Abstract:

BACKGROUND: Studies have shown that mesenchymal stem cells can reduce inflammation, promote wound healing, and reduce scar formation in wound healing. However, previous two-dimensional culture environment can lead to differences in gene expression, signal transduction, and morphology because of intracellular contact inhibition. 

OBJECTIVE: To investigate whether the ability of wound healing related factors secreted by human amniotic mesenchymal stem cells is affected by two-dimensional or three-dimensional culture environment.

METHODS: The human amniotic mesenchymal stem cells cultured by traditional enzyme digestion method were inoculated in traditional cell culture flask (two-dimensional culture group) and ShakeGel^TM 3D hydrogel (three-dimensional culture group) and induced to differentiate into adipocytes, osteoblasts, and chondrocytes, respectively. The direction of cell differentiation was determined by immunofluorescence staining. Human amniotic mesenchymal stem cells were fused to 70%-80% in two culture environments, and the growth characteristics and morphology of cells were observed under inverted phase contrast microscope and laser confocal microscope. After 24 hours of culture, relative mRNA expression of wound healing-related factors was detected by reverse transcription-quantitative polymerase chain reaction. After 48 hours of culture, the protein expression of wound healing-related factors was detected by the enzyme-linked immunosorbent assay.

RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells cultured in two-dimensional culture group were flat and spindle-shaped, which was a typical mesenchymal stem cell-like morphology. Human amniotic mesenchymal stem cells in the three-dimensional culture group were round and evenly dispersed in each layer of the hydrogel. (2) Human amniotic mesenchymal stem cells in the three-dimensional culture group exhibited the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. (3) The mRNA expression of interleukin-6, interleukin-8, epidermal growth factor, basic fibroblast growth factor, hyaluronic acid, hepatocyte growth factor, and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture group (P < 0.001, P < 0.05). There were no significant differences in the mRNA expression of interleukin-4, interleukin-10, tissue inhibitors of metalloproteinases, matrix metalloproteinase, transforming growth factor and keratinocyte growth factor between three-dimensional and two-dimensional culture groups (P > 0.05). (4) The protein expression of interleukin-6, interleukin-8, interleukin-10, epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor β1 and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture (P < 0.001, P < 0.01, P < 0.05). There were no significant differences in the protein expression of interleukin-4, tissue inhibitors of metalloproteinases, matrix metalloproteinase, and keratinocyte growth factor between two-dimensional and three-dimensional culture groups (P > 0.05). (5) These findings suggest that human amniotic mesenchymal stem cells cultured in three-dimensional hydrogel show better morphology and more encouraging paracrine effect of wound healing related factors than those cultured in traditional two-dimensional culture environment. 

Key words: hydrogel, amniotic mesenchymal stem cells, two-dimensional culture, three-dimensional culture, wound repair, paracrine effect