猪小肠黏膜下层海绵的制备及促成骨作用

目的:制备并通过体外实验评价一种新型猪小肠黏膜下层(small intestinal submucosa, SIS)海绵的基本性能,建立动物模型评价其体内促成骨能力。方法:采用冷冻干燥法制备SIS海绵,通过环境扫描电镜观察微观结构和孔径大小,比重法检测孔隙率和吸水率,万能力学试验机测试其机械性能,细胞增殖-毒性检测评估其生物相容性。通过建立比格犬动物模型评价其体内促成骨作用,将3只比格犬前磨牙拔牙窝共计18个位点随机分为3组,放置SIS海绵作为SIS海绵组、放置Bio-Oss骨粉并覆盖Bio-Gide膜作为阳性对照组,不做处理作为空白对照组,术后分别于4周和12周取材行微计算机断层扫描技术(micro computed tomography, Micro-CT)检测,采用单因素方差分析法对数据进行统计学分析,以评估SIS海绵的促成骨效果。结果:SIS海绵的平均孔径为(194.90±30.39) μm,孔隙率为92.31%±0.24%,吸水率为771.50%±40.90%,压缩弹性模量为(2.20±0.19) kPa。细胞增殖-毒性检测结果显示SIS海绵不会影响人骨髓间充质干细胞的早期增殖,Micro-CT结果显示术后4周时SIS海绵组骨体积分数(bone volume fraction, BV/TV, 52.81%±3.21%)和阳性对照组(58.30%±9.36%)显著高于空白对照组(38.65%±4.80%,P <0.05),SIS海绵组骨密度[bone mineralized density, BMD, (887.09±61.02) mg/cm³]、阳性对照组[(952.05±132.78) mg/cm³]和空白对照组[(879.29±74.27) mg/cm³] 差异无统计学意义(P >0.05);术后12周时SIS海绵组BV/TV(47.89%±3.59%)显著低于阳性对照组(60.57%±6.56%, P <0.05),与空白对照组(42.99%±2.54%)差异无统计学(P >0.05),SIS海绵组BMD[(1047±89.95) mg/cm³]和阳性对照组[(1101.37±98.85) mg/cm³]显著高于空白对照组[(890.36±79.79) mg/cm³,P <0.05]。结论:SIS海绵具有良好的理化性能和生物相容性,在犬拔牙窝成骨早期(4周)能提高新生BV/TV,在犬拔牙窝成骨后期(12周)可提高新生BMD,具有潜在促成骨应用前景。

Preparation and osteogenic effect study of small intestinal submucosa sponge

Objective: To prepare and evaluate the basic properties in vitro of a novel small intestinal submucosa (SIS) sponge, and to describe the bone formation ability of the SIS sponge in vivo. Methods: The SIS sponge was prepared by freeze-drying method. To evaluate the physicochemical properties of the sponge, electron microscope observation, porosity test, water absorption ability and mechanical property were conducted in vitro. The cytotoxicity of the SIS sponge was performed by cell counting kit-8 method. In vivo experiments, eighteen extraction sockets of premolar of three Beagle dogs were randomly divided into three groups: SIS sponge group (SIS sponge), positive control group (Bio-Oss granules and Bio-Gide membrane) and control group(no treatment). The animals were sacrificed 4 weeks and 12 weeks after operation, and micro computed tomography (Micro-CT) was applied to measure the bone volume fraction (BV/TV) and bone mineralized density (BMD). The data were analyzed with one-way ANOVA. Results: The average pore diameter of the SIS sponge was (194.90±30.39) μm, the porosity was 92.31%±0.24%, the water absorption rate was 771.50%±40.90%, and the compressive elastic modulus was (2.20±0.19) kPa. There was no significant difference in cell proliferation ability between SIS sponge and control group (P>0.05). Micro-CT quantitative results showed that BV/TV of SIS sponge group (52.81%±3.21%) and positive control group (58.30%±9.36%) were significantly higher than that of control group (38.65%±4.80%) 4 weeks after operation (P <0.05). The BMD of SIS sponge group [(887.09±61.02) mg/cm³], positive control group [(952.05±132.78) mg/cm³] and control group [(879.29±74.27) mg/cm³] showed no statistical difference 4 weeks after operation (P>0.05). The BV/TV of positive control group (60.57%± 6.56%) was significantly higher than that of SIS sponge group (47.89%±3.59%) and control group (42.99%±2.54%) 12 weeks after operation (P < 0.05). BMD of SIS sponge group [(1047±89.95) mg/cm³] and positive control group [(1101.37±98.85) mg/cm³] were significantly higher than that of control group [(890.36±79.79) mg/cm³] 12 weeks after operation (P <0.05). Conclusion: The SIS sponge has satisfying physicochemical properties and biocompatibility. The SIS sponge significantly increased bone volume fraction in the early stage of bone formation (4 weeks) and bone mineralized density in the late stage of bone formation (12 weeks).