Abstract:

Objective

To observe the effect of gelatin methacryloyl hydrogel loaded with exosomes from human adipose-derived stem cells on the proliferation and migration of human skin fibroblasts.

Methods

Human adipose-derived stem cells were isolated and cultured， and the expression of molecular markers on the surface was detected.Human adipose stem cell-derived exosomes were extracted by ultracentrifugation and identified by scanning electron microscopy and Nano-flow cytometry.Human skin fibroblasts were cultured， and immunofluorescence was performed to detect the internalization of the exosomes.The exosomes were added at concentrations of 1×10⁷/ml， 1×10⁸/ml， and 1×10⁹/ml espectively to detect the effects of exosomes on the proliferation and migration of human skin fibroblasts.GelMA hydrogels were prepared at concentrations of 5%，10%，and 15%， and the microstructures of the hydrogels were observed by scanning electron microscopy and pore size was analyzed.The in vitro degradation rate of the hydrogels was measured.ELISA was used to detect the sustained-release rate of exosomes in the hydrogel.Human skin fibroblasts were co-cultured in the hydrogel， and live-dead cell staining was performed to detect the growth and proliferation of cells in the hydrogel.

Results

The electron microscopy and nanofluidic detection showed that the acquired particles were exosomes.Fluorescently labeled human adipose stem cell-derived exosomes could be internalized by human skin fibroblasts into the cytoplasm and nucleus.The concentrations of 1×10⁷/ml，1×10⁸/ml， and 1×10⁹/ml of exosomes all promoted the proliferation and migration of human skin fibroblasts（F=3.579，P=0.021）.For proliferation， the effect of exosomes at concentrations of 1×10⁸/ml and 1×10⁹/ml was more substantial than that of 1×10⁷/ml （P=0.048，P=0.005）.After 24 hours of treatment， there was no statistically significant difference in the effect of exosomes at concentrations of 1×10⁸/ml and 1×10⁹/ml on cell proliferation （P = 0.091， P = 0.083）.The in vitro degradation rate of GelMA hydrogel at concentrations of 5%， 10% and 15% decreased with the increase of concentration， the hydrogel at a concentration of 5% was better in releasing the exosomes in a slow and sustained manner.After culturing human skin fibroblasts in the 5% GelMA hydrogel， the cells grew and proliferated favorably in the hydrogel.

Conclusion

Human skin fibroblasts can internalize exosomes from human adipose-derived stem cells to promote their proliferation and migration.GelMA hydrogel with a concentration of 5% and loaded with exosomes from human adipose-derived stem cells （1×10⁸/ml） can release exosomes slowly and continuously to promote the proliferation of human skin fibroblasts， and it can be used as a good bio-carrier for exosomes.

Key words: Adipose-derived stem cells, Exosomes, Gelatin methacryloyl, Hydrogel, Human skin fibroblasts