Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering

Human embryonic stem cells (hESC) are promising for use in regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there have been no reports on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESC were cultured to form embryoid bodies (EB), and the MSC were then migrated out of the EB. hESCd-MSC had surface markers characteristic of MSC, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSC were encapsulated in alginate at a density of 1 million cells ml−1, with an average microbead size of 207 μm. CPC contained mannitol porogen to create a porosity of 64% and 218-μm macropores, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSC encapsulated in microbeads in CPC had good viability from 1 to 21 days. ALP gene expression at 21 days was 25-fold that at 1 day. Osteocalcin (OC) at 21 days was two orders of magnitude of that at 1 day. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21 days was fivefold that at 1 day. Mineral synthesis by the encapsulated hESCd-MSC at 21 days was sevenfold that at 1 day. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSC were encapsulated in alginate microbeads in macroporous CPC, showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.