Gradient active scaffold material for osteochondral repair and its preparation method and application

[0051] Example 1

[0052] (1) Using type I collagen, calcium nitrate tetrahydrate, and diammonium hydrogen phosphate as raw materials, the pH of ammonia is adjusted to 8.0, and HA/Col powder is prepared by the co-precipitation method. In the prepared HA/Col-I composite powder, Type I collagen content is 35%, hydroxyapatite content is 65%;

[0053] (2) Use the EDC/NHS-Heparin method to construct a biologically active substance controlled release system, and soak the HA/Col-I composite powder and the pre-prepared type II collagen powder in 0.05mol/LMES buffer (pH 5.6) 30min standby, dissolve each 2.5g heparin in 300ml 0.05mol/L MES buffer containing 2gEDC and 1.5gNHS for 10min, and put the spare collagen into the MES containing heparin and EDC/NHS according to the weight ratio of collagen to heparin 1:2 In the buffer, gently shake the reaction at 37°C for 4 hours; then place the modified HA/Col-I composite powder and type II collagen powder in a 70ng/ml BMP PBS solution (containing 1.5% BSA, 4mol/ L NaCl) incubate at 37°C for 60 hours. Because the modified collagen molecules have binding sites for heparin molecules, these sites can be covalently connected with biologically active substances to make BMP It can be compounded with type I collagen and type II collagen; among them, 0.05mol/L (pH 5.6) MES buffer: accurately weigh 4.8805g MES and dissolve in 400ml deionized water, stir to dissolve, dilute to 500ml, use 2mol/L Adjust NaOH to pH 5.6, and get it;

[0054] (3) First, combine 1kg PLGA (PLGA75:25, Mw=50,000), 0.75kg HA/Col-I composite powder and 7kg 100 mesh NaCl particles, 1kg PLGA, 1kg HA/Col-I composite powder and 8kg 100 mesh size NaCl particles, 1kg PLGA, 1.33kg HA/Col-I composite powder and 9.32kg 100 mesh size NaCl particles, 1kg PLGA, 1.8kg HA/Col-I composite powder and 11.2kg 100 mesh size NaCl particles; 1kg PLGA, 2kg HA/Col-I composite powder and 7kg 40 mesh NaCl particles, 1kg PLGA, 2.6kg HA/Col-I composite powder and 8.4kg 40 mesh NaCl particles, 1kg PLGA, 3.5kg HA/Col-I composite powder and 10.5kg 40 mesh size NaCl particles, 1kg PLGA, 5.1kg HA/Col-I composite powder and 14.2kg 40 mesh size NaCl particles; and 1kg PLGA, 1kg type II collagen Powder and 8kg 50 mesh size NaCl particles, 1kg PLGA, 0.67kg type II collagen powder and 6.68kg 50 mesh size NaCl particles, 1kg PLGA, 0.43kg type II collagen powder and 5.72kg 50 mesh size NaCl particles They were mixed uniformly, and dichloromethane was added to dissolve the PLGA in the mixed particles, and then injected into the mold from bottom to top according to the hydroxyapatite content; at the same time, the biologically active substance controlled release system constructed in step (2) was introduced. The methyl chloride is dissolved and volatilized to form, the sodium chloride is dissolved in deionized water and ultrasonically cleaned to obtain a gradient scaffold material for osteochondral repair.

[0055] Such as figure 1 As shown, the cartilage layer, the calcified layer and the subchondral bone layer in the scaffold material have a layer structure, wherein the cartilage layer contains 50% type II collagen, PLGA content is 50%, and the second layer type II collagen The content is 40%, PLGA content is 60%, the third layer type II collagen content is 30%, PLGA content is 70%; the first layer type I collagen content in the calcification layer is 15%, and the hydroxyapatite content is 27.9% PLGA content is 57.1%, the second layer type I collagen content is 17.5%, hydroxyapatite content is 32.5%, PLGA content is 50%, the third layer type I collagen content is 20%, and the hydroxyapatite content is 37.1%, PLGA content is 42.9%, fourth layer type I collagen content is 22.5%, hydroxyapatite content is 41.8%, PLGA content is 35.7%; subchondral bone layer first layer type I collagen content is 23.3% , Hydroxyapatite content is 43.3%, PLGA content is 33.4%, second layer type I collagen content is 25.3%, hydroxyapatite content is 47.1%, PLGA content is 27.6%, third layer type I collagen content is 27.3%, hydroxyapatite content is 50.7%, PLGA content is 22%, fourth layer type I collagen content is 29.3%, hydroxyapatite content is 54.4%, and PLGA content is 16.3%. The pore size of the cartilage layer is 270 microns, the pore size of the calcification layer is 150 microns, and the pore size of the subchondral bone layer is 380 microns. The porosity of the cartilage layer and the calcified layer is 80%, the porosity of the subchondral bone layer is 70%, and 90% of the pores are interpenetrated.

[0056] Example 2

[0057] (1) Using type I collagen, calcium nitrate tetrahydrate, and diammonium hydrogen phosphate as raw materials, adjusting the pH value of ammonia to 8.0, preparing HA/Col powder by co-precipitation method, in the prepared HA/Col-I composite powder , The type I collagen content is 20%, and the hydroxyapatite content is 80%;

[0058] (2) Use the EDC/NHS-Heparin method to construct a biologically active substance controlled release system, and soak the HA/Col-I composite powder and the pre-prepared type II collagen powder in 0.05mol/LMES buffer (pH 5.6) 30min standby, dissolve each 3g heparin in 200ml 0.05mol/L MES buffer containing 1gEDC and 2gNHS for 10min, and put the spare collagen into the MES buffer containing heparin and EDC/NHS according to the weight ratio of collagen to heparin 1:3 In the medium, gently shake the reaction at 37°C for 4h; then put the modified HA/Col-I composite powder and type II collagen powder in a PBS solution (containing 0.5% BSA, 1mol/ml) with a concentration of 50ng/ml bFGF L NaCl) incubate at 37°C for 24h. Because the modified collagen molecules have binding sites for heparin molecules, these sites can be covalently connected with biologically active substances to make bFGF It can be compounded with type I collagen and type II collagen;

[0059] (3) First, mix 1kg PHBV (Mw=312000, HV content 5.7mol%), 1kg HA/Col-I composite powder and 8kg 120 mesh NaCl particles; 1kg PHBV, 2kg HA/Col-I composite powder and 7kg 35 mesh size NaCl particles; and 1kg PHBV, 2kg type II collagen powder and 7kg 60 mesh size NaCl particles are mixed uniformly, respectively add dichloromethane to dissolve the PHBV in the mixed particles, and then according to the content of hydroxyapatite Inject into the mold sequentially from bottom to top; at the same time, introduce the biologically active substance controlled release system constructed in step (2), dissolve and volatilize in dichloromethane, and dissolve sodium chloride in deionized water, and ultrasonically clean to obtain the osteochondral repair gradient scaffold material.

[0060] The type II collagen content in the cartilage layer of the scaffold material is 66.7%, the PHBV content is 33.3%; the type I collagen content in the calcification layer is 10%, the hydroxyapatite content is 40%, and the PHBV content is 50%; subchondral bone The type I collagen content in the layer is 13.4%, the hydroxyapatite content is 53.3%, and the PHBV content is 33.3%. The pore size of the cartilage layer is 250 microns, the pore size of the calcification layer is 120 microns, and the pore size of the subchondral bone layer is 425 microns. The porosity of the cartilage layer and the subchondral bone layer is 70%, the porosity of the calcified layer is 80%, and 85% of the pores are interpenetrated.

[0061] Example 3

[0062] (1) Using type I collagen, calcium nitrate tetrahydrate, and diammonium hydrogen phosphate as raw materials, adjusting the pH value of ammonia to 8.0, preparing HA/Col powder by co-precipitation method, in the prepared HA/Col-I composite powder , The content of type I collagen is 50%, and the content of hydroxyapatite is 50%;

[0063] (2) Use the EDC/NHS-Heparin method to construct a biologically active substance controlled release system, and soak the HA/Col-I composite powder and the pre-prepared type II collagen powder in 0.05mol/LMES buffer (pH 5.6) 30min standby, dissolve each 1g heparin in 500ml 0.05mol/L MES buffer containing 3gEDC and 0.5gNHS for 10min activation, according to the weight ratio of collagen to heparin 1:1, put the spare collagen into the MES buffer containing heparin and EDC/NHS In the solution, gently shake the reaction at 37°C for 4h; then place the modified HA/Col-I composite powder and type II collagen powder in a PBS solution with a concentration of 0.1ng/ml bFGF and 90ng/ml TGF ( Containing 2% BSA, 3mol/L NaCl) incubate at 37°C for 72h. Since the modified collagen molecule has binding sites for heparin molecules, these sites can be used to covalently interact with biologically active substances. The bond is connected, so that bFGF, TGF and type I collagen and type II collagen can be compounded;

[0064] (3) First, put 0.5kg PLA (Mw=50000), 1.5kg HA/Col-I composite powder and 8kg 120 mesh KCl particles; 0.2kg PLA, 0.8kg HA/Col-I composite powder and 9kg 35 mesh size KCl particles; and 2kg PLA, 1kg type II collagen powder and 7kg 60 mesh size KCl particles were mixed uniformly, and dichloromethane was added to dissolve the PLA in the mixed particles, and then according to the content of hydroxyapatite Inject into the mold sequentially from bottom to top; at the same time, introduce the bioactive substance controlled release system constructed in step (2), dissolve and volatilize in dichloromethane, dissolve KCl in deionized water, and ultrasonically clean to obtain the osteochondral repair gradient scaffold material.

[0065] The content of type II collagen in the cartilage layer of the scaffold material is 33.3%, and the content of PLA is 66.7%; the content of type I collagen in the calcification layer is 37.5%, the content of hydroxyapatite is 37.5%, and the content of PLA is 25%; subchondral bone The type I collagen content in the layer is 40%, the hydroxyapatite content is 40%, and the PLA content is 20%. The pore size of the cartilage layer is 250 microns, the pore size of the calcification layer is 120 microns, and the pore size of the subchondral bone layer is 425 microns. The porosity of the cartilage layer is 70%, the porosity of the subchondral bone layer is 90%, the porosity of the calcified layer is 80%, and 80% of the pores are interpenetrated.