The effects of stepped structures of hydroxyapatite surface on the mRNA expression profile of immature dendritic cells

Hydroxyapatite (HA) has significant clinical promise for the repair of bone defects and the modification of implant material interfaces. However, after implantation, there are potential risks of chronic inflammation. Dendritic cells (DCs) are key cells in regulating chronic inflammatory responses, and their phenotype and functions are influenced by the surface structure of materials. Therefore, in order to provide some theoretical guidance for the optimal design of HA surface structures, this study tried to construct stepped structures on HA surfaces and investigate the effects of stepped structures on the cytoskeletal, cellular morphology, and mRNA expression profile of immature dendritic cells (imDCs). HA dishes with stepped structures were prepared utilizing the principle of oriented attachment growth, and subsequently used to construct in vitro cell culture models. The imDCs were observed by immunofluorescence staining to characterize their cytoskeleton structures and cellular morphology. Besides, transcriptome sequencing was used to examine the changes in the mRNA expression profile of imDCs. The results indicated that the stepped structures exposed on HA surfaces would increase the spread area of imDCs and affect the cell adhesion morphology. In addition, the results of the transcriptome sequencing revealed significant alterations in the mRNA expression profile, with differentially expressed genes (DEGs) predominantly enriched in the pathways associated with the focal adhesion signal pathway, the chemokine signal pathway, and the IL-17 signal pathway. Thus, the stepped structures could modulatethe cellular morphology and the immunological functions of imDCs by modulating the aforementioned signal pathways, including the focal adhesion pathway, the chemokine signaling pathway, and the IL-17 signaling pathway, thereby affecting the immune response process. ImDCs cultured on tissue culture plates（Control）and the surfaces of HA dishes (Nano and Meso) for 24 h were used as sequencing samples. The HA dishs with stepped structures were labled as "Meso," while the HA dishs without stepped structure were labled as Nano. The sequencing samples were sent to the BGI sequencing company for transcriptome sequencing. Total RNA of imDCs cutured on tissue culture plates and the different HA dishes was extracted and transcriptome sequencing was performed. Go and KEGG enrichment analysis of differential expressed genes (DEGs) was performed. Transcripts related to Cellular immunological function were screened according to the changes of differential gene expression ≥1 and FDR≤0.05. In addition, signaling pathways linked to the immune response were explored.