Complex Tissue Regeneration Department, MERLN Institute for Technology Inspired Regenerative Medicine, Maastricht University, Maastricht, The Netherlands.
Institute for Life Science, School of Medicine, South China University of Technology, Guangzhou, China.
Tissue Eng Part C Methods. 2020 Jan;26(1):11-22. doi: 10.1089/ten.TEC.2019.0232. Epub 2019 Dec 27.
Vascularization is a critical process during bone regeneration. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of regenerated bone. One strategy for improving the survival and osteogenesis of tissue-engineered bone grafts involves the coculture of endothelial cells (ECs) with mesenchymal stromal cells (MSCs). Moreover, bone regeneration is especially challenging due to its unique structural properties with aligned topographical cues, with which stem cells can interact. Inspired by the aligned fibrillar nanostructures in human cancellous bone, we fabricated polycaprolactone (PCL) electrospun fibers with aligned and random morphology, cocultured human MSCs with human umbilical vein ECs (HUVECs), and finally investigated how these two factors modulate osteogenic differentiation of human MSCs (hMSCs). After optimizing cell ratio, a hMSCs/HUVECs ratio (90:10) was considered to be the best combination for osteogenic differentiation. Coculture results showed that hMSCs and HUVECs adhered to and proliferated well on both scaffolds. The aligned structure of PCL fibers strongly influenced the morphology and orientation of hMSCs and HUVECs; however, fiber alignment was observed to not affect alkaline phosphate (ALP) activity or mineralization of hMSCs compared with random scaffolds. More importantly, cocultured cells on both random and aligned scaffolds had significantly higher ALP activities than monoculture groups, which indicated that coculture with HUVECs provided a larger relative contribution to the osteogenesis of hMSCs compared with fiber alignment. Taken together, we conclude that coculture of hMSCs with ECs is an effective strategy to promote osteogenesis on electrospun scaffolds, and aligned fibers could be introduced to regenerate bone tissues with oriented topography without significant deleterious effects on hMSCs differentiation. This study shows the ability to grow oriented tissue-engineered cocultures with significant increases in osteogenesis over monoculture conditions. Impact statement This work demonstrates an effective method of enhancing osteogenesis of mesenchymal stromal cells on electrospun scaffolds through coculturing with endothelial cells. Furthermore, we provide the optimized conditions for cocultures on electrospun fibrous scaffolds and engineered bone tissues with oriented topography on aligned fibers. This study demonstrates promising findings for growing oriented tissue-engineered cocultures with significant increase in osteogenesis over monoculture conditions.
血管生成是骨再生过程中的一个关键过程。缺乏血管网络会导致氧气和营养供应不足，从而影响再生骨的存活。提高组织工程骨移植物的存活率和成骨能力的一种策略是将内皮细胞（ECs）与间充质基质细胞（MSCs）共培养。此外，由于骨骼具有独特的结构特性，具有定向的地形线索，干细胞可以与之相互作用，因此骨骼再生尤其具有挑战性。受人类松质骨中定向纤维状纳米结构的启发，我们制备了具有定向和随机形态的聚己内酯（PCL）电纺纤维，将人骨髓间充质干细胞（hMSCs）与人脐静脉内皮细胞（HUVECs）共培养，最后研究了这两个因素如何调节人骨髓间充质干细胞（hMSCs）的成骨分化。在优化细胞比例后，认为 hMSCs/HUVECs 比例（90:10）是成骨分化的最佳组合。共培养结果表明，hMSCs 和 HUVECs 很好地黏附和增殖在两种支架上。PCL 纤维的定向结构强烈影响 hMSCs 和 HUVECs 的形态和取向；然而，与随机支架相比，纤维取向观察到对 hMSCs 的碱性磷酸酶（ALP）活性或矿化没有影响。更重要的是，共培养细胞在随机和定向支架上的 ALP 活性均明显高于单培养组，这表明与纤维取向相比，与 HUVEC 共培养为 hMSCs 的成骨提供了更大的相对贡献。综上所述，我们得出结论，hMSCs 与 ECs 的共培养是促进静电纺丝支架上成骨的有效策略，并且可以引入定向纤维来再生具有定向拓扑结构的骨组织，而不会对 hMSCs 分化产生明显的有害影响。本研究表明，在单核培养条件下，具有显著成骨增加的定向组织工程共培养的生长能力。
