Mihaila Silvia M, Resende Margarida F, Reis Rui L, Gomes Manuela E, Marques Alexandra P
3Bs Research Group, Biomaterials, Biodegradables and Biomimetics, University of Minho, Barco, GMR, Portugal.
ICVS/3Bs, PT Government Associated Laboratory, Braga, Guimarães, Portugal.
J Tissue Eng Regen Med. 2017 Jul;11(7):1998-2013. doi: 10.1002/term.2096. Epub 2015 Oct 29.
Bone formation relies on complex processes that require well-orchestrated interactions between several cell types, such as bone-forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co-culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co-culture of microvascular-like ECs and pre-OBs, both derived from the SSEA-4 cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA-4 hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre-OBs and maintenance of the EC phenotype. Co-cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre-OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31 cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre-OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co-culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA-4 hASCs as a single-cell source is promising to attain 3D bone-like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.
骨形成依赖于复杂的过程，这些过程需要几种细胞类型之间精心协调的相互作用，如成骨细胞（OBs）和内皮细胞（ECs）。已证明它们的共培养与模拟骨微环境的特定特征相关。在这里，我们提出将源自人脂肪组织基质血管部分的SSEA-4细胞亚群（SSEA-4 hASCs）的微血管样ECs和前成骨细胞进行共培养，以确定细胞协同通讯以支持前成骨细胞完全分化和维持EC表型的条件。建立了两种细胞类型不同比例的共培养物，并在标准内皮细胞维持（ENDO）和成骨分化（OST）培养基以及它们的混合物（MIX）中维持长达21天。在整个培养过程中，根据细胞比例和培养基评估前成骨细胞的成骨成熟（碱性磷酸酶活性、骨桥蛋白和骨钙素表达、钙沉积）、EC数量的变化（CD31细胞）以及内皮表型的维持（CD31和血管性血友病因子表达、低密度脂蛋白摄取）。获得的结果表明，EC数量对前成骨细胞的成骨分化有显著影响，这取决于所使用的培养基。在ENDO培养基中未观察到成骨分化，而在OST和MIX培养基中，除了在MIX培养基中与较多数量的ECs共培养外，成骨分化达到了相似的水平。这些发现表明，使用SSEA-4 hASCs作为单一细胞来源有望获得具有促进血管化骨组织再生潜力的三维骨样模型。版权所有© 2015 John Wiley & Sons, Ltd.
