Insulin-like growth factor binding protein 7 identified in aged dental pulp by single-cell RNA sequencing.

INTRODUCTION: Aging influences the regenerative and reparative functions of dental pulp, and an in-depth and complete understanding of aged dental pulp is highly important. OBJECTIVE: This study aimed to explore the heterogeneity of young and aged dental pulp tissue via single-cell RNA sequencing (scRNA-seq), search novel markers of aged dental pulp, and further explore their mechanism. METHODS: ScRNA-seq was employed to analyze the heterogeneity of young and aged dental pulp tissue, and immunohistochemical staining was used to detect new marker Insulin-like Growth Factor Binding Protein 7 (IGFBP7) in aged dental pulp. Differentially expressed genes (DEGs) between young and aged dental pulp tissue related with senescence-associated secretory phenotype (SASP) were validated in aging model of H(2)O(2)-induced dental pulp fibroblast (DPF). The effect of IGFBP7 on cellular senescence were validated by SA-β-Gal, γ-H2AX, and F-actin cytoskeletal staining. RNA-seq was used to analyze the mechanism of IGFBP7 alleviating senescence of H(2)O(2)-induced DPFs. RESULTS: A total of 32,012 cells were sequenced from 8 dental pulp samples and categorized into 8 main clusters, including fibroblasts (FB), endothelial cells, monocytes, T cells, B cells, mesenchymal stem cells, Schwann cells, and nonmyelinating ScCs. The ratio of fibroblasts was the highest, and FB1 was the largest subcluster of fibroblasts in the young group. In aged dental pulp, the ratio of fibroblasts was relatively low, and fibroblasts had more cellular communication with other cell types in fibroblast growth factor (FGF) and insulin-like growth factor (IGF) signal pathways. IGFBP7 was significantly upregulated in the aged group. Recombinant IGFBP7 reduced the senescence of H(2)O(2)-induced DPFs. CONCLUSIONS: These findings offer insights into the mechanisms of dental pulp aging and enhance our understanding of dental pulp at the single-cell level. Further comprehensive studies are required to clarify the exact mechanisms through which IGFBP7 influences dental pulp aging.