MULTIDIMENSIONAL SPATIAL MAPPING OF EXTRACELLULAR MATRIX: CARTILAGINOUS-OSSEOUS COMPOSITE FORMATION, TENDON INTEGRATION AND VASCULARIZATION DURING SKELETAL GROWTH AND REPAIR.

Abstract

The extracellular matrix provides a crucial tissue-specific signaling hub and structural scaffold that transduces biomechanical force load into cellular responses. While this is especially important in the musculoskeletal joint, the spatial organization of extracellular matrix (ECM) assemblies has been poorly investigated. Dense supra-structures, inaccessible epitopes and complex antigen retrieval negatively affect the 3D-visualization of ECM-scaffolds in connective tissues. We have developed multiplex ECM immuno-staining techniques that now permit mapping of the entire musculoskeletal ECM with embedded cells during skeletal growth and repair. Specifically, we show that osteoblasts deposit a bony ECM on the cartilaginous template in the hypertrophic growth plate, creating a unique ECM composite that likely confers both stiffness and flexibility to neonatal bone. We demonstrate that tendon collagen I fibrils anchor along a thin, continuous tidemark at the lateral cartilaginous surface, mechanically coupling cartilage and tendon matrices at contact sites. Finally, we reveal muscle-derived laminin γ1(+) vascular basement membrane infiltration of the cartilaginous fracture callus ECM, likely facilitating nutrient delivery and structural stabilization during skeletal repair. These advances provide insight into the complexity of the ECM and its interplay with embedded cell clusters at previously unattainable spatial resolution and biological context during skeletal growth and repair.