Temperature-sensitive hydrogel and use thereof in cartilage defect repair

Abstract

A temperature-sensitive hydrogel and the use thereof in cartilage defect repair. The temperature-sensitive hydrogel comprises a chitosan-based solution, and a solute of the chitosan-based solution comprises a chitosan type solute, the chitosan type solute comprises at least one chitosan derivative, and the molecular chain of the chitosan type solute is grafted with a functional group enabling the hydrogel to be temperature-sensitive.

Description

A thermosensitive hydrogel and its application in cartilage defect repair

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202410358151.8, filed on March 27, 2024, entitled “A Thermosensitive Hydrogel and Its Application in Cartilage Defect Repair,” the entire text of which is incorporated herein by reference. Technical Field

[0003] The present application relates to the field of medical materials, and in particular, to a thermosensitive hydrogel and the application of the thermosensitive hydrogel in cartilage defect repair. Background Art

[0004] Osteoarthritis is often accompanied by cartilage defects, which lack the ability to repair naturally. Currently, bone marrow stimulation, which includes grinding and shaping, subchondral drilling, and microfracture, is commonly used to treat cartilage defects.

[0005] Among them, microfracture is a minimally invasive surgical technique performed entirely under arthroscopy and holds great promise for future use. Microfracture surgery begins by using a planer and curved curette to remove residual cartilage fragments, creating a pool-like structure to accommodate the superclot. After completely exposing the bone bed, a special arthroscopic hand awl is used to perforate the exposed subchondral plate, creating microfractures. The basic principle of microfracture is to create a rough surface in the cartilage defect area, making it easier for the superclot to adhere to the wound surface, allowing it to differentiate into stable repair tissue within the cartilage defect area, providing a suitable living environment for more bone marrow cells.

[0006] The dislodgment of super clots is one of the reasons for the failure of microfracture surgery. Currently, enhanced microfracture procedures are available on the market that use "scaffold materials" to induce in situ cartilage regeneration, stabilize microfracture clots, and reduce the risk of clot displacement. This microfracture procedure uses cartilage repair scaffolds such as collagen to fill the microfracture area. However, solid repair scaffolds often require further processing and shaping by doctors to adapt to the patient's irregular defect, which brings cumbersome procedures and inconvenience to clinical treatment.

[0007] Therefore, how to improve the success rate of microfracture surgery while simplifying the operation has become a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0008] This application aims to address, to a certain extent, one of the technical problems in the related art. To this end, this application provides a thermosensitive hydrogel and its use in cartilage defect repair. When used in cartilage defect repair, the thermosensitive hydrogel can improve the success rate of microfracture surgery while simplifying the procedure.

[0009] To achieve the above-mentioned objectives, as a first aspect of the present application, a thermosensitive hydrogel is provided, wherein the thermosensitive hydrogel comprises a chitosan-based solution, the solute of the chitosan-based solution comprises a chitosan-like solute, the chitosan-like solute comprises at least one chitosan derivative, and the molecular chain of the chitosan-like solute is grafted with functional groups that make the hydrogel thermosensitive.

[0010] In some embodiments, the temperature-sensitive functional group is selected from at least one of hydroxybutyl, hydroxypropyl, hydroxyethyl, hydroxypentyl, carboxymethyl, and carboxyethyl.

[0011] In some embodiments, the chitosan derivative is selected from at least one of hydroxybutyl chitosan, hydroxypropyl chitosan, hydroxyethyl chitosan, hydroxypentyl chitosan, carboxymethyl chitosan, and carboxyethyl chitosan.

[0012] In some embodiments, the grafting degree of the chitosan derivative is between 1.0 and 2.5.

[0013] In some embodiments, the chitosan derivative has a grafting degree between 1.6 and 2.0.

[0014] In some embodiments, the solute of the chitosan-based solution further comprises a salt capable of ionizing in the chitosan-based solution.

[0015] In some embodiments, the salt is a salt formed by a monovalent cation and a monovalent anion.

[0016] In some embodiments, the salt is selected from at least one of sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium acetate, sodium sulfate, sodium carbonate, and sodium phosphate.

[0017] In some embodiments, the mass concentration of the chitosan-based solute in the chitosan-based solution is between 1% and 8%.

[0018] In some embodiments, the weight average molecular weight of the chitosan-based solute is between 50 kDa and 1500 kDa.

[0019] In some embodiments, the molecular weight distribution of the chitosan-based solute is between 1.0 and 10.0.

[0020] In some embodiments, the molecular weight distribution of the chitosan-based solute is between 1.5 and 9.0.

[0021] In some embodiments, the pH of the chitosan-based solution is between 7.0 and 8.0.

[0022] In some embodiments, the gelation temperature of the thermosensitive hydrogel is between 20°C and 37°C.

[0023] As a second aspect of the present application, a use of a thermosensitive hydrogel in repairing cartilage defects is provided, wherein the thermosensitive hydrogel is the thermosensitive hydrogel provided in the first aspect of the present application.

[0024] In the present application, when the temperature of the thermosensitive hydrogel is below the gelling temperature point, the thermosensitive hydrogel is liquid. When the temperature of the thermosensitive hydrogel exceeds the gelling temperature point, the thermosensitive hydrogel undergoes a phase change, is converted into a solid colloid, and forms a solid three-dimensional network structure. It should be pointed out that the solid three-dimensional network structure has a certain mechanical support effect. During microfracture surgery, when the thermosensitive hydrogel is injected into the defect site of the cartilage, when the local temperature is increased to the gelling temperature point of the thermosensitive hydrogel during surgery, the thermosensitive hydrogel is converted into a colloid, and forms a solid three-dimensional network structure, which can suppress the volume shrinkage and displacement of the blood clot after microfracture surgery and improve the success rate of microfracture surgery. At the same time, it is only necessary to inject the liquid thermosensitive hydrogel into the defect site of the cartilage during microfracture surgery, without the need for additional processing, thereby reducing the complexity of microfracture surgery combined with scaffold material implantation.

[0025] These features of the present application will be disclosed in detail in the following detailed description and accompanying drawings. The best embodiments or means of the present application will be fully illustrated in conjunction with the accompanying drawings, but are not intended to limit the technical solutions of the present application. Furthermore, although multiple features, elements, and components may be present in each of the following text and accompanying drawings, they may be labeled with different symbols or numbers for convenience, but all represent components with the same or similar structure or function. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the conventional technology, the following briefly introduces the drawings required for use in the embodiments or the conventional technology descriptions. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the disclosed drawings without any creative work.

[0027] FIG1 is an infrared spectrum of chitosan and hydroxybutyl chitosan.

[0028] FIG2 is a curve showing the change of phase transition time with the concentration of HBCS in Example 7. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0030] References in this specification to "one embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with the embodiment itself can be included in at least one embodiment disclosed herein. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0031] As a first aspect of the present application, a thermosensitive hydrogel is provided, which includes a chitosan-based solution, the solute of the chitosan-based solution includes a chitosan-like solute, and the chitosan-like solute includes at least one chitosan derivative, and the molecular chain of the chitosan derivative is grafted with a functional group that makes the hydrogel thermosensitive.

[0032] The gelling temperature point of the thermosensitive hydrogel is between 4°C and 80°C. When the temperature of the thermosensitive hydrogel is below the gelling temperature point, the thermosensitive hydrogel is in a liquid state. When the temperature of the thermosensitive hydrogel exceeds the gelling temperature point, the thermosensitive hydrogel undergoes a phase change and is converted into a solid colloid, forming a solid three-dimensional network structure. It should be pointed out that the solid three-dimensional network structure has a certain mechanical support effect. During the microfracture procedure, the thermosensitive hydrogel is injected into the defective part of the cartilage. When the local temperature rises to the gelling temperature point of the thermosensitive hydrogel during the operation, the thermosensitive hydrogel is converted into a colloid and forms a solid three-dimensional network structure, which can inhibit the volume shrinkage and displacement of the blood clot after microfracture surgery and improve the success rate of microfracture surgery. In addition, it is only necessary to inject the liquid thermosensitive hydrogel into the defective part of the cartilage during the microfracture procedure, and the doctor does not need to perform additional operations on it, which reduces the complexity of microfracture surgery combined with scaffold material implantation.

[0033] In the present application, the weight average molecular weight of the chitosan and / or chitosan derivative is between 50 kDa and 2000 kDa, which can ensure that the thermosensitive hydrogel has sufficient strength and provides sufficient mechanical support after being converted into a colloid.

[0034] In the thermosensitive hydrogel, chitosan or chitosan derivatives can promote blood coagulation through the positive ion effect of amino groups, thereby improving the success rate of microfracture surgery.

[0035] As described above, the molecular chains of the chitosan derivatives are grafted with functional groups that render the hydrogel thermosensitive. These functional groups can be grafted onto the -NH2 and / or -C6-OH groups of chitosan and / or chitosan derivatives. In some embodiments, the thermosensitive functional group can be at least one of hydroxybutyl, hydroxypropyl, hydroxyethyl, hydroxypentyl, carboxymethyl, and carboxyethyl groups.

[0036] In the present application, by configuring the mass concentration of the chitosan-based solution between 0.5% and 20%, configuring the pH value of the chitosan-based solution between 7.0 and 8.0, and configuring the weight-average molecular weight of the chitosan solute between 50 kDa and 2000 kDa, a thermosensitive hydrogel with a gelation temperature between 4°C and 80°C can be obtained.

[0037] In some embodiments, the gelation temperature of the thermosensitive hydrogel is between 20° C. and 37° C. After the microfracture operation, the temperature of the surgical site returns to normal human body temperature (about 36° C.), and the thermosensitive hydrogel can undergo a phase transition and transform into a solid colloid.

[0038] Before the microfracture procedure is completed, the temperature of the thermosensitive hydrogel is the ambient temperature (eg, 15° C. to 20° C.), at which the thermosensitive hydrogel is in a liquid state.

[0039] In order to adjust the gelation temperature of the thermosensitive hydrogel between 20°C and 37°C, the molecular weight, mass concentration, grafting degree of the solute (ie, chitosan derivative) of the chitosan-based solution and the pH value of the chitosan-based solution can be adaptively adjusted.

[0040] In some embodiments, the mass concentration of the chitosan solute may be between 1% and 8%.

[0041] In some embodiments, the weight average molecular weight of the chitosan-based solute is between 50 kDa and 1500 kDa.

[0042] In the present application, chitosan solute is a polymer, therefore, chitosan solute does not have a fixed molecular weight, and chitosan solute is a mixture of multiple different molecular weights. In certain embodiments, the molecular weight distribution of the chitosan solute is between 1.0 and 10.0. In certain embodiments, the molecular weight distribution of the chitosan solute is between 1.5 and 9.0.

[0043] As described above, the chitosan-based solute may include chitosan. The degree of deacetylation is one of the most basic structural parameters for evaluating chitosan. Specifically, the degree of deacetylation refers to the ratio of the number of deacetylated glucosamine units to the total number of glucosamine units. The higher the degree of deacetylation, the more free amino groups on the chitosan molecular chain, and the better the solubility of the chitosan in acid. In some embodiments, the degree of deacetylation of the chitosan is between 50% and 100%.

[0044] In some embodiments, the degree of deacetylation of the chitosan is between 70% and 100%. In some embodiments, the degree of deacetylation of the chitosan is between 85% and 100%.

[0045] The degree of grafting of thermosensitive functional groups onto the chitosan derivative is also an important factor affecting the gelation temperature of the thermosensitive hydrogel. In some embodiments, the grafting degree of the chitosan derivative is between 1.0 and 2.5. In some embodiments, the grafting degree of the chitosan derivative is between 1.6 and 2.0.

[0046] In the present application, there is no particular limitation on the specific type of chitosan derivative. In some embodiments, the chitosan derivative is selected from at least one of hydroxybutyl chitosan, hydroxypropyl chitosan, hydroxyethyl chitosan, hydroxypentyl chitosan, carboxymethyl chitosan, and carboxyethyl chitosan.

[0047] In order to set the gelation temperature of the thermosensitive hydrogel between 20° C. and 37° C., in some embodiments, the solute of the chitosan-based solution further includes a salt that can be ionized in the chitosan-based solution.

[0048] In some embodiments, the salt is selected from salts formed by monovalent cations and monovalent anions.

[0049] In some embodiments, the salt is selected from at least one of sodium chloride (NaCl), magnesium chloride (MgCl2), potassium chloride (KCl), calcium chloride (CaCl2), sodium acetate (CH3COONa), sodium sulfate (Na2SO4), sodium carbonate (Na2CO3), and sodium phosphate (Na3PO4).

[0050] In some embodiments, the chitosan derivative can be hydroxybutyl chitosan.

[0051] By controlling the ionic salts in the hydroxybutyl chitosan solution to be formed from monovalent cations and monovalent anions, the gelling temperature and formation time of the thermosensitive hydrogel can be controlled within a range that is more conducive to surgery.

[0052] The molecular chain side chain of hydroxybutyl chitosan has more hydroxyl groups, which have certain electronegativity. When the anions in the chitosan solution are all Cl - When the reaction is carried out, different cations in the chitosan-based solution have different degrees of binding with the hydroxyl group, and different cations have different degrees of repulsion with the protonated amino group of hydroxybutyl chitosan, which affects the interaction strength between the hydroxybutyl chitosan molecular chains and ultimately affects the formation time and temperature of the gel network.

[0053] Similarly, when the cations are all Na + When the anions are mixed, there are different degrees of charge repulsion between them, and the anions also have a charge repulsion effect on the hydroxyl groups of the hydroxybutyl chitosan molecular chain. The charge attraction of different anions on the amino groups of the chitosan side chains causes the chitosan molecular chain to stretch to different degrees, thus affecting the formation time and temperature of the gel network.

[0054] By adjusting and optimizing the factors of ionic species in hydroxybutyl chitosan solution, hydroxybutyl chitosan solution can form thermosensitive hydrogel at a lower gelation temperature.

[0055] The pH value of the solution affects the gelation temperature of the thermosensitive hydrogel and the duration of phase transition. In some embodiments, the pH value of the chitosan-based solution is between 7.0 and 8.0, which helps control the gelation temperature of the thermosensitive hydrogel between 20°C and 37°C.

[0056] In some embodiments, the pH of the solution is in the range of 7.0-7.5.

[0057] In the present application, hydroxybutyl chitosan (HBCS) can be prepared by the following steps.

[0058] 1) Purification of chitosan: The crude chitosan product was dissolved in glacial acetic acid aqueous solution, filtered to remove insoluble matter, and then sodium hydroxide solution was added dropwise to the filtrate until all flocculent precipitates were precipitated. The precipitate was then washed with purified water several times until it was neutral and dried to obtain purified chitosan;

[0059] 2) Alkalization of chitosan: Add the refined chitosan to sodium hydroxide or potassium hydroxide solution and fully alkalize for 12-48 hours. After centrifugation to remove the alkali solution, the alkalized chitosan is obtained for later use;

[0060] 3) Dispersion: Add a mixture of isopropanol and water or a mixture of ethanol and water to the alkalized chitosan, and stir until the chitosan is completely dispersed in the isopropanol / ethanol aqueous solution;

[0061] 4) Hydroxybutylation reaction: 1,2-butylene oxide is added dropwise to the above solution, and the reaction is carried out under heating and stirring in a water bath at a temperature of 25°C-80°C and a reaction time of 12h-72h;

[0062] 5) pH adjustment and dialysis: After the hydroxybutylation reaction, the pH is adjusted to neutral with an acid (such as hydrochloric acid or acetic acid) until the solution is completely transparent. The reaction solution is placed in a dialysis bag with a molecular cutoff of 3500Da-50000Da and dialyzed;

[0063] 6) Impurity removal and freeze drying: insoluble impurities are removed by centrifugation / filtration, and freeze drying is performed to obtain a hydroxybutyl chitosan sample.

[0064] In some embodiments, the reaction temperature of the hydroxybutylation reaction is 35°C-63°C.

[0065] Accordingly, the method for preparing the thermosensitive hydrogel may include the following steps.

[0066] The hydroxybutyl chitosan sample prepared by the above method was weighed and dissolved in purified water to prepare an HBCS solution; the HBCS solution was placed in a 4°C refrigerator until it was completely dissolved and bubbles were eliminated, and the solution was placed in a syringe for later use.

[0067] In some embodiments, the method for preparing the thermosensitive hydrogel may further include the following steps: weighing a hydroxybutyl chitosan sample prepared by the above method, dissolving it in a saline solution (such as normal saline) to prepare an HBCS solution; placing it in a 4°C refrigerator until it is completely dissolved and bubbles are eliminated, and placing the solution in a syringe for later use.

[0068] As a second aspect of the present application, a thermosensitive hydrogel that can be used for cartilage defect repair is provided, wherein the thermosensitive hydrogel is the thermosensitive hydrogel provided in the first aspect of the present application.

[0069] As described above, the gelling temperature of the thermosensitive hydrogel is between 4°C and 80°C. When the temperature of the thermosensitive hydrogel is below the gelling temperature, the thermosensitive hydrogel is in a liquid state. When the temperature of the thermosensitive hydrogel exceeds the gelling temperature, the thermosensitive hydrogel undergoes a phase change, converts into a solid state, and forms a three-dimensional network structure. It should be pointed out that the solid three-dimensional network structure has a certain mechanical support effect. During the microfracture procedure, the thermosensitive hydrogel is injected into the open wound of the cartilage. When the temperature rises to the gelling temperature of the thermosensitive hydrogel after the operation, the liquid thermosensitive hydrogel solution is converted into a colloid to form a solid three-dimensional network structure, which can inhibit the volume shrinkage and displacement of the blood clot after microfracture surgery and improve the success rate of microfracture surgery. In addition, the liquid thermosensitive hydrogel only needs to be injected into the defective part of the cartilage during the microfracture procedure, and the doctor does not need to perform additional processing on it, which reduces the complexity of the microfracture procedure.

[0070] The thermosensitive hydrogel provided in this application can be used as a medical material / product for repairing cartilage defects to promote cartilage repair.

[0071] Example 1

[0072] A method for preparing hydroxybutyl chitosan comprises the following steps:

[0073] 1) Chitosan Purification: 20 g of crude chitosan (purchased from Aladdin, molecular weight 150 kDa) was dissolved in 1000 mL of 1% by volume glacial acetic acid aqueous solution and filtered to remove insoluble matter. 1 mol / L sodium hydroxide solution was then added dropwise to the filtrate, stirring continuously with a glass rod until all flocculent precipitates were precipitated. The precipitate was then washed several times with purified water until neutral. The precipitate was first desalted with 70% by volume ethanol, then dehydrated with 95% by volume ethanol, and dried at 50°C.

[0074] 2) Alkalization of chitosan: Weigh 1 g of refined chitosan powder and add it to 10 mL of a 50% by mass sodium hydroxide / potassium hydroxide solution. Alkalize the solution for 12 to 48 hours, then remove the alkali solution by centrifugation and set aside.

[0075] 3) Dispersion: Add 20 mL of a mixture of isopropanol / ethanol and water (V isopropanol / ethanol: V water = 1:1) to the alkalized chitosan and stir to disperse the chitosan completely in the isopropanol / ethanol aqueous solution.

[0076] 4) Hydroxybutylation reaction: 20 mL to 25 mL of 1,2-butylene oxide was added dropwise to the above solution, and the reaction was carried out under heating and stirring in a water bath at a temperature of 35°C and a reaction time of 12 h.

[0077] 5) pH adjustment and dialysis: After different reaction times, the pH is adjusted to neutral with an acid (such as hydrochloric acid, acetic acid, etc.) to completely terminate the reaction. After the solution is completely transparent, the reaction solution is placed in a dialysis bag with a molecular cutoff of 3500Da-50000Da and dialyzed for 3-7 days;

[0078] 6) Impurity removal and freeze drying: The dialyzed solution was taken out, centrifuged to remove insoluble impurities, and freeze dried to obtain a hydroxybutyl chitosan sample.

[0079] Example 2

[0080] Hydroxybutyl chitosan was prepared according to the method provided in Example 1, except that the reaction time in step 4) was 24 hours.

[0081] Example 3

[0082] Hydroxybutyl chitosan was prepared according to the method provided in Example 1, except that the reaction time in step 4) was 48 hours.

[0083] Example 4

[0084] Hydroxybutyl chitosan was prepared according to the method provided in Example 1, except that the reaction time in step 4) was 72 hours.

[0085] Example 5

[0086] Hydroxybutyl chitosan was prepared according to the method provided in Example 1, except that the reaction time in step 4) was 84 hours.

[0087] Example 6

[0088] Hydroxybutyl chitosan was prepared according to the method provided in Example 1, except that the reaction time in step 4) was 96 hours.

[0089] The products of Examples 1-6 were tested as follows.

[0090] Weigh appropriate amounts of hydroxybutyl chitosan (HBCS) and chitosan (CS), grind them with spectral grade KBr powder at room temperature, put them into a sample cup, press them into pellets, and analyze them by Fourier transform infrared spectroscopy at 400 cm -1 -4000cm -1 The infrared spectra of HBCS (shown by the dotted line) and CS (shown by the solid line) are shown in Figure 1. Compared with chitosan, the sample obtained in Example 1 has a -1 -2970cm -1 and 1462cm -1 New absorption peaks appeared at 1155cm, which are the stretching vibration absorption peak of -CH and the bending vibration absorption peak of -CH3. This shows that after the reaction with 1,2-butylene oxide, -CH3 and -CH2- are introduced into the chitosan molecular chain, that is, hydroxybutyl groups are introduced. Due to the newly introduced hydroxybutyl groups, the absorption peak at 1155cm -1 The nearby -C6-OH characteristic band weakened and almost disappeared, which proved that the hydroxybutyl group was successfully grafted onto the chitosan molecular chain and HBCS was successfully prepared.

[0091] The [C / N] atomic ratios of the freeze-dried hydroxybutyl chitosan (HBCS) samples and the chitosan raw material (CS) in Examples 1-6 were measured using an organic element analyzer (Model: Elementar Vario EL cube), and the degree of grafting (DS) and degree of deacetylation (DA, %) were calculated using the following formulas:

[0092] DA=1-([C / N] CS -6) / 2*100%,

[0093] DS=([C / N] HBCS -[C / N] CS ) / 4.

[0094] The grafting degrees DS of Examples 1 to 6 are shown in Table 1, and the raw materials DA used are shown in Table 1.

[0095] Table 1 Transition temperature and time of HBCS thermosensitive hydrogels with different hydroxybutyl grafting degrees

[0096] Example 7

[0097] Thermosensitive hydrogels were prepared using the following method.

[0098] The HBCS samples in Examples 1-6 were weighed and dissolved in purified water to prepare 2% (w / w) HBCS solutions, which were placed in a 4° C. refrigerator until completely dissolved and bubbles eliminated, and then aspirated with a syringe for later use.

[0099] The inversion method was used to characterize the transition temperature and time of the thermosensitive hydrogels of HBCS with different grafting degrees in Examples 1-6. The specific steps are as follows.

[0100] The sol-gel phase transition of HBCS was determined using the test tube inversion method. 1 mL of hydrogel was added to a 10 mL transparent sample bottle, which was placed in a 4°C-80°C water bath. Phase transition was observed every 3 seconds. The phase transition criterion was that the HBCS in the transparent sample bottle did not flow down the wall within 30 seconds after the transparent sample bottle was inverted on the table. The immersion time when the sample bottle was taken out of the water bath was the phase transition time of the hydrogel.

[0101] As shown in Table 1, as the degree of grafting increases, the sol-gel transition temperature of HBCS decreases and the transition time shortens. An increase in the degree of grafting indicates a higher content of hydroxybutyl chitosan in the resulting material. The hydroxyl groups on the hydroxybutyl chitosan side chains influence the hydrophilicity and hydrophobicity of the molecular chain, disrupting the charge repulsion between chitosan molecules and promoting the formation of a gel network. Operating room temperatures typically range from 18°C ​​to 25°C, and the ideal transition temperature for hydrogels is 20°C to 37°C. As the degree of grafting increases, the gel transition temperature becomes too low and the transition time becomes too fast, making it difficult for doctors to operate in the operating room, effectively limiting the operating time window.

[0102] Example 8

[0103] Preparation of thermosensitive hydrogels with different concentrations of HBCS.

[0104] The HBCS sample from Example 2 was weighed and dissolved in purified water to prepare 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%, 6%, 7%, and 8% (w / w) HBCS solutions, respectively. The solutions were placed in a 4°C refrigerator until completely dissolved and bubbles eliminated, and then aspirated with a syringe for later use.

[0105] The inversion method was used to characterize the transition time of the thermosensitive hydrogels of HBCS with different concentrations prepared in Example 8. The specific steps are as follows.

[0106] The sol-gel phase transition of HBCS was determined by the test tube inversion method. That is, 1 mL of hydrogel was added to a 10 mL transparent sample bottle, which was placed in a 37°C water bath. The phase transition was observed every 3 seconds. The phase transition criterion was that the HBCS in the transparent sample bottle did not flow down the wall within 30 seconds after the transparent sample bottle was inverted on the table. The immersion time when the sample bottle was taken out of the water bath was the time of the temperature-sensitive gel phase transition.

[0107] Following the above method, the phase transitions of all HBCS solutions of varying concentrations in Example 7 into gels in a 37°C water bath were observed and the phase transition times were recorded. A curve of phase transition time versus HBCS concentration was plotted, as shown in Figure 2. As HBCS concentration increased, the phase transition time gradually decreased, but the magnitude of the change became increasingly smaller, indicating a gradual equilibrium. In practical applications, increasing HBCS solubility decreases its fluidity. Therefore, to ensure the fluidity of the thermosensitive hydrogel, the HBCS concentration used was 1%-8% (w / w).

[0108] Example 9

[0109] A 0.1 mol / L saline solution was prepared, wherein the saline solution was a NaCl solution. The HBCS sample in Example 2 was weighed. A 2% HBCS solution was prepared using the 0.1 mol / L NaCl solution and fully dissolved to obtain a thermosensitive hydrogel. The thermosensitive hydrogel was placed in a 4°C refrigerator to eliminate bubbles, and the thermosensitive hydrogel after the bubbles were eliminated was aspirated with a syringe for later use.

[0110] Example 10

[0111] The thermosensitive hydrogel solution was prepared using the method in Example 9, except that the salt solution was a MgCl2 solution.

[0112] Example 11

[0113] The thermosensitive hydrogel solution was prepared using the method in Example 9, except that the salt solution was a KCl solution.

[0114] Example 12

[0115] The thermosensitive hydrogel solution was prepared using the method in Example 9, except that the salt solution was a CaCl2 solution.

[0116] Example 13

[0117] The thermosensitive hydrogel solution is prepared by the method in Example 9, except that the salt solution is a Na2SO4 solution.

[0118] Example 14

[0119] The thermosensitive hydrogel solution was prepared using the method in Example 9, except that the salt solution was a Na3PO4 solution.

[0120] Example 15

[0121] The thermosensitive hydrogel solution was prepared using the method in Example 9, except that the salt solution was a Na2CO3 solution.

[0122] Example 16

[0123] The thermosensitive hydrogel solution was prepared using the method in Example 9, except that the salt solution was a CH3COONa solution.

[0124] The inversion method was used to characterize the transition temperature and time of HBCS thermosensitive hydrogel after the introduction of different ionic salts, as follows.

[0125] The sol-gel phase transition of HBCS was determined using the test tube inversion method. 1 mL of hydrogel was added to a 10 mL transparent sample bottle, which was placed in a 20°C-80°C water bath. Phase transition was observed every 3 seconds. The phase transition criterion was that the HBCS in the transparent sample bottle did not flow down the wall within 30 seconds after the transparent sample bottle was inverted on the table. The immersion time when the sample bottle was taken out of the water bath was the phase transition time of the hydrogel.

[0126] Table 2 Transition temperature and time of HBCS thermosensitive hydrogel prepared with different ionic salt solutions

[0127] As shown in Table 2, after the introduction of salt ions, the transition temperature of the sol decreased significantly compared to the 2% solution in Example 2, which underwent phase transition after incubation at 37°C for 120 seconds. The hydrogel typically has a transition temperature of 20°C-37°C, as the operating room temperature is typically 18°C-25°C. The gel must be used in the operating room and injected into the cartilage defect. Among them, the phase transition temperatures of Na2SO4, Na3PO4, and Na2CO3 are below 20°C, which limits their clinical applications.

[0128] When the ionic salt is composed of monovalent cations and monovalent anions, the phase transition temperature and time are most suitable for clinical application.

[0129] When the types of ionic salts are NaCl, MgCl2, KCl, CaCl2, and CH3COONa, their phase transition temperatures can all meet the clinical application temperature.

[0130] The side chains of hydroxybutyl chitosan molecules have more hydroxyl groups, which have certain electronegativity. - When the cations in the solution are different, the degree of binding between different cations and hydroxyl groups is different. At the same time, the degree of repulsion between different cations and the protonated amino groups of hydroxybutyl chitosan is different, which affects the interaction strength between hydroxybutyl chitosan molecular chains, thereby affecting the formation time and temperature of the gel network. Similarly, when the cations are all Na +When the hydroxybutyl chitosan solution is heated, the HBCS solution can form a thermosensitive hydrogel at a lower gelling temperature due to the different anions' charge repulsions on the hydroxyl groups on the side chains of the chitosan chain. Furthermore, the charge attraction of different anions on the amino groups on the side chains of the chitosan chain leads to different degrees of stretching of the chitosan chain, thus affecting the formation time and temperature of the gel network. By adjusting and optimizing the ionic species of the HBCS solution, the HBCS solution can form a thermosensitive hydrogel at a lower gelling temperature.

[0131] Example 17

[0132] The HBCS sample from Example 2 was weighed and dissolved in purified water to prepare a 2% HBCS solution. The pH of the hydrogel solution was adjusted to 5.5 using 0.05M HCl and 0.05M NaOH solutions, while the NaCl concentration in the system was controlled to 0.9%. The solution was placed in a refrigerator at 4°C until completely dissolved and bubbles eliminated, thereby obtaining a thermosensitive hydrogel. The solution was aspirated with a syringe and set aside.

[0133] Example 18

[0134] Thermosensitive hydrogels were prepared using the method provided in Example 17, except that the pH of the hydrogel solution was adjusted to 6.0 using 0.05 M HCl and 0.05 M NaOH solutions.

[0135] Example 19

[0136] The thermosensitive hydrogel was prepared using the method provided in Example 17, except that the pH of the hydrogel solution was adjusted to 6.5 using 0.05 M HCl and 0.05 M NaOH solutions.

[0137] Example 20

[0138] Thermosensitive hydrogels were prepared using the method provided in Example 17, except that the pH of the hydrogel solution was adjusted to 7.0 using 0.05 M HCl and 0.05 M NaOH solutions.

[0139] Example 21

[0140] The thermosensitive hydrogel was prepared using the method provided in Example 17, except that the pH of the hydrogel solution was adjusted to 7.5 using 0.05 M HCl and 0.05 M NaOH solutions.

[0141] Example 22

[0142] The thermosensitive hydrogel was prepared using the method provided in Example 17, except that the pH of the hydrogel solution was adjusted to 8.0 using 0.05 M HCl and 0.05 M NaOH solutions.

[0143] Example 23

[0144] Thermosensitive hydrogels were prepared using the method provided in Example 17, except that the pH of the hydrogel solution was adjusted to 8.5 using 0.05 M HCl and 0.05 M NaOH solutions.

[0145] The inversion method was used to characterize the transition temperature and time of HBCS thermosensitive hydrogel at different pH values, which specifically included the following steps.

[0146] The sol-gel phase transition of HBCS was determined using the test tube inversion method. 1 mL of hydrogel was added to a 10 mL transparent sample bottle, which was placed in a 20°C-80°C water bath. Phase transition was observed every 3 seconds. The phase transition criterion was that the HBCS in the transparent sample bottle did not flow down the wall within 30 seconds after the transparent sample bottle was inverted on the table. The immersion time when the sample bottle was taken out of the water bath was the phase transition time of the hydrogel.

[0147] Table 3 shows the changes in gel transition temperature and time under different pH conditions. The lower the pH, the longer the gel transition time, reaching no transition at pH 5.5. Under neutral conditions, the transition time shortens with increasing pH. However, as the pH further increases, at the alkaline pH of 8.5, the chitosan dissolution time increases, and the phase transition temperature and time required for the solution to form a uniform gel rise, making them incompatible with clinical application. Under acidic conditions, the amino groups in hydroxybutyl chitosan are protonated and positively charged, leading to mutual repulsion between the molecular chains and preventing the formation of a gel network. Under alkaline conditions, some hydroxyl protons in the hydroxybutyl chitosan molecules are removed, preventing hydrogen bonding between molecules. This results in an increase in crosslinking temperature and time. If the solution gels too long, there is a risk of the hydrogel overflowing from the defect if the surgeon disturbs the body, preventing a satisfactory surgical outcome. Excessive transition times also waste surgical time. Therefore, a solution with a pH between 7.0 and 8.0 has a suitable gel transition time and temperature for surgical procedures.

[0148] Table 3 Transition temperature and time of HBCS thermosensitive hydrogels prepared under different pH conditions

[0149] Example 24

[0150] The method for detecting the cytotoxicity of the thermosensitive hydrogel extract comprises the following steps.

[0151] 1) Preparation of the hydrogel mixed extract: HBCS hydrogel was added to DMEM medium (extraction was performed at a ratio of gel mass to medium volume = 0.2 g / mL). The mixture was incubated in a 37°C incubator for 24 hours to obtain a hydrogel extract. The supernatant was aspirated as the 100% gel extract. Simultaneously, the 100% gel extract was diluted with DMEM medium to three different concentrations: 75%, 50%, and 25%.

[0152] 2) Cell preparation: Mouse fibroblasts (L-929) were revived and cultured in a 37°C constant temperature cell culture incubator (CO2 content 5%, relative humidity 90%). The cell culture medium was changed every 2 days. When the cells occupied 90% of the visual field under the microscope, they were digested and passaged with 0.25% trypsin. 5x10 cells were added to each well of a 96-well plate. 4 100 μL of cell suspension with a concentration of cells / mL.

[0153] 3) Control group and experimental group settings.

[0154] Positive control group: 100 μL of Dulbecco's modified eagle medium (DMEM) containing 0.5% phenol was added to each well.

[0155] Negative control group: 100 μL of DMEM medium was added to each well.

[0156] Experimental group: 100 μL of 25%, 50%, 75%, or 100% hydrogel extract was added to each well.

[0157] The experimental group and the control group were set up in 96-well plates, with 6 parallel samples in each group.

[0158] 4) Cell sample co-culture: Place the 96-well plate in a constant temperature incubator and culture the culture medium every 24 hours. After 48 hours, observe the growth of L-929 cells under an optical microscope.

[0159] 5) CCK-8 assay.

[0160] a. Aspirate the culture medium of each group and rinse with phosphate buffered saline (PBS) solution three times.

[0161] b. Prepare 10% CCK-8 solution using DMEM and CCK-8 kit.

[0162] c. Add 100 μL of the prepared CCK-8 solution to each well.

[0163] d. Continue incubating in a cell culture incubator for 4 hours.

[0164] e. Measure the absorbance of each sample at 450 nm using a microplate reader.

[0165] f. Calculate the relative proliferation rate (RGR) of cells in each well based on the absorbance value.

[0166] Relative proliferation rate (RGR) = absorbance value of the experimental group / mean absorbance value of the negative control group × 100%.

[0167] The toxicity of materials is determined according to the national biological evaluation standards for medical devices. The relevant standards are as follows.

[0168] The material toxicity level is graded as 0-1, which is qualified; grade 2 can be comprehensively evaluated based on cell morphology and cell growth density to determine whether it is qualified; and grades greater than three are unqualified.

[0169] Table 4 RGR values ​​and toxicity levels of each group in L-929 cell cytotoxicity experiment

[0170] It can be seen from Table 4 that all groups are grade 0, indicating that the HBCS hydrogel has qualified cell safety on the L-929 cell line and meets the biosafety requirements as an ideal cartilage repair material.

[0171] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0172] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present application, and these modifications and improvements fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A thermosensitive hydrogel, characterized in that The chitosan-based solution comprises a chitosan-based solution, wherein the solute of the chitosan-based solution comprises a chitosan-like solute, the chitosan-like solute comprises at least one chitosan derivative, and the molecular chain of the chitosan-like solute is grafted with a functional group that makes the hydrogel thermosensitive.

2. The thermosensitive hydrogel according to claim 1, wherein The temperature-sensitive functional group is selected from at least one of hydroxybutyl, hydroxypropyl, hydroxyethyl, hydroxypentyl, carboxymethyl, and carboxyethyl.

3. The thermosensitive hydrogel according to claim 1, wherein The chitosan derivative is at least one selected from hydroxybutyl chitosan, hydroxypropyl chitosan, hydroxyethyl chitosan, hydroxypentyl chitosan, carboxymethyl chitosan, and carboxyethyl chitosan.

4. The thermosensitive hydrogel according to claim 1, wherein The grafting degree of the chitosan derivative is between 1.0 and 2.

5.

5. The thermosensitive hydrogel according to claim 4, wherein The grafting degree of the chitosan derivative is between 1.6 and 2.

0.

6. The thermosensitive hydrogel according to claim 1, wherein The solute of the chitosan-based solution also includes a salt that can be ionized in the chitosan-based solution.

7. The thermosensitive hydrogel according to claim 6, wherein The salt is a salt formed by a monovalent cation and a monovalent anion.

8. The thermosensitive hydrogel according to claim 6, wherein The salt is selected from at least one of sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium acetate, sodium sulfate, sodium carbonate, and sodium phosphate.

9. The thermosensitive hydrogel according to claim 1, wherein In the chitosan-based solution, the mass concentration of the chitosan solute is between 1% and 8%.

10. The thermosensitive hydrogel according to claim 1, wherein The weight average molecular weight of the chitosan solute is between 50 kDa and 1500 kDa.

11. The thermosensitive hydrogel according to claim 1, wherein The molecular weight distribution of the chitosan solute is between 1.0 and 10.

0.

12. The thermosensitive hydrogel according to claim 1, wherein The pH value of the chitosan-based solution is between 7.0 and 8.

0.

13. The thermosensitive hydrogel according to any one of claims 1 to 12, wherein: The gelling temperature of the thermosensitive hydrogel is between 20°C and 37°C.

14. Application of a thermosensitive hydrogel in cartilage defect repair, characterized in that: The thermosensitive hydrogel is the thermosensitive hydrogel according to any one of claims 1 to 13.

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