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Mutations in the COL13A1 gene result in congenital myasthenic syndrome type 19 (CMS19), a disease of neuromuscular synapses and including various skeletal manifestations, particularly facial dysmorphisms. The phenotypic consequences in Col13a1 null mice (Col13a1 -/- ) recapitulate the muscle findings of the CMS19 patients. Collagen XIII (ColXIII) is exists as two forms, a transmembrane protein and a soluble molecule. While the Col13a1 -/- mice have poorly formed neuromuscular junctions, the prevention of shedding of the ColXIII ectodomain in the Col13a1 tm/tm mice results in acetylcholine receptor clusters of increased size and complexity. In view of the bone abnormalities in CMS19, we here studied the tubular and calvarial bone morphology of the Col13a1 -/- mice. We discovered several craniofacial malformations, albeit less pronounced ones than in the human disease, and a reduction of cortical bone mass in aged mice. In the Col13a1 tm/tm mice, where ColXIII is synthesized but the ectodomain shedding is prevented due to a mutation in a protease recognition sequence, the cortical bone mass decreased as well with age and the cephalometric analyses revealed significant craniofacial abnormalities but no clear phenotypical pattern. To conclude, our data indicates an intrinsic role for ColXIII, particularly the soluble form, in the upkeep of bone with aging and suggests the possibility of previously undiscovered bone pathologies in patients with CMS19., (© 2022. The Author(s).)

Objective: Knee osteoarthritis (OA) is associated with meniscal degeneration that may involve disorganization of the meniscal collagen fiber network. Our aims were to quantitatively analyze the microstructural organization of human meniscus samples in 3D using micro-computed tomography (μCT), and to compare the local microstructural organization between OA and donor samples., Method: We collected posterior horns of both medial and lateral human menisci from 10 end-stage medial compartment knee OA patients undergoing total knee replacement (medial & lateral OA) and 10 deceased donors without knee OA (medial & lateral donor). Posterior horns were dissected and fixed in formalin, dehydrated in ascending ethanol concentrations, treated with hexamethyldisilazane (HMDS), and imaged with μCT. We performed local orientation analysis of collagenous microstructure in 3D by calculating structure tensors from greyscale gradients within selected integration window to determine the polar angle for each voxel., Results: In donor samples, meniscus bundles were aligned circumferentially around the inner border of meniscus. In medial OA menisci, the organized structure of collagen network was lost, and main orientation was shifted away from the circumferential alignment. Quantitatively, medial OA menisci had the lowest mean orientation angle compared to all groups, -24° (95%CI -31 to -18) vs medial donor and -25° (95%CI -34 to -15) vs lateral OA., Conclusions: HMDS-based μCT imaging enabled quantitative analysis of meniscal collagen fiber bundles and their orientations in 3D. In human medial OA menisci, the collagen disorganization was profound with overall lower orientation angles, suggesting collagenous microstructure disorganization as an important part of meniscus degradation., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11β1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11β1 both have a role in the regulation of bone homeostasis. To study whether α11β1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11β1 as their only collagen receptor. The interaction between collagen XIII and integrin α11β1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11β1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11β1-collagen XIII interaction by crossbreeding α11 null mice (Itga11 -/- ) with mice overexpressing Col13a1 (Col13a1 oe ). When we evaluated the bone morphology by microcomputed tomography, Col13a1 oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11β1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis.

In tissue engineering, the scaffold topography influences the adhesion, proliferation, and function of cells. Specifically, the interconnected porosity is crucial for cell migration and nutrient delivery in 3D scaffolds. The objective of this study was to develop a 3D porous composite scaffold for musculoskeletal tissue engineering applications by incorporating barium titanate nanoparticles (BTNPs) into a poly-L/D-lactide copolymer (PLDLA) scaffold using the breath figure method. The porous scaffold fabrication utilised 96/04 PLDLA, dioleoyl phosphatidylethanolamine (DOPE), and different types of BTNPs, including uncoated BTNPs, Al 2 O 3 -coated BTNPs, and SiO 2 -coated BTNPs. The BTNPs were incorporated into the polymer scaffold, which was subsequently analysed using field emission scanning electron microscopy (FE-SEM). The biocompatibility of each scaffold was tested using ovine bone marrow stromal stem cells. The cell morphology, viability, and proliferation were evaluated using FE-SEM, LIVE/DEAD staining, and Prestoblue assay. Porous 3D composite scaffolds were successfully produced, and it was observed that the incorporation of uncoated BTNPs increased the average pore size from 1.6 μm (PLDLA) to 16.2 μm (PLDLA/BTNP). The increased pore size in the PLDLA/BTNP scaffolds provided a suitable porosity for the cells to migrate inside the scaffold, while in the pure PLDLA scaffolds with their much smaller pore size, cells elongated on the surface. To conclude, the breath figure method was successfully used to develop a PLDLA/BTNP scaffold. The use of uncoated BTNPs resulted in a composite scaffold with an optimal pore size while maintaining the honeycomb-like structure. The composite scaffolds were biocompatible and yielded promising structures for future tissue engineering applications., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Relationships between composition, structure and constituent-specific functional properties of human articular cartilage at different stages of osteoarthritis (OA) are poorly known. We established these relationships by comparison of elastic, viscoelastic and fibril-reinforced poroelastic mechanical properties with microscopic and spectroscopic analysis of structure and composition of healthy and osteoarthritic human tibial cartilage (n = 27). At a low frequency (0.005 Hz), proteoglycan content correlated negatively and collagen content correlated positively with the phase difference (i.e. tissue viscosity). At a high-frequency regime (> 0.05 Hz), proteoglycan content correlated negatively and collagen orientation angle correlated positively with the phase difference. Proteoglycans were lost in the early and advanced OA groups compared to the healthy group, while the superficial collagen orientation angle was greater only in the advanced OA group compared to the healthy group. Simultaneously, the initial fibril network modulus (fibril pretension) was smaller in the early and advanced OA groups compared to the healthy group. These findings suggest different mechanisms contribute to cartilage viscosity in low and high frequencies, and that the loss of superficial collagen pretension during early OA is due to lower tissue swelling (PG loss), while in advanced OA, both collagen disorganization and lower swelling modulate the collagen fibril pretension.

In this study, the rabbit model with anterior cruciate ligament transection (ACLT) was used to investigate early degenerative changes in cartilage using multiparametric quantitative magnetic resonance imaging (qMRI). ACLT was surgically induced in the knees of skeletally mature New Zealand White rabbits (n = 14). ACL transected and contralateral knee compartments-medial femur, lateral femur, medial tibia, and lateral tibia-were harvested 2 (n = 8) and 8 weeks (n = 6) postsurgery. Twelve age-matched nonoperated rabbits served as control. qMRI was conducted at 9.4 T and included relaxation times T 1 , T 2 , continuous-wave T 1ρ (CWT 1ρ ), adiabatic T 1ρ (AdT 1ρ ), adiabatic T 2ρ (AdT 2ρ ), and relaxation along a fictitious field (T RAFF ). For reference, quantitative histology and biomechanical measurements were carried out. Posttraumatic changes were primarily noted in the superficial half of the cartilage. Prolonged T 1 , T 2 , CWT 1ρ , and AdT 1ρ were observed in the lateral femur 2 and 8 weeks post-ACLT, compared with the corresponding control and contralateral groups (P < .05). Collagen orientation was significantly altered in the lateral femur at 2 weeks post-ACLT compared with the corresponding control group. In the medial femur, all the studied relaxation time parameters, except T RAFF , were increased 8 weeks post-ACLT, as compared with the corresponding contralateral and control groups (P < .05). Similarly, significant proteoglycan loss was observed in the medial femur at 8 weeks following surgery (P < .05). Multiparametric MRI demonstrated early degenerative changes primarily in the superficial cartilage with T 1 , T 2 , CWT 1ρ , and AdT 1ρ sensitive to cartilage changes at 2 weeks after surgery., (© 2020 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

Key Points: Extracellular matrix is highly remodelled in obesity and associates with the development of metabolic disorders, such as insulin resistance. Previously, we have shown that the lack of specific collagen XVIII isoforms impairs adipocyte differentiation in mice. Here, we show that mice lacking the medium and long isoforms of collagen XVIII develop insulin resistance and glucose intolerance and show elevated serum triglycerides and fat accumulation in the liver. We report that collagen XVIII-deficient mice have increased heat production at low temperatures. These results reveal a new role for collagen XVIII in the regulation of glucose and lipid metabolism, and they expand the understanding of the development of metabolic disorders., Abstract: Liver and adipose tissues play important roles in the regulation of systemic glucose and lipid metabolism. Extracellular matrix synthesis and remodelling are significantly altered in these tissues in obesity and type 2 diabetes. Collagen XVIII is a ubiquitous extracellular matrix component, and it occurs in three isoforms which differ in terms of molecular size, domain structure and tissue distribution. We recently showed that, in mice, the lack of collagen XVIII, and especially its medium and long isoforms, leads to reduced adiposity and dyslipidaemia. To address the metabolic consequences of these intriguing observations, we assessed whole-body glucose homeostasis in mice challenged with a high-fat diet and in normal physiological conditions. We observed that, in the high caloric diet, the overall adiposity was decreased by 30%, serum triglyceride values were threefold higher and the steatotic area in liver was twofold larger in collagen XVIII knockout mice compared with controls. We demonstrated that mice lacking either all three collagen XVIII isoforms, or specifically, the medium and long isoforms develop insulin resistance and glucose intolerance. Furthermore, we found that ablation of collagen XVIII leads to increased heat production in low temperatures and to reduction of the high blood triglyceride levels of the knockout mice to the level of wild-type mice. Our data indicate that collagen XVIII plays a role in the regulation of glucose tolerance, insulin sensitivity and lipid homeostasis, principally through its ability to regulate the expansion of the adipose tissue. These findings advance the understanding of metabolic disorders., (© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Objective: To develop and perform ex vivo 3D imaging of meniscus posterior horn microstructure using micro-computed tomography (μCT), and to compare specimens from healthy references against end-stage osteoarthritis (OA) using conventional section-based histology and qualitative μCT., Design: We retrieved human medial and lateral menisci from 10 deceased donors without knee OA (healthy references) and medial and lateral menisci from 10 patients having total knee replacement for medial compartment OA. Meniscal posterior horns were dissected and fixed in formalin. One subsection underwent hexamethyldisilazane (HMDS) treatment and μCT imaging. Pauli's histopathological scoring was performed for 3 other subsections. The differences in histopathological scores were estimated using mixed linear regression, resulting in fixed effects estimates for within-knee comparisons and adjusted for age and body mass index for between-subjects comparisons., Results: 3D visualization with μCT qualitatively revealed similar microstructural changes in the posterior horns as conventional histology. The mean histopathological score was higher for medial menisci from OA knees vs both medial reference menisci (mean difference [95% CI], 3.9 [2.6,5.3]), and lateral menisci from OA knees (3.9 [2.9,5.0]). The scores were similar between lateral menisci from OA knees and lateral reference menisci (0.8 [-0.6,2.2]), and between medial and lateral reference menisci (0.8 [-0.3,1.9])., Conclusions: HMDS-based μCT protocol allows unique 3D visualization of meniscus microstructures. Posterior horns of medial menisci from medial compartment OA knees had higher histopathological scores than both the lateral posterior horns from the same OA knees and medial reference menisci, suggesting a strong association between meniscus degradation and unicompartmental knee OA., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Osteoporosis is the most common degenerative bone disease that occurs when the balance of bone production and resorption is perturbed. Loss of bone mass or alteration in its quality leads to significant weakening of the bones and subsequently to higher fracture risk. Collagen XIII (ColXIII) is a conserved transmembrane protein expressed in many mesenchymal tissues. Here we show that ColXIII is a regulator of bone remodeling niche. In this study, we found that ColXIII expression is significantly upregulated in osteoporotic patients. In view of that, we studied bone homeostasis in ColXIII-overexpressing mice (Col13a1 oe ) up to 72 weeks of age and observed a cortical bone overgrowth followed by a drastic bone loss, together with increased bone vascularization. Moreover, our results demonstrate that the ColXIII-derived ectodomain enhances angiogenesis through β1-integrins and the JNK pathway. Consequently, these data suggest that ColXIII has a role in age-dependent cortical bone deterioration with possible implications for osteoporosis and fracture risk., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Articular cartilage constituents (collagen, proteoglycans, fluid) are significantly altered during osteoarthritis (OA). A fibril-reinforced poroelastic (FRPE) material model can separate the contribution of each constituent on the mechanical response of cartilage. Yet, these properties and their OA related alterations are not known for human tibial cartilage. To answer this gap in the knowledge, we characterized the FRPE as well as elastic and viscoelastic properties of healthy and osteoarthritic human tibial cartilage. Tibial osteochondral explants (n = 27) harvested from 7 cadavers were mechanically tested in indentation followed by a quantification of elastic, viscoelastic and FRPE properties. Then they were histopathologically OARSI graded for the severity of OA. FRPE modeling revealed that non-fibrillar matrix modulus was higher in the healthy group compared to the early OA (p = 0.003) and advanced OA (p < 0.001) groups. The initial fibril network modulus was also higher in the healthy group compared to the early OA (p = 0.009) and advanced OA (p < 0.001) groups. The permeability correlated with the OARSI grade (p = 0.002, r = 0.56). For the first time, the FRPE properties were characterized for human tibial cartilage. This knowledge is crucial to improve the accuracy of computational knee joint models.

Objective: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (μCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs)., Method: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified., Results: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (r s  = -0.600, P = 0.018), entropy of patterns (EP) (r s  = -0.648, P = 0.018), homogeneity index (HI) (r s  = 0.555, P = 0.032)) and tidemark roughness (TMR) (r s  = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (r p  = 0.876, P < 0.0001; r p  = 0.665, P = 0.007, respectively) and channel density (CD) (r p  = 0.680, P = 0.011; r p  = 0.582, P = 0.023, respectively). TMR was associated with CAF (r p  = 0.926, P < 0.0001) and average channel size (r p  = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples., Conclusion: We introduced a μ-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: The aims of this study were: to 1) develop a novel sample processing protocol to visualize human articular cartilage (AC) chondrons using micro-computed tomography (μCT), 2) develop and validate an algorithm to quantify the chondron morphology in 3D, and 3) compare the differences in chondron morphology between intact and osteoarthritic AC., Method: The developed protocol is based on the dehydration of samples with hexamethyldisilazane (HMDS), followed by imaging with a desktop μCT. Chondron density and depth, as well as volume and sphericity, were calculated in 3D with a custom-made and validated algorithm employing semi-automatic chondron selection and segmentation. The quantitative parameters were analyzed at three AC depth zones (zone 1: 0-10%; zone 2: 10-40%; zone 3: 40-100%) and grouped by the OARSI histological grades (OARSI grades 0-1.0, n = 6; OARSI grades 3.0-3.5, n = 6)., Results: After semi-automatic chondron selection and segmentation, 1510 chondrons were approved for 3D morphometric analyses. The chondrons especially in the deeper tissue (zones 2 and 3) were significantly larger (P < 0.001) and less spherical (P < 0.001), respectively, in the OARSI grade 3-3.5 group compared to the OARSI grade 0-1.0 group. No statistically significant difference in chondron density between the OARSI grade groups was observed at different depths., Conclusion: We have developed a novel sample processing protocol for chondron imaging in 3D, as well as a high-throughput algorithm to semi-automatically quantify chondron/chondrocyte 3D morphology in AC. Our results also suggest that 3D chondron morphology is affected by the progression of osteoarthritis (OA)., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Rheumatoid arthritis (RA) is characterised by chronic inflammation leading to articular bone and cartilage damage. Despite recent progress in RA management, adverse effects, lack of efficacy and economic barriers to treatment access still limit therapeutic success. Therefore, safer and less expensive treatments that control inflammation and bone resorption are needed. We have previously shown that celastrol is a candidate for RA treatment. We have observed that it inhibits both interleukin (IL)-1β and tumor necrosis factor (TNF) in vitro, and that it has anti-inflammatory properties and ability to decrease synovial CD68+ macrophages in vivo. Herein our goal was to evaluate the effect of celastrol in local and systemic bone loss., Methods: Celastrol was administrated intraperitoneally at a dose of 1 µg/g/day to female Wistar adjuvant-induced arthritic rats. Rats were sacrificed after 22 days of disease progression, and blood, femurs, tibiae and paw samples were collected for bone remodelling markers quantification, 3-point bending test, micro-CT analysis, nanoindentation and Fourier transform infrared spectroscopy measurements, and immunohistochemical evaluation., Results: We have observed that celastrol preserved articular structures and decreased the number of osteoclasts and osteoblasts present in arthritic joints. Moreover, celastrol reduced tartrate-resistant acid phosphatase 5b, procollagen type 1 amino-terminal propeptide and C terminal crosslinked telopeptide of type II collagen serum levels. Importantly, celastrol prevented bone loss and bone microarchitecture degradation. Celastrol also preserved bone nanoproperties and mineral content. Additionally, animals treated with celastrol had less fragile bones, as depicted by an increase in maximum load and yield displacement., Conclusions: These results suggest that celastrol reduces both bone resorption and cartilage degradation, and preserves bone structural properties., Competing Interests: Competing interests: None declared.

Research is ongoing to develop drug therapies to manage osteoarthritis (OA) and articular cartilage (AC) injuries. However, means to deliver drug to localized AC lesions are highly limited and not clinically available. This study investigates the capability of laser ultrasound (laser-induced plasma sound source) to deliver agents (methylene blue, MB, in PBS) into bovine AC. Treatment samples (n = 10) were immersed in MB solution simultaneously with LU exposure, while adjacent control 1 tissue (n = 10) was pre-treated with LU followed by immersion in MB and adjacent control 2 tissue (n = 10) was only immersed in MB. AC exposed (n = 22) or not exposed (n = 27) to LU were characterized for anomalies in structure, composition, viability or RNA expression. Optically detected MB content was significantly (p < 0.01) higher in treatment samples up to a depth of 500 µm from AC surface as compared to controls. No major unwanted short-term effects on AC structure, proteoglycan or collagen contents, chondrocyte viability or RNA expression levels were detected. In conclusion, LU can deliver agents into AC without major short-term concerns on safety. LU could reveal new strategies for the development of localized drug therapies in AC.

Contrast-enhanced micro-computed tomography (CEμCT) with phosphotungstic acid (PTA) has shown potential for detecting collagen distribution of articular cartilage. However, the selectivity of the PTA staining to articular cartilage constituents remains to be elucidated. The aim of this study was to investigate the dependence of PTA for the collagen content in bovine articular cartilage. Adjacent bovine articular cartilage samples were treated with chondroitinase ABC and collagenase to degrade the proteoglycan and the collagen constituents in articular cartilage, respectively. Enzymatically degraded samples were compared to the untreated samples using CEμCT and reference methods, such as Fourier-transform infrared imaging. Decrease in the X-ray attenuation of PTA in articular cartilage and collagen content was observed in cartilage depth of 0-13% and deeper in tissue after collagen degradation. Increase in the X-ray attenuation of PTA was observed in the cartilage depth of 13-39% after proteoglycan degradation. The X-ray attenuation of PTA-labelled articular cartilage in CEμCT is associated mainly with collagen content but the proteoglycans have a minor effect on the X-ray attenuation of the PTA-labelled articular cartilage. In conclusion, the PTA labeling provides a feasible CEμCT method for 3D characterization of articular cartilage., Competing Interests: The authors have declared that no competing interests exist.

The changes in chemical composition of human articular cartilage (AC) caused by osteoarthritis (OA) were investigated using Fourier transform infrared microspectroscopy (FTIR-MS). We demonstrate the sensitivity of FTIR-MS for monitoring compositional changes that occur with OA progression. Twenty-eight AC samples from tibial plateaus were imaged with FTIR-MS. Hyperspectral images of all samples were combined for K-means clustering. Partial least squares regression (PLSR) analysis was used to compare the spectra with the OARSI grade (histopathological grading of OA). Furthermore, the amide I and the carbohydrate regions were used to estimate collagen and proteoglycan contents, respectively. Spectral peak at 1338 cm(-1) was used to estimate the integrity of the collagen network. The layered structure of AC was revealed using the carbohydrate region for clustering. Statistically significant correlation was observed between the OARSI grade and the collagen integrity in the superficial (r = -0.55) and the deep (r = -0.41) zones. Furthermore, PLSR models predicted the OARSI grade from the superficial (r = 0.94) and the deep (r = 0.77) regions of the AC with high accuracy. Obtained results suggest that quantitative and qualitative changes occur in the AC composition during OA progression, and these can be monitored by the use of FTIR-MS.

Perfluorooctanoic acid (PFOA) is a ubiquitous and persistent environmental chemical, which has been used extensively due to its stability and surface tension-lowering properties. Toxicological effects include induction of neonatal mortality and reproductive toxicity. In this study, pregnant C57BL/6 mice were exposed orally to 0.3mg PFOA/kg/day throughout pregnancy, and female offspring were studied at the age of 13 or 17months. Morphometrical and biomechanical properties of femurs and tibias were analyzed with micro-computed tomography and 3-point bending, and bone PFOA concentrations were determined by mass spectrometry. The effects of PFOA on bone cell differentiation were studied in osteoclasts from C57BL/6 mice and in the MC3T3 pre-osteoblast cell line. PFOA exposed mice showed increased femoral periosteal area as well as decreased mineral density of tibias. Biomechanical properties of these bones were not affected. Bone PFOA concentrations were clearly elevated even at the age of 17months. In osteoblasts, low concentrations of PFOA increased osteocalcin (OCN) expression and calcium secretion, but at PFOA concentrations of 100μM and above osteocalcin (OCN) expression and calcium secretion were decreased. The number of osteoclasts was increased at all PFOA concentrations tested and resorption activity dose-dependently increased from 0.1-1.0μM, but decreased at higher concentrations. The results show that PFOA accumulates in bone and is present in bones until the old age. PFOA has the potential to influence bone turnover over a long period of time. Therefore bone is a target tissue for PFOA, and altered bone geometry and mineral density seem to persist throughout the life of the animal., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Osteoarthritis causes changes in the subchondral bone structure and composition. Plain radiography is a cheap, fast, and widely available imaging method. Bone tissue can be well seen from plain radiograph, which however is only a 2D projection of the actual 3D structure. Therefore, the aim was to investigate the relationship between bone density- and structure-related parameters from 2D plain radiograph and 3D bone parameters assessed from micro computed tomography (µCT) ex vivo. Right tibiae from eleven cadavers without any diagnosed joint disease were imaged using radiography and with µCT. Bone density- and structure-related parameters were calculated from four different locations from the radiographs of proximal tibia and compared with the volumetric bone microarchitecture from the corresponding regions. Bone density from the plain radiograph was significantly related with the bone volume fraction (r = 0.86; n = 44; p < 0.01). Mean homogeneity index for orientation of local binary patterns (HI(angle,mean)) and fractal dimension of vertical structures (FD(Ver)) were related (p < 0.01) with connectivity density (HI(angle,mean): r = -0.73, FD(Ver): r = 0.69) and trabecular separation (HI(angle,mean): r = 0.73, FD(Ver): r = -0.70) when all ROIs were pooled together (n = 44). Bone density and structure in tibia from standard clinically available 2D radiographs are significantly correlated with true 3D microstructure of bone.

The dynamics of animal populations are greatly influenced by interactions with their natural enemies and food resources. However, quantifying the relative effects of these factors on demographic rates remains a perpetual challenge for animal population ecology. Food scarcity is assumed to limit the growth and to initiate the decline of cyclic herbivore populations, but this has not been verified with physiological health indices. We hypothesized that individuals in declining populations would exhibit signs of malnutrition-induced deterioration of physiological condition. We evaluated the association of body condition with population cycle phase in bank voles (Myodes glareolus) during the increase and decline phases of a population cycle. The bank voles had lower body masses, condition indices and absolute masses of particular organs during the decline. Simultaneously, they had lower femoral masses, mineral contents and densities. Hemoglobin and hematocrit values and several parameters known to respond to food deprivation were unaffected by the population phase. There were no signs of lymphopenia, eosinophilia, granulocytosis or monocytosis. Erythrocyte counts were higher and plasma total protein levels and tissue proportions of essential polyunsaturated fatty acids lower in the population decline. Ectoparasite load was lower and adrenal gland masses or catecholamine concentrations did not suggest higher stress levels. Food availability seems to limit the size of voles during the decline but they can adapt to the prevailing conditions without clear deleterious health effects. This highlights the importance of quantifying individual health state when evaluating the effects of complex trophic interactions on the dynamics of wild animal populations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bone is a target for high affinity aryl hydrocarbon receptor (AHR) ligands, such as dioxins. Although bone morphology, mineral density and strength are sensitive endpoints of dioxin toxicity, less is known about effects on bone microarchitecture and material properties. This study characterizes TCDD-induced modulations of bone tissue, and the role of AHR in dioxin-induced bone toxicity and for normal bone phenotype. Six AHR-knockout (Ahr(-/-)) and wild-type (Ahr(+/+)) mice of both genders were exposed to TCDD weekly for 10 weeks, at a total dose of 200μg/kgbw. Bones were examined with micro-computed tomography, nanoindentation and biomechanical testing. Serum levels of bone remodeling markers were analyzed, and the expression of genes related to osteogenic differentiation was profiled using PCR array. In Ahr(+/+) mice, TCDD-exposure resulted in harder bone matrix, thinner and more porous cortical bone, and a more compact trabecular bone compartment. Bone remodeling markers and altered expression of a number of osteogenesis related genes indicated imbalanced bone remodeling. Untreated Ahr(-/-) mice displayed a slightly modified bone phenotype as compared with untreated Ahr(+/+) mice, while TCDD exposure caused only a few changes in bones of Ahr(-/-) mice. Part of the effects of both TCDD-exposure and AHR-deficiency were gender dependent. In conclusion, exposure of adult mice to TCDD resulted in harder bone matrix, thinner cortical bone, mechanically weaker bones and most notably, increased trabecular bone volume fraction in Ahr(+/+) mice. AHR is involved in bone development of a normal bone phenotype, and is crucial for manifestation of TCDD-induced bone alterations., (© 2013.)

Exposure to polychlorinated biphenyls (PCBs) induce a broad spectrum of toxic effects in various organs including bone. The most susceptible age-groups to the toxic effects of PCBs are foetuses and infants. The aim of the present study was to quantitatively evaluate changes in bone geometry, mineral density and biomechanical properties following perinatal exposure to the PCB mixture, Aroclor 1254 (A1254), and to examine the persistence of observed bone alterations by following the offspring over time. Sprague-Dawley rat offspring were exposed to A1254 from gestational day 1 to post-natal day (PND) 23. Femur and tibia were collected on PNDs 35, 77 and 350 and were analyzed by peripheral quantitative computed tomography and biomechanical testing. At PND35, exposure to A1254 induced short, thin femur and tibia, with reduced mechanical strength of femoral neck. No treatment-related bone changes were detected in offspring at PND77 or PND350. In conclusion, the present investigation suggests that perinatal exposure to A1254 leads to shorter, thinner and weaker bones in juvenile rats at PND35, with these effects being absent at later time-points as exposure is discontinued. The results indicate that the observed bone effects are mainly driven by the dioxin-like congeners, although it cannot exclude the contribution of the non dioxin-like congeners to the exposure outcome., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Phosphatases are essential for the mineralization of the extracellular matrix within the skeleton. Their precise identities and functions however remain unclear. PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase involved in the generation of inorganic phosphate for bone mineralization. It is highly expressed at sites of mineralization in bone and cartilage. The bones of Phospho1(-/-) mice are hypomineralized, bowed and present with spontaneous greenstick fractures at birth. In this study we show that PHOSPHO1 is essential for mechanically competent mineralization that is able to withstand habitual load. Long bones from Phospho1(-/-) mice did not fracture during 3-point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1(-/-) tibiae were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1(-/-) tibia. The altered dihydroxylysinonorleucine/hydroxylysinonorleucine and pyridinoline/deoxypyridinoline collagen crosslink ratios indicated possible changes in lysyl hydroxylase-1 activity and/or bone mineralization status. The bone formation and resorption markers, N-terminal propeptide and C-terminal telopeptide of Type I collagen, were both increased in Phospho1(-/-) mice and this we associated with increased bone remodeling during fracture repair or an attempt to remodel a mechanically competent bone capable of withstanding physiological load. In summary these data indicate that Phospho1(-/-) bones are hypomineralized and, consequently, are softer and more flexible. An inability to withstand physiological loading may explain the deformations noted. We hypothesize that this phenotype is due to the reduced availability of inorganic phosphate to form hydroxyapatite during mineralization, creating an undermineralized yet active bone., (Copyright © 2011 Elsevier Inc. All rights reserved.)

It has been established that almost 40% of postmenopausal women in the United States have osteopenia and models to study its prevention are thus urgently needed. Bears (Ursus spp.) displaying winter sleep were previously introduced as promising models to study the treatment of disuse-induced bone loss. The present study examined the potential of another analogous model species, the raccoon dog (Nyctereutes procyonoides), in bone research. Similar to bears, raccoon dogs display prolonged passivity and catabolism in winter, but they are of a moderate body mass, easy to handle and reared on farms. Wild specimens (n=51) were hunted in winter 2007-2008. The bone mineral density of femoral diaphysis and neck was examined with peripheral quantitative computed tomography, after which their mechanical properties were tested with the three-point bending and femoral neck loading tests. A subsample of the specimens was analyzed histologically. While the body mass of the raccoon dogs decreased from 7.0±0.3 to 4.5±0.2 kg (-36%) during winter, the bone mass and biomechanical properties remained unchanged despite of heavy wintertime catabolism similar to bears. Thus, the cortical mineral density remained at approximately 1400 mg/cm(3), the trabecular mineral density at 450 mg/cm(3) and the maximum load of the femoral neck at 700 N. However, in histological samples, the proportion of osteoid perimeter vs. mineralized bone perimeter decreased during wintering. A possible mechanism of bone mass preservation is the endocrine status of overwintering raccoon dogs, which could participate in preventing bone loss., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Arctic inhabitants consume large proportions of fish and marine mammals, and are therefore continuously exposed to levels of environmental toxicants, which may produce adverse health effects. Fetuses and newborns are the most vulnerable groups. The aim of this study was to evaluate changes in bone geometry, mineral density, and biomechanical properties during development following perinatal exposure to a mixture of environmental contaminants corresponding to maternal blood levels in Canadian Arctic human populations. Sprague-Dawley rat dams were dosed with a Northern Contaminant Mixture (NCM) from gestational day 1 to postnatal day (PND) 23. NCM contains 27 contaminants comprising polychlorinated biphenyls, organochlorine pesticides, and methylmercury. Femurs were collected on PND 35, 77 and 350, and diaphysis was analyzed by peripheral quantitative computed tomography and three-point bending test, while femoral neck was assessed in an axial loading experiment. Dose-response modeling was performed to establish the benchmark dose (BMD) for the analyzed bone parameters. Exposure to the high dose of NMC resulted in short and thin femur with reduced mechanical strength in offspring at PND35. BMD of femur length, cortical area, and stiffness were 3.2, 1.6, and 0.8 mg/kg bw/d, respectively. At PND77 femur was still thin, but at PND350 no treatment-related bone differences were detected. This study provides new insights on environmental contaminants present in the maternal blood of Canadian Arctic populations, showing that perinatal exposure induces bone alterations in the young offspring. These findings could be significant from a health risk assessment point of view.

Dioxins are known to decrease bone strength, architecture and density. However, their detailed effects on bone material properties are unknown. Here we used nanoindentation methods to characterize the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on nanomechanical behaviour of bone matrix. Pregnant rats were treated with a single intragastric dose of TCDD (1 microg/kg) or vehicle on gestational day 11. Tibias of female offspring were sampled on postnatal day (PND) 35 or 70, scanned at mid-diaphysis with pQCT, and evaluated by three-point bending and nanoindentation. TCDD treatment decreased bone mineralization (p<0.05), tibial length (p<0.01), cross-sectional geometry (p<0.05) and bending strength (p<0.05). Controls showed normal maturation pattern between PND 35 and 70 with decreased plasticity by 5.3% and increased dynamic hardness, storage and complex moduli by 26%, 13% and 12% respectively (p<0.05), while similar maturation was not observed in TCDD-exposed pups. In conclusion, for the first time, we demonstrate retardation of bone matrix maturation process in TCDD-exposed animals. In addition, the study confirms that developmental TCDD exposure has adverse effects on bone size, strength and mineralization. The current results in conjunction with macromechanical behaviour suggest that reduced bone strength caused by TCDD is more associated with the mineralization and altered geometry of bones than with changes at the bone matrix level., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The effects of lifelong physical exercise on the composition, structure and mechanical properties of bone are not well understood. Earlier, we found that voluntary physical exercise improved various properties of bone in maturing male mice up to 6 months of age. In this study, we extended the previous study to 18 months. Half of the mice (total N=144) had access to running wheels while half were kept sedentary. The collagen network was assessed biochemically and by tensile testing of decalcified bone. The mineralized femur was analyzed with pQCT and three-point-bending of the diaphysis and neck-strength-test. The proximal tibia was analyzed with microCT. The bone collagen revealed inferior tensional properties with aging and the mineralized femur demonstrated decreased stiffness with age. In the running mice, tensile properties and the BMD were reduced at 18 months of age compared to the sedentary mice. In contrast, the stiffness of both the diaphysis and femoral neck was higher, and trabecular architecture and structure were improved in the running mice. In summary, the results suggest that lifelong exercise training of male mice results in more beneficial effects on intact mineralized bone in both the diaphysis and epiphysis than on bone collagenous matrix.

This study characterized bone structure, composition, and mechanical properties in growing male mice. The development of the collagen network during maturation was monitored, and the effect of voluntary physical exercise was investigated. We hypothesized that increased bone loading from exercise would increase the amount and improve the properties of the collagen network during growth and maturation. Half of the mice (total n = 168) had access to running wheels, while half were kept sedentary. Weight and running activity were recorded, and groups of mice were killed at 1, 2, 4, and 6 months of age. The collagen network was assessed by biochemical evaluation of collagen content and cross-links and by tensile testing of decalcified bone. Mineralized femur was analyzed with pQCT and three-point-bending and femoral neck-strength tests. After 6 months, the exercising mice had 10% lower body weight than the sedentary group. There was no difference in the amount of collagen or collagen cross-links, while tensile testing had higher breaking force and stiffness of the collagen network in runners after 4 months but not after 6 months. The bone mineral density and cross-sectional area were higher in the running group after 6 months. Runners also showed higher breaking force and stiffness of the diaphysis and the femoral neck at 2 and 6 months. The significant modulation of mechanical properties of the collagen network without any change in collagen content indicates that physical exercise improves properties of the collagen network in maturing bone. The improvement after exercise of the properties of mineralized bone appears to be more pronounced and long-lasting compared to the early improved properties of the collagen network.