Heterogeneity of the osteocyte lacuno-canalicular network architecture and material characteristics across different tissue types in healing bone

Various tissue types, including fibrous connective tissue, bone marrow, cartilage, woven and lamellar bone coexist in healing bone. Similar to all bone tissue type, healing bone contains a lacuno-canalicular network (LCN) housing osteocytes that are known to orchestrate bone remodeling in healthy bone by sensing mechanical strains and translating them into biochemical signals. The structure of the LCN is also hypothesized to influence mineralization processes. Hence, the aim of the present study was to visualize and correlate spatial variations in the LCN topology with mineral characteristics, within and at the interfaces of the different tissue types that comprise healing bone. We applied a correlative multi-method approach to visualize the LCN architecture and quantify mineral particle size and orientation within healing femoral bone in a mouse osteotomy model (26 weeks old C57BL/6 mice). This approach revealed structural differences across several length scales during endochondral ossification within the following regions: calcified cartilage, bony callus, cortical bone and the transition zone between the cortical region and callus that developed during 21 days after the osteotomy. In this transition zone, we observed a continuous convergence of mineral characteristics and osteocyte lacunae shape as well as discontinuities in the lacunae volume and LCN connectivity. The bony callus exhibits a 34% higher lacunae number density with 40% larger lacunar volume compared to cortical bone. The presented correlations between LCN architecture and mineral characteristics improves our understanding of how bone develops during healing and may indicate a contribution of osteocytes to bone (re)modeling

The mechanoresponse of bone is closely related to the osteocyte lacunocanalicular network architecture

The explanation of how bone senses and adapts to mechanical stimulation still relies on hypotheses. The fluid flow hypothesis claims that a load-induced fluid flow through the lacunocanalicular network can be sensed by osteocytes, which reside within the network structure. We show that considering the network architecture results in a better prediction of bone remodeling than mechanical strain alone. This was done by calculating the fluid flow through the lacunocanalicular network in bone volumes covering the complete cross-sections of mouse tibiae, which underwent controlled in vivo loading. The established relationship between mechanosensitivity and network architecture in individual animals implies possibilities for patient-specific therapies. A new connectomics approach to analyze lacunocanalicular network properties is necessary to understand skeletal mechanobiology.Organisms rely on mechanosensing mechanisms to adapt to changes in their mechanical environment. Fluid-filled network structures not only ensure efficient transport but can also be employed for mechanosensation. The lacunocanalicular network (LCN) is a fluid-filled network structure, which pervades our bones and accommodates a cell network of osteocytes. For the mechanism of mechanosensation, it was hypothesized that load-induced fluid flow results in forces that can be sensed by the cells. We use a controlled in vivo loading experiment on murine tibiae to test this hypothesis, whereby the mechanoresponse was quantified experimentally by in vivo micro-computed tomography (µCT) in terms of formed and resorbed bone volume. By imaging the LCN using confocal microscopy in bone volumes covering the entire cross-section of mouse tibiae and by calculating the fluid flow in the three-dimensional (3D) network, we could perform a direct comparison between predictions based on fluid flow velocity and the experimentally measured mechanoresponse. While local strain distributions estimated by finite-element analysis incorrectly predicts preferred bone formation on the periosteal surface, we demonstrate that additional consideration of the LCN architecture not only corrects this erroneous bias in the prediction but also explains observed differences in the mechanosensitivity between the three investigated mice. We also identified the presence of vascular channels as an important mechanism to locally reduce fluid flow. Flow velocities increased for a convergent network structure where all of the flow is channeled into fewer canaliculi. We conclude that, besides mechanical loading, LCN architecture should be considered as a key determinant of bone adaptation.Raw microscopy data have been deposited in the Open Access Data Repository of the Max Planck Society (https://edmond.mpdl.mpg.de/imeji/collection/0fK7DWn6fkD13hs). All study data are included in the article and SI Appendix

Alterations of bone material properties in growing Ifitm5/BRIL p.S42 knock-in mice, a new model for atypical type VI osteogenesis imperfecta

IntroductionOsteogenesis imperfecta (OI) is a heterogenous group of heritable connective tissue disorders characterized by high bone fragility due to low bone mass and impaired bone material properties. Atypical type VI OI is an extremely rare and severe form of bone dysplasia resulting from a loss-of-function mutation (p.S40L) in IFITM5/BRIL, the causative gene of OI type V and decreased osteoblast secretion of pigment epithelium-derived factor (PEDF), as in OI type VI. It is not yet known which alterations at the material level might lead to such a severe phenotype. We therefore characterized bone tissue at the micrometer level in a novel heterozygous Ifitm5/BRIL p.S42L knock-in murine model at 4 and 8 weeks of age.MethodsWe evaluated in female mice, total body size, femoral and lumbar bone mineral density (BMD) by dual-energy X-ray absorptiometry. In the femoral bone we examined osteoid deposition by light microscopy, assessed bone histomorphometry and mineralization density distribution by quantitative backscattered electron imaging (qBEI). Osteocyte lacunae were examined by qBEI and the osteocyte lacuno-canalicular network by confocal laser scanning microscopy Vasculature was examined indirectly by qBEI as 2D porosity in cortex, and as 3D porosity by micro-CT in third trochanter. Collagen orientation was examined by second harmonic generation microscopy. Two-way ANOVA was used to discriminate the effect of age and genotype.ResultsIfitm5/BRIL p.S42L female mice are viable, do not differ in body size, fat and lean mass from wild type (WT) littermates but have lower whole-body, lumbar and femoral BMD and multiple fractures. The average and most frequent calcium concentration, CaMean and CaPeak, increased with age in metaphyseal and cortical bone in both genotypes and were always higher in Ifitm5/BRIL p.S42L than in WT, except CaMean in metaphysis at 4 weeks of age. The fraction of highly mineralized bone area, CaHigh, was also increased in Ifitm5/BRIL p.S42L metaphyseal bone at 8 weeks of age and at both ages in cortical bone. The fraction of lowly mineralized bone area, CaLow, decreased with age and was not higher in Ifitm5/BRIL p.S42L, consistent with lack of hyperosteoidosis on histological sections by visual exam. Osteocyte lacunae density was higher in Ifitm5/BRIL p.S42L than WT, whereas canalicular density was decreased. Indirect measurements of vascularity revealed a higher pore density at 4 weeks in cortical bone of Ifitm5/BRIL p.S42L than in WT and at both ages in the third trochanter. Importantly, the proportion of bone area with disordered collagen fibrils was highly increased in Ifitm5/BRIL p.S42L at both ages.ConclusionsDespite normal skeletal growth and the lack of a collagen gene mutation, the Ifitm5/BRIL p.S42L mouse shows major OI-related bone tissue alterations such as hypermineralization of the matrix and elevated osteocyte porosity. Together with the disordered lacuno-canalicular network and the disordered collagen fibril orientation, these abnormalities likely contribute to overall bone fragility