Single-cell RNA sequencing reveals early cell dynamics of MSC-based therapy in long bone critical-size defects in mice.

Background: Bone defects resulting from various causes present significant challenges in obtaining robust bone healing. This clinical scenario is particularly difficult in cases involving large bone defects, often leading to delayed union or non-union. Autogenous bone graft is the gold standard, but it is limited by the quantity and quality of available bone. Mesenchymal stem cells (MSCs) have shown promise in enhancing bone defect healing; however, the mechanisms by which MSCs modify the local bone microenvironment and interact with other cells early in the healing process are not fully understood. Elucidating and modulating the early biological events relevant to the healing of bone defects could lead to novel therapies to obtain a more expeditious and complete outcome. Methods: Critical-size femoral defects were created in 10 to 12-week-old BALB/c male mice and fixed with an external fixation device. Four weeks after the generation of the defect, secondary surgeries were performed. Mice were randomized into three groups based on the secondary surgery: Empty group - surgery was performed without implanting scaffolds or cells. Sc group - a 2 mm diameter cylindrical microribbon (μRB) scaffold was implanted into the defect site. Sc + MSC group - a scaffold embedded with MSCs was implanted into the bone defect site. One week after the secondary surgeries, the entire tissue within the bone defect site was harvested for single-cell RNA sequencing (scRNA-seq). Results: Uniform manifold approximation and projection (UMAP) plots with quality filtered cells from three groups were used to identify the cell distributions in the defects. We identified thirteen populations and annotated each cluster using UMAP with Louvain clustering on combined single cells of three groups based on marker gene expression. Different cell compositions were revealed, especially the proportion of various types of immune cells in the Sc vs Sc + MSC groups. MSCs and osteoblastic lineage cells (MSC/Osteo), and osteoclasts were almost exclusively found in the Sc + MSC group. Differential gene expression and pathway analysis in major cell populations identified immune cell changes and inflammatory changes in the presence of implanted MSCs. Cell-cell communications revealed a greater number of interactions between different cell types in the Sc and Sc + MSC groups. More interactions among MSCs, macrophages, and T cells were observed in Sc + MSC groups. MSC demonstrated the highest outgoing interaction strength in all groups. Conclusions: In the critical-size bone defect model, a combination of MSCs with μRB scaffolds showed an increased presence of mesenchymal lineage cells and promoted the further recruitment of macrophages and osteoclasts at 1 week. This alteration in the local immune landscape and microenvironment could enhance the cellular dynamics of critical cell populations that are important to osteogenesis. Optimizing this cellular crosstalk early in the healing process could potentially augment MSC-based therapies for subsequent bone regeneration of critical-size bone defects. The translational potential of this article: The results of our study provide a detailed transcriptional roadmap for local immune modulation by MSCs in scaffolds, supporting the optimization of robust strategies for MSC-based treatments of long bone critical-size defects in future clinical applications.