Bionic epiphyseal plate and preparation method and application thereof

Abstract

The invention provides a bionic epiphyseal plate and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The gelatin microspheres are used as cell carriers, the surface area is large, and cells can obtain sufficient nutrition support; the method comprises the following steps: by taking hMSC microspheres as a basic construction unit, carrying out static incubation under the stimulation of recombinant IHH protein, and driving the hMSC microspheres to spontaneously aggregate, fuse and self-assemble into the bionic epiphyseal plate of which internal cells are in a proliferation and differentiation balance state without depending on a mold and an external force condition. The cell state, matrix morphology and mechanical properties of the biomimetic epiphyseal plate are similar to those of a natural epiphyseal plate, the biomimetic epiphyseal plate has obvious supporting effect on cells and adaptability to mechanical microenvironment of injury sites, has dual functions of preventing bone bridges and promoting cartilage regeneration, and can be used as a candidate filler for repairing large-area epiphyseal plate defects. Compared with a traditional method for preparing a epiphyseal plate replacement scaffold, the method has higher cartilage formation induction efficiency, and the induction period is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a bionic epiphyseal plate and a preparation method and application thereof. Background Art

[0002] The epiphyseal plate is a wavy specialized cartilage tissue located between the secondary ossification center and the metaphysis, which drives bone growth. The epiphyseal plate chondrocytes are mechanically fragile and easily physically damaged. Due to the limited regenerative capacity of epiphyseal plate chondrocytes, they are easily replaced by bone tissue during the repair process to produce a "bone bridge", causing local growth stagnation, which in turn leads to serious complications such as angular deformity and limb length inequality.

[0003] At present, the commonly used clinical treatment method is bone bridge resection combined with material filling, and the fillers are mostly autologous fat, bone cement and some polymer materials. However, this inert filling mainly prevents the formation of bone bridges, and it is difficult to regenerate epiphyseal cartilage, which will eventually lead to uneven bilateral bone growth in the later stage. Therefore, the ideal epiphyseal repair strategy is to prevent bone bridges and regenerate cartilage, but traditional technology is currently difficult to meet both requirements.

[0004] At present, traditional technologies still have the following problems: (1) The use of stem cell spheres for chondrogenic induction culture has the problems of low chondrogenic induction efficiency and core necrosis; (2) Traditional chondrogenic induction culture schemes are mostly carried out using mature induction reagents, including transforming growth factor β3 and dexamethasone, which have a long induction cycle and poor efficiency; (3) Traditional microsphere culture mostly uses bioreactors for dynamic incubation, but dynamic culture may lead to reduced contact between cell microspheres and reduce the fusion ability of microspheres; after the microsphere incubation is completed, the microspheres are polymerized and shaped using molds or external forces, and the interaction effect between microspheres is not fully mobilized, and dynamic culture is not conducive to the chondrogenic differentiation of MSCs; (4) The effectiveness of traditional repair strategies is mostly verified using central or small-area marginal epiphyseal defect models of rats or rabbits. The preparation process of this model is simple and the repair difficulty is relatively low. It is difficult to reproduce and verify the large-area epiphyseal defect cases often encountered in clinical practice, and its guiding value for future translational applications is not high.

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

[0006] Therefore, how to use developmental engineering technology to improve the survival rate of induced cells and the efficiency of chondrogenic differentiation, shorten the induction cycle, and improve the repair effect of bionic epiphyseal plates is of great significance for the repair of high-difficulty epiphyseal plate injuries. Summary of the invention

[0007] The purpose of the present invention is to provide a bionic epiphyseal plate and its preparation method and application, which can prepare a bionic epiphyseal plate with the dual functions of preventing bone bridges and promoting cartilage regeneration, and is expected to be one of the candidate fillers for repairing epiphyseal defects.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a bionic epiphyseal plate, which is obtained by inducing human bone marrow mesenchymal stem cells through recombinant IHH protein.

[0010] The present invention also provides a method for preparing a bionic epiphyseal plate, comprising the following steps:

[0011] (1) adding a microslide containing gelatin microspheres to a low-adhesion culture plate, and inoculating a human bone marrow mesenchymal stem cell suspension on the microslide containing gelatin microspheres to obtain hMSC microspheres;

[0012] (2) Add hMSC chondrogenic differentiation medium to a low-adhesion culture plate containing hMSC microspheres, and simultaneously add a recombinant IHH protein solution for incubation and static culture to obtain a bionic epiphyseal plate.

[0013] Preferably, the concentration of the human bone marrow mesenchymal stem cell solution in step (1) is 2 to 4×10 6 / mL; the number of the gelatin microspheres is 80,000 to 100,000; the inoculation volume of the human bone marrow mesenchymal stem cell solution on each microslide is 200 to 400 μL;

[0014] Preferably, the concentration of the human bone marrow mesenchymal stem cell solution in step (1) is 2×10 6 / mL; the number of gelatin microspheres on the microslide is 90,000; the inoculation volume of the human bone marrow mesenchymal stem cell solution on each microslide is 300 μL.

[0015] Preferably, the concentration of the IHH protein solution in step (2) is 50 to 200 ng / mL.

[0016] Preferably, the concentration of the IHH protein solution in step (2) is 100 ng / mL.

[0017] Preferably, the static culture time in step (2) is 15 to 17 days.

[0018] Preferably, the static culture time in step (2) is 16 days, and the chondrogenic induction medium and IHH protein need to be replaced every 2 days during the static culture.

[0019] The present invention also provides a bionic epiphyseal plate or the bionic epiphyseal plate prepared by the preparation method of the bionic epiphyseal plate for use in repairing epiphyseal plate defects and in a novel articular cartilage repairing bracket.

[0020] The beneficial effects of the bionic epiphyseal plate of the present invention compared with the prior art are as follows:

[0021] (1) The present invention uses gelatin microspheres as cell carriers, which have a large surface area and can provide sufficient nutritional support for cells; hMSC microspheres are used as the basic construction unit, and static incubation is performed under the stimulation of recombinant IHH protein to drive the hMSC microspheres to spontaneously aggregate, fuse and self-assemble into a bionic epiphyseal plate, and the internal cells are in a state of equilibrium between proliferation and differentiation. Compared with traditional preparation technology, this static preparation system has a higher chondrogenic induction efficiency and shortens the induction period.

[0022] (2) Unlike the prior art, the present invention does not rely on molds and external forces, but aims to enhance the activity of cells themselves, and has stronger adaptability and vitality than traditional epiphyseal plate replacement scaffolds.

[0023] (3) The bionic epiphyseal plate prepared by the present invention contains cells in two states at the same time, namely stem cells in the proliferation cycle and chondrocytes that have differentiated. This multicellular state is similar to the natural epiphyseal plate, while the current epiphyseal plate replacement scaffolds only contain a single cell type. Therefore, this multicellular differentiation order is obviously advanced.

[0024] (4) The present invention is the first to use a short-term (16 days) IHH protein static incubation system 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 technologies, it saves experimental costs and manpower investment, while reducing the uncontrollable risks brought by long-term in vitro operations.

[0025] (5) The cell state, matrix morphology and mechanical properties of the bionic epiphyseal plate provided by the present invention are similar to those of the natural epiphyseal plate. Compared with the traditional epiphyseal plate replacement scaffold, it has obvious advantages in supporting cells and adapting to the mechanical microenvironment of the injury site, and is expected to be one of the candidate fillers for repairing large-area epiphyseal defects.

[0026] (6) The bionic epiphyseal plate provided by the present invention effectively prevents angular deformity and tibial shortening caused by epiphyseal cartilage defects. Compared with the published literature, the bionic epiphyseal plate is used in one of the most serious animal models in the field of epiphyseal repair, and it also has the dual functions of preventing bone bridges and promoting cartilage regeneration, filling the technical gap of large-area epiphyseal defects that are difficult to repair, and is expected to provide a candidate strategy for the repair of epiphyseal injuries in children. It also has a profound impact on the promotion of bionic epiphyseal plates in other application scenarios, including injuries to the epiphysis, backbone, spine, joints, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Cell characterization of hMSC microspheres in Experimental Example 1, where A is live-dead staining, green is live cells, and red is dead cells; B is Tunel staining, blue is cell nuclei, and green is Tunel-positive apoptotic cells; C is the live-dead cell ratio; D is the apoptotic cell ratio;

[0029] Figure 2 The self-assembly of hMSC microspheres in Experimental Example 1, wherein the upper part is the process of inducing the self-assembly of hMSC microspheres; the lower part is the appearance of the self-assembly product, i.e., the bionic epiphyseal plate;

[0030] Figure 3 The chondrogenic differentiation of the bionic epiphyseal plate in Experimental Example 1, where A is the SOX9 staining image after 16 days of induction; B is the SOX9 statistical result after 16 days of induction; C is the SOX9 staining image after 21 days of induction; D is the SOX9 statistical result after 21 days of induction. Red represents SOX9 positive staining, and blue represents cell nuclei.

[0031] Figure 4 The cell status in the bionic epiphyseal plate induced for 16 days in Experimental Example 1, where the blue one is the cell nucleus, the red one is Ki67, representing the proliferating cells, and the green one is CD29, representing the hMSC that has not undergone chondrogenic differentiation;

[0032] Figure 5 The cartilage matrix deposition in the bionic epiphyseal plate of Experimental Example 1, wherein A is the result of Safranin O-Fast Green staining after 16 days of induction; B is the result of Safranin O-Fast Green staining after 21 days of induction; C is the statistical result of the Safranin O-positive staining area after 16 days and 21 days of induction, wherein the red color is the Safranin O-positive area, representing the cartilage matrix;

[0033] Figure 6 The microscopic morphology of the bionic epiphyseal plate and gelatin microspheres in Experimental Example 1, where A is an electron microscope image after 16 days of induction; B is an electron microscope image after 21 days of induction; C is a surface electron microscope image of gelatin microspheres without cells; D is an image of the rat rib epiphyseal plate reported in the literature

[0034] Figure 7The gene expression of the sample induced for 16 days in Experimental Example 1, wherein A is ACAN, B is COL2A1, C is COMP, and D is PTHrP, which are all chondrocyte-specific markers; E is IHH, and F is COL10A1, which are all markers of chondrocyte hypertrophy and maturation;

[0035] Figure 8 The mechanical properties of the bionic epiphyseal plate of Experimental Example 1, where A is an example of an indentation curve (100 ng / mL IHH group); B is a heat map of the mechanical properties of each group of samples; C is a statistical result of the elastic modulus of each group of samples; D is the elastic modulus data of the tibial epiphyseal plate of young rabbits reported in the literature;

[0036] Fig. 9 Schematic diagram of the in vivo efficacy verification scheme of the bionic epiphyseal plate in Experimental Example 2, wherein A is the experimental grouping and surgical scheme; B is the epiphyseal defect modeling process;

[0037] Fig.10 This is the repair effect of the bionic epiphyseal plate on large epiphyseal defects in Experiment 2, where A is the appearance of the tibia sample 4 weeks after epiphysis resection, with a total of 7 biological replicates, the left side of each group is a healthy tibia, and the right side is a damaged tibia; B is a schematic diagram of the calculation of angular deformity, the left side is the autologous healthy control, and the right side is the damaged tibia; C is the statistical result of angular deformity; D is the statistical result of tibia length

[0038] Fig.11 This is the experimental example 2: the bionic epiphyseal plate inhibits bone bridges and promotes cartilage regeneration, where A is the Safranin O-fast green staining 4 weeks after surgery, red is cartilage tissue, green is bone tissue; B is COLII immunofluorescence staining, red is positive, indicating cartilage matrix deposition;

[0039] Fig.12 This is comparative example 1, in which the "chondrocyte spheroid method" was used to induce hMSC to differentiate into cartilage, wherein A is Safranin O staining, and the red staining indicates the cartilage matrix deposition area; B is SOX9 immunofluorescence staining, and the red color indicates differentiated chondrocytes; and C is Tunel staining, and the green color indicates cells that have undergone apoptosis. DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below, and the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. If specific techniques or conditions are not specified in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0041] The present invention prepares a bionic epiphyseal plate and performs comprehensive characterization on it. Then, young large-eared white rabbits are selected as research subjects, and a large epiphyseal defect with a width of 5 mm, a depth of 5 mm, and a height of 2 mm is made on the inner side of the proximal end of the right tibia. A difficult injury model is constructed to verify the effectiveness of the bionic epiphyseal plate. Experiments have shown that the bionic epiphyseal plate of the present application is used in one of the most serious animal models in the field of epiphyseal repair, and has the dual functions of preventing bone bridges and promoting cartilage regeneration, filling the technical gap that large epiphyseal defects are difficult to repair.

[0042] Example 1

[0043] In Example 1 of the present invention, a bionic epiphyseal plate is prepared, and the specific method is as follows:

[0044] (1) Preparation of hMSC microspheres

[0045] A. Culture of hMSCs:

[0046] Human bone marrow mesenchymal stem cells (hMSC, OriCell) were cultured in an incubator at 37°C and 5% CO2. When the cells were about 80% confluent, the culture medium was aspirated and washed twice with PBS solution, 1 mL of 0.05% trypsin was added for digestion and resuspending, and then passaged at a ratio of 1:3 and continued to be cultured in a 37°C / 5% CO2 incubator. When the hMSC expanded to a confluence rate of about 85%, the cells were digested and the density was adjusted to 2×10 6 / mL to obtain a cell resuspension for later use.

[0047] B. Composite of hMSC and gelatin microspheres:

[0048] A microslide (about 90,000 gelatin microspheres, China Huakan Biotechnology Co., Ltd.) was added to each well of a low-adhesion six-well plate, 300 μL of the mixed cell resuspension was aspirated and slowly dripped onto the microslide, and then 2 mL of PBS solution was added to the pores of the culture dish to prevent the evaporation of the culture medium and affect the cell state, and then placed in the incubator for 2 hours. After 2 hours, the hMSC and the gelatin microspheres were completely attached, and hMSC microspheres were obtained.

[0049] (2) Preparation of bionic epiphyseal plate

[0050] A. Chondrogenic differentiation of hMSC microspheres

[0051] 10 μg of IHH (Indian hedgehog) protein powder was centrifuged at 3000 rpm for 3 min. In a biosafety cabinet, 1 mL of sterile PBS solution was added to the IHH protein packaging tube to prepare a 10 μg / mL IHH stock solution, which was divided into 50 200 μL EP tubes and stored in a -20 degree refrigerator for later use.

[0052] Prepare hMSC chondrogenic differentiation medium (OriCell, China) under light-proof conditions, slowly add 5 mL of the medium into the hMSC microsphere culture plate at each well, and gently stir the hMSC microspheres with a 200 μL pipette tip to fully separate and evenly distribute them in the culture plate without obvious clumps.

[0053] B. Preparation of bionic epiphyseal plate based on static self-assembly

[0054] In the chondrogenic induction medium of hMSC microspheres, 10 μg / mL of IHH stock solution was added to the concentration of IHH in each well to 100 ng / mL, and the cells were placed in a 37-degree incubator for incubation. The static culture state was maintained for 16 days, and the chondrogenic induction medium and IHH protein were replaced every 2 days. This culture system does not use molds or other external forces (such as centrifugation). The low-adhesion six-well plate is used to prevent the hMSC microspheres from settling and adhering to the wall. The hMSC microspheres statically suspended in the culture medium gradually fuse and assemble into a whole as the culture time increases. After 16 days of incubation in the incubator, they are fully formed, translucent and elastic, which is the bionic epiphyseal plate.

[0055] Test Example 1

[0056] Experimental Example 1 of the present invention comprehensively characterized the bionic epiphyseal plate, and the specific steps are as follows:

[0057] Four groups of bionic epiphyseal plates were prepared by the method of Example 1. The difference between these four groups of bionic epiphyseal plates and Example 1 was that the final concentration of IHH protein in each group was 0 ng / mL (Control, functional control group of IHH protein), 25 ng / mL, 100 ng / mL, and 300 ng / mL, respectively. Subsequent experiments were carried out on the obtained four groups of bionic epiphyseal plates.

[0058] (1) Live and dead staining of hMSC microspheres

[0059] The live-dead staining kit was purchased from Huakan Biotechnology with the item number R002-50. In the preparation process of the bionic epiphyseal plate based on static self-assembly in step (2) of Example 1, the live-dead staining of cells was performed 72 hours after the addition of the IHH solution. Use a pipette tip to draw a mixture of 25-50 μL of hMSC microspheres and culture medium in four groups of 0 ng / mL, 25 ng / mL, 100 ng / mL, and 300 ng / mL, place it in a 96-well plate, let it stand, and discard the supernatant after the microspheres settle. Add 100 μL of the staining working solution and mix well, and incubate at 37°C in the dark for about 1 hour. After incubation, discard the supernatant, draw 1 mL of PBS to wash, repeat this step twice, and add an appropriate amount of PBS solution to keep the microspheres in a moist environment. Then observe and take pictures under a fluorescence microscope. The results are as follows. Figure 1 shown.

[0060] (2) Cell apoptosis staining

[0061] Before the bionic epiphyseal plate is applied, its internal cell viability needs to be tested. Therefore, frozen sections of each group induced for 14 days were taken, and after rewarming, proteinase K was diluted to 20 μg / mL with P0106 ​​immunostaining washing solution, 37°C, 30 minutes. PBS was washed 3 times, 5 minutes each time. Staining working solution (TdT enzyme: fluorescent labeling solution = 1:9) was added and incubated at 37°C for 1 hour. PBS was washed 3 times, 5 minutes each time. The sections were sealed with DAPI anti-fluorescence quenching sealing solution, and observed and photographed with a fluorescence microscope. The results are shown as follows: Figure 1 shown.

[0062] Figure 1 It was shown that after hMSCs were combined with gelatin microspheres, hMSCs adhered to the pore surface inside the microspheres, e.g. Figure 1 As shown in A, most hMSCs in the microspheres are in good survival condition and are green, and only a small number of red hMSCs are dead, as shown by the red arrows. Figure 1 The cell live-dead staining statistics in C show that green live cells account for more than 93% of the total cell number. Figure 1 Medium B and Figure 1 The cell apoptosis staining results in D showed that there were only a small number of dead cells in each group, indicating that gelatin microspheres and IHH protein at a concentration of 25-300 ng / mL did not cause obvious toxicity to cells. The hMSC microspheres were in good condition and could be used to prepare bionic epiphyseal plates.

[0063] Photographs were taken to record the self-assembly process of hMSC microspheres in the 100 ng / mL IHH group during culture, as well as the status of the four groups of finished products. The results are shown in Figure 2 shown.

[0064] Figure 2 It was shown that when hMSC microspheres were placed in a chondrogenic induction medium for static incubation, the microspheres spontaneously gathered toward the center of the culture dish and gradually fused and assembled into a whole. Figure 2 The lower part 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 characterizing it, this patent named it as a bionic epiphyseal plate, especially the 100ng / mL IHH group has the best bionic performance. The entire induction process does not involve artificial centrifugal polymerization, nor does it use molds. The hMSC microspheres can spontaneously aggregate and fuse under static culture conditions.

[0065] (3) Immunofluorescence staining (SOX9 single staining and Ki67 / CD29 double staining)

[0066] Remove the frozen sections and return to room temperature, wash with PBS three times, 5 minutes each time. Circle the tissue with a tissue pen, add strong immunofluorescence permeabilization solution to cover the tissue, permeabilize at room temperature for 10-15 minutes, and wash with PBS three times, 5 minutes each time. Block with immunofluorescence staining blocking solution at 37°C for 30min-1h, and wash once with PBS. Prepare CD29 / KI67 dilution solution at a ratio of 1:100 / 1:200 (the dilution ratio of SOX9 is 1:200), add it to the tissue, and incubate at 4°C overnight. Wash with PBS three times, 5 minutes each time. Prepare the corresponding secondary antibody dilution solution at a ratio of 1:200 (if CD29 is mouse-derived, use Cy3 anti-mouse secondary antibody), add it to the tissue, and incubate at 37°C for 1h. Wash with PBS three times, 10 minutes each time. Seal the slice with DAPI anti-fluorescence quenching sealing solution, observe and take pictures with a fluorescence microscope, and the results are as follows. Figure 3 , 4 shown.

[0067] SOX9 is one of the specific markers of chondrocytes. Uninduced hMSC microspheres do not express SOX9. If SOX9 is expressed, it means 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 increased significantly after stimulation with different concentrations of IHH protein for 16 days, especially the induction efficiency of 100 ng / mL IHH protein was the highest, reaching about 58%. Figure 3 C and 3D show that the number of SOX9-positive cells is greater at 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 increase significantly. In summary, the proportion of positive cells in the Control group increased from 22% at 16 days to about 40% at 21 days, while 100ng / mL IHH reached 58% at 16 days of induction, indicating that 100ng / mL IHH protein significantly shortened the chondrogenic induction cycle of hMSC microspheres, accelerated and promoted the chondrogenic differentiation of hMSC microspheres, thereby saving time and economic costs and reducing the risk of operation-induced contamination. The proportion of SOX9-positive cells in the 100ng / mL IHH group did not change significantly at 16 and 21 days of induction, but the number of SOX9-positive cells increased significantly, suggesting that the cells in this cycle are in a dynamic balance of proliferation and differentiation, which is partially similar to the cell state of the natural epiphyseal plate, and further experimental evidence will be provided in the future.

[0068] CD29 is a specific marker for hMSCs, and Ki67 can be used to detect cells with proliferation potential. Figure 4It shows that some cells in each group of bionic epiphyseal plates have not differentiated into chondrocytes, and they maintain their stemness and have proliferation potential. The bionic epiphyseal plate constructed by the static culture system can encapsulate some undifferentiated stem cells, so that the external cells can be exposed to the induction factors to differentiate into chondrocytes, while the internal cells have a low concentration of induction factors and continue to maintain their stemness. That is, both chondrocytes and undifferentiated stem cells exist in the bionic epiphyseal plate.

[0069] (4) Safranin O-Fast Green Staining

[0070] The self-assembled products (bionic epiphyseal plates) incubated in vitro for 16 days and 21 days were frozen and sectioned. The sections of each group were rewarmed to 37 degrees Celsius, and fresh Weigert stain solution (Weigert A and B were prepared in a ratio of 1:1, prepared immediately before use) was added for staining for 3-5 minutes, and then washed with water. Differentiation was performed with acidic differentiation solution for 15 seconds, and the sections were washed with distilled water for 10 minutes. Fast green staining solution was used for 5 minutes. The sections were washed with weak acid solution for 10-15 seconds and dried. Safranin staining solution was added for 5-10 minutes. Dehydration was performed with gradient ethanol (95% alcohol twice, 30 seconds each time; anhydrous ethanol twice, 1 minute each time), transparentization was performed with xylene (3 times, 2 minutes each time), and optical resin was used to seal the sections. The results are shown in the figure. Figure 5 .

[0071] Figure 5 The results showed that IHH protein significantly promoted the deposition of cartilage matrix of bionic epiphyseal plate, especially 100ng / mL IHH had a more uniform and extensive Safranin O positive staining area after short-term induction for 16 days, which was similar to the SOX9 staining results.

[0072] (5) Scanning electron microscope to detect microstructure

[0073] The self-assembled products (bionic epiphyseal plates) incubated in vitro for 16 days and 21 days were washed three times with pure water to remove surface debris, transferred to a 12-well plate and fixed with 4% paraformaldehyde for 2 hours. After fixation, they were washed three times with PBS solution, each time for 5 minutes. The residual moisture was absorbed and placed in a -80 degree refrigerator for 2 hours. The lid of the 12-well plate was opened and freeze-dried in a freeze dryer for 12 hours. After that, the lid was covered and wrapped with one or two layers of sealing film to prevent moisture from affecting the scanning results. The samples were placed in the gold spraying chamber for gold spraying. The samples were placed in the sample chamber according to the mark, adjusted to find the position, observed and photographed at 200 times and 10,000 times the field of view, respectively. The results are as follows Figure 6 shown.

[0074] Figure 6 A in the middle shows that the surface of each group of bionic epiphyseal plates is covered with a large amount of granular substances, and its microscopic morphology is similar to that of rat rib epiphyseal plates reported in previous studies ( Figure 6 D). Since the surface structure of gelatin microspheres is relatively smooth ( Figure 6C), so it can be determined that the granular material is derived from hMSC loaded with gelatin microspheres. The induction time was extended to 21 days. Figure 6 As shown in B, a large amount of crystalline substances exist on the surface of the sample stimulated by high concentration of IHH, indicating that under long-term in vitro induction conditions, the scaffold treated with high concentration of IHH has the risk of mineral deposition. Based on the above results, the present invention will no longer consider the epiphyseal plate construction system with 21 days of induction.

[0075] (6) qPCR detection of gene expression in the bionic epiphyseal plate after 16 days of induction

[0076] Extract RNA from the biomimetic epiphyseal plate according to the kit instructions and reverse transcribe it into cDNA for later use. Dilute each group of cDNA (0ng / mL IHH group, 25ng / mL IHH group, 100ng / mL IHH group, 300ng / mL IHH group) to an appropriate concentration. Use GAPDH as the internal reference gene and prepare a 10μL reaction system: 5μL TB Green, 1μL cDNA, 0.4μL F primer, 0.4μL R primer, 3.2μL H2O. Set the program, set the appropriate parameters, start the run, and obtain and analyze the data results as shown in Figure 7 Primers (COL 2A1 / ACAN / PTHrP / COMP / COL10A1 / IHH / GAPDH, etc.) were synthesized by Shanghai Bioengineering, and the sequences are shown in the table below:

[0077] Table 1 Primer sequences

[0078]

[0079] Figure 7 The results showed that IHH protein stimulation, especially the 100ng / mL IHH group, significantly increased the expression of chondrogenesis-related genes, including ACAN, COL2A1, COMP, and PTHrP, which was similar to the results of Safranin O staining and SOX9 immunofluorescence. Since the risk of ossification may increase after chondrocyte hypertrophy and maturation, no significant changes in hypertrophy genes (IHH and COL10A1) were observed after short-term stimulation of IHH protein. These results indicate that short-term treatment with IHH protein can improve the chondrogenic efficiency of bionic epiphyseal plates without the risk of hypertrophy and calcification, and is expected to be used as an alternative filler for epiphyseal defects.

[0080] (7) Nanoindentation test of bionic epiphyseal plate

[0081] Nanoindentation testing can be used to evaluate the mechanical properties of natural or artificial samples. Select a probe with a stiffness of 48N / m, a radius of 27.5μm, and a factor value of 1.5, place a confocal dish on the stage, use a Pasteur pipette to draw PBS and drip it along the middle of the black and transparent part of the probe, making sure that the probe is also in a wet state to reduce errors, and add it to about 2 / 3 of the water level of the confocal dish. Set the single-point indentation parameters, click "run experiment", and the red line in the upper right corner is a straight line for the best result. Add glue to the dish, and use tweezers to fix the bionic epiphyseal plate to the glue surface to ensure that there is no movement during the test (this step is especially critical). Select multi-point testing, that is, a 5×5 dot matrix, and measure 25 positions of each sample. Set the indentation interval to 50μm, then start running the program and record the data. The results are as follows Figure 8 shown.

[0082] Figure 8 It can be seen in Figure A that the indentation curve is smooth, indicating that the testing method of the present invention is appropriate and the result is reliable. Figure 8 Figures B and C in Figure 8 show that the mechanical properties of samples pretreated with 300 ng / mL IHH are polarized, indicating that high-concentration IHH stimulation may change the arrangement or type of the extracellular matrix, thereby causing premature hardening of some sites of the sample and a risk of calcification when used in vivo.

[0083] The existing technology also uses nanoindentation to test the mechanical properties of the tibial epiphyseal plate of young rabbits ( Figure 8 D), which proves that the lower limit of its modulus is about 60 KPa, while the modulus of the bionic epiphyseal plate prepared by the present invention is about 58 KPa (100 ng / mL IHH group), indicating that the mechanical properties of the bionic epiphyseal plate are close to those of the natural epiphyseal plate. The matching of mechanical properties is conducive to the rapid adaptation of the bionic epiphyseal plate to the mechanical microenvironment of the defect, which is of great significance for improving its therapeutic effect.

[0084] Test Example 2

[0085] Experimental Example 2 of the present invention detects the in vivo application effect of the bionic epiphyseal plate, and the specific steps are as follows:

[0086] (1) Construction of epiphyseal defect model

[0087] The bionic epiphyseal plate of Example 1 was used as the experimental group, and the Control group of 0 ng / mL IHH of Experimental Example 1 was used as the Blank control group.

[0088] Rabbits were anesthetized by injecting 3% sodium pentobarbital into the ear vein, and the anesthetic was administered at a dose of 1 mL / kg. After checking that the rabbit had no corneal reflex, skin preparation was performed, and the hair within a radius of 5 cm around the surgical site on the inside of the right knee joint of the rabbit was shaved to expose the skin of the surgical site. According to surgical principles, the skin preparation area was disinfected with iodine cotton balls. From the inside to the outside, disinfection was performed 3 times, and a hole towel was laid. Lidocaine was injected subcutaneously in the surgical area to produce a 2cm×1cm skin mound, and massaged until the lidocaine was absorbed. The skin was incised, the subcutaneous tissue was bluntly separated, and the severed blood vessels were compressed and hemostatic forceps were used to stop bleeding. The white strip tendon on the inside of the knee joint was freed and fixed with a retractor to fully expose the medial tibial growth plate of the rabbit's right lower limb. The growth plate was destroyed with a pointed scalpel, and a lesion strip of about 2 mm high, 5 mm wide, and 5 mm deep was formed along the growth plate. Take a certain amount of implant material, fill it into the damaged strip, suture the muscle to completely cover the defect site, and prevent the graft from falling out (see Fig. 9 ). The skin was sutured and the rabbit was sent back to the animal room for further breeding after it fully recovered. Penicillin 20,000 units / kg body weight was injected intramuscularly for 3 consecutive days after surgery to prevent infection. Four weeks after surgery, the experimental rabbits were killed by air embolism, and the injured and healthy tibiae were removed and fixed in 4% paraformaldehyde.

[0089] (2) Evaluation of repair effect 4 weeks after surgery

[0090] A. General Analysis

[0091] Carefully remove the connective tissues such as muscles, tendons and ligaments attached to the injured and healthy tibiae, and be careful not to injure the articular surfaces on both sides. First, draw a tangent along the proximal articular surface of the injured tibia (right limb tibia), and then draw a straight line from the proximal and distal joint centers. The two straight 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 of the autologous body as a control, draw two lines in the same way, and the angle at the corresponding position is recorded as α. Among them, α is the normal angle, which is generally above 90 degrees. Since the epiphyseal plate injury is located on the inner side of the proximal tibia, the loss of growth potential on the medial side will inevitably lead to a lower medial articular surface, thereby reducing the β angle. Based on this, the α angle minus the β angle is the angle difference of the tibiae on both sides, that is, the angle of angular deformity. In addition, use a vernier caliper to measure the length of the tibia on the injured and healthy sides, respectively, accurate to two decimal places. In principle, epiphyseal plate injury leads to epiphyseal plate function loss, which in turn induces limb shortening. Therefore, the length of the healthy side tibia minus the length of the injured side tibia is the intuitive evaluation parameter of tibial shortening, that is, the difference in tibial length. Fig.10 shown.

[0092] Fig.10The epiphyseal defect was obtained 4 weeks after surgery. It can be seen with the naked eye that almost all samples in the Blank group had obvious angular deformity, indicating that the epiphyseal defect model was successfully constructed and relatively stable. The bionic epiphyseal plates pretreated with 0ng / mL IHH and 100ng / mL IHH significantly alleviated the angular deformity and tibial shortening deformity ( Fig.10 C, Fig.10 D), in which the 100 ng / mL IHH group was superior to the 0 ng / mL IHH group.

[0093] B. Safranin O-fast green staining

[0094] Safranin O-Fast Green staining: Prepare 4 groups of paraffin sections separately, dewax and dehydrate the paraffin sections of each group. Add fresh Weigert stain solution (Weigert A solution and B solution are prepared in a ratio of 1:1, and are prepared immediately before use) to stain for 3-5 minutes, and wash with water. Differentiate with acidic differentiation solution for 15 seconds, and wash with distilled water for 10 minutes. Dye with Fast Green stain solution for 5 minutes. Wash the sections with weak acid solution for 10-15 seconds and dry them. Add safranin stain solution for 5-10 minutes. Dehydrate with gradient ethanol (95% alcohol twice, 30 seconds each time; anhydrous ethanol twice, 1 minute each time), make it transparent with xylene (3 times, 2 minutes each time), and seal with optical resin. The results are as follows. Fig.11 The principle of Safranin O-Fast Green staining is that the cartilage matrix combines with the basic dye Safranin O to show red, and the bone matrix combines with the acidic dye Fast Green to show green or blue.

[0095] Fig.11 Middle A shows that the defect site in the Blank group was filled with a large amount of blue new bone tissue, the amount of new bone in the 0ng / mL IHH group was reduced, and the 100ng / mL group had the least new bone formation and was filled with a large amount of red-stained cartilage tissue.

[0096] C. Immunofluorescence staining: Take the dewaxed and dehydrated sections, circle the tissue with a histochemical pen, add pepsin to cover the tissue, incubate at 37°C for 1h, and wash 3 times with PBS, 5 minutes each time. Block with immunofluorescence staining blocking solution at 37°C for 30min-1h, and wash once with PBS. Prepare COLⅡ dilution solution at a ratio of 1:100, add it to the tissue surface, and incubate at 4°C overnight. Wash 3 times with PBS, 5 minutes each time. Prepare the corresponding secondary antibody dilution solution at a ratio of 1:200 (if COLⅡ is mouse-derived, use Cy3 anti-mouse secondary antibody), add it to the tissue, and incubate at 37°C for 1h. Wash 3 times with PBS, 10 minutes each time. Seal the slice with DAPI anti-fluorescence quenching sealing solution, observe and take pictures with a fluorescence microscope, and the results are as follows: Fig.11 shown.

[0097] Fig.11Middle B shows that the COL II-positive area at the defect site in the 100 ng / mL group was the largest compared to the other two groups. Since COL II is a specific marker for chondrocytes, this indicates that the 100 ng / mL group had the best cartilage regeneration effect.

[0098] Comparative Example 1

[0099] Comparative Example 1 of the present invention uses chondrocytes (conventional technology) to induce the method of Example 1. The difference from Example 1 is that Comparative Example 1 does not use gelatin microsphere microslides and IHH protein. The obtained product is subjected to safranin O staining, SOX9 immunofluorescence staining and Tunel staining in Experimental Example 1. The results are as follows: Fig.12 shown.

[0100] Fig.12 Middle A shows that Safranin O staining is uneven, and the positive staining area is mostly concentrated on the outside of the chondrocyte. Fig.12 Figure B shows the staining result of SOX9, a specific marker of chondrocytes. The red SOX9 indicates that these cells have differentiated into chondrocytes. It can be seen that chondrocytes are mostly distributed outside the chondrocytes, while the cell density in the center of the chondrocytes is low, and there are almost no SOX9-positive cells. Tunel staining was then used to observe the cell apoptosis of the chondrocytes. Fig.12 As shown in C, there are a large number of apoptotic cells inside the chondrocytes. The above results show that the cell density of the chondrocyte induction system is high, and the outside of the chondrocytes can fully contact the culture medium to maintain vitality and differentiate into chondrocytes, while the cells inside the chondrocytes are difficult to obtain sufficient nutrition and apoptosis occurs. It can be seen that the existing technology has the disadvantages of low efficiency and core necrosis, and cannot overcome the problems of cell apoptosis and uneven induction.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bionic epiphyseal plate, characterized in that: The bionic epiphyseal plate is obtained by inducing human bone marrow mesenchymal stem cells through recombinant IHH protein.

2. A method for preparing the bionic epiphyseal plate according to claim 1, characterized in that: The steps include: (1) adding a gelatin microsphere microslide to a low-adhesion culture plate, and inoculating a human bone marrow mesenchymal stem cell suspension on the gelatin microsphere microslide to obtain hMSC microspheres; (2) Add hMSC chondrogenic differentiation medium to a low-adhesion culture plate containing hMSC microspheres, and simultaneously add a recombinant IHH protein solution for incubation and static culture to obtain a bionic epiphyseal plate.

3. The method for preparing the bionic 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 to 4×10 6 / mL; the number of the gelatin microspheres is 80,000 to 100,000; and the inoculation volume of the human bone marrow mesenchymal stem cell solution on each microslide is 200 to 400 μL.

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

5. The method for preparing the bionic epiphyseal plate according to claim 2, characterized in that: After the IHH protein solution is added in step (2), the final concentration of the IHH protein is 50 to 200 ng / mL.

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

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

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

9. Use of the bionic epiphyseal plate according to claim 1 or the bionic epiphyseal plate prepared by the preparation method of the bionic epiphyseal plate according to any one of claims 2 to 8 in repairing epiphyseal defects and novel articular cartilage repair scaffolds.

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