| Recent development of healing technology has triggered the application of cells to improve wound healing. Mesenchymal stromal cells (MSCs) have played a dominant role in the strategy of cell therapy for wound healing. Adipose tissue is easy to obtain, making adipose-derived stem cells (ASCs) an ideal source for researchers and plastic surgeons. However, low cell engraftment of MSCs in the lesion has always been a major disadvantage of all kinds of diseases application of single cell suspension. Advances in the development of tissue engineering and biomaterial matrices have sparked improvement of cell delivery methods to augment the therapeutic potential of MSCs for sink wound. We previously developed a microcryogel fabrication technology to produce injectable mini scaffold which could provide cells with3D matrix environment cues. These injectable microcryogels are ideal carriers for site-targeted minimal invasive cell therapy. Here, we assume that GMs provide ASCs a suitable micro niche which may amplify the function of ASCs. Meanwhile, we hypothesis that ASCs loaded GMs could be injected to the deep layer of the wound as well as spray on the wound for multi-site and multi-layer contact and realize instant treatment effects through direct ASCs-lesion contact.Objective1. ASCs were seeded into microcryogels and compared to ASCs grown in standard culture conditions.2. An excisonal wound healing nude mice model was used to compare the ability of Microcryogels-ASCs constructs versus ASCs alone to accelerate wound healing.Results1. About65%of the total cells could finally linger in the GMs after1.5h of incubation no matter how the initial cell density differed.2. When load the cells at the density of1×107/ml, the proliferation rate showed difference at day7(P<0.05), indicating the massive inner space for cell proliferation in3D environment. 3. Apoptosis was induced by200umol/L or500umol/L hydrogen peroxide for4hrs at the second day of cell seeding. When the concentration is500umol/L, cells in the microcryogels were well protected (P<0.05).4. Sternness related genes Oct4and Nanog exhibited upregulation and showed differences between two groups2h and12h after cell seeding. SOX2showed difference12h after cell seeding (P<0.05).5. Gene expression of VEGF, bFGF and PDGFbb were significantly increased at48h with hASCs in GMs compared to2D culture. The expression of EGF showed no significant difference. These results were further confirmed by cytokine analysis in the conditioned medium after48-hours culture by ELISA. All of the four cytokines showed improved secretion at some time points with hASCs in GMs compared to2D cultur. The amount of cytokines in the supernatant also increased with time (P<0.05).6. Wounds treated with Microcryogels-ASCs showed significantly accelerated healing. Bioluminescence imaging and FACS analysis of luciferase+/GFP+hASCs indicated that stem cells delivered within the microcryogels remained viable longer and demonstrated enhanced engraftment efficiency.7. Wounds treated with Microcryogels-hASCs demonstrated significantly enhanced angiogenesis, which was associated with increased levels of angiogenic cytokines within the wounds.Conclusions1. These findings demonstrate the capacity of microcryogels to enhance MSC delivery to cutaneous wounds potentially by preserving cellematrix interactions, localizing cells within wounds and enhancing stem cell properties.2. Biomimetic microcryogels provide a functionalized niche for the in vivo delivery of MSCs which accelerates normal wound healing and promotes neovascularization. |
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