技术领域technical field
本发明涉及生物医学复合材料，具体涉及一种磁性纳米多孔复合骨组织工程支架材料及其制备方法。The invention relates to biomedical composite materials, in particular to a magnetic nanoporous composite bone tissue engineering scaffold material and a preparation method thereof.
背景技术Background technique
骨组织工程的研究和发展为临床上骨缺损的修复和治疗提供了崭新的思路，并逐渐引起研究者的兴趣。其中，三维多孔支架材料是骨组织工程的重要基础，它不仅起支撑作用，保持原有组织的形状，而且还起到模板作用，为细胞提供赖以寄宿、生长、分化和增殖的场所，从而引导受损组织的再生和控制再生组织的结构。但目前骨组织工程支架的研究和应用仍面临着许多问题，如材料体内降解不可控，细胞繁殖效率低，降解产物引起炎性反应，细胞去分化等。尤其是在力学性能方面，单一的使用聚合物或陶瓷材料作为支架材料存在强度弱或脆性大的问题，都无法满足承力骨修复的要求。The research and development of bone tissue engineering provides a new idea for the repair and treatment of clinical bone defects, and gradually arouses the interest of researchers. Among them, the three-dimensional porous scaffold material is an important basis for bone tissue engineering. It not only acts as a support to maintain the shape of the original tissue, but also acts as a template, providing a place for cells to lodge, grow, differentiate and proliferate, thereby Guides the regeneration of damaged tissue and controls the structure of the regenerated tissue. However, the research and application of bone tissue engineering scaffolds still face many problems, such as uncontrollable degradation of materials in vivo, low cell reproduction efficiency, inflammatory reactions caused by degradation products, and dedifferentiation of cells. Especially in terms of mechanical properties, the single use of polymer or ceramic materials as scaffold materials has the problem of weak strength or high brittleness, which cannot meet the requirements of load-bearing bone repair.
羟基磷灰石(Hydroxy-apatite,简称HA）是最常见的一种生物活性材料，它具有与人体骨组织相似的无机成分，是目前公认的具有较好生物相容性和骨传导性的生物活性陶瓷材料。但HA人工骨仍存在着力学性能差、不能承重、缺乏骨诱导活性、骨渗入深度有限、不能完全降解等较多缺点。目前已有纳米羟基磷灰石（Nano-HA）应用于临床骨缺损的修复。但因骨骼本身是一种具有很高韧性和硬度的天然纳米复合材料，它主要由纳米级的片状羟基磷灰石结晶分散在骨胶原骨架中组成，而骨胶原和羟基磷灰石本身都不能作为结构材料，因此以Nano-HA作为单一成分的人工骨仍然不能满足理想人工骨的要求。Hydroxyapatite (Hydroxy-apatite, HA for short) is the most common bioactive material. active ceramic material. However, HA artificial bone still has many shortcomings such as poor mechanical properties, inability to bear weight, lack of osteoinductive activity, limited bone penetration depth, and incomplete degradation. At present, nano-hydroxyapatite (Nano-HA) has been applied to the repair of clinical bone defects. However, because the bone itself is a natural nanocomposite material with high toughness and hardness, it is mainly composed of nanoscale sheet-like hydroxyapatite crystals dispersed in the bone collagen skeleton, and bone collagen and hydroxyapatite themselves are both It cannot be used as a structural material, so the artificial bone with Nano-HA as a single component still cannot meet the requirements of an ideal artificial bone.
聚乳酸(Polylactic acid,简称PLLA)是目前组织工程研究和应用最为广泛的一类材料，这类材料无毒，无抗原性，具有良好的可降解吸收性、生物安全性和力学强度，可以通过控制成份含量来调节材料的降解速度，使产品性质的重复性和力学性能达到较高水平。除了作为手术缝合线和药物缓释载体外，PLLA也用作医疗缝合补强材料、骨折内固定材料和组织工程材料。Polylactic acid (PLLA) is currently the most widely used material in tissue engineering research and application. This type of material is non-toxic, non-antigenic, and has good biodegradable absorption, biosafety and mechanical strength. It can be passed through Control the content of ingredients to adjust the degradation rate of materials, so that the repeatability and mechanical properties of product properties can reach a higher level. In addition to being used as surgical sutures and drug sustained-release carriers, PLLA is also used as medical suture reinforcement materials, fracture internal fixation materials and tissue engineering materials.
随着生物工程和生物医学的发展,经常需要一些功能性材料。如单分散、微米级、功能性高分子粒子,磁性纳米粒子具有比表面积大、吸附性强、凝集作用大以及表面具有反应能力等特异性质而得到了广泛的应用。而且能在外加磁场中很方便地与介质分离，可作为分离材料和载体，用于细胞分离、固定化酶、免疫分析、靶向药物等方面。With the development of bioengineering and biomedicine, some functional materials are often needed. Such as monodisperse, micron-sized, functional polymer particles, and magnetic nanoparticles have been widely used because of their specific properties such as large specific surface area, strong adsorption, strong agglutination, and surface reactivity. Moreover, it can be easily separated from the medium in an external magnetic field, and can be used as a separation material and carrier for cell separation, immobilized enzymes, immune analysis, targeted drugs, and the like.
发明内容Contents of the invention
本发明的目的是为了解决目前骨组织工程支架存在的缺陷，满足支架的性能要求，将Nano-HA、PLLA和Fe2O3复合制备三维多孔的Nano-HA/PLLA/Fe2O3人工骨，提供一种磁性纳米多孔复合骨组织工程支架材料及其制备方法。The purpose of the present invention is to solve the defects existing in the current bone tissue engineering scaffolds, meet the performance requirements of the scaffolds, and prepare a three-dimensional porous Nano-HA/PLLA/Fe2 O3 artificial bone by compounding Nano-HA, PLLA and Fe2 O3 Provided are a magnetic nanoporous composite bone tissue engineering scaffold material and a preparation method thereof.
本发明的方案为：The scheme of the present invention is:
磁性纳米多孔复合骨组织工程支架材料，所述支架材料是由纳米羟基磷灰石、聚乳酸和磁性粒子复合而成的支架材料。The magnetic nanoporous composite bone tissue engineering support material is a support material composed of nano-hydroxyapatite, polylactic acid and magnetic particles.
优选地，所述纳米羟基磷灰石、聚乳酸和磁性粒子的质量比为纳米羟基磷灰石：聚乳酸：磁性粒子＝1～5：20：1～5。Preferably, the mass ratio of nano-hydroxyapatite, polylactic acid and magnetic particles is nano-hydroxyapatite:polylactic acid:magnetic particles=1-5:20:1-5.
优选地，所述纳米羟基磷灰石、聚乳酸和磁性粒子的质量比为纳米羟基磷灰石：聚乳酸：磁性粒子＝3：20：3。Preferably, the mass ratio of nano-hydroxyapatite, polylactic acid and magnetic particles is nano-hydroxyapatite:polylactic acid:magnetic particles=3:20:3.
优选地，所述磁性粒子为Fe2O3。Preferably, the magnetic particles are Fe2 O3 .
本发明还提供所述磁性纳米多孔复合骨组织工程支架材料的制备方法，该方法包括以下步骤：The present invention also provides a preparation method of the magnetic nanoporous composite bone tissue engineering scaffold material, the method comprising the following steps:
将Nano-HA、Fe2O3和PLLA材料在50～70℃下混合，超声波震荡，然后电磁搅拌至Nano-HA和Fe2O3完全分散均匀得到凝胶；然后将凝胶转入-5～-3℃下进行低温成型并填入生物材料即得多孔纳米复合骨组织工程支架材料。Mix Nano-HA, Fe2 O3 and PLLA materials at 50-70°C, ultrasonically oscillate, and then stir electromagnetically until Nano-HA and Fe2 O3 are completely dispersed and uniform to obtain a gel; then transfer the gel to -5 Perform low-temperature molding at ~-3°C and fill with biomaterials to obtain porous nanocomposite bone tissue engineering scaffold materials.
优选地，包括以下步骤：Preferably, the following steps are included:
（1）将PLLA溶解于有机溶剂里，60℃水浴中搅拌形成PLLA/有机溶剂的均相溶液；将不同质量比的Nano-HA和Fe2O3加入上述溶液中，超声波震荡各15min、电磁搅拌至Nano-HA和Fe2O3完全分散均匀制得凝胶；(1) Dissolve PLLA in an organic solvent and stir in a water bath at 60°C to form a homogeneous solution of PLLA/organic solvent; add Nano-HA and Fe2 O3 with different mass ratios to the above solution, ultrasonically oscillate for 15 minutes each, and electromagnetically Stir until Nano-HA and Fe2 O3 are completely dispersed and evenly prepared into a gel;
（2）将凝胶转入低温冷冻快速成型仪中-4℃条件下进行低温快速成型,通过RIPF工艺成形具有一定间隔的平行的冰线，将配制好的生物材料填入冰线间的间隔，将填了生物材料后的表面刮平，使得冰线和生物材料交替排列；在正交方向成形下一层冰线，重复以上步骤；(2) Transfer the gel into a low-temperature freezing rapid prototyping apparatus for low-temperature rapid prototyping at -4°C, and form parallel ice lines with a certain interval through the RIPF process, and fill the prepared biological materials into the intervals between the ice lines , scrape the surface filled with biomaterials, so that the ice lines and biomaterials are alternately arranged; form the next layer of ice lines in the orthogonal direction, and repeat the above steps;
（3）成形后，将冰和生物材料构成的实体拿到冷藏室，冰融化后再将产品置于真空冷冻干燥机去除有机溶剂；风干后即得多孔纳米复合骨组织工程支架材料。(3) After forming, the entity composed of ice and biological materials is taken to the refrigerator, and after the ice melts, the product is placed in a vacuum freeze dryer to remove organic solvents; after air drying, the porous nanocomposite bone tissue engineering scaffold material is obtained.
优选地，所述步骤（1）中有机溶剂是1,4-二氧六环。Preferably, the organic solvent in the step (1) is 1,4-dioxane.
优选地，所述Nano-HA通过如下方法制得：Preferably, the Nano-HA is prepared by the following method:
将硝酸钙与磷酸铵的水溶液进行化学合成，合成过程中，加入氨水调整溶液的pH值为8～13，添加分散剂，搅拌使其沉淀完全，然后经洗涤、过滤，将沉淀物在80～120℃干燥，再在600～800℃温度下烧结2～3小时，得到Nano-HA粉末。The aqueous solution of calcium nitrate and ammonium phosphate is chemically synthesized. During the synthesis process, ammonia water is added to adjust the pH value of the solution to 8-13, a dispersant is added, stirred to make the precipitation complete, and then washed and filtered to remove the precipitate at 80-13. Dry at 120°C, and then sinter at 600-800°C for 2-3 hours to obtain Nano-HA powder.
优选地，所述PLLA通过如下方法制得：Preferably, the PLLA is prepared by the following method:
①丙交酯的制备：取乳酸与ZnO粉末，逐渐升温使乳酸脱水变为丙交酯；然后升温抽真空蒸出淡黄色丙交酯单体，再以乙酸乙酯为溶剂反复洗涤、重结晶提纯，抽滤，真空干燥，得纯净丙交酯单体；①Preparation of lactide: Take lactic acid and ZnO powder, gradually raise the temperature to dehydrate the lactic acid into lactide; then raise the temperature and vacuumize to evaporate the light yellow lactide monomer, and then use ethyl acetate as solvent to repeatedly wash and recrystallize Purification, suction filtration, and vacuum drying to obtain pure lactide monomer;
②PLLA的合成：纯净丙交酯单体，加入辛酸亚锡引发剂，常温下真空干燥一定时间后，在硅油浴中浸泡3～5min，密封反应瓶，升温至单体刚熔化时反复摇动反应瓶至混合均匀，聚合完成后取出反应瓶；② Synthesis of PLLA: pure lactide monomer, add stannous octoate initiator, vacuum dry at room temperature for a certain period of time, soak in a silicone oil bath for 3 to 5 minutes, seal the reaction bottle, and shake the reaction bottle repeatedly when the temperature rises until the monomer just melts To mix evenly, take out the reaction bottle after the polymerization is completed;
③PLLA的纯化：粗制PLLA中加入二氯甲烷使其完全溶解，过滤，滤液中加入甲醇至沉淀完全，再过滤，甲醇洗涤，真空干燥，得到白色纯PLLA。③Purification of PLLA: Add dichloromethane to the crude PLLA to dissolve it completely, filter, add methanol to the filtrate until the precipitation is complete, filter again, wash with methanol, and dry in vacuum to obtain white pure PLLA.
本发明利用低温快速成型技术将Nano-HA、PLLA和磁性纳米粒子Fe2O3复合制备三维多孔的Nano-HA/PLLA/Fe2O3人工骨，该三维多孔的磁性纳米人工骨支架材料综合了Nano-HA、PLLA和Fe2O3三者的优点，使其在良好骨传导性和生物相容性的基础上，提高柔韧性和生物降解性，改善生物力学性能，更加有利于骨细胞的粘附生长和血管化，必将极大地提高骨缺损处移植人工骨的愈合速度及效果，是一种理想的新型纳米复合人工骨材料。The present invention utilizes low-temperature rapid prototyping technology to compound Nano-HA, PLLA and magnetic nanoparticles Fe2 O3 to prepare a three-dimensional porous Nano-HA/PLLA/Fe2 O3 artificial bone. The three-dimensional porous magnetic nano-artificial bone scaffold material is comprehensive Combining the advantages of Nano-HA, PLLA and Fe2 O3 , on the basis of good osteoconductivity and biocompatibility, it improves flexibility and biodegradability, improves biomechanical properties, and is more beneficial to bone cells Adhesive growth and vascularization will greatly improve the healing speed and effect of artificial bone transplanted in bone defects, and it is an ideal new nanocomposite artificial bone material.
本发明采用磁性粒子做为复合材料，磁性纳米粒子Fe2O3具有磁响应性及超顺磁性，可以在恒定磁场下聚集和定位、在交变磁场下吸收电磁波产热。由于磁性纳米粒子具有和细胞表面结合的能力，这就使在外加磁场的条件下控制和调节细胞的功能成为可能。磁性纳米粒子以平均每个细胞20pg的浓度聚集在间充质干细胞内，在外加磁场的作用下，磁性纳米粒子可显著促进骨髓间充质干细胞的增殖。而且人间充质干细胞在磁性纳米粒子和外加磁场的作用下具有分化为成骨细胞，脂肪细胞和软骨细胞的能力，磁性纳米粒子和磁场联合作用能够促进骨修复。The invention adopts magnetic particles as composite materials, and the magnetic nano-particleFe2O3 has magnetic responsiveness and superparamagnetism, can gather and locate under aconstant magnetic field, and absorb electromagnetic waves to generate heat under an alternating magnetic field. Because magnetic nanoparticles have the ability to bind to the cell surface, it is possible to control and adjust the function of cells under the condition of an external magnetic field. The magnetic nanoparticles are gathered in the mesenchymal stem cells at an average concentration of 20 pg per cell, and under the action of an external magnetic field, the magnetic nanoparticles can significantly promote the proliferation of the bone marrow mesenchymal stem cells. Moreover, human mesenchymal stem cells have the ability to differentiate into osteoblasts, adipocytes and chondrocytes under the action of magnetic nanoparticles and an external magnetic field, and the combined action of magnetic nanoparticles and magnetic fields can promote bone repair.
具体实施方式Detailed ways
以下提供本发明优选的具体实施方式，以助于进一步说明本发明，但本发明的保护范围并不仅限于这些实施例。Preferred specific embodiments of the present invention are provided below to help further illustrate the present invention, but the protection scope of the present invention is not limited to these examples.
本实施例磁性纳米多孔复合骨组织工程支架材料的制备方法技术方案如下：The technical scheme of the preparation method of the magnetic nanoporous composite bone tissue engineering scaffold material in this embodiment is as follows:
1.采用溶胶-絮凝法合成Nano-HA。1. Synthesis of Nano-HA by sol-flocculation method.
2.采用开环聚合法合成PLLA。2. PLLA was synthesized by ring-opening polymerization.
3.采用低温快速成型技术合成磁性纳米多孔复合支架材料。3. The magnetic nanoporous composite scaffold material was synthesized by low temperature rapid prototyping technology.
上述制备方法中，所述Nano-HA的合成方法如下：In the above preparation method, the synthesis method of the Nano-HA is as follows:
将硝酸钙与磷酸铵的水溶液进行化学合成，合成过程中，加入氨水调整溶液的pH值为8～13，添加分散剂，搅拌使其沉淀完全，然后经洗涤、过滤，将沉淀物在80～120℃干燥，在600～800℃温度下烧结2～3小时，得到粉末粒径小于100nm、与人体骨组织成份相似的Nano-HA粉末。The aqueous solution of calcium nitrate and ammonium phosphate is chemically synthesized. During the synthesis process, ammonia water is added to adjust the pH value of the solution to 8-13, a dispersant is added, stirred to make the precipitation complete, and then washed and filtered to remove the precipitate at 80-13. Dry at 120°C and sinter at 600-800°C for 2-3 hours to obtain Nano-HA powder with a particle size of less than 100nm and a composition similar to human bone tissue.
上述制备方法中，所述PLLA的合成方法如下：In the above-mentioned preparation method, the synthetic method of described PLLA is as follows:
①丙交酯的制备：取乳酸与ZnO粉末，逐渐升温使乳酸脱水变为丙交酯；然后升温抽真空蒸出淡黄色丙交酯单体，再以乙酸乙酯为溶剂反复洗涤、重结晶提纯，抽滤，真空干燥，得无色片状纯丙交酯晶体。①Preparation of lactide: Take lactic acid and ZnO powder, gradually raise the temperature to dehydrate the lactic acid into lactide; then raise the temperature and vacuumize to evaporate the light yellow lactide monomer, and then use ethyl acetate as solvent to repeatedly wash and recrystallize Purify, filter with suction, and dry in vacuo to obtain colorless flaky pure lactide crystals.
②PLLA的合成：纯净丙交酯单体，加入辛酸亚锡引发剂，常温下真空干燥，然后在硅油浴中浸泡3-5min，密封反应瓶，升温至单体刚熔化时反复摇动反应瓶至混合均匀，然后记时聚合完成后取出反应瓶。② Synthesis of PLLA: pure lactide monomer, add stannous octoate initiator, vacuum dry at room temperature, then soak in a silicone oil bath for 3-5min, seal the reaction bottle, and shake the reaction bottle repeatedly until the monomer is just melted. Evenly, then take out the reaction bottle after timing polymerization is completed.
③PLLA的纯化：粗制PLLA中加入二氯甲烷使其完全溶解，过滤，滤液中加入甲醇至沉淀完全，再过滤，甲醇洗涤，真空干燥，得到白色纯PLLA。③Purification of PLLA: Add dichloromethane to the crude PLLA to dissolve it completely, filter, add methanol to the filtrate until the precipitation is complete, filter again, wash with methanol, and dry in vacuum to obtain white pure PLLA.
④PLLA分子量的测定：称取一定量的PLLA溶解于三氯甲烷中，用乌氏粘度法测定，计算相对粘均分子质量。④ Determination of molecular weight of PLLA: Weigh a certain amount of PLLA and dissolve it in chloroform, measure it by Ubbelohde's viscosity method, and calculate the relative viscosity-average molecular mass.
上述制备方法中，所述磁性纳米多孔复合支架材料的合成方法如下：In the above preparation method, the synthesis method of the magnetic nanoporous composite scaffold material is as follows:
将不同质量比例的Nano-HA、块状PLLA材料以及磁性纳米粒子Fe2O3共2g，使Nano-HA和Fe2O3在复合材料中的比例分别为(10％、20％、30％、40％、50%)，采用低温快速成型技术制作成立方体状多孔支架材料(25mm×25mm×25mm)。具体步骤如下：2g of Nano-HA, bulk PLLA materials and magnetic nanoparticles Fe2 O3 in different mass ratios, so that the ratios of Nano-HA and Fe2 O3 in the composite material are (10%, 20%, 30% , 40%, 50%), using low-temperature rapid prototyping technology to make a cube-shaped porous scaffold material (25mm×25mm×25mm). Specific steps are as follows:
①将PLLA溶解于有机溶剂(1,4-二氧六环)，并于60℃水浴中搅拌配制PLLA/1,4-二氧六环均相溶液；将不同质量比的Nano-HA和Fe2O3加入上述溶液中，超声波震荡各15min、电磁搅拌12小时后至Nano-HA和Fe2O3完全分散均匀制得凝胶。① Dissolve PLLA in an organic solvent (1,4-dioxane), and stir in a 60°C water bath to prepare a PLLA/1,4-dioxane homogeneous solution; Nano-HA and Fe in different mass ratios2 O3 was added to the above solution, ultrasonically oscillated for 15 minutes each, and electromagnetically stirred for 12 hours until the Nano-HA and Fe2 O3 were completely dispersed and uniform to obtain a gel.
②成型后的凝胶转入低温冷冻快速成型仪中-4℃条件下进行低温快速成型,通过RIPF工艺成形具有一定间隔的平行的冰线，将配制好的生物材料填入冰线间的间隔，将填了生物材料后的表面刮平，使得冰线和生物材料交替排列。在正交方向成形下一层冰线，重复以上步骤。②The formed gel is transferred to a low-temperature freezing rapid prototyping apparatus at -4°C for low-temperature rapid prototyping, forming parallel ice lines with a certain interval through the RIPF process, and filling the prepared biological materials into the intervals between the ice lines , scrape the surface filled with biomaterials so that the ice lines and biomaterials are arranged alternately. Shape the next layer of ice lines in the orthogonal direction and repeat the above steps.
③成形后，将冰和生物材料构成的实体拿到冷藏室，冰融化剩下的空间即为骨组织支架所需的纵横交错、彼此连通的大孔。③ After forming, take the entity composed of ice and biological materials to the refrigerator, and the space left by the melting of the ice is the criss-cross and interconnected macropores required by the bone tissue scaffold.
④得到的样品置于真空冷冻干燥机去除有机溶剂；风干2天后取出样品，有机溶剂升华后支架材料留下了大量更为微细的孔洞，表面喷金后经电镜扫描，并测量孔隙率。④The obtained sample was placed in a vacuum freeze dryer to remove the organic solvent; the sample was taken out after air-drying for 2 days. After the sublimation of the organic solvent, the scaffold material left a large number of finer pores. After the surface was sprayed with gold, it was scanned by an electron microscope and the porosity was measured.
本发明所述制备的磁性纳米多孔复合支架材料是通过低温快速成型技术制备的一种新型的复合人工骨支架材料，经对其进一步的生物力学测试，生物降解性能测试，生物相容性研究得出一种最佳比例的新型人工骨支架材料。The magnetic nanoporous composite scaffold material prepared by the present invention is a new type of composite artificial bone scaffold material prepared by low temperature rapid prototyping technology, which is obtained through further biomechanical testing, biodegradation performance testing, and biocompatibility research. A new type of artificial bone scaffold material with an optimal ratio was developed.
1、磁性纳米多孔（Nano-HA/PLLA/Fe2O3）复合人工骨的生物力学测试，采用三点抗弯法在生物力学测试机上测定各个比例的磁性纳米多孔（Nano-HA/PLLA/Fe2O3）复合人工骨材料的弹性模量1. Biomechanical test of magnetic nanoporous (Nano-HA/PLLA/Fe2 O3 ) composite artificial bone, using three-point bending method to measure the magnetic nanoporous (Nano-HA/PLLA/Fe 2 O 3 ) Elastic modulus of Fe2 O3 ) composite artificial bone materials
表1磁性纳米多孔（Nano-HA/PLLA/Fe2O3）复合人工骨力学性能比较(n=8，)Table 1 Comparison of mechanical properties of magnetic nanoporous (Nano-HA/PLLA/Fe2 O3 ) composite artificial bones (n=8, )
2、磁性纳米多孔（Nano-HA/PLLA/Fe2O3）复合人工骨的扫描电镜观，各个比例的磁性纳米多孔（Nano-HA/PLLA/Fe2O3）复合人工骨的扫描电镜结果（5000×）：多孔网状结构，孔内壁粗糙,孔间以微孔相通。2. Scanning electron microscope observation of magnetic nanoporous (Nano-HA/PLLA/Fe2 O3 ) composite artificial bone, scanning electron microscope results of various ratios of magnetic nanoporous (Nano-HA/PLLA/Fe2 O3 ) composite artificial bone (5000×): Porous network structure, the inner walls of the pores are rough, and the pores are connected by micropores.
3、磁性纳米多孔（Nano-HA/PLLA/Fe2O3）复合人工骨的细胞相容性3. Cytocompatibility of magnetic nanoporous (Nano-HA/PLLA/Fe2 O3 ) composite artificial bone
3.1细胞增殖度测定3.1 Determination of cell proliferation
将兔骨髓间充质干细胞（rBMSCs）与磁性纳米多孔（Nano-HA/PLLA/Fe2O3）复合人工骨材料浸提液共培养，测定rBMSCs的增殖能力。结果提示Nano-HA/PLLA/Fe2O3复合人工骨材料浸提液对rBMSCs细胞增殖无明显影响。Rabbit bone marrow mesenchymal stem cells (rBMSCs) were co-cultured with magnetic nanoporous (Nano-HA/PLLA/Fe2 O3 ) composite artificial bone material extract, and the proliferation ability of rBMSCs was determined. The results indicated that the extract of Nano-HA/PLLA/Fe2 O3 composite artificial bone material had no significant effect on the proliferation of rBMSCs.
表2各组细胞培养不同时间点吸光度值、相对增殖度及毒性分级(n=6)Table 2 The absorbance value, relative proliferation degree and toxicity grade of each group of cell culture at different time points (n=6)
3.2细胞形态及生长状况3.2 Cell morphology and growth status
根据细胞形态学标准，Nano-HA/PLLA/Fe2O3复合人工骨材料无细胞毒性，具有很好的细胞相容性。According to the cell morphology standard, the Nano-HA/PLLA/Fe2 O3 composite artificial bone material has no cytotoxicity and good cytocompatibility.
3.2.1材料浸提液与细胞共培养结果：3d后细胞突充分伸展，呈多角形；5d后细胞间隙减少，细胞呈多角形，梭形；7d后细胞间隙进一步减少，相互接触，细胞呈多角形，梭形。3.2.1 Results of co-cultivation of material extracts and cells: after 3 days, the cell processes are fully stretched and appear polygonal; after 5 days, the intercellular spaces are reduced, and the cells are polygonal and spindle-shaped; Polygonal, fusiform.
3.2.2材料与细胞共培养结果，倒置显微镜下观察（100×）：随着培养时间增加，支架材料周围细胞正常分裂增长，并规则排列。3.2.2 Results of co-cultivation of materials and cells, observed under an inverted microscope (100×): As the culture time increases, the cells around the scaffold material divide and grow normally, and are arranged regularly.
3.2.3材料与细胞共培养1、3、5天后，扫描电镜检测结果：随着培养时间增加，支架材料上附着细胞逐渐增多，并可见孔隙内细胞突触，5天后材料表面已完全被细胞覆盖。3.2.3 After 1, 3, and 5 days of co-cultivation of the material and cells, the results of scanning electron microscopy: with the increase of the culture time, the number of cells attached to the scaffold material gradually increased, and cell synapses in the pores were visible. After 5 days, the surface of the material was completely covered by cells. cover.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.
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