Thrombospondin-2 deficiency in growing mice alters bone collagen ultrastructure and leads to a brittle bone phenotype

Abstract

Thrombospondin-2 (TSP2) is a matricellular protein component of the bone extracellular matrix. Long bones of adult TSP2-deficient mice have increased endosteal bone thickness due to expansion of the osteoblast progenitor cell pool, and these cells display deficits in osteoblastic potential. Here, we investigated the effects of TSP2 deficiency on whole bone geometric and mechanical properties in growing 6-wk-old male and female wild-type and TSP2-knockout (KO) mice. Microcomputed tomography and mechanical testing were conducted on femora and L2 vertebrae to assess morphology and whole bone mechanical properties. In a second series of experiments, femoral diaphyses were harvested from wild-type and TSP2-KO mice. Detergent-soluble type I collagen content was determined by Western blot of right femora. Total collagen content was determined by hydroxyproline analysis of left femora. In a third series of experiments, cortical bone was dissected from the anterior and posterior aspects of the femoral middiaphysis and imaged by transmission electron microscopy to visualize collagen fibrils. Microcomputed tomography revealed minimal structural effects of TSP2 deficiency. TSP2 deficiency imparted a brittle phenotype on cortical bone. Femoral tissue mineral density was not affected by TSP2 deficiency. Instead, transmission electron microscopy revealed less intensely stained collagen fibrils with altered morphology in the extracellular matrix assembled by osteoblasts on the anterior surface of TSP2-KO femora. Femoral diaphyseal bone displayed comparable amounts of total collagen, but the TSP2-KO bones had higher levels of detergent-extractable type I collagen. Together, our data suggest that TSP2 is required for optimal collagen fibrillogenesis in bone and thereby contributes to normal skeletal tissue quality.

Teriparatide Treatment Improves Bone Defect Healing Via Anabolic Effects on New Bone Formation and Non-Anabolic Effects on Inhibition of Mast Cells in a Murine Cranial Window Model

“…Olfactory receptor 56 Thbs2 a Thrombospondin 2, regulation of bone collagen ultrastructure, (35,36) regulation on angiogenic cell function (37)…”

Thrombospondins 1 and 2 affect lysyl oxidase protein and collagen matrix maturation in cortical bone of growing male and female mice via non-redundant pathways

“…Data presented here suggest that TSP2-dependent regulation of LOX contributes, at least in part to the phenotype of TSP2-/-osteoblasts and bone tissue (10,11). Thus, LOPP accounted for a higher percentage of total soluble LOX content in TSP2-/-cultures.…”

“…More robust removal of the LOX-pro-peptide suggests increased LOX activation under conditions of TSP-2 deficiency. However, this possibility seems unlikely given that TSP2-/-osteoblasts and bone ECM display reductions in insoluble cross-linked collagen content ( Figure 5), as well as deficits in collagen fibrillogenesis and matrix maturation (10,11). Alternatively, TSP2 might affect the fate or function of the liberated LOPP (26,28) or facilitate functional interactions between mature active LOX and collagen.…”

“…Detergent-soluble proteins were extracted as we have described (7,11). Briefly, bone tissues were crushed and homogenized on ice with a polytron in lysis buffer containing 50 mM Tris, 150 mM NaCl, 2mM EGTA, 10% NP40, 10 mM Na pyrophosphate decahydrate plus a protease inhibitor cocktail of 1mM PMSF, 5 mM sodium fluoride, 1 mM sodium orthovanadate and the Complete Protease Inhibitor Cocktail (Roche).…”

“…When they are cultured under osteogenic conditions, primary marrow-derived mesenchymal stem cells (MSC) harvested from TSP2-/-mice assemble a mature, insoluble ECM with reduced total collagen content (10). In femoral diaphysis of growing TSP2-/-mice, collagen fibril morphology is altered and detergent soluble type I collagen content is elevated despite WT equivalent total hydroxyproline content (11). These data suggest that TSP2 facilitates collagen matrix maturation and point to the possibility of impaired collagen crosslinking in the TSP2-deficient osteoblast ECM.…”