Joensuu K, Uusitalo L, Alm J J, Aro H T, Hentunen T A, Heino T J
Department of Cell Biology and Anatomy, Kiinamyllynkatu 10 C3, 20520 Turku, Finland.
Department of Cell Biology and Anatomy, Kiinamyllynkatu 10 C3, 20520 Turku, Finland.
Orthop Traumatol Surg Res. 2015 May;101(3):381-6. doi: 10.1016/j.otsr.2015.01.014. Epub 2015 Mar 23.
Despite recent advances in bone tissue engineering, efficient bone formation and vascularization remains a challenge for clinical applications.
The aim of this study was to investigate if the osteoblastic differentiation of human mesenchymal stromal cells (MSCs) can be enhanced by co-culturing them with peripheral blood (PB) mononuclear cells (MNCs), with and without vascular endothelial growth factor (VEGF), a coupling factor of bone formation and angiogenesis.
Human bone marrow (BM) derived MSCs were co-cultured with PB-MNCs in osteogenic medium with or without VEGF. Osteoblastic differentiation and mineral deposition were studied by staining for alkaline phosphatase (ALP), and von Kossa, respectively, and measurements for ALP activity and calcium concentration (Ca). Cell proliferation was assayed with Alamar blue. The mechanism(s) were further studied by Transwell(®) cell culture experiments.
Both ALP and mineralization (von Kossa and Ca) were significantly higher in the MSC-MNC co-cultures compared to plain MSC cultures. VEGF alone had no effect on osteoblastic differentiation of MSCs, but further enhanced differentiation in co-culture settings. The mechanism was shown to require cell-cell contact between MSCs and MNCs and the factors contributing to further differentiation appear to be soluble. No differences were observed in cell proliferation.
Our study demonstrates that the in vitro ALP activity and mineralization of human BM-MSCs is more efficient in the presence of PB-MNCs, and exogenously added VEGF further enhances the stimulatory effect. This indicates that PB-MNCs could be a potential cell source in development of co-culture systems for novel tissue engineering applications for enhanced bone healing.
尽管骨组织工程领域近来取得了进展，但有效的骨形成和血管生成对于临床应用而言仍是一项挑战。
本研究的目的是探究将人间充质基质细胞（MSCs）与外周血（PB）单个核细胞（MNCs）共培养，无论有无骨形成与血管生成的偶联因子血管内皮生长因子（VEGF），是否能够增强MSCs的成骨分化。
将人骨髓（BM）来源的MSCs与PB-MNCs在添加或不添加VEGF的成骨培养基中进行共培养。分别通过碱性磷酸酶（ALP）染色、冯科萨染色研究成骨分化和矿物质沉积，并测定ALP活性和钙浓度（Ca）。用阿拉玛蓝检测细胞增殖。通过Transwell®细胞培养实验进一步研究其机制。
与单纯的MSC培养相比，MSC-MNC共培养中ALP和矿化（冯科萨染色和Ca）显著更高。单独的VEGF对MSCs的成骨分化没有影响，但在共培养条件下进一步增强了分化。结果表明该机制需要MSCs和MNCs之间的细胞-细胞接触，且促成进一步分化的因子似乎是可溶的。在细胞增殖方面未观察到差异。
我们的研究表明，在存在PB-MNCs的情况下，人BM-MSCs的体外ALP活性和矿化更有效，并且外源性添加的VEGF进一步增强了刺激作用。这表明PB-MNCs可能是开发用于增强骨愈合的新型组织工程应用的共培养系统中的一种潜在细胞来源。
