Impact of Hydrogel Stiffness on Differentiation of Human Adipose-Derived Stem Cell Microspheroids

Abstract

Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered 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 tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although 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. This 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-to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although 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 indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatinbased hydrogels show promising potential for future applications in cartilage or bone TE.

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids

“…Although this lack of Ki67 staining, light microscopic evaluation showed the cellular outgrowth. Similar results were reported when spheroids of human adipose-derived MSC were encapsulated in 5, 7.5, and 10 w/v% gelMA hydrogels: sprouting was observed and was more pronounced in the softest gels (Žigon-Branc et al, 2019). In this study, outgrowth of cells was especially seen in the softer gelMA hydrogels crosslinked with Li-TPO-L as a PI.…”

“…The constructs with lower stiffness resulted in a downregulation of collagen I and an increase in collagen II production. Žigon-Branc et al (2019) also demonstrated that the extent of chondrogenic differentiation of MSC spheroids was more pronounced in softer hydrogels.…”

Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression

“…The present results indicated that cellular viability was maintained in the presence of FGF-4, while osteogenic differentiation of stem cell spheroids was enhanced, at least partially by regulation of RUNX2 and BGLAP expression. In a previous study, RUNX2 and BGLAP were selected as markers for osteogenesis (38). RUNX2 is a molecular biomarker for osteoblastic differentiation and is able to induce the expression and synthesis of BGLAP (43).…”

“…Spheroids of mesenchymal stem cells also expressed higher transcription factors that regulate stemness compared with monolayer cultures, along with higher alkaline phosphatase activity and enhanced expression of osteogenesis-associated genes ( 37 ). In another previous study, encapsulation of stem cell microspheroids was performed using gelatin-based hydrogels and it was demonstrated to have promising potential for bone or cartilage tissue engineering ( 38 ).…”

Methacrylation increase growth and differentiation of primary human osteoblasts for gelatin hydrogels

“…However, when microspheroids were placed in softer Gel-MOD, these started to outgrow and interconnect within a few days, and their protrusion was slower or more limited in stiffer Gel-MOD hydrogels. [32]. Previous studies have shown a significant influence on stem cell migration, proliferation, and differentiation by varying stiffness of twodimensional (2D) or 3D substrates [33].…”