纳米硅酸镁锂功能化聚己内酯膜对骨髓间充质干细胞介导骨再生修复的作用研究

doi:10.13591/j.cnki.kqyx.2025.08.002

摘要/Abstract

摘要：

目的制备纳米硅酸镁锂功能化聚己内酯(nanosilicate functionalized polycaprolactone,PCL/LAP)电纺膜,评价其对骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)介导骨再生修复的作用。方法通过静电纺丝技术制备PCL/LAP电纺膜,并与大鼠BMSCs细胞共培养,通过细胞骨架染色、活/死细胞染色和CCK-8法检测PCL/LAP电纺膜的细胞相容性。利用划痕实验、Transwell迁移实验和qRT-PCR检测细胞迁移相关基因Pdgf、Tgfβ和Mmp2表达,评估PCL/LAP电纺膜对BMSCs迁移能力的影响。采用碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色和qRT-PCR检测成骨分化基因(Alp、Col1a1、Runx2、Bglap和Bmp2)与成血管相关基因(Angpt1、Fgf2和Vegfa)的表达水平,Western blot检测RUNX2蛋白表达,评估PCL/LAP电纺膜对BMSCs成骨分化与成血管的调控作用。PCL和PCL/LAP电纺膜条件培养液分别作用血管内皮细胞EAhy926,通过qRT-PCR检测成血管相关基因(KDR、ENOS和HIF1A)的表达。结果 BMSCs在PCL/LAP电纺膜表面黏附良好,细胞活力无明显影响(P>0.05)。PCL/LAP不仅促进BMSCs增殖(P<0.05)、迁移,还能增强ALP活性和促进矿化结节形成(P<0.05),上调成骨分化相关基因与蛋白的表达(P<0.05)。PCL/LAP电纺膜可促进BMSCs高表达成血管相关因子基因(P<0.05),间接调控血管内皮细胞成血管分化基因的表达(P<0.05)。结论 PCL/LAP电纺膜具有优异的生物相容性,可促进BMSCs的增殖、迁移、成骨分化及其介导的成血管分化,有望作为屏障膜应用于骨缺损的再生修复治疗。

关键词: 屏障膜, 聚己内酯, 硅酸镁锂, 骨髓间充质干细胞, 骨再生

Abstract:

Objective To fabricate nanosilicate functionalized polycaprolactone (PCL/LAP) electrospun membrane and evaluate its role in bone marrow mesenchymal stem cells (BMSCs)-induced bone repairing. Methods The PCL/LAP electrospun membranes were fabricated via electrospinning technology and co-cultured with rat BMSCs. The cytocompatibility of the membranes was evaluated through cytoskeleton staining, live/dead cell staining and CCK-8 assay. The migration capacity of BMSCs was assessed using scratch assay, Transwell migration experiments and expression of migration-related genes (Pdgf and Tgfβ) was evaluated by qRT-PCR. The osteogenic differentiation and pro-angiogenesis potential were determined by alkaline phosphatase (ALP) staining, alizarin red staining, expression levels of osteogenesis-related genes (Alp, Col1a1, Runx2, Bglap and Bmp2) and angiogenesis-related genes (Angpt1, Fgf2 and Vegfa) along with RUNX2 protein expression. PCL and PCL/LAP electrospun membranes conditioned medium was subsequently used to stimulate vascular endothelial cells (EAhy926). The expression of angiogenesis-associated genes (KDR, ENOS and HIF1A) was quantified by qRT-PCR. Results BMSCs adhered well to the surface of the PCL/LAP membranes, with no significant impact on cell viability (P>0.05). PCL/LAP membranes not only promoted the proliferation (P<0.05), migration (P<0.05), but also enhanced ALP activity and mineralized nodule formation (P<0.05), increased osteogenic differentiation gene and protein expression (P<0.05) of BMSCs. Moreover, PCL/LAP promoted the expression of angiogenic genes of BMSCs (P<0.05), to indirectly regulate angiogenesis-related gene expression in vascular endothelial cells (P<0.05). Conclusion PCL/LAP electrospun membranes exhibit excellent biocompatibility and can promote proliferation, migration, osteogenic differentiation and BMSC-mediated angiogenic differentiation, showing great potential for bone defect repairing as barrier membrane.

Key words: barrier membrane, polycaprolactone, nanosilicate, bone marrow mesenchymal stem cells, bone regeneration

中图分类号:

R318.08

肖隆, 胡伟强, 林旭馨, 何梦娇, 骆凯, 许雄程. 纳米硅酸镁锂功能化聚己内酯膜对骨髓间充质干细胞介导骨再生修复的作用研究[J]. 口腔医学, 2025, 45(8): 567-575.

XIAO Long, HU Weiqiang, LIN Xuxin, HE Mengjiao, LUO Kai, XU Xiongcheng. Effects of nanosilicate functionalized polycaprolactone membrane on bone mesenchymal stem cells-induced bone repairing[J]. Stomatology, 2025, 45(8): 567-575.

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引物名称	序列(5'-3')
GAPDH	F:CGGCAAGTTCAACGGCACAGTCAAGG R:ACGACATACTCAGCACCAGCATCACC
Alp	F:ACAAGGTGGTGGACGGTGAAC R:CGTGAAGCAGGTGAGCCATAGG
Col1a1	F:CAACAGACTGGCAACCTCAAGAAG R:CACAAGCGTGCTGTAGGTGAATC
Bglap	F:GGACCCTCTCTCTGCTCACTCTG R:ACCTTACTGCCCTCCTGCTTGG
Bmp2	F:AAAGCGTCAAGCCAAACACAAAC R:ACATCACTGAAGTCCACATACAAAGG
Pdgf	F:AAAGGCAAGCACCGAAAG R:GAACAACATTATCACTCCAAGG
Runx2	F:ACCAGCAGCACTCCATATCTCTAC R:CTTCCATCAGCGTCAACACCAT
Tgfβ	F:GGACCTCGGCTGGAAGTG R:CCTTGCTGTACTGCGTGTC
Mmp2	F:CCCCTTCACTTTTCTGGGCA R:CATCAGGGGAGGGCCCATAG

引物名称	序列(5'-3')
Vegfa	F:CGTCCTGTGTGCCCCTAAT R:TGGCTTTGGTGAGGTTTGAT
Fgf2	F:CTGGCTATGAAGGAAGATGGAC R:CGGTAAGTGTTGTAGTTATTGGAC
Angpt1	F:TATGGATGTGAATGAAGGAGGATGG R:ACTGCCTCTGACTGGTTATTGC
KDR	F:CGCAGAGTGAGGAAGGAGGAC R:CCGTAGGATGATGACAAGAAGTAGC
ENOS	F:TTGTCTGCGGCGATGTTACC R:GCGTATGCGGCTTGTCACC
HIF1A	F:AGGACACAGATTTAGACTTGGAGATG R:CAGTGGTAGTGGTGGCATTAGC