Ex vivo regional gene therapy is a promising tissue-engineering strategy for bone regeneration: osteogenic mesenchymal stem cells (MSCs) can be genetically modified to express an osteoinductive stimulus (e.g., bone morphogenetic protein-2), seeded onto an osteoconductive scaffold, and then implanted into a bone defect to exert a therapeutic effect. Compared to recombinant human BMP-2 (rhBMP-2), which is approved for clinical use, regional gene therapy may have unique benefits related to the addition of MSCs and the sustained release of BMP-2. However, the cellular and transcriptional mechanisms regulating the response to these two strategies for BMP-2 mediated bone regeneration are largely unknown. Here, for the first time, we performed single-cell RNA sequencing (10x Genomics) of hematoma tissue in six rats with critical-sized femoral defects that were treated with either regional gene therapy or rhBMP-2. Our unbiased bioinformatic analysis of 2393 filtered cells in each group revealed treatment-specific differences in their cellular composition, transcriptional profiles, and cellular communication patterns. Gene therapy treatment induced a more robust chondrogenic response, as well as a decrease in the proportion of fibroblasts and the expression of profibrotic pathways. Additionally, gene therapy was associated with an anti-inflammatory microenvironment; macrophages expressing canonical anti-inflammatory markers were more common in the gene therapy group. In contrast, pro-inflammatory markers were more highly expressed in the rhBMP-2 group. Collectively, the results of our study may offer insights into the unique pathways through which ex vivo regional gene therapy can augment bone regeneration compared to rhBMP-2. Furthermore, an improved understanding of the cellular pathways involved in segmental bone defect healing may allow for the further optimization of regional gene therapy or other bone repair strategies.

Osteonecrosis of the femoral head (ONFH) remains a devastating complication of glucocorticoid therapy and alcohol abuse, yet the pathophysiology is poorly understood. Using single-cell RNA sequencing, we analyzed femoral head specimens from patients undergoing total hip arthroplasty due to steroid-induced ONFH, alcohol-induced ONFH, or hip osteoarthritis (OA). Our comprehensive analysis across conditions revealed etiology-specific cellular compositions and transcriptional profiles. Steroid-induced ONFH demonstrated a distinctive immunological signature characterized by a neutrophil-predominant, elevated innate-to-adaptive immune cell ratio compared to alcohol-induced ONFH or OA. T cell analysis revealed impaired maturation in steroid-induced ONFH, with abundant naïve T cells but reduced mature CD4+ T cells and CD4+ memory T cells compared to both alcohol-induced ONFH and OA. In addition, compared to alcohol-induced ONFH, steroid-induced ONFH was associated with an increase in exhausted/dysfunctional CD8+ T cells which suggests an impaired response to stress and reflects chronic immune dysfunction. Within stromal cell populations, steroid-induced ONFH demonstrated an increase in pericytes, endothelial cells, and mesenchymal cell with osteogenic features as compared to both alcohol-induced ONFH or OA. These findings collectively reveal novel cellular mechanisms underlying steroid-induced ONFH which may contribute to further investigation of etiology-specific pathogenesis and treatment strategies. LAY SUMMARY: Osteonecrosis of the femoral head is a painful condition where bone tissue in the hip joint dies, often associated with steroid medications or alcohol use. We analyzed the gene expression of thousands of cells from hip tissue samples to understand how these two risk factors contribute to bone death, and how they compare to hip degeneration seen in osteoarthritis. Each condition is characterized by unique patterns of immune response and cellular dysfunction. Despite causing similar hip problems, steroids and alcohol may damage bone through unique biological pathways, suggesting that patients may benefit from different treatments depending on the underlying cause.