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. 2018 Sep;176:13–23. doi: 10.1016/j.biomaterials.2018.05.032

Fig. 4.

Fig. 4

HD cultures secrete a glycoprotein-rich PCM and take on characteristics of quiescent cells. (A) Representative micrographs of Ki67 staining of hMSC in 1:0.75 hydrogels after 72 h. Significantly more hMSC were positive for Ki67 in LD (n = 250) than HD (n ≥ 500) cultures (P < 0.001). (B) Quantification of hMSC volume in LD and HD cultures (n ≥ 20, ***P < 0.001). (C) Profile plots showing the heavy fraction of a selection of structural proteins, or (D) glycoproteins synthesized by LD and HD cultures of hMSC encapsulated in 1:0.375 and 1:0.75 S-HA-PEGDA hydrogels (two technical replicates, TR) after 72 h in culture. (E) Representative micrographs showing staining for lumican and versican secreted by hMSC in LD and HD cultures in 1:0.375 hydrogels after 72 h. (F) Representative micrographs showing Ki67 staining of hMSC in HD cultures within 1:0.75 hydrogels after treatment with paclitaxel (PTX+) or vehicle control (PTX-) for 72 h. Significantly more hMSC were positive for Ki67 in the presence of paclitaxel than vehicle controls (n ≥ 500, P < 0.001). (G) Representative micrographs showing staining for lumican and versican secreted by hMSC in 1:0.75 hydrogels treated with paclitaxel or the vehicle control after 72 h. (H) Gene expression analyses (normalized to undifferentiated controls) in HD cultures of hMSC for markers of adipogenesis (PPARγ, C/EPBα) in 1:0.375, and (I) osteogenesis (RUNX2, BGLAP) in 1:0.75 hydrogels 72 h after encapsulation. Cultures were treated with paclitaxel (outlined grey) or with the vehicle control (n ≥ 8, **P < 0.01, ***P < 0.001). (J) Schematic highlighting potential means by which HD cultures could regulate hMSC response compared to LD cultures. In LD cultures, hMSC rely on cues from the hydrogel, secrete PCM and interact with it to differentiate. HD culture may drive quiescence via an indirect effect mediated by the formation of interacting PCM and their sequestration of signaling molecules. In (A) and (F) scale bar = 10 μm. In (E) and (G) scale bar = 100 μm, insets = 10 μm. In (A) and (F) a Fisher's exact test was used to detect statistical significance. In (B), and (H)–(I) plots show mean + SD and a two-tailed Mann-Whitney test was used to detect statistical significance.