Pre-Culture of Human Mesenchymal Stromal Cells in Spheroids Reduces Hypertrophic Differentiation <i> in vitro</i> and Facilitates Chondrogenesis at a Low Total Cell Count Upon Embedding in Biomaterials

Abstract

Material assisted cartilage tissue engineering approaches have limited application in cartilage treatment due to hypertrophic and fibrocartilaginous tissue formation and the high cell count needed. The aim of this study was to investigate the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. hMSCs were pre-cultured in spheroids (250 cells/spheroid) for three days in chondrogenic differentiation media. Spheroids were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels at four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content at a cell density of 5 Mio cells/ml (1’000 spheroids/sample). Macro-pellets derived from hMSCs (250’000 cells, MP) or hMSC spheroids (250’000 cells/1’000 spheroids, SP) served as controls. hMSCs differentiation (day 21) was analyzed using quantification of glycosaminoglycans (GAGs), gene expression analysis and (immuno-) histological stainings. Embedding of hMSC spheroids in THA-Col biomaterials induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and production of GAGs. A lower THA content led to a more pronounced chondrogenic phenotype compared to a higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures (MP, SP) showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in MP culture compared to SP group.This study indicated a positive benefit of pre-culturing hMSCs in spheroids with respect to hypertrophic gene expression compared to the gold standard pellet culture without compromising chondrogenic matrix production. Funding: This work is part of the osteochondral defect collaborative research program supported by the AO Foundation. F.S. received the AO Foundation Fellowship in Health Sciences and Technology from ETH Zurich Foundation. Declaration of Interests: The authors have no competing of interests. Ethics Approval Statement: Full ethical approval (Inselspital Bern - Bern Req-2016-00141, University hospital Freiburg - EKFreiburg: 135/14).