Restoring Bone Regeneration in Critical Size Defects through Local Delivery of Angiogenic Factors

Critical size bone defects represent a significant challenge worldwide, often leading to persistent pain and physical disability that profoundly impact patients' quality of life and mental well-being.To address the intricate and complex repair processes involved in these defects, we performed single-cell RNA sequencing and revealednotable shifts in cellular populations within regenerative tissue. Specifically, we observed a decrease in progenitor lineage cells and endothelial cells, coupled with an increase in fibrotic lineage cells and pro-inflammatory cells within regenerative tissue. Furthermore, our analysis of differentially expressed genes and associated signaling pathway at the single-cell level highlighted impaired angiogenesis as a central pathway in critical size bone defects, notably influenced by reduction of Spp1 and Cxcl12 expression. This deficiency was particularly pronounced in progenitor lineage cells and myeloid lineage cells, underscoring its significance in the regeneration process. In response to these findings, we developed an innovative approach to enhance bone regeneration in critical size bone defects. Our fabrication process involves the integration of electrospun PCL fibers with electrosprayed PLGA microspheres carrying Spp1 and Cxcl12. This design allows for the gradual release of Spp1 and Cxcl12 in vitro and in vivo. To evaluate the efficacy of our approach, we applied locally delivered Spp1 and Cxcl12 embedded PCL scaffolds in a murine model of critical size bone defects. Our results demonstrated restored angiogenesis, accelerated bone regeneration, alleviated pain responses and improved mobility in treated mice. Overall design: The regenerative tissues from the defect gap were harvested at 3 weeks post-surgery and dissociated in 2 mg/mL Collagenase P and 2 mg/mL pronase at room temperature for 1 hour. The erythrocytes were removed using anti-GYPA and Streptavidin Microbubbles. Subsequently, the cell suspension was filtered through a 30 µm strainer to yield a single-cell suspension. Two sets of single-cell libraries were prepared: one from cells derived from a 1 mm bone defect (control, n=3) and the other from cells obtained from a 3 mm bone defect (nonunion, n=3). For each sample, ten thousand cells were loaded for processed to generate single-cell mRNA libraries using the Chromium Single Cell 3' kit (v3.1 Chemistry, 10× Genomics Inc). These libraries were barcoded, purified, and sequenced in a 2 × 150-bp paired-end configuration on an Illumina NovaSeq platform.