Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels

Abstract

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Figure 1

hMSC matrix interactions and fate choice within photopolymerized MeHA hydrogels. a, Schematic of RGD conjugation to and photopolymerization of MeHA. b,d,e, Representative brightfield images of hMSCs within MeHA gels following b, 7 days mixed osteogenic/adipogenic media incubation, d, 7 days osteogenic media incubation, or e, mixed osteogenic/adipogenic media with encapsulated hMSCs transfected with constitutively active ROCK (ROCKΔ3). Oil red O (ORO) stains neutral lipids (adipogenesis) red and fast blue RR stains alkaline phosphatase (ALP, osteogenesis) purple. c–e, Percentage differentiation of hMSCs toward osteogenic or adipogenic lineages for these same groups. f, Representative brightfield images and percentage differentiation of hMSCs following 7 days mixed media incubation within MeHA hydrogels (with hMSCs incubated with primary anti-CD44 antibodies prior to encapsulation), MeAlg hydrogels, or MeDex hydrogels of elastic modulus corresponding to osteogenesis in the physically crosslinked alginate system (~20 kPa). For all mixed media groups, the percentage differentiation was significantly different between osteogenesis and adipogenesis (p < 0.001, t test). Error bars represent standard errors for the mean. Scale bars: b,d,e,f 100 µm, 5 µm (inset).

Figure 2

MeMaHA sequential crosslinking schematic, characterization & proteolytic degradation kinetics. a, MeMaHA chemical structure (m = .755, n = .14, o = .105). b, Schematic of sequential crosslinking of MeMaHA using a primary addition and (nominally) secondary radical polymerization to create “−UV” and “D0 UV” hydrogels, respectively. c, ¹H NMR spectra (D₂O) showing uncrosslinked MeMaHA polymer, −UV and D0 UV hydrogels, respectively. d, Degradation kinetics of −UV and D0 UV hydrogels in the presence of 20 nM MMP-2. For all timepoints % HA release was greater from −UV relative to D0 UV gels (p < 0.01, t test). Error bars represent standard errors for the mean.

Figure 3

MeMaHA hydrogel structure-dependent hMSC matrix interactions & fate choice. a,f, Representative 3D TFM images of hMSCs, b,g, average drift-corrected bead displacements within 15 µm of the cell surface (*p < 0.001, t test), and c,h, average circularity of hMSCs within −UV or D0 UV cells (*p < 0.001, t test), following a–c, 7 days incubation in growth media or f–h, an additional 14 days incubation in mixed osteogenic/adipogenic media. d–e, Representative staining for hMSC vinculin (green), actin (red), and nuclei (blue) in d −UV and e D0 UV gels. i–j, hMSC differentiation following 14 d mixed media incubation. i(i),j(i), percentage differentiation of hMSCs toward osteogenic or adipogenic lineages in i(i) −UV or j(i) D0 UV hydrogels (^#p < 0.005, t test). i(ii–iii)–j(ii–iii), Representative bright field and fluorescent images of hMSCs; i(ii),j(ii), staining for ALP (osteogenesis) and lipid droplets (adipogenesis), or i(iii),j(iii), immunocytochemistry for osteocalcin (OC, osteogenesis) and fatty acid binding protein (FABP, adipogenesis) in i(ii–iii), −UV or j(ii–iii), D0 UV hydrogels, respectively. Error bars represent standard errors for the mean. Scale bars: a,f, 10 µm; d,e, 15 µm; i(ii),j(ii), 25 µm; i(iii),j(iii), 20 µm.

Figure 4

Delayed secondary crosslinking re-directs hMSC matrix interactions & fate choice without altering cell shape. a, Schematic of delayed UV exposure following 7 d growth media incubation. b(i),d(i), Representative TFM images, b(ii),d(ii), hydrogel deformations (^*p < 0.001, t test), and b(iii),d(iii) circularity of hMSCs within MeMaHA hydrogels following b, D7 UV exposure or d, D7 UV exposure and an additional 14 d mixed media incubation. c, HA release from D7 UV versus −UV hydrogels (normalized to total HA content) in the presence of 20 nM MMP-2. e(i), Representative brightfield image of a D7 UV hydrogel, with encapsulated hMSCs stained for ALP (osteogenesis) and lipid droplet (adipogenesis), and e(ii), relative frequency of lineage commitment within D7 UV hydrogels following 14 d mixed media incubation (^*p < 0.001, ^#p < 0.005, t test). Error bars represent standard errors for the mean. Scale bars: b,d, 25 µm; e(i),10 µm.

Figure 5

hMSC matrix interactions & lineage commitment upon pharmacologically induced changes in cytoskeletal tension. a, Cell-induced bead displacements (^*p < 0.001, ^#p < 0.005 relative to 2.5 wt%, t test) and b, circularity analysis of hMSCs within 2.5 wt% −UV MeMaHA gels following 7 d growth media incubation with or without daily 10 µM Y-27632, or within 1.5 wt% D7 UV MeMaHA gels plus one additional day growth media incubation. c–d, Representative TFM images of hMSCs within 2.5 wt%, −UV MeMaHA hydrogels following 7 d growth media incubation either c, without or d, with daily 10 µM Y-27632. e, Representative TFM image of an hMSC within a 1.5 wt%, D7 UV hydrogel. f, Cell-induced bead displacements (^*p < 0.001 relative to 2.5 wt%, t test), g, circularity analysis, and h, percentage differentiation fate of hMSCs toward osteogenic or adipogenic lineages within 2.5 wt% −UV MeMaHA gels following an additional 14 d mixed media incubation. i–j, Representative bright field images of these same groups stained for ALP (osteogenesis); lipid-containing cells (red arrows) appear yellow. Error bars represent standard errors for the mean. Scale bars: c–e, 25 µm; i–j, 50 µm.