Single-Cell Analysis of Molecular Mechanisms in Rapid Antler Osteogenesis During Growth and Ossification Stages

Antlers, as the only fully regenerable bone tissue in mammals, serve as an exceptional model for investigating bone growth, mineralization, articular cartilage repair, and the pathophysiology of osteoporosis. Nevertheless, the exact molecular mechanisms governing osteogenesis, particularly the dynamic cellular interactions and signaling pathways coordinating these processes, remain poorly characterized. This study used single-cell RNA sequencing (scRNA-seq) on the 10× Genomics Chromium platform, combined with bulk-RNA sequencing results, to comprehensively analyze molecular regulatory mechanisms in rapid antler osteogenesis. The results showed that eight cell types were identified in sika deer antler during the growth and ossification stages: mesenchymal, chondrocyte, osteoblast, pericyte, endothelial, monocyte/macrophage, osteoclast, and NK cells. Chondrocytes were predominantly found during the growth stage, while osteoblasts were more abundant during the ossification stage. Mesenchymal cells were subclassified into three subcategories: MSC_1 (<i>VCAN</i> and <i>SFRP2</i>), MSC_2 (<i>TOP2A</i>, <i>MKI67</i>), and MSC_3 (<i>LYVE1</i> and <i>TNN</i>). MSC_3 was predominantly present during the growth stage. During the growth stage, MSC_1 and MSC_2 upregulated genes related to vasculature development (<i>COL8A1</i>, <i>NRP1</i>) and cell differentiation (<i>PTN</i>, <i>SFRP2</i>). During the ossification stage, these subcategories upregulated genes involved in the positive regulation of p53 class mediator signal transduction (<i>RPL37</i>, <i>RPL23</i>, <i>RPS20</i>, and <i>RPL26</i>), osteoblast differentiation (<i>SPP1</i>, <i>IBSP</i>, <i>BGLAP</i>), and proton-motive ATP synthesis (<i>NDUFA7</i>, <i>NDUFB3</i>, <i>NDUFA3</i>, <i>NDUFB1</i>). Endothelial cells were categorized into five subpopulations: Enc_1 (<i>SPARCL1</i>, <i>VWF</i>), Enc_2 (<i>MCM5</i>), Enc_3 (<i>ASPM</i>, <i>MKI67</i>), Enc_4 (<i>SAT1</i>, <i>CXCL12</i>), and Enc_5 (<i>ZFHX4</i>, <i>COL6A3</i>). Combined scRNA-seq and bulk RNA-seq analysis revealed that the ossification stage's upregulation genes included osteoclast- and endothelial cell-specific genes, while the growth stage's upregulation genes were mainly linked to collagen organization, osteoblast differentiation, mitotic cell cycle, and chondrocyte differentiation. Overall, this study offers a detailed single-cell analysis of gene expression patterns in antlers during the growth and ossification stages, providing insights into the molecular mechanisms driving rapid osteogenesis.