Recapitulation of mesenchymal cell condensation using human mesenchymal stem cell-collagen microspheres.

Abstract

Mesenchymal cell condensation is a critical transitional stage that precedes cartilage or bone formation. A microencapsulation technique was previously established to entrap mesenchymal stem cells (MSC) in collagen microspheres. Here, we hypothesize that the MSC-induced gel contraction mimics the mesenchymal cell condensation and thereby regulates MSC differentiation. The effect of cell density and timing for chondrogenesis induction was investigated. In general, gene expression of SOX9 was upregulated at early time points (day 0–2) while RUNX2 was gradually increased. COL2A1 was constantly expressed in intermediate cell density and was increased at later time points in high cell density but decreased in low cell density. COL1A1 was downregulated except the intermediate cell density with constant expression. COL10A1 was constantly expressed except the low cell density with a decreasing trend. Osterix was gradually upregulated and peak at day 7. However, aggrecan and ALP expression was lower than the monolayer throughout the culturing period. Nuclear localization of Sox9 was observed at day 0 and day 1 whereas nuclear localization of Runx2 was observed from day 0 to day 7 in low and intermediate cell densities but only at day 0 in high cell density. Deposition of collagen II and collagen X were increased with time and cell density. Upon chondrogenesis, gene expression of SOX9, COL2A1, and aggrecan were higher in MSC-collagen microspheres when chondrogenesis was induced at day 3 than that at day 0 and day 1. This work suggests the importance of timing of induction in developing protocols for stem cell chondrogenesis.