目的 构建丝素蛋白(SF)、细菌纤维素(BCNR)、羟基磷灰石(HAp)复合骨再生支架并评价其促成骨活性的价值.方法 将HAp颗粒、BCNR和骨形态发生蛋白2(BMP2)依次加入SF水溶液中,搅拌均匀后倒入不同大小的模具,-25 ℃下处理24 h,冷冻成型,通过冻干机将复合支架冻干.将SF与BCNR不同质量比的复合支架设置为A组(2∶1)、B组(4∶1)、C组(6∶1),将未加入BMP2的无活性复合支架设置为D组.扫描电镜检测支架的表面形貌和孔隙结构;压汞仪检测支架的孔隙率;万能材料试验机压缩支架,获得应力-应变曲线,分析支架的压缩强度和杨氏模量.将永生化小鼠胚胎成纤维细胞(iMEF)接种到A组、B组、C组及D组复合支架上.细胞接种4、8d后,细胞活/死染色检测各组活细胞和死细胞比例;细胞计数试剂盒8(CCK-8)实验检测各组细胞的增殖活性;碱性磷酸酶(ALP)染色检测各组染色阳性细胞;ALP活性检测观察各组细胞的ALP活性.选取15只雌性SD大鼠,构建大鼠肌袋异位成骨模型,植入不同SF与BCNR质量比的复合支架及未加入BMP2的无活性复合支架,分别为A'组(2∶1)、B'组(4∶1)、C'组(6∶1)和D'组,另设假手术组,每组3只.假手术组在切开皮肤、钝性分离股四头肌肌肉,于肌肉中形成肌袋后,仅缝合肌袋及皮肤,不植入支架,其他四组在肌袋内植入对应的支架,并缝合肌袋及皮肤.术后2、4周行X线片检查,观察各组骨形成情况.术后4周,收集植入的支架与组织复合物,进行病理组织切片、HE染色和Masson染色,观察各组成骨情况.另对组织切片进行免疫组化染色,观察各组成骨标志物Ⅰ型胶原蛋白(COL1)和骨桥蛋白(OPN)的表达情况.结果 扫描电镜显示,相较于A组和C组,B组片层结构和微孔结构更加规则、均匀;孔隙率分析结果表明,B组和C组孔隙率分别为(89.752±1.866)％和(84.257±1.013)％,均高于A组的(81.171±1.268)％(P＜0.05或0.01),而C组孔隙率低于B组(P＜0.01).力学性能检测结果显示,B组和 C 组压缩强度分别为(0.373±0.009)MPa 和(0.403±0.017)MPa,均高于 A 组的(0.044±0.003)MPa(P＜0.01),B组和C组杨氏模量分别为(7.413±0.094)MPa和(9.515±0.615)MPa,均高于A组的(1.881±0.036)MPa(P＜0.01),而C组压缩强度和杨氏模量均高于B组(P＜0.05或0.01).细胞活/死染色结果显示,细胞接种4 d后,B组死细胞明显少于A组、C组和D组;细胞接种8 d后,B组活细胞最多,死细胞最少.CCK-8实验结果显示,细胞接种4 d后,A组和B组细胞增殖活性分别为0.474±0.009 和 0.545±0.018,均高于 D 组的 0.394±0.016(P＜0.01),C 组细胞增殖活性为 0.419±0.005,与D组差异无统计学意义(P＞0.05),而A组和C组细胞增殖活性均低于B组(P＜0.01);细胞接种8 d后,B组细胞增殖活性为1.290±0.021,高于D组的1.047±0.011(P＜0.01),C组细胞增殖活性为0.794±0.032,低于D组(P＜0.01),A组细胞增殖活性为1.086±0.020,与D组差异无统计学意义(P＞0.05),而A组和C组细胞增殖活性均低于B组(P＜0.01).ALP染色结果显示,细胞接种4、8d后,相较于D组,A组、B组和C组有更多的阳性细胞,B组阳性细胞多于A组和C组.ALP活性检测结果显示,细胞接种4d后,A组、B组和C组细胞ALP活性分别为1.399±0.071、1.934±0.011、1.565±0.034,均高于D组的0.082±0.003(P＜0.01),而A组和C组细胞ALP活性均低于B组(P＜0.01);细胞接种8 d后,A组、B组和C组细胞ALP活性分别为2.602±0.055、3.216±0.092、2.145±0.170,均高于D组的0.101±0.001(P＜0.01),而A组和C组细胞ALP活性均低于B组(P＜0.01).X线片检查结果显示,术后2周,假手术组、D'组、A'组和C'组均无明显骨形成,而B'组有明显骨形成;术后4周,A'组、B'组和C'组均有明显骨形成,B'组骨形成明显多于其他两组,而假手术组和D'组均无明显骨形成.HE染色和Masson染色结果显示,术后4周,B'组形成大量均匀分布的新生骨组织,而A'组和C'组只在局部有少量新生骨组织,D'组仅有部分组织长入,且无明显新生骨组织,B'组形成的新生骨组织的成熟度比A'组和C'组更高.免疫组化染色结果显示,B'组COL1和OPN阳性染色均较A'组和C'组更多.COL1和OPN表达强度分析结果显示,A'组、B'组和C'组COL1表达强度分别为2.822±0.384、22.810±2.435、12.480±0.912,OPN表达强度分别为 1.545±0.081、5.374±0.121、2.246±0.116,B'组和C'组COL1、OPN表达强度均高于A'组(P＜0.01),而C'组COL1和OPN表达强度均低于B'组(P＜0.01).结论 基于SF、BCNR和HAp成功构建复合骨再生支架,其中SF和BCNR质量比为4:1的复合支架具有均匀孔隙结构、高孔隙率、良好的力学性能和生物相容性、优异的体外促成骨性能,还具有优异的骨诱导性和骨传导性.
Fabrication of the composite scaffolds for bone regeneration and verification of their value in muscle pouch osteogenic activity in rats
Objective To fabricate the composite scaffolds for bone regeneration with silk fibroin(SF),bacterial cellulose nanofibers(BCNR)and hydroxyapatite(HAp)and evaluate their osteogenic activity.Methods HAp particles,BCNR and bone morphogenetic protein-2(BMP2)were added into SF aqueous solution in turn,poured into molds of different sizes after being mixed evenly and processed at-25℃ for 24 hours to obtain frozen molds,and the composite scaffolds were frozen-dried by freezing-drying machine.The composite scaffolds with different mass ratios of SF and BCNR were divided into groups A(2∶1),B(4∶1)and C(6∶1),and the inactive composite scaffolds without BMP2 fell into group D.The surface morphology and pore structure of the scaffolds were detected by scanning electron microscopy.The porosity of the scaffolds was measured by mercury intrusion porosimeter.The stress-strain curve was obtained by using the universal material testing machine to compress the scaffolds,with which their compressive strength and Young's modulus were analyzed.Immortalized mouse embryonic fibroblasts(iMEF)were inoculated on the composite scaffolds of group A,B,C and D.At 4 and 8 days after cell inoculation,the proportion of alive and dead cells in each group was detected by cell survival/death staining;the cell counting kit-8(CCK-8)was used to detect cell proliferation activity in each group;the positive staining cells were detected in each group by alkaline phosphatase(ALP)staining;the ALP activity was observed in each group with ALP activity detection.A total of 15 female SD rats were selected to establish osteogenesis models with ectopic muscle bag.The composite scaffolds implanted with different SF/BCNR mass ratios and the inactive composite scaffolds without BMP2 fell into group A'(2∶1),B'(4∶1),C'(6∶1)and D'respectively,and a sham operation group was set at the same time,with 3 rats in each groups.In the sham operation group,the muscle bag and skin were sutured without scaffold implantation after the incision of skin,the blunt separation of the quadriceps muscle,and the formation of muscle bag in the muscle.In the other four groups,the corresponding scaffolds were implanted in the muscle bag and the muscle bag and skin were sutured.X-ray examination was performed at 2 and 4 weeks after operation to observe the osteogenesis in each group.At 4 weeks after operation,the implanted scaffolds and tissue complexes were collected by pathological tissue sectioning,HE staining and Masson staining,and for observing the osteogenesis by in each group.Immunohistochemical staining was also performed on the tissue sections to observe the expression of osteogenic markers type Ⅰ collagen(COL1)and osteopontin(OPN)in each group.Results Scanning electron microscopy showed that the lamellar and micropore structures of group B were more regular and uniform than those of groups A and C.The porosity rate analysis showed that the porosity rates of groups B and C were(89.752±1.866)％and(84.257±1.013)％respectively,higher than that of group A[(81.171±1.268)％](P＜0.05 or 0.01),with the porosity rate of group C lower than that of group B(P＜0.01).The mechanical property test showed that the compressive strengths of groups B and C were(0.373±0.009)MPa and(0.403±0.017)MPa respectively,higher than that of group A[(0.044±0.003)MPa](P＜0.01),and the Young's moduli of groups B and C were(7.413±0.094)MPa and(9.515±0.615)MPa respectively,higher than that of group A[(1.881±0.036)MPa](P＜0.01),with the compressive strength and Young's modulus of group C higher than those of group B(P＜0.05 or 0.01).The cell survival/death staining showed that the number of dead cells of group B was significantly smaller than that of groups A,C and D at 4 days after cell inoculation,and that group B had the most living cells and the fewest dead cells at 8 days after cell inoculation.The results of CCK-8 experiment showed that at 4 days after cell inoculation,the cell proliferation activity of groups A and B was 0.474±0.009 and 0.545±0.018 respectively,higher than 0.394±0.016 of group D(P＜0.01);the cell proliferation activity of group C was 0.419±0.005,with no significant difference from that of group D(P＞0.05),while the cell proliferation activity of groups A and C were both lower than that of group B(P＜0.01).At 8 days after cell inoculation,the cell proliferation activity of group B was 1.290±0.021,higher than 1.047±0.011 of group D(P＜0.01);the cell proliferation activity of group C was 0.794±0.032,lower than that of group D(P＜0.01);the cell proliferation activity of group A was 1.086±0.020,with no significant difference from that of group D(P＞0.05);the cell proliferation activity of groups A and C was lower than that of group B(P＜0.01).At 4 and 8 days after cell inoculation,ALP staining showed that more positive cells were found in groups A,B and C when compared with group D,and more positive cells were found in group B than in groups A and C.At 4 days after cell inoculation,the ALP activity detection showed that the ALP activity of groups A,B and C was 1.399±0.071,1.934±0.011 and 1.565±0.034 respectively,higher than 0.082±0.003 of group D(P＜0.01),while the ALP activity of groups A and C was lower than that of group B(P＜0.01).At 8 days after cell inoculation,the cell activity of groups A,B and C was 2.602±0.055,3.216±0.092 and 2.145±0.170 respectively,higher than 0.101±0.001 of group D(P＜0.01),while the ALP activity of groups A and C was lower than that of group B(P＜0.01).X-ray examination results showed that at 2 weeks after operation,no obvious osteogenesis was observed in the sham operation group,group D',A'and C',while it was observed in group B'.At 4 weeks after operation,obvious osteogenesis was observed in group A',B'and C',with significantly more osteogenesis in group B'than in the other two groups,while there was no obvious osteogenesis in the sham operation group and group D'.At 4 weeks after operation,the HE staining and Masson staining showed that a large number of uniformly distributed new bone tissue was formed in group B',while only a small amount of new bone tissue was found locally in groups A'and C',and only part of new tissue was found to grow in group D'with no obvious new bone tissue observed.The maturity of new bone tissue formed in group B'was higher than that in group A'and C'.Immunohistochemical staining showed more COL1 and OPN positive staining in group B'when compared with groups A'and C'.The expression intensity analysis of COL1 and OPN showed that in groups A',B'and C',the expression intensity of COL1 was 2.822±0.384,22.810±2.435 and 12.480±0.912 respectively and the expression intensity of OPN was 1.545±0.081,5.374±0.121 and 2.246±0.116 respectively,with higher expression intensity of COL1 and OPN in groups B'and C'than that in group A'(P＜0.01)and lower expression intensity of COL1 and OPN in group C'than that in B'group(P＜0.01).Conclusions The composite scaffold for bone regeneration is successfully fabricated with SF,BCNR and HAp.The composite scaffold with a mass ratio of SF to BCNR of 4:1 has uniform pore structure,high porosity,good mechanical properties and biocompatibility,excellent pro-osteogenic properties in vitro,as well as excellent osteo-inductivity and osteo-conductivity.
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