A modular approach to the engineering of a centimeter-sized bone tissue construct with human amniotic mesenchymal stem cells-laden microcarriers

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.