REGENERATIONFIELD OF THE DISCLOSURE

[0001] The present disclosure relates to chitosan / thrombin formulations that may be used for tissue regeneration. The formulations may be solid (lyophilized) or liquid format. The formulations may be mixed with blood-derived components such as, but not limited to platelet-rich plasma, plasma powder, fibrinogen and combinations thereof. The formulations may also comprise cells, such as, but not limited to keratinocytes, fibroblasts and melanocytes.BACKGROUND

[0002] Freeze-dried formulations of chitosan that were designed to be reconstituted in autologous platelet-rich plasma (PRP), a blood-derived component, were previously developed [1], These formulations may be injected into the body following a surgical procedure where they solidify in situ and contribute to soft tissue repair [2-7], However, chitosan-PRP implants solidify fairly slowly, especially when reconstituted with human PRP. When applied directly after mixing, the wound’s wet environment may hinder the biomaterial from adhering to the site of injection. In addition, solidification time varies greatly between patients; Clot reaction times may range from ~10 min up to 40 min. In a rotator cuff multicenter, prospective, randomized clinical trial (ClinicalTrials.gov, NCT number NCT05333211 , Sponsor: ChitogenX Inc., Kirkland, QC, Canada), the biomaterial was required to be incubated in a syringe for 30-45 min before reaching a viscosity suitable for injection at the repaired site. There remains a need to develop new chitosan-based formulations that will solidify more quickly and predictably. A more rapid and controlled implant solidification would facilitate its handling, its administration, and ultimately its clinical implementation. This disclosure describes how thrombin, a coagulation factor, may be incorporated into chitosan formulations. In addition, the use of autologous PRP introduces variability to the product composition because it is highly dependent on the patients’ initial blood composition and on the isolation procedure. There remains a need to develop new chitosan-based formulations that will be mixed with commercially available blood-derived components that are well characterized and consistent. This disclosure describes how plasma powders and fibrinogen may be incorporated to chitosan formulations that containthrombin. These formulations are intended for tissue regeneration and may also comprise cells.

[0003] Most prior art relates to chitosan-based materials designed to initiate hemostasis and control hemorrhage in skin would healing applications. A porous chitosan sponge was coated with self-assembled thrombin / tannic acid films [8], A puff pastry-like chitosan / konjac glucomannan matrix was loaded with thrombin-occupied microporous starch particles [9], Fibrinogen and thrombin aerosol were deposited over the surface of 3D composite scaffolds of collagen / chitosan

[0010] , Fibrinogen and thrombin were added on top of cross-linked collagen / chitosan porous scaffolds

[0011] , Cross-linked chitosan films were coated with thrombin

[0012] , Decellularized liver extracellular matrix was loaded with two natural polymers (oxidized cellulose and chitosan) in association with thrombin

[0013] ,

[0004] Two manuscripts describe the use of thrombin in conjunction with chitosanglycerol phosphate (GP) / blood implants [14, 15], These manuscripts describe how thrombin accelerates chitosan-GP / blood solidification in vitro and how thrombin may be deposited to the surface of a cartilage defect in vivo to promote hemostasis prior to treating the defect with chitosan-GP / blood.SUMMARY

[0005] In one aspect, there is provided a tissue regeneration (including wound healing) multi-component biomaterial composition and methods for preparing and using the same are disclosed. The tissue regeneration biomaterial composition comprises chitosan, thrombin, and a lyoprotectant, such as but not limited to trehalose, which work together to promote tissue regeneration including wound healing by accelerating solidification, facilitating gelation, and providing a supportive matrix for cell growth.

[0006] In one alternative, the chitosan component has a specific molecular weight (Mn) and degree of deacetylation (DDA), which are relevant for its physicochemical properties and biodegradability. The thrombin component accelerates the solidification of chitosan and blood derived products, while the trehalose acts as a lyoprotectant.

[0007] The tissue regeneration biomaterial composition may be reconstituted with platelet-rich plasma (PRP), plasma powder, fibrinogen and combinations thereof to form an implant. Additionally, cells such as fibroblasts, melanocytes and keratinocytes may be incorporated into the biomaterial to enhance its therapeutic properties.

[0008] Methods are also disclosed for preparing the biomaterial, including mixing chitosan with thrombin, trehalose, and calcium chloride, freeze-drying, and then reconstituting with PRP, plasma powder, fibrinogen and combinations thereof. The reconstituted biomaterial may be used to deliver cells to a site in need of treatment, such as a wound or tissue defect.

[0009] The composition and methods offer advantages over existing biomaterials, including improved injectable biomaterials with quick and predictable solidification that may be mixed with well characterized commercially available blood-derived products and that may deliver cells for improved tissue repair.

[0010] In one alternative, there is provided a tissue regeneration multi-component biomaterial composition, comprising: a chitosan component having a number average molar mass (Mn) of about 32- 47 kDa and a degree of deacetylation (DDA) of about 80-90%; a thrombin component present in an amount effective to accelerate the solidification of the chitosan component mixed with at least one blood-derived product; and a trehalose component present in a lyoprotectant effective amount.

[0011] In one alternative, the thrombin component is present in an amount from about 0.25 to 20 NIH / mL.

[0012] In one alternative, the tissue regeneration multi-component biomaterial composition is reconstituted with PRP, plasma powder, fibrinogen and combinations thereof to form an implant.

[0013] In one alternative, the plasma powder further comprises fibrinogen present in an amount from about 2.5 to 20 mg / mL a fibrinogen concentration.

[0014] In one alternative, the tissue regeneration multi-component biomaterial composition further comprises at least one cell.

[0015] In one alternative, the at least one cell is selected from the group consisting of keratinocytes, fibroblasts, melanocytes and combinations thereof.

[0016] In one alternative, there is provided a method for preparing a biomaterial comprising:

[0017] mixing chitosan with thrombin, trehalose, CaC , and freeze-drying to form a freeze-dried cake;

[0018] reconstituting the freeze-dried cake with PRP, plasma powder, fibrinogen and combinations thereof; and

[0019] allowing the mixture to solidify upon mixing forming an implant.

[0020] In one alternative said chitosan has a Mn of between 32-47 kDa and a DDA of between 80-90%.

[0021] In another alternative, there is provided a method for delivering cells to a site in need of treatment, comprising:

[0022] preparing a biomaterial described herein;

[0023] mixing the biomaterial with a cell suspension; and

[0024] applying the mixture to a tissue site.

[0025] In one alternative, the chitosan component is present in an amount ranging from 1 -4% w / v.

[0026] In one alternative, the trehalose component is present in an amount ranging from 0.5-1.5% w / v.

[0027] In another alternative, there is provided a tissue regeneration multi-component biomaterial composition, wherein the chitosan component is present in an amount ranging from 1-1 .5% w / v.

[0028] In another alternative, there is provided a tissue regeneration multi-component biomaterial composition, further comprising CaCl2 in an amount ranging from 42-63 mM.

[0029] In another alternative, there is provided a tissue regeneration multi-component biomaterial composition with a clotting time from 0.5 - 10.2 minutes.

[0030] In another alternative, there is provided a tissue regeneration multi-component biomaterial composition selected from the group consisting of:

[0031] a) 1 % w / v chitosan having a Mn of 39 kDa, a DDA of 82%; 1 % w / v trehalose;42 mM CaC ; thrombin selected from 0.25. 0.5 and 1 NIH / mL and PRP;

[0032] b) 1 % w / v chitosan having a Mn of 32 kDa, a DDA of 85%; 1 % w / v trehalose;42 mM CaC ; thrombin selected from 0.5, 1 , 2, 5 and 10 NIH / mL; fibrinogen selected from 2.5 and 10 mg / mL and plasma powder suspension;

[0033] c) 1 % w / v chitosan having a Mn selected from 37 and 47 kDa, a DDA selected from 83 and 90%; 1 % w / v trehalose; 42 mM CaC ; 5 NIH / mL thrombin; 10 mg / mL fibrinogen and plasma powder suspension;

[0034] d) Chitosan selected from 1 and 2% w / v having a Mn selected from 36 and 37 kDa and a DDA selected from 84 and 85%; 1 % w / v trehalose; 42 mM CaCl2; 10 NIH / mL thrombin; 10 mg / mL fibrinogen and 600000 cells / mL of keratinocytes;

[0035] e) Chitosan selected from 1 and 1.5% w / v having a Mn of 36 kDa and a DDA of 84%; trehalose selected from 1 and 1 .5% w / v; CaCL selected from 42 and 63 mM; thrombin selected from 2.5, 5 and 10 NIH / mL and 10 mg / mL fibrinogen;

[0036] f) 1 % w / v chitosan having a Mn of 37 kDa and a DDA of 83%; 1 % w / v trehalose;42 mM CaCh; 20 NIH / mL thrombin; 20 mg / mL fibrinogen and skin cells;

[0037] g) 1 % w / v chitosan having a Mn of 41 kDa and a DDA of 83%; 1 % w / v trehalose;42 mM CaCh; 20 NIH / mL thrombin;20 mg / mL fibrinogen and fibroblasts; and

[0038] h) 1 % w / v chitosan having a Mn of 39 kDa and a DDA of 83%; 1% w / v trehalose; 42 mM CaCh; 20 NIH / mL thrombin;20 mg / mL fibrinogen and skin cells.

[0039] In one alternative, the wound healing multi-component biomaterial compositions exhibit a clotting time of:

[0040] For composition a) 3.02 mins;

[0041] For composition b) from 28.6 mins to 9.6 mins with increasing thrombin with 2.5 mg / mL fibrinogen and from 10.2 mins to 0.5 mins with increasing thrombin with 10 mg / mL fibrinogen;

[0042] For composition c) between 0.4-0.7 mins for 90% DDA chitosan, and between 0.8-1.1 mins for 83% DDA chitosan;

[0043] For composition d) 3 mins;

[0044] For composition e) between 0.6 -5.75 mins for 1 % w / v chitosan and between 1 .75-8.9 mins for 1 .5% w / v chitosan;

[0045] For composition f) 2 mins; and

[0046] For composition g) 2 mins.

[0047] In yet another alternative, there is provided method of regenerating tissue at a site in need of tissue regeneration, comprising: applying a tissue regeneration multicomponent biomaterial composition as described herein, to the site in need of tissue regeneration.

[0048] In one alternative, said site in need of tissue regeneration is selected from a wound, surgical site and tissue defect.BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A-1 depicts a lyophilized material containing thrombin and thrombeolastograph curves of material with and without thrombin

[0050] Figure 1A-2 depicts clot reaction time, maximal amplitude and K value versus thrombin concentration of various formulations

[0051] Figure 1 B depicts the Force versus time to push 500 pL of the biomaterial of various thrombin concentrations through a syringe

[0052] Figure 1C-1 and 1 C-2 depict rheological results of biomaterials with varying thrombin concentration

[0053] Figure 1 D depicts stiffness and force at equilibrium of biomaterials with varying thrombin concentration

[0054] Figure 1 E depicts quantification of biomaterial adherence to tendon tissues

[0055] Figure 2A depicts cakes prepared with chitosan and thrombin of varying concentrations

[0056] Figure 2B depicts thromboelastograph curves of chitosan cakes with thrombin and 2.5 mg / mL or 10 mg / mL of fibrinogen

[0057] Figure 2C depicts thromboelastograph data of chitosan cakes with thrombin and 2.5 mg / mL of fibrinogen

[0058] Figure 2D depicts thromboelastograph data of chitosan cakes with thrombin and 10 mg / mL of fibrinogen

[0059] Figure 3A depicts chitosan cakes with varying degree of deacetylation of chitosan

[0060] Figure 3B depicts thromboelastograph curves of chitosan cakes of 83% DDA and 90% DDA with 5 NIH / mL thrombin and 10 mg / mL of fibrinogen

[0061] Figure 3C depicts clot reaction time and clot maximal amplitude of 83% DDA chitosan cakes with 5 NIH / mL thrombin and 10 mg / mL of fibrinogen

[0062] Figure 3D depicts clot reaction time and clot maximal amplitude of 90% DDA chitosan cakes with 5 NIH / mL thrombin and 10 mg / mL of fibrinogen

[0063] Figure 3E depicts stained sections from clots produced with 83% DDA chitosan and thrombin

[0064] Figure 3F depicts stained sections from clots produced with 90% DDA chitosan and thrombin

[0065] Figure 4A depicts confocal microscopic images of chitosan / fibrin clots containing cells after 1 day

[0066] Figure 4B depicts confocal microscopic images of chitosan / fibrin clots containing cells after ? days

[0067] Figure 4C depicts chitosan formulations on rat skin

[0068] Figure 4D depicts paraffin sections of treated rat skin

[0069] Figure 5A depicts thromboelastograph curves of various chitosan / thrombin / fibrinogen formulations

[0070] Figure 5B depicts clot reaction time and maximal amplitude versus thrombin concentration of various 1.0% chitosan / thrombin / fibrinogen formulations

[0071] Figure 5C depicts clot reaction time and maximal amplitude versus thrombin concentration of various 1.5% chitosan / thrombin / fibrinogen formulations

[0072] Figure 6A depicts chitosan / fibrin formulation containing a cell suspension isolated from cadaveric rat skin at delivery and after clotting at 2 minutes

[0073] Figure 6B depicts hematoxylin and Eosin-stained paraffin section of a chitosan / fibrin clot containing cells isolated from cadaveric rat skin. Black arrows point to cells

[0074] Figure 7A depicts confocal microscopy images of Live-Dead stained Rat-1 fibroblasts in the chitosan / fibrin clots at 1 , 3 and 7 days post-clotting. White arrows point to cells

[0075] Figure 7B depicts results of the Presto Blue assay showing increased Rat-1 fibroblast metabolic activity with time in culture

[0076] Figure 7C depicts paraffin sections of a chitosan / fibrin clot containing Rat-1 fibroblasts on rat skin stained with Hematoxylin and Eosin. Black arrows point to cells

[0077] Figure 8A depicts splinted wounds on rat skin treated with a chitosan / fibrinogen formulation containing autologous cells (top panels) or with autologous cells only (bottom panels) at different time points post-surgery

[0078] Figure 8B depicts % wound closure at different time points post-surgery.DETAILED DESCRIPTION

[0079] Referring now to FIG. 1 A-1 , there is depicted one alternative of a) the biomaterial in a vial, which is a lyophilized formulation of chitosan, designed to be mixed with platelet-rich plasma (PRP) to create a hybrid biomaterial that coagulates in situ. To ensure rapid coagulation in situ during arthroscopic surgery, thrombin was added to the formulation. FIG. 1A-1 b) depicts typical thromboelastograph curves obtained for biomaterial with and without thrombin. FIG. 1A-1c) depicts typical thromboelastograph curves obtained for PRP with and without thrombin.

[0080] Referring now to FIG. 1A-2d), there is depicted clot reaction time (R) (time needed to reach an amplitude of 2 mm) of formulations with and without thrombin at various times after storage at room temperature. It is evident the clot reaction times of formulations with thrombin are statistically shorter than clot reaction times of formulations without thrombin. FIG. 1A-2e) depicts clot maximal amplitude (MA) in mm (indicative of clot strength) and FIG. 1A-2f) depicts the K value in minutes (time needed after R to reach an amplitude of 20 mm) with a p-value < 0.05. 0 indicates the mean value. Note that statistically significant differences were also observed for the R values between samples with and without thrombin issued from different timepoints but are not indicated on the figure for clarity’s sake. For the biomaterial with and without thrombin at t=0, n=7 and n=3, at t=1 month, n=6 and n=3 and at t=2 months, n=2 and n=3. For PRP controls with and without thrombin, n=6 and n=2.

[0081] Referring now to FIG. 1 B, there is depicted the force in Newtons (N) (lines indicate mean values and envelopes indicate min and max values) versus time in minutes necessary to push 500 microlitre (pL) of the biomaterial out of a 10 cc-syringe equipped with a spinal 18-gauge needle every two minutes, 20 times (N=6 and n=2). The 0.5 NIH / mL solution allows for easy ejection of the biomaterial as the force required for biomaterial ejection remains below 20N for at least 6 minutes. This makes it easier to manipulate than the 1.0 NIH / mL solution, for which a force exceeding 20 N is needed for biomaterial ejection after 4-5 minutes.

[0082] Referring now to FIG. 1C-1 there is depicted rheological results. At FIG. 1 C-1 a) Vertical black line indicates the beginning of the test (t=0 minutes). On the right of this vertical line: Time sweeps mean raw data for storage (G’, full lines) and loss (G”,dotted lines) modulus of biomaterial with thrombin (0.0, 0.25, 0.5, 1.0 NIH / ml) for a displacement of 1 % and a frequency of 5 rad / sec. On the left of the vertical black line: data were extrapolated based on the first 3 minutes of the test for thrombin concentration of 0.5 and 1.0 NIH / mL to determine their gelation point. The equation y = A + BeRtwas used for the fit, where R>0, y is either the loss or the storage modulus and t is the time. Black dots: gelation point (G’=G”) for each thrombin concentration. FIG. 1 C-1 b) is a Zoom of FIG. 1 C-1 a) for t = -1 to 3 minutes.

[0083] Referring now to FIG. 1C-2c) there is depicted time to gelation versus thrombin concentration. The black dots show the time needed to reach the gelation point for biomaterials with each thrombin concentration (0.0, 0.25, 0.5, 1.0 NIH / ml). In order to account for the time elapsed between the rehydration of the biomaterial and the start of the test, 1.25 minutes were added to the experimental time observed in FIG. 1C- 1 b). The experimental data were fitted using the equation y = AeRx, where R<0 (solid curves), y is the time to gelation and x is the thrombin concentration. FIG. 1C-2d) depicts the results of phase angle (5°) for biomaterials (0.0, 0.25, 0.5 or 1 .0 NIH / ml of thrombin) over 60 minutes. 5 = arctan (G” / G’). The experimental data was fitted using the equation 6 = A + BeRx, where R<0 (solid curves), and x is the thrombin concentration. FIG. 1C-2e) depicts values of G’ (Pa) and FIG. 1C-2f) depicts values of G” (Pa) for biomaterials with or without thrombin (0.0, 0.25, 0.5, 1 .0 NIH / ml) at 1 , 2, 5, 7 and 10 minutes (• are experimental data). The values of G’ and G” (Pa) at those times were empirically fitted with the equation y = A - BeRx, where R<0 (solid curves), and x is the thrombin concentration. For results presented in FIG. 1C-1a) and FIG. 1 C-2d), n=2 and n=2. For results presented in FIG. 1C-1 b), FIG. 1C-2c), FIG. 1C- 2e) and FIG. 1 C-2f), n=5 and n=2.

[0084] Referring now to FIG. 1 D, there is depicted the results of two experiments for the average stiffness and force at equilibrium in response to an indentation compressive strain (amplitude of 0.7 mm - 10% of sample’s height -, speed of 0.07mm / s) for i) biomaterials with different concentrations of thrombin (0.0, 0.25, 0.5 and 0.1 NIH / mL) and ii) PRP. The presence of additional thrombin appears to leave unchanged the mechanical properties of the completely solidified biomaterial. Both the stiffness and the force at equilibrium remained stable across every thrombin concentration tested. A difference is however noticeable between the stiffness of the biomaterial and the stiffness of PRP, indicating that the presence of Chitosan (CS)increases the resistance of the biomaterial to compression. CS does not appear to have an impact on the equilibrium force. n=2 and n=3.

[0085] Referring now to FIG. 1 E, there is depicted the quantification of the adherence of the biomaterial to tendon tissues when soaked in 0.9% NaCI for biomaterial with and without 0.5 NIH / mL of thrombin. After soaking, a photo of the NaCI solution was taken and the amount of biomaterial detached from the tendon was quantified by its red channel weight. FIG. 1 E a) shows the results when the biomaterial was injected on the tendon immediately after reconstitution with PRP and left for 0, 2, 5 or 7 minutes. The tendon was then soaked in 0.9% NaCI. FIG. 1 E b) shows the results when the biomaterial was left for 0, 2, 5 or 7 minutes in a 10cc syringe before being placed on the tendon. A 2-minute wait was then observed before soaking the tendon in 0.9% NaCI. FIG. 1 E c) is a photograph of the aspect of the biomaterial just after its injection on the tendon, before soaking. FIG. 1 E d) is a photograph when no waiting time is observed after injection of the biomaterial, the biomaterial almost completely dissolved in 0.9% NaCI when soaked. FIG 1 E e) is a photograph of the tendon when a 5-minute waiting time is observed after injection of the biomaterial containing 0.5 NIH / mL of thrombin. The biomaterial does not dissolve when soaked in 0.9% NaCI and its aspect on the tendon seems unchanged. For results presented in FIG. 1 E a) and FIG. 1 E b), n=2 and n=2.

[0086] Referring now to FIG. 2A, there is depicted photographs of various chitosan cakes in vials wherein the cakes were prepared with chitosan number average molar mass ( / Wn) 32 kDa and 85% degree of deacetylation (DDA) and different concentrations of thrombin from 0.5 NIH / mL to 10 NIH / mL thrombin.

[0087] Referring now to FIG. 2B, there is depicted thromboelastography (TEG) tracings for chitosan cakes containing thrombin of FIG. 2A mixed with plasma powder suspensions containing 2.5 mg / mL fibrinogen and 10 mg / mL.

[0088] Referring now to FIG. 2C, there is depicted clot reaction times and maximal amplitude data for chitosan cakes containing thrombin mixed with plasma powder suspensions containing 2.5 mg / mL fibrinogen.

[0089] Referring now to FIG. 2D, there is depicted clot reaction times and maximal amplitude data for chitosan cakes containing thrombin mixed with plasma powder suspensions containing 10 mg / mL fibrinogen.

[0090] Referring now to FIG. 3A, there is depicted photographs of chitosan cakes prepared with chitosan number average molar mass ( / Wn) 37 kDa and 83% degree of deacetylation (DDA) (FIGS. 3A a and b) and chitosan number average molar mass ( / Wn) 47 kDa and 90% degree of deacetylation (DDA) (FIGS. 3A c and d) with 5 NIH / mL thrombin.

[0091] Referring now to FIG. 3B, there is depicted TEG tracings for chitosan cakes containing 83% DDA and 90% DDA of chitosan containing 5 NIH / mL thrombin with plasma powder suspensions (PT-0001 and PT-0321) containing 10 mg / mL fibrinogen.

[0092] Referring now to FIG. 3C, there is depicted clot reaction time and clot maximal amplitude data for chitosan cakes of 83% DDA with chitosan number average molar mass ( / Wn) 37 kDa and 5 NIH / mL thrombin mixed with two different plasma powder suspensions (PT-0001 and PT-0321 ) containing 10 mg / mL fibrinogen.

[0093] Referring now to FIG. 3D, there is depicted clot reaction time and clot maximal amplitude data for chitosan cakes of 90% DDA with chitosan number average molar mass ( / Wn) 47 kDa and 5 NIH / mL thrombin mixed with two different plasma powder suspensions (PT-0001 and PT-0321 ) containing 10 mg / mL fibrinogen.

[0094] Referring now to FIG. 3E, there is depicted Cibacron Red-stained sections from clots produced with chitosan number average molar mass ( / Wn) 37 kDa and 83% DDA and 5 NIH / mL thrombin.

[0095] Referring now to FIG. 3F, there is depicted Cibacron Red-stained sections from clots produced with chitosan number average molar mass ( / Wn) 47 kDa and 90% DDA and 5 NIH / mL thrombin.

[0096] Referring now to FIG. 4A, there is depicted confocal microscopy images of Live- Dead stained cells in the chitosan / fibrin clots at 1-day post-clotting. Each panel shows a single Z-slice from a 1 .61 mm2 area within a clot that had a total size of 1 .9 cm2 X 4 mm. The white arrows point to cells in the clots.

[0097] Referring now to FIG. 4B, there is depicted confocal microscopy images of Live- Dead stained cells in the chitosan / fibrin clots at 7-days post-clotting. Each panel shows a single Z-slice from a 1 .61 mm2 area within a clot that had a total size of 1 .9 cm2 X 4 mm. The white arrows point to cells in the clots.

[0098] Referring now to FIG. 4C, there is depicted images of rat skin after different chitosan formulations were applied on rat skin by solution viscosity spray or solution viscosity drip and the clotting times

[0099] Referring now to FIG. 4D, there is depicted images of paraffin sections stained with Hematoxylin and Eosin identifying the chitosan / fibrin and the keratinocytes.

[0100] Referring now to FIG. 5A, there is depicted TEG curves for formulations with 1 % w / v and 1.5% w / v chitosan containing thrombin at three different concentrations, 2.5, 5, and 10 NIH / mL, mixed with a 10 mg / mL fibrinogen solution.

[0101] Referring now to FIG. 5B, there is depicted TEG data clot reaction time and maximal amplitude for formulations with 1 % w / v chitosan containing thrombin at three different concentrations, 2.5, 5, and 10 NIH / mL, mixed with a 10 mg / mL fibrinogen solution.

[0102] Referring now to FIG. 5C, there is depicted TEG data clot reaction time and maximal amplitude for formulations with 1 .5% w / v chitosan containing thrombin at three different concentrations, 2.5, 5, and 10 NIH / mL, mixed with a 10 mg / mL fibrinogen solution.

[0103] Referring now to FIG. 6A, there is depicted Chitosan / fibrin formulation containing a cell suspension of Example 6 at delivery (left side photo) and after clotting at 2 minutes (right side photo).

[0104] Referring now to FIG. 6B, there is depicted the presence of cells in the solid clot (left side photo) and at a higher magnification (right side photo) of Example 6.

[0105] Referring now to FIG. 7A, there is depicted the live cells in the clots formed from the formulation of Example 7 and live cells increased over time from day 1 to day 7.

[0106] Referring now to FIG. 7B, there is depicted the change in metabolic activity in the clot from day 1 to day 7.

[0107] Referring now to FIG. 7C, there is depicted the fibroblast cells visible in the clots on the rat skin (at various levels of magnification).

[0108] Referring now to FIG. 8A, there is depicted the change in wound healing from day 0 to day 23 treated with chitosan / fibrin with autologous cells along the top panel and from day 0 to day 22 treated with autologous cells only along the bottompanel. It is clear the chitosan / fibrin with autologous cells healed much quicker that autologous cells treatment alone.

[0109] Referring now to FIG. 8B, there is depicted the percent wound closure with chitosan / fibrin and autologous cells formulation (test article) versus treatment with autologous cells alone. It is clear the test article would closure was greater over periods of time compared to the autologous cell treatment alone.EXAMPLE 11- Preparation of freeze-dried chitosan-thrombin formulations

[0110] Chitosan formulations containing 1 % w / v chitosan number average molar mass ( / Wn) 39 kDa and 82% degree of deacetylation (DDA) with 1 % w / v trehalose, 42 mM CaCl2, 0.01 % bovine serum albumin (BSA) and different concentrations of thrombin (0.25 NIH / mL, 0.5 NIH / mL and 1 NIH / mL) were prepared.

[0111] All vials were then freeze-dried through the following cycle using a Millrock freeze-dryer (Laboratory series LD85S3): 1 ) step freezing (to 20 °C then isothermal for 15 min, to 5°C then isothermal for 30 min, to -5 °C then isothermal for 30 min) then ramp freezing to -40°C at -1 °C / min, then isothermal for 2h; 2) ramp heating at 0.5°C / min up to 8°C at 76 mTorr, then isothermal for 650 min; 3) ramp heating at 0.15°C / min until 40°C at 76 mTorr, then isothermal for6h. A less aggressive cycle was initially used, heating up to only 25°C during step 3 (secondary drying), but for 10 hours. However, no differences were observed in the resulting chitosan formulations, and the cycle heating up to 40°C was preferred for the rest of the study.

[0112] Freeze-dried chitosan-thrombin cakes were slightly retracted from the vial walls for all thrombin concentrations (Figure 1 A-1a).4-lsolation of platelet-rich plasma

[0113] Commercial sterile citrated sheep blood (Cedarlane, Product N°CL2581- 500C) was used to isolate the leucocyte-rich PRP. Briefly, whole blood was distributed in 10 mL aliquots in 15 mL Falcon tubes, then centrifuged at 800 g for 20 minutes (unforced deceleration). The supernatant, buffy coat and first 1-2mm of erythrocytes were collected in new 15 mL Falcon tubes, and a second centrifugation was then performed at 600 g for 10 minutes (unforced deceleration).5- Rehydration of the chitosan-thrombin formulations

[0114] Freeze-dried chitosan-thrombin cakes were reconstituted in an isovolume of PRP by vigorous manual shaking for 30 sec.

[0115] Freeze-dried chitosan-thrombin formulations rapidly solubilized in PRP without any apparent influence by the thrombin concentration.6- Assessment of solidification properties of chitosan-thrombin formulations by thromboelastography

[0116] Following the force, clotting and indentation testing described below, the chitosan formulation containing 0.5 NIH / mL of thrombin was chosen for further testing. A stability study was conducted for a period of two months, and thrombin activity was assessed at three specific time points (t = 0, 28, and 56 days) using thromboelastography (TEG). All the samples were stored at room temperature. 360 pL of chitosan-thrombin-PRP formulation was loaded in a TEG Model 5000, Haemoscope Corp., Niles, IL, USA and TEG tracings acquired.

[0117] Thrombin had a statistically significant impact on the biomaterial’s clot reaction time compared to the biomaterial without thrombin (control) over a period of up to 56 days (Figure 1A-2 d-f). The average clot reaction time (R) for the control biomaterial was 4.04 min, while biomaterial with 0.5 NIH / mL of thrombin had an average R of 3.02 min over the three time points. MA had a consistent value of approximately 67 mm at all time points, irrespective of the thrombin concentration. No significant differences in clot reaction time were observed between samples with thrombin at each time point, suggesting that no activity loss occurred during this 2- month period.7- Assessment of the force required to eject the biomaterial

[0118] The protocol to measure the force required to eject the biomaterial was implemented on the multi-axis mechanical tester Mach-1 , Model V500css (Biomomentum Inc., Laval, QC, Canada), with a 25 kg uniaxial load cell and a flat circular indenter with a diameter of 30 mm. The material was placed in a syringe and 0.5 mL of the biomaterial was pushed every 2 min, 10 times, at a speed of 1 mm / s (resulting in 3 s of movement). Force data was saved at a frequency of 100 Hz for the whole duration of the test.

[0119] The 0.5 NIH / mL solution allows for easy ejection of the biomaterial as the force required for biomaterial ejection remains below 20 N for at least 6 min (Figure1 B). This makes it easier to manipulate than the 1.0 NIH / mL solution, for which a force exceeding 20 N is needed for biomaterial ejection after 4-5 min.8- Assessment ofclotting properties of the biomaterial through rheology measurements

[0120] Clotting properties were measured using a Physica MCR 501 rheometer (Anton Paar, Graz, Austria). Concentric cylinders with rough geometry were used for measurements (CC17 / T200 / SS / p, cup diameter of 18.08 mm, and bob diameter of 16.66 mm). Rheological properties were measured during a time sweep over the course of either 1 h or 15 min at a fixed 1 % strain and a fixed 5 rad / s frequency. All measurements were performed at 37°C.

[0121] The gelation point (cross-over between G' and G") occurred on average at 4.15 min after rehydration of freeze-dried samples in PRP for the controls without thrombin and after 2.40, 1.03, and 0.35 min for the formulations containing, respectively, 0.25, 0.5, and 1.0 NIH / mL of thrombin (Figures 1C-1 and 1C-2).9- Assessment of hybrid-clot mechanical properties through indentation

[0122] The indentation protocol used to assess clot mechanical properties was implemented on the multi-axis mechanical tester Mach-1 , Model V500css (Biomomentum Inc., Laval, QC, Canada) with a 150 g uniaxial load cell and a spherical indenter with a radius of 1 ,5mm. For each sample, the surface of the biomaterial was found by lowering the probe at a speed of 0.05 mm / s until a force of 0.1 gf was detected. A single indentation of 0.7 mm was then performed at a velocity of 0.07 mm / s. Force data were saved at a frequency of 100 Hz during the indentation and the relaxation that followed, until the relaxation rate reached 1 gf / min (slope measured for 10 s).

[0123] Despite its ability to increase the speed of the coagulation process, additional thrombin does not seem to have a significant impact on the mechanical properties of the fully coagulated clot, as the force at equilibrium and stiffness were stable regardless of the thrombin concentration tested, and no correlation was observed between either of them and thrombin concentration (Figure 1 D).10- Assessment of hybrid-clot homogeneity through histology and MASQH algorithm

[0124] Clots were fixed in 10% neutral buffered formalin (NBF) before being dehydrated, cleared, paraffin embedded, sectioned and stained with Cibacron Brilliant Red / Weigert iron hematoxylin. The homogeneity of the chitosan distribution in the hybrid clot was evaluated using the MASQH algorithm

[0016] ,

[0125] The histology of the clots confirmed that chitosan was evenly distributed among blood components in the presence of thrombin. A MASQH homogeneity score of 0.92 ± 0.03 was computed for chitosan-PRP clots without thrombin and of 0.93 ± 0.02 for chitosan-PRP clots with 0.5 NIH / mL.11- Assessment of biomaterial adhesion to tendon tissues using an ex vivo test

[0126] Ex vivo evaluation of the chitosan-thrombin-PRP formulations adhesion to tendon tissues was performed using bovine tendons obtained from a local butcher shop. Prior to testing, tendons were defrosted and heated to 37°C in a 0.9% NaCI bath. Right after mixing a chitosan-thrombin cake with PRP, the formulation was retrieved from its vial with a 10 mL syringe and an 18 G needle and a waiting time varying between 0 and 7 min was observed to mimic the time taken by the surgeon between mixing the biomaterial and its use. Next, 3 mL of the biomaterial was delivered to the tendon, and a second waiting time varying between 0 and 7 min was observed. The tendon and adhered biomaterial were submerged in a 1 L 0.9% NaCI bath and twisted in the fluid for 1 min to simulate the presence of physiological fluids and movement of the shoulder. The quantity of biomaterial remaining on the tendon was then visually observed (qualitative evaluation). The color of the 0.9% NaCI bath was quantitatively evaluated through image analysis: A photograph of the bath was taken, and the weight of its red channel was computed using the Imaged software.

[0127] Most of the biomaterial dissolved in the fluid after 1 min in the absence of a waiting period, while most of it remained on the tendon after a 5-min waiting period (Figure 1 E).12- Assessment of thrombin activity in freeze-dried chitosan formulations

[0128] The activity of thrombin in the CS formulations was quantified before FD and in CS FD cakes using a fluorometric thrombin activity assay kit (Abeam, ab197006). FD samples were rehydrated in water instead of PRP. All samples were diluted at 1 / 2 and 1 / 5 in assay buffer. As per Abeam protocol, 6 standards were used to construct a standard curve, with concentrations ranging from 0.26 NIH / mL to 1.3 NIH / mL. Fluorescence was measured at Ex / Em = 350 / 450 at 37°C in a kinetic modeevery 2 minutes for 60 minutes using an Infinite M200 reader (Tecan Trading AG, Switzerland).

[0129] An activity loss of -20-25% was observed after lyophilization for chitosan formulations containing 0.5 NIH / mL of thrombin.13- Rotator cuff repair model

[0130] A rotator cuff repair study in 24 skeletally mature New Zealand White rabbits was initiated. Supraspinatus tendons were bilaterally transected and repaired with transosseous suturing. In each animal, one shoulder was additionally treated with freeze-dried chitosan-thrombin reconstituted in autologous PRP. Necropsies have been performed and analysis is ongoing.14- Summary of Example 1

[0131] This work demonstrated that thrombin has the potential to accelerate the solidification of chitosan-PRP implants. Thrombin may be added to the chitosan formulation before freeze-drying, and a balance may be achieved to obtain a biomaterial that is both fast to coagulate and easy to handle / inject. Lyophilized thrombin inside the chitosan cakes was stable for 2 months at room temperature.EXAMPLE 21- Preparation of freeze-dried chitosan-thrombin formulations

[0132] Chitosan formulations containing 1 % w / v chitosan number average molar mass ( / Wn) 32 kDa and 85% degree of deacetylation (DDA) with 1 % w / v trehalose, 42 mM CaCh, 0.01 % BSA and different concentrations of thrombin (0.5 NIH / mL, 1 NIH / mL, 2 NIH / mL, 5 NIH / mL and 10 NIH / mL) were freeze-dried as described above in Example 1 .

[0133] All freeze-dried chitosan-thrombin cakes were slightly retracted from the vial walls (Figure 2A).2- Rehydration of the chitosan-thrombin formulations

[0134] Plasma powder was resuspended in 0.9% NaCI containing either 2.5 mg / mL or 10 mg / mL fibrinogen. Freeze-dried chitosan-thrombin cakes were reconstituted in an isovolume of suspended plasma powder by vigorous manual shaking for 20 sec.

[0135] All freeze-dried cakes were soluble in suspended plasma powder.3- Assessment of solidification properties of chitosan-thrombin formulations by thromboelastography

[0136] 360 pL of each sample was loaded in a sample cup and the TEG test was allowed to run until maximal amplitude was reached.

[0137] All mixtures clotted. For plasma powder suspensions containing 2.5 mg / mL fibrinogen, clot reaction time decreased from 28.6 minutes to 9.6 minutes with increasing thrombin concentrations (Figure 2C). Clots were soft and had low maximal amplitudes. For plasma powder suspensions containing 10 mg / mL fibrinogen, clot reaction time decreased from 10.2 minutes to 0.5 minutes with increasing thrombin concentrations (Figure 2D). Clots were much stiffer and had greater maximal amplitudes.4- Assessment of hybrid-clot homogeneity through histology

[0138] Clots were fixed in 10% neutral buffered formalin (NBF) before being dehydrated, cleared, paraffin embedded, sectioned and stained with Cibacron Brilliant Red / Weigert iron hematoxylin.

[0139] Plasma powder particles were distributed throughout the clots, within a homogenous mixture of chitosan and fibrin fibers.5- Assessment of biomaterial delivery to tendon tissues using an ex vivo test

[0140] For ex vivo work, delivery was on top of bovine tendon. Prior to testing, tendons were defrosted and heated to 37°C in a PBS bath. 2 mL mixture was aspirated from the Ortho-R vial into a 5-cc syringe through a dispensing pin. Half the volume of the mixture (1 mL) was immediately injected on top of a bovine tendon.

[0141] For plasma powder suspensions containing 2.5 mg / mL fibrinogen, the mixtures containing 0.5 to 5 NIH / mL of thrombin were liquid at time 0, while the mixture containing 10 NIH / mL of thrombin was already starting to solidify at time 0. For plasma powder suspensions containing 10 mg / mL fibrinogen, the mixtures containing 0.5 and 1 NIH / mL of thrombin were liquid at time 0, the mixture containing 2 NIH / mL of thrombin was already solidifying at time 0, and the mixtures containing 5 and 10 NIH / mL of thrombin were already gel-like at time 0.6-Summary of example 2

[0142] This work demonstrated that freeze-dried chitosan formulations that also contain lyophilized thrombin may be reconstituted with plasma powder-fibri nogensuspensions to form injectable implants that solidify. Increasing the thrombin concentration in the cakes decreases solidification time post-mixing. Mixtures solidify more quickly when more fibrinogen is used to resuspend the plasma powder and the clots are stiffer. Plasma powder particles are distributed throughout the solid clots.EXAMPLE 31- Preparation of freeze-dried chitosan-thrombin formulations

[0143] Two chitosans were used in this example: 1) Chitosan number average molar mass ( / Wn) 37 kDa and 83% degree of deacetylation (DDA) and 2) Chitosan number average molar mass ( / Wn) 47 kDa and 90% degree of deacetylation (DDA). Chitosan formulations containing 1 % w / v chitosan with 1 % w / v trehalose, 42 mM CaC , 0.01 % BSA and 5 NIH / mL thrombin were freeze-dried as described above in Example 1 . Different formats of cakes were prepared and tested in triplicate.

[0144] All freeze-dried chitosan-thrombin cakes were slightly retracted from the vial walls (Figure 3A).2- Rehydration of the chitosan-thrombin formulations

[0145] Plasma powder was resuspended in 0.9% NaCI containing 10 mg / mL fibrinogen. Freeze-dried chitosan-thrombin cakes were reconstituted in an isovolume of suspended plasma powder by vigorous manual shaking for 20 sec.

[0146] All freeze-dried cakes were soluble in suspended plasma powder.3- Assessment of solidification properties of chitosan-thrombin formulations by thromboelastography

[0147] 360 pL of each sample was loaded in a sample cup and the TEG test was allowed to run until maximal amplitude was reached.

[0148] Coagulation kinetics were repeatable for the different formulations (Figures 3B-3D). Clot reaction time was slightly shorter for the formulations containing chitosan 90% (0.4-0.7 min) than for the formulations containing chitosan 83% (0.8-1.1 min). Clot maximal amplitude was slightly higher for the formulations containing chitosan 90% (average 49.7 mm) than for the formulations containing chitosan 83% (average 43.6 mm).4- Assessment of hybrid-clot homogeneity through histology

[0149] Clots were fixed in 10% neutral buffered formalin (NBF) before being dehydrated, cleared, paraffin embedded, sectioned and stained with Cibacron Brilliant Red / Weigert iron hematoxylin.

[0150] Plasma powder particles were distributed throughout the clots, within a scaffold made of fibrin and chitosan (Figures 3E and 3F).5- Assessment of biomaterial delivery to tendon tissues using an ex vivo test

[0151] For ex vivo work, delivery was on top of bovine tendon. Prior to testing, tendons were defrosted and heated to 37°C in a PBS bath. Mixtures were aspirated from the Ortho-R vials into 3-cc syringes through a dispensing pin. Mixtures were injected on top of bovine tendon every 30 seconds through an 18-gauge needle

[0152] All the mixtures were already starting to coagulate at the first injection, but the mixtures containing chitosan 90% DDA were more gel-like than the mixtures containing chitosan 83% DDA at time 0. At times 2 min or 2 min 30 sec, the mixtures had become too solid to be extruded easily through an 18-gauge needle.6-Summary of example 3

[0153] This work demonstrated that chitosans with different number average molar masses and degrees of deacetylation (DDA) may be used to prepare chitosan- thrombin cakes of different formats. Results were repeatable and formulations consistently coagulated within a few minutes post-mixing to produce solid clots that contain plasma powder particles distributed within a fibrin / chitosan network.EXAMPLE 41- Preparation of chitosan-thrombin formulations

[0154] Chitosan formulations containing 4% w / v or 2% w / v chitosan number average molar mass ( / Wn) 36 kDa and 84% degree of deacetylation (DDA) with 1 % w / v trehalose, 42 mM CaCl2 were prepared.

[0155] Freeze-dried chitosan cakes containing 1 % w / v chitosan number average molar mass ( / Wn) 37 kDa and 85% degree of deacetylation (DDA) with 1 % w / v trehalose, 42 mM CaC were reconstituted at ! or % of their original volume in sterile water for injection, in order to obtain solutions with 4% w / v or 2% w / v chitosan.

[0156] Stock thrombin solution (100 NIH / mL) was added to each chitosan solution to make chitosan solutions with thrombin at 20 NIH / mL.2- Preparation of cell suspensions in a buffer containing fibrinogen

[0157] Fibrinogen was added to Ringer’s lactate and mixed at 37°C to dissolve to make a Ringer’s solution with 20 mg / mL fibrinogen.

[0158] A primary keratinocyte suspension (P3) was added to the Ringer’s lactate-fibrinogen and gently swirled to mix well (to obtain a final concentration of 600 000 cells / mL).3- Mixing of chitosan-thrombin with cell suspensions

[0159] Chitosan-thrombin formulations and the keratinocyte-Ringer’s lactatefibrinogen suspensions were mixed at a 1 :1 ratio using dual syringe systems equipped with either a tip with a static mixer (for the 4% w / v chitosan solutions) or with a spraying tip (for the 2% chitosan solutions).4- Cell culture

[0160] For cell culture, delivery was in 24-well plates (0.5 mL chitosan formulation + 0.5 mL keratinocyte-Ringer’s lactate-fibrinogen suspension). Formulations were allowed to clot and were then covered with supplemented dermal cell medium. The formulations were cultured for 7 days.

[0161] The chitosan formulations containing thrombin / fibrinogen solidified within 3 minutes following application in the cell culture vessels. Keratinocytes were visible within the clots.

[0162] Live-Dead solution was prepared as per manufacturer’s instructions and mixed at a 1 :1 ratio with culture medium. Culture medium was removed from the wells and replaced with 0.4 mL Live-Dead / medium solution. Plates were covered in aluminum paper and incubated for 15 minutes at room temperature. For imaging with the confocal microscope, 6 mm biopsy punches were used to extract cores and the cores were placed on 35 mm Mattek chambers for imaging.

[0163] Live cells were detected in the chitosan / fibrin clots. Live cells were more numerous in the chitosan / fibrin clots at day 7 when compared to day 1 (Figures 4A and 4B)

[0164] 100 pL Presto Blue solution was added to each well. Plates were incubated at 37°C for 1 h. Absorbance of the Presto Blue / culture medium solutions was read in cuvettes at 570 nm using 600 nm as a reference wavelength.

[0165] Metabolic activity was higher in the chitosan / fibrin clots that contained cells versus clots without cells at day 1. Metabolic activity in the chitosan / fibrin clots was higher at day 7 versus day 1 .5- Assessment of biomaterial delivery to rat skin using an ex vivo test

[0166] For ex vivo work, delivery was on cadaveric rat (0.5 mL chitosan formulation + 0.5 mL keratinocyte-Ringer’s lactate-fibrinogen suspension). The skin of the rat was shaved and the skin was scratched with a pointy metal implement to create wounds prior to delivery.

[0167] The high viscosity chitosan formulations clotted within 3 minutes of application and the lower viscosity chitosan formulations clotted within 1 minute of application (Figure 4C). The high viscosity clots applied with the drip method were thicker than the lower viscosity clots applied with the spray method. The chitosan / fibrin clots remained in place on the skin when the animal was tilted.

[0168] Skin samples were fixed in 10% neutral buffered formalin, histoprocessed and stained with Hematoxylin and Eosin.

[0169] Chitosan / fibrin clots containing keratinocytes were visible on histological sections of rat skin (Figure 4D).6-Summary of example 4

[0170] This work demonstrated that chitosan formulations with fibrinogen / thrombin may be used to deliver primary keratinocytes. The keratinocytes remained alive and metabolically active after delivery in tissue culture vessels for up to 7 days. The formulations coagulated within a few minutes after delivery to rat skin and remained in place when the animal was tilted. The more viscous chitosan formulations coagulated a bit slower than the less viscous formulations and must be applied with a drip method, and not by spraying, which led to a thicker coating.EXAMPLE 51- Preparation of chitosan-thrombin formulations

[0171] Chitosan formulations containing 3% w / v or 2% w / v chitosan number average molar mass ( / Wn) 36 kDa and 84% degree of deacetylation (DDA) with 3% w / v or 2% w / v trehalose, 126.6 mM or 84.4 mM CaCL were prepared.

[0172] Stock thrombin solution (100 NIH / mL) was added to each chitosan solution to make chitosan solutions with thrombin at 2.5, 5 or 10 NIH / mL.2- Preparation of fibrinogen solution

[0173] Fibrinogen was added to Ringer’s lactate and mixed at 37°C to dissolve to make a Ringer’s solution with 20 mg / mL fibrinogen.3- Mixing of chitosan-thrombin with fibrinogen solution

[0174] Chitosan-thrombin formulations and the Ringer’s lactate-fibrinogen solution were mixed at a 1 :1 ratio using dual syringe systems equipped with a tip with a static mixer. This led to formulations of 1.5% w / v or 1 % w / v chitosan final concentration.4- Assessment of solidification properties of chitosan-thrombin formulations by thromboelastography

[0175] 360 pL of each sample was loaded in a sample cup and the TEG test was allowed to run until maximal amplitude was reached.

[0176] Clot reaction time decreased with the increase of thrombin concentration (Figures 5A, 5B, 5C). Clot reaction times were shorter for the formulations containing 1 % w / v chitosan (0.6-5.75 min) than for the formulations containing 1.5 % w / v chitosan (1.75-8.9 min). For the same thrombin concentration, clot maximal amplitude was higher for the formulations containing 1 % w / v chitosan (22-24 mm) than for the formulations containing 1.5% w / v chitosan (17-21 mm) (Figures 5A, 5B, 5C).5-Summary of example 5

[0177] This work demonstrated that chitosan formulations with fibrinogen / thrombin may be used to obtain formulations that solidify quickly (less than 6 minutes for formulations with 1 % chitosan). The more viscous chitosan formulations coagulated a bit slower than the less viscous formulations (all formulations coagulated in less than 9 minutes). This work also demonstrated that less viscous formulations produced clots with greater maximal amplitude than the clots obtained with more viscous formulations.EXAMPLE 67- Preparation of chitosan-thrombin formulations

[0178] Freeze-dried chitosan cakes containing 1 % w / v chitosan number average molar mass ( / Wn) 37 kDa and 83% degree of deacetylation (DDA) with 1 % w / v trehalose, 42 mM CaC were reconstituted at % of their original volume in sterile water for injection, in order to obtain solutions with 2% w / v chitosan.

[0179] Stock thrombin solution (200 NIH / mL) was added to the chitosan solutions in order to obtain thrombin concentration of 40 NIH / mL.2- Isolation of cell suspension from rat skin

[0180] 8-mm biopsy punches were used to collect skin biopsies from a cadaveric rat.

[0181] The biopsies were rinsed in 10 mL Ringer’s lactate, two biopsies per tube.

[0182] The biopsies were placed in a Petri dish and scissors were used to remove the subcutaneous fat tissue.

[0183] 0.5% Trypsin-EDTA solution was pre-heated at 37°C in a small bacterial oven.

[0184] Two 8-mm biopsies were placed in each Trypsin-containing tube and the tubes were placed at 37°C.

[0185] The tubes were incubated for 30 minutes.

[0186] The biopsies were rinsed in 10 mL Ringer’s lactate to get rid of residual enzyme.

[0187] Syringes with 0.9 mL Ringer’s lactate were prepared.

[0188] The biopsies were placed on a Petri dish and a few drops of Ringer’s lactate were ejected onto the biopsies from the syringes.

[0189] The epidermis and then the dermis were scraped with the scalpel blade until the dermis had nearly disintegrated.

[0190] The remaining volume of Ringer’s lactate was emptied from the syringe on the Petri dish to get all the cells in one corner of the dish.

[0191] The cell suspension was aspirated up and down to make sure that all cells were collected.

[0192] The cell suspension was filtered through a cell strainer (40 pm).3- Preparation of cell suspensions in a buffer containing fibrinogen

[0193] Fibrinogen was added to Ringer’s lactate and mixed at 37°C to dissolve to make a Ringer’s solution with 60 mg / mL fibrinogen.

[0194] 200 pL isolated cell suspension was mixed with 400 pL Fg (60 mg / mL).4- Mixing of chitosan-thrombin with cell suspensions

[0195] Chitosan-thrombin formulations and the isolated Celis-Ringer’s lactatefibrinogen suspensions were mixed at a 1 : 1 ratio using a dual syringe system equipped with a tip with a static mixer.5- Assessment of solidification and histology

[0196] The syringes were attached to the blending connecter with static mixer.

[0197] The first two drops were ejected and discarded.

[0198] Then, two drops at a time were ejected in Petrie dishes.

[0199] Solidification time was documented by tilting the Petrie dishes (Figure 6A).

[0200] The clots were fixed in 10% NBF when they were solid.

[0201] The clots were processed for paraffin embedding, sectioning and staining with hematoxylin and eosin (Figure 6B).6-Summary of example 6

[0202] This work demonstrated that chitosan formulations with fibrinogen / thrombin can be used to deliver cell suspensions isolated from rat skin. Solidification occurred in about 2 minutes and cells were present in the solid matrix.EXAMPLE 71- Preparation of chitosan-thrombin formulations

[0203] Freeze-dried chitosan cakes containing 1 % w / v chitosan number average molar mass ( / Wn) 41 kDa and 83% degree of deacetylation (DDA) with 1 % w / v trehalose, 42 mM CaCh were reconstituted at % of their original volume in sterile water for injection, in order to obtain solutions with 2% w / v chitosan.

[0204] Stock thrombin solution (200 NIH / mL) was added to the chitosan solutions in order to obtain thrombin concentration of 40 NIH / mL.2- Preparation of cell suspensions in a buffer containing fibrinogen

[0205] Fibrinogen was added to Ringer’s lactate and mixed at 37°C to dissolve to make a Ringer’s solution with 60 mg / mL fibrinogen.

[0206] A Rat-1 fibroblast suspension (P4) containing 1.8 X 106cells / mL in Ringer’s lactate was prepared.

[0207] 200 pL Rat-1 cell suspension was mixed with 400 pL Fg (60 mg / mL).3- Mixing of chitosan-thrombin with cell suspensions

[0208] Chitosan-thrombin formulations and the Rat-1 fibroblast-Ringer’s lactate-fibrinogen suspensions were mixed at a 1 :1 ratio using a dual syringe system equipped with a tip with a static mixer.4- Cell culture

[0209] For cell culture, delivery was in 48-well plates. Formulations were allowed to clot and were then covered with 0.5 mL DMEM-High glucose supplemented with 10% FBS. The formulations were cultured for 7 days.

[0210] The chitosan formulations containing thrombin / fibrinogen solidified within a few minutes following application in the cell culture vessels. Rat-1 cells were visible within the clots.

[0211] Live-Dead solution was prepared as per manufacturer’s instructions and mixed at a 1 :1 ratio with culture medium. Culture medium was removed from the wells and replaced with 0.4 mL Live-Dead / medium solution. Plates were covered in aluminum paper and incubated for 15 minutes at room temperature. The clots were placed on 35 mm Mattek chambers for imaging with the confocal microscope.

[0212] Live cells were detected in the chitosan / fibrin clots. Live cells were more numerous in the chitosan / fibrin clots at day 7 when compared to days 3 and 1 (Figures 7A).

[0213] 80 pL Presto Blue solution was added to each well. Plates were incubated at 37°C for 1 h. Absorbance of the Presto Blue / culture medium solutions was read in cuvettes at 570 nm using 600 nm as a reference wavelength.

[0214] Metabolic activity in the chitosan / fibrin clots was higher at day 7 versus days 3 and 1 (Figure 7B).5- Assessment of biomateral delivery to rat skin using an ex vivo test

[0215] For ex vivo work, delivery was on a cadaveric rat.

[0216] Skin samples were fixed in 10% neutral buffered formalin, histoprocessed and stained with hematoxylin and eosin.

[0217] Chitosan / fibrin clots containing Rat-1 fibroblasts were visible on histological sections of rat skin (Figure 7C).6-Summary of example 7

[0218] This work demonstrated that chitosan formulations with fibrinogen / thrombin can be used to deliver Rat-1 fibroblasts. The fibroblasts remained alive and metabolically active after delivery in tissue culture vessels up to 7 days.EXAMPLE 81- Preparation of chitosan-thrombin formulations

[0219] Freeze-dried chitosan cakes containing 1 % w / v chitosan number average molar mass ( / Wn) 39 kDa and 83% degree of deacetylation (DDA) with 1 % w / v trehalose, 42 mM CaC were reconstituted at % of their original volume in sterile water for injection, in order to obtain solutions with 2% w / v chitosan.

[0220] Stock thrombin solution (200 NIH / mL) was added to the chitosan solutions in order to obtain thrombin concentration of 40 NIH / mL.2- Rat skin defect model

[0221] Male Sprague-Dawley rates aged 4 weeks (>100 g) were used for the study.

[0222] Biopsy punches (8mm in diameter) were used to create a circular wound pattern, followed by forceps and scissors to pull the skin to remove the tissue sample.

[0223] A retaining silicon ring was placed around the wound and secured with six sutures.

[0224] Six wounds were treated with a chitosan / fibrin / autologous cell formulation and 6 wounds were treated with autologous cells only.3- Isolation of cell suspension from rat skin

[0225] Two 8-mm biopsies were collected per rat.

[0226] The biopsies were rinsed in 10 mL Ringer’s lactate, two biopsies per tube.

[0227] The biopsies were placed in a Petri dish and scissors were used to remove the subcutaneous fat tissue.

[0228] 0.5% T rypsin-EDTA solution was pre-heated at 37°C in a small bacterial oven.

[0229] Two 8-mm biopsies were placed in each T rypsin-containing tube and the tubes were placed at 37°C.

[0230] The tubes were incubated for 30 minutes.

[0231] The biopsies were rinsed in 10 mL Ringer’s lactate to get rid of residual enzyme.

[0232] Syringes with 0.9 mL Ringer’s lactate were prepared.

[0233] The biopsies were placed on a Petri dish and a few drops of Ringer’s lactate were ejected onto the biopsies from the syringes.

[0234] The epidermis and then the dermis were scraped with the scalpel blade until the dermis had nearly disintegrated.

[0235] The remaining volume of Ringer’s lactate was emptied from the syringe on the Petri dish to get all the cells in one corner of the dish.

[0236] The cell suspension was aspirated up and down to make sure that all cells were collected.

[0237] The cell suspension was filtered through a cell strainer (40 pm).4- Preparation of cell suspensions in a buffer containing fibrinogen

[0238] Fibrinogen was added to Ringer’s lactate and mixed at 37°C to dissolve to make a Ringer’s solution with 60 mg / mL fibrinogen.

[0239] 200 pL isolated cell suspension was mixed with 400 pL Fg (60 mg / mL).5- Mixing of chitosan-thrombin with cell suspensions and application to skin defects

[0240] Chitosan-thrombin formulations and the isolated Cell-Ringer’s lactatefibrinogen suspensions were mixed at a 1 : 1 ratio using a dual syringe system equipped with a tip with a static mixer.

[0241] Two drops were applied to the skin defects.6- Assessment of wound closure

[0242] Bandages were changed every 3-6 days and pictures of the wounds were acquired along with a ruler (Figure 8A).

[0243] % Wound closure was measured using Image J (Figure 8B).7-Summary of example 8

[0244] This work demonstrated that chitosan formulations with fibrinogen / thrombin can be used to deliver autologous cells to skin defects. Wound closure was faster when chitosan formulations were used to deliver the autologous cells compared to delivering the autologous cells by themselves.REFERENCES1. Chevrier, A., et aL, Injectable chitosan-platelet-rich plasma (PRP) implants to promote tissue regeneration: In vitro properties, in vivo residence, degradation, cell recruitment and vascularization. J Tissue Eng Regen Med, 2018. 12(1): p. 217-228.2. Depres-Tremblay, G., et aL, Freeze-dried chitosan -platelet-rich plasma implants improve supraspinatus tendon attachment in a transosseous rotator cuff repair mode! in the rabbit. J Biomed App, 2019. 33(6): p. 792-807.3. Depres-Tremblay, G., et aL, Freeze-dried chitosan-platelet-rich plasma implants for rotator cuff tear repair: Pilot ovine studies. ACS Biomat Sci Eng, 2018. 4(11): p. 3737-3746.4. Chevrier, A., M.B. Hurtig, and M. Lavertu, Chitosan-Platelet-Rich Plasma Implants Improve Rotator Cuff Repair in a Large Anima! Model: Pivotal Study. Pharmaceutics, 2021. 13(1955): p. 1-17.5. Chevrier, A., M.B. Hurtig, and M. Lavertu, Chitosan-platelet-rich plasma implants improve rotatorcuff repair in a large anima! model: Pilot study. Biomed App, 2022. 37(2): p. 183-194.6. Ghazi zadeh, L., et aL, Freeze-dried chitosan-PRP injectable surgical implants for meniscus repair: Pilot feasibility studies in ovine models. Reg Med Ther, 2017. 1(1): p. 16-29.7. Dwivedi, G., et aL, Injectable freeze-dried chitosan-platelet-rich-plasma implants improve marrow-stimulated cartilage repair in a chronic-defect rabbit mode!. J Tissue Eng Regen Med, 2019. 13(4): p. 599-611.8. Huang, X.-f., et aL, A novel chitosan-based sponge coated with self-assembled thrombin / tannic acid multilayer films as a hemostatic dressing. Chinese Journal of Polymer Science, 2015. 33(2): p. 284-290.9. Shi, Z., et aL, Puff pastry-Hke chitosan / konjac g / ucomannan matrix with thrombin-occupied microporous starch particles as a composite for hemostasis. Carbohydr Polym, 2020. 232: p. 115814.10. Tripathi, D., et aL, Fabrication of Three-Dimensional Bioactive Composite Scaffolds for Hemostasis and Wound Healing. AAPS PharmSciTech, 2021. 22(4): p. 138.11. Han, C.M., et al., Application of coiiagen-chitosan / fibrin glue asymmetric scaffolds in skin tissue engineering. J Zhejiang Univ Sci B, 2010. 11(7): p. 524-30. 12. Lee, M.H., et al., Hemostatic Patches Based on Crosslinked Chitosan Films Applied inInterventional Procedures. 2021. 13(15).13. Ibne Mahbub, M.S., et aL, Decellularized liver extracellular matrix and thrombin loaded biodegradable TOCN / Chitosan nanocomposite for hemostasis and wound healing in rat liver hemorrhage model. Int J Biol Macromol, 2023. 225: p. 1529-1542. 14. Marchand, C., et aL, Solidification mechanisms of chitosan-giyceroi phosphate / biood implant for articular cartilage repair. Osteoarthritis Cartilage, 2009. 17(7): p. 953-960.15. Marchand, C., et aL, Microdrilled Cartilage Defects Treated with Thrombin-Solidified Chitosan / Biood Implant Regenerate a More Hyaline, Stable, and Structurally Integrated Osteochondral Unit Compared to Drilled Controls. Tissue Engineering Part A, 2012. 18(5-6): p. 508-519.16. Milano, F., et aL, Robust Segmentation-Free Algorithm for Homogeneity Quantification in Images. IEEE Trans Image Process, 2021. 30: p. 5533-5544.

Claims

CLAIMS1 . A tissue regeneration multi-component biomaterial composition, comprising: a) a chitosan component having a number average molar mass (Mn) of about 32-47 kilodalton (kDa) and a degree of deacetylation (DDA) of about 80-90%; b) a thrombin component present in an amount effective to accelerate the solidification of the chitosan component with a blood-derived product mixture; and c) a trehalose component present in a lyoprotectant effective amount.

2. The tissue regeneration multi-component biomaterial composition of claim 1 , wherein the thrombin component is present in an amount from about 0.25 to 20 NIH / mL.

3. The tissue regeneration multi-component biomaterial composition of claim 1 or 2 reconstituted with platelet-rich plasma (PRP), plasma powder, fibrinogen and combinations thereof to form an implant.

4. The tissue regeneration multi-component biomaterial composition of claim 3, wherein the plasma powder comprises fibrinogen present in an amount from about 2.5 to 20 mg / mL a fibrinogen concentration.

5. The tissue regeneration multi-component biomaterial composition of any one of claims 1 to 4 further comprising at least one cell.

6. The tissue regeneration multi-component biomaterial composition of claim 5, wherein the at least one cell is selected from the group consisting of keratinocytes, fibroblasts, melanocytes and combinations thereof.

7. A method for preparing a tissue regeneration biomaterial comprising: a) mixing chitosan with thrombin, trehalose, CaCL, and freeze-drying to form a freeze-dried cake; b) reconstituting the freeze-dried cake with at least one of PRP, plasma powder, fibrinogen and combinations thereof forming a mixture; and c) allowing the mixture to solidify upon mixing forming an implant.

8. The method of claim 7, wherein said chitosan has a Mn of between 32-47 kDa and a DDA of between 80-90%.

9. A method for delivering cells to a site in need of treatment, comprising:a) preparing a biomaterial according to any one of claims 1 to 6; b) mixing the biomaterial with a cell suspension to form a mixture; and c) applying the mixture to a tissue site.

10. The tissue regeneration multi-component biomaterial composition of any one of claims 1 to 6, wherein the chitosan component is present in an amount ranging from 1-4% w / v.

11. The tissue regeneration multi-component biomaterial composition of any one of claims 1 to 6 and 10, wherein the trehalose component is present in an amount ranging from 0.5-1 .5% w / v.

12. The tissue regeneration multi-component biomaterial composition of any one of claims 1 to 6, wherein the chitosan component is present in an amount ranging from 1-1 .5% w / v.

13. The tissue regeneration multi-component biomaterial composition of any one of claims 1 to 6, further comprising CaCh in an amount ranging from 42-63 mM.

14. The tissue regeneration multi-component biomaterial composition of any one of claims 1 to 6 with a clotting time from 0.5 - 10.2 minutes.

15. A tissue regeneration multi-component biomaterial composition selected from the group consisting of: a) 1 % w / v chitosan having a Mn of 39 kDa, a DDA of 82%; 1 % w / v trehalose; 42 mM CaCh; thrombin selected from 0.25, 0.5 and 1 NIH / mL and PRP; b) 1 % w / v chitosan having a Mn of 32 kDa, a DDA of 85%; 1 % w / v trehalose; 42 mM CaC ; thrombin selected from 0.5, 1 , 2, 5 and 10 NIH / mL; fibrinogen selected from 2.5 and 10 mg / mL and plasma powder suspension; c) 1 % w / v chitosan having a Mn selected from 37 and 47 kDa, a DDA selected from 83 and 90%; 1 % w / v trehalose; 42 mM CaCh; 5 NIH / mL thrombin; 10 mg / mL fibrinogen and plasma powder suspension; d) Chitosan selected from 1 and 2% w / v having a Mn selected from 36 and 37 kDa and a DDA selected from 84 and 85%; 1 % w / v trehalose; 42 mM CaCL;10 NIH / mL thrombin; 10 mg / mL fibrinogen and 600000 cells / mL of keratinocytes;e) Chitosan selected from 1 and 1 .5% w / v having a Mn of 36 kDa and a DDA of 84%; trehalose selected from 1 and 1 .5% w / v; CaCl2 selected from 42 and 63 mM; thrombin selected from 2.5, 5 and 10 NIH / mL and 10 mg / mL fibrinogen; f) 1 % w / v chitosan having a Mn of 37 kDa and a DDA of 83%; 1 % w / v trehalose; 42 mM CaCl2; 20 NIH / mL thrombin;20 mg / mL fibrinogen and skin cells; g) 1 % w / v chitosan having a Mn of 41 kDa and a DDA of 83%; 1 % w / v trehalose;42 mM CaCL; 20 NIH / mL thrombin;20 mg / mL fibrinogen and fibroblasts; and h) 1 % w / v chitosan having a Mn of 39 kDa and a DDA of 83%; 1 % w / v trehalose; 42 mM CaCL; 20 NIH / mL thrombin;20 mg / mL fibrinogen and skin cells.

16. The tissue regeneration multi-component biomaterial compositions of claim 15 wherein said compositions exhibit a clotting time of: a) For composition a) 3.02 mins; b) For composition b) from 28.6 mins to 9.6 mins with increasing thrombin with 2.5 mg / mL fibrinogen and from 10.2 mins to 0.5 mins with increasing thrombin with 10 mg / mL fibrinogen; c) For composition c) between 0.4-0.7 mins for 90% DDA chitosan, and between 0.8-1.1 mins for 83% DDA chitosan; d) For composition d) 3 mins; e) For composition e) between 0.6 -5.75 mins for 1 % w / v chitosan and between 1 .75-8.9 mins for 1 .5% w / v chitosan; f) For composition f) 2 mins; and g) For composition g) 2 mins.

17. A method of regenerating tissue at a site in need of tissue regeneration, comprising: applying a tissue regeneration multi-component biomaterial composition of any one of claims 1-6 and 10-15 to the site in need of tissue regeneration.

18. The method of claim 17 wherein said site in need of tissue regeneration is selected from a wound, surgical site and tissue defect.

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