A berberine / chondrocyte / injectable hydrogel biomimetic material, and a preparation method and use thereof

Abstract

The present application belongs to the field of biomedical technology, and relates to a berberine / chondrocyte / injectable hydrogel biomimetic material, a preparation method and application thereof. The present application combines phosphoric acid modified methylacrylate chitosan injectable hydrogel (scaffold material), chondrocyte (seed cell) and berberine (bioactive substance) into one to form a berberine / chondrocyte / injectable hydrogel biomimetic cartilage scaffold material. The material can realize long-time effect of berberine on chondrocyte, is prepared into a cartilage scaffold, can act on residual chondrocyte and new chondrocyte around the cartilage scaffold, realizes regeneration and repair of cartilage tissue on the basis of climbing material provided by the hydrogel, and has a "1+1+1>3" effect, thereby providing a new strategy for cartilage defect treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a berberine / chondrocyte / injectable hydrogel biomimetic material, its preparation method, and its uses. Background Technology

[0002] Articular cartilage is a transparent cartilage covering the surface of joints, composed of chondrocytes and extracellular matrix. Its main functions are lubrication and shock absorption. Due to the lack of blood vessels and nerves, it is easily damaged by trauma, tumors, inflammation, etc., and is difficult to heal. With the advancement of medical technology and the increasingly serious aging of the population in my country and the world, the number of patients with cartilage defects is also increasing. This not only affects the quality of life of patients and causes disability, but also places a heavy burden on patients' families and even national healthcare. Therefore, articular cartilage repair is a challenging problem for orthopedic surgeons and has received increasing attention. Only by restoring the morphology, biology, and physicochemical properties of articular cartilage to near-normal levels can joint function be reconstructed. This presents both an opportunity and a challenge for clinical medical professionals.

[0003] Medical materials are a rapidly developing field that has emerged in recent years as an interdisciplinary area of ​​medicine and materials science. Scaffold materials, seed cells, and bioactive substances are the three essential elements of medical materials, and high-performance medical materials combining these three elements are attracting increasing attention. Developing and commercializing high-performance biomimetic cartilage materials has become a new approach to promoting cartilage defect repair in the medical field, and if properly developed, it will benefit a wide range of patients.

[0004] The foundation of medical materials lies in scaffold materials. Good medical scaffold materials should possess excellent biosafety, biocompatibility, and biodegradability, and be readily available and relatively inexpensive. Chitosan-based hydrogels play a crucial role in cartilage defects. Chitosan is produced by deacetylating chitin, which is widely distributed, ranking second in abundance in nature, making it readily available. Chitosan is insoluble in water and common organic solvents, and its modified forms are widely used in the medical field. Chitosan itself is an oligosaccharide composed of 2-10 glucosamine molecules, readily degraded in vivo, and possesses immunomodulatory, antioxidant, and free radical scavenging properties. The degraded components are non-toxic to organisms. Furthermore, chitosan swells upon absorbing water, exhibiting strong water absorption. Chitosan-based bioscaffolds facilitate cell adhesion, proliferation, and differentiation, allowing cells to grow more effectively on its surface and form biologically functional complexes.

[0005] Seed cells are also closely related to cartilage defects. Based on their differentiation, they can be divided into differentiated seed cells (such as chondrocytes, osteoblasts, and fibroblasts) and undifferentiated seed cells (such as BMSCs, ADSCs, and synovial mesenchymal stem cells (SMSCs)). In cartilage repair, the most important cells are chondrocytes, which are the only cells in cartilage tissue, and the type II collagen and aggrecan they secrete are essential substances for cartilage repair.

[0006] Bioactive substances play a crucial role in cartilage defect repair, and they are diverse, such as the natural drug berberine. Recent studies have shown that berberine plays an important role in cartilage defect repair, promoting repair by regulating the expression of target factors in related signaling pathways. Berberine, also known as coptisine, is a quaternary ammonium alkaloid isolated from the traditional Chinese medicine Coptis chinensis, and is also the main antibacterial component of Coptis chinensis. Berberine itself has a bitter taste and appears as yellow needle-like crystals. It was first obtained by M.-E. Chavalier and G. Pertain in 1826 from the bark of Xanthoxylon clava. The chemical formula of berberine is C63-C64 ... 20 H 18 NO4 has a molecular weight of 336.36, a melting point of 204-205℃, and a density of 1.117 g / cm³. 3 Berberine is soluble in hot water, slightly soluble in water or ethanol, very slightly soluble in chloroform, and insoluble in ether. It is widely distributed in nature, found in approximately 4 families and 10 genera. As a plant antibiotic, berberine plays an important role in orthopedic, gastrointestinal, and cardiovascular diseases.

[0007] Cartilage defects are a challenging clinical problem for orthopedic surgeons, often accompanied by localized pain and functional impairment, and in severe cases, disability. Research into this condition is urgently needed. The current gold standard for cartilage defect repair is autologous or allogeneic cartilage transplantation, which involves disadvantages such as secondary surgery, high costs, complex procedures, rejection reactions, and significant patient discomfort. Novel biomimetic cartilage materials that combine scaffold materials, seed cells, and bioactive substances have attracted considerable attention in recent years. However, the application of such composite biomimetic cartilage materials is extremely limited in current technologies. Most suffer from drawbacks such as the inability of the load-bearing material to function for extended periods, insufficient scaffold material rigidity or inability to provide the necessary porosity for new tissue angiogenesis, and the inability of seed cells to induce new tissue regeneration. Furthermore, their safety and reliability need improvement. Summary of the Invention

[0008] This invention addresses the problems of existing technologies by providing a berberine / chondrocyte / injectable hydrogel biomimetic material, its preparation method, and its applications. This invention innovatively combines phosphate-modified methacrylic anhydride chitosan injectable hydrogel (scaffold material), chondrocytes (seed cells), and berberine (bioactive substance) into a single berberine / chondrocyte / injectable hydrogel biomimetic cartilage scaffold material. This material enables the long-term action of berberine on chondrocytes, and when prepared as a cartilage scaffold, it can act on surrounding residual and newly formed chondrocytes. Based on the adhesion substances provided by the hydrogel, it achieves cartilage tissue regeneration and repair, resulting in a synergistic effect ("1+1+1>3"), providing a new strategy for the treatment of cartilage defects.

[0009] The technical solution of the present invention is as follows:

[0010] First, this invention provides a berberine / chondrocyte / injectable hydrogel biomimetic material, the technical solution of which is: seeding ATDC5 chondrocytes pretreated with berberine (BBR) into a solid gel formed by an injectable hydrogel; the specific preparation method includes:

[0011] (1) Berberine (BBR) pretreatment of ATDC5 chondrocytes: ATDC5 chondrocytes were stimulated for 12-36 h in DMEM / F12 medium containing 6-36 μmol / L berberine, 10% FBS and 1% antibiotics, and then centrifuged to obtain pretreated cells.

[0012] (2) Seeding ATDC5 chondrocytes into a solid gel formed by injectable hydrogel: The injectable hydrogel was injected into a syringe to form a solid gel block, cut into small pieces, lyophilized, sterilized, and placed in a well plate or culture dish. The pretreated cells were counted using a cell counting chamber and a cell suspension was prepared (using DMEM / F12 medium with 10% FBS and 1% penicillin antibiotics) at a concentration of 3-5 × 10⁻⁵ cells / mL. 6 100 μL of cell suspension at a concentration of cells / mL was dropped onto the lyophilized solid gel block.

[0013] In step (1), the stimulation time is preferably 16-32 hours, more preferably 24 hours.

[0014] The concentration of berberine in the culture medium described in step (1) is preferably 12-36 μmol / L, more preferably 12-24 μmol / L, and most preferably 24 μmol / L.

[0015] In step (2), the preferred cell concentration is 5 × 10⁻⁶. 6 Cells / mL.

[0016] In step (2), the hydrogel is preferably cut into small pieces in cylindrical shape, and more preferably into cylindrical shapes with a diameter of 0.6 cm and a height of 0.4 cm.

[0017] The injectable hydrogel can be produced using existing technologies, particularly the injectable, crack-resistant, biodegradable supramolecular hydrogel described in invention patent ZL202010574779.3. This hydrogel modifies natural polysaccharides with methacrylic anhydride and phosphoric acid or phosphonic acid, forming supramolecular bonds with metal particles to prepare the injectable, crack-resistant hydrogel. The multifunctional interactions within the hydrogel, such as hydrogen bonds, hydrophilic-hydrophobic interactions, electrostatic interactions, and van der Waals interactions, along with the supramolecular bonds, endow the supramolecular hydrogel with crack-resistant properties. It can be compressed to a certain extent (compressive strain greater than 85%) without fracturing, and after soaking in aqueous solution for a period of time, it can recover to near its initial height. The phosphate groups can form metal ligand supramolecular bonds with the metal particles, making the hydrogel injectable. Especially when combined with ATDC5 chondrocytes pretreated with berberine (BBR) as described in this invention, the ATDC5 chondrocytes exhibit good activity on the hydrogel, and the hydrogel carrying ATDC5 chondrocytes shows significant in vitro safety.

[0018] This invention further protects the use of the above-mentioned berberine / chondrocyte / injectable hydrogel biomimetic material in cartilage scaffolds;

[0019] The biomimetic material obtained by this invention can serve as a cartilage scaffold, providing a microenvironment after berberine stimulation to the cartilage tissue at the defect site, and enhancing the ability of berberine to bind with chondrocytes. Using hydrogel as a scaffold carrier, it fills the cartilage defect while providing adhesion material for chondrocytes. As new cartilage tissue forms, the scaffold material gradually degrades, ultimately forming complete new cartilage tissue. This reduces the risk of secondary surgery and infection associated with autologous cartilage repair, the cumbersome procedures, insufficient donor cartilage, and rejection reactions from allogeneic or xenogeneic cartilage, thus lowering the pain, risks, and costs associated with cartilage defect treatment. Attached Figure Description

[0020] Figure 1 To compare the overall morphological images of knee cartilage defect patients at 4, 8, and 12 weeks post-surgery in different treatment groups of the experimental cases;

[0021] Figure 2 To compare the imaging assessment and 3D reconstruction of Micro CT in different treatment groups in the experimental cases.

[0022] Figure 3 HE staining images of the heart, liver, spleen, lungs and kidneys of mice in different treatment groups 12 weeks after surgery in the comparative experimental cases.

[0023] Figure 4To compare the histological evaluation of HE staining at 4, 8, and 12 weeks post-surgery in the experimental cases.

[0024] Figure 5 To compare the expression of COL II in cartilage damage in experimental cases and perform related quantitative analysis.

[0025] Figure 6 To compare the expression of SOX9 in cartilage damage in experimental cases and perform related quantitative analysis.

[0026] Figure 7 To compare the expression of AGC in cartilage damage in experimental cases and perform related quantitative analysis.

[0027] in:

[0028] Figure 1 In the group, A consists of a, f, and k; B consists of b, g, and I; C consists of c, h, and m; D consists of d, I, and n; and E consists of e, j, and o.

[0029] Figure 2 In the table, * represents comparison with group A; # represents comparison with group B. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; #, P < 0.05; ##, P < 0.01; ###, P < 0.001; ####, P < 0.001.

[0030] Figure 5 In the table, * represents comparison with group A; # represents comparison with group B. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ##, P < 0.01; ###, P < 0.001; ####, P < 0.001.

[0031] Figure 6 In the table, * represents comparison with group A; # represents comparison with group B. *, P < 0.05; ****, P < 0.0001; #, P < 0.05; ##, P < 0.01; ###, P < 0.001.

[0032] Figure 7 In the table, * represents comparison with group A; # represents comparison with group B. **, P < 0.01; ***, P < 0.001; #, P < 0.05; ##, P < 0.01; ###, P < 0.001; ####, P < 0.001. Detailed Implementation

[0033] The present invention will now be described in detail through specific embodiments. The scope of the present invention is not limited to these specific embodiments.

[0034] in:

[0035] ATDC5 chondrocytes were purchased from Shanghai Anwei Biotechnology Co., Ltd., and cultured in DMEM / F12 medium containing 10% fetal bovine serum and penicillin antibiotics at 37°C in an incubator containing 5% CO2.

[0036] Unless otherwise specified, the experimental techniques used in the following embodiments and comparative examples were all performed using the methods described below:

[0037] Cell resuscitation

[0038] (1) Prepare centrifuge tubes, pipettes, pipette tips, culture dishes, etc. After preheating the water bath to 37°C, put the culture medium into the water bath to preheat.

[0039] (2) Take out the frozen ATDC5 chondrocytes from the -80℃ freezer, put the cryovial into a 37℃ water bath to thaw the contents of the cryovial quickly, open the cryovial in a clean bench, add 1mL of culture medium, gently pipette and mix well, then transfer to a 15mL centrifuge tube, add another 3mL of culture medium, mix well again, put it into a centrifuge and centrifuge at 1500rpm for 3min.

[0040] (3) After centrifugation, discard the supernatant, add 1 mL of culture medium to the centrifuge tube, gently pipette the cell pellet to mix it into a cell suspension, transfer the cell suspension into a culture dish with a pipette, add sufficient culture medium, gently shake the cell culture dish to distribute the cells evenly, and place the cells in an incubator at 37°C and 5% CO2 for culture.

[0041] Cell cryopreservation

[0042] (1) When the growth density of ATDC5 chondrocytes reaches 80%-90% under a microscope, use a pipette to aspirate the culture medium from the culture dish, add preheated PBS buffer, shake and clean it, then use a pipette to remove the PBS, and repeat twice.

[0043] (2) Add 2 mL of preheated trypsin to the culture dish, shake well, and incubate in an incubator for 1-2 min. Observe the cells under a microscope. When the digested cells are fully suspended, add 4 mL of preheated culture medium, gently pipette to mix, and then use a pipette to transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1500 rpm for 3 minutes.

[0044] (3) After centrifugation, remove the supernatant as much as possible, add 1 mL of cell cryopreservation solution, and gently pipette to disperse the cells evenly in the cryopreservation solution. Transfer the cryopreservation solution to a cryovial, and then freeze the cryovial at -80°C for use in subsequent experiments.

[0045] CCK8 Experiment:

[0046] (1) Culture the cells of the experimental group and the control group, digest the cells with preheated trypsin, stop digestion with preheated culture medium, centrifuge at 1500 rpm for 3 min, add 1 mL of culture medium to fully resuspend the cells, take 10 μL of cell suspension and add it to a cell counting plate, count the number of cells with a cell counter, and then plate them in 96-well plates, with 6 wells in each group and about 3000 to 5000 cells per well.

[0047] (2) After cell adhesion, cells were divided into 4 groups according to LPS stimulation (control group, 2 μg / mL, 4 μg / mL, 6 μg / mL), 6 groups according to BBR stimulation (control group, 3 μmol / L, 6 μmol / L, 12 μmol / L, 24 μmol / L, 36 μmol / L), and further grouped according to the optimal concentration of LPS for ATDC5 chondrocytes (control group, LPS group, 3 μmol / L BBR+LPS, 6 μmol / L BBR+LPS, 12 μmol / L BBR+LPS, 24 μmol / L BBR+LPS, 36 μmol / L BBR+LPS). The culture medium was discarded at 12, 24, 36, and 48 h after LPS stimulation, 24 h after BBR stimulation, and 24 h after stimulation at the optimal concentration. 100 μL of culture medium and 10 μL of [unclear text - possibly a continuation of the previous sentence] were added to each well. Add 10 μL of CCK-8 reagent to a 96-well plate and incubate for 1 hour. Measure the OD value at 450 nm using a microplate reader.

[0048] Calcein-AM / PI live / dead cell staining assay

[0049] (1) Culture the experimental group cells and the control group cells, digest the cells with preheated trypsin, neutralize and terminate the trypsin digestion in the culture medium, centrifuge at 1500 rpm for 3 min, and seed the cells into 96-well plates at a density of 3000 cells per well. After observing the cells under a microscope, ATDC5 chondrocytes were treated with LPS and BBR concentrations selected according to CCK8.

[0050] (2) After removing the Calcein-AM / PI kit (Tongren Chemical) from the -20℃ freezer, allow it to thaw at room temperature. Once fully thawed, prepare a 15mL centrifuge tube under light-protected conditions and add 5mL of PBS buffer, then add 10μL of Calcein-AM stock solution and 15μL of PI stock solution, and mix well to prepare the working solution. At this point, the concentration of Calcein-AM is 2μmol / L, and the concentration of PI is 4.5μmol / L.

[0051] (3) After stimulation with BBR and LPS, the operation was carried out in the dark, the cell culture medium was discarded, the cells were washed repeatedly with PBS 3 times, Calcein-AM / PI working solution was added, and the cells were incubated in an incubator for 15 min.

[0052] (4) After incubation, the cells were detected under a fluorescence microscope using different lasers. Live cells showed yellow-green fluorescence, while dead cells showed red fluorescence.

[0053] Preparation of injectable hydrogels (refer to invention patent ZL202010574779.3):

[0054] (1) Fill a beaker with 300 mL of deionized water and add 3 mL of acetic acid solution. Weigh 3 g of chitosan and dissolve it in the acetic acid solution. Stir the mixture in the beaker using a stir bar on a magnetic stirrer to accelerate the dissolution of chitosan. After the chitosan is fully dissolved, add 1.1 mL of MA and react at room temperature for 24 h.

[0055] (2) Weigh 20.787g of MES and dissolve it in 150mL of deionized water. Weigh 1.17g of PS and dissolve it in the above MES solution. Weigh 3.51g of EDC and 1.17g of NHS and dissolve them in the above PS solution.

[0056] (3) The solution obtained in (1) was added dropwise to the solution in (2), and the reaction was carried out on ice. The reaction was carried out at room temperature for 24 hours.

[0057] (4) After the reaction, pour the resulting solution into a dialysis bag and clamp the dialysis bag with a clip. Dialyze in deionized water for 1 week, changing the deionized water 3 times a day.

[0058] (5) Pour the solution from the dialysis bag into a large dish and freeze it in a -80°C freezer. After it is completely frozen, put the large dish into a freeze dryer to freeze dry. After about 3 days, you will get the freeze-dried CSMAP.

[0059] (6) Weigh CSMAP solution at 20 g / mL and dissolve it in deionized water. Add 10 mg / mL MA, 0.4 mg / mL BIS and 1 mg / mL I2959 to the solution and stir thoroughly. Then, draw up the solution with a syringe and place the syringe under ultraviolet light to crosslink into a gel.

[0060] (7) After cross-linking, the syringe was placed in a -20°C freezer and frozen. After freezing, it was cut into appropriately sized hydrogels with a knife and soaked in deionized water for 2 days to remove any unreacted compounds. Finally, it was placed in a -80°C freezer and then freeze-dried in a vacuum freeze dryer to obtain the final hydrogel. It was then stored in a vacuum drying oven for use in subsequent experiments.

[0061] Example 1

[0062] Preparation of berberine / chondrocytes / injectable hydrogel biomimetic materials:

[0063] (1) Berberine (BBR) pretreatment of ATDC5 chondrocytes: ATDC5 chondrocytes were stimulated with a culture medium containing 24 μmol / L berberine for 24 h, and then centrifuged to obtain pretreated cells;

[0064] (2) ATDC5 chondrocytes were seeded into solid gels formed by injectable hydrogels: Injectable hydrogels were injected into a 2.5 mL syringe to form solid gel blocks, which were then cut into hydrogels with a diameter of 0.6 cm and a height of 0.4 cm, lyophilized, sterilized, and placed in well plates. The pretreated cells were then seeded at a rate of 5 × 10⁻⁶ cells / well. 6 100 μL of cell suspension at a concentration of cells / mL was dropped onto the lyophilized solid gel block.

[0065] Examples 2-4

[0066] The preparation method is the same as in Example 1, except that the different parameters are shown in the table below:

[0067]

[0068] Comparative Example 1

[0069] ATDC5 chondrocytes were directly seeded into an injectable hydrogel-formed solid gel: After sterilization, the injectable hydrogel-formed solid gel was placed in a 24-well plate, and ATDC5 chondrocytes were seeded at a rate of 5 × 10⁶ cells / well. 6 100 μL of cell suspension at a concentration of cells / mL was dropped onto the lyophilized solid gel block.

[0070] Comparative experimental cases

[0071] Seventy-five healthy 6-week-old C57Bl / 6 mice were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. and housed in cages, receiving water and food. Animal modeling (Reference: Bethan L Thomas, Suzanne E Eldridge, Babak Nosrati, Mario Alvarez, Anne-Sophie Thorup, Giovanna Nalesso, Sara Caxaria, Aida Barawi, James Gnicholson, Mauro Perretti, Carles Gaston-Massuet, Costantino Pitzalis, Alison Maloney, Adrian Moore, Ray Jupp, Francesco Dell'Accio. WNT3A-loaded exosomes enable cartilage repair[J]. J Extracell) In Vesicles, 2021, 10(7):e12088., mice were anesthetized with isoflurane and fixed on the operating table. The skin of the right knee joint was prepared and the right knee joint was disinfected with povidone-iodine three times. The patellar ligament was exposed at the right joint and cut from the medial side. The patella was dislocated and the knee joint was fully exposed. A hand drill (about 0.8 mm in diameter) was used to drill a hole in the non-weight-bearing area of ​​the femoral condyle of the right knee joint. The hole was drilled to the depth of the cartilage and subchondral bone, and the defect diameter was about 0.8 mm and the depth was 1 mm. Mice were randomly divided into group A (n=72) and group A' (n=3). The 72 mice in group A were further randomly divided into groups B, C, D, and E (n=18 each). After undergoing the following repair treatment, they were fed normally. Knee joint specimens from 6 rats in each of groups B, C, D, and E were collected at 4, 8, and 12 weeks for relevant observation experiments. Group A' was used as a normal control only at 12 weeks for in vivo safety analysis.

[0072] Group A was the normal control group. The left knee joint of 72 mice was sham-operated by simply cutting the synovial bursa without any other treatment and then suturing.

[0073] Group A' received no treatment and was fed according to standard practices.

[0074] In group B mice, a hole was drilled by hand in the non-weight-bearing area of ​​the femoral condyle of the right knee joint. The hole was drilled to the depth of the cartilage and subchondral bone, causing bleeding. The defect diameter was approximately 0.8 mm and the depth was 1 mm (this was the cartilage defect group).

[0075] In addition to the treatment given to mice in group B, group C mice received an injection of hydrogel to fill the cartilage defects.

[0076] In addition to the treatment of mice in group B, mice in group D were implanted with the material prepared in comparison example 1 to fill the cartilage defects.

[0077] In addition to the methods used in Group B, mice in Group E were implanted with the biomimetic material prepared in Example 1 to fill the cartilage defects.

[0078] (1) Gross specimen observation

[0079] Four weeks post-surgery, Group B ( Figure 1 b) A small amount of tissue fills the defect, but the defect still exists. In groups C and D, some tissue can be observed, but the defect is still not fully repaired. The defect in group C is irregular, while that in group D is more irregular than that in group C. Figure 1 c and d) have more regenerated tissue, group E ( Figure 1 e) There is transparent soft tissue proliferation around the defect, but the defect area is still visible at the edge.

[0080] Eight weeks post-surgery, Group B ( Figure 1 g) Slight repair was observed, but concavity remains. Group C ( Figure 1 h) Translucent tissue proliferation is visible around the defect, and the defect boundary is visible. Group D ( Figure 1 i) Repairing tissue to fill the defect, but the surface is irregular. Group E ( Figure 1 j) The repaired tissue at the defect site is clearly connected to the surrounding tissue, with blurred boundaries.

[0081] 12 weeks post-surgery, Group B ( Figure 1 l) There is tissue repair, but local defects are still visible. Group C ( Figure 1 m) has largely filled the missing area, but the boundary is still clearly visible. Group D ( Figure 1 n) The surface is nearly smooth and almost identical to the surrounding tissue. Group E ( Figure 1 o) Repair the tissue to resemble the surrounding tissue.

[0082] (2) Micro CT assessment

[0083] The relevant bone parameters changed with the treatment intervention, as shown in representative images from MicroCT. Figure 2 In the middle. For example Figure 2 As shown, at 4 weeks, the BV / TV, Tb.N, and Tb.Th levels in group E were higher than in other groups and closer to those in group A, while Tb.Sp showed a gradually decreasing trend in groups B, C, D, and E. The same trend was observed at 8 weeks and 12 weeks. Therefore, this indicates that our novel material may induce subchondral bone remodeling, thus playing an important role in cartilage defect repair.

[0084] (3) In vivo safety analysis

[0085] To verify whether hydrogel implantation in the knee joints of mice had any effect on the internal organs, we harvested the heart, liver, spleen, lungs, and kidneys of mice at 12 weeks of age and stained them with hematoxylin and eosin (HE). Group A' served as the normal control group. Figure 3As shown, the results indicated that no significant abnormalities were observed in the heart, liver, spleen, lungs, and kidneys of the mice. This demonstrates that the hydrogel exhibits good biocompatibility in mice.

[0086] (4) Evaluation of HE staining for in vivo cartilage regeneration

[0087] HE staining is used to observe the structure of femoral cartilage tissue in mice. For example... Figure 4 :

[0088] At 4 weeks, in group E, the cartilage defect nearly healed over time, with tissue filling and subchondral bone reconstruction, but gaps remained. In group B, the defect did not show healing and no tissue filling. In groups C and D, fibrous tissue repair was observed.

[0089] At 8 weeks, group E showed subchondral bone reconstruction and tissue repair. Group B had a small amount of fibrous tissue filling, group C had fibrous tissue filling but still had some defects, and group D had relatively dense tissue filling that was close to the edge.

[0090] At 12 weeks, group E showed tissue growth, a tendency for defect repair, subchondral bone reconstruction, and a nearly smooth surface. Defects persisted in group B. Group C showed disordered cell distribution, loose tissue, and subchondral bone reconstruction. Group D showed subchondral bone reconstruction, with no obvious structural defects.

[0091] (5) Immunohistochemical staining

[0092] Immunohistochemical staining was used to detect the expression of cartilage-related proteins. The specific detection method was as follows: (1) The slides to be stained were baked in an oven for 6 hours and then cooled to room temperature before staining; (2) The slides were dewaxed to water using xylene and graded alcohol; (3) An immunohistochemical pen was used to draw a circle 3 mm away from the tissue area to be tested, avoiding drawing on the tissue; (4) Trypsin antigen retrieval solution was added to the area circled by the immunohistochemical pen and incubated at 37°C for 30-40 min; (5) The slides were washed 3 times with PBS for 5 min each time. Endogenous peroxidase inhibitor was added to the circled area and incubated for 10 min; (6) The slides were washed 3 times with PBS for 5 min each time. Non-specific staining inhibitor was added to the circled area and incubated for 10 min; (7) The slides were washed 3 times with PBS for 5 min each time. The corresponding primary antibody (SOX9, AGC, COLII) was added to the circled area and incubated overnight at 4°C; (8) The slides were washed 3 times with PBS for 5 min each time. Add biotin-labeled goat anti-mouse / rabbit, mouse, rabbit IgG polymer or rabbit anti-goat IgG polymer to the designated area and incubate for 10 min; (9) Wash the sections three times with PBS, 5 min each time. Add streptomycin antibiotic protein-peroxidase to the designated area and incubate for 10 min; (10) Wash the sections three times with PBS, 5 min each time. Remove PBS and add DAB for color development. After color development, place the sections in PBS to stop the color development; (11) Counterstain with hematoxylin for 2 min, differentiate with hydrochloric acid for 2-3 s. Rinse with tap water to return to blue. Dehydrate using a stepwise ethanol gradient and clear with xylene. Mount with neutral resin.

[0093] The higher the expression levels of COLII, SOX9, and AGC proteins, the better the cartilage tissue repair and the closer it is to normal cartilage.

[0094] like Figure 5 As shown, COL II expression was detected at 4 weeks, 8 weeks, and 12 weeks: at 4 weeks, COL II expression in groups D and E was higher than that in group B, and the difference was statistically significant. At 8 weeks, the expression in group E was higher than that in group B, and the difference was statistically significant. At 12 weeks, the expression in groups C, D, and E was increased compared with group B, and all differences were statistically significant.

[0095] like Figure 6 As shown, SOX9 expression was detected at 4 weeks, 8 weeks, and 12 weeks: At 4 weeks, SOX9 expression in groups C and E was higher than that in group B, and the difference was statistically significant. At 8 weeks, SOX9 expression in group E was higher than that in group B, and the difference was statistically significant. At 12 weeks, SOX9 expression in groups D and E was increased compared to group B, and the difference was statistically significant.

[0096] like Figure 7As shown, AGC expression was detected at 4 weeks, 8 weeks, and 12 weeks: At 4 weeks, AGC expression in groups D and E was higher than that in group B, and the difference was statistically significant. At 8 weeks, AGC expression in groups C, D, and E was higher than that in group B, and the difference was statistically significant. At 12 weeks, AGC expression in groups D and E was increased compared to group B, and the difference was statistically significant.

[0097] In summary, the berberine / chondrocyte / injectable hydrogel biomimetic material of this invention can serve as a cartilage scaffold, providing a long-term microenvironment for the cartilage tissue at the defect site after berberine stimulation, and chondrocytes with superior berberine-chondrocyte binding function. The hydrogel acts as a scaffold carrier, filling the cartilage defect while providing adhesion material for chondrocytes. As new cartilage tissue forms, the scaffold material gradually degrades, ultimately forming complete new cartilage tissue. This reduces the pain, risks, and costs of treatment for patients with cartilage defects, and avoids the drawbacks of autologous or allogeneic cartilage harvesting.

[0098] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0099] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A berberine / chondrocyte / injectable hydrogel biomimetic material, characterized in that, Specific preparation methods include: (1) Berberine (BBR) pretreatment of ATDC5 chondrocytes: ATDC5 chondrocytes were stimulated for 12-36 h in DMEM / F12 medium containing 12-36 μmol / L berberine in 10% FBS and 1% double antibiotics, and then centrifuged to obtain pretreated cells. (2) Seeding ATDC5 chondrocytes into a solid gel formed by injectable hydrogel: Injectable hydrogel is injected into a syringe to form a solid gel block, which is then cut into small pieces, lyophilized, sterilized, and placed in well plates or culture dishes. The pretreated cells are counted and a cell suspension is prepared at 3-5 × 10⁻⁵. 6 100 μL of cell suspension at a concentration of cells / mL was dropped onto the lyophilized solid gel block; The injectable hydrogel is a phosphate-modified methacrylic anhydride chitosan injectable hydrogel. It is prepared by modifying natural polysaccharides with methacrylic anhydride and phosphoric acid or phosphonic acid, and then combining them with metal particles to form supramolecular bonds, thus creating an injectable hydrogel that is resistant to cracking and degradable supramolecular.

2. The berberine / chondrocyte / injectable hydrogel biomimetic material according to claim 1, characterized in that, The stimulation time in step (1) is 16-32 hours.

3. A berberine / chondrocyte / injectable hydrogel biomimetic material according to claim 1 or 2, characterized in that, The stimulation time in step (1) is 24 hours.

4. The berberine / chondrocyte / injectable hydrogel biomimetic material according to claim 1, characterized in that, The concentration of berberine in the culture medium described in step (1) is 12-24 μmol / L.

5. The berberine / chondrocyte / injectable hydrogel biomimetic material according to claim 1, characterized in that, The concentration of berberine in the culture medium described in step (1) is 24 μmol / L.

6. The berberine / chondrocyte / injectable hydrogel biomimetic material according to claim 1, characterized in that, In step (2), the preferred cell concentration is 5 × 10⁻⁶. 6 Cells / mL.

7. The berberine / chondrocyte / injectable hydrogel biomimetic material according to claim 1, characterized in that, In step (2), the hydrogel is cut into small cylindrical pieces.

8. A berberine / chondrocyte / injectable hydrogel biomimetic material according to claim 1 or 7, characterized in that, In step (2), the hydrogel is cut into small pieces, which are cylindrical in diameter and height of 0.6 cm and 0.4 cm respectively.

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