The cortical microenvironment drives early immune organization and controls early osteoclastogenesis in bone healing

We employed single-cell RNA sequencing using the 10x Genomics platform to investigate the cellular dynamics and early immune-stromal interactions during fracture healing in young and aged mice. Tissue-resident and infiltrating cell populations were collected from spatially defined bone marrow and cortical compartments from intact femurs as well as day 3 adn day 7 post-osteotomy. In totall, over 178,000 cells were profiled without prior enrichment or cell sorting, capturing the full diversity of immune and stromal cells involved in early bone regeneration. This dataset provides a comprehensive, unbiased atlas of the early fracture healing response and offers novel insights into the spatial regulation of osteoclastogenesis and the alterations with age. Overall design: Young (12-week-old) and aged (52-week-old) C57BL/6J mice underwent standardized femoral osteotomy and were sacrificed at day 3 adn day 7 post-osteotomy. Cells were isolated separately from the bone marrow and cortex compartments of the injured femur, using marrow flushing and enzymatic digestion, respectively. Samples from intact contralateral femurs and age-matched uninjured mice served as additional controls. A total of 30 mice were used to collect tissues (n=5, per age and timepoint). Single-cell suspensions were processed during the 10x Genomics Chromium platform with 3' v3 chemistry. To enable sample multiplexing, cells were stained with hashtag oligonucleotide (HTO) antibodies prior to pooling and library generation. Sample abbreviations: BM = Bone marrow, CB = Cortical bone, H = Hematoma.