single cell RNA sequencing (scRNA-seq) of mouse first molar and adjacent alveolar bone tissues with and without orthodontic loading

Single-cell RNA sequencing (scRNA-seq) was performed on mouse first molar and adjacent alveolar bone tissues (within 1 mm, buccal and palatal sides). Two groups were analyzed: non-loaded control and orthodontic tooth movement (OTM) after 1 day. The study aims to reveal early cellular and transcriptional changes induced by orthodontic force. Overall design: Healthy C57BL/6J mice (6–8 weeks old, 50 g) were used in this study. A nickel–titanium coil spring was attached to the maxillary right first molar and maxillary incisors using a 0.010-inch stainless steel ligature to provide mesial orthodontic force (50 g constant force) on the right first molar. The contralateral (left) first molar, with springs attached but without force application, served as the control. On day 1 after force application, the upper right maxillary first molar was extracted following removal of the orthodontic appliance. Alveolar bone tissues within 1 mm of the extraction socket (buccal and palatal sides), with gingiva carefully removed, were immediately collected and rinsed in ice-cold PBS containing 1% FBS. Tissues from each condition were pooled, minced into 1–2 mm fragments, and digested with 2 mL GEXSCOPE® Bone Tissue Dissociation Solution at 37 °C for 60 min. The digested suspension was filtered and centrifuged, followed by removal of red blood cells using 2 mL GEXSCOPE® RBC lysis buffer at 25 °C for 10 min. Single-cell suspensions were then obtained and processed for scRNA-seq using the 10x Genomics platform.Samples were pooled from 10 mice for each condition prior to sequencing.