仿生矿化后的丝素电纺复合支架体内成骨性能的实验研究

Bone regeneration ability of electrospun composite scaffolds after biomimetic mineralization:an in vivo study

目的探讨仿生矿化后的丝素电纺复合支架体内修复颅骨缺损的能力。方法利用静电纺丝技术制备丝素电纺支架(非矿化组),通过模拟体液(SBF)浸泡法对支架仿生矿化(矿化组),扫描电镜观察其微观形貌。18只8周龄的雄性SD大鼠购自南京医科大学动物实验中心,采用随机数字表示法分为3组,分别为矿化组、非矿化组和对照组,每组6只。构建SD大鼠颅骨缺损模型,在直径为5 mm的骨缺损区分别植入矿化组及非矿化组支架,对照组不作处理,分别于术后4、8周取材,通过微计算机断层扫描技术(micro-CT)、苏木精-伊红(HE)及马松染色(Masson染色)比较评估不同支架的体内骨再生情况。应用GraphPad Prism 8统计软件分析,采用单因素方差分析。结果扫描电镜结果显示仿生矿化后的丝素电纺支架表面形成明显的羟基磷灰石矿化层。动物实验结果显示,术后4周和8周,通过对CT三维重建、HE及Masson染色的观察以及对骨体积分数(BV/TV),骨小梁数(Tb.N),骨小梁厚度(TB.Th)和骨小梁间隙(TB.Sp)定量指标的分析结果显示,在矿化组和非矿化组均能观察到新骨的形成,且矿化组的修复效果均优于非矿化组,差异有统计学意义[矿化组、非矿化组及对照组BV/TV在4周时分别为(22.880±2.324)、(12.600±1.965)、(4.967±1.580)%,F=61.838,P<0.05,8周时分别为(45.770±4.433)、(29.400±4.086)、(19.310±2.272)%,F=38.686,P<0.05;Tb.N在4周时分别为(0.029±0.001)、(0.019±0.003)、(0.008±0.003)mm-1,F=52.890,P<0.05,8周时分别为(0.053±0.002)、(0.037±0.003)、(0.023±0.001)mm-1,F=171.433,P<0.05;TB.Sp在4周时分别为(10.810±0.179)、(11.350±0.098)、(11.730±0.163)μm,F=27.655,P<0.05,8周时分别为(8.792±0.175)、(10.060±0.339)、(11.150±0.275)μm,F=56.807,P<0.05]。结论仿生矿化后的丝素电纺复合支架能有效促进骨再生,有望用于骨组织工程。

Objective To study the osteogenesis efficiency of electrospun composite scaffolds after biomimetic mineralization in vivo.Methods Silk fibroin electrospun scaffolds(non-mineralized groups)were prepared by electrospinning technology.The scaffolds were biomimetic and mineralized by simulated body fluid(SBF)soaking method(mineralized groups).The microcosmic appearance of the scaffolds was observed by scanning electron microscope.Eighteen 8-week-old male Sprague-Dawley(SD)rats(provided by the Animal Experiment Center of Nanjing Medical University)were randomly divided into 3 groups:mineralized groups,non-mineralized groups and control groups.The rat models of skull bone defects were established and the scaffolds were implanted.The repair effect of composite scaffolds on bone defect was detected by micro computed tomography(micro CT)scan,hematoxylin and eosin(HE)and Masson staining at 4 and 8 weeks after surgery.GraphPad Prism 8 statistical software was used for analysis,and one-way analysis of variance was used.Results The results of scanning electron microscope showed that an obvious hydroxyapatite mineralized layer was formed on the surface of the silk fibroin electrospun scaffolds after biomimetic mineralization.The results of CT,histological staining and analysis of bone volume/total volume(BV/TV),trabecular number(Tb.N),trabecular thickness(TB.Th)showed that the mineralized groups could more effectively promote the repair of the bone defects as compared with non-mineralized groups at various time points[for the mineralized groups,non-mineralized groups and control groups,BV/TVat 4 weeks:(22.880±2.324),(12.600±1.965),(4.967±1.580)%,F=61.838,P<0.05;8 weeks:(45.770±4.433),(29.400±4.086),(19.310±2.272)%,F=38.686,P<0.05.for Tb.N at 4 weeks:(0.029±0.001),(0.019±0.003),(0.008±0.003)mm-1,F=52.890,P<0.05;at 8 weeks:(0.053±0.002),(0.037±0.003),(0.023±0.001)mm-1,F=171.433,P<0.05.for TB.Sp at 4 weeks:(10.810±0.179),(11.350±0.098),(11.730±0.163)μm,F=27.655,P<0.05;at 8 weeks:(8.792±0.175),(10.060±0.339),(11.150±0.275)μm,F=56.807,P<0.05].Conclusion The electrospun composite scaffolds after biomimetic mineralization can effectively promote bone regeneration and are expected to be used in bone tissue engineering.