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Abstract：
OBJECTIVE: To evaluate the efficacy of a modified sericin hydrogel scaffold loaded with dexamethasone (SMH-CD/DEX) scaffold for promoting bone defect healing by stimulating anti-inflammatory macrophage polarization.
METHODS: The light-curable SMH-CD/DEX scaffold was prepared using dexamethasone-loaded NH2-β-cyclodextrin (NH2-β-CD) and sericin hydrogel and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), biocompatibility assessment and drug release test. THP-1 macrophages incubated with the scaffold were examined for protein expressions of iNOS and Arg-1, mRNA expressions of IL-6, Il-10, Arg-1 and iNOS, and surface markers CD86 and CD206 using Western blotting, RT-qPCR, and flow cytometry. In a co-culture system of human periodontal ligament stem cells (HPDLSCs) and THP-1 macrophages, the osteogenic ability of the stem cells incubated with the scaffold was evaluated by detecting protein expressions of COL1A1 and Runx2 and expressions of ALP, Runx2, OCN and BMP2 mRNA, ALP staining, and alizarin red staining. In a rat model of mandibular bone defect, the osteogenic effect of the scaffold was assessed by observing bone regeneration using micro-CT and histopathological staining.
RESULTS: In THP-1 macrophages, incubation with SMH-CD/DEX scaffold significantly enhanced protein expressions of Arg-1 and mRNA expressions of IL-10 and Arg-1 and lowered iNOS protein expression and IL-6 and iNOS mRNA expressions. In the co-culture system, SMH-CD/DEX effectively increased the protein expressions of COL1A1 and Runx2 and mRNA expressions of ALP and BMP2 in HPDLSCs and promoted their osteogenic differentiation. In the rat models, implantation of SMH-CD/DEX scaffold significantly promoted bone repair and bone regeneration in the bone defect.
CONCLUSIONS: The SMH-CD/DEX scaffold capable of sustained dexamethasone release promotes osteogenic differentiation of stem cells and bone defect repair in rats by regulating M2 polarization.
METHODS: The light-curable SMH-CD/DEX scaffold was prepared using dexamethasone-loaded NH2-β-cyclodextrin (NH2-β-CD) and sericin hydrogel and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), biocompatibility assessment and drug release test. THP-1 macrophages incubated with the scaffold were examined for protein expressions of iNOS and Arg-1, mRNA expressions of IL-6, Il-10, Arg-1 and iNOS, and surface markers CD86 and CD206 using Western blotting, RT-qPCR, and flow cytometry. In a co-culture system of human periodontal ligament stem cells (HPDLSCs) and THP-1 macrophages, the osteogenic ability of the stem cells incubated with the scaffold was evaluated by detecting protein expressions of COL1A1 and Runx2 and expressions of ALP, Runx2, OCN and BMP2 mRNA, ALP staining, and alizarin red staining. In a rat model of mandibular bone defect, the osteogenic effect of the scaffold was assessed by observing bone regeneration using micro-CT and histopathological staining.
RESULTS: In THP-1 macrophages, incubation with SMH-CD/DEX scaffold significantly enhanced protein expressions of Arg-1 and mRNA expressions of IL-10 and Arg-1 and lowered iNOS protein expression and IL-6 and iNOS mRNA expressions. In the co-culture system, SMH-CD/DEX effectively increased the protein expressions of COL1A1 and Runx2 and mRNA expressions of ALP and BMP2 in HPDLSCs and promoted their osteogenic differentiation. In the rat models, implantation of SMH-CD/DEX scaffold significantly promoted bone repair and bone regeneration in the bone defect.
CONCLUSIONS: The SMH-CD/DEX scaffold capable of sustained dexamethasone release promotes osteogenic differentiation of stem cells and bone defect repair in rats by regulating M2 polarization.
摘要：
目的：评价负载地塞米松的丝胶凝胶支架(SMH-CD/DEX)通过刺激抗炎巨噬细胞极化促进骨缺损愈合的效果。
方法：使用负载地塞米松的NH2-β-环糊精（NH2-β-CD）和丝胶凝胶制备光固化SMH-CD/DEX支架，并通过扫描电子显微镜（SEM）进行表征，傅里叶变换红外光谱(FTIR),生物相容性评价和药物释放试验。检测与支架孵育的THP-1巨噬细胞的iNOS和Arg-1蛋白表达，IL-6，IL-10，Arg-1和iNOS的mRNA表达，和表面标记CD86和CD206使用蛋白质印迹，RT-qPCR,和流式细胞术。在人牙周膜干细胞（HPDLSCs）和THP-1巨噬细胞的共培养系统中，通过检测COL1A1和Runx2蛋白的表达和ALP的表达来评价与支架孵育的干细胞的成骨能力，Runx2、OCN和BMP2mRNA，ALP染色,和茜素红染色。在大鼠下颌骨缺损模型中,支架的成骨作用通过显微CT和组织病理学染色观察骨再生来评估。
结果：在THP-1巨噬细胞中，与SMH-CD/DEX支架孵育显着增强了Arg-1的蛋白表达以及IL-10和Arg-1的mRNA表达，并降低了iNOS蛋白表达以及IL-6和iNOSmRNA表达。在共同文化体系中，SMH-CD/DEX可有效增加HPDLSCs中COL1A1和Runx2的蛋白表达以及ALP和BMP2的mRNA表达，促进其成骨分化。在大鼠模型中,植入SMH-CD/DEX支架可显著促进骨缺损的骨修复和骨再生。
结论：可持续释放地塞米松的SMH-CD/DEX支架通过调节M2极化促进大鼠干细胞成骨分化和骨缺损修复。
方法：使用负载地塞米松的NH2-β-环糊精（NH2-β-CD）和丝胶凝胶制备光固化SMH-CD/DEX支架，并通过扫描电子显微镜（SEM）进行表征，傅里叶变换红外光谱(FTIR),生物相容性评价和药物释放试验。检测与支架孵育的THP-1巨噬细胞的iNOS和Arg-1蛋白表达，IL-6，IL-10，Arg-1和iNOS的mRNA表达，和表面标记CD86和CD206使用蛋白质印迹，RT-qPCR,和流式细胞术。在人牙周膜干细胞（HPDLSCs）和THP-1巨噬细胞的共培养系统中，通过检测COL1A1和Runx2蛋白的表达和ALP的表达来评价与支架孵育的干细胞的成骨能力，Runx2、OCN和BMP2mRNA，ALP染色,和茜素红染色。在大鼠下颌骨缺损模型中,支架的成骨作用通过显微CT和组织病理学染色观察骨再生来评估。
结果：在THP-1巨噬细胞中，与SMH-CD/DEX支架孵育显着增强了Arg-1的蛋白表达以及IL-10和Arg-1的mRNA表达，并降低了iNOS蛋白表达以及IL-6和iNOSmRNA表达。在共同文化体系中，SMH-CD/DEX可有效增加HPDLSCs中COL1A1和Runx2的蛋白表达以及ALP和BMP2的mRNA表达，促进其成骨分化。在大鼠模型中,植入SMH-CD/DEX支架可显著促进骨缺损的骨修复和骨再生。
结论：可持续释放地塞米松的SMH-CD/DEX支架通过调节M2极化促进大鼠干细胞成骨分化和骨缺损修复。
