Abstract
BackgroundAngiogenesis plays an important role in tissue repair and regeneration, and conditioned medium (CM) derived from mesenchymal stem cells (MSC-CM) possesses pro-angiogenesis. Nevertheless, the profile and concentration of growth factors in MSC-CM remain to be optimized. Fibroblast growth factor-2 (FGF-2) has been proven to be an effective angiogenic factor. Thus, the aim of this study was to verify whether FGF-2 gene overexpression optimized CM from human gingival mesenchymal stem cells (hGMSCs) and whether such optimized CM possessed more favorable pro-angiogenesis effect.MethodsFirst, FGF-2 gene-modified hGMSCs were constructed using lentiviral transfection technology (LV-FGF-2+-hGMSCs) and the concentration of angiogenesis-related factors in LV-FGF-2+-hGMSC-CM was determined by ELISA. Then, human umbilical vein endothelial cells (HUVECs) were co-cultured for 3 days with LV-FGF-2+-hGMSC-CM, and the expression level of placenta growth factor (PLGF), stem cell factor (SCF), vascular endothelial growth factor receptor 2 (VEGFR2) in HUVECs were determined by qRT-PCR, western blot, and cellular immunofluorescence techniques. The migration assay using transwell and in vitro tube formation experiments on matrigel matrix was conducted to determine the chemotaxis and angiogenesis enhanced by LV-FGF-2+-hGMSC-CM. Finally, NOD-SCID mice were injected with matrigel mixed LV-FGF-2+-hGMSC-CM, and the plug sections were analyzed by immunohistochemistry staining with anti-human CD31 antibody.ResultsLV-FGF-2+-hGMSC-CM contained significantly more FGF-2, vascular endothelial growth factor A (VEGF-A), and transforming growth factor β (TGF-β) than hGMSC-CM. HUVECs pretreated with LV-FGF-2+-hGMSC-CM expressed significantly more PLGF, SCF, and VEGFR2 at gene and protein level than hGMSC-CM pretreated HUVECs. Compared with hGMSC-CM, LV-FGF-2+-hGMSC-CM presented significantly stronger chemotaxis to HUVECs and significantly strengthened HUVECs mediated in vitro tube formation ability. In vivo, LV-FGF-2+-hGMSC-CM also possessed stronger promoting angiogenesis ability than hGMSC-CM.ConclusionsOverexpression of FGF-2 gene promotes hGMSCs paracrine of angiogenesis-related growth factors, thereby obtaining an optimized conditioned medium for angiogenesis promotion.
Highlights
Angiogenesis plays an important role in tissue repair and regeneration, and conditioned medium (CM) derived from mesenchymal stem cells (MSC-CM) possesses pro-angiogenesis
Results human gingival mesenchymal stem cells (hGMSCs) culture and identification The hGMSCs were obtained by tissue block digestionlimited dilution method (Fig. 1a, b)
LV-Fibroblast growth factor-2 (FGF-2) transfection promotes FGF-2 gene expression and FGF-2, vascular endothelial growth factor A (VEGF-A), and transforming growth factor β (TGF-β) paracrine of hGMSCs The lentivirus vector pHBLV-CMV-MCS-3FLAG-EF1ZsGreen-T2A-PURO-FGF-2 was successfully constructed as shown by the map of the plasmid (Additional file 1)
Summary
Angiogenesis plays an important role in tissue repair and regeneration, and conditioned medium (CM) derived from mesenchymal stem cells (MSC-CM) possesses pro-angiogenesis. Periodontitis, bone fractures, malformations, and surgical removal of tumors can cause oral and maxillofacial bone defects and interfere with normal function and configuration. Regeneration of these damaged bone tissue has become an important clinical problem [1]. Bone grafts, including autografts, allografts, xenografts, and synthetic grafts, are the primary treatment modalities [2, 3]. These bone grafts are limited in clinical application for varied reasons. The new options are needed to improve bone regeneration outcomes
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