Endosteal Bone Mineralisation In The Proximal Tibia of Mice With And Without Conditional E11 Knockout.

Abstract

Endosteal Bone Mineralisation In The Proximal Tibia of Mice With And Without Conditional E11 Knockout. A Boyde 1, *, KA Staines2, DJ Buttle3, AA Pitsillides4, C Farquharson2 1Oral Growth and Development Barts and The London School of Medicine and Dentistry QMUL, London, UK 2Roslin Institute and R(D)SVS, The University of Edinburgh, Easter Bush, Midlothian, UK 3Department of Infection and Immunity, The University of Sheffield, Sheffield, UK 4Comparative Biomedical Sciences, Royal Veterinary College, London, UK. Introduction The transmembrane glycoprotein E11 is expressed during early osteocyte commitment, but details of its role(s) in osteoblast to osteocyte transition have yet to be ascertained. Here, we studied whether E11 expression influences bone formation and mineralisation rates in regions where new bone forms rapidly at cortical endosteal surfaces in the proximal tibia, exploiting mice harbouring a conditional deletion of E11 in late osteoblasts. Methods Bones were obtained from 6 week-old ‘wild-type’ (Cre+/-E11wt/wt), ‘control’ (Cre-/-E11co/co) and ‘knockout’ (Cre+/-E11co/co) mice which had received intraperitoneal calcein injections at 9 days and 2 days prior to sacrifice. Tibio-fibulae were embedded in PMMA, and blocks finished to TS surfaces 3mm from the growth plate. Confocal fluorescence (CSLM) montage imaging used a Leica SPI inverted microscope, 10/0.3 objective, excitation 488nm, emission > 492nm. Double label intervals were measured using ImageJ. A 63/1.3 objective with 532nm excitation and > 534nm emission was used for detailed study of osteocytes and calcein distribution. Results and Conclusions Measured mineral apposition rates varied considerably around the tibial TS with, typically, endosteal formation in every sector. At 6 to 10µm/day, values were most consistent and uniform along the relatively flat postero-medial endosteum. Periosteal formation is normally only found at the medial edge and antero-medial surface. Using less efficient excitation of calcein at 532nm gave images which revealed labelled fronts to be composed of large numbers of spherical features consistent with matrix vesicle initiated mineralisation. We found no effect of E11 deletion on bone mineral apposition rates or osteocyte lacunar and canalicular morphology. Most of the rapidly formed endosteal bone is lamellar with more than one lamella formed per day. We present a new paradigm of bone formation wherein mineralisation is initiated within an ordered collagen matrix via matrix vesicles, contrasting with both woven bone and slowly forming mature lamellar bone.