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.
Conclusion: 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.
目的: 探究改性丝胶蛋白水凝胶(SMH-CD/DEX)调节巨噬细胞极化促进骨缺损修复的能力。
方法: 将地塞米松搭载于丝胶蛋白水凝胶构建SMH-CD/DEX水凝胶。通过扫描电镜、傅里叶红外光谱、细胞活性实验和释放曲线测定，对SMHCD/DEX水凝胶的形貌、化学性质及生物活性进行表征分析。通过Western blot、RT-qPCR、流式细胞术检测THP-1巨噬细胞中iNOS和Arg-1蛋白表达量，IL-6、IL-10、Arg-1、iNOS基因表达水平及CD86、CD206的表达比例。在共培养条件下通过Western blot，RT-qPCR检测hPDLSCs人牙周膜干细胞COL1A1和Runx2蛋白的表达量，ALP、Runx2、OCN、BMP2基因表达量，并进行碱性磷酸酶染色和茜素红染色。在大鼠下颌骨骨缺损模型植入水凝胶，通过Micro-CT扫描和组织病理染色分析新骨形成情况。
结果: 通过主客体相互作用与化学偶联改性丝胶蛋白水凝胶，成功构建SMH-CD/DEX水凝胶。SMH-CD/DEX水凝胶能够有效提高巨噬细胞M2极化相关标志物的IL-10及Arg-1的表达量(P < 0.001)；并降低M1极化相关标志物IL-6及iNOS表达(P < 0.001)。在共培养体系中，SMH-CD/DEX水凝胶可提高人牙周膜干细胞的成骨分化相关标志物ALP、BMP2 (P < 0.05)及COL1A1(P < 0.001)、Runx2表达量(P < 0.01)。碱性磷酸酶染色和茜素红染色结果显示，SMH-CD/DEX水凝胶能够有效促进人牙周膜干细胞成骨分化。Micro-CT及HE、Masson染色结果显示，SMH-CD/DEX水凝胶有效促进骨缺损再生修复(P < 0.05)。
结论: SMH-CD/DEX水凝胶能够调节巨噬细胞M2极化促进干细胞成骨分化并促进骨缺损愈合。
Keywords: bone regeneration; drug delivery; human periodontal ligament stem cells; immunoregulation; macrophage.