Freeman Fiona E, Stevens Hazel Y, Owens Peter, Guldberg Robert E, McNamara Laoise M
1 Biomedical Engineering, Centre for Biomechanics Research (BMEC), National University of Ireland Galway , Galway, Ireland .
2 George W. Woodruff School of Mechanical Engineering, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia .
Tissue Eng Part A. 2016 Oct;22(19-20):1176-1190. doi: 10.1089/ten.TEA.2015.0339. Epub 2016 Sep 28.
In vitro bone regeneration strategies that prime mesenchymal stem cells (MSCs) with chondrogenic factors, to mimic aspects of the endochondral ossification process, have been shown to promote mineralization and vascularization by MSCs both in vitro and when implanted in vivo. However, these approaches required the use of osteogenic supplements, namely dexamethasone, ascorbic acid, and β-glycerophosphate, none of which are endogenous mediators of bone formation in vivo. Rather MSCs, endothelial progenitor cells, and chondrocytes all reside in proximity within the cartilage template and might paracrineally regulate osteogenic differentiation. Thus, this study tests the hypothesis that an in vitro bone regeneration approach that mimics the cellular niche existing during endochondral ossification, through coculture of MSCs, endothelial cells, and chondrocytes, will obviate the need for extraneous osteogenic supplements and provide an alternative strategy to elicit osteogenic differentiation of MSCs and mineral production. The specific objectives of this study were to (1) mimic the cellular niche existing during endochondral ossification and (2) investigate whether osteogenic differentiation could be induced without the use of any external growth factors. To test the hypothesis, we evaluated the mineralization and vessel formation potential of (a) a novel methodology involving both chondrogenic priming and the coculture of human umbilical vein endothelial cells (HUVECs) and MSCs compared with (b) chondrogenic priming of MSCs alone, (c) addition of HUVECs to chondrogenically primed MSC aggregates, (d-f) the same experimental groups cultured in the presence of osteogenic supplements and (g) a noncoculture group cultured in the presence of osteogenic growth factors alone. Biochemical (DNA, alkaline phosphatase [ALP], calcium, CD31, vascular endothelial growth factor [VEGF]), histological (alcian blue, alizarin red), and immunohistological (CD31) analyses were conducted to investigate osteogenic differentiation and vascularization at various time points (1, 2, and 3 weeks). The coculture methodology enhanced both osteogenesis and vasculogenesis compared with osteogenic differentiation alone, whereas osteogenic supplements inhibited the osteogenesis and vascularization (ALP, calcium, and VEGF) induced through coculture alone. Taken together, these results suggest that chondrogenic and vascular priming can obviate the need for osteogenic supplements to induce osteogenesis of human MSCs in vitro, while allowing for the formation of rudimentary vessels.
体外骨再生策略是通过用软骨生成因子预处理间充质干细胞（MSC）来模拟软骨内成骨过程的某些方面，已证明这种策略在体外以及植入体内时都能促进MSC的矿化和血管生成。然而，这些方法需要使用成骨补充剂，即地塞米松、抗坏血酸和β-甘油磷酸酯，而这些都不是体内骨形成的内源性介质。相反，MSC、内皮祖细胞和软骨细胞都在软骨模板内相邻存在，并且可能通过旁分泌调节成骨分化。因此，本研究检验了这样一个假设：通过将MSC、内皮细胞和软骨细胞共培养来模拟软骨内成骨过程中存在的细胞微环境的体外骨再生方法，将无需额外的成骨补充剂，并提供一种诱导MSC成骨分化和矿物质生成的替代策略。本研究的具体目标是：（1）模拟软骨内成骨过程中存在的细胞微环境；（2）研究在不使用任何外部生长因子的情况下是否能诱导成骨分化。为了验证这一假设，我们评估了（a）一种既包括软骨生成预处理又包括人脐静脉内皮细胞（HUVEC）与MSC共培养的新方法的矿化和血管形成潜力，并将其与（b）单独对MSC进行软骨生成预处理、（c）将HUVEC添加到经软骨生成预处理的MSC聚集体中、（d - f）在存在成骨补充剂的情况下培养的相同实验组以及（g）仅在存在成骨生长因子的情况下培养的非共培养组进行比较。进行了生化分析（DNA、碱性磷酸酶[ALP]、钙、CD31、血管内皮生长因子[VEGF]）、组织学分析（阿尔新蓝、茜素红）和免疫组织学分析（CD31），以研究在不同时间点（1、2和3周）的成骨分化和血管生成情况。与单独的成骨分化相比，共培养方法增强了成骨和血管生成，而成骨补充剂则抑制了单独通过共培养诱导的成骨和血管生成（ALP、钙和VEGF）。综上所述，这些结果表明，软骨生成和血管预处理可以在体外诱导人MSC成骨时无需成骨补充剂，并允许形成原始血管。
