In-situ osteochondral regeneration and repair stent and preparation method thereof

Abstract

The invention relates to an in-situ osteochondral regeneration and repair scaffold and a preparation method thereof, the in-situ osteochondral regeneration and repair scaffold 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 modulus is 6-13KPa, and the aperture is 130-140 [mu] m; the porosity of the connecting layer is not lower than 95%, the Young modulus is 4-19 KPa, and the aperture is 50-120 [mu] m; the porosity of the subchondral bone layer is not lower than 95%, the Young modulus is 4-19 KPa, and the aperture is 300-400 microns. The in-situ osteochondral regeneration and repair scaffold prepared by the method has good biocompatibility, is beneficial to attachment, migration and proliferation of cartilage cells and bone marrow mesenchymal stem cells, does not cause immunological rejection, provides a better scaffold environment for repair after osteochondral implantation, and has a good application prospect in the field of in-situ osteochondral regeneration and repair, and experiments prove that the in-situ osteochondral regeneration and repair scaffold has a good application prospect in the field of in-situ osteochondral regeneration and repair. The in-situ osteochondral regeneration and repair scaffold does not affect cell migration; animal experiments prove that the in-situ osteochondral regeneration and repair scaffold has a good repair effect on rat osteochondral defects.

Description

Technical Field

[0001] The present invention relates to the technical field of osteocartilage scaffolds, and in particular to an in-situ osteocartilage regeneration and repair scaffold and a preparation method thereof. Background Art

[0002] Articular cartilage is a highly differentiated connective tissue that can withstand stress, reduce shock and act as a lubricant. However, acute trauma and degenerative osteoarthritis can easily cause damage and pathology to the osteochondral cartilage. Due to its special characteristics of no blood vessels and nerves and low metabolism, articular cartilage is difficult to self-repair. It is currently a common orthopedic disease in clinical practice, with an increasing incidence year by year and extremely challenging to treat.

[0003] At present, the clinical methods for treating cartilage defects include: microfracture, bone plate drilling, arthroscopic grinding and lavage, and autologous or allogeneic cartilage transplantation. The first three methods mainly use the repair function of subchondral bone to generate fibrocartilage with poor performance, and the last method uses autologous cartilage or exogenous cartilage to transplant to the defect site for repair. This method has the disadvantages of secondary surgery and limited sources. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an in situ bone cartilage regeneration and repair scaffold and a preparation method thereof.

[0005] The technical solution of the present invention is:

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

[0007] S1. Preparing cartilage layer precursor solution, subchondral bone layer precursor solution and connection layer precursor solution;

[0008] S2. The cartilage layer precursor solution is placed in an X-axis temperature gradient mold for freezing to obtain a cartilage layer frozen body, and the subchondral bone layer precursor solution is placed in a Y-axis temperature gradient mold for freezing to obtain a subchondral bone layer frozen body;

[0009] S3. Place the subchondral bone layer frozen body into the connected X and Y axis temperature gradient mold, add the connection layer precursor liquid, and finally place the cartilage layer frozen body, and finally freeze-dry to obtain the in situ bone cartilage regeneration repair scaffold.

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

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

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

[0013] Further, step S3 includes:

[0014] S301. Pre-dry the connecting layer precursor solution in an environment of 20°C to 40°C for 30 to 45 minutes to obtain a dried connecting layer precursor solution;

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

[0016] Furthermore, the method further includes calcifying the subchondral bone layer in the in situ osteocartilage regeneration and repair scaffold obtained in step S3.

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

[0018] 1) preparing a floating plate, wherein a fixing hole is provided on the floating plate for the in situ osteochondral regeneration and repair scaffold to pass through, wherein 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 protrudes from the upper end surface 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) floating the floating plate with the in situ osteochondral regeneration and repair scaffold in a calcium chloride ethanol solution, soaking the portion of the subchondral bone layer extending from the lower end surface of the floating plate in the calcium chloride ethanol solution for 45 to 60 minutes and then soaking in an anhydrous ethanol solution for 5 to 10 minutes to obtain a primary calcium scaffold;

[0020] 3) taking out the pre-calcified scaffold prepared in step 2), floating the floating plate with the pre-calcified scaffold in a dipotassium hydrogen phosphate aqueous solution, soaking the part of the pre-calcified scaffold extending from the lower end surface of the floating plate in the dipotassium hydrogen phosphate aqueous solution for 5 to 30 minutes, and then soaking in deionized water for 5 to 10 minutes to obtain a pre-calcified scaffold;

[0021] 4) Taking out the pre-calcified scaffold prepared in step 3), floating the floating plate with the pre-calcified scaffold in a bionic calcium ion buffer, culturing in an environment of 36.5° C. to 38° C. for 1 to 3 days, and obtaining a calcified in situ osteochondral regeneration and repair scaffold.

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

[0023] Furthermore, the bionic calcium ion buffer 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 bone cartilage regeneration and repair scaffold prepared according to the above method.

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

[0026] 1. The present invention relates to an in situ osteochondral regeneration and repair scaffold and a preparation method thereof. In the in situ osteochondral regeneration and repair scaffold prepared by the method of the present 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 6-13KPa, and the pore size is 130-140μm; the porosity of the connecting layer is not less than 95%, the Young's modulus is 4-19KPa, and the pore size is 50-120μm; the main fiber direction of the subchondral bone layer is perpendicular to the outer surface of the joint and the fiber arrangement is relatively sparse, the porosity of the subchondral bone layer is not less than 95%, the Young's modulus is 4-19KPa, and the pore size is 300-400μm. The method of the present invention controls the orientation and porosity of the pores in the repair scaffold, and when the patient is rehabilitated, there is enough space for the corresponding level of bone fiber cells to attach and grow.

[0027] 2. The in situ osteochondral regeneration and repair scaffold and preparation method thereof of the present invention use silk fibroin and hydroxyapatite as FDA-approved biomaterials, have good biocompatibility, are conducive to the attachment, migration and proliferation of chondrocytes and bone marrow mesenchymal stem cells, do not cause immune rejection, provide a better scaffold environment for the repair of osteochondral implantation, and are more conducive to the recovery of patients; and experiments have shown that the in situ osteochondral regeneration and repair scaffold does not affect the migration of cells; animal experiments have shown that the in situ osteochondral regeneration and repair scaffold has a good repair effect on rat osteochondral defects. The preparation method of the present invention is simple, has high economic benefits, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a scanning electron micrograph (100 μm) of the in situ osteochondral regeneration and repair scaffold structure prepared in Example 1 of the present invention;

[0029] Figure 2 This is a scanning electron microscopic 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 The biocompatibility detection structure of the in situ osteochondral regeneration and repair scaffold and cell co-culture in Example 2 of the present invention;

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

[0032] Figure 5 This is a physical picture of the in situ osteochondral regeneration and repair scaffold in Example 4 of the present invention repairing rat osteochondral damage. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technicians in this field can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0034] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0035] Sources:

[0036] Silkworm cocoons were purchased from Guangxi Sericulture Science Research Institute. Silkworm cocoons were used to prepare silk fibroin solution by the following method: silkworm cocoons were degummed in 0.5% sodium bicarbonate alkaline solution to obtain silk fibroin fibers, the silk fibroin fibers were dissolved in 9.3 M 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] Rats were purchased from Chongqing Labite Biotechnology Co., Ltd.

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

[0040] The remaining reagents in this example were all analytical grade reagents from MacLean Reagent Company.

[0041] Example 1 Example 1 Preparation of in situ bone cartilage regeneration repair scaffold

[0042] Take 10 ml of 70 mg / ml silk fibroin solution and 10 ml of 70 mg / ml polyurethane solution, mix them ultrasonically for 5 minutes as the cartilage layer precursor solution. Take 10 ml of 70 mg / ml silk fibroin solution and 1.6 g of hydroxyapatite powder, then add 10 ml of 70 mg / ml polyurethane solution, stir mechanically for 5 minutes as the connection layer precursor solution. Take 10 ml of 70 mg / ml silk fibroin solution and 0.8 g of hydroxyapatite powder, then add 20 ml of 70 mg / ml polyurethane solution, stir mechanically for 5 minutes as the subchondral bone layer precursor solution. Take 10ml of cartilage layer precursor solution, 5ml of connection layer precursor solution, and 10ml of subchondral bone layer precursor solution, pour the cartilage layer precursor solution and subchondral bone layer precursor solution into the X-axis temperature gradient mold and the Y-axis temperature gradient mold respectively, pre-freeze for 60min in a -15℃ environment, and then freeze for 12h in a -80℃ environment. After freezing, use the freeze-drying method to obtain the cartilage layer frozen body and the subchondral bone layer frozen body. First, place the connection layer precursor solution in a 20℃ environment for pre-drying for 30min, place the subchondral bone layer frozen body into the connected X- and Y-axis temperature gradient molds, add the connection layer precursor solution, and finally place the cartilage layer frozen body, pre-freeze for 120min in a -30℃ environment, and then freeze for 8h in a -80℃ environment. After being completely frozen, transfer it to a laboratory freeze dryer and freeze-dry for 72h to obtain a primary in situ osteochondral regeneration and repair scaffold.

[0043] Prepare a floating plate, the thickness of which does not exceed 1 / 3 of the thickness of the subchondral bone layer. A fixing hole is provided on the floating plate 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 floating plate, 1 / 3 of the length of the subchondral bone layer extends out of the upper end surface of the floating plate. Float the floating plate with the in situ osteochondral regeneration and repair scaffold in a 1% calcium chloride ethanol solution. After the portion of the subchondral bone layer extending from the lower end surface of the floating plate is soaked in the calcium chloride ethanol solution for 45 minutes, it is transferred to an anhydrous ethanol solution and soaked for 10 minutes to obtain a primary calcium scaffold. Then, float the primary calcium scaffold with the floating plate in a 2% dipotassium hydrogen phosphate aqueous solution. After the portion of the primary calcium scaffold extending from the lower end surface of the floating plate is soaked in the dipotassium hydrogen phosphate aqueous solution for 20 minutes, it is transferred to deionized water and soaked for 10 minutes. min to obtain a pre-calcified scaffold; after each drying, the steps of the initial calcified scaffold and the pre-calcified scaffold were repeated 10 times, and the floating plate with the calcified scaffold was floated in a bionic calcium ion buffer and cultured in a 37°C environment for 2 days. The bionic calcium ion buffer contained, in 1 L, NaCl (9.642 g), NaHCO3 (0.426 g), KCl (0.27 g), K2HPO4·3H2O (0.2772), MgCl2·6H2O (0.3732 g), 1M HCl aqueous solution (45.6 mL), CaCl2·2H2O (0.441 g), Na2SO4·10H2O (0.0852 g), and NH2C(CH2OH)3 (Tris, 7.3416 g), and finally a calcified complete in situ osteochondral regeneration and repair scaffold was obtained.

[0044] In this example, the prepared in situ osteochondrocyte regeneration and repair scaffold was subjected to cell adhesion and cell proliferation tests. The in situ osteochondrocyte regeneration and repair scaffold was sterilized at high temperature, cut into small pieces, and co-cultured with chondrocytes in a 24-well plate. The structure of the in situ osteochondrocyte regeneration and repair scaffold is as follows: Figure 1As shown, it can be seen from the figure that the bracket has a porous structure. And after testing, the cartilage layer contains a number of first attachment holes parallel to the outer surface of the joint position and at least one end passes through the outer wall surface of the cartilage layer, the porosity of the cartilage layer is not less than 85%, the Young's modulus is 6-13KPa, and the pore size of the first attachment hole is 130-140μm; the transition connection layer contains a number of third attachment holes extending from the surface opening on one side of the transition connection layer close to the subchondral bone layer into the transition connection layer and extending in an uncertain direction, the porosity of the connection layer is not less than 95%, the Young's modulus is 4-19KPa, and the pore size of the third attachment holes is 50-120μm; the subchondral bone layer contains a number of second attachment holes perpendicular to the outer surface of the joint position and extending from the surface opening on one side 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 4-19KPa, and the pore size of the second attachment holes is 300-400μm. Scanning electron microscopy images of chondrocyte adhesion and growth on the scaffold 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 compatibility.

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

[0046] The in situ osteochondrocyte 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 replaced 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 osteochondrocyte regeneration and repair scaffold on the activity, differentiation, adhesion, proliferation and other growth conditions of mesenchymal stem cells, which were observed under a confocal fluorescence microscope after cell live and dead staining.

[0047] Test results see Figure 3 As shown in the figure, it can be seen that bone marrow mesenchymal stem cells not only have good adhesion and growth on the surface of the in situ osteochondral regeneration and repair scaffold, but also enter the scaffold through the pores and grow inside the scaffold. The cell live and dead display has good activity, and the stem cell spreading is good, which is conducive to the adhesion, migration and proliferation of stem cells, providing a better scaffold environment for the repair of osteochondral after implantation.

[0048] Example 3 Verification of 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, shake it at 37°C for 14d, and extract the exudate of the scaffold. Co-culture the obtained extract with mouse chondrocytes, add 2.5ml of culture medium and 100,000 chondrocytes in a 6-well plate, add 200μl of extract after the cells adhere to the wall, and place 3 parallel samples in each group in a cell culture incubator for incubation. When the cell coverage rate is close to 95%, scratch it at 45 degrees with a 1ml gun tip, and then continue to culture. After 12h, observe the migration of cells.

[0050] In this example, the cell migration of the prepared in situ osteochondral regeneration and repair scaffold was tested by cell scratch experiment. The experimental results are as follows: Figure 4 As shown in the figure, it can be seen that the in situ osteochondral regeneration and repair scaffold has little effect on cell migration.

[0051] Example 4 Analysis of the experimental effect of in situ osteochondral 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 chute defect of 7-week-old rats. The rat knee joint defect model was a cylindrical defect model with a diameter of 1.5 mm or 2 mm in the femoral chute of the knee joint. The rats were not restricted in diet and exercise. After 8 weeks of normal feeding, the experimental rats were killed, the knee joints were removed, and the defect healing was observed. Figure 5 As shown, the in situ osteochondral regeneration and repair scaffold has a good repair effect on rat osteochondral defects.

[0053] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments. On the basis of the present invention, some modifications or improvements can be made thereto, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing an in situ osteochondral regeneration and repair scaffold, characterized in that: The following steps are involved: S1. Preparing cartilage layer precursor solution, subchondral bone layer precursor solution and connection layer precursor solution; S2. The cartilage layer precursor solution is placed in an X-axis temperature gradient mold for freezing to obtain a cartilage layer frozen body, and the subchondral bone layer precursor solution is placed in a Y-axis temperature gradient mold for freezing to obtain a subchondral bone layer frozen body; S3. Place the subchondral bone layer frozen body into the connected X and Y axis temperature gradient mold, add the connection layer precursor liquid, and finally place the cartilage layer frozen body, and finally freeze-dry to obtain the in situ bone cartilage regeneration repair scaffold.

2. The preparation method according to claim 1, characterized in that: The cartilage layer precursor solution in step S1 is obtained by mixing and stirring a silk fibroin solution with a concentration of 50 to 100 mg / ml and a polyurethane with a concentration of 50 to 100 mg / ml, wherein the volume ratio of the silk fibroin solution to the polyurethane is 1:

1.

3. The preparation method according to claim 1, characterized in that: The subchondral bone precursor solution in step S1 is prepared by stirring and mixing a silk fibroin solution with a concentration of 50 to 100 mg / ml and hydroxyapatite powder, and then injecting a polyurethane with a concentration of 50 to 100 mg / ml and stirring and mixing them evenly. The volume ratio of the silk fibroin solution: polyurethane is 1:2, and the mass ratio of the silk fibroin solution: hydroxyapatite powder is (10 to 20):

1.

4. The preparation method according to claim 1, characterized in that: The connecting layer precursor liquid in step S1 is prepared by stirring and mixing a silk fibroin solution with a concentration of 50 to 100 mg / ml and hydroxyapatite powder, and then injecting a polyurethane with a concentration of 50 to 100 mg / ml and stirring and mixing them evenly. The volume ratio of the silk fibroin solution: polyurethane is 1:1, and the mass ratio of the silk fibroin solution: hydroxyapatite powder is (5 to 10):

1.

5. The preparation method according to any one of claims 1 to 4, characterized in that: Step S3 includes: S301. Pre-dry the connecting layer precursor solution in an environment of 20°C to 40°C for 30 to 45 minutes to obtain a dried connecting layer precursor solution; S302. After placing the subchondral bone layer frozen body into the connected X and Y axis temperature gradient mold, add the dried connection layer precursor liquid, and then place the cartilage layer frozen body, first pre-freeze in a -50℃ to -30℃ environment for 60 to 180 minutes, then freeze in a -80℃ to -60℃ environment for 6 to 10 hours, and finally freeze-dry to obtain the in situ bone cartilage regeneration repair scaffold.

6. The preparation method according to claim 1, characterized in that: The method also includes calcifying the subchondral bone layer in the in situ osteocartilage regeneration and repair scaffold obtained in step S3.

7. The preparation method according to claim 6, characterized in that: The specific steps of the calcification treatment are: 1) preparing a floating plate, wherein a fixing hole is provided on the floating plate for the in situ osteochondral regeneration and repair scaffold to pass through, wherein 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 protrudes from the upper end surface of the floating plate, and the thickness of the floating plate does not exceed 1 / 3 of the thickness of the subchondral bone layer; 2) floating the floating plate with the in situ osteochondral regeneration and repair scaffold in a calcium chloride ethanol solution, soaking the portion of the subchondral bone layer extending from the lower end surface of the floating plate in the calcium chloride ethanol solution for 45 to 60 minutes and then soaking in an anhydrous ethanol solution for 5 to 10 minutes to obtain a primary calcium scaffold; 3) taking out the pre-calcified scaffold prepared in step 2), floating the floating plate with the pre-calcified scaffold in a dipotassium hydrogen phosphate aqueous solution, soaking the part of the pre-calcified scaffold extending from the lower end surface of the floating plate in the dipotassium hydrogen phosphate aqueous solution for 5 to 30 minutes, and then soaking in deionized water for 5 to 10 minutes to obtain a pre-calcified scaffold; 4) Taking out the pre-calcified scaffold prepared in step 3), floating the floating plate with the pre-calcified scaffold in a bionic calcium ion buffer, culturing in an environment of 36.5° C. to 38° C. for 1 to 3 days, and obtaining a calcified in situ osteochondral regeneration and repair scaffold.

8. The preparation method according to claim 7, characterized in that: Before step 4), step 2) and step 3) are cycled 3-10 times.

9. The preparation method according to claim 8, characterized in that: The bionic calcium ion buffer 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.

10. An in situ bone cartilage regeneration and repair scaffold prepared according to the method of any one of claims 1 to 9.

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