Tong Xiaojie, Chen Jin, Wang Renqin, Hou Dan, Wu Gang, Liu Chang, Pathak Janak Lal
School and Hospital of Stomatology, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Medical University, Guangzhou 510182, China.
Department of Oral and Maxillofacial Surgery/Pathology, Amsterdam UMC and Academic Center for Dentistry Amsterdam (ACTA), Amsterdam Movement Science, Vrije Universiteit Amsterdam, 1081 LA Amsterdam, The Netherlands.
Bioengineering (Basel). 2023 Oct 20;10(10):1227. doi: 10.3390/bioengineering10101227.
The combination of hyaluronic acid (HA) and BMP-2 has been reported to promote bone regeneration. However, the interaction of endothelial cells and bone marrow mesenchymal stem cells (BMSCs) during HA + BMP-2 treatment is not fully understood. This study aimed to analyze the direct effect of HA, as well as the paracrine effect of HA-treated endothelial cells, on the BMP-2-mediated osteogenic differentiation of BMSCs. The angiogenic differentiation potential of HA at different molecular weights and different concentrations was tested. The direct effect of HA, as well as the indirect effect of HA-treated human umbilical cord endothelial cells (HUVECs, i.e., conditioned medium (CM)-based co-culture) on the BMP-2-mediated osteogenic differentiation of BMSCs was analyzed using alkaline phosphatase (ALP) staining and activity, alizarin red S (ARS) staining, and RT-qPCR of osteogenic markers. Angiogenic differentiation markers were also analyzed in HUVECs after treatment with HA + BMP-2. The bone regeneration potential of BMP-2 and HA + BMP-2 was analyzed in a rat ectopic model. We found that 1600 kDa HA at 300 µg/mL promoted tube formation by HUVECs in vitro and upregulated the mRNA expression of the angiogenic markers CD31, VEGF, and bFGF. HA inhibited, but conditioned medium from HA-treated HUVECs promoted, the BMP-2-mediated osteogenic differentiation of BMSCs, as indicated by the results of ALP staining and activity, ARS staining, and the mRNA expression of the osteogenic markers RUNX-2, ALP, COLI, and OPN. HA + BMP-2 (50 ng/mL) upregulated the expression of the angiogenesis-related genes VEGF and bFGF in HUVECs and bone regeneration in vivo compared to BMP-2 treatment. In conclusion, the paracrine effect of hyaluronic acid-treated endothelial cells promotes BMP-2-mediated osteogenesis, suggesting the application potential of HA + BMP-2 in bone tissue engineering.
据报道，透明质酸（HA）与骨形态发生蛋白-2（BMP-2）联合使用可促进骨再生。然而，在HA + BMP-2治疗过程中，内皮细胞与骨髓间充质干细胞（BMSCs）之间的相互作用尚未完全明确。本研究旨在分析HA的直接作用以及HA处理的内皮细胞的旁分泌作用对BMP-2介导的BMSCs成骨分化的影响。测试了不同分子量和不同浓度的HA的血管生成分化潜能。使用碱性磷酸酶（ALP）染色及活性、茜素红S（ARS）染色和成骨标志物的逆转录定量聚合酶链反应（RT-qPCR），分析HA的直接作用以及HA处理的人脐静脉内皮细胞（HUVECs，即基于条件培养基（CM）的共培养）对BMP-2介导的BMSCs成骨分化的间接作用。在用HA + BMP-2处理后的HUVECs中，也对血管生成分化标志物进行了分析。在大鼠异位模型中分析了BMP-2和HA + BMP-2的骨再生潜能。我们发现，300 μg/mL的1600 kDa HA可促进HUVECs在体外形成管腔，并上调血管生成标志物CD31、血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）的mRNA表达。如ALP染色及活性、ARS染色以及成骨标志物RUNX-2、ALP、I型胶原蛋白（COLI）和骨桥蛋白（OPN）的mRNA表达结果所示，HA抑制BMP-2介导的BMSCs成骨分化，但HA处理的HUVECs的条件培养基可促进其成骨分化。与BMP-2治疗相比，HA + BMP-2（50 ng/mL）上调了HUVECs中血管生成相关基因VEGF和bFGF的表达以及体内的骨再生。总之，透明质酸处理的内皮细胞的旁分泌作用促进了BMP-2介导的成骨作用，提示HA + BMP-2在骨组织工程中的应用潜力。
