3.3 Effect of DP on macrophage polarization in vitro
CCK-8 analysis results showed that both BMSCs and RAW264.7 cells cultured on each scaffold material maintained similar proliferation activities, with no significant differences found among the groups (Figure 3A&B). Flow cytometric evaluation indicated upregulated expression of the M2-macrophage marker CD206 as well as downregulated expression of the M1-macrophage marker CD86 among RAW264.7 cells in the DP group (Figure 3C). qRT-PCR results further confirmed that the expression of the M2 macrophage-related gene ARG-1 was significantly higher, while that of the M1 macrophage-related geneiNOS was much lower in the DP group than in other two groups (Figure 3D). Additionally, similar trends in the secretion levels of cytokines IL-10 and TNF-α among the groups were confirmed by ELISA evaluation (Figure 3E).
The transcriptomes of RAW264.7 cells cultured on COL and DP scaffolds were further analyzed. Transcription of 3092 genes in these cells was altered after their culture on DP (|log2fold change| >1, false discovery rate [FDR] <0.05), of which 1405 genes were upregulated and 1687 were downregulated (Figure 3F&G). GO enrichment analysis showed that most of the over-expressed genes were associated with regulation of the inflammatory response, antigen processing and presentation, cytokine activity, the major histocompatibility complex (MHC) protein complex, as well as regulation of angiogenesis and vasculature development (Figure 3H). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the over-expressed pathways were associated with inflammatory modulation, including the p53 signaling pathway, NF-kappa B signaling pathway, and IL-17 signaling pathway (Figure 3I).