Rao Rameshwar R, Ceccarelli Jacob, Vigen Marina L, Gudur Madhu, Singh Rahul, Deng Cheri X, Putnam Andrew J, Stegemann Jan P
Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
Acta Biomater. 2014 Jul;10(7):3091-7. doi: 10.1016/j.actbio.2014.03.010. Epub 2014 Mar 18.
Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0-20 mg ml(-1)), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml(-1) HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml(-1) HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 2 0mg ml(-1) HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.
内皮细胞（EC）与间充质干细胞（MSC）共培养可在受限的三维胶原蛋白/纤维蛋白（COL/FIB）复合水凝胶中形成强大的血管网络。然而，当此类水凝胶通过细胞介导的重塑作用发生收缩时，形成内皮网络的能力就会丧失。在本研究中，我们在受限和不受限的COL/FIB基质中创建了人EC与人MSC的共培养体系，并系统地添加了纳米颗粒羟基磷灰石（HA，0 - 20 mg ml⁻¹），这是一种已被证明具有促血管生成作用的类骨矿物质。对培养7天的构建体进行凝胶收缩、血管网络形成和力学性能检测。在体外，在无HA的受限COL/FIB构建体中观察到强大的内皮网络形成，但添加5、10或20 mg ml⁻¹ HA会显著抑制这种反应。在不受限的基质中，纯COL/FIB构建体中网络形成被消除，但1.25或2.5 mg ml⁻¹ HA可挽救这种情况，而更高剂量则再次抑制血管生成。HA以剂量依赖的方式抑制凝胶收缩，这与内皮网络形成无关。HA影响凝胶的初始硬度，但凝胶重塑消除了这种影响。在小鼠皮下植入含0、2.5或20 mg ml⁻¹ HA的COL/FIB，导致植入部位灌注增加，不同材料之间无显著差异。第7天的组织学检查显示宿主和人CD31染色的脉管系统浸润植入物。这些发现与骨科组织工程材料和支架的设计相关，在骨科组织工程中，再生需要血管生成和矿化相的形成。
