Gene expression profile at single cell level of alveolar bone during orthodontic tooth movement

We used single cell RNA sequencing (SCRNA-seq) to analyze the diffenrence in cell population in the alveolar bone before and after orthodntic tooth movement Healthy C57BL/6J mice (6-8 weeks old) weighing 20-25g were used. A nickel-titanium coil spring was attached to the maxillary right first molar and maxillary incisors via a 0.020-inch stainless steel ligature in order to provide mesial orthodontic force on the first molar. The constant force was maintained at 25g. For the control group, no force was applied after spring attachment.Twelve mice were randomly divided into the orthodontic tooth movement group and control group for scRNA-seq. On day 7 after force application, the upper right maxillary first molar was extracted following the removal of the orthodontic appliance. Alveolar bone within 1mm of the extraction socket was immediately collected and cleansed with ice-cold PBS containing 1% FBS. Alveolar bone was pooled from 6 mice per group. Tissue was minced into 1–2 mm size and digested with 2 ml GEXSCOPE® Bone Tissue Dissociation Solution for 60min at 37℃. Samples were then filtered and centrifuged. Single-cell suspensions were obtained after removal of red cells with 2 mL GEXSCOPE® red blood cell lysis buffer at 25°C for 10 min and later analyzed using scRNAseq.