Distinct differences in hypoxic responses between human oral mucosa and skin fibroblasts in a 3D collagen matrix

Abstract

The differences between oral mucosa and skin wound healing involving hypoxic responses of fibroblasts are poorly elucidated. In this study, we aimed to study the different hypoxic responses between oral and skin fibroblasts embedded in a three-dimensional (3D) collagen matrix to address the early stage of wound healing. Primary oral mucosa fibroblasts (OMFs) obtained from the retromolar area and skin fibroblasts (SFs) obtained from the abdomen were cultured in the 3D ‘floating model’ under either 21%, 5% or 1% O₂ for 2 days. Cell viability under hypoxia was higher in the OMFs than in the SFs. Collagen gel contraction was suppressed under hypoxic conditions in both fibroblasts, consistent with the reduction of alpha smooth muscle actin expression, except for SFs under 1% O₂. Subsequently, their gene expression profiles between 21 and 1% O₂ concentrations were compared via microarray technology, and the expression profiles of the extracellular matrix (ECM)-associated proteins, including matrix metalloproteinases and collagens, were evaluated. The OMFs were more susceptible to 1% O₂, and more of their genes were downregulated than the SFs’. Although the production and expression levels of ECM-associated proteins in both fibroblasts diminished under hypoxia, those levels in OMFs were significantly higher than those in SFs. In the case of single origin OMFs and SFs, our findings suggest that OMFs possess a higher baseline production capacity of several ECM-associated proteins than SFs, except type III collagen. The intrinsic hypoxic responses of OMFs may be attributed to a more favourable wound healing in oral mucosa.

Appendices

Appendix 1

A list of 207 and 560 upregulated and downregulated genes in OMFs, a list of 62 and 43 upregulated and downregulated genes in SFs and a list of 62 and 47 upregulated and downregulated genes in both OMFs and SFs, at 1% O₂ concentration, respectively.

Table 1 Upregulated genes in OMFs

Table 2. Downregulated genes in OMFs

Table 3. Upregulated genes in SFs

Table 4. Downregulated genes in SFs

Table 5. Upregulated genes in OMFs and SFs

Table 6. Downregulated genes in OMFs and SFs

Appendix 2

Gene annotation enrichment analysis of hypoxia-upregulated genes in OMFs cultured in the ‘floating model’ was performed using DAVID Bioinformatics Resources 6.8. The biological process terms were extracted when the p-value was <0.01. The asterisks indicate the GO terms related to ECM remodelling, hypoxic response and cell viability. The annotation gene lists of these GO terms are shown in the following Appendix 6.

Table 7.

Appendix 3

Gene annotation enrichment analysis of hypoxia-downregulated genes in OMFs cultured in the ‘floating model’ was performed using DAVID Bioinformatics Resources 6.8. The biological process terms were extracted when the p-value was <0.01. The asterisks indicate the GO terms related to ECM remodelling, hypoxic response and cell viability. The annotation gene lists of these GO terms are shown in the following Appendix 7.

Table 8.

Appendix 4

Gene annotation enrichment analysis of hypoxia-upregulated genes in SFs cultured in the ‘floating model’ was performed using DAVID Bioinformatics Resources 6.8. The biological process terms were extracted when the p-value was <0.01. The asterisks indicate the GO terms related to ECM remodelling, hypoxic response and cell viability. The annotation gene lists of these GO terms are shown in the following Appendix 8.

Table 9.

Appendix 5

Gene annotation enrichment analysis of hypoxia-downregulated genes in SFs cultured in the ‘floating model’ was performed using DAVID Bioinformatics Resources 6.8. The biological process terms were extracted when the p-value was <0.01. The asterisks indicate the GO terms related to ECM remodelling, hypoxic response and cell viability. The annotation gene lists of these GO terms are shown in the following Appendix 9.

Table 10.

Appendix 6

Based on a functional annotation chart of hypoxia-upregulated genes in OMFs cultured in the ‘floating model’, genes related to ECM remodelling, hypoxic response and cell viability (indicated by eight asterisks in Appendix 2) were listed.

Table 11. Negative regulation of apoptotic process

Table 12. Apoptotic process

Table 13. Positive regulation of cell migration

Table 14. Cellular response to hypoxia

Table 15. Cell proliferation

Table 16. Regulation of apoptotic process

Table 17. Response to hypoxia

Table 18. Negative regulation of intrinsic apoptotic signaling pathway

Appendix 7

Based on a functional annotation chart of hypoxia-downregulated genes in OMFs cultured in the ‘floating model’, genes related to ECM remodelling, hypoxic response and cell viability (indicated by five asterisks in Appendix 3) were listed.

Table 19. Regulation of apoptotic process

Table 20. Extracellular matrix organization

Table 21. Apoptotic signaling pathway

Table 22. Apoptotic process

Table 23. Regulation of apoptotic process

Appendix 8

Based on a functional annotation chart of hypoxia-upregulated genes in SFs cultured in the ‘floating model’, genes related to hypoxic response (indicated by two asterisks in Appendix 4) were listed.

Table 24. Response to hypoxia

Table 25. Cellular response to hypoxia

Appendix 9

Based on a functional annotation chart of hypoxia-downregulated genes in SFs cultured in the ‘floating model’, genes related to ECM remodelling and cell viability (indicated by two asterisks in Appendix 5) were listed.

Table 26. Negative regulation of cell proliferation

Table 27. Positive regulation of collagen biosynthetic process