高分子三维支架材料通过诱导外周巨噬细胞的极化促进成骨前体细胞分化

doi:10.13701/j.cnki.kqyxyj.2024.03.004

摘要/Abstract

摘要： 目的:考察硫酸软骨素-壳聚糖-羟基磷灰石(SF-CS-HA)三维支架材料对小鼠巨噬细胞极化的影响,并进一步研究骨免疫微环境调控对小鼠颅顶骨前体细胞在骨皮质外骨生成的影响。方法:制备SF-CS-HA三维支架材料,将支架材料与小鼠巨噬细胞RAW264.7共培养,流式细胞术检测巨噬细胞的M2型分化情况,RT-qPCR检测炎症因子基因的表达,Western blot检测TGF-β蛋白表达的变化。将巨噬细胞与SF-CS-HA三维支架材料共孵育后,制备条件培养基,比较完全培养基和条件培养基对小鼠颅顶骨前体细胞MC3T3-E1(3T3)增殖、迁移、ALP活性及形态变化,并通过Western blot检测骨生成相关蛋白的表达。最后,通过动物模型验证SF-CS-HA材料对于小鼠颅骨骨量增加的影响。结果:扫描电镜观察显示,SF-CS-HA三维支架材料表面和内部都具有均匀的空腔结构。流式细胞术检测结果显示,SF-CS-HA三维支架材料能够促进小鼠外周巨噬细胞向M2型极化。RT-qPCR和Western blot结果显示,SF-CS-HA三维支架材料提高了IL-10基因和TGF-β蛋白的表达量,降低了IL-1β基因的表达量。且SF-CS-HA三维支架能够促进MC3T3-E1细胞的增殖,显著提高ALP活性,增加细胞的迁移能力,提高细胞骨架蛋白Factin的生成。而条件培养基(骨免疫微环境调控下)对于MC3T3-E1细胞增殖和迁移能力的提升更为显著。此外,Western blot结果显示,骨免疫微环境调控下,骨生成蛋白OPN、COLA1、RUNX2、OCN和TGF-β显著增加。动物实验结果显示,SF-CS-HA材料能够在体内促进小鼠颅骨的新骨形成。结论:SF-CS-HA三维支架材料能够促进小鼠外周巨噬细胞的极化,且SF-CS-HA能够调控骨免疫微环境,促进骨细胞生成,在骨皮质外达到骨增量。

关键词: 三维支架材料, 巨噬细胞, 极化, 共培养, 颅顶骨前体细胞

Abstract: Objective: To investigate the effect of three-dimensional scaffold SF-CS-HA on the polarization of mouse macrophages, and explore the impact of bone immunomodulatory microenvironment regulation on the extracortical bone formation of mouse calvarial precursor cells. Methods: SF-CS-HA was prepared and co-cultured with mouse macrophages RAW264.7. Flow cytometry was performed to detect the polarization of macrophages into M2 phenotype. RT-qPCR was used to measure the expression of inflammatory cytokine genes, and western blot was conducted to evaluate the expression of TGF-β. After co-incubation of macrophages with SF-CS-HA 3D, conditioned medium was prepared. The proliferation, migration, ALP activity, and morphological changes of mouse calvarial precursor cells MC3T3-E1 were compared between complete medium and conditioned medium. Western blot was performed to detect the expression of bone formation-related proteins. An animal model was used to verify the effect of SF-CS-HA on the increase of skull bone mass in mice. Results: Scanning electron microscopy observations revealed that SF-CS-HA had a uniform porous structure both on the surface and internally. Flow cytometry analysis showed that SF-CS-HA promoted the polarization of peripheral macrophages towards M2 phenotype. RT-qPCR and western blot results demonstrated that SF-CS-HA increased the expression of IL-10 gene and TGF-β protein, while decreased the expression of IL-1β gene. Additionally, SF-CS-HA promoted the proliferation of MC3T3-E1 cells, significantly increased ALP activity, enhanced cell migration, and increased the production of cytoskeletal protein Factin. Furthermore, conditioned medium exhibited more significant enhancement in the proliferation and migration capabilities of MC3T3-E1 cells. Moreover, Western blot indicated that under the regulation of the bone immune microenvironment, the expression of bone formation proteins OPN, COLA1, RUNX2, OCN, and TGF-β increased significantly. The results of animal experiments showed that SF-CS-HA could promote new bone formation in the mouse skull in vivo. Conclusion: SF-CS-HA 3D scaffold can promote the polarization of peripheral macrophages and regulate the bone immune microenvironment, thereby promoting osteogenic differentiation and bone formation in the cortical bone exogenesis.

Key words: 3D scaffold material, macrophages, polarization, co-culture, calvarial precursor cells

刘鹬, 黄伟琨, 梁羽. 高分子三维支架材料通过诱导外周巨噬细胞的极化促进成骨前体细胞分化[J]. 口腔医学研究, 2024, 40(3): 206-213.

LIU Yu, HUANG Weikun, LIANG Yu. Polymeric Three-dimensional Scaffold Promotes Osteogenic Precursor Cells Differentiation through Induction of Peripheral Macrophages Polarization[J]. Journal of Oral Science Research, 2024, 40(3): 206-213.

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