Abstract
Three-dimensional (3D) cell constructs are expected to provide osteoinductive materials to develop cell-based therapies for bone regeneration. The proliferation and spontaneous aggregation capability of induced pluripotent stem cells (iPSCs) thus prompted us to fabricate a scaffold-free iPSC construct as a transplantation vehicle. Embryoid bodies of mouse gingival fibroblast-derived iPSCs (GF-iPSCs) were seeded in a cell chamber with a round-bottom well made of a thermoresponsive hydrogel. Collected ball-like cell constructs were cultured in osteogenic induction medium for 30 days with gentle shaking, resulting in significant upregulation of osteogenic marker genes. The constructs consisted of an inner region of unstructured cell mass and an outer osseous tissue region that was surrounded by osteoblast progenitor-like cells. The outer osseous tissue was robustly calcified with elemental calcium and phosphorous as well as hydroxyapatite. Subcutaneous transplantation of the GF-iPSC constructs into immunodeficient mice contributed to extensive ectopic bone formation surrounded by teratoma tissue. These results suggest that mouse GF-iPSCs could facilitate the fabrication of osteoinductive scaffold-free 3D cell constructs, in which the calcified regions and surrounding osteoblasts may function as scaffolds and drivers of osteoinduction, respectively. With incorporation of technologies to inhibit teratoma formation, this system could provide a promising strategy for bone regenerative therapies.
Highlights
Regeneration of large bone defects caused by trauma, tumor resection, or severe alveolar ridge resorption in dentistry is still a clinical challenge that awaits efficient tissue engineering protocols to achieve sufficient regeneration [1, 2]
We previously reported that an osteogenic induction method for mouse gingival fibroblasts (GFs)-derived induced pluripotent stem cells (iPSCs) (GF-iPSCs) in embryoid bodies (EBs) was advantageous for osteogenesis, as the resulting iPSCs showed significantly higher calcium production capacity than mesenchymal stem cells (MSCs) during osteogenic differentiation [17]
When the RA-treated EBs were cultured in the roundbottom wells of the pNIPAAm gel chamber for two days, the EBs aggregated to form ball-like 3D cell constructs with the same diameter as the wells (1.5 mm) (Figure 1(c): inset)
Summary
Regeneration of large bone defects caused by trauma, tumor resection, or severe alveolar ridge resorption in dentistry is still a clinical challenge that awaits efficient tissue engineering protocols to achieve sufficient regeneration [1, 2]. Recent approaches to fabricating tissue-engineered bone rely on the osteoinductive ability of transplanted cells seeded in exogenous scaffolds [3, 4]. Biomaterial scaffolds facilitate three-dimensional (3D) culture of osteogenic/progenitor cells ex vivo, they have been associated with immunogenicity, unsatisfactory biological activity, enhanced inflammatory reactions, and uncontrollable cell-biomaterial interactions [5]. A scaffold-free approach, in which biomimetic 3D bone tissues are fabricated as cell constructs, could be an attractive alternative for generation of tissueengineered transplants. Bone marrow-derived mesenchymal stem cells (MSCs) are currently the most popular cell source because of their easy collection and preferential. Stem Cells International Floating culture ES medium 2 days +RA 2 days EB +RA 2 days (a). Osteogenic induction medium 30 days (e) (f ) Shaker (b)
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