Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation

Abstract

The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

Figure 1. Fabrication and characterization of void-forming hydrogels

(a). Schematic of the strategy to create void-forming hydrogels. Porogens (red) and mesenchymal stem cells (green) are co-encapsulated into a bulk hydrogel (grey). Pores (white) form within the intact bulk hydrogel due to porogen degradation, allowing cell deployment out of the material and into damaged tissues. Note that the rate of cell migration out of the material is expected to be a function of the distance of the cells from the newly formed pores. (b-e). Characterization of void-forming hydrogels. (b). Confocal micrographs of aminofluorescein-labeled porogens (green) within a rhodamine labeled bulk gel (red), over the time-course of porogen degradation. (c-d). Quantative analysis of the total level of fluorescein, proportional to porogen density, either (c) remaining within gels, or (d) dissolved into media bathing gels. Gels were dissolved into EDTA at set time points to quantify remaining label. (e). Relative shear modulus G’ of void-forming hydrogels as a function of volume fraction of porogen, 1 week after hydrogel fabrication. Values of G’ are normalized to the value obtained for a standard hydrogel (no porogen) at day 1. Effects of porogen volume fraction on composite shear modulus were significant (p < 0.05, 1-way ANOVA). (f). Morphology of Calcein-AM stained mMSC in standard hydrogels (top) or in void-forming hydrogels (bottom) at day 4 and 10 after encapsulation (dotted blue line denotes void location). To right: 3D projections of Calcein-AM stained cells within either standard gels (top) or void-forming gels (bottom) after 40 days of in vitro culture. (g). Representative confocal micrograph of mMSC stained with phalloidin (green, with Hoescht 33342 nuclear counterstain, blue) in situ within standard (top) and void forming gel (bottom) at day 7. (h) Representative micrographs depicting Ki67 expression (green, with Hoescht 33342 nuclear counterstain, blue) in 10µm cryosections of mMSC in either standard gels (top) or void-forming gels (bottom) at day 7. (i) 24 hr ³H-thymidine incorporation by mMSC either in standard gels or void-forming hydrogels 1 week after encapsulation. (j) 24 hr ³H-thymidine incorporation by mMSC in void-forming hydrogels wherein the bulk component had a varied level of integrin-binding RGD peptides. RGD density had a significant effect on ³H-incoproration (1-way ANOVA). Error bars are SD, n = 4 scaffolds. * p < 0.05, **** p < 0.001, 2-tailed t-test. Scale bars: b,c: 1mm (b inset: 200µm); f,h: 100µm; g: 20µm.

Figure 2. Manipulating Stem Cell Osteogenesis and Proliferation by Controlling the Elasticity of the Bulk Phase of Void-Forming Hydrogels

(a). ³H-thymidine incorporation in last 24 hr by mMSC, after 7 days of culture in void-forming hydrogels, as a function of the elastic modulus of the bulk component. RGD density of gels was constant (375µM). (b). Analysis of Alkaline Phosphatase (ALP; osteogenic biomarker) activity, normalized to the DNA density of mMSC deployed from void forming hydrogels of varying bulk elastic modulus (375µM RGD). Analysis was performed on cells that were released between days 7 and 14 of culture under osteogenic conditions. Bulk gel elasticity had significant effects (one-way ANOVA) on both proliferation and ALP activity. (c-d). Immunoblot analysis of MAPK phosphorylation (anti Phospho-p44/42 MAPK, Thr202/Tyr204) and total MAPK expression for mMSC within void-forming hydrogels as a function of bulk component elasticity, 7 days after gel formation, as depicted by (c) a representative blot and (d) quantitative analysis. GAPDH was used as a loading control for Western Blots. (e-h). Analysis of (e,f-g,h) Collagen I expression (green; Hoescht 33342 nuclear counterstain, blue) via antibody staining, and analysis of mineralization (f) via Von Kossa staining, of mMSC within void-forming hydrogels of varying bulk component elasticity, after 14 days of culture under osteogenic conditions. (g-h). Quantification of average Collagen I fluorescence signal from 16 cellular regions within the material either (g) without or (h) with normalization to the number of nuclei in each region. Matrix elasticity had a significant effect on Collagen I levels (ANOVA). Error bars: SD, n = 3–5 biologic replicates. * p < 0.05, compared to 5 kPa condition, Holm Bonferroni test, or p < 0.05 by 2-way t-test with Holm Bonferonni correction for multiple comparisons. Scale bars: d,e: 400µm.

Figure 3. Controlling cell deployment kinetics from void-forming hydrogels in vitro and in vivo

(a). Kinetic analysis of murine mesenchymal stem cell (mMSC) deployment either from () the bulk phase of void-forming hydrogels, or from standard nanoporous hydrogels (▲).The black line denotes the number of cells initially encapsulated into each scaffold. Difference in net cell deployment between the two types of hydrogels was statistically significant (p < 0.01, 2-tailed t-test) at all time-points. (b-c). Kinetics of mMSC deployment from the bulk phase of void-forming hydrogels as a function of porogen degradation rate, as manipulated by controlling (b) the degree of oxidation of polymers used to form porogens (3% (▲), 5% () or 7.5% ()), or (c) the concentration of calcium (25mM (●), 50mM () or 100mM ()) used to crosslink porogens. Net cell deployment was significantly greater from materials with porogens comprised by 7.5% degree of oxidation (p < 0.001 at all time points after day 0 by Holm-Bonferonni) compared to deployment from materials with either 3 or 5% degree of oxidation. Each degree of porogen crosslinking yielded a level of net deployment that was statistically unique amongst the different materials tested at all time points after day 0 (Holm-Bonferonni test). (d) Analysis of net mMSC deployment at day 7, as a function of the elasticity of bulk component of void-forming gels. Elasticity had a significant effect (1-way ANOVA) on cell deployment. (e). Cumulative cell deployment for mMSC after 1 week of culture in void-forming gels with varying density of RGD peptides. (f). Representative images of Nu/J mice either 7 (top) or 30 (bottom) days after injection of standard (left) or pore-forming hydrogels (right) containing mCherry-expressing mMSC into the subcutaneous tissues of Nu/J mice. (g). Total radiant efficiency (proportional to cell number) from mCherry-mMSC injected within the following hydrogels: void-forming gels with porogens crosslinked with either 100mM () or 50mM () Ca²⁺, or within standard hydrogels (▲). Release of cells from either void-forming hydrogel yielded significantly more release than from a standard hydrogel at all time-points beginning at day 10, and altering porogen fabrication yielded a significant effect on radiant efficiency beginning on day 17 (p < 0.05, 2-tailed t-test). RGD density was fixed at 187µM in the bulk gel phase. (h). Total radiant efficiency resulting from mCherry-mMSC injected within void-forming hydrogels in which the RGD concentration was either ()187µM or () 750µM within the bulk phase, or within standard hydrogels (▲). Cell transplantation within either void-forming hydrogel type led to substantially higher total radiant efficiency at all time points after day 12 (187µM RGD) or 19 (750µM RGD), compared to transplantation within standard hydrogels. The difference in total radiant efficiency was affected by the density of RGD presented by the bulk phase beginning on day 24 (p < 0.05). (i-j) Representative micrographs of tissues in Nude rat cranial defects one week after transplanting mCherry-mMSC with either i) void-forming or j) standard hydrogels. mCherry antigen was probed with DAB chromogen. Error bars are SEM, n = 3–4 scaffolds (in vitro studies) or n = 4–8 scaffolds (in vivo studies). Scale bar: g,h: 100µm.

Figure 4. Matrix elasticity regulates mesenchymal stem cell mediated bone regeneration

(a-b). Analysis of bone regeneration in cranial defects due to hMSC transplantation via saline bolus, standard hydrogels or void-forming hydrogels. (a). Representative Micro-Computed Tomographic (µCT) images of regeneration in cranial defects in nude rats 12 weeks after introducing human MSC (hMSC) in saline (Cells Alone), within standard hydrogels or within void-forming hydrogels. (b). Quantitative analysis of the total volume of newly formed bone tissue using µCT. (c-j). Analysis of bone regeneration in cranial defects due to hMSC transplanted in void-forming hydrogels with different bulk-component elastic moduli. (c). Representative Micro-Computed Tomographic (µCT) images of regeneration in nude rat cranial defects 12 weeks after hMSC delivery in void-forming hydrogels of varying bulk component moduli. (d-e). Quantitative analysis of (d) total volume and (e) average bone mineral density of regenerated bone. (f). Representative histologic analysis of new bone formation and remaining polymer with Hematoxyln-Eosin staining. (g). Representative Masson’s Trichrome staining depicting new bone formation. (h-j). High resolution micrographs depicting trichrome staining of a portion of newly regenerated tissue derived from hMSC transplanted in void forming gels with a bulk modulus of 60 kPa. (h) Entire sub-section. (i-j). High resolution images depicting (i) osteoblast-like cells at the edge of newly forming tissue, and (j) osteocyte-like cells in the central part of newly formed tissues (denoted by black arrows). Error bars: SD, n = 4–5. ** p < 0.05, 2-tailed t-test. Scale bars: f-h: 100µm; i-j: 20µm.