Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs in vitro to boost their bone forming capacity in vivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks in vivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days in vitro improves the quantity and quality of bone formation in vivo. This study highlights the need for optimizing in vitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation.

中文翻译：

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工程构建物的生物反应器培养时间会影响间充质干细胞的骨形成

植入生物材料支架中的间充质干细胞（MSC）的灌注培养为细胞存活提供了营养，增强了细胞外基质的沉积，并增加了成骨细胞的分化。然而，关于在体外适当增加细胞构建物的灌注持续时间以增强其在体内的骨形成能力尚无共识。。我们通过在直接灌注生物反应器中的大孔复合支架中培养人MSC来研究这种现象，并在XYZ振荡器上比较了它们在连续动态培养条件下对支架的响应。连续灌注生物反应器中的细胞播种比静态播种导致更均匀的MSC分布。我们在21天的生物反应器培养物中观察到了所有复合支架中类似的钙沉积，无论其孔径大小如何。与动态培养中的支架相比，灌注支架在其后达到稳定的培养物中最多显示14天的DNA含量和成骨标记物表达。然后，我们评估了当将MSC种植的支架植入鼠异位部位时，灌注培养持续时间对骨形成的影响。在第2周，人类MSC持续存在于所有支架中在体内，我们观察到在每种性别下，在灌注下培养7天的构建体相对于培养1天的构建体，新血管形成增加。植入后8周，与培养1或7天和无细胞构建物相比，在培养14天的生物反应器构建物中，我们观察到更大的骨体积分数，骨矿物质密度，组织向内生长，胶原蛋白密度和成骨细胞标志物。综上所述，这些数据表明，在灌注培养下在体外培养MSC至少14天可改善体内骨形成的数量和质量。这项研究强调了体外优化的必要性 工程构建体的生物反应器培养持续时间，以达到所需的骨形成水平。