An in-situ osteochondral regeneration repair scaffold and a preparation method thereof

Abstract

The application relates to an in-situ osteochondral regeneration and repair support and a preparation method thereof. The in-situ osteochondral regeneration and repair support prepared by the method comprises a cartilage layer, a connecting layer and a subchondral bone layer. The porosity of the cartilage layer is not less than 85%, the Young's modulus is 6-13 KPa, and the pore size is 130-140 mu m. The porosity of the connecting layer is not less than 95%, the Young's modulus is 4-19 KPa, and the pore size is 50-120 mu m. The porosity of the subchondral bone layer is not less than 95%, the Young's modulus is 4-19 KPa, and the pore size is 300-400 mu m. The in-situ osteochondral regeneration and repair support prepared by the method has good biocompatibility, is beneficial to the adhesion, migration and proliferation of cartilage cells and bone marrow mesenchymal stem cells, cannot cause immune rejection, provides a better support environment for the repair after osteochondral implantation, and does not affect the migration of cells through experiments. The in-situ osteochondral regeneration and repair support has a good repair effect on the osteochondral defects of rats through animal experiments.

Description

Technical Field

[0001] This invention relates to the field of osteocartilage scaffold technology, and in particular to an in situ osteocartilage regeneration and repair scaffold and its preparation method. Background Technology

[0002] Articular cartilage is a highly differentiated connective tissue that can withstand stress, cushion shocks, and provide lubrication. However, acute trauma and degenerative osteoarthritis easily cause damage and lesions to articular cartilage. Due to its unique characteristics such as being avascular and nerveless and having low metabolism, articular cartilage is difficult to repair itself, making it a common orthopedic disease in clinical practice. Its incidence is increasing year by year, and its treatment is extremely challenging.

[0003] Currently, clinical treatments for cartilage defects include: microfracture surgery, bone plate drilling, arthroscopic grinding and irrigation, and autologous or allogeneic cartilage transplantation. The first three methods mainly utilize the repair function of subchondral bone to generate fibrocartilage with poor performance. The last method uses autologous or exogenous cartilage transplanted to the defect site for repair, but this method has disadvantages such as requiring a second surgery and having limited cartilage sources. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes an in-situ osteochondral regeneration and repair scaffold and its preparation method.

[0005] The technical solution of this invention is as follows:

[0006] A method for preparing an in-situ osteochondral regeneration and repair scaffold includes the following steps:

[0007] S1. Prepare cartilage layer precursor solution, subchondral bone layer precursor solution and connecting layer precursor solution;

[0008] S2. Place the cartilage layer precursor solution into an X-axis temperature gradient mold and freeze to obtain a frozen cartilage layer. Place the subchondral bone layer precursor solution into a Y-axis temperature gradient mold and freeze to obtain a frozen subchondral bone layer.

[0009] S3. After placing the subchondral bone layer cryobody into the connected X and Y axis temperature gradient mold, add the connecting layer precursor solution, and finally place the cartilage layer cryobody in it. Finally, freeze-dry to obtain an in-situ osteochondral regeneration and repair scaffold.

[0010] Further, the cartilage layer precursor solution in step S1 is obtained by mixing and stirring a silk fibroin solution with a concentration of 50-100 mg / ml and a polyurethane solution with a concentration of 50-100 mg / ml, wherein the volume ratio of the silk fibroin solution to the polyurethane is 1:1.

[0011] Further, the subchondral bone layer precursor solution in step S1 is prepared by mixing a silk fibroin solution with a concentration of 50-100 mg / ml and hydroxyapatite powder evenly, and then injecting a polyurethane solution with a concentration of 50-100 mg / ml and mixing evenly. The volume ratio of the silk fibroin solution to polyurethane is 1:2, and the mass ratio of the silk fibroin solution to hydroxyapatite powder is (10-20):1.

[0012] Further, the precursor solution for the connecting layer in step S1 is prepared by mixing a silk fibroin solution with a concentration of 50-100 mg / ml and hydroxyapatite powder evenly, and then injecting a polyurethane solution with a concentration of 50-100 mg / ml and mixing evenly. The volume ratio of the silk fibroin solution to polyurethane is 1:1, and the mass ratio of the silk fibroin solution to hydroxyapatite powder is (5-10):1.

[0013] Further, step S3 includes:

[0014] S301. Place the pre-dried binder layer solution in an environment of 20℃~40℃ for 30~45min to obtain the dried binder layer pre-dried solution.

[0015] S302. After placing the subchondral bone layer cryobody into the connected X and Y axis temperature gradient mold, add the dried connecting layer precursor liquid, then place the cartilage layer cryobody in, pre-freeze it in an environment of -50℃ to -30℃ for 60 to 180 minutes, then freeze it in an environment of -80℃ to -60℃ for 6 to 10 hours, and finally freeze-dry it to obtain an in situ osteochondral regeneration and repair scaffold.

[0016] Furthermore, it also includes calcification treatment of the subchondral bone layer in the in-situ osteochondral regeneration and repair scaffold obtained in step S3.

[0017] Furthermore, the specific steps of the calcification treatment are as follows:

[0018] 1) Prepare a floating plate, wherein the floating plate has a fixing hole for the in situ osteochondral regeneration and repair scaffold to pass through, and when the in situ osteochondral regeneration and repair scaffold is placed on the floating plate, 1 / 5 to 1 / 2 of the length of the subchondral bone layer extends out of the upper end face of the floating plate, and the thickness of the floating plate does not exceed 1 / 3 of the thickness of the subchondral bone layer.

[0019] 2) The float plate with the in situ osteochondral regeneration and repair scaffold is floated in calcium chloride ethanol solution. The part of the subchondral bone layer that extends from the lower end face of the float plate is soaked in calcium chloride ethanol solution for 45-60 minutes and then soaked in anhydrous ethanol solution for 5-10 minutes to obtain the primary calcium scaffold.

[0020] 3) Take out the precalcium scaffold prepared in step 2), float the float plate with the precalcium scaffold in the dipotassium hydrogen phosphate aqueous solution, and soak the part of the precalcium scaffold that protrudes from the lower surface of the float plate in the dipotassium hydrogen phosphate aqueous solution for 5 to 30 minutes, and then transfer it to deionized water for 5 to 10 minutes to obtain the precalcium scaffold.

[0021] 4) Take out the pre-calcified scaffold obtained in step 3), float the floating plate with the pre-calcified scaffold in the biomimetic calcium ion buffer, and culture it in an environment of 36.5℃~38℃ for 1~3 days to obtain the calcified in situ osteochondral regeneration and repair scaffold.

[0022] Furthermore, before step 4), repeat steps 2) and 3) 3-10 times.

[0023] Furthermore, the biomimetic calcium ion buffer solution described in S404 is a mixture of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, HCl aqueous solution, CaCl2·2H2O, Na2SO4·10H2O, and NH2C(CH2OH)3.

[0024] The in-situ osteochondral regeneration and repair scaffold was prepared according to the above method.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. This invention relates to an in situ osteochondral regeneration and repair scaffold and its preparation method. In the in situ osteochondral regeneration and repair scaffold prepared by the method of this invention, the main fiber direction of the cartilage layer is parallel to the outer surface of the joint, and the fibers are relatively dense. The porosity of the cartilage layer is not less than 85%, the Young's modulus is 6–13 kPa, and the pore size is 130–140 μm. The porosity of the connecting layer is not less than 95%, the Young's modulus is 4–19 kPa, and the pore size is 50–120 μm. The main fiber direction of the subchondral bone layer is perpendicular to the outer surface of the joint, and the fibers are relatively sparsely arranged. The porosity of the subchondral bone layer is not less than 95%, the Young's modulus is 4–19 kPa, and the pore size is 300–400 μm. The method of this invention, by controlling the orientation and porosity of the pores within the repair scaffold, provides sufficient space for the attachment and growth of corresponding layers of osteofibroblasts during patient rehabilitation.

[0027] 2. This invention provides an in situ osteochondral regeneration and repair scaffold and its preparation method. The silk fibroin and hydroxyapatite used are FDA-approved biomaterials with good biocompatibility, facilitating the attachment, migration, and proliferation of chondrocytes and bone marrow mesenchymal stem cells without causing immune rejection. This provides a better scaffold environment for repair after osteochondral implantation, thus promoting patient recovery. Furthermore, experiments have demonstrated that the in situ osteochondral regeneration and repair scaffold does not affect cell migration. Animal experiments have shown that the in situ osteochondral regeneration and repair scaffold has a good repair effect on osteochondral defects in rats. The preparation method of this invention is simple, economically efficient, and has broad application prospects. Attached Figure Description

[0028] Figure 1 A scanning electron microscope image (100 μm) of the in situ osteochondral regeneration and repair scaffold structure prepared in Example 1 of the present invention;

[0029] Figure 2 Scanning electron microscope image (2 μm) of the in situ osteochondral regeneration and repair scaffold prepared in Example 1 of the present invention and co-cultured with cells;

[0030] Figure 3 This is the structure for biocompatibility testing of in situ osteochondral regeneration and repair scaffold and cell co-culture in Embodiment 2 of the present invention;

[0031] Figure 4 The results of the cell scratch experiment in Example 3 of this invention;

[0032] Figure 5 This is a photograph of the in situ osteochondral regeneration and repair scaffold used in Example 4 of the present invention to repair osteochondral damage in rats. Detailed Implementation

[0033] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0034] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0035] Source of materials:

[0036] Silkworm cocoons were purchased from the Guangxi Sericultural Research Institute. Silk fibroin solution was prepared from the silkworm cocoons using the following method: the silkworm cocoons were degummed in a 0.5% sodium bicarbonate alkaline solution to obtain silk fibroin fibers. The silk fibroin fibers were dissolved in a 9.3M lithium bromide solution and dialyzed for 3 days to obtain the silk fibroin solution.

[0037] The dialysis bags were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0038] The rats were purchased from Chongqing Lepit Biotechnology Co., Ltd.

[0039] The polyurethane resin was purchased from Guangzhou Guanzhi Co., Ltd., model C303.

[0040] All other reagents used in this embodiment are analytical grade reagents from McLean Reagent Company.

[0041] Example 1 illustrates the preparation of an in situ osteochondral regeneration and repair scaffold.

[0042] Take 10 ml of a 70 mg / ml silk fibroin solution and 10 ml of a 70 mg / ml polyurethane solution, and sonicate for 5 minutes to prepare the cartilage layer precursor solution. Take 10 ml of a 70 mg / ml silk fibroin solution and 1.6 g of hydroxyapatite powder, mix them, and then add 10 ml of a 70 mg / ml polyurethane solution, and mechanically stir for 5 minutes to prepare the connecting layer precursor solution. Take 10 ml of a 70 mg / ml silk fibroin solution and 0.8 g of hydroxyapatite powder, mix them, and then add 20 ml of a 70 mg / ml polyurethane solution, and mechanically stir for 5 minutes to prepare the subchondral bone layer precursor solution. Take 10 ml of cartilage layer precursor solution, 5 ml of connecting layer precursor solution, and 10 ml of subchondral bone layer precursor solution. Pour the cartilage layer precursor solution and the subchondral bone layer precursor solution into X-axis and Y-axis temperature gradient molds respectively, pre-freeze at -15℃ for 60 min, and then freeze at -80℃ for 12 h. After freezing, use freeze-drying to obtain cartilage layer and subchondral bone layer cryosomes. First, pre-dry the connecting layer precursor solution at 20℃ for 30 min. After placing the subchondral bone layer cryosome into the connected X and Y-axis temperature gradient molds, add the connecting layer precursor solution, and finally add the cartilage layer cryosome. Pre-freeze at -30℃ for 120 min, then freeze at -80℃ for 8 h. After complete freezing, transfer to a laboratory freeze dryer and freeze-dry for 72 h to obtain a primary in-situ osteochondral regeneration and repair scaffold.

[0043] Prepare a float plate, the thickness of which does not exceed 1 / 3 of the thickness of the subchondral bone layer. The float plate has fixation holes for the in-situ osteochondral regeneration and repair scaffold to pass through. When the in-situ osteochondral regeneration and repair scaffold is placed on the float plate, 1 / 3 of the length of the subchondral bone layer extends beyond the upper end face of the float plate. Float the float plate with the in-situ osteochondral regeneration and repair scaffold in a 1% calcium chloride ethanol solution. After soaking the portion of the subchondral bone layer extending from the lower end face of the float plate in the calcium chloride ethanol solution for 45 minutes, it is transferred to anhydrous ethanol solution and soaked for 10 minutes to obtain a precalcified scaffold. Then, float the precalcified scaffold with the float plate in a 2% dipotassium hydrogen phosphate aqueous solution. After soaking the portion of the precalcified scaffold extending from the lower end face of the float plate in the dipotassium hydrogen phosphate aqueous solution for 20 minutes, it is transferred to deionized water and soaked for 10 minutes. The pre-calcified scaffold was obtained by drying. After each drying, the steps of obtaining the pre-calcified scaffold and the initial calcified scaffold were repeated 10 times. The float plate with the calcified scaffold was floated in a biomimetic calcium ion buffer and cultured at 37°C for 2 days. The biomimetic calcium ion buffer, per 1L, contained NaCl (9.642g), NaHCO3 (0.426g), KCl (0.27g), K2HPO4·3H2O (0.2772g), MgCl2·6H2O (0.3732g), 1M HCl aqueous solution (45.6mL), CaCl2·2H2O (0.441g), Na2SO4·10H2O (0.0852g), and NH2C(CH2OH)3 (Tris, 7.3416g). Finally, a complete in-situ osteochondral regeneration and repair scaffold with calcification was obtained.

[0044] This embodiment also tested the cell adhesion and cell proliferation of the prepared in situ osteochondral regeneration and repair scaffold. After high-temperature sterilization, the in situ osteochondral regeneration and repair scaffold was cut into small pieces and co-cultured with chondrocytes in 24-well plates. The structure of the in situ osteochondral regeneration and repair scaffold is as follows. Figure 1As shown in the figure, the bracket has a porous structure. Furthermore, upon testing, the cartilage layer contains several first attachment pores parallel to the outer surface of the joint position, with at least one end penetrating the outer wall surface of the cartilage layer. The porosity of the cartilage layer is not less than 85%, the Young's modulus is 6–13 kPa, and the pore diameter of the first attachment pores is 130–140 μm. The transition connecting layer contains several third attachment pores extending into the transition connecting layer from an opening on the side surface of the transition connecting layer near the subchondral bone layer, with an indefinite extension direction. The porosity of the connecting layer is not less than 95%, the Young's modulus is 4–19 kPa, and the pore diameter of the third attachment pores is 50–120 μm. The subchondral bone layer contains several second attachment pores perpendicular to the outer surface of the joint position, extending from an opening on the side surface of the subchondral bone layer away from the cartilage layer. The porosity of the subchondral bone layer is not less than 95%, the Young's modulus is 4–19 kPa, and the pore diameter of the second attachment pores is 300–400 μm. Scanning electron micrographs of chondrocyte adhesion and growth on the scaffold are shown below. Figure 2 As shown, chondrocytes can adhere and grow on the scaffold, indicating that the scaffold is non-toxic to chondrocytes, can promote cell proliferation and growth, and has good biocompatibility.

[0045] Example 2: Co-culture of in situ osteochondral regeneration and repair scaffold with bone marrow mesenchymal stem cells

[0046] The in situ osteochondral regeneration and repair scaffold prepared in Example 1 was sterilized and co-cultured with mouse bone marrow mesenchymal stem cells. The cell culture medium was changed every 3 days. After 7 days, the survival of bone marrow mesenchymal stem cells on the scaffold was observed. The purpose was to detect the effect of the in situ osteochondral regeneration and repair scaffold on the growth of mesenchymal stem cells, such as activity, differentiation, adhesion, and proliferation. Cell liveness and death staining was performed and observed under a confocal fluorescence microscope.

[0047] See test results Figure 3 As shown in the figure, bone marrow mesenchymal stem cells not only adhere well to and grow on the surface of the in situ osteocartilage regeneration and repair scaffold, but also enter the scaffold through pores to grow inside. The cell viability and cell death indicators show good activity, and the stem cells spread well, which is conducive to the adhesion, migration and proliferation of stem cells, providing a better scaffold environment for repair after osteocartilage implantation.

[0048] Example 3 verifies the effect of in situ osteochondral regeneration and repair scaffold extract on cell migration.

[0049] Take 1g of the in situ osteochondral regeneration and repair scaffold prepared in Example 1, soak it in 10ml of cell culture medium, and shake it at 37℃ for 14 days. Extract the exudate from the scaffold. Co-culture the obtained extract with mouse chondrocytes. Add 2.5ml of culture medium and 100,000 chondrocytes to a 6-well plate. After the cells adhere, add 200μl of extract. Each group has 3 replicates. Incubate in a cell culture incubator. When the cell coverage is close to 95%, make a 45-degree scratch with a 1ml pipette tip, and then continue culturing. Observe the cell migration after 12 hours.

[0050] In this embodiment, the cell scratch assay was used to detect cell migration in the prepared in situ osteochondral regeneration and repair scaffold. The experimental results are as follows: Figure 4 As shown in the figure, the in situ osteochondral regeneration and repair scaffold has virtually no impact on cell migration.

[0051] Example 4: Analysis of the In Vivo Effect of In Situ Osteochondrial Regeneration and Repair Scaffold in Rats

[0052] The in-situ osteochondral regeneration and repair scaffold prepared in Example 1 was sterilized and implanted into the knee joint groove defect of 7-week-old rats. The rat knee joint defect model was a cylindrical defect model with a diameter of 1.5 mm and 2 mm created in the femoral groove of the knee joint. The rats were not subject to dietary or exercise restrictions and were normally fed for 8 weeks. After that, the experimental rats were sacrificed, the knee joints were removed, and the defect healing was observed. The results are as follows: Figure 5 As shown, this in situ osteochondral regeneration and repair scaffold has a good repair effect on osteochondral defects in rats.

[0053] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above. Modifications or improvements can be made to the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing an in-situ osteochondral regeneration repair scaffold, characterized in that, The method comprises the following steps: S1. preparing a cartilage layer precursor solution, a subchondral bone layer precursor solution, and a connecting layer precursor solution; The cartilage layer precursor solution is prepared by mixing and stirring a silk fibroin solution with a concentration of 50-100 mg / mL and a polyurethane with a concentration of 50-100 mg / mL, wherein the volume ratio of the silk fibroin solution to the polyurethane is 1:1; The subchondral bone layer precursor solution is prepared by mixing and stirring a silk fibroin solution with a concentration of 50-100 mg / mL and hydroxyapatite powder, and then injecting and stirring a polyurethane with a concentration of 50-100 mg / mL, wherein the volume ratio of the silk fibroin solution to the polyurethane is 1:2, and the mass ratio of the silk fibroin solution to the hydroxyapatite powder is (10-20):1; The connecting layer precursor solution is prepared by mixing and stirring a silk fibroin solution with a concentration of 50-100 mg / mL and hydroxyapatite powder, and then injecting and stirring a polyurethane with a concentration of 50-100 mg / mL, wherein the volume ratio of the silk fibroin solution to the polyurethane is 1:1, and the mass ratio of the silk fibroin solution to the hydroxyapatite powder is (5-10):1; S2. freezing the cartilage layer precursor solution in an X-axis temperature gradient mold to obtain a cartilage layer frozen body, and freezing the subchondral bone layer precursor solution in a Y-axis temperature gradient mold to obtain a subchondral bone layer frozen body; S3. placing the subchondral bone layer frozen body into the connected X-axis and Y-axis temperature gradient molds, adding the connecting layer precursor solution, and then placing the cartilage layer frozen body, and finally performing freeze-drying and calcium treatment on the subchondral bone layer to obtain an in-situ bone cartilage regeneration and repair scaffold.

2. The production method according to claim 1, characterized by, Step S3 comprises: S301. pre-drying the connecting layer precursor solution in an environment with a temperature of 20-40°C for 30-45 min to obtain a dried connecting layer precursor solution; S302. placing the subchondral bone layer frozen body into the connected X-axis and Y-axis temperature gradient molds, adding the dried connecting layer precursor solution, and then placing the cartilage layer frozen body, pre-freezing in an environment with a temperature of -50°C to -30°C for 60-180 min, freezing in an environment with a temperature of -80°C to -60°C for 6-10 h, and finally freeze-drying to obtain the in-situ bone cartilage regeneration and repair scaffold.

3. The preparation method according to claim 1, characterized in that, The specific steps of the calcium treatment are as follows: 1) preparing a floating plate, wherein a fixing hole is formed on the floating plate for the in-situ bone cartilage regeneration and repair scaffold to pass through, and when the in-situ bone cartilage regeneration and repair scaffold is placed on the floating plate, 1 / 5-1 / 2 of the length of the subchondral bone layer protrudes from the upper end surface of the floating plate, and the thickness of the floating plate is not more than 1 / 3 of the thickness of the subchondral bone layer; 2) floating the floating plate with the in-situ bone cartilage regeneration and repair scaffold in a calcium chloride ethanol solution, and immersing the part of the subchondral bone layer protruding from the lower end surface of the floating plate in the calcium chloride ethanol solution for 45-60 min and then in anhydrous ethanol solution for 5-10 min to obtain a primary calcium scaffold. 3) taking out the primary calcium scaffold prepared in step 2), floating the float plate with the primary calcium scaffold in the aqueous dipotassium hydrogen phosphate solution, immersing the part of the primary calcium scaffold protruding from the lower end surface of the float plate in the aqueous dipotassium hydrogen phosphate solution for 5-30 min, and then immersing in deionized water for 5-10 min to obtain a pre-calcium scaffold; 4) taking out the pre-calcium scaffold prepared in step 3), floating the float plate with the pre-calcium scaffold in a biomimetic calcium ion buffer, and culturing in an environment at 36.5-38°C for 1-3 days to obtain a calcified in-situ bone cartilage regeneration and repair scaffold.

4. The production method according to claim 3, characterized by, Before step 4), steps 2) and 3) are cycled 3-10 times.

5. The preparation method according to claim 3, characterized in that, The biomimetic calcium ion buffer in step 4) is a mixed solution of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, HCl aqueous solution, CaCl2·2H2O, Na2SO4·10H2O and NH2C(CH2OH)3.

6. The in-situ bone cartilage regeneration and repair scaffold prepared by the preparation method in any one of claims 1-5.

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