Acceleration of burn wound healing by micronized amniotic membrane seeded with umbilical cord-derived mesenchymal stem cells

Abstract

Umbilical cord-derived mesenchymal stem cells (UC-MSC) are promising candidates for wound healing. However, the low amplification efficiency of MSC in vitro and their low survival rates after transplantation have limited their medical application. In this study, we fabricated a micronized amniotic membrane (mAM) as a microcarrier to amplify MSC in vitro and used mAM and MSC (mAM-MSC) complexes to repair burn wounds. Results showed that MSC could live and proliferate on mAM in a 3D culture system, exhibiting higher cell activity than in 2D culture. Transcriptome sequencing of MSC showed that the expression of growth factor-related, angiogenesis-related, and wound healing-related genes was significantly upregulated in mAM-MSC compared to traditional 2D-cultured MSC, which was verified via RT-qPCR. Gene ontology (GO) analysis of differentially expressed genes (DEGs) showed significant enrichment of terms related to cell proliferation, angiogenesis, cytokine activity, and wound healing in mAM-MSC. In a burn wound model of C57BL/6J mice, topical application of mAM-MSC significantly accelerated wound healing compared to MSC injection alone and was accompanied by longer survival of MSC and greater neovascularization in the wound.

Keywords: Amniotic membrane; Burn wound healing; Microcarrier; Umbilical cord-derived mesenchymal stem cells.

Graphical abstract

Fig. 1

Fabrication of mAM. (A) General view of the cleaned amniotic membrane. (B) DAPI images of amniotic membrane before and after decellularization. (C) Bright field images of mAM at different magnifications.

Fig. 2

Characteristics of mAM-MSC. (A) SEM images showing the morphology of mAM and mAM-MSC at different magnifications. Red arrows indicate microscopic MSC. (B) Immunofluorescence images of phalloidin indicating the cytoskeleton of MSC on mAM cultured for 3 and 7 days. (C) Comparing cell proliferation rate of MSC in traditional 2D plate and on mAM by CCK8 assay. (D) Live/dead staining of MSC seeded on mAM on day 1, 3 and 7 using fluorescence microscopy. ∗P ​< ​0.05 and ∗∗P ​< ​0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Bioinformatic analysis of RNA-seq. (A) Sample-to-Sample cluster analysis of sequencing samples in mAM-MSC group and MSC group; C1–C3 refer to three samples in MSC group, and S1–S3 refer to three samples in mAM-MSC group. (B) Statistical graph of DEGs in the two groups. (C) Heat map of gene expression levels of DEGs in the two groups. (D and E) The GO and KEGG enrichment analysis of DEGs of mAM-MSC group vs. MSC group. (F) GSEA based on RNA-Seq data. P value ​< ​0.05 and fold change >1.5 or fold change <0.5 was set as the criteria.

Fig. 4

Angiogenesis-related genes were significantly enriched in mAM-MSC group. (A) Heatmap of genes within some GO terms with angiogenesis functions according to GSEA results. (B) Heatmap of gene expression level of some growth factors based on RNA-seq analysis. (C) RT-qPCR identified the mRNA level of some factors. Error bars represent mean ​± ​SD; n ​= ​3 independent experiments. Significance was determined using t-test, ∗P ​< ​0.05, ∗∗P ​< ​0.01 and ∗∗∗P ​< ​0.001.

Fig. 5

Biological effects of mAM-MSC on HUVECs in vitro. (A) Representative images of transwell assay. (B) Representative images of tube formation assay. (C) Quantification of HUVECs migrated in each group. (D–E) Quantitative analysis of nodes and total length formed in the three groups. NC: high-glucose DMEM. ∗P ​< ​0.05, ∗∗P ​< ​0.01 and ∗∗∗P ​< ​0.001.

Fig. 6

mAM assisted survival of MSC on burn wound. (A) Representative bioluminescence images in two groups on day 0, 3, 7 and 9. MSC alone was injected into the left wound, and mAM-MSC was applied on the right. (B) Quantitative analysis of the photons on wounds in two groups on day 0, 3, 7 and 9. ∗P ​< ​0.05 and ∗∗P ​< ​0.01.

Fig. 7

mAM-MSC accelerated burn wound healing. (A) Schematic diagrams of the burn wound model and treatment. (B) Gross photos of the wound area in four groups on day 0, 3, 7 and 11; (C) Statistical column chart of the remaining wound area in each group. The data are presented as mean ​± ​SD. n ​= ​5. ∗∗∗P ​< ​0.001 and ∗∗∗∗P ​< ​0.0001.

Fig. 8

mAM-MSC accelerated burn wound healing by H&E staining results. (A) Images of H&E staining of the wound tissue sections in four groups on day 11. Black arrows referred to wound edges. Scale bar: 1.25 ​mm. (B) Quantitative analysis of wound edge on day 11. N ​= ​5. Significance was determined using one-way ANOVA. ∗∗∗∗P ​< ​0.0001.

Fig. 9

CD31 staining results and mRNA level of factors in vivo. (A–B) Images of immunohistochemical staining of CD31 of the wound tissue sections in four groups on day 11 and quantitative analysis of the capillary hpf. Error bars represent mean ​± ​SD; n ​= ​5. Significance was determined using one-way ANOVA. (C) RT-qPCR identifies the mRNA level of some factors of the wounds on day 7. Significance was determined using one-way ANOVA. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001 and ∗∗∗∗P ​< ​0.0001.