WO2021219083A1 - 双层骨软骨组织修复支架及其制备方法 - Google Patents

双层骨软骨组织修复支架及其制备方法 Download PDF

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WO2021219083A1
WO2021219083A1 PCT/CN2021/091001 CN2021091001W WO2021219083A1 WO 2021219083 A1 WO2021219083 A1 WO 2021219083A1 CN 2021091001 W CN2021091001 W CN 2021091001W WO 2021219083 A1 WO2021219083 A1 WO 2021219083A1
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layer
double
tissue repair
osteochondral tissue
concentration
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French (fr)
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范代娣
刘凯强
段志广
严建亚
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陕西巨子生物技术有限公司
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Priority to AU2021262336A priority Critical patent/AU2021262336A1/en
Priority to US17/922,125 priority patent/US20230173140A1/en
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    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
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Definitions

  • the invention belongs to the technical field of biomedical materials, and specifically relates to a double-layer bone cartilage tissue repair scaffold and a preparation method thereof.
  • Osteochondrial defects are common diseases of joints, including articular cartilage and subchondral bone defects. There are many reasons for osteochondral damage, such as sports injuries, traffic accidents, and various degenerative diseases. In addition, with age, the natural wear and tear of cartilage tissue can easily cause osteoarthritis, which is also the main cause of osteochondral damage. Due to physiological characteristics, cells in cartilage tissue can hardly be regenerated, so cartilage reconstruction is a huge challenge. As cartilage damage progresses, it usually expands to the subchondral bone, making the repair of cartilage and subchondral tissue a necessary condition.
  • Recombinant collagen is a new type of genetically engineered protein.
  • the mRNA of human collagen can be reverse transcribed into cDNA by recombinant E. coli BL21. After digestion, specific stitching and ligation, it is introduced into E. coli for high-density fermentation. After separation, purification and preparation.
  • recombinant collagen also has the advantages of good water solubility, low immunogenicity, good product stability and no potential virus risks. Because of these characteristics, recombinant collagen has been widely used in various aspects of biomedical engineering, including the preparation of soft tissue fillers, hemostatic sponges, and vascular stents.
  • Sodium hyaluronate is a linear polysaccharide composed of 250-25,000 repeating disaccharide units. It is the most abundant component in cartilage and an important aggrecan component, which organizes the chondrocyte cytoplasmic matrix into an elastic structure. Therefore, the hydrogel based on sodium hyaluronate is one of the most promising natural biomaterials for osteochondral tissue engineering and cartilage tissue engineering. According to the mineral composition of the calcified cartilage interface, hydroxyapatite is the best choice of scaffold material for the bone cartilage interface.
  • the present invention provides a double-layer osteochondral tissue repair scaffold and a preparation method thereof.
  • a method for preparing a double-layer osteochondral tissue repair scaffold characterized in that the method comprises:
  • Preparing a first material liquid including recombinant collagen, sodium hyaluronate and hydroxyapatite;
  • Preparing a second feed solution including recombinant collagen and sodium hyaluronate;
  • a cross-linking agent is added to the gel-like double-layer structure for cross-linking.
  • the method for preparing a double-layer osteochondral tissue repair scaffold according to item 3 characterized in that the concentration of recombinant collagen in the first material solution ranges from 90 to 120 mg/mL, and the second material solution contains The concentration of recombinant collagen ranges from 60 to 90 mg/mL.
  • the second material liquid is poured into the upper layer of the first gel-like body and left to stand to obtain a gel-like double-layer structure.
  • the first material liquid is poured into the upper layer of the second gel-like body and left to stand to obtain a gel-like double-layer structure.
  • the method for preparing a double-layer osteochondral tissue repair scaffold according to any one of items 1 to 9, wherein the process of adding a cross-linking agent to the gel-like double-layer structure for cross-linking includes: The double-layer structure is immersed in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution.
  • a double-layer osteochondral tissue repair scaffold prepared according to the method for preparing a double-layer osteochondral tissue repair scaffold according to any one of items 1 to 14.
  • a double-layer osteochondral tissue repair scaffold characterized in that the double-layer osteochondral tissue repair scaffold comprises:
  • the first layer is made of raw materials including recombinant collagen, sodium hyaluronate and hydroxyapatite;
  • the second layer is made of raw materials including recombinant collagen and sodium hyaluronate.
  • the double-layer osteochondral tissue repair scaffold prepared by the present invention has excellent mechanical properties, good biocompatibility, and suitable degradation rate. After the scaffold material is degraded, it can be reused as a raw material for new bone formation, thereby realizing osteochondral Organizational repair.
  • Figure 1 is a schematic diagram of the structure of the double-layer osteochondral tissue repair scaffold of the present invention.
  • the present invention relates to a method for preparing a double-layer osteochondral tissue repair scaffold.
  • the method includes:
  • Preparing a first material liquid including recombinant collagen, sodium hyaluronate and hydroxyapatite;
  • Preparing a second feed solution including recombinant collagen and sodium hyaluronate;
  • a cross-linking agent is added to the gel-like double-layer structure for cross-linking.
  • recombinant collagen is a new type of genetically engineered protein.
  • the mRNA of human collagen can be reverse transcribed into cDNA by recombinant E. coli BL21, which is digested, sutured and connected, and then introduced into E. coli for high-density fermentation , And then separated, purified and prepared.
  • recombinant collagen refers to the recombinant collagen described in claim 1 of Chinese Patent Application Publication CN1371919A, which has a triple-stranded, triple-helix structure, and can be used for example the genetic engineering disclosed in Chinese Patent Application Publication CN1371919A Expression method to prepare.
  • the concentration of recombinant collagen in the first material liquid and the second material liquid are different. Further, the concentration of recombinant collagen in the first feed solution is higher than the concentration of recombinant collagen in the second feed solution.
  • the concentration of recombinant collagen in the first feed solution ranges from 90 to 120 mg/mL, for example, it can be 90 mg/mL, 95 mg/mL, 100 mg/mL, 105 mg/mL, 110 mg/mL , 115mg/mL, 120mg/mL.
  • the concentration of the recombinant collagen in the second feed solution ranges from 60 to 90 mg/mL, for example, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL .
  • the concentration of sodium hyaluronate in the first liquid is the same as that of the second liquid. Further, the concentration of sodium hyaluronate in the first material liquid and the second material liquid ranges from 8 to 15 mg/mL, for example, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL.
  • the concentration of hydroxyapatite in the first feed solution ranges from 30 to 60 mg/mL, for example, it can be 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL. mL, 55mg/mL, 60mg/mL.
  • the process of freeze-drying the first material liquid and the second material liquid to form a gel-like double-layer structure includes: pouring the first material liquid into a mold, cooling and standing still , To obtain a first gel-like body; pour the second material liquid into the upper layer of the first gel-like body, and let it stand still to obtain a gel-like double-layer structure. That is, the first material liquid forms the bottom layer of the gel-like double-layer structure, that is, the gel-like body formed first.
  • the process of freeze-drying the first material liquid and the second material liquid to form a gel-like double-layer structure includes: pouring the second material liquid into a mold, cooling and standing still , To obtain a second gel-like body; pour the first material liquid into the upper layer of the second gel-like body, and let it stand to obtain a gel-like double-layer structure. That is, the second material liquid forms the bottom layer of the gel-like double-layer structure, that is, the gel-like body formed first.
  • the molecular weight of the recombinant collagen is 80kD to 110kD, for example, it can be 80kD, 85kD, 90kD, 95kD, 97kD, 100kD, 110kD.
  • the molecular weight of the sodium hyaluronate is 80kD to 150kD, for example, it can be 80kD, 90kD, 100kD, 110kD, 120kD, 130kD, 140kD, 150kD.
  • the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
  • concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 30-60 mmol/mL.
  • it can be 30mmol/mL, 40mmol/mL, 50mmol/mL, 60mmol/mL.
  • the said cross-linking conditions are cross-linking for 24 to 72 hours, and washing with pure water for 3 to 5 times.
  • the method further comprises freeze-drying, packaging and sterilizing the double-layer gel after cross-linking the double-layer gel.
  • the invention also provides a layer cartilage tissue repair scaffold prepared by the above method.
  • the present invention also provides a double-layer osteochondral tissue repair scaffold.
  • the double-layer osteochondral tissue repair scaffold includes a first layer 1 and a second layer 2, wherein the first layer 1 is composed of recombinant collagen , Sodium hyaluronate and hydroxyapatite, the second layer 2 is made of raw materials including recombinant collagen and sodium hyaluronate.
  • the porosity of the first layer 1 is 80%-97%.
  • the porosity of the second layer 2 is 58% to 86%.
  • the pore size of the first layer 1 is 100-200 ⁇ m, for example, it may be 100 ⁇ m, 110 ⁇ m, 120 ⁇ m, 130 ⁇ m, 140 ⁇ m, 150 ⁇ m, 160 ⁇ m, 170 ⁇ m, 180 ⁇ m, 190 ⁇ m, 200 ⁇ m.
  • the pore size of the second layer 2 is 50-80 ⁇ m, for example, it may be 50 ⁇ m, 60 ⁇ m, 70 ⁇ m, or 80 ⁇ m.
  • the thickness and width of the first layer 1 and the second layer 2 can be adjusted according to actual needs.
  • the thickness of the first layer 1 is 2 to 4 mm
  • the thickness of the second layer 2 is 3 to 6 mm.
  • the double-layer osteochondral tissue repair scaffold of this embodiment simulates the physiological structure and composition of natural osteochondral.
  • the upper layer uses recombinant collagen and sodium hyaluronate to repair the cartilage layer; the lower layer uses recombinant collagen and sodium hyaluronate It is the organic phase, and the nano-hydroxyapatite is the inorganic phase, which is used to repair the subchondral bone layer. After it is freeze-dried, it is made by cross-linking under the action of a cross-linking agent, which is 95% of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride weak.
  • the first liquid which includes recombinant collagen, sodium hyaluronate and hydroxyapatite.
  • the recombinant collagen, sodium hyaluronate, and hydroxyapatite are added to the non-pyrogenic water, and after uniformly stirring, the first liquid is obtained.
  • the concentration of recombinant collagen is 100mg/mL
  • the molecular weight of recombinant collagen is 97kD
  • the concentration of sodium hyaluronate is 10mg/mL
  • the molecular weight of sodium hyaluronate is 100kD
  • the concentration of hydroxyapatite is 50mg/mL.
  • a second feed solution which includes recombinant collagen and sodium hyaluronate.
  • the recombinant collagen and sodium hyaluronate are added to the pyrogen-free water, and the second liquid is obtained after uniformly stirring.
  • the concentration of recombinant collagen is 80 mg/mL
  • the molecular weight of recombinant collagen is 97 kD
  • the concentration of sodium hyaluronate is 10 mg/mL
  • the molecular weight of sodium hyaluronate is 100 kD.
  • Example 2 The difference between Example 2 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 95mg/mL, the molecular weight of recombinant collagen is 100kD, the concentration of sodium hyaluronate is 9mg/mL, and the concentration of sodium hyaluronate is 9mg/mL.
  • the molecular weight is 90kD, the concentration of hydroxyapatite is 45mg/mL; in the second feed solution, the concentration of recombinant collagen is 85mg/mL, the molecular weight of recombinant collagen is 100kD, the concentration of sodium hyaluronate is 9mg/mL, hyaluronic acid The molecular weight of sodium is 90kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Example 3 The difference between Example 3 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 105mg/mL, the molecular weight of recombinant collagen is 95kD, the concentration of sodium hyaluronate is 12mg/mL, and the concentration of sodium hyaluronate is 12mg/mL.
  • the molecular weight is 110kD, and the concentration of hydroxyapatite is 55mg/mL; in the second liquid, the concentration of recombinant collagen is 75mg/mL, the molecular weight of recombinant collagen is 95kD, the concentration of sodium hyaluronate is 12mg/mL, hyaluronic acid The molecular weight of sodium is 110kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Example 4 The difference between Example 4 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 120mg/mL, the molecular weight of recombinant collagen is 105kD, the concentration of sodium hyaluronate is 8mg/mL, and the concentration of sodium hyaluronate is 8mg/mL.
  • the molecular weight is 80kD, and the concentration of hydroxyapatite is 40mg/mL; in the second liquid, the concentration of recombinant collagen is 70mg/mL, the molecular weight of recombinant collagen is 105kD, the concentration of sodium hyaluronate is 8mg/mL, hyaluronic acid The molecular weight of sodium is 80kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Example 5 The difference between Example 5 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 115mg/mL, the molecular weight of recombinant collagen is 80kD, the concentration of sodium hyaluronate is 15mg/mL, and the concentration of sodium hyaluronate is 15mg/mL.
  • the molecular weight is 120kD, and the concentration of hydroxyapatite is 30mg/mL; in the second feed solution, the concentration of recombinant collagen is 90mg/mL, the molecular weight of recombinant collagen is 80kD, and the concentration of sodium hyaluronate is 15mg/mL, hyaluronic acid
  • the molecular weight of sodium is 120kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Example 6 The difference between Example 6 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 110 mg/mL, the molecular weight of recombinant collagen is 110 kD, the concentration of sodium hyaluronate is 14 mg/mL, and the concentration of sodium hyaluronate is 14 mg/mL.
  • the molecular weight is 130kD, and the concentration of hydroxyapatite is 60mg/mL; in the second liquid, the concentration of recombinant collagen is 65mg/mL, the molecular weight of recombinant collagen is 110kD, the concentration of sodium hyaluronate is 14mg/mL, hyaluronic acid
  • the molecular weight of sodium is 130kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Example 7 The difference between Example 7 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 90mg/mL, the molecular weight of recombinant collagen is 85kD, the concentration of sodium hyaluronate is 13mg/mL, and the concentration of sodium hyaluronate is 13mg/mL.
  • the molecular weight is 150kD, and the concentration of hydroxyapatite is 35mg/mL; in the second feed solution, the concentration of recombinant collagen is 60mg/mL, the molecular weight of recombinant collagen is 85kD, and the concentration of sodium hyaluronate is 13mg/mL, hyaluronic acid
  • the molecular weight of sodium is 150kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 80mg/mL, the molecular weight of recombinant collagen is 120kD, the concentration of sodium hyaluronate is 5mg/mL, and the concentration of sodium hyaluronate is 5mg/mL.
  • the molecular weight is 130kD, and the concentration of hydroxyapatite is 70mg/mL; in the second feed solution, the concentration of recombinant collagen is 100mg/mL, the molecular weight of recombinant collagen is 120kD, the concentration of sodium hyaluronate is 5mg/mL, hyaluronic acid The molecular weight of sodium is 130kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 130mg/mL, the molecular weight of recombinant collagen is 70kD, the concentration of sodium hyaluronate is 18mg/mL, and the concentration of sodium hyaluronate is 18mg/mL.
  • the molecular weight is 170kD, and the concentration of hydroxyapatite is 40mg/mL; in the second liquid, the concentration of recombinant collagen is 60mg/mL, the molecular weight of recombinant collagen is 70kD, and the concentration of sodium hyaluronate is 18mg/mL, hyaluronic acid
  • the molecular weight of sodium is 170kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that in the first liquid, the concentration of recombinant collagen is 90 mg/mL, the molecular weight of recombinant collagen is 90 kD, the concentration of sodium hyaluronate is 10 mg/mL, and the concentration of sodium hyaluronate is 90 mg/mL.
  • the molecular weight is 60kD, and the concentration of hydroxyapatite is 20mg/mL; in the second liquid, the concentration of recombinant collagen is 110mg/mL, the molecular weight of recombinant collagen is 90kD, and the concentration of sodium hyaluronate is 10mg/mL, hyaluronic acid
  • the molecular weight of sodium is 60kD.
  • Other reaction conditions are the same as in Example 1. See Table 1 for details.
  • the double-layer osteochondral tissue repair scaffold samples prepared in the above examples and comparative examples were selected, and a 500N sensor was used through the electronic universal material testing machine (INSTRON 5565) carry out testing.
  • the double-layer osteochondral scaffold sample was made into a cylindrical shape with a diameter of 15mm and a height of 10mm, and the compression performance of the tissue engineering cartilage scaffold was measured at a loading rate of 10mm/min. After the measurement, the compressive stress, compressive strain and Compression modulus of elasticity and other data.
  • the elastic modulus (E) is determined by the slope of the stress-strain curve obtained for each sample.
  • the double-layer osteochondral tissue repair scaffold materials prepared in the above examples and comparative examples were selected for relevant cytotoxicity tests.
  • the cytotoxicity test of the scaffold material is to evaluate the potential hazards of the material. This test uses the MTT method to detect the cytotoxicity of the scaffold material.
  • the hBMSC cells were cultured at 37°C with a CO 2 concentration of 5.0%.
  • the second-generation hBMSC cells are grown to 70%, they are digested with 3mL trypsin (filtered with a membrane with a pore size of 0.22 ⁇ m) and seeded in a 96-well cell culture plate at a cell density of 3 ⁇ 10 4 cells/mL. Inoculate 100 ⁇ L per well. Put the cell culture plate with the cells in the incubator for 24 hours, aspirate the culture medium, and use every 8 holes as a group of parallel samples.
  • the control group uses 100 ⁇ L complete culture medium to culture the cells, and the test group uses 100 ⁇ L scaffolds.
  • the extract of the material is used to culture the cells.
  • the double-layer osteochondral scaffold prepared by the present invention was selected for animal experiments, specifically:
  • the New Zealand white rabbit was anesthetized under general anesthesia, and the osteochondral defect (3.0mm in diameter, 5.0mm in depth) was polished in the pulley groove of the rabbit's right leg with a dental grinder, and then a porous double-layer osteochondral scaffold of the same size as the defect was implanted
  • the rabbits were randomly divided into 2 groups: the stent group and the control group (defect only). The rabbits were euthanized 12 weeks after the operation, the tissue repair was observed, and the percentage of osteochondral repair was calculated.
  • the double-layer osteochondral tissue repair scaffold of Examples 1-7 can be compressed to more than 70%, and its elasticity model The amount is about 2MPa. After 20 cycles of compression, the recovery performance of the double-layer osteochondral scaffold material is good, and the mechanical properties fully meet the needs of the human body.
  • the numerical ranges listed in the present invention all include the data at the two end points of the numerical range, as well as each specific value in the numerical range, and the numerical value can be arbitrarily combined with the end points to form a new small range.

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Abstract

本发明涉及一种双层骨软骨组织修复支架的制备方法，包括：配制第一料液，第一料液中包括重组胶原蛋白、透明质酸钠和羟基磷灰石；配制第二料液，第二料液中包括重组胶原蛋白和透明质酸钠；对第一料液和第二料液冷冻干燥并形成凝胶状双层结构；对凝胶状双层结构加入交联剂进行交联。本发明还涉及一种双层骨软骨组织修复支架，包括：由包括重组胶原蛋白、透明质酸钠和羟基磷灰石在内的原料制成的第一层；由包括重组胶原蛋白和透明质酸钠在内的原料制成的第二层。本发明制备得到的双层骨软骨组织修复支架具有优异的机械性能、良好的生物相容性、适宜的降解速率，且支架材料降解后可作为新骨生成的原材料被再次利用，从而实现骨软骨组织的修复。

Description

双层骨软骨组织修复支架及其制备方法 技术领域
本发明属于生物医用材料技术领域，具体涉及一种双层骨软骨组织修复支架及其制备方法。
背景技术
骨软骨缺损是关节的常见疾病，包括关节软骨和软骨下骨缺损。骨软骨损伤的原因很多，例如运动损伤、交通事故和各种退行性疾病。另外，随着年龄的增长，软骨组织的自然磨损很容易引起骨关节炎，这也是骨软骨损伤的主要原因。由于生理特性，软骨组织中的细胞几乎不能再生，因此，软骨重建是一个巨大的挑战。随着软骨损伤的进展，它通常扩展到软骨下骨，使得软骨和软骨下组织的修复成为必要条件。
关节软骨和软骨下骨在生理结构上紧密相连，彼此相互作用形成相互依存的功能单元。据报道，没有健康的软骨下骨的支持，软骨不能自发修复。因此，对于骨软骨疾病应同时进行软骨下骨的修复，以重建软骨层。
近年来，组织工程支架的植入已被认为是治疗骨软骨缺损的有效策略。已经出现了许多基于生物材料的软骨缺损修复方法，这些方法对骨软骨损伤具有一定的治愈作用。但研究重点主要集中在缺损部位的软骨区域，软骨下骨和钙化软骨区域往往被忽略。现在已经认识到软骨下骨损伤可以在退行性关节疾病的发生和发展中产生重要的影响。因此，为了完成骨软骨缺损的修复，必须在缺损部位再生健康的软骨下骨。如今，发展用于骨软骨再生的生物活性多层支架被认为是一种理想的策略。这些材料通常由单独的软骨层和骨修复层组成，这些支架并不能真正解决骨软骨组织多层的修复问题。另外，提高双层支架的稳定性并在不同层之间实现良好的界面仍然是一项重大挑战。
发明内容
重组胶原蛋白是一种新型的基因工程蛋白，可通过重组大肠杆菌BL21 将人胶原蛋白的mRNA逆转录成cDNA，其经过酶切、特定缝合和连接后，导入大肠杆菌中进行高密度发酵，之后进行分离、纯化制备得到。除了胶原蛋白的优点外，重组胶原蛋白还具有水溶性好、免疫原性低、产品稳定性好且无病毒隐患的优点。由于这些特性，重组胶原蛋白已广泛应用于生物医学工程的各个方面，包括制备软组织填充物、止血海绵和血管支架等。透明质酸钠是由250-25,000个重复的二糖单元组成的线性多糖，是软骨中含量最多的成分，并且是重要的聚集蛋白聚糖成分，将软骨细胞质基质组织成弹性结构。因此，基于透明质酸钠的水凝胶是骨软骨组织工程和软骨组织工程应用最有前途的天然生物材料之一。根据钙化软骨界面的矿物质组成，羟基磷灰石是骨软骨界面支架材料的最佳选择。
针对现有软骨组织修复支架存在的问题，本发明提供一种双层骨软骨组织修复支架及其制备方法。
具体的，本发明提供的技术方案如下：
1.一种双层骨软骨组织修复支架的制备方法，其特征在于，所述方法包括：
配制第一料液，所述第一料液中包括重组胶原蛋白、透明质酸钠和羟基磷灰石；
配制第二料液，所述第二料液中包括重组胶原蛋白和透明质酸钠；
对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构；
对所述凝胶状双层结构加入交联剂进行交联。
2.根据项1所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液和第二料液中重组胶原蛋白的浓度不同。
3.根据项2所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中重组胶原蛋白的浓度高于所述第二料液中重组胶原蛋白的浓度。
4.根据项3所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中重组胶原蛋白的浓度范围为90～120mg/mL，所述第二料液中重组胶原蛋白的浓度范围为60～90mg/mL。
5.根据项1所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中透明质酸钠的浓度与所述第二料液相同。
6.根据项5所述的双层骨软骨组织修复支架的制备方法，其特征在于， 所述第一料液和所述第二料液中透明质酸钠的浓度范围为8～15mg/mL。
7.根据项1所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中羟基磷灰石的浓度范围为30～60mg/mL。
8.根据项1所述的双层骨软骨组织修复支架的制备方法，其特征在于，对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构的过程包括：
将所述第一料液倒入模具，冷却静置，得到第一凝胶状体；
将所述第二料液倒入所述第一凝胶状体的上层，静置，得到凝胶状双层结构。
9.根据项1所述的双层骨软骨组织修复支架的制备方法，其特征在于，对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构的过程包括：
将所述第二料液倒入模具，冷却静置，得到第二凝胶状体；
将所述第一料液倒入所述第二凝胶状体的上层，静置，得到凝胶状双层结构。
10.根据项1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述重组胶原蛋白的分子量为80kD～110kD。
11.根据项1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述透明质酸钠的分子量为80kD～150kD。
12.根据项1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐。
13.根据项1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，对所述凝胶状双层结构加入交联剂进行交联的过程包括将凝胶状双层结构浸入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液中。
14.根据项1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液浓度为30～60mmol/mL。
15.一种根据项1至14任一项所述的双层骨软骨组织修复支架的制备方法制得的双层骨软骨组织修复支架。
16.一种双层骨软骨组织修复支架，其特征在于，所述双层骨软骨组织修复支架包括：
第一层，所述第一层由包括重组胶原蛋白、透明质酸钠和羟基磷灰石在内的原料制成；
第二层，所述第二层由包括重组胶原蛋白和透明质酸钠在内的原料制成。
17.根据项16所述的双层骨软骨组织修复支架，其特征在于，所述第一层的孔隙率为80％～97％，所述第二层的孔隙率为58％～86％。
18.根据项16所述的双层骨软骨组织修复支架，其特征在于，所述第一层的孔径为50～80μm，所述第二层的孔径为100～200μm。
19.根据项16所述的双层骨软骨组织修复支架，其特征在于，所述第一层的厚度为2～4mm，所述第二层的厚度为3～6mm。
本发明制备得到的双层骨软骨组织修复支架具有优异的机械性能、良好的生物相容性、适宜的降解速率，且支架材料降解后可作为新骨生成的原材料被再次利用，从而实现骨软骨组织的修复。
附图说明
图1为本发明双层骨软骨组织修复支架的结构示意图。
附图标记：1-第一层，2-第二层。
具体实施方式
本发明涉及一种双层骨软骨组织修复支架的制备方法，所述方法包括：
配制第一料液，所述第一料液中包括重组胶原蛋白、透明质酸钠和羟基磷灰石；
配制第二料液，所述第二料液中包括重组胶原蛋白和透明质酸钠；
对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构；
对所述凝胶状双层结构加入交联剂进行交联。
其中，重组胶原蛋白是一种新型的基因工程蛋白，可通过重组大肠杆菌BL21将人胶原蛋白的mRNA逆转录成cDNA，其经过酶切、特定缝合和连接后，导入大肠杆菌中进行高密度发酵，之后进行分离、纯化制备得到。在本说明书中，重组胶原蛋白是指中国专利申请公开CN1371919A的权利要求1所述的重组胶原蛋白，其具有三链、三螺旋结构，其可以采用例如 该中国专利申请公开CN1371919A中公开的基因工程表达方法来制备。
在一个具体的实施方式中，所述第一料液和第二料液中重组胶原蛋白的浓度不同。进一步的，所述第一料液中重组胶原蛋白的浓度高于所述第二料液中重组胶原蛋白的浓度。
在一个具体的实施方式中，所述第一料液中重组胶原蛋白的浓度范围为90～120mg/mL，例如可以是90mg/mL、95mg/mL、100mg/mL、105mg/mL、110mg/mL、115mg/mL、120mg/mL。所述第二料液中重组胶原蛋白的浓度范围为60～90mg/mL，例如可以是60mg/mL、65mg/mL、70mg/mL、75mg/mL、80mg/mL、85mg/mL、90mg/mL。
在一个具体的实施方式中，所述第一料液中透明质酸钠的浓度与所述第二料液相同。进一步的，所述第一料液和所述第二料液中透明质酸钠的浓度范围为8～15mg/mL，例如可以是8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL。
在一个具体的实施方式中，所述第一料液中羟基磷灰石的浓度范围为30～60mg/mL，例如可以是30mg/mL、35mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL。
在一个具体的实施方式中，对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构的过程包括：将所述第一料液倒入模具，冷却静置，得到第一凝胶状体；将所述第二料液倒入所述第一凝胶状体的上层，静置，得到凝胶状双层结构。即所述第一料液形成凝胶状双层结构的底层，也即首先形成的凝胶状体。
在一个具体的实施方式中，对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构的过程包括：将所述第二料液倒入模具，冷却静置，得到第二凝胶状体；将所述第一料液倒入所述第二凝胶状体的上层，静置，得到凝胶状双层结构。即所述第二料液形成凝胶状双层结构的底层，也即首先形成的凝胶状体。
在一个具体的实施方式中，所述重组胶原蛋白的分子量为80kD～110kD，例如可以是80kD、85kD、90kD、95kD、97kD、100kD、110kD。
在一个具体的实施方式中，所述透明质酸钠的分子量为80kD～150kD，例如可以是80kD、90kD、100kD、110kD、120kD、130kD、140kD、150kD。
在一个具体的实施方式中，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐。进一步的，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液浓度为30～60mmol/mL。例如可以是30mmol/mL、40mmol/mL、50mmol/mL、60mmol/mL。所述的交联条件为交联24～72h，用纯水清洗3～5次。
在一个具体的实施方式中，所述方法还包括将所述双层凝胶交联后，对所述双层凝胶进行冷冻干燥、包装灭菌。
本发明还提供了由上述方法制备的层软骨组织修复支架。
本发明还提供一种双层骨软骨组织修复支架，如图1所示，所述双层骨软骨组织修复支架包括第一层1和第二层2，其中第一层1由包括重组胶原蛋白、透明质酸钠和羟基磷灰石在内的原料制成，第二层2由包括重组胶原蛋白和透明质酸钠在内的原料制成。
在一个具体的实施方式中，第一层1的孔隙率为80％～97％。
在一个具体的实施方式中，第二层2的孔隙率为58％～86％。
在一个具体的实施方式中，第一层1的孔径为100～200μm，例如可以为100μm、110μm、120μm、130μm、140μm、150μm、160μm、170μm、180μm、190μm、200μm。
在一个具体的实施方式中，第二层2的孔径为50～80μm，例如可以为50μm、60μm、70μm、80μm。
第一层1和第二层2的厚度和宽度可以根据实际的需要进行调节。在一个具体的实施方式中，第一层1的厚度为2～4mm，第二层2的厚度为3～6mm。
实施例1
本实施例的双层骨软骨组织修复支架是通过模拟天然骨软骨的生理结构与组成，上层采用重组胶原蛋白及透明质酸钠，用于修复软骨层；下层采用重组胶原蛋白与透明质酸钠为有机相，以纳米羟基磷灰石为无机相，用于修复软骨下骨层。将其冷冻干燥后，在交联剂作用下进行交联制成，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的95％乙醇溶液。
本实施例的双层骨软骨组织修复支架的制备方法具体包括：
(1)配制第一料液，第一料液包括重组胶原蛋白、透明质酸钠和羟基 磷灰石。具体的，将重组胶原蛋白、透明质酸钠和羟基磷灰石加入到无热源水中，搅拌均匀后，得到第一料液。其中，重组胶原蛋白的浓度为100mg/mL，重组胶原蛋白的分子量为97kD，透明质酸钠浓度为10mg/mL，透明质酸钠的分子量为100kD，羟基磷灰石浓度为50mg/mL。
(2)配制第二料液，第二料液包括重组胶原蛋白和透明质酸钠。具体的，将重组胶原蛋白和透明质酸钠加入到无热源水中，搅拌均匀后，得到第二料液。其中，重组胶原蛋白的浓度为80mg/mL，重组胶原蛋白的分子量为97kD，透明质酸钠浓度为10mg/mL，透明质酸钠的分子量为100kD。
(3)将1mL第一料液倒入Φ15*10的柱状模具中，在4℃下冷却30min后得到第一凝胶状体；将2mL的第二料液倒入第一凝胶状体的上层，在室温下静置40min，得到凝胶状双层结构。
(4)将得到的凝胶状双层结构，进行冷冻干燥，然后用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的乙醇(95％)溶液进行交联36h(30～60mmol/mL)，其中交联剂用量为50mmol/mL。交联反应结束后，用纯化水清洗3～5次，去除残留的交联剂。清洗后冷冻干燥，并封装灭菌，得到双层骨软骨组织修复支架。
实施例2
实施例2与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为95mg/mL，重组胶原蛋白的分子量为100kD，透明质酸钠浓度为9mg/mL，透明质酸钠的分子量为90kD，羟基磷灰石浓度为45mg/mL；第二料液中，重组胶原蛋白的浓度为85mg/mL，重组胶原蛋白的分子量为100kD，透明质酸钠浓度为9mg/mL，透明质酸钠的分子量为90kD。其他反应条件与实施例1相同，详情见表1。
实施例3
实施例3与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为105mg/mL，重组胶原蛋白的分子量为95kD，透明质酸钠浓度为12mg/mL，透明质酸钠的分子量为110kD，羟基磷灰石浓度为55mg/mL；第二料液中，重组胶原蛋白的浓度为75mg/mL，重组胶原蛋白的分子量为95kD，透明质酸钠浓度为12mg/mL，透明质酸钠的分子量为110kD。其他反应条件与实施例1相同，详情见表1。
实施例4
实施例4与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为120mg/mL，重组胶原蛋白的分子量为105kD，透明质酸钠浓度为8mg/mL，透明质酸钠的分子量为80kD，羟基磷灰石浓度为40mg/mL；第二料液中，重组胶原蛋白的浓度为70mg/mL，重组胶原蛋白的分子量为105kD，透明质酸钠浓度为8mg/mL，透明质酸钠的分子量为80kD。其他反应条件与实施例1相同，详情见表1。
实施例5
实施例5与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为115mg/mL，重组胶原蛋白的分子量为80kD，透明质酸钠浓度为15mg/mL，透明质酸钠的分子量为120kD，羟基磷灰石浓度为30mg/mL；第二料液中，重组胶原蛋白的浓度为90mg/mL，重组胶原蛋白的分子量为80kD，透明质酸钠浓度为15mg/mL，透明质酸钠的分子量为120kD。其他反应条件与实施例1相同，详情见表1。
实施例6
实施例6与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为110mg/mL，重组胶原蛋白的分子量为110kD，透明质酸钠浓度为14mg/mL，透明质酸钠的分子量为130kD，羟基磷灰石浓度为60mg/mL；第二料液中，重组胶原蛋白的浓度为65mg/mL，重组胶原蛋白的分子量为110kD，透明质酸钠浓度为14mg/mL，透明质酸钠的分子量为130kD。其他反应条件与实施例1相同，详情见表1。
实施例7
实施例7与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为90mg/mL，重组胶原蛋白的分子量为85kD，透明质酸钠浓度为13mg/mL，透明质酸钠的分子量为150kD，羟基磷灰石浓度为35mg/mL；第二料液中，重组胶原蛋白的浓度为60mg/mL，重组胶原蛋白的分子量为85kD，透明质酸钠浓度为13mg/mL，透明质酸钠的分子量为150kD。其他反应条件与实施例1相同，详情见表1。
对比例1
对比例1与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为80mg/mL，重组胶原蛋白的分子量为120kD，透明质酸钠浓度为5mg/mL，透明质酸钠的分子量为130kD，羟基磷灰石浓度为70mg/mL；第 二料液中，重组胶原蛋白的浓度为100mg/mL，重组胶原蛋白的分子量为120kD，透明质酸钠浓度为5mg/mL，透明质酸钠的分子量为130kD。其他反应条件与实施例1相同，详情见表1。
对比例2
对比例2与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为130mg/mL，重组胶原蛋白的分子量为70kD，透明质酸钠浓度为18mg/mL，透明质酸钠的分子量为170kD，羟基磷灰石浓度为40mg/mL；第二料液中，重组胶原蛋白的浓度为60mg/mL，重组胶原蛋白的分子量为70kD，透明质酸钠浓度为18mg/mL，透明质酸钠的分子量为170kD。其他反应条件与实施例1相同，详情见表1。
对比例3
对比例3与实施例1的不同在于，第一料液中，重组胶原蛋白的浓度为90mg/mL，重组胶原蛋白的分子量为90kD，透明质酸钠浓度为10mg/mL，透明质酸钠的分子量为60kD，羟基磷灰石浓度为20mg/mL；第二料液中，重组胶原蛋白的浓度为110mg/mL，重组胶原蛋白的分子量为90kD，透明质酸钠浓度为10mg/mL，透明质酸钠的分子量为60kD。其他反应条件与实施例1相同，详情见表1。
表1实施例和对比例的具体反应条件
Figure PCTCN2021091001-appb-000001
对实施例1-7和对比例1-3制备得到的双层骨软骨组织修复支架样品进 行性能测试。
(1)力学性能试验
为了验证所制备的双层骨软骨组织修复支架材料的力学性能，选取上述实施例和对比例所制备的双层骨软骨组织修复支架样品，通过电子万能材料试验机(INSTRON 5565)使用500N的传感器进行测试。具体为：将双层骨软骨支架样品制成直径为15mm、高度为10mm的圆柱状，以10mm/min的加载速率对组织工程软骨支架进行压缩性能测量，测量结束后采集压缩应力，压缩应变和压缩弹性模量等数据。弹性模量(E)是通过为每个样品获得的应力-应变曲线的斜率确定的。
(2)细胞毒性试验
为了验证所制备的双层骨软骨修复支架材料的生物学相容性，选取上述实施例和对比例所制备的双层骨软骨组织修复支架材料样品进行相关的细胞毒性试验
支架材料的细胞毒性检测是对材料的潜在危害进行评估，本试验通过MTT法来检测支架材料的细胞毒性。首先制备支架材料的浸提液，即选取本发明上述实施例所制备的经Co-60辐射灭菌的无菌双层骨软骨修复支架材料2.0g，加入20mL DMEM完全培养液，37℃恒温箱中浸提72±2h，制得100mg/mL的支架浸提液。
在37℃，CO 2浓度为5.0％的条件下培养hBMSC细胞。培养二代hBMSC细胞长到70％时，以3mL的胰蛋白酶(用孔径为0.22μm的膜过滤)溶液进行消化，以3×10 4cells/mL的细胞密度接种于96孔细胞培养板中，每孔接种100μL。将接有细胞的细胞培养板放入培养箱培养24h后，吸去培养液，并以每8个孔为一组平行样，对照组是用100μL完全培养液来培养细胞，试验组用100μL支架材料的浸提液来培养细胞，培养7天之后吸去培养液，于每孔中加入50μL MTT溶液和100μL新鲜完全培养液，在37℃下CO 2培养箱中孵育3h后取出，吸出培养液加150μL的DMSO，摇床孵育15～20分钟，使用酶标仪于450nm处测定吸光值，计算出hBMSC细胞的相对增殖率。
(3)骨软骨缺损模型修复试验
为了验证本发明制备的双层骨软骨支架对骨软骨缺损的修复效果，选取上述事实例和对比例所制备的双层骨软骨支架进行动物实验，具体为：
将新西兰大白兔全身麻醉，使用牙科打磨机在兔子的右腿的滑车槽中打磨出骨软骨缺损(直径3.0mm，深度5.0mm)，然后将和缺损同样大小的多孔双层骨软骨支架植入缺损中，将兔子随机分为2组：支架组，对照组(仅缺损)。手术后12周将兔子安乐死，观察组织修复情况，计算骨软骨的修复百分率。
上述力学性能试验、细胞毒性试验和骨软骨缺损模型修复试验的结果如表2。
表2实施例和对比例的不同试验结果
Figure PCTCN2021091001-appb-000002
通过对力学性能试验、细胞毒性试验和骨软骨缺损修复试验的结果进行分析，由表2可以看出，实施例1-7的双层骨软骨组织修复支架可以压缩至70％以上，其弹性模量约为2MPa，经过20次循环压缩，双层骨软骨支架材料的回复性能良好，机械性能完全符合人体的需求。
通过细胞学实验，结果显示本发明实施例1-7制备的双层骨软骨组织修复支架材料浸提液的细胞相对增殖率达到100％以上或将近100％，细胞毒性评价为0级。
本发明制得的双层骨软骨组织修复支架用于骨软骨缺损修复的动物实验表明：实施例1-7制备的无菌多孔骨修复支架的动物骨软骨修复率明显高于对比例制备的支架的修复率，尤其是实施例1-5的动物骨软骨修复率均在90％以上，这说明动物骨软骨的缺损被均匀的软骨样组织填平，新生组织与周围正常软骨连接良好，表明这些双层骨软骨组织修复支架对骨软骨缺损有良好的修复作用。
本申请接受各种修改和可替换的形式，具体的实施方式已经在附图中借助于实施例来显示并且已经在本申请详细描述。但是，本申请不意在受限于公开的特定形式。相反，本申请意在包括本申请范围内的所有修改形式、等价物、和可替换物，本申请的范围由所附权利要求及其法律等效物限定。
在本发明中列举的数值范围均包括该数值范围的两个端点的数据，也包括该数值范围中具体的每一个数值，并且该数值可以与端点任意组合组成新的小范围。

Claims (19)

  1. 一种双层骨软骨组织修复支架的制备方法，其特征在于，所述方法包括：
    配制第一料液，所述第一料液中包括重组胶原蛋白、透明质酸钠和羟基磷灰石；
    配制第二料液，所述第二料液中包括重组胶原蛋白和透明质酸钠；
    对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构；
    对所述凝胶状双层结构加入交联剂进行交联。
  2. 根据权利要求1所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液和第二料液中重组胶原蛋白的浓度不同。
  3. 根据权利要求2所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中重组胶原蛋白的浓度高于所述第二料液中重组胶原蛋白的浓度。
  4. 根据权利要求3所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中重组胶原蛋白的浓度范围为90～120mg/mL，所述第二料液中重组胶原蛋白的浓度范围为60～90mg/mL。
  5. 根据权利要求1所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中透明质酸钠的浓度与所述第二料液相同。
  6. 根据权利要求5所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液和所述第二料液中透明质酸钠的浓度范围为8～15mg/mL。
  7. 根据权利要求1所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述第一料液中羟基磷灰石的浓度范围为30～60mg/mL。
  8. 根据权利要求1所述的双层骨软骨组织修复支架的制备方法，其特征在于，对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构的过程包括：
    将所述第一料液倒入模具，冷却静置，得到第一凝胶状体；
    将所述第二料液倒入所述第一凝胶状体的上层，静置，得到凝胶状双层结构。
  9. 根据权利要求1所述的双层骨软骨组织修复支架的制备方法，其特征 在于，对所述第一料液和所述第二料液冷冻干燥并形成凝胶状双层结构的过程包括：
    将所述第二料液倒入模具，冷却静置，得到第二凝胶状体；
    将所述第一料液倒入所述第二凝胶状体的上层，静置，得到凝胶状双层结构。
  10. 根据权利要求1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述重组胶原蛋白的分子量为80kD～110kD。
  11. 根据权利要求1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述透明质酸钠的分子量为80kD～150kD。
  12. 根据权利要求1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐。
  13. 根据权利要求1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，对所述凝胶状双层结构加入交联剂进行交联的过程包括将凝胶状双层结构浸入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液中。
  14. 根据权利要求1至9任一项所述的双层骨软骨组织修复支架的制备方法，其特征在于，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液浓度为30～60mmol/mL。
  15. 一种根据权利要求1至14任一项所述的双层骨软骨组织修复支架的制备方法制得的双层骨软骨组织修复支架。
  16. 一种双层骨软骨组织修复支架，其特征在于，所述双层骨软骨组织修复支架包括：
    第一层，所述第一层由包括重组胶原蛋白、透明质酸钠和羟基磷灰石在内的原料制成；
    第二层，所述第二层由包括重组胶原蛋白和透明质酸钠在内的原料制成。
  17. 根据权利要求16所述的双层骨软骨组织修复支架，其特征在于，所述第一层的孔隙率为80％～97％，所述第二层的孔隙率为58％～86％。
  18. 根据权利要求16所述的双层骨软骨组织修复支架，其特征在于，所述第一层的孔径为50～80μm，所述第二层的孔径为100～200μm。
  19. 根据权利要求16所述的双层骨软骨组织修复支架，其特征在于，所述第一层的厚度为2～4mm，所述第二层的厚度为3～6mm。
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CN114949371A (zh) * 2022-04-12 2022-08-30 重庆大学 修复关节骨软骨缺损的双层多孔支架及其制备方法
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