Cartilage extracellular matrix microcapsules, tissue engineering scaffolds and preparation methods thereof

Abstract

The invention discloses a kind of chondrocyte extracellular matrix microcapsule, tissue engineering scaffold and their preparation method, belong to the field of tissue engineering. The chondrocyte extracellular matrix microcapsule of the present invention includes a core material and a wall material wrapped outside the core material; the mass ratio of the wall material to the core material is preferably 1: (1-3), and the wall material is chitosan; the core material includes growth factor, adsorbent, emulsifier, dispersant and base oil; the mass ratio of the growth factor, adsorbent, emulsifier, dispersant and base oil is (0.01-0.03): (1-10): (1-6): (1-4): (2-6). The tissue engineering scaffold prepared with chondrocyte extracellular matrix microcapsule has excellent mechanical properties, high water absorption, high pore size, no biological toxicity, not only provides a microenvironment for cell survival, but also can continuously promote the differentiation and proliferation of BMSC into chondrocytes, promote the regeneration and repair of cartilage.

Description

Technical Field

[0001] The invention belongs to the field of tissue engineering, and in particular relates to a cartilage cell extracellular matrix microcapsule, a tissue engineering scaffold and a preparation method thereof. Background Art

[0002] Articular cartilage is a kind of hyaline cartilage rich in type II collagen and glycosaminoglycans, which plays a role in bearing mechanical loads and lubricating joints during joint activities. Unlike other tissues, cartilage tissue does not have blood vessels or nerves, and its self-repair ability after degeneration and injury is extremely limited. It is difficult to repair after the injury reaches 3mm, and when the injury reaches 6mm, it will cause damage to the underlying bone. At present, the main surgical methods for treating articular cartilage injuries include microfracture, autologous chondrocyte implantation (ACI), mosaicplasty, and allogeneic cartilage transplantation. However, these methods all have certain limitations, cannot completely restore the original autologous cartilage, and the prognosis is uncertain. Based on this, implanting a suitable tissue engineering scaffold provides a better solution to this problem.

[0003] Cartilage tissue engineering is to culture and expand cartilage seed cells in vitro with the participation of some factors that are conducive to cell differentiation and proliferation. A certain amount of seed cells are cultured on a biomaterial scaffold and then transplanted to the cartilage defect site to form new cartilage tissue. After reshaping and integration with the body tissue, the damaged cartilage can be regenerated and repaired. The content of cartilage tissue engineering mainly consists of three aspects: seed cells, biomaterial scaffolds, and growth factors. Among them, the biomaterial scaffold, as a temporary substitute for the extracellular matrix, provides a three-dimensional geometric shape for the engineered cartilage tissue and a microenvironment for the survival of seed cells. It is the key to the success of cartilage tissue engineering.

[0004] At present, the materials used in biological scaffolds mainly include natural and synthetic materials. Synthetic polymers include polyglycolic acid, polylactic acid, polyglycolic acid complex, polyvinyl alcohol, etc., and natural polymers include cartilage extracellular matrix, agarose, gelatin, hyaluronic acid, etc. Among them, cartilage extracellular matrix is ​​widely used in tissue engineering scaffold materials due to its good biosafety and biocompatibility as well as cell recognition and adhesion sites.

[0005] Existing tissue engineering scaffolds only passively provide a microenvironment for cells to survive, and their performance is relatively poor, and they cannot promote the regeneration and differentiation of cartilage well. Summary of the invention

[0006] In view of this, the first purpose of the present invention is to provide a chondrocyte extracellular matrix microcapsule with a sustained release function, which can provide nutrition for chondrocyte seed cells for a long time, maintain the growth of chondrocyte seed cells in the cartilage defect site, and achieve the regeneration and repair of damaged cartilage.

[0007] The second purpose of the present invention is to provide a method for preparing the above-mentioned cartilage extracellular matrix microcapsules.

[0008] The third purpose of the present invention is to propose a cartilage extracellular matrix tissue engineering scaffold, which has excellent mechanical properties, high water absorption, high pore size, and no biological toxicity. It not only provides a microenvironment for cells to survive, but also can continuously promote the differentiation and proliferation of BMSCs into chondrocytes, thereby promoting the regeneration and repair of cartilage.

[0009] The fourth object of the present invention is to provide a method for preparing the above-mentioned cartilage extracellular matrix tissue engineering scaffold.

[0010] The cartilage extracellular matrix microcapsule of the present invention comprises a core material and a wall material wrapped outside the core material; the mass ratio of the wall material to the core material is preferably 1:(1-3), more preferably 1:(1-2.5), further preferably 1:(1.2-2), and most preferably 1:(1.4-1.6).

[0011] The wall material of the cartilage cell extracellular matrix microcapsule provided by the present invention is chitosan; the chitosan has a deacetylation degree of ≥90% and a molecular weight of 20-40w;

[0012] The core material includes growth factor, adsorbent, emulsifier, dispersant and base oil; the mass ratio of the growth factor, adsorbent, emulsifier, dispersant and base oil is (0.01-0.03):(1-10):(1-6):(1-4):(2-6); the most preferred ratio is 0.02:6:3:2:3.

[0013] The growth factors in the core material of the present invention include TPF-β1 and / or BMP-4. Preferably, the growth factors include TPF-β1 and BMP-4, which are compounded at a mass ratio of 1:1. The present invention adopts a compounding method of TPF-β1 and BMP-4 to improve the repair of cartilage damage by the scaffold, wherein TPF-β1 can promote the Sox-9 gene expression of bone marrow mesenchymal stem cells and the synthesis of cartilage extracellular matrix, and can promote the metabolism of chondrocytes; BMP-4 can increase the output of extracellular matrix, inhibit cell hypertrophy, and promote stem cell differentiation into cartilage; however, the production factor is easily inactivated, so a carrier is required to store the growth factor and release it slowly to increase the action time of the growth factor; the ultrastructure of the extracellular matrix is ​​a natural and excellent carrier of growth factors such as TPF-β1 and BMP-4. Using the extracellular matrix as an adsorbent for growth factors can effectively prolong the survival time of the growth factors.

[0014] The adsorbent is cartilage extracellular matrix, and preferably, the particle size of the cartilage extracellular matrix is ​​800-1200 meshes.

[0015] Preferably, the cartilage extracellular matrix of the present invention is derived from cattle, and the method for preparing the cartilage extracellular matrix comprises the following steps:

[0016] S1. Take the kneecap of a cow and open the cavity under sterile conditions. Use a scalpel to remove the cartilage without removing the subchondral bone.

[0017] S2. Soak and wash with PBS to remove blood and debris on the surface of the cartilage;

[0018] S3. Soak in 0.5 mol / L sodium hydroxide solution for inactivation, with a solid-liquid ratio of 1:5-1:10, m / V, at 4°C for 24-48 hours;

[0019] S4. Freeze the cartilage in liquid nitrogen and then grind it. The freezing time is 2-4 minutes. The grinding process is 1500 rpm and the grinding time is 1-2 minutes.

[0020] S5. Remove fat and cell membrane, add degreasing liquid, the material-liquid ratio is 1:3-1:5, m / V is oscillated at 37°C for 48 hours, and the liquid is changed every 12 hours. The degreasing liquid ratio is as follows:

[0021] 3-8 parts of sodium carbonate, 0.1-0.5 parts of EDTA-4Na, 1-3 parts of sodium bicarbonate, 0.5-1 parts of SDS, 0.5-1 parts of tritonx-100, 3-5 parts of isopropanol, 0.1-0.3 parts of AEBSF, 82.2-92.8 parts of purified water.

[0022] The degreasing solution uses a combination of weakly alkaline sodium carbonate and sodium bicarbonate for degreasing, which can effectively remove lipids in cartilage tissue and is relatively mild. The added SDS and triton x-100 can change the permeability of cell membranes, thereby rupturing cells, and can also increase the solubility of lipids. At the same time, EDTA-4Na is used as a heavy metal scavenger in the present invention, which can effectively reduce the heavy metal content in the obtained extracellular matrix and ensure the biological safety of the extracellular matrix.

[0023] S6. DNase and RNase digestion: the decellularized cartilage tissue was treated with a digestion enzyme mixture at a solid-liquid ratio of 1:1-1:2, m / V. The digestion enzyme mixture contained 50-100 U / ml DNase and 0.5-2 U / ml RNase. The mixture was placed in a constant temperature shaker at 37°C for 12-24 hours with an oscillation frequency of 100-180 rpm.

[0024] S7. repeatedly washing the decellularized cartilage tissue with water, freeze-drying it, and obtaining a solid cartilage extracellular matrix;

[0025] S8. Grind the solid extracellular matrix of cartilage after freezing with liquid nitrogen, the freezing time is 2-4 minutes, the grinding process is 1500 rpm, the grinding time is 1-2 minutes, after the extracellular matrix of cartilage returns to room temperature, place it in a 50°C constant temperature drying oven for 0.5-1h, first pass it through an 800-mesh sieve, take the powder under the sieve, then pass it through a 1200-mesh sieve, take the powder on the sieve, repeat the above operation 3-5 times, and finally obtain the extracellular matrix powder of cartilage according to the present invention as an adsorbent;

[0026] The cartilage extracellular matrix prepared by the method of the present invention can effectively remove lipids and cells in cartilage tissue, and can preserve collagen and glycosaminoglycans to a greater extent, and maintain the complete morphology, histology and ultrastructure of the extracellular matrix.

[0027] The emulsifier is Span 80 and / or Span 20.

[0028] The dispersant is preferably polyvinyl alcohol; the alcoholysis degree of the polyvinyl alcohol is preferably 98% to 100%; the viscosity of the polyvinyl alcohol is preferably 5-6 mPa.s; the polyvinyl alcohol preferably has a volatile matter of ≤5.0, a purity of ≥93.5, and an ash content of ≤0.7; the pH value of the polyvinyl alcohol is preferably 5 to 7; in the present invention, the most preferred polyvinyl alcohol is Aladdin brand, with the product number P139533.

[0029] The base oil is sweet almond oil.

[0030] The method for preparing cartilage extracellular matrix microcapsules of the present invention comprises:

[0031] S1) Preparation of core material solution: sequentially mix growth factor and adsorbent in PBS to obtain core material PBS solution; S2) Preparation of oil phase: mix emulsifier, dispersant and sweet almond oil and heat to obtain oil phase; S3) Preparation of wall material solution: add chitosan into 0.1 mol / L acetic acid aqueous solution, fully dissolve and filter to obtain wall material solution; S4) mix and stir the core material aqueous solution and the wall material solution, add the oil phase and continue stirring to obtain cartilage extracellular matrix microcapsule solution; S5) spray dry the cartilage extracellular matrix microcapsule solution to obtain the desired cartilage extracellular matrix microcapsules.

[0032] Specifically, in the above preparation method, the specific operation process of step S1 is as follows: first, the growth factor is added to PBS and stirred. The stirring speed during this process is preferably 500-800 rpm, and the stirring time is preferably 10-30 min, more preferably 15-25 min, and more preferably 20 min; then the adsorbent is slowly added and stirred. The stirring speed during this process is preferably 100-300 rpm; the time is preferably 20-50 min, more preferably 25-40 min, and more preferably 30 min, thereby obtaining a core material PBS solution.

[0033] The emulsifier, dispersant and sweet almond oil are mixed and heated to obtain an oil phase; the temperature of the mixed heating is preferably 50°C to 90°C, more preferably 60°C to 80°C, and more preferably 70°C; the time of the mixed heating is preferably 20 to 50 min, more preferably 20 to 40 min, and more preferably 30 min; the speed of the mixed stirring is preferably 500 to 800 rpm.

[0034] In step S3, chitosan is mixed and stirred with 0.1 mol / L acetic acid aqueous solution to obtain a wall material solution; the mass ratio of chitosan to acetic acid aqueous solution is preferably 1:(2-5), more preferably 1:(3-4); the rotation speed during the mixing and stirring process is preferably 500-800 rpm; the time is preferably 20-50 min, more preferably 20-40 min, and more preferably 30 min.

[0035] In step S4, when the core material aqueous solution and the wall material solution are mixed and stirred, the mixing and stirring speed is preferably 500 to 800 rpm, and the time is preferably 20 to 50 min, more preferably 20 to 40 min, and more preferably 30 min. Then, the oil phase is added and stirred continuously to obtain the cartilage cell extracellular matrix microcapsules. The stirring speed during this process is preferably 2000 to 2500 rpm, and the time is preferably 1 to 4 h, and more preferably 2 to 3 h.

[0036] The invention discloses a chondrocyte extracellular matrix tissue engineering scaffold, which comprises the following raw materials by weight: 0.5-1 part of chondrocyte extracellular matrix microcapsules, 0.5-1.5 parts of soluble chondrocyte extracellular matrix, 0.5-1.5 parts of carboxymethyl chitosan, 0.5-1.5 parts of gelatin, 1-2 parts of hydrochloric acid, 0.05-0.15 parts of inorganic salt and 92.35-96.95 parts of pure water.

[0037] Among them, cartilage extracellular matrix microcapsules have been introduced in detail above.

[0038] The soluble chondrocyte extracellular matrix is ​​obtained by further enzymatic hydrolysis of chondrocyte extracellular matrix powder, and the specific preparation process includes the following steps:

[0039] S1, prepare pepsin solution, the pepsin concentration is 0.1-0.3%, and use hydrochloric acid to adjust the pH to 1-3, preferably, 1.5-2.5, most preferably 1.8;

[0040] S2. Fully mix the cartilage extracellular matrix powder and the pepsin solution, with a solid-liquid ratio of 1:5-1:8, m / V, and oscillate at a constant temperature for 12-24 hours;

[0041] S3, centrifugation and filtration, taking the supernatant, adding 30% hydrogen peroxide solution to terminate the reaction;

[0042] S4. Freeze-drying the solution after the reaction is terminated to obtain the desired soluble extracellular matrix.

[0043] The concentration of the hydrochloric acid is 1 mol / L, and the inorganic salt is one or a mixture of two of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate.

[0044] The preparation method of the cartilage extracellular matrix tissue engineering scaffold comprises the following steps:

[0045] S1. Preparation of tissue engineering scaffold solution:

[0046] a. Take pure water, add hydrochloric acid and inorganic salt, start stirring, stirring time 10-20min, stirrer speed 300-600r / min;

[0047] b. Add carboxymethyl chitosan and continue stirring for 10-20 minutes at a stirring speed of 600-800 r / min;

[0048] c. Add gelatin, raise the temperature to 45-60°C, continue stirring for 10-20 minutes, and the mixer speed is 600-800r / min;

[0049] d. After cooling naturally to room temperature, add 0.5-1.5 parts of soluble chondrocyte extracellular matrix and continue stirring for 5-10 minutes at a stirring speed of 300-600 r / min;

[0050] e. Add 0.5-1 part of chondrocyte extracellular matrix microcapsules and continue stirring for 5-10 minutes at a stirring speed of 100-200 r / min;

[0051] S2, molding:

[0052] a. Weigh a specified amount of S1 tissue engineering scaffold solution and add it to the mold;

[0053] b. Pre-cool the mold in a 4-6℃ refrigerator for 10-20 minutes, then put it in a -20℃ refrigerator for 1-2 hours, and finally put it in a -80℃ refrigerator for deep cooling for 1-2 hours;

[0054] c. Place the frozen mold into a freeze dryer for freeze drying at -80°C for 12-24 hours;

[0055] S3, cross-linking: using carbodiimide and succinimide or glutaraldehyde as cross-linking agents, cross-linking at 4°C for 24-48h;

[0056] S4, freeze-drying: The cross-linked scaffold is repeatedly rinsed with purified water, and after the rinsing is completed, freeze-dried to finally obtain the cartilage extracellular matrix tissue engineering scaffold of the present invention.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The scaffold prepared by the present invention has excellent mechanical properties. The compressive strength of normal human cartilage is 1MPa. The compressive strength of the scaffold prepared by the present invention is better than that of cartilage itself. Therefore, after being implanted into the human body, it can provide good mechanical support. Moreover, the water absorption rate of the scaffold of the present invention is very high, which can reach more than 1800%, and the porosity is ≥85%. These properties can provide a very suitable environment for cell growth. At the same time, it can also continuously release growth factors to promote the proliferation and differentiation of bone marrow mesenchymal stem cells. Through testing, it can be seen that the scaffold of the present invention has no biological toxicity and can be applied to the treatment of organisms. After treatment, the cartilage grows well and basically completely covers the defect site, and the treatment effect is very excellent. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a product photo of the tissue engineering scaffold prepared in Example 1;

[0060] Figure 2 is a SEM image of the tissue engineering scaffold of Example 1;

[0061] Figure 3 The MTT test results of the tissue engineering scaffold in Example 1;

[0062] Figure 4 The sustained release curve of the growth factor TFP-β1 in each embodiment;

[0063] Figure 5 This is a picture of rabbit knee cartilage recovery in the blank control group of the animal experiment for 6 weeks;

[0064] Figure 6 This is a picture of cartilage recovery in the rabbit knees in the microfracture treatment group after 6 weeks of animal experiment;

[0065] Figure 7 This is a picture of the recovery of rabbit knee cartilage in the animal experiment after 6 weeks using the engineering scaffold of Example 1. DETAILED DESCRIPTION

[0066] In order to allow those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below in conjunction with the accompanying drawings.

[0067] The invention discloses a chondrocyte extracellular matrix tissue engineering scaffold, which comprises the following raw materials by weight: 0.5-1 part of chondrocyte extracellular matrix microcapsules, 0.5-1.5 parts of soluble chondrocyte extracellular matrix, 0.5-1.5 parts of carboxymethyl chitosan, 0.5-1.5 parts of gelatin, 1-2 parts of 1 mol / L hydrochloric acid, 0.05-0.15 parts of inorganic salts and 92.35-96.95 parts of pure water.

[0068] Among them, the chondrocyte extracellular matrix microcapsules include a core material and a wall material wrapped outside the core material; the mass ratio of the wall material to the core material is preferably 1:(1-3), more preferably 1:(1-2.5), more preferably 1:(1.2-2), and most preferably 1:(1.4-1.6).

[0069] The wall material of the chondrocyte extracellular matrix microcapsule provided by the present invention is chitosan; the chitosan has a deacetylation degree of ≥90% and a molecular weight of 20-40w; the core material comprises growth factor, adsorbent, emulsifier, dispersant and base oil (preferably sweet almond oil); the mass ratio of the growth factor, adsorbent, emulsifier, dispersant and base oil is (0.01-0.03):(1-10):(1-6):(1-4):(2-6); the most preferred ratio is 0.02:6:3:2:3.

[0070] The growth factors in the core material of the present invention include TPF-β1 and / or BMP-4. Preferably, the growth factors include TPF-β1 and BMP-4, which are compounded at a mass ratio of 1:1. The present invention adopts a compounding method of TPF-β1 and BMP-4 to improve the repair of cartilage damage by the scaffold, wherein TPF-β1 can promote the Sox-9 gene expression of bone marrow mesenchymal stem cells and the synthesis of cartilage extracellular matrix, and can promote the metabolism of chondrocytes; BMP-4 can increase the output of extracellular matrix, inhibit cell hypertrophy, and promote stem cell differentiation into cartilage; however, the production factor is easily inactivated, so a carrier is required to store the growth factor and release it slowly to increase the action time of the growth factor; the ultrastructure of the extracellular matrix is ​​a natural and excellent carrier of growth factors such as TPF-β1 and BMP-4. Using the extracellular matrix as an adsorbent for growth factors can effectively prolong the survival time of the growth factors.

[0071] The adsorbent is cartilage extracellular matrix, and preferably, the particle size of the cartilage extracellular matrix is ​​800-1200 meshes.

[0072] Preferably, the cartilage extracellular matrix of the present invention is derived from cattle, and the method for preparing the cartilage extracellular matrix comprises the following steps:

[0073] S1. Take the kneecap of a cow and open the cavity under sterile conditions. Use a scalpel to remove the cartilage without removing the subchondral bone.

[0074] S2. Soak and wash with PBS to remove blood and debris on the surface of the cartilage;

[0075] S3. Soak in 0.5 mol / L sodium hydroxide solution for inactivation, with a solid-liquid ratio of 1:5-1:10, m / V, at 4°C for 24-48 hours;

[0076] S4. Freeze the cartilage in liquid nitrogen and then grind it. The freezing time is 2-4 minutes. The grinding process is 1500 rpm and the grinding time is 1-2 minutes.

[0077] S5. Remove fat and cell membrane, add degreasing liquid, the material-liquid ratio is 1:3-1:5, m / V is oscillated at 37°C for 48 hours, and the liquid is changed every 12 hours. The degreasing liquid ratio is as follows:

[0078] 3-8 parts of sodium carbonate, 0.1-0.5 parts of EDTA-4Na, 1-3 parts of sodium bicarbonate, 0.5-1 parts of SDS, 0.5-1 parts of tritonx-100, 3-5 parts of isopropanol, 0.1-0.3 parts of AEBSF, 82.2-92.8 parts of purified water.

[0079] The degreasing solution uses a combination of weakly alkaline sodium carbonate and sodium bicarbonate for degreasing, which can effectively remove lipids in cartilage tissue and is relatively mild. The added SDS and triton x-100 can change the permeability of cell membranes, thereby rupturing cells, and can also increase the solubility of lipids. At the same time, EDTA-4Na is used as a heavy metal scavenger in the present invention, which can effectively reduce the heavy metal content in the obtained extracellular matrix and ensure the biological safety of the extracellular matrix.

[0080] S6. DNase and RNase digestion: the decellularized cartilage tissue was treated with a digestion enzyme mixture at a solid-liquid ratio of 1:1-1:2, m / V. The digestion enzyme mixture contained 50-100 U / ml DNase and 0.5-2 U / ml RNase. The mixture was placed in a constant temperature shaker at 37°C for 12-24 hours with an oscillation frequency of 100-180 rpm.

[0081] S7. repeatedly washing the decellularized cartilage tissue with water, freeze-drying it, and obtaining a solid cartilage extracellular matrix;

[0082] S8. Grind the solid extracellular matrix of cartilage after freezing with liquid nitrogen, the freezing time is 2-4 minutes, the grinding process is 1500 rpm, the grinding time is 1-2 minutes, after the extracellular matrix of cartilage returns to room temperature, place it in a 50°C constant temperature drying oven for 0.5-1h, first pass it through an 800-mesh sieve, take the powder under the sieve, then pass it through a 1200-mesh sieve, take the powder on the sieve, repeat the above operation 3-5 times, and finally obtain the extracellular matrix powder of cartilage according to the present invention as an adsorbent;

[0083] The cartilage extracellular matrix prepared by the method of the present invention can effectively remove lipids and cells in cartilage tissue, and can preserve collagen and glycosaminoglycans to a greater extent, and maintain the complete morphology, histology and ultrastructure of the extracellular matrix.

[0084] The emulsifier is Span 80 and / or Span 20.

[0085] The dispersant is preferably polyvinyl alcohol; the alcoholysis degree of the polyvinyl alcohol is preferably 98% to 100%; the viscosity of the polyvinyl alcohol is preferably 5-6 mPa.s; the polyvinyl alcohol preferably has a volatile matter of ≤5.0, a purity of ≥93.5, and an ash content of ≤0.7; the pH value of the polyvinyl alcohol is preferably 5 to 7; in the present invention, the most preferred polyvinyl alcohol is Aladdin brand, with the product number P139533.

[0086] The method for preparing cartilage extracellular matrix microcapsules of the present invention comprises:

[0087] S1) Preparation of core material solution: sequentially mix growth factor and adsorbent in PBS to obtain core material PBS solution; S2) Preparation of oil phase: mix emulsifier, dispersant and sweet almond oil and heat to obtain oil phase; S3) Preparation of wall material solution: add chitosan into 0.1 mol / L acetic acid aqueous solution, fully dissolve and filter to obtain wall material solution; S4) mix and stir the core material aqueous solution and the wall material solution, add the oil phase and continue stirring to obtain cartilage extracellular matrix microcapsule solution; S5) spray dry the cartilage extracellular matrix microcapsule solution to obtain the desired cartilage extracellular matrix microcapsules.

[0088] Specifically, in the above preparation method, the specific operation process of step S1 is as follows: first, the growth factor is added to PBS and stirred. The stirring speed during this process is preferably 500-800 rpm, and the stirring time is preferably 10-30 min, more preferably 15-25 min, and more preferably 20 min; then the adsorbent is slowly added and stirred. The stirring speed during this process is preferably 100-300 rpm; the time is preferably 20-50 min, more preferably 25-40 min, and more preferably 30 min, thereby obtaining a core material PBS solution.

[0089] The emulsifier, dispersant and sweet almond oil are mixed and heated to obtain an oil phase; the temperature of the mixed heating is preferably 50°C to 90°C, more preferably 60°C to 80°C, and more preferably 70°C; the time of the mixed heating is preferably 20 to 50 min, more preferably 20 to 40 min, and more preferably 30 min; the speed of the mixed stirring is preferably 500 to 800 rpm.

[0090] In step S3, chitosan is mixed and stirred with 0.1 mol / L acetic acid aqueous solution to obtain a wall material solution; the mass ratio of chitosan to acetic acid aqueous solution is preferably 1:(2-5), more preferably 1:(3-4); the rotation speed during the mixing and stirring process is preferably 500-800 rpm; the time is preferably 20-50 min, more preferably 20-40 min, and more preferably 30 min.

[0091] In step S4, when the core material aqueous solution and the wall material solution are mixed and stirred, the mixing and stirring speed is preferably 500 to 800 rpm, and the time is preferably 20 to 50 min, more preferably 20 to 40 min, and more preferably 30 min. Then, the oil phase is added and stirred continuously to obtain the cartilage cell extracellular matrix microcapsules. The stirring speed during this process is preferably 2000 to 2500 rpm, and the time is preferably 1 to 4 h, and more preferably 2 to 3 h.

[0092] The soluble chondrocyte extracellular matrix is ​​obtained by further enzymatic hydrolysis of the chondrocyte extracellular matrix, and the specific preparation process includes the following steps:

[0093] S1, prepare pepsin solution, the pepsin concentration is 0.1-0.3%, and use hydrochloric acid to adjust the pH to 1-3, preferably, 1.5-2.5, most preferably 1.8;

[0094] S2. Fully mix the cartilage extracellular matrix powder and the pepsin solution, with a solid-liquid ratio of 1:5-1:8, m / V, and oscillate at a constant temperature for 12-24 hours;

[0095] S3, centrifugation and filtration, taking the supernatant, adding 30% hydrogen peroxide solution to terminate the reaction;

[0096] S4. Freeze-drying the solution after the reaction is terminated to obtain the desired soluble extracellular matrix.

[0097] The concentration of the hydrochloric acid is 1 mol / L, and the inorganic salt is one or a mixture of two of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate.

[0098] The method for preparing the cartilage extracellular matrix tissue engineering scaffold of the present invention comprises the following steps:

[0099] S1. Preparation of tissue engineering scaffold solution:

[0100] a. Take pure water, add hydrochloric acid and inorganic salt, start stirring, stirring time 10-20min, stirrer speed 300-600r / min;

[0101] b. Add carboxymethyl chitosan and continue stirring for 10-20 minutes at a stirring speed of 600-800 r / min;

[0102] c. Add gelatin, raise the temperature to 45-60°C, continue stirring for 10-20 minutes, and the mixer speed is 600-800r / min;

[0103] d. After cooling naturally to room temperature, add 0.5-1.5 parts of soluble chondrocyte extracellular matrix and continue stirring for 5-10 minutes at a stirring speed of 300-600 r / min;

[0104] e. Add 0.5-1 part of chondrocyte extracellular matrix microcapsules and continue stirring for 5-10 minutes at a stirring speed of 100-200 r / min;

[0105] S2, molding:

[0106] a. Weigh a specified amount of S1 tissue engineering scaffold solution and add it to the mold;

[0107] b. Pre-cool the mold in a 4-6℃ refrigerator for 10-20 minutes, then put it in a -20℃ refrigerator for 1-2 hours, and finally put it in a -80℃ refrigerator for deep cooling for 1-2 hours;

[0108] c. Place the frozen mold into a freeze dryer for freeze drying at -80°C for 12-24 hours;

[0109] S3, cross-linking: using carbodiimide and succinimide or glutaraldehyde as cross-linking agents, cross-linking at 4°C for 24-48h;

[0110] S4, freeze-drying: The cross-linked scaffold is repeatedly rinsed with purified water, and after the rinsing is completed, freeze-dried to finally obtain the cartilage extracellular matrix tissue engineering scaffold of the present invention.

[0111] The present application is further described below with reference to Examples 1-5.

[0112] Example 1

[0113] The cartilage extracellular matrix tissue engineering scaffold of this embodiment includes the following components in parts by weight:

[0114] 0.5 parts by weight of chondrocyte extracellular matrix microcapsules, 1 part by weight of soluble chondrocyte extracellular matrix, 1 part by weight of carboxymethyl chitosan, 1 part by weight of gelatin, 1 part by weight of 1 mol / L hydrochloric acid, 0.05 parts by weight of inorganic salt (sodium chloride), and 95.45 parts by weight of pure water;

[0115] The preparation method of the cartilage extracellular matrix tissue engineering scaffold comprises the following steps:

[0116] S1. Preparation of tissue engineering scaffold solution: a. Take pure water, add hydrochloric acid and inorganic salt, start stirring, stir for 10 minutes, and stir at a speed of 400 r / min; b. Add carboxymethyl chitosan, continue stirring for 10 minutes, and stir at a speed of 600 r / min; c. Add gelatin, heat to 50°C, continue stirring for 20 minutes, and stir at a speed of 700 r / min; d. After naturally cooling to room temperature, add soluble chondrocyte extracellular matrix, continue stirring for 5 minutes, and stir at a speed of 400 r / min; e. Add chondrocyte extracellular matrix microcapsules, continue stirring for 5 minutes, and stir at a speed of 100 r / min;

[0117] S2, molding: a. Take the above tissue engineering scaffold solution and add it to the mold; b. Put the mold into a 4-6℃ refrigerator for precooling for 20 minutes, then put it into a -20℃ refrigerator for 2 hours, and finally put it into a -80℃ refrigerator for deep cooling for 1 hour; c. Put the frozen mold into a freeze dryer for freeze drying, the freeze dryer temperature is -80℃, and the freeze drying time is 24 hours;

[0118] S3, cross-linking: using 50 mM carbodiimide and 10 mM succinimide as cross-linking agents, cross-linking at 4 °C for 24 h;

[0119] S4, freeze-drying: The cross-linked scaffold is repeatedly rinsed with purified water, and after the rinsing is completed, freeze-dried to finally obtain the cartilage extracellular matrix tissue engineering scaffold of the present invention.

[0120] The cartilage cell extracellular matrix microcapsules include a core material and a wall material wrapped outside the core material, and the mass ratio of the wall material to the core material is preferably 1:1.5; the wall material is chitosan, the chitosan deacetylation degree is ≥90%, and the molecular weight is 20-40w; the core material includes: growth factor, adsorbent, emulsifier, dispersant and base oil (sweet almond oil); the mass ratio of the growth factor, adsorbent, emulsifier, dispersant and base oil is 0.02:6:3:2:3;

[0121] The growth factor is TPF-β1 and BMP-4 in a 1:1 ratio; the adsorbent is cartilage cell extracellular matrix; the cartilage cell extracellular matrix is ​​derived from cattle, and the particle size of the cartilage cell extracellular matrix is ​​800-1200 meshes.

[0122] The method for preparing cartilage extracellular matrix as an adsorbent comprises the following steps:

[0123] S1. Take the kneecap of the cow and open the cavity under sterile conditions. Use a scalpel to remove the cartilage without removing the subchondral bone.

[0124] S2. Soak and wash with PBS to remove blood and debris on the surface of the cartilage;

[0125] S3, soak inactivate with 0.5 mol / L sodium hydroxide solution, with a solid-liquid ratio of 1:5 m / V, and soak at 4°C for 48 hours;

[0126] S4, freezing the cartilage in liquid nitrogen, and then grinding it, the freezing time is 3 minutes, the grinding process is 1500 rpm, and the grinding time is 2 minutes;

[0127] S5, remove fat and cell membrane, add degreasing liquid, the solid-liquid ratio is 1:3, m / V is oscillated at a constant temperature of 37°C for 48 hours, and the liquid is changed every 12 hours; the degreasing liquid has the following formula: 5 parts of sodium carbonate, 0.2 parts of EDTA4Na, 2 parts of sodium bicarbonate, 1 part of SDS, 1 part of tritonx-100, 5 parts of isopropanol, 0.1 parts of AEBSF, and 85.7 parts of purified water.

[0128] S6, DNase and RNase digestion, the decellularized cartilage tissue was treated with a digestion enzyme mixture, the material-liquid ratio was 1:1, m / V, the digestion enzyme mixture contained 50U / ml DNase and 1U / ml RNase, and the mixture was placed in a constant temperature shaker at 37°C for 24 hours with an oscillation frequency of 150rpm;

[0129] S7, repeatedly washing the decellularized cartilage tissue with water, and freeze-drying it to obtain a cartilage extracellular matrix solid;

[0130] S8, the chondrocyte extracellular matrix of step S7 is cryo-ground with liquid nitrogen, the freezing time is 3 minutes, the grinding process is 1500 rpm, the grinding time is 2 minutes, after the chondrocyte extracellular matrix returns to room temperature, it is placed in a 50° C. constant temperature drying oven for 1 hour, first passed through an 800-mesh sieve, the powder under the sieve is taken, and then passed through a 1200-mesh sieve, the powder on the sieve is taken, and the above operation is repeated 3-5 times to finally obtain the chondrocyte extracellular matrix powder of the present invention;

[0131] The emulsifier is Span 80 and / or Span 20.

[0132] The dispersant is preferably polyvinyl alcohol; the alcoholysis degree of the polyvinyl alcohol is preferably 98% to 100%; the viscosity of the polyvinyl alcohol is preferably 5-6 mPa·s; the polyvinyl alcohol preferably has a volatile matter of ≤5.0, a purity of ≥93.5, and an ash content of ≤0.7; the pH value of the polyvinyl alcohol is preferably 5 to 7; in the present invention, the most preferred polyvinyl alcohol is Aladdin's P139533.

[0133] The present invention also provides a method for preparing cartilage extracellular matrix microcapsules, comprising: S1, core material solution preparation: mixing growth factors and adsorbents in PBS to obtain a core material PBS solution; S2, oil phase preparation: mixing an emulsifier, a dispersant and sweet almond oil and heating them to obtain an oil phase; S3, wall material solution preparation: adding chitosan to a 0.1 mol / L acetic acid aqueous solution, fully dissolving and filtering to obtain a wall material solution; S4, mixing the core material aqueous solution and the wall material solution, adding the oil phase and continuing to stir to obtain a cartilage extracellular matrix microcapsule solution; S5, spray-drying the cartilage extracellular matrix microcapsule solution to obtain the desired microcapsules.

[0134] In this embodiment, in the above preparation method, the specific operation process of preparing the core material solution is as follows: first, the growth factor is stirred and diluted in PBS to the required content, the stirring speed is preferably 600r / min, and the time is preferably 20min; then the adsorbent is slowly added and stirred, the stirring speed is preferably 100r / min, and the time is preferably 30min, to obtain the core material PBS solution.

[0135] During the oil phase preparation process, the temperature for mixing and heating the emulsifier, dispersant and sweet almond oil is preferably 70° C., and the mixing and heating time is preferably 30 min; the stirring speed during the mixing process is preferably 800 rpm.

[0136] During the preparation of the wall material solution, the mass ratio of chitosan to acetic acid aqueous solution is preferably 1:(3-4); the rotation speed during the mixing process is preferably 500 rpm, and the mixing time is preferably 30 min.

[0137] The core material aqueous solution and the wall material solution are mixed and stirred at a speed of preferably 500 rpm for 30 min, and then the oil phase is added and stirred continuously at a speed of preferably 2500 rpm for 2 h.

[0138] The soluble chondrocyte extracellular matrix is ​​obtained by further enzymatic hydrolysis of the chondrocyte extracellular matrix, and the preparation process comprises the following steps:

[0139] S1. Prepare a pepsin solution with a pepsin concentration of 0.2% and adjust the pH to 1.8 using hydrochloric acid;

[0140] S2, fully mix the cartilage extracellular matrix powder and pepsin solution, with a solid-liquid ratio of 1:5, m / V, and oscillate at a constant temperature for 24 hours;

[0141] S3, centrifugation and filtration, taking the supernatant, adding 30% hydrogen peroxide solution to terminate the reaction;

[0142] S4. Freeze-dry the terminated solution to obtain the desired soluble extracellular matrix.

[0143] Example 2

[0144] The cartilage extracellular matrix tissue engineering scaffold of this embodiment includes the following components in parts by weight:

[0145] 0.5 parts of chondrocyte extracellular matrix microcapsules, 0.5 parts of soluble chondrocyte extracellular matrix, 0.5 parts of carboxymethyl chitosan, 0.5 parts of gelatin, 1 part of 1 mol / L hydrochloric acid, 0.05 parts of inorganic salt (sodium chloride), and 96.95 parts of pure water.

[0146] The preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in this example is the same as the preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in Example 1.

[0147] In addition, other aspects, such as the composition and preparation method of the chondrocyte extracellular matrix microcapsules, the soluble chondrocyte extracellular matrix, the material selection of each component, etc., are consistent with those in Example 1.

[0148] Example 3

[0149] The cartilage extracellular matrix tissue engineering scaffold of this embodiment includes the following components in parts by weight:

[0150] 0.5 parts of chondrocyte extracellular matrix microcapsules, 0.7 parts of soluble chondrocyte extracellular matrix, 0.8 parts of carboxymethyl chitosan, 0.7 parts of gelatin, 1 part of 1 mol / L hydrochloric acid, 0.05 parts of inorganic salt (sodium chloride), and 96.25 parts of pure water.

[0151] The preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in this example is the same as the preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in Example 1.

[0152] In addition, other aspects, such as the composition and preparation method of the chondrocyte extracellular matrix microcapsules, the soluble chondrocyte extracellular matrix, the material selection of each component, etc., are consistent with those in Example 1.

[0153] Example 4

[0154] The cartilage extracellular matrix tissue engineering scaffold of this embodiment includes the following components in parts by weight:

[0155] 0.5 parts of chondrocyte extracellular matrix microcapsules, 1.2 parts of soluble chondrocyte extracellular matrix, 1.2 parts of carboxymethyl chitosan, 1.2 parts of gelatin, 1.5 parts of 0.1 mol / L hydrochloric acid, 0.05 parts of inorganic salt (sodium chloride), and 94.35 parts of pure water.

[0156] The preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in this example is the same as the preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in Example 1.

[0157] In addition, other aspects, such as the composition and preparation method of the chondrocyte extracellular matrix microcapsules, the soluble chondrocyte extracellular matrix, the material selection of each component, etc., are consistent with those in Example 1.

[0158] Example 5

[0159] The cartilage extracellular matrix tissue engineering scaffold of this embodiment includes the following components in parts by weight:

[0160] 0.8 parts of chondrocyte extracellular matrix microcapsules, 1 part of soluble chondrocyte extracellular matrix, 1 part of carboxymethyl chitosan, 1 part of gelatin, 1 part of 1 mol / L hydrochloric acid, 0.05 parts of inorganic salt (sodium chloride), and 95.15 parts of pure water.

[0161] The preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in this example is the same as the preparation method of the chondrocyte extracellular matrix tissue engineering scaffold in Example 1.

[0162] In addition, other aspects, such as the composition and preparation method of the chondrocyte extracellular matrix microcapsules, the soluble chondrocyte extracellular matrix, the material selection of each component, etc., are consistent with those in Example 1.

[0163] Taking Example 1 as an example, the cartilage extracellular matrix tissue engineering scaffold prepared by the present invention is as follows Figure 1 As shown in Figure 2, after scanning electron microscopy observation, it can be seen that the scaffold has a spatial reticular structure with a pore size of more than 100um and a large gap, which is conducive to the migration and adhesion of cells inside the scaffold. Figure 2 shown.

[0164] Performance Testing

[0165] Water absorption rate refers to GB / T 1034-2008

[0166] The sample is immersed in 23℃ distilled water and placed at a specified temperature for a certain period of time. The mass difference between the test sample at the beginning of the experiment and after water absorption is expressed as the percentage of the mass difference to the initial mass.

[0167] Calculation formula

[0168] c=(m2-m1) / m1*100

[0169] In the formula

[0170] C—water absorption mass fraction of the sample, expressed in %;

[0171] m1—weight of the sample before immersion and after drying, in mg;

[0172] m2—weight of the sample after immersion, in mg;

[0173] Table 1

[0174] Example 1 Example 2 Example 3 Example 4 Example 5 Water absorption (%) 2200 1804 1980 2315 2235

[0175] Compression strength refers to standard GB / T1041-2008

[0176] The calculation formula is:

[0177] σ=F / A

[0178] Where:

[0179] σ—stress parameter, in megapascals (MPa);

[0180] F—test force, in Newton (N);

[0181] A—Original area of ​​the sample, in square millimeters (mm 2 );

[0182] Table 2

[0183] Example 1 Example 2 Example 3 Example 4 Example 5 Compression strength(MPa) 1.84 1.46 1.65 2.21 1.98

[0184] Porosity reference standard YY / T 1616-2018

[0185] Select a test tube with a scale and fill it with a certain amount of anhydrous ethanol to measure the volume of anhydrous ethanol V1; take 8 stent samples and immerse them in anhydrous ethanol to fully immerse them, connect negative pressure to suck out the remaining air in the sample, so that the anhydrous ethanol is fully filled in the porous stent until no bubbles escape, and obtain the total volume of the stent and anhydrous ethanol V2; take out the stent soaked with anhydrous ethanol and read the value to obtain the remaining anhydrous ethanol volume V3; test each sample 3 times and take the average value.

[0186] Calculation formula

[0187] E = (V1-V3) / (V2-V3), where:

[0188] V1—initial volume of anhydrous ethanol;

[0189] V2—the volume of anhydrous ethanol in the test tube after the porous support is fully filled with anhydrous ethanol;

[0190] V3—the volume of anhydrous ethanol remaining after the stent filled with anhydrous ethanol is taken out;

[0191] Table 3

[0192] Example 1 Example 2 Example 3 Example 4 Example 5 Porosity (%) 87.4 92.5 90.1 95.3 86.8

[0193] MTT toxicity test refers to GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5 In Vitro Cytotoxicity Test" and uses the MTT method to determine the potential cytotoxicity of the sample. Confirm whether the material extract can cause a toxic reaction after contacting the cultured cells.

[0194] Evaluation Criteria

[0195] (1) The viability of the 50% sample extract should be at least equal to or higher than that of the 100% sample extract; otherwise, the experiment should be repeated.

[0196] (2) The lower the cell viability %, the greater the potential cytotoxicity.

[0197] (3) The cell viability was less than 70% of the blank group, indicating that the sample had potential cytotoxicity.

[0198] (4) The cell viability % of the 100% test sample extract is the final result.

[0199] The test results are as follows Figure 3 shown.

[0200] Growth factor sustained release

[0201] Add 0.01g of cartilage extracellular matrix tissue engineering scaffold material into 1mL of phosphate buffered saline PBS (pH 7.4), and then shake at 80rpm at room temperature (20℃~25℃). After a certain period of sustained release, centrifuge at 10000rcf for 5 minutes, take the supernatant and save it, and add an equal amount of fresh PBS buffer (pH 7.4) to continue the sustained release; take multiple supernatant samples in this way during the sustained release period. Use an ELISA kit to determine the concentration of growth factor TGF-β1 in the supernatant sample. Test 3 samples in each group and take the average result. See the results. Figure 4 It can be seen that the sustained release time of the engineering stent of the present invention can reach an ultra-long 42 days.

[0202] Small animal experiments

[0203] Test method:

[0204] S1. Induction of anesthesia: New Zealand rabbits were used as experimental animals, and xylazine hydrochloride injection was used to induce anesthesia in the experimental rabbits by intramuscular injection;

[0205] S2. Modeling: Use a 4mm diameter dental drill to drill a cartilage defect model with a diameter of 4mm and a depth of 2mm on the femoral cartilage of the knee joint, and rinse the wound with saline;

[0206] S3. Drug treatment: The engineering scaffold of Example 1 was placed in the knee cartilage defect model of New Zealand rabbits, sutured, and the recovery was observed. At the same time, a blank control group without treatment and a microfracture treatment group were set up to compare the joint recovery of the three groups.

[0207] The scaffold prepared by the present invention has excellent mechanical properties. The compressive strength of normal human cartilage is 1MPa. The compressive strength of the scaffold prepared by the present invention is better than that of the cartilage itself. Therefore, after being implanted into the human body, it can provide better mechanical support. In addition, the water absorption rate of the scaffold of the present invention is very high, which can reach more than 1800%, and the porosity is ≥85%. These properties can provide a very suitable environment for cell growth. At the same time, it can also continuously release growth factors to promote the proliferation and differentiation of bone marrow mesenchymal stem cells. By conducting a cell growth test, it can be seen that the scaffold of the present invention has no biological toxicity and can be used in the treatment of organisms; Figure 5-7 It can be seen that without treatment or with microfracture treatment, only fibrous cartilage will be formed in the defect area. However, after treatment with the scaffold of the present invention, the cartilage grows well and basically completely covers the defect area, and the treatment effect is very excellent.

[0208] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. A cartilage extracellular matrix microcapsule, characterized in that: It is composed of a core material and a wall material wrapped around the core material; The mass ratio of the wall material to the core material is 1:(1-3); The wall material is chitosan; The core material is a growth factor, an adsorbent, an emulsifier, a dispersant and a base oil; the mass ratio of the growth factor, the adsorbent, the emulsifier, the dispersant and the base oil is (0.01-0.03): (1-10): (1-6): (1-4): (2-6); The growth factors in the core material include TPF-β1 and BMP-4, which are compounded at a mass ratio of 1:1; The adsorbent is bovine cartilage extracellular matrix, and the preparation method of the cartilage extracellular matrix comprises the following steps: S1. Take the cow kneecap, open the cavity under sterile conditions, and remove the cartilage using a scalpel; S2. Soak and wash with PBS to remove blood and debris on the surface of the cartilage; S3. Soak in 0.5 mol / L sodium hydroxide solution for inactivation, with a solid-liquid ratio of 1:5-1:10, m / V, at 4°C for 24-48 hours; S4. Freeze the cartilage in liquid nitrogen and then grind it. The freezing time is 2-4 minutes. The grinding process is 1500 rpm and the grinding time is 1-2 minutes. S5. Remove fat and cell membrane, add degreasing solution, the material-liquid ratio is 1:3-1:5, m / V, oscillate at a constant temperature of 37°C for 48 hours, and change the solution every 12 hours. The degreasing solution has the following composition: 3-8 parts of sodium carbonate, 0.1-0.5 parts of EDTA-4Na, 1-3 parts of sodium bicarbonate, 0.5-1 parts of SDS, 0.5-1 parts of triton x-100, 3-5 parts of isopropanol, 0.1-0.3 parts of AEBSF, and 82.2-92.8 parts of purified water; S6. DNase and RNase digestion: the decellularized cartilage tissue was treated with a digestion enzyme mixture at a solid-liquid ratio of 1:1-1:2, m / V. The digestion enzyme mixture contained 50-100 U / ml DNase and 0.5-2 U / ml RNase. The mixture was placed in a constant temperature shaker at 37°C for 12-24 hours with an oscillation frequency of 100-180 rpm. S7. repeatedly washing the decellularized cartilage tissue with water, freeze-drying it, and obtaining a solid cartilage extracellular matrix; S8. Freeze the solid chondrocyte extracellular matrix with liquid nitrogen and then grind it. The freezing time is 2-4 minutes. The grinding process is 1500 rpm and the grinding time is 1-2 minutes. After the chondrocyte extracellular matrix returns to room temperature, place it in a 50°C constant temperature drying oven for 0.5-1h, first pass it through an 800-mesh sieve, take the powder under the sieve, then pass it through a 1200-mesh sieve, take the powder on the sieve, repeat the above operation 3-5 times to finally obtain the chondrocyte extracellular matrix powder.

2. The cartilage extracellular matrix microcapsule according to claim 1, characterized in that The mass ratio of the wall material to the core material is 1:(1-2.5).

3. The cartilage extracellular matrix microcapsule according to claim 2, characterized in that: The mass ratio of the wall material to the core material is 1:(1.2-2).

4. The chondrocyte extracellular matrix microcapsule according to claim 3, characterized in that: The mass ratio of the wall material to the core material is 1:(1.4-1.6).

5. The cartilage extracellular matrix microcapsule according to claim 1, characterized in that: The mass ratio of the growth factor, adsorbent, emulsifier, dispersant and base oil is 0.02:6:3:2:

3.

6. The cartilage extracellular matrix microcapsule according to claim 1, characterized in that: The emulsifier is Span 80 and / or Span 20; The base oil is sweet almond oil; The dispersant is polyvinyl alcohol; the alcoholysis degree of the polyvinyl alcohol is 98% to 100%; the viscosity of the polyvinyl alcohol is 5-6 mPa.s; the volatile matter of the polyvinyl alcohol is ≤5.0, the purity is ≥93.5, and the ash content is ≤0.7; and the pH value of the polyvinyl alcohol is 5 to 7.

7. The method for preparing cartilage extracellular matrix microcapsules according to claim 1, characterized in that: The steps include: S1. Preparation of core material solution: sequentially mixing growth factors and adsorbents in PBS to obtain a core material PBS solution; S2, oil phase preparation: mixing and heating the emulsifier, dispersant and sweet almond oil to obtain an oil phase; S3, wall material solution preparation: chitosan is added into 0.1 mol / L acetic acid aqueous solution, fully dissolved and filtered to obtain a wall material solution; S4, mixing the core material aqueous solution and the wall material solution, adding the oil phase and continuing to stir to obtain a cartilage extracellular matrix microcapsule solution; S5. Spray-drying the chondrocyte extracellular matrix microcapsule solution to obtain the desired chondrocyte extracellular matrix microcapsules.

8. The method for preparing cartilage extracellular matrix microcapsules according to claim 7, characterized in that: The specific operation process of step S1 is as follows: first, the growth factor is added to PBS and stirred, during which the stirring speed is 500 to 800 rpm and the stirring time is 10 to 30 minutes; then the adsorbent is slowly added and stirred, during which the stirring speed is 100 to 300 rpm and the stirring time is 20 to 50 minutes, thereby obtaining a core material PBS solution; In step S2, the emulsifier, dispersant and base oil are mixed and heated to obtain an oil phase; the temperature of the mixed heating is 50°C to 90°C; the time of the mixed heating is 20 to 50 minutes; the speed of the mixed stirring is 500 to 800 rpm; In step S3, chitosan is mixed with 0.1 mol / L acetic acid aqueous solution to obtain a wall material solution; the mass ratio of chitosan to acetic acid aqueous solution is 1:(2-5); the speed during the mixing and stirring process is 500-800 rpm; the time is 20-50 min; In step S4, when the core material aqueous solution and the wall material solution are mixed and stirred, the mixing and stirring speed is 500-800 rpm, and the time is 20-50 min; then the oil phase is added and continued stirring to obtain cartilage extracellular matrix microcapsules. During this process, the stirring speed is continued at 2000-2500 rpm, and the time is 1-4 h.

9. A cartilage extracellular matrix tissue engineering scaffold, characterized in that: The invention comprises the following raw materials by weight: 0.5-1 part of the chondrocyte extracellular matrix microcapsules according to any one of claims 1 to 6, 0.5-1.5 parts of soluble chondrocyte extracellular matrix, 0.5-1.5 parts of carboxymethyl chitosan, 0.5-1.5 parts of gelatin, 1-2 parts of hydrochloric acid, 0.05-0.15 parts of inorganic salts, and 92.35-96.95 parts of pure water; the soluble chondrocyte extracellular matrix is ​​obtained by further enzymolysis of bovine chondrocyte extracellular matrix with a pepsin solution.

10. The cartilage extracellular matrix tissue engineering scaffold according to claim 9, characterized in that: The preparation process of soluble cartilage extracellular matrix includes the following steps: S1. Prepare a pepsin solution with a pepsin concentration of 0.1-0.3%, and adjust the pH to 1-3 using hydrochloric acid; S2. Fully mix the bovine cartilage extracellular matrix powder and the pepsin solution, with a solid-liquid ratio of 1:5-1:8, m / V, and oscillate at a constant temperature for 12-24 hours; S3, centrifugation and filtration, taking the supernatant, adding 30% hydrogen peroxide solution to terminate the reaction; S4. Freeze-drying the solution after the reaction is terminated to obtain the desired soluble extracellular matrix.

11. The cartilage extracellular matrix tissue engineering scaffold according to claim 9, characterized in that: The concentration of the hydrochloric acid is 1 mol / L; The inorganic salt is one or a mixture of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate.

12. The method for preparing a cartilage extracellular matrix tissue engineering scaffold according to claim 9, characterized in that: The following steps are involved: S1. Preparation of tissue engineering scaffold solution: a. Take pure water, add hydrochloric acid and inorganic salt, start stirring, stirring time 10-20min, stirrer speed 300-600r / min; b. Add carboxymethyl chitosan and continue stirring for 10-20 minutes at a stirring speed of 600-800 r / min; c. Add gelatin, raise the temperature to 45-60°C, continue stirring for 10-20 minutes, and the mixer speed is 600-800r / min; d. After cooling naturally to room temperature, add 0.5-1.5 parts of soluble chondrocyte extracellular matrix and continue stirring for 5-10 minutes at a stirring speed of 300-600 r / min; e. Add 0.5-1 part of chondrocyte extracellular matrix microcapsules and continue stirring for 5-10 minutes at a stirring speed of 100-200 r / min; S2, molding: a. Weigh a specified amount of S1 tissue engineering scaffold solution and add it to the mold; b. Pre-cool the mold in a 4-6℃ refrigerator for 10-20 minutes, then put it in a -20℃ refrigerator for 1-2 hours, and finally put it in a -80℃ refrigerator for deep cooling for 1-2 hours; c. Place the frozen mold into a freeze dryer for freeze drying at -80°C for 12-24 hours; S3, cross-linking: using carbodiimide and succinimide or glutaraldehyde as cross-linking agents, cross-linking at 4°C for 24-48h; S4. Freeze-drying: The cross-linked scaffold is repeatedly rinsed with purified water, and after the rinsing is completed, freeze-dried to finally obtain the cartilage extracellular matrix tissue engineering scaffold.

Images