Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon emb e dding in biomaterials to generate cartilage microtissues

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (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 x 10 (6) cells/ml (2 x 10(4) spheroids/ml). Macropellets derived from single hMSCs (2.5 x 10 5 cells, ScMP) or hMSC spheroids (2.5 x 10 (5) cells, 10 (3) spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs . Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 x 10 (6) /ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct.

Automated summary

This study investigates the potential of embedding hMSC spheroids in biomaterials to promote cell differentiation. The results show that embedding hMSC spheroids in THA-Col hydrogels induces chondrogenic differentiation, with lower THA content leading to a more pronounced chondrogenic phenotype. The study suggests that pre-culturing hMSC spheroids at low cell count can be a promising approach for cartilage tissue engineering.