1 Translational Center Wuerzburg "Regenerative Therapies in Oncology and Musculoskeletal Disease," Wuerzburg, Germany .
2 Fujifilm Manufacturing Europe B.V., LJ Tilburg, The Netherlands .
Tissue Eng Part A. 2017 Sep;23(17-18):946-957. doi: 10.1089/ten.TEA.2016.0436. Epub 2017 Jul 11.
The development of scaffold formulations based on extracellular matrix (ECM)-inspired synthetic materials constitutes an important resource for the advance of cell-based therapies in bone tissue engineering approaches, where both cell and scaffold implantation are often needed. Culturing cells on porous microcarriers (MCs) allows cell expansion in a three-dimensional microenvironment and constitutes a possible solution for minimally invasive cell and scaffold simultaneous delivery, but the reduced pore dimension and pore interconnection diameter of several commercially available MCs limits de facto cell ingrowth, and ultimately their suitability for in vivo cell delivery. In this study we investigated the potential of a new macroporous MC based on a collagen I-based recombinant peptide (Cellnest™) for C2C12 cells and human bone marrow-derived mesenchymal stromal cells (hBMSCs) expansion and we analyzed the influence of dehydrothermal (DHT), hexamethylene diisocyanate (HMDIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking strategies on cell vitality, proliferation, and hBMSCs differentiation. We established a double emulsification protocol for the manufacturing of MCs characterized by external pores of 20-40 μm diameter, 73% porosity, and 20 ± 3 μm pore interconnection diameter supporting cell ingrowth and proliferation into the MC. MCs cross-linked with DHT and HMDIC supported higher cell proliferation comparing to a commercially available equivalent over the course of 7 days and resulted in higher cell yield by day 28. Moreover, while hBMSCs expansion on Cellnest-MCs did not lead to a significant upregulation of the early markers of osteogenic differentiation Col1a1 and Runx2, their differentiation potential into the osteogenic lineage was preserved when cultured in differentiation medium, as confirmed by mineralized ECM deposition. We believe that Cellnest-MCs will help in reaching clinically relevant cell quantities and ultimately help in accelerating the translation of cell-based therapies for bone tissue engineering in the clinical practice.
基于细胞外基质 (ECM) 启发的合成材料的支架配方的开发为基于细胞的治疗在骨组织工程方法中的进展提供了重要资源，其中细胞和支架的植入通常都是需要的。在多孔微载体 (MC) 上培养细胞允许在三维微环境中进行细胞扩增，并且是微创细胞和支架同时输送的一种可能解决方案，但几种市售 MC 的减小的孔径和孔连通直径实际上限制了细胞的向内生长，并且最终限制了它们用于体内细胞输送的适用性。在这项研究中，我们研究了一种基于胶原蛋白 I 的重组肽（CellnestTM）的新型大孔 MC 对 C2C12 细胞和人骨髓间充质基质细胞（hBMSCs）扩增的潜力，并分析了去水热处理（DHT）、六亚甲基二异氰酸酯（HMDIC）和 1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）交联策略对细胞活力、增殖和 hBMSCs 分化的影响。我们建立了一种双重乳化方案来制造 MCs，其特征在于具有 20-40 μm 直径的外部孔、73%的孔隙率和 20±3 μm 的孔连通直径，支持细胞向内生长和增殖到 MC 中。与商业上等效的 MC 相比，用 DHT 和 HMDIC 交联的 MC 在 7 天的过程中支持更高的细胞增殖，并在第 28 天产生更高的细胞产量。此外，虽然 hBMSCs 在 Cellnest-MCs 上的扩增不会导致成骨分化的早期标志物 Col1a1 和 Runx2 的显著上调，但当在分化培养基中培养时，它们向成骨谱系分化的潜力得以保留，这一点得到了矿化细胞外基质沉积的证实。我们相信，Cellnest-MCs 将有助于达到临床相关的细胞数量，并最终有助于加速基于细胞的治疗在骨组织工程中的临床转化。
