Small molecules modified biomimetic gelatin/hydroxyapatite nanofibers constructing an ideal osteogenic microenvironment with significantly enhanced cranial bone formation.
Small molecules modified biomimetic gelatin/hydroxyapatite nanofibers constructing an ideal osteogenic microenvironment with significantly enhanced cranial bone formation.
复制标题
小分子修饰仿生明胶/羟基磷灰石纳米纤维构建理想的成骨微环境，显着增强颅骨形成
DOI：
10.2147/ijn.s174553
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发表时间：
2018
影响因子：
8
通讯作者：
Yang B
中科院分区：
文献类型：
--
作者：
Li D;Zhang K;Shi C;Liu L;Yan G;Liu C;Zhou Y;Hu Y;Sun H;Yang B
背景：骨不连的修复是全世界面临的重大临床挑战。构建提供骨传导和骨诱导信号的成骨微环境是一种领先的策略。材料与方法采用静电纺丝法制备了抗坏血酸(AA)和水合甘油磷酸二钠(β-GP)修饰的仿生明胶/羟基磷灰石(GH)纳米纤维支架。然后用N-羟基磺基琥珀酰亚胺钠盐(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)对支架进行交联。用扫描电子显微镜(SEM)对未交联型和交联型支架的形貌进行了表征。用傅里叶变换红外光谱(FT-IR)对小分子与生长激素支架的相互作用模型进行了评价。采用四甲基偶氮唑盐比色法、阿拉马蓝和CCK8比色法检测不同交联型支架材料的生物相容性。采用定量逆转录聚合酶链式反应(qRT-PCR)检测支架上培养的骨髓基质细胞(BMSCs)成骨基因的表达。最后，将该交联支架植入大鼠颅骨缺损模型中，以评估体内的成骨效果。结果扫描电子显微镜结果显示，各种支架均呈细胞外基质(ECM)样的纤维多孔结构。(FT-IR)分析表明，AA和β-GP与生长激素支架共价键合。四甲基偶氮唑蓝、阿拉马蓝和CCK8检测表明，所有支架均能很好地支持BMSCs的生长。定量逆转录聚合酶链式反应结果显示，第7天，碱性磷酸酶和Runx2在GH/A/B支架上的表达水平分别是GH组的3.5倍和1.5倍。AA-和β-GP修饰的GH支架能显著诱导成骨基因的表达，且具有时间特异性。重要的是，AA和β-GP在体外协同促进成骨细胞分化，在体内显著诱导骨再生。结果显示，AA和β-GP复合修饰的生长激素纳米纤维支架可以作为引导骨再生的模板，6周时骨缺损基本完全修复(94.28%±5.00%)。此外，单独的AA和β-GP修饰的生长激素纳米纤维支架在体内12周分别修复了62.95%±9.39%和66.56%±18.45%的骨缺损。此外，AA和β-gp在体内具有抗炎作用。结论AA、β-GP和GH纳米纤维为骨再生创造了良好的骨传导和骨诱导微环境。证明AA和β-GP复合修饰的生长激素纳米纤维是一种多功能的骨组织工程支架材料。
Background Repair of nonunion critical-sized bone defects is a significant clinical challenge all over the world. Construction of osteogenic microenvironment that provides osteoconductive and osteoinductive signals is a leading strategy. Materials and methods In the present study, ascorbic acid (AA) and β-glycerophosphate disodium salt hydrate (β-GP) modified biomimetic gelatin/hydroxyapatite (GH) nanofibrous scaffolds were developed by electrospinning. Then the scaffolds were crosslinked by N-hydroxysulfo-succinimide sodium salt (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). The morphology of the non-crosslinked and crosslinked scaffolds was evaluated by scanning electron microscope (SEM). Fourier transform infrared spectroscopy (FT-IR) was used to assess the interacting model between the small molecules and GH scaffold. Then MTT, Alamar Blue, and CCK8 assays were used to investigate the biocompatibility of the various crosslinked scaffolds. Subsequently, the osteogenic genes expression of bone marrow stromal cells (BMSCs) cultured on the scaffolds were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Finally, the crosslinked scaffolds were implanted in a rat calvarial defect model to assess the osteogenic effects in vivo. Results SEM results showed that the various scaffolds presented extracellular matrix (ECM)-like fibrous porous structure. (FT-IR) spectrum indicated that AA and β-GP were covalently bonded with GH scaffolds. The MTT, Alamar Blue, and CCK8 assays demonstrated that all the scaffolds can support BMSCs’ growth well. The qRT-PCR results showed that the expression level of Alp and Runx2 in BMSCs on GH/A/B scaffold was about 3.5- and 1.5-fold, respectively, compared with that of GH group on day 7. The results also showed that AA- and β-GP-modified GH scaffolds can significantly induce the higher levels of osteogenic gene expression in a temporal specific manner. Importantly, AA and β-GP synergistically promoted osteoblast differentiation in vitro and dramatically induced bone regeneration in vivo. Impressively, AA and β-GP dual modified GH nanofibrous scaffold could serve as a template for guiding bone regeneration and the bone defects were almost repaired completely (94.28%±5.00%) at 6 weeks. Besides, single AA or β-GP-modified GH nanofibrous scaffolds could repair 62.95%±9.39% and 66.56%±18.45% bone defects, respectively, at 12 weeks in vivo. In addition, AA and β-GP exhibit an anti-inflammatory effect in vivo. Conclusion Our data highlighted that, AA, β-GP, and GH nanofibers created a fine osteoconductive and osteoinductive microenvironments for bone regeneration. We demonstrated that AA and β-GP dual modified GH nanofiber is a versatile bone tissue engineering scaffold.