CN106806940A - A kind of preparation method of nano hydroxylapatite doped porous Bionics Bone support - Google Patents

本发明提供了一种掺杂纳米羟基磷灰石的多孔仿生骨支架的制备方法，该方法以透明质酸、明胶和纳米羟基磷灰石为原料，采用冷冻干燥法和化学交联法，制备出具备良好机械强度的透明质酸-明胶-纳米羟基磷灰石交联复合多孔支架。采用该方法制备的多孔复合支架孔径均一、孔壁薄、孔隙贯穿，具有很好的细胞粘附能力和机械强度，具有骨传导性。本发明的优点在于该复合支架更加真实的模拟骨组织微环境，通过调节各组分的浓度、体积比及冷冻干燥条件，可调控支架的孔径、孔隙率、降解率、吸水率、机械强度等参数；所用材料均具备良好的生物相容性，形成的支架满足骨组织的内部多孔结构；材料来源充足，制备工艺简单。The invention provides a method for preparing a porous bionic bone scaffold doped with nano-hydroxyapatite. The method uses hyaluronic acid, gelatin and nano-hydroxyapatite as raw materials, and adopts a freeze-drying method and a chemical cross-linking method to prepare A hyaluronic acid-gelatin-nano-hydroxyapatite cross-linked composite porous scaffold with good mechanical strength was obtained. The porous composite scaffold prepared by the method has uniform pore diameter, thin pore wall and penetrating pores, has good cell adhesion ability and mechanical strength, and has osteoconductivity. The advantage of the present invention is that the composite scaffold more realistically simulates the microenvironment of bone tissue. By adjusting the concentration, volume ratio and freeze-drying conditions of each component, the pore size, porosity, degradation rate, water absorption rate, mechanical strength, etc. of the scaffold can be adjusted. parameters; the materials used have good biocompatibility, and the formed scaffold meets the internal porous structure of bone tissue; the source of the material is sufficient, and the preparation process is simple.

一种掺杂纳米羟基磷灰石的多孔仿生骨支架的制备方法Preparation method of porous biomimetic bone scaffold doped with nano-hydroxyapatite

技术领域：Technical field:

本发明涉及新型生物材料和组织工程技术领域，具体涉及一种掺杂纳米羟基磷灰石的多孔仿生骨支架的制备方法。The invention relates to the technical field of novel biomaterials and tissue engineering, in particular to a method for preparing a porous bionic bone scaffold doped with nano-hydroxyapatite.

背景技术：Background technique:

本发明以组织工程为依托。骨组织工程是临床上修复骨缺损的有效途径之一，近年来受到国内外学者的广泛关注。骨组织工程的三要素或四要素，主要包括支架材料、种子细胞、细胞与支架材料的整合以及植入物与体内微环境的整合，其过程是指将种子细胞在体外进行扩增培养，然后将扩增的细胞移植到具有良好生物相容性、可降解性的支架材料上继续培养，使细胞粘附在支架上形成细胞-支架复合物；将该复合物植入骨缺损部位，随着支架的降解和被吸收，植入的细胞在体内不断增殖并分泌细胞外基质，最终形成骨组织，从而达到修复创伤和重建功能的目的。支架材料作为骨组织工程的重要组成部分，为细胞获取营养、生长和代谢提供了良好的微环境。骨组织工程的发展提供了一种组织再生的技术手段，将改变外科传统的“以创伤修复创伤”的治疗模式，迈入无创伤修复的新阶段。The present invention is based on tissue engineering. Bone tissue engineering is one of the effective ways to repair bone defects clinically, and has attracted extensive attention from scholars at home and abroad in recent years. The three or four elements of bone tissue engineering mainly include scaffold materials, seed cells, the integration of cells and scaffold materials, and the integration of implants and in vivo microenvironment. The process refers to the expansion and culture of seed cells in vitro, and then The amplified cells are transplanted to a scaffold material with good biocompatibility and degradability to continue culturing, so that the cells adhere to the scaffold to form a cell-scaffold complex; the complex is implanted into the bone defect site, with The degradation and absorption of the scaffold, the implanted cells continue to proliferate in the body and secrete extracellular matrix, and finally form bone tissue, so as to achieve the purpose of repairing trauma and reconstructing functions. As an important part of bone tissue engineering, scaffold materials provide a good microenvironment for cells to obtain nutrients, grow and metabolize. The development of bone tissue engineering provides a technical means of tissue regeneration, which will change the traditional surgical treatment mode of "repairing trauma with trauma" and enter a new stage of non-trauma repair.

骨组织工程支架材料分为人工合成高分子材料和天然高分子材料两大类。合成高分子材料主要有聚乳酸、聚己内酯、聚乙烯醇等。其最显著特点是具有良好的力学和加工性能，能满足许多组织工程的要求，显示出良好的成骨效应。缺点是难降解，残余的溶剂对机体产生不利影响。天然高分子材料有胶原、明胶、透明质酸、壳聚糖、羟基磷灰石等，它们均无毒，具备良好的生物相容性及细胞亲和性，形成的支架可仿生机体细胞外基质，为细胞增殖、分化提供仿生3D空间。Scaffold materials for bone tissue engineering are divided into two categories: synthetic polymer materials and natural polymer materials. Synthetic polymer materials mainly include polylactic acid, polycaprolactone, polyvinyl alcohol, etc. Its most notable feature is that it has good mechanical and processing properties, can meet the requirements of many tissue engineering, and shows a good osteogenic effect. The disadvantage is that it is difficult to degrade, and the residual solvent will have an adverse effect on the body. Natural polymer materials include collagen, gelatin, hyaluronic acid, chitosan, hydroxyapatite, etc., all of which are non-toxic, have good biocompatibility and cell affinity, and the scaffold formed can mimic the extracellular matrix of the body , providing a bionic 3D space for cell proliferation and differentiation.

透明质酸属蛋白多糖，广泛存在于身体组织的基质中，是骨组织的重要有机成分。透明质酸具有高度的吸水能力和固有的膨胀性，在生物介质中透明质酸能够促进细胞渗透进入支架内部，透明质酸可以通过与成骨细胞表面特异性受体α整合素亚单位紧密结合，增强细胞迁移、黏附、增殖和分化来诱导新骨形成，也可以诱导间充质细胞分化从而加速形成新骨。明胶是胶原蛋白部分水解的产物，无毒、几乎无抗原性，并且仍然保留着胶原的细胞粘附信号肽序列RGD，能够提高细胞粘附能力，生物降解性和生物相容性好。采用明胶制备的骨支架具有一系列优良的特性，如提高抗压强度和细胞粘附能力，可促进细胞的粘附、增殖、分化等行为。明胶-羟基磷灰石复合支架中成骨细胞的粘附、增殖能力，碱性磷酸酶活性和骨钙蛋白表达能力很强。羟基磷灰石同骨的无机成分相近，有较好的生物相容性和骨传导性，可与人体骨形成牢固的化学键合，因而被认为是一种最具潜力的人体硬组织替换材料。人工合成的纳米羟基磷灰石具有更强的生物活性，其生物力学强度比天然羟基磷灰石好。Hyaluronic acid is a proteoglycan, which widely exists in the matrix of body tissues and is an important organic component of bone tissue. Hyaluronic acid has a high water absorption capacity and inherent expansibility. In biological media, hyaluronic acid can promote cell penetration into the interior of the scaffold. Hyaluronic acid can be tightly combined with the specific receptor α integrin subunit on the surface of osteoblasts. , enhance cell migration, adhesion, proliferation and differentiation to induce new bone formation, and can also induce mesenchymal cell differentiation to accelerate the formation of new bone. Gelatin is a product of partial hydrolysis of collagen, non-toxic, almost non-antigenic, and still retains the cell adhesion signal peptide sequence RGD of collagen, which can improve cell adhesion, and has good biodegradability and biocompatibility. Bone scaffolds prepared with gelatin have a series of excellent properties, such as improving compressive strength and cell adhesion ability, and can promote cell adhesion, proliferation, differentiation and other behaviors. The adhesion, proliferation, alkaline phosphatase activity and osteocalcin expression of osteoblasts in gelatin-hydroxyapatite composite scaffolds are very strong. The inorganic composition of hydroxyapatite is similar to that of bone, it has good biocompatibility and osteoconductivity, and can form a strong chemical bond with human bone, so it is considered to be the most potential human hard tissue replacement material. Synthetic nano-hydroxyapatite has stronger biological activity, and its biomechanical strength is better than natural hydroxyapatite.

近年来，透明质酸、明胶和纳米羟基磷灰石因其独特的理化性质和生理功能，在医学方面已得到广泛应用。文献已报道采用透明质酸-明胶、明胶-羟基磷灰石、透明质酸-羟基磷灰石等为材料构建的支架，而透明质酸-明胶-纳米羟基磷灰石复合支架的制备却尚未报道，因此综合透明质酸、明胶的理化和生理特性，羟基磷灰石的骨传导特性，本发明制备了一种多孔透明质酸-明胶-纳米羟基磷灰石仿生骨支架。In recent years, hyaluronic acid, gelatin, and nano-hydroxyapatite have been widely used in medicine because of their unique physical and chemical properties and physiological functions. The literature has reported scaffolds constructed from hyaluronic acid-gelatin, gelatin-hydroxyapatite, hyaluronic acid-hydroxyapatite, etc., but the preparation of hyaluronic acid-gelatin-nano-hydroxyapatite composite scaffolds has not yet been reported. According to the report, a porous hyaluronic acid-gelatin-nanometer hydroxyapatite biomimetic bone scaffold was prepared by combining the physical, chemical and physiological properties of hyaluronic acid and gelatin, and the osteoconductive properties of hydroxyapatite.

发明内容Contents of the invention

本发明的目的是提供一种掺杂纳米羟基磷灰石的多孔仿生骨支架的制备方法，该方法制备过程稳定，产物形貌均一可控，操作简单。The purpose of the present invention is to provide a preparation method of a porous bionic bone scaffold doped with nano-hydroxyapatite, the preparation process of which is stable, the appearance of the product is uniform and controllable, and the operation is simple.

本发明采用冷冻干燥法和化学交联相结合，制备多孔透明质酸-明胶-纳米羟基磷灰石仿生复合骨支架，具体步骤如下：The present invention adopts the combination of freeze-drying method and chemical crosslinking to prepare porous hyaluronic acid-gelatin-nanometer hydroxyapatite biomimetic composite bone scaffold, and the specific steps are as follows:

(1)、分别配置透明质酸和明胶水溶液，搅拌去除气泡，将上述两种溶液混合，配置成透明质酸-明胶溶液；(1), respectively configure hyaluronic acid and gelatin aqueous solution, stir to remove air bubbles, mix the above two solutions, and configure hyaluronic acid-gelatin solution;

(2)、将纳米羟基磷灰石超声分散处理后，加入到步骤(1)的混合溶液中，形成纳米羟基磷灰石的透明质酸-明胶悬浮液；(2), after the nano-hydroxyapatite is ultrasonically dispersed, it is added to the mixed solution of step (1) to form a hyaluronic acid-gelatin suspension of nano-hydroxyapatite;

(3)、将步骤(1)所得脱泡的透明质酸-明胶溶液和步骤(2)所得脱泡的透明质酸-明胶-纳米羟基磷灰石混合溶液注入到模具中，预冷冻至结晶，移入冷冻干燥机中冷冻干燥，即得到复合支架；(3), pour the degassed hyaluronic acid-gelatin solution obtained in step (1) and the degassed hyaluronic acid-gelatin-nano hydroxyapatite mixed solution obtained in step (2) into the mold, and pre-freeze until crystallization , moved into a freeze dryer and freeze-dried to obtain a composite scaffold;

(4)、将冻干后的复合支架取出，并转移至24孔板，加入化学交联剂，室温交联，确保交联完全；将所有支架反复冲洗至中性，将支架在预冷冻至结晶，再次冷冻干燥即得交联后的复合多孔仿生骨支架。(4) Take out the lyophilized composite scaffold, transfer it to a 24-well plate, add a chemical cross-linking agent, and cross-link at room temperature to ensure that the cross-linking is complete; wash all the scaffolds repeatedly to neutrality, and pre-freeze the scaffold to crystallization, freeze-drying again to obtain a cross-linked composite porous bionic bone scaffold.

本发明所述多孔仿生骨支架各组分均为天然生物材料，透明质酸和明胶具有良好的生物相容性，为细胞提供粘附位点和三维多孔结构；纳米羟基磷灰石是人骨骼的重要无机组分，具有良好的生物活性和骨传导作用，同时提高了支架的力学性能。相比于透明质酸、明胶和透明质酸-明胶支架，纳米羟基磷灰石的引入，提高了该复合支架的力学性能和骨传导性。Each component of the porous bionic bone scaffold described in the present invention is a natural biomaterial, and hyaluronic acid and gelatin have good biocompatibility, and provide adhesion sites and a three-dimensional porous structure for cells; nano-hydroxyapatite is a human bone It has good biological activity and osteoconduction, and at the same time improves the mechanical properties of the scaffold. Compared with hyaluronic acid, gelatin and hyaluronic acid-gelatin scaffolds, the introduction of nano-hydroxyapatite improved the mechanical properties and osteoconductivity of the composite scaffold.

本发明所述多孔仿生骨支架制备过程中，纳米羟基磷灰石经超声分散处理，防止纳米颗粒在透明质酸-明胶溶液中聚团；溶液混合后尽快注入模具，防止纳米颗粒沉降，造成纳米颗粒在溶液中分散不均。In the preparation process of the porous bionic bone scaffold of the present invention, the nano-hydroxyapatite is ultrasonically dispersed to prevent the agglomeration of the nanoparticles in the hyaluronic acid-gelatin solution; the solution is injected into the mold as soon as possible after the solution is mixed to prevent the sedimentation of the nanoparticles, resulting in nano The particles are not uniformly dispersed in the solution.

本发明所述多孔仿生骨支架采用化学交联方法制备，交联剂为碳化二亚胺和N-羟基琥珀酰亚胺的乙醇水混合溶液(优选pH＝5)，交联温度为室温，交联时间为6～48h。The porous bionic bone scaffold of the present invention is prepared by a chemical cross-linking method, the cross-linking agent is an ethanol-water mixed solution of carbodiimide and N-hydroxysuccinimide (preferably pH=5), the cross-linking temperature is room temperature, and the cross-linking agent is The connection time is 6~48h.

本发明所述多孔仿生骨支架的制备过程中，通过调节透明质酸、明胶、纳米羟基磷灰石的浓度、质量百分比及冷冻干燥条件(预冷冻时间、冻干时间)和化学交联时间等，可制备出具有不同孔径、孔隙率、降解率、吸水率和机械强度的复合支架。In the preparation process of the porous bionic bone scaffold of the present invention, by adjusting the concentration, mass percentage and freeze-drying conditions (pre-freezing time, freeze-drying time) and chemical cross-linking time of hyaluronic acid, gelatin, nano-hydroxyapatite, etc. , composite scaffolds with different pore sizes, porosity, degradation rate, water absorption and mechanical strength can be prepared.

本发明所述掺杂纳米羟基磷灰石的多孔仿生骨支架的制备方法，其特征在于：The preparation method of the porous bionic bone scaffold doped with nano-hydroxyapatite of the present invention is characterized in that:

步骤(1)中配置的透明质酸溶液浓度为5mg/ml～15mg/ml，明胶溶液浓度为5mg/ml～0.2g/ml；上述两种溶液混合时，透明质酸溶液和明胶溶液的体积比为1:9～9:1。The concentration of the hyaluronic acid solution configured in step (1) is 5mg/ml～15mg/ml, and the concentration of the gelatin solution is 5mg/ml～0.2g/ml; when the above two solutions are mixed, the volume of the hyaluronic acid solution and the gelatin solution The ratio is 1:9～9:1.

步骤(2)所述混合溶液中纳米羟基磷灰石的质量浓度为1～50mg/ml。The mass concentration of nano-hydroxyapatite in the mixed solution in step (2) is 1-50 mg/ml.

步骤(3)、(4)中预冷冻温度在-20℃～-80℃之间；冷冻干燥时间为12～48h。The pre-freezing temperature in steps (3) and (4) is between -20° C. to -80° C.; the freeze-drying time is 12 to 48 hours.

步骤(4)中所有支架用PBS反复冲洗至中性。In step (4), all scaffolds were washed repeatedly with PBS until neutral.

采用本发明所述方法制得的多孔仿生骨支架孔径为90～240μm，孔隙率＞90％，细胞平均粘附率为80％，具备一定的吸水能力和降解能力。The porous bionic bone scaffold prepared by the method of the invention has a pore diameter of 90-240 μm, a porosity of more than 90 percent, an average cell adhesion rate of 80 percent, and certain water absorption and degradation capabilities.

本发明所述多孔仿生骨支架的降解实验采用溶菌酶检测，溶菌酶的浓度为0.05～0.5mg/ml(pH＝5.2)，降解时间为5周。The degradation experiment of the porous bionic bone scaffold of the present invention is detected by lysozyme, the concentration of lysozyme is 0.05-0.5 mg/ml (pH=5.2), and the degradation time is 5 weeks.

本发明所述多孔仿生骨支架随着透明质酸含量的增加，孔径和孔隙率增加，吸水率减小，降解率增加；所述多孔仿生骨支架随着纳米羟基磷灰石含量的增加，孔径和孔隙率减小，吸水率减小，降解率增加。With the increase of the content of hyaluronic acid, the porous biomimetic bone scaffold of the present invention increases the pore size and porosity, decreases the water absorption rate, and increases the degradation rate; And the porosity decreases, the water absorption decreases, and the degradation rate increases.

采用本发明所述方法制备的透明质酸-明胶-纳米羟基磷灰石多孔复合支架孔径均一、孔壁薄、孔隙贯穿，具有很好的细胞粘附能力和机械强度，具有骨传导性。本发明的优点在于该复合支架能够更加真实的模拟骨组织微环境，通过调节各组分的浓度、体积比及冷冻干燥条件，可调控支架的孔径、孔隙率、降解率、吸水率、机械强度等参数；所用材料均具备良好的生物相容性，形成的支架满足骨组织的内部多孔结构；材料来源充足，制备工艺简单。The hyaluronic acid-gelatin-nanometer hydroxyapatite porous composite scaffold prepared by the method of the invention has uniform pore size, thin pore wall and penetrating pores, has good cell adhesion ability and mechanical strength, and has osteoconductivity. The advantage of the present invention is that the composite scaffold can more realistically simulate the microenvironment of bone tissue, and by adjusting the concentration, volume ratio and freeze-drying conditions of each component, the pore diameter, porosity, degradation rate, water absorption rate, and mechanical strength of the scaffold can be adjusted. and other parameters; the materials used have good biocompatibility, and the formed scaffold meets the internal porous structure of bone tissue; the source of the material is sufficient, and the preparation process is simple.

附图说明Description of drawings

图1：掺杂纳米羟基磷灰石的多孔仿生骨支架的制备示意图。Figure 1: Schematic diagram of the preparation of porous biomimetic bone scaffolds doped with nano-hydroxyapatite.

图2：不同组分掺杂纳米羟基磷灰石的多孔仿生骨支架的扫描电镜图片。Figure 2: Scanning electron microscope images of porous biomimetic bone scaffolds doped with nano-hydroxyapatite with different components.

图3：不同组分掺杂纳米羟基磷灰石的多孔仿生骨支架的降解率。Figure 3: Degradation rates of porous biomimetic bone scaffolds doped with nano-hydroxyapatite with different components.

图4：掺杂纳米羟基磷灰石的多孔仿生骨支架的傅里叶红外图谱。Figure 4: Fourier transform infrared spectrum of the porous biomimetic bone scaffold doped with nano-hydroxyapatite.

图5：复合支架与成骨前体细胞共培养的扫描电镜图片。Figure 5: Scanning electron micrographs of composite scaffolds co-cultured with osteogenic precursor cells.

图6：复合支架与成骨前体细胞共培养的ALP染色图片。Figure 6: ALP staining pictures of composite scaffolds co-cultured with osteogenic precursor cells.

具体实施方式detailed description

实施例1：Example 1:

掺杂纳米羟基磷灰石的多孔透明质酸(5mg/ml)-明胶(5mg/ml)复合支架的制备：Preparation of porous hyaluronic acid (5mg/ml)-gelatin (5mg/ml) composite scaffold doped with nano-hydroxyapatite:

在60℃水浴下，将透明质酸、明胶分别溶于三蒸水制得5mg/ml透明质酸溶液和5mg/ml明胶混合液；搅拌去除气泡，将上述两种溶液混合，配置成体积比为1:9和9:1的透明质酸-明胶溶液；In a water bath at 60°C, dissolve hyaluronic acid and gelatin in three-distilled water respectively to obtain a 5 mg/ml hyaluronic acid solution and a 5 mg/ml gelatin mixture; stir to remove air bubbles, mix the above two solutions, and configure the volume ratio 1:9 and 9:1 hyaluronic acid-gelatin solutions;

纳米羟基磷灰石超声分散处理后，按不同质量比加入到上述溶液中(其中一种溶液不添加纳米羟基磷灰石)，形成不含纳米羟基磷灰石的透明质酸-明胶悬浮液、含有1mg/ml纳米羟基磷灰石和50mg/ml纳米羟基磷灰石的透明质酸-明胶悬浮液；After nano-hydroxyapatite is ultrasonically dispersed, it is added to the above solutions according to different mass ratios (one of the solutions does not add nano-hydroxyapatite) to form a hyaluronic acid-gelatin suspension without nano-hydroxyapatite, Hyaluronic acid-gelatin suspension containing 1 mg/ml nano-hydroxyapatite and 50 mg/ml nano-hydroxyapatite;

将上述透明质酸-明胶、透明质酸-明胶-纳米羟基磷灰石混合液静置除泡，注入模具，-80℃预冷冻3h以上，真空冷冻干燥24h，即得到不同比例复合支架。The above-mentioned hyaluronic acid-gelatin and hyaluronic acid-gelatin-nano hydroxyapatite mixtures were left to defoam, poured into the mold, pre-frozen at -80°C for more than 3 hours, and vacuum freeze-dried for 24 hours to obtain composite scaffolds with different proportions.

冻干后的复合支架用水溶性碳化二亚胺和N-羟基琥珀酰亚胺进行化学交联，交联剂使支架发生官能团反应。将冻干后的三组复合支架小心取出，将支架转移至24孔板，每孔加入2mL含50mM碳化二亚胺、20mM N-羟基琥珀酰亚胺的95％乙醇-5％水混合溶液(pH＝5)，室温交联24h，确保交联完全；交联后吸出交联剂，将所有支架用PBS反复冲洗至中性；将支架在-80℃预冷冻3h，再次冷冻干燥即得交联后的复合支架。The freeze-dried composite scaffold is chemically cross-linked with water-soluble carbodiimide and N-hydroxysuccinimide, and the cross-linking agent makes the scaffold react with functional groups. The three groups of composite scaffolds after freeze-drying were carefully taken out, and the scaffolds were transferred to a 24-well plate, and 2 mL of 95% ethanol-5% water mixed solution containing 50 mM carbodiimide and 20 mM N-hydroxysuccinimide was added to each well ( pH=5), cross-linked at room temperature for 24 hours to ensure complete cross-linking; after cross-linking, the cross-linking agent was aspirated, and all scaffolds were washed repeatedly with PBS until neutral; Combined composite scaffold.

实施例2：Example 2:

掺杂纳米羟基磷灰石的多孔透明质酸(15mg/ml)-明胶(0.2g/ml)复合支架的制备：Preparation of porous hyaluronic acid (15mg/ml)-gelatin (0.2g/ml) composite scaffold doped with nano-hydroxyapatite:

在60℃水浴下，将透明质酸、明胶分别溶于三蒸水制得15mg/ml透明质酸溶液和0.2g/ml明胶混合液；搅拌去除气泡，将上述两种溶液混合，配置成体积比为1:9和9:1的透明质酸-明胶溶液；In a water bath at 60°C, dissolve hyaluronic acid and gelatin in triple-distilled water to obtain a 15 mg/ml hyaluronic acid solution and a 0.2 g/ml gelatin mixture; stir to remove air bubbles, mix the above two solutions, and prepare a volume Hyaluronic acid-gelatin solutions with ratios of 1:9 and 9:1;

纳米羟基磷灰石超声分散处理后，按不同质量比加入到上述溶液中(其中一种溶液不添加纳米羟基磷灰石)，形成不含纳米羟基磷灰石的透明质酸-明胶悬浮液、含有1mg/ml纳米羟基磷灰石和50mg/ml纳米羟基磷灰石的透明质酸-明胶悬浮液；After nano-hydroxyapatite is ultrasonically dispersed, it is added to the above solutions according to different mass ratios (one of the solutions does not add nano-hydroxyapatite) to form a hyaluronic acid-gelatin suspension without nano-hydroxyapatite, Hyaluronic acid-gelatin suspension containing 1 mg/ml nano-hydroxyapatite and 50 mg/ml nano-hydroxyapatite;

将上述透明质酸-明胶、透明质酸-明胶-纳米羟基磷灰石混合液静置除泡，注入模具，-80℃预冷冻3h以上，真空冷冻干燥24h，即得到不同比例复合支架。The above-mentioned hyaluronic acid-gelatin and hyaluronic acid-gelatin-nano hydroxyapatite mixtures were left to defoam, poured into the mold, pre-frozen at -80°C for more than 3 hours, and vacuum freeze-dried for 24 hours to obtain composite scaffolds with different proportions.

冻干后的复合支架用水溶性碳化二亚胺和N-羟基琥珀酰亚胺进行化学交联，交联剂使支架发生官能团反应。将冻干后的三组复合支架小心取出，将支架转移至24孔板，每孔加入2mL含50mM碳化二亚胺、20mM N-羟基琥珀酰亚胺的95％乙醇-5％水混合溶液(pH＝5)，室温交联24h，确保交联完全；交联后吸出交联剂，将所有支架用PBS反复冲洗至中性；将支架在-80℃预冷冻3h，再次冷冻干燥即得交联后的复合支架。The freeze-dried composite scaffold is chemically cross-linked with water-soluble carbodiimide and N-hydroxysuccinimide, and the cross-linking agent makes the scaffold react with functional groups. The three groups of composite scaffolds after freeze-drying were carefully taken out, and the scaffolds were transferred to a 24-well plate, and 2 mL of 95% ethanol-5% water mixed solution containing 50 mM carbodiimide and 20 mM N-hydroxysuccinimide was added to each well ( pH=5), cross-linked at room temperature for 24 hours to ensure complete cross-linking; after cross-linking, the cross-linking agent was aspirated, and all scaffolds were washed repeatedly with PBS until neutral; Combined composite scaffold.

图1为掺杂纳米羟基磷灰石的多孔仿生骨支架的制备流程示意图。复合支架的扫描电镜如图2所示，扫描电镜观察复合支架呈多孔结构，孔隙及内部结构均匀，孔内部贯穿性很高，大孔内部有许多小孔，有利于营养物质和代谢物质在支架内部的传输。支架表面及孔壁粗糙，孔壁较薄，有利于细胞粘附。由表1和图3可知，复合支架的孔径、孔隙率、吸水率、粘附率、降解率各不相同。上述参数均与复合支架各组分的最终质量浓度有关。随着透明质酸含量的增加，复合支架的孔径和孔隙率增加，吸水率降低，粘附率增加，降解率增加；所述复合支架随着纳米羟基磷灰石含量的增加，孔径和孔隙率减小，吸水率减小，降解率增加，粘附率变化不大。傅里叶红外图谱显示仿生骨支架中纳米羟基磷灰石掺杂成功(图4)。Figure 1 is a schematic diagram of the preparation process of the porous biomimetic bone scaffold doped with nano-hydroxyapatite. The scanning electron microscope of the composite support is shown in Figure 2. The scanning electron microscope shows that the composite support has a porous structure, the pores and internal structure are uniform, the internal penetration of the pores is very high, and there are many small holes inside the large pores, which is conducive to the flow of nutrients and metabolites in the support. Internal transmission. The surface of the scaffold and the pore wall are rough, and the pore wall is thinner, which is conducive to cell adhesion. It can be seen from Table 1 and Figure 3 that the pore size, porosity, water absorption rate, adhesion rate, and degradation rate of the composite scaffolds are different. The above parameters are all related to the final mass concentration of each component of the composite scaffold. As the content of hyaluronic acid increases, the pore size and porosity of the composite scaffold increase, the water absorption rate decreases, the adhesion rate increases, and the degradation rate increases; Decrease, the water absorption rate decreases, the degradation rate increases, and the adhesion rate changes little. The Fourier transform infrared spectrum showed that nano-hydroxyapatite was successfully doped in the bionic bone scaffold (Fig. 4).

实施例3：Example 3:

掺杂纳米羟基磷灰石的多孔仿生骨支架的细胞培养以及分析评价：Cell culture and analysis evaluation of porous biomimetic bone scaffold doped with nano-hydroxyapatite:

将实验室自行设计并制备的复合支架灭菌处理，支架经75％乙醇浸泡3h，紫外照射1h，浸泡入含10％双抗的PBS深度灭菌。采用Ⅰ型胶原进行表面修饰后上述9组复合支架接种成骨前体细胞，接种密度为2×10⁶个/mL，待6h细胞贴壁后补加培养基，细胞培养7天后，进行细胞死活、凋亡等方面的相关染色，细胞增殖、分化检测，Image Pro软件分析。由图5可知，成骨前体细胞呈团簇或单细胞状态分布于支架的孔隙和表面，细胞与支架、细胞与细胞之间相互连接。成骨前体细胞与复合支架共培养至第3天，只有加诱导培养基的复合支架组表达碱性磷酸酶(ALP)，其他组均为表达ALP；培养至第7天，对照组和复合支架组均表达ALP；ALP表达的关系如下：加诱导培养基的复合支架组＞未加诱导培养基的复合支架组＞对照组，说明复合支架与诱导培养基协同作用促进成骨前体细胞分化为成骨细胞的能力(图6)。因此，本发明掺杂纳米羟基磷灰石的多孔仿生骨支架可作为一种骨组织工程支架材料。The composite scaffold designed and prepared by the laboratory was sterilized. The scaffold was soaked in 75% ethanol for 3 hours, irradiated with ultraviolet light for 1 hour, and soaked in PBS containing 10% double antibody for deep sterilization. After surface modification with type Ⅰ collagen, the above 9 groups of composite scaffolds were inoculated with osteoblast precursor cells at a seeding density of 2×10 ⁶ cells/mL. After 6 hours of cell attachment, the medium was added. After 7 days of cell culture, the cells were killed and activated. , apoptosis and other related staining, cell proliferation, differentiation detection, Image Pro software analysis. It can be seen from Figure 5 that the osteoblast precursor cells are distributed in the pores and surfaces of the scaffold in the state of clusters or single cells, and the cells and the scaffold, and the cells are connected to each other. The osteoblast precursor cells were co-cultured with the composite scaffold until the third day, only the composite scaffold group with induction medium expressed alkaline phosphatase (ALP), and the other groups expressed ALP; All the scaffold groups expressed ALP; the relationship of ALP expression was as follows: the composite scaffold group with induction medium > the composite scaffold group without induction medium > the control group, indicating that the composite scaffold and induction medium synergistically promoted the differentiation of osteogenic precursor cells The ability to become osteoblasts (Figure 6). Therefore, the porous biomimetic bone scaffold doped with nano-hydroxyapatite of the present invention can be used as a scaffold material for bone tissue engineering.

表1不同组分的多孔仿生骨支架的孔径、孔隙率、吸水率、粘附率Table 1 Pore diameter, porosity, water absorption, adhesion rate of porous bionic bone scaffolds with different components

其中HA：透明质酸，G：明胶水溶液，HAP：羟基磷灰石，括号中的比例关系为体积比，百分数指羟基磷灰石在质酸-明胶悬浮液中的质量百分比。Among them, HA: hyaluronic acid, G: gelatin aqueous solution, HAP: hydroxyapatite, the proportion relationship in brackets is the volume ratio, and the percentage refers to the mass percentage of hydroxyapatite in the hyaluronic acid-gelatin suspension.

上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。The above-mentioned embodiments are only to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.