低温3D打印联合冷冻干燥技术制备组织工程骨支架的研究

CYTOCOMPATIBILITY AND PREPARATION OF BONE TISSUE ENGINEERING SCAFFOLD BY COMBINING LOW TEMPERATURE THREE DIMENSIONAL PRINTING AND VACUUM FREEZE-DRYING TECHNIQUES

目的探讨采用低温3D打印联合冷冻干燥技术制备组织工程骨支架的方法,评价支架细胞相容性。方法取新鲜牛肌腱及家蚕生丝分别制备胶原蛋白(collagen,COL)、丝素蛋白(silk fibroin,SF)。采用计算机辅助软件Solid Works2014设计大小为9 mm×9 mm×3 mm、孔径为500μm的支架模型,以质量比9∶3∶2的COL、SF和纳米羟基磷灰石(nano-hydroxyapatite,n HA)混合物为原料,采用低温3D打印联合冷冻干燥技术构建COL/SF/n HA复合支架。大体观察结合扫描电镜观测支架形态及表面结构;采用力学试验机测量支架压缩位移、压缩应力及弹性模量,分析力学性能;将支架与MC3T3-E1细胞共培养1、7、14、21 d,倒置显微镜及扫描电镜观察细胞生长情况,MTT法检测细胞增殖情况,RT-PCR及Western blot检测MC3T3-E1细胞COLⅠ、ALP及骨钙素(osteocalcin,OCN)基因及蛋白表达情况。结果低温3D打印联合冷冻干燥技术成功制备COL/SF/n HA复合支架,扫描电镜示支架为大孔及微孔共存的3D多孔径立体结构。支架弹性模量为(344.783 07±40.728 55)k Pa,压缩应力为(0.062 15±0.007 15)MPa,压缩位移为(0.958 41±0.000 84)mm。共培养后倒置显微镜、扫描电镜观察及MTT检测均显示,随时间延长,支架表面大量细胞黏附,伸展充分,大孔孔壁上细胞生长良好,数量逐渐增多。RTPCR及Western blot检测示,MC3T3-E1细胞COLⅠ、ALP及OCN基因及蛋白均表达。结论低温3D打印联合冷冻干燥技术可精确控制支架微观结构,得到大孔、微孔共存的组织工程骨支架,且细胞相容性良好,为下一步骨缺损修复研究奠定基础。

Objective To study the preparation and cytocompatibility of bone tissue engineering scaffolds by combining low temperature three dimensional（3D） printing and vacuum freeze-drying techniques. Methods Collagen（COL）and silk fibroin（SF） were manufactured from fresh bovine tendon and silkworm silk. Solid Works2014 was adopted to design bone tissue engineering scaffold models with the size of 9 mm×9 mm×3 mm and pore diameter of 500 μm. According to the behavior of composite materials that low temperature 3D printing equipment required, COL, SF, and nano-hydroxyapatite（n HA）at a ratio of 9 ∶ 3 ∶ 2 and low temperature 3D printing in combination with vacuum freeze-drying techniques were accepted to build COL/SF/n HA composite scaffolds. Gross observation and scanning electron microscope（SEM） were applied to observe the morphology and surface structures of composite scaffolds. Meanwhile, compression displacement, compression stress, and elasticity modulus were measured by mechanics machine to analyze mechanical properties of composite scaffolds. The growth and proliferation of MC3T3-E1 cells were evaluated using SEM, inverted microscope, and MTT assay after cultured for 1, 7, 14, and 21 days on the composite scaffolds. The RT-PCR and Western blot techniques were adopted to detect the gene and protein expressions of COL I, alkaline phosphatase（ALP）, and osteocalcin（OCN） in MC3T3-E1 cells after 21 days. Results COL/SF/n HA composite scaffolds were successfully prepared by low temperature 3D printing technology and vacuum freeze-drying techniques; the SEM results showed that the bionic bone scaffolds were 3D polyporous structures with macropores and micropores. The mechanical performance showed that the elasticity modulus was（344.783 07±40.728 55） kPa; compression displacement was（0.958 41±0.000 84） mm; and compression stress was（0.062 15±0.007 15） MPa. The results of inverted microscope, SEM, and MTT method showed that a large number of cells adhered to the surface with full extension and good cells growth inside the macropores, which demonstrated a satisfactory proliferation rate of the MC3T3-E1 cells on the composite scaffolds. The RT-PCR and Western blot electrophoresis revealed gene expressions and protein synthesis of COL I, ALP, and OCN in MC3T3-E1 cells. Conclusion Low temperature 3D printing in combination with vacuum freeze-drying techniques could realize multi-aperture coexisted bionic bone tissue engineering scaffolds and control the microstructures of composite scaffolds precisely that possess good cytocompatibility. It was expected to be a bone defect repair material, which lays a foundation for further research of bone defect.