State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai 200237, PR China.
Biomaterials. 2011 Oct;32(30):7532-42. doi: 10.1016/j.biomaterials.2011.06.054. Epub 2011 Jul 20.
Tissue engineering of clinical-relevant large tissue constructs remains a big challenge due to the mass transfer limit. A modular approach via the assembling of modular tissues thus eliminating the mass transfer limit holds great promise for fabricating centimeter-sized constructs. In the present study, we investigated the feasibility of using microcarriers seeded with adult mesenchymal stem cells (MSCs) to fabricate a large bone tissue. It was demonstrated that human amniotic MSCs (hAMSCs) were efficiently seeded onto CultiSpher S microcarriers (made of porcine gelatin) in a spinner flask and quickly proliferated while retaining a great viability. Within a total culture period of 28 days, using a two-stage culture strategy, hAMSCs-laden microcarriers with a high cell density were prepared at the first stage and the cells were then directly induced to undergo osteogenic differentiation in the same culture flask. During this cultivation process, the aggregation of cell-laden microcarriers was apparent, which resulted in aggregates of 700-800 μm, a size permissive for maintaining high cell viability. The osteogenic differentiation of hAMSCs on microcarriers was confirmed with increased mineral deposition (Alizarin red S staining and quantification of calcium content), ALP activity as well as gene expression of osteogenic markers (collagen type I and osteocalcin). These modular bone-like tissues were used as building blocks to fabricate a macroscopic bone construct in a cylindrical perfusion culture chamber (2 cm in diameter). After a 7-day perfusion culture, these modular tissues readily assembled into a centimeter-sized construct (diameter × height: 2 cm × 1 cm). Both good cell viability and fairly homogenous distribution of cellular content and bone-characteristic ECM within the macrotissue were elaborated. This paper provided a proof-of-concept study for modularly engineering clinical-relevant large tissue replacements with cell-laden microcarriers.
由于传质限制，临床相关大组织构建的组织工程仍然是一个巨大的挑战。通过组装模块化组织来实现模块化方法，从而消除传质限制，为制造厘米级构建体提供了巨大的前景。在本研究中，我们研究了使用接种有成人间充质干细胞（MSCs）的微载体来构建大骨组织的可行性。结果表明，人羊膜 MSCs（hAMSCs）可有效地接种到旋转瓶中的 CultiSpher S 微载体（由猪明胶制成）上，并在保持高活力的同时快速增殖。在总共 28 天的培养期内，使用两阶段培养策略，在第一阶段制备高细胞密度的负载 hAMSCs 的微载体，然后在同一培养瓶中直接诱导细胞进行成骨分化。在培养过程中，细胞负载微载体的聚集是明显的，导致聚集物为 700-800 μm，这一大小允许保持高细胞活力。通过增加矿物质沉积（茜素红 S 染色和钙含量的定量）、碱性磷酸酶活性以及成骨标志物（I 型胶原和骨钙素）的基因表达，证实了 hAMSCs 在微载体上的成骨分化。这些模块化的骨样组织被用作构建块，在圆柱形灌注培养室（直径 2 cm）中构建宏观骨构建体。在 7 天的灌注培养后，这些模块化组织很容易组装成一个厘米级的构建体（直径×高度：2 cm×1 cm）。在大组织中，细胞活力良好，细胞内容物和具有骨特征的细胞外基质的分布也相当均匀。本文为使用负载细胞的微载体模块化工程临床相关大组织替代物提供了概念验证研究。
