Scaffold-Free Osteochondral Engineering Using Embryonic-Derived Mesenchymal Stem Cell Spheroids.

Abstract

In this study, we explored whether embryonic stem cell-derived mesenchymal stem cell (ES-MSC) cellular spheroids in combination with a closed chamber system could be used to create scaffold-free cartilage and endochondral graft tissues. ES-MSC cellular spheroids were cultured in chondrogenic medium for 3-4 days and seeded into a customizable Net Mold chamber system (NCS) and cultured in chondrogenic medium for an additional 18 days to fuse and form a single tissue construct. To assess potential for cartilage repair, cellular spheroids were matured in the NCS for only 7 days before implantation into ex vivo human cartilage defects. To engineer osteochondral tissues, cellular spheroids were initially cultured in chondrogenic medium for 14 days, seeded into one well of the NSC, and cultured together in osteogenic medium for 21 days. For the chondrogenic phase, cellular spheroids were initially cultured in chondrogenic medium for 14 days before seeding in an NCS chamber, adjacent to the osteogenic spheroids. The combined osteogenic and chondrogenic constructs were cultured in serum-free medium for an additional 3 weeks. Cellular spheroids cultured in the NCS developed into neocartilage tissues expressing cartilage-associated genes (COL2A1, ACAN, and COMP) and stained positive for cartilage matrix molecules (glycosaminoglycan and collagen type II). The cartilage-like constructs that were implanted into cartilage defects created in ex vivo osteoarthritic (OA) tissue resulted in repair tissue with an elastic modulus of 46 ± 6 kPa that was histologically integrated with the explant tissues. Spheroids cultured in osteogenic medium produced tissues that were positive for von Kossa stain and for osteopontin immunostaining. Pre-differentiation in chondrogenic and osteogenic medium before placing in the NCS resulted in fused cartilage and bone-like constructs with regional production of chondrogenic and mineralized matrix (Alizarin Red S, von Kossa, and osteopontin positive). Spheroids in stacked NCS chambers produced osteochondral neotissues up to 2 mm in thickness. Our results indicate the potential for cellular spheroids, from a clinically relevant ES-MSC source, to generate scaffold-free chondrogenic or osteochondrogenic graft tissues.