An artificial epiphyseal plate and a preparation method and application thereof

Abstract

The application provides a kind of bionic epiphyseal plate and its preparation method and application, belong to the technical field of biological medical material.The present application uses gelatin microspheres as cell carrier, which has large surface area, and cells can obtain sufficient nutritional support;hMSC microspheres are used as basic building blocks, and static incubation is carried out under the stimulation of recombinant IHH protein, which drives hMSC microspheres to spontaneously aggregate, fuse and self-assemble into a kind of bionic epiphyseal plate with internal cells in proliferation and differentiation balance state.The cell state, matrix morphology and mechanical properties of the bionic epiphyseal plate are similar to those of natural epiphyseal plate, and the support effect on cells and the adaptability to the mechanical microenvironment of the damage site are obvious, which has the dual functions of preventing bone bridge and promoting cartilage regeneration, and can be used as a candidate filler for large-area epiphyseal plate defect repair.Compared with the traditional method of preparing epiphyseal plate replacement scaffold, the method of the present application has higher chondrogenic induction efficiency and shortens the induction period.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a biomimetic epiphyseal plate, its preparation method, and its application. Background Technology

[0002] The epiphyseal plate is a wavy, specialized cartilage tissue located between the secondary ossification center and the metaphysis, driving bone growth. Epiphyseal plate chondrocytes are mechanically fragile and highly susceptible to physical damage. Due to their limited regenerative capacity, they are easily replaced by bone tissue during the repair process, creating "bone bridges," which can lead to local growth arrest and serious complications such as angular deformities and limb length discrepancies.

[0003] Currently, the commonly used clinical treatment is bone bridge resection combined with filler material. The filler materials are often autologous fat, bone cement, and some polymer materials. However, this inert filler primarily prevents bone bridge formation but hinders epiphyseal cartilage regeneration, ultimately leading to uneven bone growth on both sides later on. Therefore, the ideal epiphyseal plate repair strategy is to prevent bone bridges and regenerate cartilage, but traditional techniques currently struggle to achieve both simultaneously.

[0004] Currently, traditional techniques still have the following problems: (1) Using stem cell progenitor spheres for chondrogenic induction culture results in low chondrogenic induction efficiency and core necrosis; (2) Traditional chondrogenic induction culture programs mostly use mature induction reagents, including transforming growth factor β3 and dexamethasone, which have long induction cycles and poor efficiency; (3) Traditional microsphere culture mostly uses bioreactors for dynamic incubation, but dynamic culture may reduce the contact between cell microspheres and reduce the fusion ability of microspheres; after microsphere incubation, the microspheres are polymerized and shaped by molds or external forces, and the interaction effect between microspheres is not fully mobilized, and dynamic culture is not conducive to chondrogenic differentiation of MSCs; (4) The effectiveness verification of traditional repair strategies mostly uses central or small-area marginal epiphyseal plate defect models of rats or rabbits. The preparation process of this model is simple and the repair difficulty is low. It is difficult to reproduce and verify the large-area epiphyseal plate defect cases that are often faced in clinical practice, and the guiding value for future translational applications is not high.

[0005] With the development of interdisciplinary fields related to medicine, a tissue engineering technique that simulates development—developmental engineering—has brought hope for the active filling of epiphyseal plate injuries. Unlike the top-down manufacturing strategy of traditional tissue engineering, developmental engineering focuses on the spontaneous aggregation and assembly of stem and progenitor cells, using cell clusters as basic units to construct large-scale grafts. This bottom-up advanced manufacturing technology aims to achieve functional regeneration of organs.

[0006] Therefore, how to utilize developmental engineering technology to improve the survival rate and chondrogenic differentiation efficiency of induced cells, while shortening the induction period and improving the repair effect of biomimetic epiphyseal plates, is of great significance for the repair of complex epiphyseal plate injuries. Summary of the Invention

[0007] The purpose of this invention is to provide a biomimetic epiphyseal plate, its preparation method and application, which can produce a biomimetic epiphyseal plate with dual functions of preventing bone bridging and promoting cartilage regeneration, and is expected to be one of the candidate fillers for epiphyseal plate defect repair.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a biomimetic epiphyseal plate, which is obtained by inducing human bone marrow mesenchymal stem cells with recombinant IHH protein.

[0009] This invention also provides a method for preparing a biomimetic epiphyseal plate, comprising the following steps: (1) Add a microcarrier containing gelatin microspheres to a low-adhesion culture plate, and seed human bone marrow mesenchymal stem cell suspension onto the microcarrier containing gelatin microspheres to obtain hMSC microspheres; (2) Add hMSC chondrogenic differentiation induction medium to a low adhesion culture plate containing hMSC microspheres, and add recombinant IHH protein solution for incubation and static culture to obtain a biomimetic epiphyseal plate.

[0010] Preferably, the concentration of the human bone marrow mesenchymal stem cell solution in step (1) is 2~4×10⁻⁶. 6 The number of gelatin microspheres is 80,000 to 100,000; the seeding volume of human bone marrow mesenchymal stem cell solution on each microcarrier is 200 to 400 μL. Preferably, the concentration of the human bone marrow mesenchymal stem cell solution in step (1) is 2 × 10⁻⁶. 6 The number of gelatin microspheres on the microcarrier is 90,000; the seeding volume of human bone marrow mesenchymal stem cell solution on each microcarrier is 300 μL.

[0011] Preferably, the concentration of the IHH protein solution in step (2) is 50~200 ng / mL.

[0012] Preferably, the concentration of the IHH protein solution in step (2) is 100 ng / mL.

[0013] Preferably, the static culture time in step (2) is 15 to 17 days.

[0014] Preferably, the static culture time in step (2) is 16 days. During static culture, a new chondrogenic induction medium and IHH protein need to be replaced every 2 days.

[0015] The present invention also provides a biomimetic epiphyseal plate or a method for preparing a biomimetic epiphyseal plate, and the application of the prepared biomimetic epiphyseal plate in epiphyseal plate defect repair and novel articular cartilage repair scaffold.

[0016] The beneficial effects of the biomimetic epiphyseal plate described in this invention compared with the prior art are as follows: (1) This invention uses gelatin microspheres as cell carriers, which have a large surface area, allowing cells to obtain sufficient nutritional support. Using hMSC microspheres as the basic building block, static incubation is performed under the stimulation of recombinant IHH protein, driving the hMSC microspheres to spontaneously aggregate, fuse, and self-assemble into a biomimetic epiphyseal plate, with the internal cells in a state of balanced proliferation and differentiation. Compared to traditional preparation techniques, this static preparation system has higher chondrogenic induction efficiency and a shorter induction period.

[0017] (2) Unlike existing technologies, this invention does not rely on molds and external forces, and aims to enhance the activity of cells themselves, and has stronger adaptability and vitality than traditional epiphyseal plate replacement scaffolds.

[0018] (3) The biomimetic epiphyseal plate prepared by the present invention contains two types of cells at the same time: stem cells in the proliferation cycle and differentiated chondrocytes. This multicellular state is similar to that of the natural epiphyseal plate, while the current epiphyseal plate replacement scaffold only contains a single cell type. Therefore, this multicellular differentiation order has obvious advantages.

[0019] (4) This invention is the first to use a short-term (16-day) static incubation system of IHH protein to stimulate the fusion and assembly potential of hMSCs in microspheres. This exogenous IHH protein greatly shortens the in vitro induction time of hMSCs and significantly improves their chondrogenic differentiation efficiency. Compared with traditional techniques, it saves experimental costs and manpower, while reducing the uncontrollable risks brought about by long-term in vitro operations.

[0020] (5) The biomimetic epiphyseal plate provided by this invention has similar cell state, matrix morphology and mechanical properties to the natural epiphyseal plate. Compared with traditional epiphyseal plate replacement scaffolds, it has obvious advantages in supporting cells and adapting to the mechanical microenvironment of the damage site, and is expected to be one of the candidate fillers for repairing large-area epiphyseal plate defects.

[0021] (6) The biomimetic epiphyseal plate provided by this invention effectively prevents angular deformities and tibial shortening caused by epiphyseal cartilage defects. Compared with published literature, this biomimetic epiphyseal plate has been used in one of the most severe animal models in the field of epiphyseal repair, and has the dual functions of preventing bone bridges and promoting cartilage regeneration. It fills the technical gap of large-area epiphyseal plate defects that are difficult to repair, and is expected to provide a candidate strategy for the repair of epiphyseal plate injuries in children. It also has a profound impact on the promotion of biomimetic epiphyseal plates in other application scenarios, including injuries to the epiphysis, diaphysis, spine, and joints. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Cell characterization of hMSC microspheres in Experiment Example 1: A represents live / dead staining, with green for live cells and red for dead cells; B represents TUNEL staining, with blue for cell nuclei and green for TUNEL-positive apoptotic cells; C represents the ratio of live to dead cells; and D represents the percentage of apoptotic cells. Figure 2 This is the self-assembly of hMSC microspheres in Experiment Example 1. The upper part shows the process of inducing the self-assembly of hMSC microspheres; the lower part shows the appearance of the self-assembled product, i.e., the biomimetic epiphyseal plate. Figure 3 The images show the chondrogenic differentiation of the biomimetic epiphyseal plate in Experiment Example 1. A shows the SOX9 staining image after 16 days of induction; B shows the SOX9 statistical results after 16 days of induction; C shows the SOX9 staining image after 21 days of induction; and D shows the SOX9 statistical results after 21 days of induction. Red indicates SOX9-positive staining, and blue represents the cell nucleus.

[0024] Figure 4 The cell state in the bionic epiphyseal plate induced for 16 days in Experiment Example 1 is shown. Blue represents cell nuclei, red represents Ki67, which represents proliferating cells, and green represents CD29, which represents hMSCs that have not differentiated into chondrocytes. Figure 5 The image shows the cartilage matrix deposition in the biomimetic epiphyseal plate of Experiment Example 1. A shows the results of Safranin O-Fixed Green staining after 16 days of induction; B shows the results of Safranin O-Fixed Green staining after 21 days of induction; and C shows the statistical results of the Safranin O positive staining area after 16 and 21 days of induction, where red represents the Safranin O positive area, representing the cartilage matrix. Figure 6 The images show the microstructure of the biomimetic epiphyseal plate and gelatin microspheres in Experiment 1. A is an electron micrograph after 16 days of induction; B is an electron micrograph after 21 days of induction; C is an electron micrograph of the surface of the gelatin microspheres without cell loading; and D is an image of the rat rib epiphyseal plate reported in the literature. Figure 7 The gene expression of the sample induced for 16 days in Experiment 1 is shown. A is ACAN, B is COL2A1, C is COMP, and D is PTHrP, all of which are chondrocyte-specific markers. E is IHH and F is COL10A1, both of which are markers of chondrocyte hypertrophy and maturation. Figure 8The mechanical properties of the biomimetic epiphyseal plate in Experiment 1 are shown in Figure A, where A is an example of the indentation curve (100 ng / mL IHH group); B is a thermogram of the mechanical properties of each group of samples; C is the statistical results of the elastic modulus of each group of samples; and D is the elastic modulus data of the tibial epiphyseal plate of young rabbits reported in the literature. Figure 9 This is a schematic diagram of the in vivo efficacy verification protocol for the bionic epiphyseal plate in Experiment Example 2, where A represents the experimental grouping and surgical plan; B represents the epiphyseal defect modeling process. Figure 10 To illustrate the repair effect of the biomimetic epiphyseal plate on large epiphyseal defects in Experiment Example 2, A shows the appearance of tibial tissue harvested 4 weeks after epiphyseal resection, with 7 biological replicates in total. In each group, the left side is the healthy tibia and the right side is the damaged tibia; B is a schematic diagram of angular deformity calculation, with the left side being the autologous healthy control and the right side being the damaged tibia; C shows the statistical results of angular deformity; and D shows the statistical results of tibial length. Figure 11 To illustrate Experiment 2, the biomimetic epiphyseal plate inhibits bone bridging and promotes cartilage regeneration. In Figure A, Safranin O-Fixed Green staining was performed 4 weeks post-surgery, with red representing cartilage tissue and green representing bone tissue. In Figure B, COL II immunofluorescence staining was performed, with red indicating a positive result, representing cartilage matrix deposition. Figure 12 For Comparative Example 1, hMSCs were induced to differentiate into chondrocytes using the "chondroid ball method". In the figure, A is Safranin O staining, with red staining indicating the area of ​​chondrocyte matrix deposition; B is SOX9 immunofluorescence staining, with red staining indicating differentiated chondrocytes; and C is TUNEL staining, with green staining indicating apoptotic cells. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0026] This invention fabricated a biomimetic epiphyseal plate and comprehensively characterized it. Then, using young white rabbits as the research subject, a large epiphyseal defect measuring 5mm wide, 5mm deep, and 2mm high was created on the medial side of the proximal end of the right tibia to construct a high-difficulty injury model to verify the effectiveness of the biomimetic epiphyseal plate. Experiments demonstrate that the biomimetic epiphyseal plate of this application has been used in one of the most severe animal models in the field of epiphyseal repair, and simultaneously possesses the dual functions of preventing bone bridging and promoting cartilage regeneration, filling the technological gap in the difficult repair of large-area epiphyseal plate defects.

[0027] Example 1 Example 1 of this invention prepared a biomimetic epiphyseal plate, and the specific method is as follows: (1) Preparation of hMSC microspheres A. Culture of hMSCs: Human bone marrow mesenchymal stem cells (hMSCs, OriCell) were cultured in an incubator at 37°C and 5% CO2. When the cells reached approximately 80% confluence, the culture medium was aspirated, and the cells were washed twice with PBS. 1 mL of 0.05% trypsin was added for digestion, and the cells were resuspended. The cells were then passaged at a 1:3 ratio and cultured again in an incubator at 37°C and 5% CO2. When the hMSCs expanded to approximately 85% confluence, the cells were digested and the density was adjusted to 2 × 10⁶ cells / years. 6 Cells / mL were collected to obtain a cell resuspension for later use.

[0028] B. Combination of hMSCs with gelatin microspheres: One microcarrier (approximately 90,000 gelatin microspheres, Huakan Biotechnology Co., Ltd., China) was placed in each well of a low-adhesion six-well plate. 300 μL of well-mixed cell resuspension was slowly added dropwise to the microcarrier. 2 mL of PBS solution was then added to the wells of a culture dish to prevent culture medium evaporation from affecting cell state. The plate was then incubated for 2 hours. After 2 hours, the hMSCs and gelatin microspheres were completely adhered, yielding the hMSC microspheres.

[0029] (2) Preparation of biomimetic epiphyseal plates A. Chondrogenic differentiation of hMSC microspheres Centrifuge 10 μg of IHH (Indian hedgehog) protein powder at 3000 rpm for 3 min. Under biosafety cabinet conditions, add 1 mL of sterile PBS solution to the IHH protein packaging tube to prepare 10 μg / mL IHH stock solution. Aliquot the solution into 50 200 μL EP tubes and store at -20 degrees Celsius for later use.

[0030] Prepare hMSC chondrogenic differentiation induction medium (OriCell, China) under light-protected conditions. Slowly add the medium at a rate of 5 mL per well to the hMSC microsphere culture plate. Gently disperse the hMSC microspheres with the tip of a 200 μL pipette to ensure they are fully separated and evenly distributed within the culture plate without any obvious clumps.

[0031] B. Preparation of biomimetic epiphyseal plates based on static self-assembly In the chondrogenic induction medium for hMSC microspheres, 10 μg / mL of IHH stock solution was added to bring the IHH concentration in each well to 100 ng / mL. The medium was then incubated at 37°C for 16 days in a static state, with the medium and IHH protein replaced every 2 days. This culture system does not use molds or other external forces (such as centrifugation). Low-adhesion six-well plates prevent hMSC microspheres from settling and adhering to the well walls. The statically suspended hMSC microspheres in the medium gradually fuse and assemble into a single unit over time. After 16 days of incubation, the microspheres are fully formed, translucent, and elastic, thus resembling a biomimetic epiphyseal plate.

[0032] Experimental Example 1 Experimental Example 1 of this invention comprehensively characterized the biomimetic epiphyseal plate. The specific steps are as follows: Four groups of biomimetic epiphyseal plates were prepared using the method described in Example 1. The difference between these four groups of biomimetic epiphyseal plates and those in Example 1 was that the final concentrations of IHH protein in each group were 0 ng / mL (Control, functional control group of IHH protein), 25 ng / mL, 100 ng / mL, and 300 ng / mL, respectively. Subsequent experiments were conducted on the four groups of biomimetic epiphyseal plates.

[0033] (1) Cell viability staining of hMSC microspheres The live / dead staining kit was purchased from Huakan Biotechnology, catalog number R002-50. In step (2) of Example 1, during the preparation of the biomimetic epiphyseal plate based on static self-assembly, live / dead staining was performed 72 h after adding IHH solution. Using a pipette tip, 25-50 μL of each of the four groups (0 ng / mL, 25 ng / mL, 100 ng / mL, and 300 ng / mL) of hMSC microspheres and culture medium were placed in a 96-well plate, allowed to stand, and the supernatant was discarded after the microspheres settled. 100 μL of staining working solution was added and mixed well, and incubated at 37°C in the dark for approximately 1 hour. After incubation, the supernatant was discarded, and 1 mL of PBS was used for washing. This step was repeated twice, with an appropriate amount of PBS solution added to keep the microspheres in a humid environment. The microspheres were then observed and photographed under a fluorescence microscope. The results are as follows: Figure 1 As shown.

[0034] (2) Cell apoptosis staining Before applying the biomimetic epiphyseal plate, the viability of its internal cells needs to be tested. Therefore, frozen sections from each group induced for 14 days were taken, thawed, and then diluted with P0106 ​​immunostaining washing buffer to 20 μg / mL. The sections were incubated at 37°C for 30 minutes. They were then washed three times with PBS for 5 minutes each time. Staining working solution (TdT enzyme: fluorescent labeling solution = 1:9) was added and incubated at 37°C for 1 h. The sections were washed three times with PBS for 5 minutes each time. The sections were mounted with mounting solution containing DAPI to inhibit fluorescence quenching, observed and photographed under a fluorescence microscope. The results are shown below. Figure 1 As shown.

[0035] Figure 1 The results show that after combining hMSCs with gelatin microspheres, the hMSCs adhere to the pore surface inside the microspheres, as shown in the diagram. Figure 1 As shown in Figure A, most hMSCs within the microspheres are in good condition and appear green. Only a small number of red hMSCs have died, as indicated by the red arrows. Figure 1 The statistical results of cell liveness and death staining in C showed that green live cells accounted for more than 93% of the total number of cells. Figure 1 China B and Figure 1 The apoptosis staining results of the D cells showed that only a small number of dead cells were present in each group, indicating that the gelatin microspheres and IHH protein at a concentration of 25-300 ng / mL would not cause significant toxicity to the cells. The hMSC microspheres were in good condition and could be used to prepare biomimetic epiphyseal plates.

[0036] The self-assembly process of hMSC microspheres in the 100 ng / mL IHH group during the culture was documented by photographs, along with the finished product status of the four groups. The results are as follows: Figure 2 As shown.

[0037] Figure 2 The results showed that when hMSC microspheres were placed in chondrogenic induction medium for static incubation, the microspheres spontaneously gathered towards the center of the culture dish and gradually fused and assembled into a whole. Figure 2 The lower half shows the appearance of each group of products, which are translucent and elastic. The appearance of this self-assembled product is similar to cartilage. After characterization, this patent named it a biomimetic epiphyseal plate, especially the 100 ng / mL IHH group, which showed the best biomimetic performance. The entire induction process did not involve artificial centrifugation polymerization or the use of molds. The hMSC microspheres could spontaneously aggregate and fuse under static culture conditions.

[0038] (3) Immunofluorescence staining (SOX9 single staining and Ki67 / CD29 double staining) Remove frozen sections and allow them to reach room temperature. Wash three times with PBS for 5 minutes each time. Circle the tissue with a histochemical pen, cover the tissue with strong immunofluorescence permeation buffer, and permeate at room temperature for 10-15 minutes. Wash three times with PBS for 5 minutes each time. Block with immunofluorescence staining blocking solution at 37°C for 30 minutes to 1 hour, and wash once with PBS. Prepare CD29 / KI67 dilution buffer (SOX9 dilution ratio is 1:200) at a ratio of 1:100 / 1:200, add it to the tissue, and incubate overnight at 4°C. Wash three times with PBS for 5 minutes each time. Prepare the corresponding secondary antibody dilution buffer at a ratio of 1:200 (e.g., use Cy3 anti-mouse secondary antibody if CD29 is mouse-derived), add it to the tissue, and incubate at 37°C for 1 hour. Wash three times with PBS for 10 minutes each time. Mount with mounting buffer containing DAPI for anti-fluorescence quenching, observe and photograph under a fluorescence microscope. The results are as follows. Figure 3 , 4As shown.

[0039] SOX9 is one of the specific markers of chondrocytes. Uninduced hMSC microspheres do not express SOX9; if SOX9 is expressed, it indicates that these cells have been induced into chondrocytes. Figure 3 As shown in A and 3B, compared with the Control group, the proportion of SOX9 positive cells in each group was significantly increased after 16 days of stimulation with different concentrations of IHH protein, especially the induction efficiency of 100 ng / mL IHH protein, which reached about 58%. Figure 3 C and 3D analyses showed a higher number of SOX9-positive cells after 21 days of induction, but except for the Control group, the proportion of positive cells in the other groups (compared to 16 days) did not significantly increase. In summary, the proportion of positive cells in the Control group increased from 22% at 16 days to approximately 40% at 21 days, while 100 ng / mL IHH reached 58% at 16 days of induction. This indicates that 100 ng / mL IHH protein significantly shortens the chondrogenic induction cycle of hMSC microspheres, accelerating and promoting chondrogenic differentiation, thus saving time and economic costs and reducing the risk of operational contamination. The proportion of SOX9-positive cells in the 100 ng / mL IHH group did not change significantly between 16 and 21 days of induction, but the number of SOX9-positive cells increased significantly, suggesting that cells in this cycle are in a dynamic balance of proliferation and differentiation, showing some similarity to the cell state of the natural epiphyseal plate. Further experiments are needed to confirm this.

[0040] CD29 is a specific marker for hMSCs, while Ki67 can be used to detect cells with proliferative potential. Figure 4 The results showed that each group of biomimetic epiphyseal plates contained some undifferentiated chondrocytes, maintaining stem cell characteristics and proliferative potential. The biomimetic epiphyseal plates constructed using a static culture system could encapsulate some undifferentiated stem cells. This allowed external cells to access inducing factors and differentiate into chondrocytes, while the internal cells maintained a low concentration of inducing factors, thus preserving stem cell characteristics. In other words, the biomimetic epiphyseal plates simultaneously contained both chondrocytes and undifferentiated stem cells.

[0041] (4) Safranin O-Fast Green staining Frozen sections of the self-assembled products (biomimetic epiphyseal plates) incubated for 16 and 21 days were prepared. Each group of sections was warmed to 37°C, and fresh Weigert staining solution (Weigert A and B solutions prepared 1:1, freshly prepared and used) was added for 3-5 min, followed by rinsing with water. Differentiation was performed in acidic differentiation solution for 15 s, followed by rinsing with distilled water for 10 min. Fast Green staining was applied for 5 min. Sections were washed with a weak acid solution for 10-15 s and air-dried. Safranin staining solution was added for 5-10 min. Dehydration was performed using a gradient of ethanol (95% ethanol twice, 30 s each time; anhydrous ethanol twice, 1 min each time), followed by clearing with xylene (3 times, 2 min each time). The sections were then mounted with optical resin. The results are shown below. Figure 5 .

[0042] Figure 5 The results showed that IHH protein significantly promoted the deposition of cartilage matrix in the biomimetic epiphyseal plate. In particular, 100 ng / mL IHH showed a relatively uniform and extensive Safranin O positive staining area after 16 days of short-term induction, similar to the results of SOX9 staining.

[0043] (5) Scanning electron microscopy to detect microstructure The self-assembled products (biomimetic epiphyseal plates) incubated for 16 and 21 days were washed three times with pure water to remove surface impurities, transferred to 12-well plates, and fixed with 4% paraformaldehyde for 2 hours. After fixation, they were washed three times with PBS solution for 5 minutes each time. Residual moisture was absorbed, and the plates were frozen at -80°C for 2 hours. The 12-well plates were then opened and freeze-dried for 12 hours. Afterward, the plates were closed and sealed with one or two turns of sealing film to prevent moisture from affecting the scanning results. The samples were then placed in the gold sputtering chamber for gold sputtering. The samples were placed in the sample chamber according to the markings, adjusted to the correct position, and observed and photographed at 200x and 10000x magnification. The results are as follows. Figure 6 As shown.

[0044] Figure 6 As shown in Figure A, the surface of each group of biomimetic epiphyseal plates was covered with a large amount of granular material, and its microstructure was similar to that of rat rib epiphyseal plates reported in previous studies. Figure 6 (D). Due to the relatively smooth surface structure of gelatin microspheres ( Figure 6 (C), therefore, it can be determined that this particulate material originates from hMSCs loaded with gelatin microspheres. Extending the induction time to 21 days yielded the following results: Figure 6 As shown in Figure B, the sample surface stimulated with high concentrations of IHH exhibits a large amount of crystalline material, indicating that under long-term in vitro induction conditions, scaffolds treated with high concentrations of IHH pose a risk of mineral deposition. Based on these results, this invention will no longer consider using an epiphyseal plate construction system induced for 21 days.

[0045] (6) qPCR detection of gene expression in the bionic epiphyseal plate induced for 16 days RNA was extracted from the biomimetic epiphyseal plate according to the kit instructions and reverse transcribed into cDNA for later use. The cDNA from each group (0 ng / mL IHH group, 25 ng / mL IHH group, 100 ng / mL IHH group, 300 ng / mL IHH group) was diluted to appropriate concentrations. Using GAPDH as an internal control gene, a 10 μL reaction mixture was prepared: 5 μL TB Green, 1 μL cDNA, 0.4 μL F primer, 0.4 μL R primer, and 3.2 μL H2O. The program was set, appropriate parameters were set, and the reaction was started. The data results were obtained and analyzed as follows: Figure 7 As shown. Primers ( COL 2A1 / ACAN / PTHrP / COMP / COL10A1 / IHH / GAPDH (etc.) were synthesized by Shanghai Sangon Biotech, and the sequence is shown in the table below: Table 1 Primer Sequences

[0046] Figure 7 The results showed that IHH protein stimulation, especially in the 100 ng / mL IHH group, significantly increased the expression of chondrogenic genes, including ACAN , COL2A1 , COMP and PTHrP, Similar to the results of safranin O staining and SOX9 immunofluorescence, short-term stimulation with IHH protein did not reveal significant changes in hypertrophy genes (IHH and COL10A1), despite the potential for increased ossification risk due to chondrocyte hypertrophy and maturation. These results suggest that short-term treatment with IHH protein can enhance the chondrogenic efficiency of the biomimetic epiphyseal plate without the risk of hypertrophy and calcification, making it a promising alternative filler for epiphyseal plate defects.

[0047] (7) Nanoindentation test of biomimetic epiphyseal plate Nanoindentation testing can be used to evaluate the mechanical properties of natural or artificial samples. A probe with a stiffness of 48 N / m, a radius of 27.5 μm, and a factor value of 1.5 was selected. A confocal dish was placed on the stage. PBS was added along the middle of the black and transparent sections of the probe using a Pasteur dropper, ensuring the probe was also wetted to reduce error, and filling the confocal dish to approximately 2 / 3 full. Single-point indentation parameters were set; the optimal result was a straight line (red line in the upper right corner) after clicking "run experiment". Glue was added to the dish, and the biomimetic epiphyseal plate was fixed to the glue surface with tweezers to ensure no movement during testing (this step is crucial). Multi-point testing was selected, i.e., a 5×5 dot matrix, measuring 25 locations for each sample. The indentation interval was set to 50 μm, and the program was then run and data recorded. The results are shown below. Figure 8 As shown.

[0048] Figure 8 As can be seen in Figure A, the indentation curve is smooth, indicating that the test method of this invention is appropriate and the results are reliable. Figure 8 In B and C, the mechanical properties of samples pretreated with 300 ng / mL IHH showed a polarization, indicating that high concentrations of IHH stimulation may alter the arrangement or type of the extracellular matrix, leading to premature hardening of some sites in the sample and posing a risk of calcification when applied in vivo.

[0049] Existing technology also uses nanoindentation to test the mechanical properties of the tibial epiphyseal plate of young rabbits. Figure 8 The modulus of the biomimetic epiphyseal plate prepared in this invention is approximately 58 kPa (100 ng / mL IHH group), which indicates that the mechanical properties of the biomimetic epiphyseal plate are close to those of the natural epiphyseal plate. The matching of mechanical properties is conducive to the rapid adaptation of the biomimetic epiphyseal plate to the mechanical microenvironment of the defect site, which is of great significance for improving its therapeutic effect.

[0050] Experimental Example 2 Test Example 2 of this invention examined the in vivo application effect of the bionic epiphyseal plate. The specific steps are as follows: (1) Construction of epiphyseal defect model The biomimetic epiphyseal plate of Example 1 was used as the experimental group, and the biomimetic epiphyseal plates of 0 ng / mL IHH and 100 ng / mL IHH in Example 1 were used as the experimental group, while the Blank group was used as the control group.

[0051] Rabbits were anesthetized by injecting 3% sodium pentobarbital into the marginal ear vein at a dosage of 1 mL / kg. After confirming the absence of corneal reflex, the skin was prepared by shaving the hair within a 5 cm radius around the surgical site on the medial side of the right knee joint to expose the skin. Following surgical principles, the prepared area was disinfected with iodine-soaked cotton balls, three times from the inside out, and a drape was applied. Lidocaine was injected subcutaneously into the surgical area to create a 2 cm × 1 cm wheal, which was massaged until absorbed. The skin was incised, and the subcutaneous tissue was bluntly dissected. Hemostasis was achieved by applying pressure to the severed blood vessels using hemostatic forceps. The strip-shaped white tendon on the medial side of the knee joint was freed and fixed with a retractor, fully exposing the medial growth plate of the tibia in the right hind limb. The growth plate was destroyed with a pointed scalpel, creating a lesion band approximately 2 mm high, 5 mm wide, and 5 mm deep. A certain amount of implant material is taken and filled into the damaged strip. The muscle is then sutured to completely cover the defect site to prevent graft dislodgement (see...). Figure 9 The skin was sutured, and the rabbit was returned to the animal house for continued rearing after it had fully recovered. For three consecutive days post-surgery, the rabbits received intramuscular injections of penicillin at 20,000 units / kg body weight to prevent infection. Four weeks post-surgery, the experimental rabbits were euthanized using the air embolization method, and the injured and healthy tibias were removed and fixed in 4% paraformaldehyde solution.

[0052] (2) Evaluation of repair effect 4 weeks after surgery A. General Analysis Carefully remove the connective tissue, including muscles, tendons, and ligaments, attached to the tibia on both the injured and healthy sides, taking care not to damage the articular surfaces on both sides. First, draw a tangent line along the proximal articular surface of the injured tibia (right limb tibia). Then, draw a straight line from the center of the proximal and distal joints. The two lines intersect at the proximal end of the tibia, and the angle on the medial side (opposite to the fibula) is recorded as β. Using the healthy tibia as a reference, draw two lines in the same way, and record the angle at the corresponding position as α. Here, α is the normal angle, generally above 90 degrees. Since the epiphyseal plate injury is located on the medial side of the proximal tibia, the loss of growth potential on the medial side will inevitably lead to a lower medial articular surface, thus making the β angle smaller. Based on this, the difference between the α angle and the β angle is the angle difference between the two tibias, i.e., the angle of angular deformity. In addition, measure the length of the tibia on the injured and healthy sides separately using calipers, accurate to two decimal places. Epiphyseal plate injury, in principle, leads to epiphyseal plate dysfunction, thereby inducing limb shortening. Therefore, the difference between the length of the healthy tibia and the length of the injured tibia is the direct evaluation parameter for tibial shortening, i.e., the difference in tibial length. The results are as follows: Figure 10 As shown.

[0053] Figure 10 The images show that samples were taken 4 weeks after epiphyseal plate defect surgery. Grossly, almost all samples in the Blank group exhibited significant angular deformity, indicating that the epiphyseal plate defect model was successfully constructed and relatively stable. Biomimetic epiphyseal plates pretreated with 0 ng / mL IHH and 100 ng / mL IHH significantly alleviated angular deformity and tibial shortening deformity. Figure 10 C, Figure 10 (D), where the 100 ng / mL IHH group was superior to the 0 ng / mL IHH group.

[0054] B. Safranin O-Fast Green Staining Safranin O-Fix Green Staining: Four groups of paraffin sections were prepared, and each group was dewaxed and dehydrated. Fresh Weigert staining solution (prepared 1:1 with Weigert A and B solutions, used immediately after preparation) was added and stained for 3-5 min, followed by rinsing with water. Differentiation was performed with acidic differentiation solution for 15 s, followed by rinsing with distilled water for 10 min. Filtration with Fast Green staining solution was performed for 5 min. The sections were washed with a weak acid solution for 10-15 s and air-dried. Safranin staining solution was added for 5-10 min. Dehydration was performed using a gradient of ethanol (95% ethanol twice, 30 s each time; anhydrous ethanol twice, 1 min each time), followed by clearing with xylene (3 times, 2 min each time), and mounting with optical resin. The results are shown below. Figure 11 As shown. The principle of Safranin O-Fix Green staining is that cartilage matrix combines with the basic dye Safranin O to produce a red color, while bone matrix combines with the acidic dye Fast Green to produce a green or blue color.

[0055] Figure 11 The results showed that in the Blank group, the defect was filled with a large amount of blue new bone tissue, the amount of new bone was reduced in the 0 ng / mL LHIHH group, and the 100 ng / mL group had the least amount of new bone formation and was filled with a large amount of red-stained cartilage tissue.

[0056] C. Immunofluorescence staining: After dewaxing and dehydration, the tissue was circled with a histochemical pen, and pepsin was added to cover the tissue. The tissue was incubated at 37°C for 1 hour, followed by washing three times with PBS for 5 minutes each time. Immunofluorescence blocking solution was applied at 37°C for 30 minutes to 1 hour, followed by washing once with PBS. A 1:100 dilution of COL II was prepared and added to the tissue surface, incubated overnight at 4°C. The tissue was washed three times with PBS for 5 minutes each time. A 1:200 dilution of the corresponding secondary antibody was prepared (e.g., if COL II is mouse-derived, Cy3 anti-mouse secondary antibody was used), added to the tissue, and incubated at 37°C for 1 hour. The tissue was washed three times with PBS for 10 minutes each time. The slides were mounted with a DAPI-containing anti-fluorescence quenching mounting solution, observed and photographed under a fluorescence microscope. The results are as follows: Figure 11 As shown.

[0057] Figure 11 The results showed that the 100 ng / mL group had the widest positive area of ​​COL II in the defect site compared to the other two groups. Since COL II is a specific marker of chondrocytes, this indicates that the 100 ng / mL group had the best cartilage regeneration effect.

[0058] Comparative Example 1 Comparative Example 1 of this invention utilizes cartilage microspheres (conventional technique) and follows the method of Example 1 for induction. Unlike Example 1, Comparative Example 1 does not use gelatin microsphere microcarriers or IHH protein. The resulting product was subjected to Safranin O staining, SOX9 immunofluorescence staining, and TUNEL staining as described in Example 1. The results are as follows... Figure 12 As shown.

[0059] Figure 12 The A-value shows uneven staining of safranin O, with the positively stained areas mostly concentrated on the outer part of the cartilaginous spheres. Figure 12 Image B shows the staining results of SOX9, a specific marker for chondrocytes. The red SOX9 indicates that these cells have differentiated into chondrocytes. Chondrocytes are mostly distributed on the outer surface of the chondrocyte sphere, while the cell density in the center of the chondrocyte sphere is low, and almost no SOX9-positive cells are observed. Subsequently, TUNEL staining was used to observe cell apoptosis in the chondrocyte sphere. Figure 12 As shown in Figure C, numerous apoptotic cells were observed inside the chondrocytes. These results indicate that the chondrocyte induction system has a high cell density, allowing the outer surface of the chondrocytes to fully contact the culture medium, maintain their viability, and differentiate into chondrocytes. However, cells inside the chondrocytes struggle to obtain sufficient nutrients and undergo apoptosis. Therefore, the existing technology suffers from low efficiency and core necrosis, failing to overcome the problems of apoptosis and uneven induction.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic epiphyseal plate, characterized in that, Includes the following steps: (1) Add gelatin microsphere microcarriers to a low-adhesion culture plate, and seed human bone marrow mesenchymal stem cell suspension onto the gelatin microsphere microcarriers to obtain hMSC microspheres; (2) Add hMSC chondrogenic differentiation induction medium to a low adhesion culture plate containing hMSC microspheres, and add recombinant IHH protein solution for incubation and static culture to obtain a biomimetic epiphyseal plate.

2. The method for preparing the biomimetic epiphyseal plate according to claim 1, characterized in that, The concentration of the human bone marrow mesenchymal stem cell solution in step (1) is 2~4×10⁻⁶. 6 The number of gelatin microspheres is 80,000 to 100,000; the seeding volume of human bone marrow mesenchymal stem cell solution on each microcarrier is 200 to 400 μL.

3. The method for preparing the biomimetic epiphyseal plate according to claim 2, characterized in that, The concentration of the human bone marrow mesenchymal stem cell solution in step (1) is 2 × 10⁻⁶. 6 The number of gelatin microspheres on the microcarrier is 90,000; the seeding volume of human bone marrow mesenchymal stem cell solution on each microcarrier is 300 μL.

4. The method for preparing the biomimetic epiphyseal plate according to claim 1, characterized in that, The final concentration of IHH protein after adding the IHH protein solution in step (2) is 50~200 ng / mL.

5. The method for preparing the biomimetic epiphyseal plate according to claim 1, characterized in that, The concentration of the IHH protein solution in step (2) is 100 ng / mL.

6. The method for preparing the biomimetic epiphyseal plate according to claim 1, characterized in that, The static culture time in step (2) is 15 to 17 days.

7. The method for preparing the biomimetic epiphyseal plate according to claim 6, characterized in that, The static culture time in step (2) is 16 days. During static culture, the chondrogenic induction medium and IHH protein need to be replaced every 2 days.

8. A biomimetic epiphyseal plate prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the biomimetic epiphyseal plate of claim 8 in epiphyseal plate defect repair and articular cartilage repair scaffolds.

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