Unique Spatial Transcriptomic Profiling of the Murine Femoral Fracture Callus: A Preliminary Report.

Fracture callus formation is a dynamic stage of bone activity and repair with precise, spatially localized gene expression. Metastatic breast cancer impairs fracture healing by disrupting bone homeostasis and imparting an altered genomic profile. Previous sequencing techniques such as single-cell RNA and in situ hybridization are limited by missing spatial context and low throughput, respectively. We present a preliminary approach using the Visium CytAssist spatial transcriptomics platform to provide the first spatially intact characterization of genetic expression changes within an orthopedic model of impaired fracture healing. Tissue slides prepared from BALB/c mice with or without MDA-MB-231 metastatic breast cancer cells were used. Both unsupervised clustering and histology-based annotations were performed to identify the hard callus, soft callus, and interzone for differential gene expression between the wild-type and pathological fracture model. The spatial transcriptomics platform successfully localized validated genes of the hard (Dmp1, Sost) and soft callus (Acan, Col2a1). The fibrous interzone was identified as a region of extensive genomic heterogeneity. MDA-MB-231 samples demonstrated downregulation of the critical bone matrix and structural regulators that may explain the weakened bone structure of pathological fractures. Spatial transcriptomics may represent a valuable tool in orthopedic research by providing temporal and spatial context.

Bone-derived extracellular matrix hydrogel from thrombospondin-2 knock-out mice for bone repair.

Bone extracellular matrix (ECM) has been shown to mimic aspects of the tissue's complex microenvironment, suggesting its potential role in promoting bone repair. However, current ECM-based therapies suffer from limitations such as inefficient scale-up, lack of mechanical integrity, and sub-optimal efficacy. Here, we fabricated hydrogels from decellularized ECM (dECM) from wild type (WT) and thrombospondin-2 knockout (TSP2KO) mouse bones. TSP2KO bone ECM hydrogel was found to have distinct mechanical properties and collagen fibril assembly from WT. Furthermore, TSP2KO hydrogel promoted mesenchymal stem cell (MSC) attachment, spreading, and invasion in vitro. Similarly, it promoted formation of tube-like structures by human umbilical vein endothelial cells (HUVECs). When applied to a murine calvarial defect model, TSP2KO hydrogel enhanced repair, in part, due to increased angiogenesis. Our study suggests the pro-angiogenic therapeutic potential of TSP2KO bone ECM hydrogel in bone repair. STATEMENT OF SIGNIFICANCE: The study describes the first successful preparation of a novel hydrogel made from decellularized mouse bones. Bones from wild-type mice and mice lacking thrombospondin 2 (TSP2) were used to fabricate the gels. Hydrogels from TSP2KO bones have unique characteristics in structure and biomechanics. These gels interacted well with cells in vitro and helped repair damaged bone in a mouse model. Therefore, TSP2KO bone-derived hydrogel has translational potential for accelerating repair of bone defects that are otherwise difficult to heal. This study not only creates a new material with promise for healing, but also validates tunability of native biomaterials by genetic engineering.