Chitosan temperature-sensitive gel composition for treating osteoarthritis and preparation method therefor

Abstract

A chitosan temperature-sensitive gel composition for treating osteoarthritis and a preparation method therefor. The composition comprises 0.5%-5% of a chitosan temperature-sensitive gel and 106-107 / mL of stem cells. Mixing mesenchymal stem cells and the chitosan temperature-sensitive gel and then injecting same into articular cavities can exert the joint lubricating effect of the chitosan gel itself, and the mesenchymal stem cells can secrete a large number of inflammation inhibitory cytokines, and can also promote cartilage cell regeneration, thereby effectively treating osteoarthritis.

Description

Chitosan thermosensitive adhesive composition for treating osteoarthritis and its preparation method Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a chitosan thermosensitive adhesive composition for treating osteoarthritis, its preparation method, and its uses. Background Technology

[0002] Osteoarthritis, commonly known as "bone hyperplasia" or "bone spurs," is a degenerative disease characterized by degeneration and destruction of articular cartilage and bone hyperplasia. It is also called osteoarthritis, degenerative arthritis, senile arthritis, hypertrophic arthritis, etc. Currently, routine treatments for osteoarthritis patients include oral anti-inflammatory analgesics and glucosamine-based drugs, intra-articular injection of sodium hyaluronate, physical therapy, and surgery. Surgical treatment mainly involves arthroscopy (endoscopy) or open surgery. While these methods can temporarily relieve pain, the long-term effects are not ideal.

[0003] With the advent and development of cell gene therapy technology, the use of mesenchymal stem cell (MSC) injections to treat osteoarthritis has become a novel treatment method. MSCs can secrete large amounts of anti-inflammatory factors and promote chondrocyte regeneration, making them more effective in treating osteoarthritis. However, in practice, the effect of simply using stem cell injections to treat osteoarthritis is not ideal. The main reason is that stem cells flow and diffuse within the joint cavity, resulting in a short time of action at the affected site, thus greatly weakening the treatment effect. Furthermore, a single large-dose injection of high-concentration MSCs can actually stimulate the release of inflammatory factors from MSCs, exacerbating inflammation.

[0004] Therefore, to address the above issues, it is necessary to develop a carrier that can support the growth of mesenchymal stem cells (MSCs) and remain at the affected site for an extended period after injection. This carrier needs to be safe, biodegradable, and able to effectively balance the interaction between the active ingredients of inflammatory drugs and mesenchymal stem cells (MSCs). Summary of the Invention

[0005] To address the shortcomings of current mesenchymal stem cell (MSC) injections for treating osteoarthritis, where stem cells migrate and diffuse within the joint cavity, resulting in a short duration of action at the affected site and failing to provide long-term therapeutic effects, this invention provides a composite therapeutic product for osteoarthritis comprising chitosan thermosensitive gel and stem cells, along with its preparation method. The aim is to achieve safe and long-lasting efficacy for effective treatment of osteoarthritis. This therapeutic product utilizes the thermosensitive properties of chitosan gel; at body temperature, the mixture of the anti-inflammatory stem cells and the thermosensitive gel solidifies in situ within the joint cavity, ensuring that the therapeutic drugs and cells can remain at the lesion or injury site for an extended period, thus achieving a prolonged therapeutic and repairing effect. To achieve the above objectives, this invention first provides a thermosensitive gel composition for treating osteoarthritis.

[0006] Specifically, the temperature-sensitive adhesive composition comprises 0.5%-5% chitosan temperature-sensitive adhesive and 10 6 -10 7 / mL stem cells.

[0007] Preferably, the chitosan thermosensitive adhesive includes one or more of hydroxypropyl chitosan, hydroxypropyl chitin, hydroxybutyl chitosan, and hydroxybutyl chitin.

[0008] More preferably, the chitosan thermosensitive adhesive is hydroxypropyl chitin.

[0009] Preferably, the temperature-sensitive adhesive composition further includes an inflammation inhibitor, which includes one or more of the following: icariin, prostaglandins, interleukins, tumor necrosis factor, astragalus polysaccharides, and nuclear factor-κB (NF-κB).

[0010] Preferably, the stem cells are human mesenchymal stem cells (MSCs).

[0011] The present invention also provides a method for preparing the aforementioned temperature-sensitive adhesive composition, comprising:

[0012] S1: Add chitosan powder to physiological saline, stir to dissolve the powder, filter, and obtain chitosan thermosensitive adhesive solution;

[0013] S2: The fully cultured mesenchymal stem cells (MSCs) are centrifuged and enriched, then slowly resuspended with physiological saline, centrifuged and enriched again to obtain mesenchymal stem cells (MSCs).

[0014] S3: In a sterile environment, the chitosan thermosensitive adhesive prepared in S1 is mixed with an inflammation inhibitor and slowly injected into the mesenchymal stem cells (MSCs) prepared in S2. The mixture is gently blown to ensure that the mesenchymal stem cells (MSCs) are fully mixed with the blend solution, thus obtaining the final therapeutic product of chitosan thermosensitive adhesive.

[0015] Preferably, the chitosan in step S1 includes one or more of hydroxypropyl chitosan, hydroxypropyl chitin, hydroxybutyl chitosan, and hydroxybutyl chitin.

[0016] Preferably, step S1 also includes filtering or dialysis using a 200-mesh sieve or a dialysis bag.

[0017] Preferably, the inflammation inhibitor in step S1 includes at least one of icariin, prostaglandin, interleukin, tumor necrosis factor, astragalus polysaccharide, and nuclear factor-κB.

[0018] Preferably, a solvent is used in step S1, which includes: deionized water, double-distilled water, physiological saline or cell culture medium.

[0019] Preferably, in step S1, the concentration of the temperature-sensitive adhesive is 0.5%-5% (by mass / volume) and the concentration of the inflammation inhibitor is 10 ng / mL-25 μg / mL.

[0020] Preferably, the concentration of mesenchymal stem cells (MSCs) in step S3 is 10. 6 -10 7 / mL;

[0021] Preferably, steps S1, S2 and S3 are all carried out at 2-10℃.

[0022] The present invention also provides the use of the above-described thermosensitive adhesive composition in the preparation of arthritis medicaments.

[0023] Preferably, the thermosensitive adhesive composition for treating osteoarthritis is administered by injection.

[0024] Compared with the prior art, the thermosensitive adhesive composition for treating osteoarthritis described in this invention has the following beneficial effects:

[0025] 1. The composite therapeutic product for osteoarthritis, which combines chitosan thermosensitive adhesive, inflammation inhibitor, and stem cell therapy, provided by this invention, is quick, simple, green, and environmentally friendly in preparation. Furthermore, this composite therapeutic product is in a liquid state under in vitro conditions, which facilitates industrial production and filling.

[0026] 2. The composite therapeutic product for osteoarthritis provided by this invention, which combines chitosan thermosensitive gel, inflammation inhibitor, and stem cell therapy, is liquid below the phase transition temperature (low temperature), making it easy to inject. After injection into animals or humans, due to the thermosensitive properties of chitosan gel, the composite therapeutic product rapidly transforms into a semi-solid gel in vivo. This avoids the loss of effective ingredients caused by the fluidity of liquids, which leads to short retention time of drug active ingredients and cells at the affected area and the need for frequent administration. It is an excellent sustained-release drug delivery system that can achieve long-term maintenance of therapeutic effect after a single injection, improve patient compliance, and reduce the economic burden on patients.

[0027] 3. The preferred embodiment of the present invention provides a composite treatment product for osteoarthritis, which combines chitosan thermosensitive gel, inflammation inhibitor, and stem cell therapy. The inflammation inhibitor and mesenchymal stem cells are mixed with chitosan thermosensitive gel and injected into the joint cavity. On the one hand, the chitosan gel itself lubricates the joint; on the other hand, the mesenchymal stem cells secrete a large amount of anti-inflammatory factors. Combined with the inflammation inhibitor, this effectively suppresses the inflammatory response in the joint, alleviates symptoms, and slows disease progression. Simultaneously, the mesenchymal stem cells can also promote chondrocyte regeneration, leading to a more effective treatment of osteoarthritis. Attached Figure Description

[0028] Figure 1 is an electron microstructure diagram of the hydroxypropyl chitin prepared in Example 1 of this application;

[0029] Figure 2 shows the thermosensitive properties test of the hydroxypropyl chitin prepared in Example 1 of this application;

[0030] Figure 3 is a rat in vivo imaging image of the hydroxypropyl chitin combined stem cells prepared in Example 1 of this application;

[0031] Figure 4 is a magnetic resonance imaging of the rat bone joint of the hydroxypropyl chitin combined stem cells prepared in Example 1 of this application.

[0032] Figure 5 is an in vivo anatomical diagram of the rat bone joint of the hydroxypropyl chitin combined stem cells prepared in Example 1 of this application.

[0033] Figure 6 shows safranin staining of rat cartilage tissue containing hydroxypropyl chitin combined stem cells prepared in Example 1 of this application;

[0034] Figure 7 shows the distribution of inflammatory cells and chondroitin in rats of the hydroxypropyl chitin combined stem cells prepared in Example 1 of this application.

[0035] Figure 8 is a rat in vivo imaging image of the hydroxypropyl chitin combined stem cells and the inflammation inhibitor prepared in Example 2 of this application.

[0036] Figure 9 is a magnetic resonance imaging of the rat bone joints of hydroxypropyl chitin combined stem cells and inflammation inhibitors prepared in Example 2 of this application.

[0037] Figure 10 shows the ICRS scores of rat articular cartilage containing hydroxypropyl chitin combined stem cells and inflammation inhibitors prepared in Example 2 of this application.

[0038] Figure 11 shows the concentration detection results of PEG2 inflammatory factor in the rat bone joints of the hydroxypropyl chitin combined stem cells and the inflammatory inhibitor prepared in Example 2 of this application.

[0039] Figure 12 shows the concentration detection results of NO inflammatory factors in the rat bone joints of the hydroxypropyl chitin combined stem cells and the inflammatory inhibitor prepared in Example 2 of this application.

[0040] Figure 13 shows the concentration detection results of TNFα inflammatory factor in the rat bone joints of the hydroxypropyl chitin combined stem cells and the inflammatory inhibitor prepared in Example 2 of this application.

[0041] Figure 14 shows the concentration detection results of IL-1β inflammatory factor in the rat bone joints of the hydroxypropyl chitin combined stem cells and the inflammatory inhibitor prepared in Example 2 of this application. Detailed Implementation

[0042] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0043] This invention provides a thermosensitive adhesive composition for treating osteoarthritis, comprising 0.5%-5% chitosan thermosensitive adhesive and 10 6 -10 7 / mL stem cells.

[0044] Specifically, this treatment product utilizes the thermosensitive properties of chitosan gel. At body temperature, the stem cells (preferred embodiment also includes an inflammation inhibitor) used for treatment are mixed with the thermosensitive gel and solidify in situ into a gel within the joint cavity. This ensures that the therapeutic drugs and cells can remain at the lesion or injury site for a long time, thereby achieving a long-term treatment and repair effect.

[0045] Preferably, the chitosan thermosensitive adhesive comprises: hydroxypropyl chitosan, hydroxypropyl chitin, hydroxybutyl chitosan, and hydroxybutyl chitin. The phase transition temperatures of each component are 15-30°C.

[0046] More preferably, the chitosan thermosensitive adhesive is hydroxypropyl chitin.

[0047] Specifically, the above-mentioned method for preparing chitosan thermosensitive adhesive includes (taking hydroxypropyl chitin as an example): the thermosensitive hydroxypropyl chitin has a molecular weight of 15,000-300,000 Da and a degree of deacetylation (DD) of 35%-65%, and the preparation method of the hydroxypropyl chitin includes:

[0048] S1. Using chitosan with a degree of deacetylation ≥ 85% as raw material, disperse it in an alkaline solution at -10℃ to -25℃ and freeze for 12-36 hours. React it with propylene oxide at 0-25℃ to obtain hydroxypropyl chitosan;

[0049] S2. The product from step S1 reacts with acetic anhydride under low temperature and alkaline conditions to generate a thermosensitive hydroxypropyl chitosan solution. After neutralization with HCl, the solution is dialyzed with triple-distilled water to remove salt particles. Then, it is eluted with ethanol at 0-5℃ and freeze-dried to obtain thermosensitive hydroxypropyl chitosan.

[0050] Furthermore, most of the free amino groups in the thermosensitive hydroxypropyl chitosan structure are blocked by acetyl groups, which reduces the number of positive charges carried in the molecule and is beneficial to cell survival and proliferation.

[0051] Furthermore, the alkaline conditions are achieved using an alkaline solution, which includes, but is not limited to, sodium hydroxide solution, potassium hydroxide solution, or urea with a mass concentration of 10%-20%.

[0052] Furthermore, in step S1, the mass ratio of chitosan to propylene oxide is 1:5-1:15, and the reaction is carried out at 0-25°C with stirring for 12-48 hours, so that the degree of hydroxypropyl substitution (DS) (number of hydroxypropyl residues on chitosan glucosamine residues: number of glucosamine residues) is between 0.5 and 1.2.

[0053] Furthermore, in step S1, isopropanol needs to be added before adding propylene oxide, and the mixture is stirred at 0-25°C for 12-48 hours. The mass ratio of chitosan to isopropanol is 1:8-1:12.

[0054] Furthermore, in step S2, the mass ratio of hydroxypropyl chitosan to acetic anhydride is between 4:1 and 1:1.

[0055] The present invention will be further explained below through specific embodiments.

[0056] Example 1

[0057] Preparation of Hydroxypropyl Chitosan Thermosensitive Adhesive Composition 1

[0058] S1: Under sterile conditions at 2-10℃, add hydroxypropyl chitosan powder to double-distilled water, stir thoroughly to dissolve the powder, filter, and obtain hydroxypropyl chitosan thermosensitive adhesive solution, as shown in Figure 1 (containing color developer).

[0059] S2: Under sterile conditions at 2-10℃, fully cultured mesenchymal stem cells (MSCs) are centrifuged and enriched, then slowly resuspended with physiological saline, centrifuged and enriched again to obtain mesenchymal stem cells (MSCs), and plasmids carrying luciferase gene marker DNA are transferred into these injectable mesenchymal stem cells.

[0060] S3: Under sterile conditions at 2-10℃, slowly inject the hydroxypropyl chitin thermosensitive gel prepared in S1 into the mesenchymal stem cells (MSCs) prepared in S2, and gently pipette to thoroughly mix the MSCs with the thermosensitive gel solution, obtaining a MSC concentration of 10. 6 -10 7 Therapeutic products containing hydroxypropyl chitosan thermosensitive gel composition per mL.

[0061] Example 2

[0062] Preparation of Hydroxypropyl Chitosan Thermosensitive Adhesive Composition 2

[0063] S1: Under sterile conditions at 2-10℃, add hydroxypropyl chitosan powder to double-distilled water, stir thoroughly to dissolve the powder, filter, and obtain hydroxypropyl chitosan thermosensitive adhesive solution.

[0064] S2: Under sterile conditions at 2-10℃, fully cultured mesenchymal stem cells (MSCs) are centrifuged and enriched, then slowly resuspended with physiological saline, centrifuged and enriched again to obtain mesenchymal stem cells (MSCs), and plasmids carrying luciferase gene marker DNA are transferred into these injectable mesenchymal stem cells.

[0065] S3: Under sterile conditions at 2-10℃, the hydroxypropyl chitin thermosensitive gel prepared in S1 was mixed with astragalus polysaccharide to form a solution, which was then slowly injected into the mesenchymal stem cells (MSCs) prepared in S2. The mixture was gently pipetted to ensure thorough mixing of the MSCs with the solution, resulting in a MSC concentration of 10. 6 -10 7 Therapeutic products containing hydroxypropyl chitosan thermosensitive gel composition per mL.

[0066] Example 3

[0067] Structure and temperature-sensitive properties testing of thermosensitive adhesive

[0068] The hydroxypropyl chitin prepared in step S1 of Example 1 was added to double-distilled water to prepare solutions of 1%, 1.5%, 2%, and 4%. The structure of the hydroxypropyl chitin powder and solutions of different concentrations were observed under an electron microscope, and the results are shown in Figure 1. It can be seen that the 1-4% hydroxypropyl chitin solutions all have a porous structure, which can support the growth of stem cells; among them, the pores are more uniform when the concentration of hydroxypropyl chitin is 1.5%.

[0069] The 1%, 1.5%, 2%, and 4% solutions prepared above were photographed at 4°C; then photographed at 37°C, and their structures are shown in Figure 2. It can be seen that at 4°C (below the phase transition temperature), the thermosensitive adhesive of this application is in a liquid state; while when the temperature is raised to 37°C (phase transition temperature), the thermosensitive adhesive solidifies into a gel state.

[0070] Example 4

[0071] Biological test cases

[0072] Preparation before experiment

[0073] Rat preparation: SPF-grade nine-week-old healthy rats were purchased from Beijing Huafukang Biotechnology Co., Ltd., and rats with osteoarthritis were artificially induced by surgical or drug methods.

[0074] Experimental Groups: The experiment was divided into a surgical modeling group and a drug-based modeling group. In the surgical modeling group, healthy rats were labeled Ctrl-O; Modeling group: Op; Modeling + thermosensitive gel group: Op + CH; Modeling + mesenchymal stem cell group: Op + MSC; Modeling + thermosensitive gel + mesenchymal stem cell group: Op + CH + MSC; Modeling + thermosensitive gel + Astragalus polysaccharide + mesenchymal stem cells: Op + CH + APS + MSC. In the drug-based modeling group, healthy rats were labeled Ctrl-P; Modeling group: Pp; Modeling + thermosensitive gel group: Pp + CH; Modeling + mesenchymal stem cell group: Pp + MSC; Modeling + thermosensitive gel + mesenchymal stem cell group: Pp + CH + MSC; Modeling + thermosensitive gel + Astragalus polysaccharide + mesenchymal stem cells: Pp + CH + APS + MSC.

[0075] Detection methods

[0076] In vivo imaging: In vivo imaging experiments were performed on rats in each group at the corresponding time after drug administration. The experimental rats were administered an aqueous solution of sodium fluorescein, and general anesthesia was performed 10 minutes later. The general anesthetized rats were placed in a small animal in vivo imaging system for photography. After the photography was completed, the rats were removed and returned to their cages to recover.

[0077] Magnetic resonance imaging: Rats were cultured for 10 days. On the 10th day, each group of rats was given general anesthesia and placed in an MRI scanner for imaging. After the imaging was completed, the rats were removed and returned to their cages to wait for recovery.

[0078] Detection of inflammatory cells and chondrocyte polysaccharides: After culturing rats for ten days, they were sacrificed, and cartilage tissue from the affected area of ​​the osteoarthritis model was taken (cartilage tissue from the corresponding joint of the healthy group was taken). Paraffin sections were prepared, and then stained with HE (hematoxylin-eosin staining method) and observed under a microscope.

[0079] Safranin staining of cartilage tissue: After culturing rats for ten days, they were sacrificed, and cartilage tissue from the affected area of ​​the osteoarthritis model was taken (cartilage tissue from the corresponding joint of the healthy group was taken). The tissue sections were stained with safranin, observed under a microscope, and photographed.

[0080] Inflammatory factor detection: After culturing rats for ten days, they were sacrificed, and cartilage tissue from the affected area of ​​the osteoarthritis model was taken (cartilage tissue from the corresponding joint of the healthy group was taken). The samples were processed according to the instructions of different inflammatory factor detection kits, and the content of inflammatory factors was detected.

[0081] Example 1: Experimental study on the therapeutic effect of the composition on osteoarthritis in rats.

[0082] Injection administration: Hydroxypropyl chitosan thermosensitive gel obtained in step S1 of Example 1 was injected into the osteoarthritis lesions of rats in the model + thermosensitive gel group (Op+CH and Pp+CH); mesenchymal stem cells obtained in step S2 of Example 1 were injected into the osteoarthritis lesions of rats in the model + mesenchymal stem cell group (Op+MSC and Pp+MSC); and a combination of hydroxypropyl chitosan thermosensitive gel and stem cells obtained in step S3 of Example 1 was injected into the osteoarthritis lesions of rats in the model + thermosensitive gel + mesenchymal stem cell group (Op+CH+MSC and Pp+CH+MSC). Healthy rats (Ctrl-O and Ctrl-P) and model rats (Op and Pp) were not treated.

[0083] The in vivo imaging detection method was used, and the results are shown in Figure 3. Figure 3 shows that since the healthy group, model group, and thermosensitive gel group did not receive mesenchymal stem cells, no colorimetric results were observed in the in vivo imaging. The stem cell group and the thermosensitive gel + stem cell group received live mesenchymal stem cells containing luciferase gene-labeled DNA plasmids, so the arthritis lesions in the in vivo imaging showed red, green, and blue colorimetric results as shown in Figure 3. Red indicates areas with dense and highly active mesenchymal stem cells containing luciferase gene-labeled DNA plasmids, while green and blue indicate areas with decreased cell concentration and activity due to diffusion and apoptosis of the aforementioned mesenchymal stem cells. The in vivo imaging results indicate that the mesenchymal stem cells in the thermosensitive gel + stem cell group remained in the rat joint cavity for approximately 2 days longer than those in the stem cell group alone.

[0084] The results of the nuclear magnetic resonance (NMR) examination are shown in Figure 4. Figure 4 shows that 10 days after injection, significant cartilage thickening was observed in the rats in the model + thermosensitive gel + mesenchymal stem cell group. This indicates that the combined treatment product of chitosan thermosensitive gel + stem cells for osteoarthritis presented in this application is more effective than mesenchymal stem cells alone.

[0085] The rats were euthanized, dissected, and bone and joint tissues were collected and photographed. The results are shown in Figure 5. It can be seen that the osteoarthritis tissues of rats injected with thermosensitive colloid + mesenchymal stem cells showed relatively significant repair.

[0086] The cartilage tissue was stained with safranin, and the results are shown in Figure 6. Compared to using mesenchymal stem cell solution alone, using chitosan thermosensitive gel + mesenchymal stem cells can effectively increase the thickness of cartilage tissue in diseased joints.

[0087] The inflammatory cells and cartilage polysaccharides were detected using the same methods, and the results are shown in Figure 7. The top two columns show HE staining, and the bottom two columns show Safranin-Fix Green staining. Red represents polysaccharides, and green represents bone tissue. As shown in Figure 1, the combination of thermosensitive colloid and stem cells reduced the number of inflammatory cells and increased the content and distribution of proteoglycans in the osteoarthritis lesions of rats.

[0088] Example 2: Therapeutic effect of the composition on osteoarthritis in rats

[0089] Injection administration: Hydroxypropyl chitosan thermosensitive gel obtained in step S1 of Example 1 was injected into the osteoarthritis lesions of rats in the modeling + thermosensitive gel group (Op+CH and Pp+CH); mesenchymal stem cells obtained in step S2 of Example 1 were injected into the osteoarthritis lesions of rats in the modeling + mesenchymal stem cell group (Op+MSC and Pp+MSC); a combination of hydroxypropyl chitosan thermosensitive gel and stem cells obtained in step S3 of Example 1 was injected into the osteoarthritis lesions of rats in the modeling + thermosensitive gel + mesenchymal stem cell group (Op+CH+MSC and Pp+CH+MSC); a compound therapeutic product for treating osteoarthritis, consisting of hydroxypropyl chitosan thermosensitive gel, astragalus polysaccharide, and stem cells obtained in step S3 of Example 2, was injected into the osteoarthritis lesions of rats in the modeling + thermosensitive gel + astragalus polysaccharide + mesenchymal stem cell group (Op+CH+APS+MSC and Pp+CH+APS+MSC). Healthy rats (Ctrl-O and Ctrl-P) and modeling rats (Op and Pp) were not treated.

[0090] The in vivo imaging detection method was used, and the results are shown in Figure 8. Figure 8 shows that since the healthy group, diseased group, and thermosensitive gel group did not receive mesenchymal stem cells, the in vivo imaging did not show color development results. The stem cell group, thermosensitive gel + stem cell group, and thermosensitive gel + Astragalus polysaccharide + stem cell group received live mesenchymal stem cells containing luciferase gene marker DNA plasmids. Therefore, the arthritis lesions in the in vivo imaging showed red, green, and blue color development results as shown in Figure 8. Red indicates areas with dense and highly active mesenchymal stem cells containing luciferase gene marker DNA plasmids, while green and blue indicate areas with decreased cell concentration and activity due to diffusion and apoptosis of the aforementioned mesenchymal stem cells. From the in vivo imaging results, the mesenchymal stem cells in the thermosensitive gel + Astragalus polysaccharide + stem cell group remained in the rat joint cavity for approximately twice as long as those in the stem cell group alone, and for approximately 2 days longer than those in the thermosensitive gel + stem cell group.

[0091] The results of the nuclear magnetic resonance (NMR) test are shown in Figure 9. Figure 9 shows that 10 days after injection, significant cartilage thickening was observed in the rats in the model + thermosensitive gel + astragalus polysaccharide + mesenchymal stem cell group. This indicates that the combined treatment product of chitosan thermosensitive gel + inflammation inhibitor + stem cells for osteoarthritis provided in this application is more effective than the use of mesenchymal stem cells alone.

[0092] Rats were euthanized, dissected, and bone and joint tissues were collected. The articular cartilage damage was scored according to the ICRS standard, and the results are shown in Figure 10. It can be seen that the ICRS score decreased significantly, indicating that the cartilage was repaired.

[0093] The inflammatory factors were detected using the prescribed method, and the results are shown in Figures 11-14. It can be seen that after using the compound treatment product of Example 2, the levels of various inflammatory factors decreased, indicating that the composition provided by this invention has a certain inhibitory effect on inflammation.

[0094] The animal experiments in this study all showed that, within the same time frame, the combined treatment product of chitosan thermosensitive gel, inflammation inhibitor, and stem cell therapy for osteoarthritis had a better effect on inhibiting inflammation at the joint site and increasing cartilage tissue thickness than mesenchymal stem cells alone; at the same time, the combined treatment product of chitosan thermosensitive gel, inflammation inhibitor, and stem cell therapy for osteoarthritis had a longer duration of action than mesenchymal stem cells alone.

[0095] It should be understood that the above detailed description of the technical solutions of the present invention with reference to preferred embodiments is illustrative and not restrictive. Those skilled in the art can modify the technical solutions described in the embodiments or make equivalent substitutions for some of the technical features based on reading this specification; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A thermosensitive adhesive composition for treating osteoarthritis, characterized in that, Includes 0.5%-5% chitosan thermosensitive adhesive and 10 6 -10 7 / mL stem cells, wherein the chitosan thermosensitive gel includes hydroxypropyl chitin.

2. The temperature-sensitive adhesive composition according to claim 1, characterized in that, The chitosan thermosensitive adhesive further includes one or more of hydroxypropyl chitosan, hydroxybutyl chitosan, and hydroxybutyl chitin, preferably hydroxypropyl chitin.

3. The temperature-sensitive adhesive composition according to claim 1, characterized in that, The thermosensitive adhesive composition also includes an inflammation inhibitor, which includes one or more of the following: icariin, prostaglandins, interleukins, tumor necrosis factor, astragalus polysaccharides, and nuclear factor-κB.

4. The temperature-sensitive adhesive composition according to claim 1, characterized in that, The stem cells mentioned are human mesenchymal stem cells.

5. A method for preparing the thermosensitive adhesive composition according to any one of claims 1-4, comprising: S1: Add chitosan thermosensitive adhesive powder to physiological saline, mix, filter, and obtain chitosan thermosensitive adhesive solution; S2: The fully cultured mesenchymal stem cells are centrifuged and enriched, then suspended in physiological saline, centrifuged and enriched again to obtain the mesenchymal stem cells to be injected. S3: In a sterile environment, the chitosan thermosensitive adhesive solution prepared in S1 is mixed with an inflammation inhibitor and injected into the mesenchymal stem cells to be injected prepared in S2. The mixture is gently blown to ensure that the mesenchymal stem cells to be injected are fully mixed with the mixed solution.

6. The preparation method according to claim 5, characterized in that, Steps S1, S2, and S3 are all performed at 2-10℃.

7. The preparation method according to claim 5, characterized in that, Step S1 also includes filtering or dialysis using a 200-mesh sieve or a dialysis bag.

8. The preparation method according to claim 5, characterized in that, The inflammation inhibitor in step S1 includes at least one of the following: icariin, prostaglandin, interleukin, tumor necrosis factor, astragalus polysaccharide, and nuclear factor-κB, and the concentration of the inflammation inhibitor is preferably 10 ng / mL to 25 μg / mL.

9. Use of the thermosensitive adhesive composition according to any one of claims 1-4 in the preparation of an arthritis medicament.

10. The use according to claim 9, characterized in that, The temperature-sensitive adhesive composition is administered via injection.

Images