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S. Zigon-Branc, M. Markovic, J. Van Hoorick, S. Van Vlierberghe, P. Dubruel, E. Zerobin, S. Baudis, A. Ovsianikov:
"Impact of hydrogel stiffness on differentiation of human adipose-derived stem cell microspheroids";
Tissue Engineering, Parts A, B, & C, 5 (2019), 1; 1369 - 1381.

Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered (TE) constructs, as they have tunable mechanical properties, are compatible with different types of cells and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone TE-related studies were performed by utilizing a single cell encapsulation approach. Even though multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. The present study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584 or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3-5 week culture period in the control, chondrogenic or osteogenic medium. While in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicates that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels shows promising potential for future applications in cartilage or bone TE.

Hydrogels, three-dimensional (3D) tissue-engineered (TE) constructs, tunable mechanical properties compatible with different types of cells

http://dx.doi.org/10.1089/ten.TEA.2018.0237

https://www.ncbi.nlm.nih.gov/pubmed/30632465