背景:牙槽骨缺损的有效治疗仍然是牙科治疗中的主要问题。本研究的目的是开发一种纤维蛋白和魔芋葡甘聚糖(KGM)复合水凝胶作为鼻粘膜来源的外胚层间充质干细胞(EMSC)的骨形成支架,用于牙槽骨缺损的再生,并研究了嵌入水凝胶中的黑磷纳米颗粒(BPNs)的成骨加速作用。
方法:从大鼠鼻粘膜中分离原代EMSC,用于牙槽骨修复。纤维蛋白和KGM以不同的比例制备了拟骨水凝胶支架,通过力学性能和生物相容性分析确定最佳配比。然后,将最佳水凝胶与BPNs整合以获得BPNs/纤维蛋白-KGM水凝胶,并评价其对体外成骨EMSCs的影响。探讨水凝胶的体内成骨增强作用,将BPNs/纤维蛋白-KGM支架联合EMSC植入大鼠牙槽骨缺损模型。显微计算机断层扫描(CT),组织学检查,采用实时定量聚合酶链反应(RT-qPCR)和免疫印迹技术评价骨形态和骨再生成骨相关基因的表达。
结果:KGM的添加改善了纤维蛋白水凝胶的机械性能和生物降解特性。体外,含有BPNs的复合水凝胶被证明是生物相容性的,并且能够通过上调矿化和碱性磷酸酶的活性来增强EMSC的成骨作用。在体内,显微CT分析和组织学评估表明,植入EMSCs-BPNs/纤维蛋白-KGM水凝胶的大鼠表现出最佳的骨重建。与模型组相比,包括骨桥蛋白在内的成骨基因的表达(Opn,p<0.0001),骨钙蛋白(Ocn,p<0.0001),型胶原蛋白(Col,p<0.0001),骨形态发生蛋白2(Bmp2,p<0.0001),Smad1(p=0.0006),runt相关转录因子2(Runx2,p<0.0001)均显著上调。
结论:含纤维蛋白KGM的EMSCs/BPNs水凝胶通过有效上调成骨相关基因的表达促进大鼠牙槽骨缺损的修复,促进骨基质的形成和矿化。
BACKGROUND: Effective treatments for the alveolar bone defect remain a major concern in dental therapy. The objectives of this study were to develop a fibrin and konjac glucomannan (KGM) composite hydrogel as scaffolds for the osteogenesis of nasal mucosa-derived ectodermal mesenchymal stem cells (EMSCs) for the regeneration of alveolar bone defect, and to investigate the osteogenesis-accelerating effects of black phosphorus nanoparticles (BPNs) embedded in the hydrogels.
METHODS: Primary EMSCs were isolated from rat nasal mucosa and used for the alveolar bone recovery. Fibrin and KGM were prepared in different ratios for osteomimetic hydrogel scaffolds, and the optimal ratio was determined by mechanical properties and biocompatibility analysis. Then, the optimal hydrogels were integrated with BPNs to obtain BPNs/fibrin-KGM hydrogels, and the effects on osteogenic EMSCs in vitro were evaluated. To explore the osteogenesis-enhancing effects of hydrogels in vivo, the BPNs/fibrin-KGM scaffolds combined with EMSCs were implanted to a rat model of alveolar bone defect. Micro-computed tomography (CT), histological examination, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were conducted to evaluate the bone morphology and expression of osteogenesis-related genes of the bone regeneration.
RESULTS: The addition of KGM improved the mechanical properties and biodegradation characteristics of the fibrin hydrogels. In vitro, the BPNs-containing compound hydrogel was proved to be biocompatible and capable of enhancing the osteogenesis of EMSCs by upregulating the mineralization and the activity of alkaline phosphatase. In vivo, the micro-CT analysis and histological evaluation demonstrated that rats implanted EMSCs-BPNs/fibrin-KGM hydrogels exhibited the best bone reconstruction. And compared to the model group, the expression of osteogenesis genes including osteopontin (Opn, p < 0.0001),
osteocalcin (Ocn, p < 0.0001), type collagen (Col , p < 0.0001), bone morphogenetic protein-2 (Bmp2, p < 0.0001), Smad1 (p = 0.0006), and runt-related transcription factor 2 (Runx2, p < 0.0001) were all significantly upregulated.
CONCLUSIONS: EMSCs/BPNs-containing fibrin-KGM hydrogels accelerated the recovery of the alveolar bone defect in rats by effectively up-regulating the expression of osteogenesis-related genes, promoting the formation and mineralisation of bone matrix.