Bone tissue characterization of a mouse model of atypical type VI osteogenesis imperfecta reveals hypermineralization of the bone matrix, elevated osteocyte lacunar density and altered vascularity

Introduction Osteogenesis imperfecta (OI) is a very heterogeneous connective tissue disorder characterized by low bone mass and high skeletal fragility. A common feature of almost all forms of osteogenesis imperfecta (OI) is a hypermineralized bone matrix. Null mutations in SERPINF1, encoding the potent antiangiogenic factor PEDF, cause type VI OI with excessive osteoid formation but concomitantly very elevated matrix mineralization. Recently, an extremely severe OI case has been reported with reduced PEDF secretion by osteoblasts, similar to OI type VI, but with normal PEDF serum levels. This atypical type VI OI was caused by a loss-of-function (p.S40L) in IFITM5 the causative gene for type V OI. Six cases have been reported since then, but the genotype-phenotype correlation is still unclear. To achieve further insights into the bone material properties at tissue level and micro meter scale, vascularization and thus pathophysiology of atypical type VI OI, we investigated a new knock-in (KI) mouse model carrying a leucine substitution for the BRIL p.Serine42 residue.

Methods We analyzed longitudinal sections of distal femurs of 8 weeks-old heterozygous male mutants (KI, n = 10) and wild-types (WT, n = 9) using quantitative backscattered electron imaging (qBEI) with a scanning electron microscope (DSM962, Zeiss). Bone mineralization density distribution (BMDD) was measured in cancellous metaphyseal bone and midshaft cortical bone. The qBEI images were used to evaluate the osteocyte lacunae sections (OLS) in cortical bone and the structural histomorphometric parameters in cancellous bone. We used X-ray microcomputed tomography (micro-CT) to evaluate vascularization in the femoral third trochanter.

Results qBEI revealed that bone matrix mineralization was significantly increased and more heterogenous in KI compared to WT cancellous (CaPeak: +2.38 %, P = 0.0331) and cortical bone (CaPeak: +2.81 %, P = 0.0085; CaMean: +2.48 %, P = 0.0023; CaWidth: +11.24 %, P < 0.0001, CaHigh: +51 %, P = 0.0027). We further observed in KI mice an increased OLS density (+23.11 %, P < 0.0001) and decreased OLS mean area and perimeter (-20.25 %, P < 0.0001; -13 %, P < 0.0001, respectively) versus WT. Histomorphometry revealed no changes of mineralized BV/TV, BS/TV, Tb.N and Tb.Th between the two genotypes. Micro-CT analyzes yielded increased pore volume/bone volume in KI (+14.28 %, P = 0.044) mirroring increased vascularity.

Discussion Our new mouse model for a typical type VI OI has elevated bone matrix mineralization as in other forms of OI. The increased bone vascularization is consistent with defective PEDF secretion in bone as reported in affected patients. Further analysis of osteoblasts function and osteoid formation, collagen orientation and mineral particle characterization will provide additional insights in atypical OI type VI.

Keywords Atypical OI type VI, Mineralization

Korrespondenzadresse Ghazal Hedjazi, Ludwig Boltzmann Institute of osteology, Osteology, Kundratstr. 37, 1120 Vienna Austria

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