Detached osteoclasts in the granulation tissue surrounding sequestra in medication-related osteonecrosis of the jaw

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious complication of anti-resorptive agents used for osteoporosis and bone metastasis. As the global population ages, the number of patients with MRONJ continues to rise. Resorptive osteoclasts firmly attached to bone surfaces cannot be detected using single-cell RNA-Seq (scRNA-Seq); however, we hypothesized that multinucleated giant and detached osteoclasts (DetOCs) can be detected by this method. scRNA-Seq was performed on granulation tissues surrounding sequestra from four patients with MRONJ and radicular cysts, a common odontogenic infectious disease with normal bone metabolism. Our results show that DetOCs were detected exclusively in the granulation tissues of MRONJ. These DetOCs expressed well-known osteoclast markers as well as COL27A1. Subcluster analysis revealed that SPP1+ TREM2+ macrophages differentiated into DetOCs and expressed several types of collagens. Immunostaining confirmed the expression of COL27A1 in DetOCs in MRONJ, as well as in osteoclasts in mandibular cancer. Overall, we successfully identified DetOCs in the granulation tissues surrounding sequestra in MRONJ, and our scRNA-Seq analysis suggests the potential to classify osteoclast subtypes based on the expression of collagen types. Overall design: Single-cell gene expression profiling was performed using the Chromium Fixed RNA Profiling workflow (CG000527; 10x Genomics). Library preparation involved the Chromium Fixed RNA Kit (Human Transcriptome) and the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit. Following hybridization of detection probes to RNA within fixed cells, adjacent probes were joined through a ligation reaction. Samples were then pooled to normalize cell counts across groups and processed on the Chromium X instrument, where cells were encapsulated with barcoded primer beads. Annealing and extension reactions added cell-identifying adapter sequences, and indexed primers were used for PCR amplification and library purification, generating sequencing-ready libraries.