A distinctive patchy osteomalacia characterises Phospho1‐deficient mice

Abstract

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation.

Evaluation of laser ablation microtomy for correlative microscopy of hard tissues

“…LSs of femur and proximal tibia cut in a parasagittal plane are very common in our work with mouse genetic models. We have previously reported on the combined XMT, PMMA‐BSE‐SEM, macerated 3D SEM sample study of this material (Boyde et al ., ). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral or showed arrested mineralisation fronts, as failure of fusion of the separate mineralising microvolumes within bone.…”

“…For 3D characterisation, we used X‐ray microtomography. SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which had not been previously recognised (Boyde et al ., ).…”

“…Most blocks were re-imaged using BSE SEM after iodine staining -wet, using a triiodide solution, or dry, by iodine vapour to reveal cells and uncalcified tissue like cartilage, tendon, ligament, osteoid and muscle (Boyde, 2012;Boyde et al, 2014a,b;Ley et al, 2014). Many of the samples had also been studied by confocal fluorescence and reflection imaging (Boyde et al, 2005) and several by in-house highcontrast x-ray microtomography (XMT: Boyde et al, 2014a) or standard commercial microtomography (µCT: Boyde et al, 2017).…”

How To Build a Bone: PHOSPHO1, Biomineralization, and Beyond

“…Backscattered scanning electron microscopy (BSE‐SEM) furthermore revealed generalized hypomineralization relative to wild‐type mice in transverse tibial cross‐sections of the same animals . Further analysis of the Phospho1 –/– microstructure exposed diffuse regions of hypomineralization in diaphyseal cortical and trabecular bone with large areas of osteoid accumulation . Interestingly, there was no discernible anatomical pattern between individual Phospho1 –/– animals, but regions lacking mineral exhibited small focal areas of mineral nucleation at their borders, which failed to propagate more widely.…”

PHOSPHO1, a novel skeletal regulator of insulin resistance and obesity

“…An 76 alternative candidate is the bone-specific cytosolic phosphatase; Phosphatase, Orphan 1 (PHOSPHO1) (21)(22)(23)(24)(25)(26)(27)(28)(29)(30). 77 PHOSPHO1 initiates bone matrix mineralization and PHOSPHO1 deficiency causes significant skeletal pathology, 78 bowed long bones, osteomalacia and scoliosis in early life (31)(32)(33)(34). In addition to the role of PHOSPHO1 in skeletal 79 biomineralization, PHOSPHO1 has been implicated in the regulation of energy metabolism in humans (35)(36)(37)(38).…”