A Mechanistic Investigation into Bone Bridge Formation Post-Growth Plate Injury in Rats Utilizing Single-cell Transcriptomic Sequencing and Its Relevance for Hydrogel Development in Bone Defect Repair

Introduction: Growth plate injury (GPI) can result in bone bridge formation, leading to limb length discrepancy and angular deformities in children. Bone bridge formation disrupts the normal structure of the growth plate and prematurely arrests longitudinal bone growth. However, the cellular heterogeneity and molecular mechanisms underlying this pathological process remain poorly understood. Recent advances in single-cell RNA sequencing (scRNA-seq) enable detailed characterization of complex tissue microenvironments and intercellular communication during tissue repair. Therefore, this study aimed to investigate the cellular composition and molecular mechanisms underlying bone bridge formation after growth plate injury and to explore potential therapeutic strategies based on these findings. Methods: A rat distal femoral growth plate injury model was established to investigate morphological and histological changes during bone bridge formation. ScRNA-seq was performed to characterize cellular composition and transcriptional profiles of bone bridge tissue and normal growth plate cartilage. Bioinformatic analyses, including cell clustering, differential gene expression, pathway enrichment, cell–cell communication analysis, and pseudotime trajectory analysis, were conducted to identify regulatory networks involved in bone bridge formation. Immunofluorescence staining was performed to validate Type H vessels distribution after injury. In addition, in vitro assays were conducted to evaluate the effects of Wnt5a on Type H endothelial cells proliferation, migration, and tube formation. Based on the mechanistic findings, an injectable thermosensitive hydrogel composed of hyaluronic acid, poloxamer 407, and tannic acid was developed as a potential drug delivery platform for Bone Defected. Results: ScRNA-seq revealed a complex cellular micro-environment in bone bridge tissue, including chondrocytes, osteoblasts, skeletal stem cells, Type H endothelial cells, and immune-related populations. Both endochondral and intramembranous ossification participate in the formation of the bone bridge. In addition, Type H endothelial cells, played a critical role in vascular–osteogenic coupling during bone bridge formation. Immunofluorescence staining further confirmed that after growth plate injury, Type H vessels from the metaphysis invaded the injury site and exhibited spatial proximity to newly formed bone bridge tissue. Cell–cell communication analysis indicated that skeletal stem cells secreted Wnt5a, which interacted with Mcam and Fzd6 receptors on Type H endothelial cells, suggesting Wnt5a signaling as a key regulator of endothelial activation. In vitro experiments further demonstrated that Wnt5a significantly promoted Type H endothelial cells proliferation, migration, and tube formation. Inspired by the pro-angiogenic activity of Wnt5a, an injectable thermosensitive hydrogel was developed, showing good injectability and stability. SEM analysis revealed a three-dimensional crosslinked network structure conducive to sustained drug release and cell ingrowth. Conclusion: This study provides a single-cell transcriptomic perspective on bone bridge formation following growth plate injury and highlights Type H endothelial cells, played a critical role in vascular–osteogenic coupling during bone bridge formation. Furthermore, inspired by the pro-angiogenic activity of Wnt5a, the injectable thermosensitive hydrogel offers a promising biomaterial platform for targeted therapeutic delivery and may provide a potential strategy for improving bone defected repair. Keywords: Growth Plate Injury; Bone Bridge Formation; Single-cell RNA-seq; Type H vessels; Wnt5a Signaling; Foxy5 peptide; Injectable Hydrogel; Bone Defected.