Melatonin accelerated angiogenesis activity of alginate-gelatin encapsulated endothelial progenitor cells in a mouse model of volumetric muscle loss

Volumetric muscle loss (VML) is a challenging clinical condition, resulting in the lack of appropriate function. The induction of angiomyogenesis can, in part, restore the function of injured sites using tissue engineering and stem cells. It has been documented that the combination of melatonin (Mel) and endothelial progenitor cells (EPCs) contributes to enhanced regeneration potential. Here, the angiogenesis properties of Mel-treated EPCs laden in alginate-gelatin (Alg-Gel) hydrogel were monitored in VML mice. Human EPCs were exposed to different doses of Mel, and the survival rate, NO levels, morphological changes, and angiogenesis potential were monitored using MTT, Griess assay, Transwell insert, PCR array, and western blotting in in vitro conditions. Using Mel-treated EPCs laden Alg-Gel hydrogel, the microvascular density and fibrosis were investigated using immunohistochemistry, immunofluorescence, and Masson’s trichrome staining. Data indicated dose-dependent activity of Mel on EPC viability compared to the control group after 5 days ( p < 0.05). In the presence of Mel, EPCs can produce non-toxic levels of NO, coincident with enhanced migration potential and morphological adaptation ( p < 0.05). The expression of genes such as VEGFR2 , VE-Cadherin , IGF1 , CCN1 , and FN1 related to migration and angiogenesis was upregulated in the presence of Mel ( p < 0.05). The increase of Ang-1, −2, VE-Cadherin, and vWF indicated maturation of EPCs toward mature vascular-like cells with angiocrine activity ( p < 0.05). Within the muscular tissue, EPCs exhibited potential to increase the microvascular density (vWF + and α-SMA + vessels) compared to sham and control mice ( p < 0.05). These features coincided with the reduction of fibrotic changes and collagen fiber deposition in mice that received Mel-treated EPCs ( p < 0.05). Immunofluorescence staining revealed differentiation of EPCs toward vWF + and α-SMA + -like cells, indicating promoted arteriogenesis and angiogenesis. These data highlight the superior role of Mel in the induction of EPC angiogenesis potential in VML mice.