Device and process for preparing controllable mesoporous multiphase gradient osteochondral scaffold

Abstract

The invention relates to a device and a process for preparing a controllable mesoporous multiphase gradient osteochondral scaffold. The device comprises a cylindrical body, an insert and a base, wherein the cylindrical body is connected with the base through the insert; a cylindrical groove is formed in the cylindrical body; the insert consists of a microneedle and a base which are connected with each other; a constant-temperature solution is carried in the base; the base is in contact with the constant-temperature solution in the base; the microneedles are located in the cylindrical grooves. The multiphase gradient osteochondral stent has the beneficial effects that the multiphase gradient osteochondral stent prepared by the method can simulate a complex gradient structure of a natural osteochondral tissue in structure, realizes the purpose of gradient distribution of internal material components of the stent structure, and can meet the needs of tissue damage repair of different parts of the cartilage.

Description

Technical Field

[0001] The present invention relates to the technical field of osteochondrium scaffolds, and more specifically, to a device and process for preparing a controllable mesoporous multiphase gradient osteochondrium scaffold. Background Art

[0002] Osteochondral injury is a common orthopedic disease, especially among athletes and the elderly. Cartilage is a connective tissue without blood vessels, nerves, or lymph nodes, and has good pressure resistance and shock absorption functions. Unlike other types of tissue (such as bones, skin, muscles, etc.), it is difficult for damaged cartilage to restore its original structure and function through its own healing mechanism, which makes the problem of cartilage injury more complicated and difficult to deal with.

[0003] After cartilage damage, patients will experience pain, inflammation, joint stiffness, deformity, bone spur formation, and even disability. These symptoms not only seriously affect the patient's daily life and mobility, but may also further lead to other joint diseases, such as osteoarthritis. Due to the key role of cartilage in the human movement system, its damage often requires long-term treatment and management, which brings heavy economic and psychological burdens to patients and their families.

[0004] Existing technologies and products for osteochondral repair include 1) injectable filling materials: injectable gel materials based on hyaluronic acid, PEG, PLGA, collagen, hydrogel, stem cell injection and PRP; 2) membranes or patches: cell-derived decellularized matrix membranes, or patches made based on natural decellularized matrix membranes such as porcine dermal decellularized matrix; 3) biological scaffolds: repair scaffolds made based on polymers such as polylactic-co-glycolic acid copolymer, polycaprolactone or natural bio-based materials such as collagen, hyaluronic acid, etc. These materials have not shown ideal osteochondral repair efficacy in cartilage repair. The reasons may be: 1) It is difficult for a single-layer structural design to simulate the complex multi-layer structure of osteochondral; 2) It is difficult for a single component to meet the needs of tissue repair in different regions; 3) The sparse microporous structure and disordered microporous distribution are not conducive to the migration of tissue repair cells, as well as the exchange, diffusion and circulation of nutrients and metabolic waste, thereby affecting the cartilage repair effect. 3D bioprinting technology can accurately print scaffold materials with different structures (such as pores, interlayers, etc.) and different sizes, but there are problems such as high equipment and technical requirements and high costs. In view of the above problems, it is urgent to develop new scaffold material preparation technology or devices to construct multiphase osteochondral repair materials with controllable microporous structures. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and to propose a device and process for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold.

[0006] In a first aspect, a device for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold is provided, comprising: a columnar body, an insert 3 and a base 4;

[0007] Among them, the columnar body is connected to the base 4 through the insert 3; a cylindrical groove is provided in the columnar body; the insert 3 is composed of a connected microneedle 301 and a base 302; a constant temperature solution is carried in the base 4; the base 302 is in contact with the constant temperature solution in the base 4; the microneedle 301 is located in the cylindrical groove.

[0008] Preferably, the columnar body comprises a main plate 1 and a side plate 2;

[0009] Among them, the two sides of the main board 1 are connected to the side panel 2 by screws 5; the two sides of the main board 1 are provided with a first semicircular groove; the side of the side panel 2 connected to the main board 1 is provided with a second semicircular groove; the first semicircular groove and the second semicircular groove are spliced ​​into the cylindrical groove.

[0010] In a second aspect, a process for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold material is provided, which is performed by any device described in the first aspect, comprising:

[0011] Step 1, obtaining raw materials of multiphase gradient osteochondral scaffold;

[0012] Step 2: preparing at least two gels according to the raw materials of the multiphase gradient osteochondrocyte scaffold and a certain material ratio;

[0013] Step 3, injecting one of the at least two gels into the columnar groove of the columnar body to a predetermined height;

[0014] Step 4, adding a constant temperature solution to the base 4, and under the heat conduction effect between the base 302 of the insert 3 and the microneedle 301, ice crystals spread around the microneedle 301;

[0015] Step 5, using low-temperature vacuum freeze-drying and chemical cross-linking to fix the gel in the columnar groove to obtain a multiphase gradient osteochondrocyte scaffold monolayer structure;

[0016] Step 6, repeating steps 3-5 until the at least two gels are all converted into corresponding multiphase gradient osteochondrocyte scaffold single-layer structures;

[0017] Step 7, loosen the screws 5 in sequence, remove the side plate 2, and pull out the insert 3 to obtain the multiphase gradient bone cartilage scaffold material.

[0018] Preferably, in step 2, the preparation of at least two gels comprises:

[0019] A first gel, a second gel and a third gel are prepared; the first gel corresponds to the subchondral bone layer of the multiphase gradient osteocartilage scaffold, the second gel corresponds to the middle cartilage layer of the multiphase gradient osteocartilage scaffold, and the third gel corresponds to the superficial cartilage layer of the multiphase gradient osteocartilage scaffold.

[0020] Preferably, in step 1, the raw materials of the multiphase gradient osteochondrocyte scaffold include: hyaluronic acid, cartilage acellular matrix, type I collagen and hydroxyapatite.

[0021] Preferably, in step 4, the constant temperature solution comprises liquid nitrogen and anhydrous ethanol.

[0022] Preferably, in step 5, the chemical cross-linking includes: genipin, glutaraldehyde, sodium polytriphosphate, light curing and thermal cross-linking.

[0023] Preferably, in step 2, the pore size of the multiphase gradient osteochondrocyte scaffold is controlled by adjusting the mass concentration of the gel.

[0024] Preferably, in step 4, the pore size of the multiphase gradient osteochondrocyte scaffold is controlled by adjusting the cooling rate.

[0025] In a third aspect, a multiphase gradient osteochondrium scaffold is provided, wherein the multiphase gradient osteochondrium scaffold is prepared by any process described in the third aspect.

[0026] The beneficial effects of the present invention are:

[0027] 1. The multiphase gradient osteochondrium scaffold prepared by the device and process provided by the present invention can structurally simulate the complex gradient structure of natural osteochondrium tissue, achieve the purpose of gradient distribution of material components inside the scaffold structure, and meet the needs of repairing tissue damage in different parts of the cartilage.

[0028] 2. The present invention controls the pore size inside the scaffold material by selecting different concentrations of gel ratios and cooling rates to meet the needs of different cells. In addition, the pore direction inside the material is generally longitudinal from the bottom to the top (the bottom is the part that receives the cold source). The pore direction around the microneedle is perpendicular to the microneedle, that is, the lateral direction centered on the microneedle. The longitudinal and lateral pore directions are more conducive to cell migration upward or left and right, allowing the implanted material to be more rapidly cellized by the host.

[0029] 3. The multiphase gradient osteochondrocyte scaffold material prepared by the present invention has a rich microporous structure. The macroscopic channel structure design is more conducive to the exchange, diffusion and circulation of solution nutrients and cell metabolites. The scaffold material with a microscopic porous structure is also more conducive to cell migration, improving the cell implantation effect of the bionic scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A schematic diagram of the structure of a device for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold material;

[0031] Figure 2 A schematic side view of a device for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold material;

[0032] Figure 3 A flow chart of a preparation process for preparing a controllable mesoporous multiphase gradient scaffold material;

[0033] Figure 4 This is a physical picture of the osteochondral scaffold material;

[0034] Figure 5 Schematic diagram of XRM of osteochondral scaffold material;

[0035] Figure 6 Schematic diagram of the dual-phase gradient osteochondrocyte scaffold material XRM;

[0036] Figure 7 The longitudinal sections of cartilage scaffold materials with different concentrations are shown in scanning electron microscopy images;

[0037] Figure 8 A schematic diagram of the structure and composition of natural articular cartilage;

[0038] Explanation of the reference numerals: main board 1, side board 2, insert 3, microneedle 301, base 302, base 4, screw 5. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with embodiments. The description of the following embodiments is only used to help understand the present invention. It should be noted that for ordinary persons in the art, without departing from the principle of the present invention, the present invention can also be modified in some ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0040] Embodiment 1:

[0041] The natural articular cartilage structure has a multi-layered structure, e.g. Figure 8 As shown in the figure, the natural articular cartilage structure can be divided into area A, area B and area C from top to bottom. Area A represents the cartilage area, area B represents the junction area between cartilage and subchondral bone, and area C represents the subchondral bone area. The cartilage area includes collagen fibrils a1, chondrocytes a2 and hyaline cartilage a3; the junction area between cartilage and subchondral bone includes calcified cartilage b1; and the subchondral bone area includes subchondral bone c1.

[0042] Existing osteochondral repair technologies usually use a single-layer structure or a single component, which is difficult to simulate the complex multi-layer structure of osteochondral. To solve this problem, Figure 1As shown, Example 1 of the present application provides a device for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold, comprising: a columnar body, an insert 3 and a base 4;

[0043] Among them, the columnar body is connected to the base 4 through the insert 3; a cylindrical groove is provided in the columnar body; the insert 3 is composed of a connected microneedle 301 and a base 302; a constant temperature solution (0 to -196°C) is carried in the base 4; the base is in contact with the constant temperature solution in the base 4; the microneedle 301 is located in the cylindrical groove.

[0044] Specifically, the microneedle 301 can play a placeholder role. When constructing a bone cartilage scaffold, a channel structure can be formed inside the scaffold material. The material of the microneedle 301 includes but is not limited to heat-conducting materials such as steel needles, copper needles, and stainless steel. The diameter range of the microneedle 301 is not limited to 0.1-0.5 mm. The base 302 and the microneedle 301 play a role in heat conduction. The bottom of the base 302 is placed on the cold source, and the heat will be conducted from top to bottom along the base 302, and a temperature difference will be formed. The moisture in the scaffold material will form a crystal nucleus as the temperature decreases. As the temperature further decreases, the crystal nucleus forms ice crystals and begins to grow. Using the temperature change process of the microneedle 301 from bottom to top, ice crystals are first formed and grown at the bottom of the scaffold material and derived in the upward and side directions. After freeze-drying, a bone cartilage scaffold material containing channels and having basically the same pore size can be obtained.

[0045] In addition, the main board 1 and the side board 2 are combined to form a columnar body, which is used for shaping irregular and fluid materials such as hydrogels. The structure composed of the main board and the side board includes but is not limited to a cylindrical, quadrilateral or polygonal shape. The two sides of the main board 1 are connected or disassembled by rotating the screws 5 in the forward or reverse direction; the two sides of the main board 1 are provided with a first semicircular groove; the side of the side board 2 connected to the main board 1 is provided with a second semicircular groove; the first semicircular groove and the second semicircular groove are spliced ​​into the cylindrical groove.

[0046] Embodiment 2:

[0047] Based on Example 1, Example 2 of the present application provides a preparation process of a three-phase osteochondral scaffold material, comprising:

[0048] Step 1: Select the corresponding material according to the components of the superficial cartilage, middle cartilage and subchondral bone layer.

[0049] Specifically, the gel component includes, but is not limited to, sodium alginate, sodium hyaluronate, chitosan, collagen, hydroxyapatite, calcium phosphate, hydrotalcite, etc., used alone or in combination.

[0050] Step 2: Gel 1 (superficial cartilage layer), gel 2 (middle cartilage layer) and gel 3 (subchondral bone layer) were prepared according to a certain material ratio. Figure 3 A), transfer them into syringes ( Figure 3 B).

[0051] Step 3: Install the syringe containing gel 3 on the microinjection instrument, and inject gel 3 into the columnar groove according to the pre-set program to reach a predetermined height.

[0052] Step 4: Add a constant temperature solution to the base of the device (liquid nitrogen and anhydrous ethanol can be added in proportion to obtain the required constant temperature solution). Use the heat conduction effect between the base 302 of the insert and the microneedle 301 to control the growth speed and direction of the ice crystals (the ice crystals will grow slowly at a lower temperature and spread upward and left and right around the microneedle 301).

[0053] It should be noted that, in addition to the above-mentioned method of forming ice crystals by cooling, the temperature change method can also be used to form recrystallization by heating. The temperature loading method includes but is not limited to the use of constant temperature liquid, constant temperature instrument, etc.; the temperature range is not limited to from 0 to -196°C, or from -196 to 37°C.

[0054] Step 5: Use low-temperature vacuum freeze-drying-chemical cross-linking and other processes to fix the internal structure of the scaffold material to obtain the subchondral bone layer scaffold material, which is recorded as: SD.

[0055] Specifically, the material cross-linking methods include but are not limited to chemical cross-linking such as genipin, glutaraldehyde, sodium polytriphosphate, light curing and thermal cross-linking.

[0056] Step 6, injecting gel solution 2, using the water absorption property of SD material in dry state, repeating steps 3-5, and obtaining subchondral bone layer-middle cartilage scaffold material, recorded as: SMD.

[0057] Step 7, injecting the gel 1 solution, using the water absorption characteristics of the SMD scaffold material in a dry state, repeating steps 3-5 to prepare a three-phase scaffold material of superficial cartilage layer-middle cartilage layer-subchondral bone layer.

[0058] Step 8, then loosen the screws 5, remove the side plate 2, and pull out the insert 3 in sequence to obtain the three-phase gradient bone cartilage scaffold material.

[0059] It should be noted that the above only shows the preparation of a three-phase osteochondrocyte scaffold material. By adjusting the type of gel prepared in step 2, the embodiment of the present application can also realize the preparation of other multi-phase osteochondrocyte scaffold materials, for example, the preparation of a two-phase osteochondrocyte scaffold material.

[0060] Specifically, the process provided in this embodiment is the process corresponding to the device provided in Example 1. Therefore, the parts in this embodiment that are the same or similar to those in Example 1 can be referenced to each other and will not be repeated in this application.

[0061] Embodiment 3:

[0062] Based on Example 2, Example 3 of the present application provides a more specific preparation process of a three-phase osteochondral scaffold material, including:

[0063] Step 1: Synthesis of chitosan / cartilage decellularized matrix (C-cECM).

[0064] In step 1, the preparation of chitosan solution includes: adding 5 ml of distilled water to 0.2 g of chitosan (200-800 cP) powder, and dissolving it under magnetic stirring at 37° C. for 30 minutes until it becomes clear and transparent, to obtain a chitosan solution with a mass concentration of 4%.

[0065] The preparation of cartilage decellularized matrix solution includes: adding 5 ml of distilled water to 0.2 g of cartilage decellularized matrix microcarrier material (particle size less than 100 μm), stirring at 60° C. for 1-2 hours, and obtaining a collagen solution with a mass concentration of 4%.

[0066] 4% chitosan solution and 4% cartilage decellularized matrix solution were mixed in equal proportions, and dissolved under magnetic stirring at 37° C. for 30-60 minutes to allow the two to be fully dissolved and mixed, thereby obtaining chitosan-cartilage decellularized matrix gel (C-cECM).

[0067] Step 2: Synthesis of calcium-containing type I collagen complex (Ca-Col I).

[0068] Specifically, 1.0 g of anhydrous calcium hydrogen phosphate is placed in a beaker, sealed with aluminum foil, and placed in a constant temperature drying oven at 180°C-250°C for 30-180 minutes, and after being fully cooled, 2 ml (mass concentration 1.0%) of type I collagen solution is added thereto, and magnetic stirring is used to dissolve for 30-60 minutes at 37°C to fully dissolve and mix the two, thereby obtaining a calcium-containing type I collagen complex (Ca-Col I).

[0069] Step 3: Synthesis of magnesium-containing nano-hydrotalcite (MgAl-LDH).

[0070] The preparation of the alkaline solution includes: dissolving 0.2-0.8 grams of sodium hydroxide (NaOH) in 40 milliliters of deionized water to prepare an alkaline solution.

[0071] The salt solution is prepared by dissolving 0.5-1.0 g of magnesium chloride hexahydrate (MgCl2·6H2O) and 0.1-0.5 g of aluminum chloride nonahydrate (AlCl3·9H2O) in 10 ml of deionized water to prepare MgCl2·6H2O. 2+ and Al 3+ of mixed solution.

[0072] On a magnetic stirrer, add the above metal salt solution dropwise to the alkaline solution. After the addition is complete, continue stirring the reaction for 0.5-2 hours. After the reaction is completed, collect the precipitate of the reaction product by centrifugation (4000-10000g, 5-10 minutes). Wash the precipitate several times with deionized water until the pH value of the solution is close to neutral. Reselect the washed precipitate and transfer it to a reactor, and hydrothermally heat it at 100°C for 12-18 hours to obtain a magnesium-aluminum-containing nano-hydrotalcite material (MgAl-LDH).

[0073] Step 4: Prepare the ingredients for each layer.

[0074] Preparation of superficial cartilage layer (gel 1): (4-8%) C-cECM + (0.02-0.8%) MgAl-LDH. 0.4-0.8 g C-cECM and 0.002-0.08 g MgAl-LDH were fully dissolved in 10 ml physiological saline and stored at 4°C for later use.

[0075] Preparation of cartilage middle layer (Gel 2): ​​(1-2%) C-cECM + (1-2%) Ca-Col I + (0.1-1.0%) MgAl-LDH. 0.1-0.2 g C-cECM, 0.1-0.2 g Ca-Col I and 0.01-0.1 g Mg-LDH were fully dissolved in 10 ml physiological saline and stored at 4°C for later use.

[0076] Preparation of subchondral bone layer (Gel 3): (4-8%) Ca-Col I + (0.4-2.0%) MgAl-LDH. 0.4-0.8 g Ca-Col I and 0.04-0.2 g Mg-LDH were fully dissolved in 10 ml physiological saline and stored at 4°C for later use.

[0077] Step 5: construct a three-phase gradient osteochondral scaffold.

[0078] Install the 1 ml syringe containing gel 3 into the micro-injector, and inject gel 3 into the columnar groove of the device according to the predetermined program of moving at a speed of 4 mm / min for 30 seconds. When the predetermined program is completed, add a solution of minus 5°C to minus 10°C to the base of the device. After it is fully frozen, place the device containing the scaffold material in a freeze dryer, freeze-dry for 12-24 hours, and then add 0.1-0.5mM genipin at 25-37°C for 12-24 hours to obtain the subchondral bone scaffold material (SD).

[0079] Based on the previous step, replace the 1 ml syringe containing gel 2, and inject gel 2 into the columnar groove of the device containing the SD scaffold material according to the predetermined program of moving at a speed of 2 mm / min for 15 seconds. When the predetermined program is completed, let it stand for 10-30 minutes, and add a solution of minus 10℃ to minus 20℃ to the base of the device. After it is fully frozen, place the device containing the scaffold material in a freeze dryer, freeze-dry for 12-24 hours, and then add 0.1-0.5mM genipin at 25-37℃ for 12-24 hours to obtain a biphasic gradient (middle cartilage-subchondral bone) scaffold material (SMD).

[0080] Based on the previous step, replace the 1 ml syringe containing gel 1, and inject gel 1 into the columnar groove of the device containing the SMD scaffold material according to the predetermined program of moving at a speed of 2 mm / min for 15 seconds. When the predetermined program is completed, let it stand for 30-60 minutes, and add a solution of minus 20℃ to minus 40℃ to the base of the device. After it is fully frozen, place the device containing the scaffold material in a freeze dryer, freeze dry for 12-24 hours, and then add 0.1mM-0.5mM genipin at 25-37℃ for 12-24 hours to obtain a three-phase gradient osteochondrocyte scaffold material.

[0081] It should be noted that the parts in this embodiment that are the same or similar to those in Embodiment 2 can be referenced to each other and will not be described in detail in this application.

[0082] Embodiment 4:

[0083] Based on Example 2, Example 4 of the present application provides a preparation process of a dual-phase gradient osteochondrocyte scaffold material, comprising:

[0084] Step 1: Synthesis of hyaluronic acid / cartilage decellularized matrix (Hy-cECM).

[0085] The preparation of the hyaluronic acid solution includes: adding 5 ml of distilled water to 0.4 g of hyaluronic acid (200-800 cP) powder, dissolving under magnetic stirring at 37° C. for 30 minutes until the powder becomes clear and transparent, to obtain a hyaluronic acid solution with a mass concentration of 4%.

[0086] The preparation of cartilage decellularized matrix solution includes: adding 5 ml of distilled water to 0.2 g of cartilage decellularized matrix microcarrier material (particle size less than 100 μm), stirring at 60° C. for 1-2 hours, and obtaining a collagen solution with a mass concentration of 4%.

[0087] 4% hyaluronic acid solution and 4% cartilage decellularized matrix solution were mixed in equal proportions, and dissolved under magnetic stirring at 37° C. for 30-60 minutes to allow the two to be fully dissolved and mixed, thereby obtaining hyaluronic acid-cartilage decellularized matrix gel (C-cECM).

[0088] Step 2: Synthesis of type I collagen complex (Ca-Col I).

[0089] Take 1.0g of anhydrous calcium hydrogen phosphate and place it in a beaker, seal it with aluminum foil, and place it in a constant temperature drying oven at 180℃-250℃ for 30-180 minutes. After it is fully cooled, add 2ml (mass concentration 1.0%) type I collagen solution, and dissolve it under magnetic stirring at 37℃ for 30-60 minutes to fully dissolve and mix the two to obtain calcium-containing type I collagen complex (Ca-Col I).

[0090] Step 3: Synthesis of calcium-containing nano-hydrotalcite (Mg-LDH).

[0091] The preparation of the alkaline solution includes: dissolving 0.2-0.8 grams of sodium hydroxide (NaOH) in 40 milliliters of deionized water to prepare an alkaline solution.

[0092] The salt solution is prepared by dissolving 0.5-1.0 g of CaCl2·6H2O, 0.1-0.5 g of aluminum chloride nonahydrate (AlCl3·9H2O) and 0.1-0.5 g of CaCl2·6H2O in 10 ml of deionized water to prepare MgCl2. 2+ 、Al 3+ , Ca 2+ of mixed solution.

[0093] On a magnetic stirrer, add the above metal salt solution dropwise to the alkaline solution. After the addition is complete, continue stirring the reaction for 0.5-2 hours. After the reaction is completed, collect the precipitate of the reaction product by centrifugation (4000-10000g, 5-10 minutes). Wash the precipitate several times with deionized water until the pH value of the solution is close to neutral. Reselect the washed precipitate and transfer it to a reactor, and hydroheat it at 100°C for 12-18 hours to obtain a magnesium-containing nano-hydrotalcite material (Ca-LDH).

[0094] Step 4: Prepare the ingredients for each layer.

[0095] Cartilage layer (gel armor): (2-4%) Hy-cECM + (0.01-0.6%) Ca-LDH. 0.2-0.4 g Hy-cECM and 0.001-0.06 g Ca-LDH are fully dissolved in 10 ml physiological saline and stored at 4°C for later use.

[0096] Subchondral bone layer (Gel B): (4-8%) Ca-Col I + (0.8-2.0%) Ca-LDH. 0.4-0.8 g Col I and 0.08-0.2 g Ca-LDH are fully dissolved in 10 ml of normal saline and stored at 4°C for later use.

[0097] Step 5: construct a biphasic gradient osteochondral scaffold.

[0098] Gel B is injected into the columnar groove of the device by manual injection. When the predetermined height is reached, a solution at -5°C to -10°C is added to the base of the device. After it is fully frozen, the device containing the scaffold material is placed in a freeze dryer and freeze-dried for 12-24 hours. Subsequently, 0.25-0.5% glutaraldehyde is used at room temperature for 24 hours to obtain the subchondral bone scaffold material (bone layer, BL).

[0099] Based on the previous step, slowly add gel A solution to the columnar groove of the device containing BL scaffold material. When the predetermined height is reached, stand for 10-30 minutes, and add a solution of -10℃ to -20℃ to the base of the device. After it is fully frozen, place the device containing the scaffold material in a freeze dryer and freeze-dry for 12-24 hours. Subsequently, use 0.25%-0.5% glutaraldehyde at room temperature for 24 hours to obtain a biphasic gradient osteochondrocyte scaffold material with an upper cartilage repair layer (CL) and a lower subchondral bone repair layer (BL).

[0100] It should be noted that the parts in this embodiment that are the same or similar to those in Embodiment 2 can be referenced to each other and will not be described in detail in this application.

[0101] Embodiment 5:

[0102] Based on Example 2, Example 5 of the present application provides a method for controlling the pore size of a multiphase gradient osteochondrocyte scaffold, comprising:

[0103] Step 1: Prepare decellularized cartilage matrix (cECM) with different concentrations.

[0104] Accurately weigh 0.2, 0.3, 0.4, 0.5, and 0.6 g of cartilage decellularized matrix microcarriers (particle size less than 100 μm), dissolve them in 10 ml of distilled water, and stir them thoroughly for 0.5-1.0 hours using a magnetic stirrer to obtain cartilage decellularized matrix gel solutions (cECM) with mass concentrations of 2%, 3%, 4%, 5%, and 6%, respectively.

[0105] Step 2: construct osteochondral scaffolds with different pore sizes.

[0106] 1 ml syringes containing different cECM concentrations (2%, 3%, 4%, 5%, and 6%) were sequentially installed into the microinjector, and the cECM of different concentrations was injected into different tooling slots according to the predetermined program of migration at a speed of 6 mm / min for 30 seconds, e.g. Figure 7 As shown, 2%, 3%, 4%, 5%, and 6% single-phase osteochondral scaffolds were obtained.

[0107] Subsequently, a solution of -60°C to -80°C (100 ml of liquid nitrogen plus 50 ml of anhydrous ethanol) was added to the tooling base to completely freeze the scaffold material. Finally, the tooling containing different concentrations of osteochondral scaffolds was placed in a freeze dryer and freeze-dried for 24 hours to obtain a radially consistent single-phase osteochondral scaffold material.

[0108] In addition, the embodiment of the present application can also control the pore size of the osteochondral scaffold material by the cooling rate. Specifically, when the concentration remains unchanged, the pore size of the scaffold will decrease as the cooling rate increases, that is, the faster the cooling rate, the smaller the pore size.

[0109] It should be noted that the parts in this embodiment that are the same or similar to those in Embodiment 2 can be referenced to each other and will not be described in detail in this application.

[0110] In summary, the present invention provides an apparatus and process for preparing a controllable mesoporous multiphase gradient osteochondrocyte scaffold, and carries out a bionic design for the multi-layer structure of natural osteochondrocytes, constructing a two-phase, three-phase and more equal gradient structure to meet the needs of cartilage repair for different types of cartilage damage. The central pore and peripheral pore structure inside the scaffold material can meet the exchange, diffusion and circulation of nutrients and metabolic waste in the process of cell metabolism. By means of low-temperature freezing of the solution to form ice crystals, a scaffold material with relatively consistent radial direction inside the scaffold material and gradually decreasing pore size and pore spacing is obtained, which is more conducive to cell migration to the scaffold, including a two-layer structure from bottom to top and from the central pore to the peripheral pore, etc., which will greatly improve the implantation effect of the bionic scaffold and solve the technical problems of poor implantation effect and clinical application of existing osteochondrocyte repair technology.

Claims

1. A device for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold material, characterized in that: include: Columnar body, insert 3 and base 4; Among them, the columnar body is connected to the base 4 through the insert 3; a cylindrical groove is provided in the columnar body; the insert 3 is composed of a connected microneedle 301 and a base 302; a constant temperature solution is carried in the base 4; the base 302 is in contact with the constant temperature solution in the base 4; the microneedle is located in the cylindrical groove.

2. The device for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold material according to claim 1, characterized in that: The columnar body comprises a main plate 1 and a side plate 2; Among them, the two sides of the main board 1 are connected to the side panel 2 by screws 5; the two sides of the main board 1 are provided with a first semicircular groove; the side of the side panel 2 connected to the main board 1 is provided with a second semicircular groove; the first semicircular groove and the second semicircular groove are spliced ​​into the cylindrical groove.

3. A process for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold material, characterized in that: Executed by the device of claim 1 or 2, comprising: Step 1, obtaining raw materials of multiphase gradient osteochondral scaffold; Step 2: preparing at least two gels according to the raw materials of the multiphase gradient osteochondrocyte scaffold and a certain material ratio; Step 3, injecting one of the at least two gels into the columnar groove of the columnar body to a predetermined height; Step 4, adding a constant temperature solution to the base 4, and under the heat conduction effect between the base 302 of the insert 3 and the microneedle 301, ice crystals spread around the microneedle 301; Step 5, using low-temperature vacuum freeze-drying and chemical cross-linking to fix the gel in the columnar groove to obtain a multiphase gradient osteochondrocyte scaffold monolayer structure; Step 6, repeating steps 3-5 until the at least two gels are all converted into corresponding multiphase gradient osteochondrocyte scaffold single-layer structures; Step 7, loosen the screws 5 in sequence, remove the side plate 2, and pull out the insert 3 to obtain the multiphase gradient bone cartilage scaffold material.

4. The process for preparing a controllable mesoporous multiphase gradient osteochondrocyte scaffold material according to claim 3, characterized in that: In step 2, the preparation of at least two gels comprises: A first gel, a second gel and a third gel are prepared; the first gel corresponds to the subchondral bone layer of the multiphase gradient osteocartilage scaffold, the second gel corresponds to the middle cartilage layer of the multiphase gradient osteocartilage scaffold, and the third gel corresponds to the superficial cartilage layer of the multiphase gradient osteocartilage scaffold.

5. The process for preparing a controllable mesoporous multiphase gradient osteochondrocyte scaffold material according to claim 3, characterized in that: In step 1, the raw materials of the multiphase gradient osteochondrocyte scaffold include: hyaluronic acid, cartilage decellularized matrix, type I collagen and hydroxyapatite.

6. The process for preparing a controllable mesoporous multiphase gradient osteochondrocyte scaffold material according to claim 3, characterized in that: In step 4, the constant temperature solution includes liquid nitrogen and anhydrous ethanol.

7. The process for preparing a controllable mesoporous multiphase gradient osteochondrocyte scaffold material according to claim 3, characterized in that: In step 5, the chemical cross-linking includes: genipin, glutaraldehyde, sodium polytriphosphate, light curing and thermal cross-linking.

8. The process for preparing a controllable mesoporous multiphase gradient bone cartilage scaffold material according to claim 3, characterized in that: In step 2, the pore size of the multiphase gradient osteochondrocyte scaffold is controlled by adjusting the mass concentration of the gel.

9. The process for preparing a controllable mesoporous multiphase gradient osteochondrocyte scaffold material according to claim 3, characterized in that: In step 4, the pore size of the multiphase gradient osteochondral scaffold is controlled by adjusting the cooling rate.

10. A multiphase gradient osteochondral scaffold, characterized in that: The multiphase gradient osteochondrocyte scaffold is prepared by the process described in any one of claims 3-9.

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