Growth Factor-Reinforced ECM Fabricated from Chemically Hypoxic MSC Sheet with Improved In Vivo Wound Repair Activity

Abstract

MSC treatment can promote cutaneous wound repair through multiple mechanisms, and paracrine mediators secreted by MSC are responsible for most of its therapeutic benefits. Recently, MSC sheet composed of live MSCs and their secreted ECMs was reported to promote wound healing; however, whether its ECM alone could accelerate wound closure remained unknown. In this study, Nc-ECM and Cc-ECM were prepared from nonconditioned and CoCl₂-conditioned MSC sheets, respectively, and their wound healing properties were evaluated in a mouse model of full-thickness skin defect. Our results showed that Nc-ECM can significantly promote wound repair through early adipocyte recruitment, rapid reepithelialization, enhanced granulation tissue growth, and augmented angiogenesis. Moreover, conditioning of MSC sheet with CoCl₂ dramatically enriched its ECM with collagen I, collagen III, TGF-β1, VEGF, and bFGF via activation of HIF-1α and hence remarkably improved its ECM's in vivo wound healing potency. All the Cc-ECM-treated wounds completely healed on day 7, while Nc-ECM-treated wounds healed about 85.0% ± 8.6%, and no-treatment wounds only healed 69.8% ± 9.6% (p < 0.05). Therefore, we believe that such growth factor-reinforced ECM fabricated from chemically hypoxic MSC sheet has the potential for clinical translation and will lead to a MSC-derived, cost-effective, bankable biomaterial for wound management.

Figure 1

Morphology of MSC sheets derived ECMs. Representative macroscopic images of Nc-ECMs and Cc-ECMs (a). The 7 mm diameter ECM dressings were stained with coomassie blue dye for 5 minutes (b).

Figure 2

PSR staining of MSC sheets and their derived ECMs. Imaging of MSC sheets and ECMs under light microscopy (a, c). Imaging of birefringent collagen through crossed polarization light microscopy. Large orange-red fibers were collagen I and thin blue-green filamentous fibers were collagen III (b, d). Scale bars represent 100 μm.

Figure 3

Western blot analysis of HIF-1α, collagens, and growth factors in MSC sheets and their derived ECMs. The proteins expressed in MSC sheets (a). The proteins expressed in their derived ECMs. The equal amounts of total protein in two groups were loaded for western blot analysis (b).

Figure 4

Effects of CoCl₂ on protein expression. The relative protein levels of HIF-1α, collagens, and growth factors in MSC sheets (a). The relative protein levels of collagens and growth factors in ECMs (b). ^∗p < 0.001, n = 8.

Figure 5

ECMs accelerated wound closure in a mouse model of full-thickness skin defect. Representative images of wound closure during a 7-day in vivo wound healing.

Figure 6

Quantitative analysis of wound closure. Wound closure over time was presented as percentage of healed wound area relative to initial wound area. ^∗p < 0.05; ^∗∗p < 0.001; n = 8.

Figure 7

Tissue infiltration and wound edge integration were enhanced with ECMs at day 7 postoperatively. PSR staining of wound edge (UD = uninjured dermis, GT = granulation tissue) (a). Imaging of birefringent collagen through crossed polarization light microscopy. Large orange-red fibers were collagen I and thin blue-green filamentous fibers were collagen III (b). Color maps of fiber orientations were performed using Orientation J software (c). Scale bars represent 200 μm.

Figure 8

Histological analysis of wound healing. Wound sections on 3, 5, and 7 days after operation were stained with H&E. Black arrows highlighted the epidermal tongue. Scale bars represent 1 mm.

Figure 9

ECMs accelerated granulation tissue formation and reepithelialization. Granulation tissue thickness of the center of wounds at day 7 postoperatively (a). Percentage of closure of the epidermal gap was evaluated at day 7 postoperatively (b). ^∗p < 0.05; ^∗∗p < 0.001; n = 8.

Figure 10

ECMs accelerated angiogenesis. High power field (HPF) (100x) imaging of the center in wound granulation tissues stained with MTC at day 7 postoperatively (a). Scale bars represent 200μm. Number of vessels per HPF at day 7 postoperatively (b). ^∗∗p < 0.001; n = 8.