Temperature-sensitive hydrogel and platelet-rich plasma compound as well as preparation method and application thereof

Abstract

The invention discloses a temperature-sensitive hydrogel and platelet-rich plasma compound, and belongs to the field of biomedical materials. The compound is formed by uniformly mixing a temperature-sensitive hydrogel solution and platelet-rich plasma at the temperature of 2-8 DEG C, and the mass ratio of the temperature-sensitive hydrogel solution to the platelet-rich plasma is (0.25-100): 1; the temperature-sensitive hydrogel solution is prepared from the following components in percentage by mass: 0.5 to 10 percent of anionic group substituted hydroxybutyl chitosan, 0.1 to 10 percent of osmotic pressure regulator and the balance of water. The temperature-sensitive hydrogel / platelet-rich plasma compound is uniform gel, has temperature sensitivity, and can still keep a gel state for a long time even if the compound is soaked in a large amount of PBS solution at the body temperature; growth factors in platelets in the compound can be gradually released out of the gel, and a long-term acting effect is achieved. The material can be used for preparing wound repair materials, soft tissue repair materials, cartilage repair materials, bone repair materials, tendon repair materials, osteoarthritis materials and cosmetic filling materials.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a thermosensitive hydrogel and platelet-rich plasma composite, a preparation method thereof, and an application thereof. Background Art

[0002] Hydroxybutyl chitosan is a new type of thermosensitive biomaterial developed in the past ten-odd years, with good biocompatibility and degradability. The aqueous solution of hydroxybutyl chitosan is in a solution state at low temperatures (below its gel temperature). After gradually heating up, it can turn into a non-flowable gel state after reaching a temperature higher than its gel temperature, such as reaching body temperature of 37°C. This property enables the material to be fixed and play a role for a long time after acting on the affected area. The thermosensitive hydrogel of hydroxybutyl chitosan can be used on wounds of almost any shape, including planar, wrinkled, and slit-shaped ones. After use, it turns into a non-flowable gel that adheres to the wound surface and will not flow away due to body position changes, thus achieving a long-term treatment effect and solving the problem that other liquid gel materials have high fluidity and are easy to leave the wound area that needs to be shielded.

[0003] Platelet-rich plasma (PRP) is the platelet-rich plasma part obtained by centrifugally separating human autologous blood. It is rich in growth factors and can be used to treat various diseases such as fractures, osteoarthritis, chronic wounds, and cosmetic filling. During the clinical application of PRP, it often faces the problem of flowing away with gravity on the wound surface and cannot stay for a long time, resulting in the inability to achieve the due treatment effect. Moreover, when PRP stays on the wound surface, the platelets inside it are easily activated, and the growth factors inside are released in large amounts in a short time. Only a very small part of them plays a role, and most of them are hydrolyzed by enzymes in the wound, and the action time is not long enough.

[0004] The invention patent with publication number CN116785235 A discloses a thermosensitive hydrogel for slow release of hydroxybutyl chitosan and platelet-rich plasma drugs, which mixes hydroxybutyl chitosan and platelet-rich plasma in a ratio of not less than 2:1 and forms a gel state under conditions close to body temperature, thereby reducing the treatment frequency and improving the use efficiency of PRP.

[0005] However, a hydroxybutyl chitosan hydrogel dressing and a preparation method thereof disclosed in the Chinese invention patent application with publication number CN113908329 A found that strong polar ionic osmotic pressure regulators commonly used in pharmacopoeias such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, and sodium sulfate cannot be used for compatibility with hydroxybutyl chitosan preparations under conventional conditions. This is because when hydroxybutyl chitosan is compatible with the above-mentioned strong polar substances, it is a homogeneous, transparent flowing solution at low temperatures, but turns into a white opaque gel when heated. The gel strength is low and it is easily broken into pieces under external force and will not solidify again unless it is homogenized at low temperature again.

[0006] This characteristic makes it impossible to mix hydroxybutyl chitosan hydrogel with bioactive materials containing isotonic salt concentrations, such as platelet-rich plasma (PRP), exosomes, cell culture medium, etc., in large proportions. Otherwise, precipitation will occur, causing hydroxybutyl chitosan to lose its gel properties, thereby limiting the application of hydroxybutyl chitosan in the above fields. Summary of the Invention

[0007] In view of the above problems, the first object of the present invention is to provide a composite of modified hydroxybutyl chitosan thermosensitive hydrogel and platelet-rich plasma, which can be transformed into a non-flowing gel at body temperature and can maintain the gel state for a long time, realizing the gradual release of growth factors in platelets and exerting a long-term effect. It can be used for the culture of human own cells, as a superficial and deep tissue on the body surface or an implant material in the body.

[0008] The second object of the present invention is to provide a preparation method of the above thermosensitive hydrogel and platelet-rich plasma composite, and its application in the preparation of wound repair materials, soft tissue repair materials, cartilage repair materials, bone repair materials, tendon repair materials, osteoarthritis materials, and cosmetic filling materials.

[0009] The thermosensitive hydrogel and platelet-rich plasma composite of the present invention is uniformly mixed by a thermosensitive hydrogel solution and platelet-rich plasma at 2-8°C. The mass ratio of the thermosensitive hydrogel to platelet-rich plasma is: (0.25-100):1, and the composite transforms into a gel when heated to near body temperature. The thermosensitive hydrogel solution contains 0.5%-10% by mass concentration of anion group-substituted hydroxybutyl chitosan, an osmotic pressure regulator: 0.1-10%; and water: the balance.

[0010] The preparation method of the above thermosensitive hydrogel and platelet-rich plasma composite includes the following steps:

[0011] (1) Dissolve anion group-substituted hydroxybutyl chitosan in an osmotic pressure regulator at 2°C-8°C to obtain a thermosensitive hydrogel solution with a mass percentage concentration of 0.5%-10%.

[0012] (2) Weigh the above thermosensitive hydrogel solution and platelet-rich plasma, mix them at 2°C-8°C, shake well, and heat to 37°C or implant into the filling site to obtain a solidified thermosensitive hydrogel / platelet-rich plasma composite.

[0013] Before the thermosensitive hydrogel solution in step (1) is mixed with platelet-rich plasma, it also undergoes a step of obtaining a sterile thermosensitive hydrogel after moist heat sterilization or radiation sterilization.

[0014] The osmotic pressure regulator is one or more of polyhydric alcohols such as glycerol and mannitol, or strongly polar ionic compounds such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, and phosphate buffer solution.

[0015] When the anionic group-substituted hydroxybutyl chitosan thermosensitive hydrogel of the present invention is formulated with the above-mentioned osmotic pressure regulator, a uniform solution can be obtained. Especially when formulated with an osmotic pressure regulator of a strongly polar ionic compound (such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, phosphate buffer solution, etc.), a thermosensitive solution with good performance can be obtained, and when forming a gel at body temperature, it also presents a uniform and transparent state, with high gel strength, no problems of turning white or crumbling, can be well matched with the human physiological environment, and at the same time has good compatibility with cell culture medium, can be used as a cell carrier, and is a thermosensitive cell carrier with better effects.

[0016] The anionic group-substituted hydroxybutyl chitosan derivative has the following structure:

[0017]

[0018] Among them, R1, R2, R3, and R4 are one of the following groups:

[0019] H, hydroxybutyl, anionic group, acetyl group.

[0020] The anionic group is: carboxymethyl, carboxyethyl, methylcarboxymethyl, carboxypropyl, 2-carboxypropyl, 1,3-dicarboxypropyl, carboxybutyl, 3-carboxybutyl, 2-carboxymethylpropyl, sulfonic acid group, sulfopropyl, 2-hydroxy sulfopropyl, and the group formed after the hydrogen atom on the hydroxybutyl is substituted by the above ionic groups.

[0021] Preferably, the anionic group-substituted hydroxybutyl chitosan is carboxymethyl hydroxybutyl chitosan, acetylated carboxymethyl hydroxybutyl chitosan, propionylated carboxymethyl hydroxybutyl chitosan.

[0022] The structural formulas of the above groups are as follows:

[0023] Hydroxybutyl: CH3CH2CHOHCH2-; Acetyl group: Carboxymethyl: -CH2COOH; Carboxyethyl: -CH2CH2COOH; Methylcarboxymethyl: Carboxypropyl: -CH2CH2CH2COOH; 2-Carboxypropyl: 1,3-Dicarboxypropyl: Carboxybutyl: -CH2CH2CH2CH2COOH; 3-Carboxybutyl: 2-Carboxymethylpropyl: Sulfonic acid group: -SO3H; Sulfopropyl group: -CH2CH2CH2SO3H; 2-Hydroxy sulfopropyl group: 2-Carboxymethoxybutyl: 2-Carboxyethoxybutyl:

[0024] The degree of substitution of the hydroxybutyl group is 1.0 - 4.0; the degree of substitution of the anionic group is 0.1 - 3.0.

[0025] Preferably, the degree of substitution of the hydroxybutyl group is 1.5 - 3.0; the degree of substitution of the anionic group is 0.2 - 2.0.

[0026] The anionic group-substituted hydroxybutyl chitosan derivative of the present invention is prepared by the following method:

[0027] Method 1: First, modify chitosan with an anionic group to obtain anionic-modified chitosan, then perform hydroxybutylation modification to obtain a hydroxybutyl chitosan derivative substituted with an anionic group, and then perform acylation modification to obtain the final anionic group-substituted hydroxybutyl chitosan derivative.

[0028] Method 2: First, perform hydroxybutylation modification on chitosan to obtain hydroxybutyl-substituted chitosan; then perform anionic group modification on the hydroxybutyl chitosan to obtain a hydroxybutyl chitosan derivative substituted with an anionic group, and it can also be further subjected to acylation modification to obtain the final anionic group-substituted hydroxybutyl chitosan derivative.

[0029] Method 3: First, perform hydroxybutylation modification on chitosan to obtain hydroxybutyl-substituted chitosan; then perform acylation modification to obtain an acylated hydroxybutyl chitosan derivative, and then perform anionic group modification to obtain the final anionic group-substituted hydroxybutyl chitosan derivative.

[0030] The solvent in the reaction system is one or a combination of more of water, sodium hydroxide solution, potassium hydroxide solution, urea solution, guanidine hydrochloride solution, hydrochloric acid, nitric acid, methanol, formic acid, ethanol, acetic acid, acetonitrile, n-propanol, isopropanol, n-butanol, acetone, dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dioxane.

[0031] The types of the above grafting reactions include nucleophilic substitution reaction, Schiff base reaction, Michael addition reaction and ring-opening reaction.

[0032] The reagents for the nucleophilic substitution reaction include: chloroacetic acid, 3-chloropropionic acid, 2-chloropropionic acid, 4-chlorobutyric acid, 2-methyl-2-chloropropionic acid, 5-chlorobutyric acid, 2-methyl-4-chloropropionic acid, chlorosulfonic acid, 3-chloropropanesulfonate, 3-chloro-2-hydroxypropanesulfonate.

[0033] The Schiff base reaction reagents include: glyoxylic acid, pyruvic acid, α-ketoglutaric acid; the reducing agents include: sodium borohydride, lithium aluminum hydride, lithium borohydride, potassium borohydride, sodium cyanoborohydride, borane ammonia, etc.

[0034] The Michael addition reaction reagents include: acrylic acid, methacrylic acid.

[0035] The ring-opening reaction reagents include: sodium 2,3-epoxypropylsulfonate, propanesultone.

[0036] The pH value range for the Schiff base reaction is 3 - 7, and the pH value range for the reduction reaction is 8 - 14.

[0037] The pH value range for the Michael addition reaction is 3 - 7.

[0038] The pH value for the ring-opening reaction ranges from 0 - 5 when using the reagent propanesultone; and ranges from 7 - 14 when using the reagent sodium 2,3-epoxypropylsulfonate.

[0039] The pH value of the reaction system is adjusted using an acid or a base. The base is sodium hydroxide or potassium hydroxide, and the molar concentration of the base solution is 0.01 - 15 mol / L. The acid is hydrochloric acid, sulfuric acid, acetic acid, and the concentration of the acid is 0.01 - 18.4 mol / L.

[0040] The reaction temperature is 0°C - 70°C; the reaction time is 0.5 h - 120 h.

[0041] The present invention also provides the application of the above thermosensitive hydrogel / platelet-rich plasma complex: the thermosensitive hydrogel / platelet-rich plasma complex prepared according to the above method is used for the preparation of wound repair materials, soft tissue repair materials, cartilage repair materials, bone repair materials, tendon repair materials, osteoarthritis materials, and cosmetic filling materials.

[0042] The advantages of the present invention are:

[0043] 1. The thermosensitive hydrogel / platelet-rich plasma complex of the present invention is a uniform gel with temperature sensitivity, that is, it is a flowing solution at low temperature and transforms into a non-flowing gel at body temperature. It is homogeneous in both states and there is no obvious precipitation. Even when immersed in a large amount of PBS solution at body temperature, it can still maintain the gel state for a long time.

[0044] 2. The growth factors in the platelets in the thermosensitive hydrogel / platelet-rich plasma complex of the present invention can be gradually released outside the gel, exerting a long-term effect.

[0045] 3. The thermosensitive hydrogel / platelet-rich plasma complex of the present invention, since platelet-rich plasma is extracted from its own blood, can be used to culture human body's own cells for treating various cell therapy-related diseases.

[0046] 4. The thermosensitive hydrogel / platelet-rich plasma complex of the present invention can be used for superficial body surface, deep body surface tissues or in vivo implantation. It can be used for the care of acute and chronic wounds, the repair of various soft tissues, cartilage repair, tendon repair, bone repair, etc. It can also be used as a carrier for various drugs or cells and a tissue engineering scaffold material. Description of the Drawings

[0047] Figure 1 It is a photo of the blood stratification when preparing platelet-rich plasma in Example 4 of the present invention.

[0048] Figure 2 It is a photo of the state after the sample in Example 1 of the present invention was dissolved in 0.85% sodium chloride solution at low temperature to prepare a 5% concentration solution and then heated to 37°C.

[0049] Figure 3 It is a photo of the state of the thermosensitive hydrogel in Example 6 of the present invention and the platelet-rich plasma prepared in Example 4 at the initial stage of low-temperature mixing (left), after low-temperature shaking (middle), and after heating to 37°C (right).

[0050] Figure 4 It is a photo of the state of the thermosensitive hydrogel in Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention and the platelet-rich plasma prepared in Example 4 after low-temperature mixing.

[0051] Figure 5 It is a photo of the appearance after the thermosensitive hydrogel in Example 7 of the present invention, the thermosensitive hydrogels in Comparative Example 1, Comparative Example 2, and Comparative Example 3 were respectively mixed with the platelet-rich plasma prepared in Example 4 at low temperature, and then heated to 37°C after being uniformly mixed.

[0052] Figure 6 It is a photo of the state after the thermosensitive hydrogels in Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention and the platelet-rich plasma prepared in Example 4 were heated to 37°C to form a gel after low-temperature mixing, and then soaked in PBS solution at 37°C for 3 h.

[0053] Figure 7 It is a photo of the state after the thermosensitive hydrogels in Example 6, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present invention were respectively mixed with the platelet-rich plasma prepared in Example 4 at low temperature, uniformly mixed, and then heated to 37°C, and then soaked in PBS solution at 37°C for 72 h.

[0054] Figure 8 It is a curve of the cumulative release amount of P-selectin measured by taking the soaking solution at different time points after the thermosensitive hydrogel / platelet-rich plasma complex prepared in Example 7 was soaked in PBS solution at 37°C.

[0055] Figure 9After the thermosensitive hydrogel / platelet-rich plasma complex prepared in Example 7 was soaked in PBS solution at 37°C for different time points, the soaking solutions were taken separately to test the cumulative release amount curve of transforming growth factor (TGF).

[0056] Figure 10 After the thermosensitive hydrogel / platelet-rich plasma complex prepared in Example 7 was soaked in PBS solution at 37°C for different time points, the soaking solutions were taken separately to test the cumulative release amount curve of platelet-derived growth factor (PDGF).

[0057] Figure 11 Photographs of the wound healing conditions of the thermosensitive hydrogel / platelet-rich plasma complex prepared in Example 6, the thermosensitive hydrogel alone, and normal saline at 0 days, 7 days, and 14 days in the animal experiment for repairing skin defects in diabetic rats. Detailed implementation manners

[0058] The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments.

[0059] Example 1: Preparation of propionylated carboxymethyl hydroxybutyl chitosan

[0060] (1) Prepare hydroxybutyl chitosan according to the method disclosed in patent application CN 102276756.

[0061] (2) Take 40 g of hydroxybutyl chitosan and disperse it in 150 mL of 50% sodium hydroxide solution. After dispersing evenly, add 160 mL of isopropanol solution containing 35 g of chloroacetic acid, and stir and react at 60°C for 4 h.

[0062] (3) Adjust the pH value of the reacted material to neutral with dilute hydrochloric acid or dilute sodium hydroxide solution at room temperature, and obtain 42.7 g of carboxymethyl hydroxybutyl chitosan through dialysis and freeze-drying.

[0063] (4) Dissolve 10 g of the obtained carboxymethyl hydroxybutyl chitosan in 500 mL of isopropanol aqueous solution, and stir to dissolve evenly. Add an isopropanol solution of acetic anhydride, containing 6 mL of propionic anhydride and 50 mL of isopropanol, and stir and react at 30°C for 3 h. After the reaction is completed, adjust to pH 11 with sodium hydroxide solution, and obtain partially propionylated carboxymethyl hydroxybutyl chitosan through dialysis and freeze-drying.

[0064] Example 2: Preparation of carboxymethyl hydroxybutyl chitosan

[0065] (1) Prepare hydroxybutyl chitosan according to the method disclosed in patent application CN 102276756.

[0066] (2) Take 20 g of hydroxybutyl chitosan, disperse it in 200 mL of 50% sodium hydroxide solution, add 2000 mL of dimethyl sulfoxide aqueous solution, add 130 mL of aqueous solution containing 35 g of chloroacetic acid, and stir and react at 50 °C for 4 h.

[0067] (3) Adjust the pH value of the reacted material to neutral with dilute hydrochloric acid or dilute sodium hydroxide solution at room temperature, and obtain 22.9 g of carboxymethyl hydroxybutyl chitosan through dialysis and freeze-drying.

[0068] Example 3: Preparation of acetylated carboxymethyl hydroxybutyl chitosan

[0069] Steps (1) to (3) are the same as in Example 1.

[0070] (4) Dissolve 10 g of the obtained carboxymethyl hydroxybutyl chitosan in 500 mL of isopropyl alcohol aqueous solution, stir and dissolve evenly. Add an isopropyl alcohol solution of acetic anhydride, containing 2.5 mL of acetic anhydride and 50 mL of isopropyl alcohol, and stir and react at 30 °C for 5 h. After the reaction is completed, adjust the pH to 8 with sodium hydroxide solution, and obtain partially acetylated carboxymethyl hydroxybutyl chitosan through dialysis and freeze-drying.

[0071] Example 4: Preparation of PRP using a platelet-rich plasma collector disclosed in the utility model patent with publication number CN218078428U

[0072] (1) According to the ratio of blood: anticoagulant = 10:1, take 1.5 ml of 2.5% sodium citrate solution as anticoagulant and 15 ml of venous blood into the platelet-rich plasma collector, and mix well.

[0073] (2) Place the above product in a centrifuge for centrifugation, with a centrifugal force of 1500 g and a time of 10 minutes. At this time, the solution is divided into 3 layers. The top layer is the plasma layer, the middle layer is the PRP layer, and the bottom layer is the red blood cell layer.

[0074] (3) Drain the lower-layer red blood cells downward, extract about 2 / 3 of the upper-layer liquid into a syringe, and the remaining liquid in the centrifuge tube is platelet-rich plasma (PRP), as shown in Figure 1 .

[0075] Example 5: Preparation of PRP using a platelet-rich plasma preparation device disclosed in the invention patent with publication number CN116273499A

[0076] (1) According to the ratio of 20 U / mL, mix 2 mL of heparin sodium injection (2 ml: 12,500 units) with 60.5 mL of normal saline, suck 5 mL (containing 1000 units of heparin) into the platelet-rich plasma preparation device, and then take 50 ml of human venous blood and mix well.

[0077] (2) Place the product in a centrifuge for centrifugation at a centrifugal force of 500 g for 10 minutes.

[0078] (3) Drain the red blood cells vertically downward, and subject the product to secondary centrifugation at a centrifugal force of 1400 g for 10 minutes.

[0079] (4) Remove the upper-layer plasma from the product to obtain platelet-rich plasma (PRP).

[0080] Example 6:

[0081] Dissolve 5 g of the partially propionylated carboxymethyl hydroxybutyl chitosan prepared in Example 1 in 95 ml of 0.85% sodium chloride solution at 2°C - 8°C to obtain a thermosensitive hydrogel solution with a concentration of 5%. Take this thermosensitive hydrogel solution in a bottle and heat it to 37°C to obtain a uniformly solidified colorless and transparent hydrogel, as shown in Figure 2 .

[0082] Weigh 2.0 g of this hydrogel solution and 1.0 g of the platelet-rich plasma prepared in Example 4, mix them at 2°C - 8°C, shake well, and heat to 37°C to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex, as shown in Figure 3 . It can be seen from the figure that initially (left) the thermosensitive hydrogel and platelet-rich plasma are significantly stratified. After shaking well at low temperature (middle), it becomes a light yellow homogeneous solution, and after heating to 37°C (right), it becomes a uniform light yellow solid gel.

[0083] Example 7:

[0084] Dissolve 5 g of the carboxymethyl hydroxybutyl chitosan prepared in Example 2 in 95 ml of 0.85% sodium chloride solution at 2°C - 8°C to obtain a thermosensitive hydrogel solution with a concentration of 5%. Weigh 2.0 g of this hydrogel and 1.0 g of the platelet-rich plasma prepared in Example 4, mix them at 2°C - 8°C, shake well, and heat to 37°C to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex, designated as gel complex 1, abbreviated as G1.

[0085] Example 8:

[0086] Dissolve 5 g of the carboxymethyl hydroxybutyl chitosan prepared in Example 2 in 95 ml of 0.85% sodium chloride solution at 2°C - 8°C to obtain a thermosensitive hydrogel solution with a concentration of 5%. Weigh 2.0 g of this hydrogel and 2.0 g of the platelet-rich plasma prepared in Example 5, mix them at 2°C - 8°C, shake well, and heat to 37°C to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex, designated as gel complex 2, abbreviated as G2.

[0087] Example 9:

[0088] 5 g of the partially acetylated carboxymethyl hydroxybutyl chitosan prepared in Example 3 was dissolved in 95 ml of 0.85% sodium chloride solution at 2°C - 8°C to obtain a thermosensitive hydrogel solution with a concentration of 5%. 2.0 g of this hydrogel and 3.0 g of the platelet-rich plasma prepared in Example 4 were weighed, mixed at 2°C - 8°C, shaken well, and then heated to 37°C to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex, designated as gel complex 3, abbreviated as G3.

[0089] Comparative Example 1:

[0090] 2.5 g of hydroxybutyl chitosan prepared by the method disclosed in Patent Application CN 102276756 was dissolved in 97.5 ml of 3% glycerol aqueous solution to obtain a thermosensitive hydrogel solution with a concentration of 2.5%. 2.0 g of this hydrogel and 1.0 g of the platelet-rich plasma prepared in Example 4 were weighed, mixed at 2°C - 8°C, shaken well, and then heated to 37°C to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex as gel complex 4, abbreviated as G4.

[0091] Comparative Example 2:

[0092] 2.5 g of hydroxybutyl chitosan prepared by the method disclosed in Patent Application CN 102276756 was dissolved in 97.5 ml of 3% glycerol aqueous solution to obtain a thermosensitive hydrogel solution with a concentration of 2.5%. 2.0 g of this hydrogel and 0.5 g of the platelet-rich plasma prepared in Example 4 were weighed, mixed at 2°C - 8°C, shaken well, and then heated to 37°C to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex, designated as gel complex 5, abbreviated as G5.

[0093] Comparative Example 3:

[0094] 2.5 g of hydroxybutyl chitosan prepared by the method disclosed in Patent Application CN 102276756 was dissolved in 97.5 ml of 3% glycerol aqueous solution to obtain a thermosensitive hydrogel solution with a concentration of 2.5%. 2.0 g of this hydrogel and 0.25 g of the platelet-rich plasma prepared in Example 4 were weighed, mixed at 2°C - 8°C, shaken well, and then heated to 37°C to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex, designated as gel complex 6, abbreviated as G6.

[0095] The thermosensitive hydrogels in Comparative Example 1, Comparative Example 2 and Comparative Example 3 were mixed with the platelet-rich plasma prepared in Example 4 at low temperature, and their states were as Figure 4 shown, all showing a uniform light yellow solution.

[0096] Example 10: Property test of gel complex

[0097] The thermosensitive hydrogel / platelet-rich plasma complexes prepared in Example 7, Example 8, Example 9, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were denoted as G1, G2, G3, G4, G5, and G6, respectively. The states of the above-mentioned gels and complexes in the state of the thermosensitive hydrogel alone before mixing, the state of PRP, the state during the mixing process, the states at different temperatures after mixing, and the states after soaking in PBS for different times were tested, as shown in Table 1 in detail. The state of PRP alone was a light yellow translucent homogeneous flowing liquid.

[0098] Table 1 States of different samples under different conditions

[0099]

[0100]

[0101] The above data indicate that when the gels (5%) prepared with hydroxybutyl chitosan substituted with anionic groups are mixed with PRP at mass ratios of 2:3, 1:1, and 2:1, homogeneous thermosensitive hydrogels can be formed, which can solidify at 37°C, have uniform properties, and there is no obvious change in state after soaking in PBS at 37°C for 72 h. When the thermosensitive hydrogels (2.5%) prepared with ordinary hydroxybutyl chitosan are mixed with PRP at mass ratios of 2:1, 4:1, and 8:1, the former two turn into off-white opaque gels, which are not uniform; the latter is a semi-transparent relatively uniform gel. It shows that the optimal ratio of hydroxybutyl chitosan gel to PRP for mixing is 8:1, and the proportion of PRP that can be mixed is very low. Moreover, when the thermosensitive hydrogels prepared with hydroxybutyl chitosan are mixed with PRP in various ratios and soaked in PBS at 37°C for 72 h, they will gradually shrink, crack, and precipitate, losing the gel properties.

[0102] Example 11: Release of growth factors in the thermosensitive hydrogel / platelet-rich plasma complex

[0103] 3 mL of the thermosensitive hydrogel / platelet-rich plasma complex prepared in Example 7 was soaked in 5 mL of PBS solution at 37°C for 3 h, 12 h, 24 h, 48 h, and 72 h respectively. Then, the upper extract was taken out for testing, and 5 mL of new PBS solution was added again. The contents of P-selectin, transforming growth factor (TGF), and platelet-derived growth factor (PDGF) in the extracts taken out at different time points were tested, and the cumulative release amounts at different time points were calculated, as Figures 8 - 10 shown. It can be seen from the results that the three factors are released uniformly and slowly in the gel. This indicates that the growth factors in platelets can be encapsulated and stored in the gel complex, protected, and slowly released over time, increasing the action time of the growth factors.

[0104] Example 12: Animal experiment on chronic wound repair

[0105] Part of the partially acetylated carboxymethyl hydroxybutyl chitosan derivative prepared in Example 2 was dissolved in 0.85% sodium chloride solution at 2°C - 8°C at a concentration of 5% to obtain a thermosensitive hydrogel solution. The above thermosensitive hydrogel, the thermosensitive hydrogel / platelet-rich plasma complex prepared in Example 7, and normal saline (blank group) were used for the repair test of the diabetic rat skin defect model. SD rats were fasted for more than 12 h and intraperitoneally injected with STZ at 60 mg / kg - 80 mg / kg. After injecting STZ, the drinking and eating conditions of all test rats were closely observed every day. After 7 d, the blood glucose was measured with a blood glucose meter. A fasting blood glucose greater than 16.7 mmol / L indicated successful induction of diabetes in the rats. One day after the successful construction of the diabetic rat model, a skin injury model was established. After anesthesia, 2 cm × 2 cm full-thickness skin was removed from the back of the rats, and the above three groups of materials were used for treatment. Photos were taken at different time points, and the ratio of the wound shrinkage area to the original area was calculated as the wound healing rate. The results are as Figure 11 shown. The results showed that the average healing rates of the thermosensitive hydrogel / platelet-rich plasma complex group, the thermosensitive hydrogel group, and the blank group at 7 days were 31%, 26%, and 8% respectively; and the healing rates at 14 days were 90%, 82%, and 75% respectively. The experimental results show that the simple thermosensitive hydrogel group has a good effect on promoting wound healing compared with the blank group, and the thermosensitive hydrogel / platelet-rich plasma complex group is more effective in promoting wound healing than the simple thermosensitive hydrogel group.

[0106] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Those of ordinary skill in the art can still modify or equivalently replace the specific implementation manners of the present invention with reference to the above examples. Any modifications or equivalent replacements that do not depart from the spirit and scope of the present invention are within the scope of the claims of the present invention.

Claims

1. A thermosensitive hydrogel and platelet-rich plasma complex, characterized in that: The thermosensitive hydrogel solution is uniformly mixed with platelet-rich plasma at 2-8°C, wherein the mass ratio of the thermosensitive hydrogel solution to the platelet-rich plasma is (0.25-100):1; the thermosensitive hydrogel solution contains 0.5%-10% anionic group-substituted hydroxybutyl chitosan, 0.1-10% osmotic pressure regulator, and the balance of water.

2. The thermosensitive hydrogel and platelet-rich plasma complex according to claim 1, characterized in that: The osmotic pressure regulator is glycerol, mannitol or sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium nitrate, sodium sulfate, or phosphate buffer.

3. The thermosensitive hydrogel and platelet-rich plasma complex according to claim 1, characterized in that: The anionic group-substituted hydroxybutyl chitosan has the following structure: Wherein, R1, R2, R3, and R4 are one of the following groups: H, hydroxybutyl, anionic substituent, acyl group; The anionic group is selected from carboxymethyl, carboxyethyl, methylcarboxymethyl, carboxypropyl, 2-carboxypropyl, 1,3-dicarboxypropyl, carboxybutyl, 3-carboxybutyl, 2-carboxymethylpropyl, sulfonic acid, sulfonic acid propyl, 2-hydroxysulfonic acid propyl; The acyl group is acetyl, propionyl or butyryl.

4. The thermosensitive hydrogel and platelet-rich plasma complex according to claim 1, characterized in that: The anionic group-substituted hydroxybutyl chitosan is carboxymethyl hydroxybutyl chitosan, acetylated carboxymethyl hydroxybutyl chitosan or propionylated carboxymethyl hydroxybutyl chitosan.

5. A method for preparing the thermosensitive hydrogel and platelet-rich plasma complex according to claim 1, characterized in that: The following steps are involved: (1) dissolving the anionic group-substituted hydroxybutyl chitosan in an osmotic pressure regulator at 2° C. to 8° C. to obtain a thermosensitive hydrogel solution with a mass percentage concentration of 0.5% to 10%; (2) Weigh the above thermosensitive hydrogel solution and platelet-rich plasma in a mass ratio of (0.25-100):1, mix at 2°C-8°C, shake well, raise the temperature to 37°C or implant into the filling site to obtain a solidified thermosensitive hydrogel / platelet-rich plasma complex.

6. The method for preparing the thermosensitive hydrogel and platelet-rich plasma complex according to claim 5, characterized in that: Before the thermosensitive hydrogel solution in step (1) is mixed with platelet-rich plasma, it is sterilized by wet heat sterilization or radiation sterilization to obtain a sterile thermosensitive hydrogel solution.

7. Use of the thermosensitive hydrogel and platelet-rich plasma complex of claims 1-4 in preparing wound repair materials, soft tissue repair materials, cartilage repair materials, bone repair materials, tendon repair materials, osteoarthritis materials, and cosmetic filling materials.

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