Osteogenesis 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.
We 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.
Ifitm5/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.
Despite 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.
•Ifitm5/BRIL pS42L mice, a model for atypical OI type VI, shows high bone fragility.•Bone material properties were evaluated in mutants and WT at 4 and 8 weeks of age.•Vascularity and osteocyte density decreased with age and were higher in mutants.•Osteocyte canalicular density increased with age and was lower in mutants.•At both ages, mutants show hypermineralization and disordered collagen orientation.
Osteocytes are terminally differentiated osteoblasts embedded within the bone matrix and key orchestrators of bone metabolism. However, they are generally not characterized by conventional bone ...histomorphometry because of their location and the limited resolution of light microscopy. OI is characterized by disturbed bone homeostasis, matrix abnormalities and elevated bone matrix mineralization density. To gain further insights into osteocyte characteristics and bone metabolism in OI, we evaluated 2D osteocyte lacunae sections (OLS) based on quantitative backscattered electron imaging in transiliac bone biopsy samples from children with OI type I (n = 19) and age-matched controls (
= 24). The OLS characteristics were related to previously obtained, re-visited histomorphometric parameters. Moreover, we present pediatric bone mineralization density distribution reference data in OI type I (
= 19) and controls (
= 50) obtained with a field emission scanning electron microscope. Compared to controls, OI has highly increased OLS density in cortical and trabecular bone (+50.66%, +61.73%; both
< 0.001), whereas OLS area is slightly decreased in trabecular bone (-10.28%;
= 0.015). Correlation analyses show a low to moderate, positive association of OLS density with surface-based bone formation parameters and negative association with indices of osteoblast function. In conclusion, hyperosteocytosis of the hypermineralized OI bone matrix associates with abnormal bone cell metabolism and might further impact the mechanical competence of the bone tissue.
Abstract
The enthesis allows the insertion of tendon into bone thanks to several remarkable strategies. This complex and clinically relevant location often features a thin layer of fibrocartilage ...sandwiched between tendon and bone to cope with a highly heterogeneous mechanical environment. The main purpose of this study was to investigate whether mineralized fibrocartilage and bone close to the enthesis show distinctive three-dimensional microstructural features, possibly to enable load transfer from tendon to bone. As a model, the Achilles tendon-calcaneus bone system of adult rats was investigated with histology, backscattered electron imaging and micro-computed tomography. The microstructural porosity of bone and mineralized fibrocartilage in different locations including enthesis fibrocartilage, periosteal fibrocartilage and bone away from the enthesis was characterized. We showed that calcaneus bone presents a dedicated protrusion of low porosity where the tendon inserts. A spatially resolved analysis of the trabecular network suggests that such protrusion may promote force flow from the tendon to the plantar ligament, while partially relieving the trabecular bone from such a task. Focusing on the tuberosity, highly specific microstructural aspects were highlighted. Firstly, the interface between mineralized and unmineralized fibrocartilage showed the highest roughness at the tuberosity, possibly to increase failure resistance of a region carrying large stresses. Secondly, fibrochondrocyte lacunae inside mineralized fibrocartilage, in analogy with osteocyte lacunae in bone, had a predominant alignment at the enthesis and a rather random organization away from it. Finally, the network of subchondral channels inside the tuberosity was highly anisotropic when compared to contiguous regions. This dual anisotropy of subchondral channels and cell lacunae at the insertion may reflect the alignment of the underlying collagen network. Our findings suggest that the microstructure of fibrocartilage may be linked with the loading environment. Future studies should characterize those microstructural aspects in aged and or diseased conditions to elucidate the poorly understood role of bone and fibrocartilage in enthesis-related pathologies.
A demanding task of the musculoskeletal system is the attachment of tendon to bone at entheses. This region often presents a thin layer of fibrocartilage (FC), mineralized close to the bone and ...unmineralized close to the tendon. Mineralized FC deserves increased attention, owing to its crucial anchoring task and involvement in enthesis pathologies. Here, we analyzed mineralized FC and subchondral bone at the Achilles tendon-bone insertion of rats. This location features enthesis FC anchoring tendon to bone and sustaining tensile loads, and periosteal FC facilitating bone-tendon sliding with accompanying compressive and shear forces. Using a correlative multimodal investigation, we evaluated potential specificities in mineral content, fiber organization and mechanical properties of enthesis and periosteal FC. Both tissues had a lower degree of mineralization than subchondral bone, yet used the available mineral very efficiently: for the same local mineral content, they had higher stiffness and hardness than bone. We found that enthesis FC was characterized by highly aligned mineralized collagen fibers even far away from the attachment region, whereas periosteal FC had a rich variety of fiber arrangements. Except for an initial steep spatial gradient between unmineralized and mineralized FC, local mechanical properties were surprisingly uniform inside enthesis FC while a modulation in stiffness, independent from mineral content, was observed in periosteal FC. We interpreted these different structure-property relationships as a demonstration of the high versatility of FC, providing high strength at the insertion (to resist tensile loading) and a gradual compliance at the periosteal surface (to resist contact stresses).
Mineralized fibrocartilage (FC) at entheses facilitates the integration of tendon in bone, two strongly dissimilar tissues. We focus on the structure-function relationships of two types of mineralized FC, enthesis and periosteal, which have clearly distinct mechanical demands. By investigating them with multiple high-resolution methods in a correlative manner, we demonstrate differences in fiber architecture and mechanical properties between the two tissues, indicative of their mechanical roles. Our results are relevant both from a medical viewpoint, targeting a clinically relevant location, as well as from a material science perspective, identifying FC as high-performance versatile composite.
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Not much is known about the time course of mineralization in newly formed bone from healthy individuals nor in patients with bone disease. To investigate the early phase of mineral accumulation in ...human bone, we measured the mean mineralization content between double fluorescence labels (CaYoung) with quantitative backscattered electron imaging (qBEI) in human transiliac biopsy samples. Fluorescent labels for histomorphometric evaluation were administered over two 2-day periods separated by a 10-day-free interval, 4–5 days before biopsy procedure. We compared n = 19 children with osteogenesis imperfecta type 1 (OI, 6 girls/13 boys, age-range 2.2–14.1 years) with both a reference group of n = 38 healthy children (REF, 24 girls/14 boys, age-range 1.5–20.9 years) and an age-matched subgroup (n = 17) of the latter (CON, 5 girls/12 boys, age-range 2.0–14.7 years).
We observed significantly higher levels of CaYoung in OI type 1 compared to CON and REF in both cortical bone (Ct.CaYoung, +8.3 % and +7.0 %, respectively) and cancellous bone (Cn.CaYoung, +6.5 % and +4.9 %, all p < 0.001). When comparing cortical and cancellous compartments intra-individually, we found a significantly higher Cn.CaYoung than Ct.CaYoung in REF (2.3 ± 2.9 %, p < 0.001), but not in the OI group (0.6 ± 1.6 %, not significant). In the REF group, n = 7 samples also contained double labels in primary woven bone (which is deposited at the external cortex during growth/lateral drift of the iliac crest). This primary woven bone in REF had a higher CaYoung than secondary osteonal bone (4.9 ± 2.7 %).
Our findings suggest that bone in OI has an accelerated mineral accumulation compared to healthy bone. This is reflecting the overall increased bone mineralization in OI as reported previously, and indicates that the higher levels of mineralization than those seen in healthy individuals are already achieved in OI type 1 early after onset of mineralization.
•We measured mineralization kinetics by the combination of qBEI and CLSM imaging.•The mineral content of bone between the double labels (CaYoung) was analyzed.•CaYoung was increased in young patients with OI compared to healthy children.•In healthy children but not in OI, CaYoung was higher in spongiosa than in cortex.
Collagen is a versatile structural molecule in nature and is used as a building block in many highly organized tissues, such as bone, skin, and cornea. The functionality and performance of these ...tissues are controlled by their hierarchical organization ranging from the molecular up to macroscopic length scales. In the present study, polarized Raman microspectroscopic and imaging analyses were used to elucidate collagen fibril orientation at various levels of structure in native rat tail tendon under mechanical load. In situ humidity-controlled uniaxial tensile tests have been performed concurrently with Raman confocal microscopy to evaluate strain-induced chemical and structural changes of collagen in tendon. The methodology is based on the sensitivity of specific Raman scattering bands (associated with distinct molecular vibrations, such as the amide I) to the orientation and the polarization direction of the incident laser light. Our results, based on the changing intensity of Raman lines as a function of orientation and polarization, support a model where the crimp and gap regions of collagen hierarchical structure are straightened at the tissue and molecular level, respectively. However, the lack of measurable changes in Raman peak positions throughout the whole range of strains investigated indicates that no significant changes of the collagen backbone occurs with tensing and suggests that deformation is rather redistributed through other levels of the hierarchical structure.
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