Bioactive material for promoting cartilage regeneration as well as preparation method and application thereof

Abstract

The invention discloses a bioactive material for promoting cartilage regeneration as well as a preparation method and application of the bioactive material. The bioactive material comprises 1-(1-propionylpiperidine-4-yl)-3-(4-(trifluoromethoxy) phenyl) urea (TPPU) and hydrogel prepared by taking chitosan and beta-glycerophosphate as cross-linking agents, the preparation method comprises the following steps: S1, dissolving chitosan in an acetic acid solution, uniformly mixing, and sterilizing to obtain an intermediate solution A; s2, dissolving beta-glycerophosphate in sterile water, and uniformly mixing to obtain an intermediate solution B; s3, adding a suspension liquid formed by TPPU in dimethyl sulfoxide into the intermediate liquid B, and uniformly mixing to obtain an intermediate liquid C; and S4, dropwise adding the intermediate solution A into the intermediate solution C, and uniformly mixing to obtain the bioactive material for promoting cartilage regeneration, which can be applied to preparation of products for promoting cartilage regeneration. The bioactive material prepared by the invention shows excellent cartilage formation performance, slows down the injury process of articular cartilage, is beneficial to recovery of normal structures and functions of joints, and realizes repair of injured joint tissues.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and new material technology, and in particular to a bioactive material for promoting cartilage regeneration and a preparation method and application thereof. Background Art

[0002] Temporomandibular joint osteoarthritis (TMJOA) is a joint degenerative disease that seriously affects the quality of life of patients. Its main characteristics include joint pain, joint snapping or murmurs, facial and neck muscle tenderness, and mandibular dysfunction. With the aging of the global population, the incidence of TMJOA has shown an increasing trend year by year. Studies have shown that the prevalence of this disease in patients with temporomandibular disorders (TMD) ranges from 18.01% to 84.47%, and approximately 8% to 16% of the population worldwide is affected.

[0003] The treatment of TMJOA has always been a difficult problem in the field of stomatology. Although conventional clinical treatment methods, such as psychotherapy, physical therapy, the use of occlusal stabilization plates, drug therapy, and intra-articular injection therapy, can relieve patients' symptoms to a certain extent, the long-term treatment effect of the disease is not ideal. Although surgical treatment is suitable for patients with severe joint damage and refractory severe pain, it is often difficult to obtain long-term stable effects after treatment.

[0004] Articular cartilage is an important component of the temporomandibular joint, which can effectively disperse and buffer the impact force acting on the joint surface. However, articular cartilage lacks intrinsic self-repair ability. Once damaged, cartilage is not easy to heal and may cause irreversible damage to the joint. At present, for patients with early and middle stage TMJOA, non-steroidal anti-inflammatory drugs or intra-articular injection of hyaluronic acid are mainly used clinically to temporarily relieve pain. However, these methods cannot fundamentally intervene in the degeneration process of cartilage, and the long-term treatment effect is limited, and may be accompanied by certain side effects.

[0005] Although there are case reports in the prior art about the application of 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (TPPU) in bone repair, there are significant differences in the structure and repair mechanism between cartilage and bone. Therefore, there are currently no successful cases and mechanism studies on the application of TPPU in cartilage repair. Summary of the invention

[0006] In view of the technical problems in the prior art of cartilage repair in temporomandibular joint osteoarthritis, the present invention aims to provide a bioactive material for promoting cartilage regeneration.

[0007] To achieve the above purpose, a bioactive material for promoting cartilage regeneration includes 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea and a high molecular active matrix.

[0008] Furthermore, the polymer active matrix is ​​a hydrogel prepared using chitosan and β-glycerophosphate as cross-linking agents.

[0009] Furthermore, the concentration of the 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea in the polymer active matrix is ​​3 to 4 mmol / L.

[0010] Furthermore, the mass ratio of chitosan to glycerophosphate is 1:2-3.

[0011] The present invention also provides a method for preparing a bioactive material for promoting cartilage regeneration, comprising the following steps:

[0012] S1: dissolving chitosan in acetic acid solution, mixing and sterilizing to obtain intermediate solution A;

[0013] S2: dissolve β-glycerophosphate in sterile water and mix well to obtain intermediate solution B;

[0014] S3: adding a suspension of 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea in dimethyl sulfoxide to the intermediate solution B and mixing well to obtain the intermediate solution C;

[0015] S4: Drop the intermediate solution A into the intermediate solution C, and mix well to obtain a bioactive material that promotes cartilage regeneration.

[0016] The present invention also relates to the application of the bioactive material for promoting cartilage regeneration in the preparation of products for promoting cartilage regeneration.

[0017] Furthermore, the product for promoting cartilage regeneration is a drug for treating temporomandibular arthritis.

[0018] Furthermore, the drug for treating temporomandibular arthritis is chitosan hydrogel microspheres loaded with 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.

[0019] The present invention has the following beneficial effects:

[0020] 1. The bioactive material provided by the present invention exhibits excellent cartilage-forming properties, can effectively reduce the host's immune response to the implant, and slow down the continuous damage process of articular cartilage and subchondral bone. At the same time, the material has the ability to induce cartilage reconstruction, which helps to restore the normal structure and function of the joint and ultimately achieve the repair of damaged joint tissue.

[0021] Second, the present invention verifies that under the conditions of inflammation and stress microenvironment, bioactive materials can significantly reduce the inflammatory response of M1 macrophages and effectively regulate the inflammatory immune response of joint tissues. This provides a reference for the diagnosis and prognosis analysis of TMJOA patients and the clinical transformation and application of stem cell-based tissue engineering technology in the field of temporomandibular joint (TMJ) tissue regeneration.

[0022] 3. Based on the TMJOA experimental animal model, the present invention deeply explores the feasibility of treating TMJOA by regulating the inflammatory microenvironment of the joints by regulating the levels of endogenous EETs (epoxyeicosatrienoic acids). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 These are the in vivo test results of the bioactive materials of the present invention for promoting cartilage repair in the temporomandibular joint: A. Photo of TMJOA mice; B. Technical roadmap for treating TMJOA mice with bioactive materials; C. Representative images of H&E staining and SOX9 (chondrogenesis indicator) immunohistochemical staining of TMJ tissue sections.

[0025] Figure 2 These are the results of the anti-inflammatory test of the bioactive material of the present invention: A. Representative images of HE staining of inflammatory synovial tissue of TMJOA mice at 3 weeks, 7 weeks and 11 weeks and quantitative analysis of synovial inflammation scores; B. RT-qPCR detection of the expression statistics of TNF-α and IL-1β mRNA in the joint tissue of 7w group animals; C. Immunohistochemistry staining and quantitative analysis results of IL-1β in animal tissue sections. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] A bioactive material for promoting cartilage regeneration comprises 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea and a high molecular active matrix.

[0028] Furthermore, the polymer active matrix is ​​a hydrogel prepared using chitosan and β-glycerophosphate as cross-linking agents.

[0029] Furthermore, the concentration of the 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea in the polymer active matrix is ​​3 to 4 mmol / L.

[0030] Furthermore, the mass ratio of chitosan to glycerophosphate is 1:2-3.

[0031] The present invention also provides a method for preparing a bioactive material for promoting cartilage regeneration, comprising the following steps:

[0032] S1: dissolving chitosan in acetic acid solution, mixing and sterilizing to obtain intermediate solution A;

[0033] S2: dissolve β-glycerophosphate in sterile water and mix well to obtain intermediate solution B;

[0034] S3: adding a suspension of 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea in dimethyl sulfoxide to the intermediate solution B and mixing well to obtain the intermediate solution C;

[0035] S4: Drop the intermediate solution A into the intermediate solution C, and mix well to obtain a bioactive material that promotes cartilage regeneration.

[0036] The present invention also relates to the application of the bioactive material for promoting cartilage regeneration in the preparation of products for promoting cartilage regeneration.

[0037] Furthermore, the product for promoting cartilage regeneration is a drug for treating temporomandibular arthritis.

[0038] Furthermore, the drug for treating temporomandibular arthritis is chitosan hydrogel microspheres loaded with 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.

[0039] Example 1

[0040] Preparation of TPPU-CS hydrogel bioactive materials

[0041] S1: Dissolve 0.2 g of chitosan (CS) in 8 mL of 0.1 mol / L acetic acid solution, stir thoroughly, and sterilize under high temperature and high pressure to obtain intermediate solution A;

[0042] S2: Dissolve 0.56 g of β-glycerophosphate (β-GP) in 2 mL of sterile distilled water and stir thoroughly to dissolve, to obtain intermediate solution B;

[0043] S3: Add 3 μL of a 10 μmol / L suspension of 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (TPPU) in dimethyl sulfoxide (DMSO) to the intermediate solution B, mix well, and refrigerate at 4°C to obtain the intermediate solution C;

[0044] S4: Drop the intermediate liquid A into the intermediate liquid C, stir evenly to obtain a bioactive material for promoting cartilage regeneration, which is named TPPU-CS hydrogel bioactive material.

[0045] Example 2

[0046] Preparation of TPPU-CS hydrogel bioactive materials

[0047] S1: Dissolve 0.2 g of CS in 8 mL of 0.1 mol / L acetic acid solution, stir thoroughly, and sterilize under high temperature and high pressure to obtain intermediate solution A;

[0048] S2: Dissolve 0.4 g of β-GP in 2 mL of sterile distilled water and stir thoroughly to dissolve, obtaining intermediate solution B;

[0049] S3: Add 3.5 μL of a 10 μmol / L suspension of TPPU in DMSO to the intermediate solution B, mix well, and refrigerate at 4°C to obtain the intermediate solution C;

[0050] S4: Drop the intermediate liquid A into the intermediate liquid C, stir evenly to obtain a bioactive material for promoting cartilage regeneration, which is named TPPU-CS hydrogel bioactive material.

[0051] Example 3

[0052] Preparation of TPPU-CS hydrogel bioactive materials

[0053] S1: Dissolve 0.2 g of CS in 8 mL of 0.1 mol / L acetic acid solution, stir thoroughly, and sterilize under high temperature and high pressure to obtain intermediate solution A;

[0054] S2: Dissolve 0.6 g of β-GP in 2 mL of sterile distilled water and stir thoroughly to dissolve, obtaining intermediate solution B;

[0055] S3: Add 4 μL of a 10 μmol / L suspension of TPPU in DMSO to the intermediate solution B, mix well, and refrigerate at 4°C to obtain the intermediate solution C;

[0056] S4: Drop the intermediate liquid A into the intermediate liquid C, stir evenly to obtain a bioactive material for promoting cartilage regeneration, which is named TPPU-CS hydrogel bioactive material.

[0057] The amounts of the components used in the preparation of the TPPU-CS hydrogel bioactive material in Examples 1 to 3 are shown in Table 1:

[0058] Table 1 List of dosage ratios of each component in Examples 1 to 3

[0059]

[0060] Example 4

[0061] This example is a test example of the in vivo test and anti-inflammatory test of the TPPU-CS hydrogel bioactive material prepared in Example 1 for promoting the repair of temporomandibular joint cartilage. The specific test scheme is as follows:

[0062] (1) Animal model construction and TPPU-CS hydrogel bioactive material disposal plan

[0063] like Figure 1 As shown in A, 7-week-old C57BL / 6 mice were selected as experimental subjects, and a unilateral reverse bite (UAC) model was constructed through specific surgical operations to simulate the pathological state of human temporomandibular joint osteoarthritis (TMJOA), thereby establishing a TMJOA mouse model. Figure 1 As shown in B, TMJOA mice were injected into the articular cavity from the 1st week to the 3rd week after modeling. The injection was the TPPU-CS hydrogel bioactive material prepared according to Example 1, in order to evaluate its effect in promoting the repair and regeneration of articular cartilage and anti-inflammatory effects.

[0064] (2) In vivo experimental plan to promote temporomandibular joint cartilage repair

[0065] In order to evaluate the effect of bioactive materials on the repair of temporomandibular joint cartilage, the specific steps of the cartilage repair test include: obtaining TMJ tissue sections at the 3rd, 7th and 11th weeks of modeling, and performing H&E staining to observe the morphological changes of articular cartilage; at the same time, performing immunohistochemical staining of the cartilage formation indicator SOX9 to quantitatively analyze the degree of chondrocyte proliferation and differentiation, thereby evaluating the effect of cartilage repair.

[0066] (3) Anti-inflammatory effect evaluation test plan

[0067] In order to comprehensively evaluate the anti-inflammatory effects of bioactive materials, the anti-inflammatory test was designed as follows:

[0068] First, at the 3rd, 7th, and 11th weeks of modeling, inflammatory synovial tissue was obtained for HE staining to observe the morphological changes of synovial tissue. At the same time, the severity of synovial inflammation was scored and quantitatively analyzed to evaluate the degree of relief of inflammation.

[0069] Secondly, at week 7, real-time fluorescence quantitative PCR (RT-qPCR) technology was used to detect the mRNA expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the joint tissues of the experimental animals. These inflammatory cytokines are key mediators in the pathogenesis of arthritis, and the reduction of their expression levels can reflect the anti-inflammatory effect of the material.

[0070] Finally, immunohistochemistry was used to stain IL-1β in animal tissue sections at weeks 3, 7, and 11, and quantitative analysis was performed. The anti-inflammatory effect and duration of the material were further evaluated by observing the distribution and expression intensity of IL-1β in synovial tissue.

[0071] Comparative Example 1

[0072] The only difference between this example and Example 4 is that the experimental animals used are non-immunized C57 mice of the same age, and all the steps of applying the TPPU-CS hydrogel bioactive material are replaced by applying an equal amount of physiological saline, i.e., the blank control group.

[0073] Comparative Example 2

[0074] The only difference between this example and Example 4 is that all the steps of applying the TPPU-CS hydrogel bioactive material were replaced by applying an equal amount of normal saline, also referred to as the UAC group.

[0075] Experimental results and analysis

[0076] (1) In vivo test results of bioactive materials in promoting cartilage repair in the temporomandibular joint

[0077] according to Figure 1 The experimental results shown in C deeply analyzed the changes in cartilage and bone tissue of the condyle of TMJOA mice induced by UAC, and evaluated the potential protective effect of TPPU-CS hydrogel bioactive material on this pathological process.

[0078] From the perspective of histological observation, at the 3rd, 7th, and 11th weeks after modeling, it was found through the observation of HE staining results that the condyle of TMJOA mice induced by UAC in Comparative Example 2 (UAC group) showed obvious time-dependent progression of cartilage degeneration and bone destruction. Specifically, the cartilage thickness gradually decreased, chondrocytes atrophied and the number decreased significantly, and the fibrous layer gradually strengthened. Especially at the 7th and 11th weeks after modeling, the reduction in cartilage thickness was particularly obvious compared with Comparative Example 1 (blank control group). In addition, UAC-induced mice also showed severe destruction of cartilage and bone tissue, expansion of the subchondral bone marrow cavity, and increased degree of destruction of trabeculae and collagen fibers. At the same time, Example 4 (TPPU-CS hydrogel group) was at the 3rd, 7th, and 11th weeks after modeling (i.e., the 0th, 4th, and 8th weeks after TPPU-CS hydrogel treatment), and the results showed that the treatment of TPPU-CS hydrogel active biomaterials effectively slowed down the deconstruction process of cartilage and subchondral bone in UAC-induced TMJOA mice. Compared with Comparative Example 2, the loss of articular cartilage and subchondral bone in mice in Example 4 was significantly alleviated.

[0079] From the immunohistochemical analysis, the test further evaluated the expression of cartilage synthesis indicators SOX9 and COLⅡ through immunohistochemical staining: Compared with comparative example 1 (blank control group), the expression of cartilage synthesis indicators SOX9 and COLⅡ in the UAC group mice was significantly reduced in comparative example 2 (UAC group), indicating that the cartilage synthesis ability was severely inhibited. At the same time, after the treatment of TPPU-CS hydrogel, the expression of SOX9 and COLⅡ in Example 4 (TPPU-CS hydrogel group) was significantly increased, indicating that the bioactive material can promote the synthesis and repair of cartilage.

[0080] (2) Anti-inflammatory test results of bioactive materials

[0081] like Figure 2 As shown in A, compared with control group 1 (blank control group), control group 2 (UAC group) can clearly observe the inflamed synovial tissue through HE staining tissue sections at the 7th week of modeling. Specifically, there are a large number of infiltrating inflammatory cells in the synovium, obvious accumulation of lipid droplets, formation of lymphoid follicles, and significant thickening of the synovial lining layer. These pathological changes all indicate the presence and aggravation of inflammatory response. However, in Example 4 (TPPU-CS hydrogel group), due to the use of TPPU-CS hydrogel, a bioactive material, for treatment, the inflammatory response of the synovial tissue was significantly alleviated, which was manifested by a reduction in the inflamed area, a decrease in the degree of inflammatory cell infiltration, and the relative integrity of the synovial structure was maintained.

[0082] like Figure 2As shown in Figure B, TNF-α and IL-1β are cytokines that play a core role in immune response and inflammation, and changes in their expression levels are of great significance for evaluating anti-inflammatory effects. The results of RT-qPCR showed that the expression levels of these pro-inflammatory markers IL-1β and TNF-α in the experimental mice of Comparative Example 2 (UAC group) were significantly upregulated compared with the animals that were not immunostimulated in Comparative Example 1 (blank control group), which further confirmed that UAC treatment triggered a strong inflammatory response. However, in Example 4 (TPPU-CS hydrogel group), due to the application of TPPU-CS hydrogel bioactive materials, the expression levels of these inflammation-related cytokines were statistically significantly reduced, indicating that TPPU-CS hydrogel has a significant anti-inflammatory effect and can effectively inhibit the production of inflammatory cytokines in arthritic animals.

[0083] Figure 2 The immunohistochemical staining results in C further support the above conclusions. The mice in comparative example 2 (UAC group) showed significant IL-1β positive staining in the synovial area and subchondral bone area, indicating that there was a large amount of IL-1β expression in this area, which is consistent with the aggravated inflammatory response. In contrast, the IL-1β positive staining in these areas of the mice in Example 4 (TPPU-CS hydrogel group) was significantly weakened, further confirming the effectiveness of the TPPU-CS hydrogel bioactive material in reducing inflammatory responses and inhibiting the expression of inflammatory cytokines.

[0084] In summary, TPPU-CS hydrogel bioactive material has a significant protective effect on UAC-induced osteochondral damage and can slow down the process of TMJOA cartilage degradation. At the same time, TPPU-CS hydrogel bioactive material showed significant anti-inflammatory effect in arthritis animal models, which can effectively reduce the inflammatory response of synovial tissue, reduce the expression level of inflammatory cytokines, and improve the pathological state of joint tissue.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bioactive material for promoting cartilage regeneration, characterized in that: The biologically active material comprises 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea and a high molecular active matrix.

2. The bioactive material for promoting cartilage regeneration according to claim 1, characterized in that: The polymer active matrix is ​​a hydrogel prepared by using chitosan and beta-glycerophosphate as crosslinking agents.

3. The bioactive material for promoting cartilage regeneration according to claim 2, characterized in that: The concentration of the 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea in the polymer active matrix is ​​3-4 mmol / L.

4. The bioactive material for promoting cartilage regeneration according to claim 3, characterized in that: The mass ratio of chitosan to glycerophosphate is 1:2-3.

5. The method for preparing a bioactive material for promoting cartilage regeneration according to claim 4, characterized in that: The following steps are involved: S1: dissolving chitosan in acetic acid solution, mixing and sterilizing to obtain intermediate solution A; S2: dissolve β-glycerophosphate in sterile water and mix well to obtain intermediate solution B; S3: adding a suspension of 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea in dimethyl sulfoxide to the intermediate solution B and mixing well to obtain the intermediate solution C; S4: Drop the intermediate solution A into the intermediate solution C, and mix well to obtain a bioactive material that promotes cartilage regeneration.

6. Use of the bioactive material for promoting cartilage regeneration according to any one of claims 1 to 5 in the preparation of a product for promoting cartilage regeneration.

7. Use of the bioactive material for promoting cartilage regeneration according to claim 6 in the preparation of a product for promoting cartilage regeneration, characterized in that: The product for promoting cartilage regeneration is a medicine for treating temporomandibular arthritis.

8. Use of the bioactive material for promoting cartilage regeneration according to claim 7 in the preparation of a product for promoting cartilage regeneration, characterized in that: The medicine for treating temporomandibular arthritis is chitosan hydrogel microspheres loaded with 1-(1-propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea.

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