While cell morphology has been recognized as a fundamental regulator of cell behavior, few studies have measured the complex cell morphological changes of chondrocytes using quantitative cell morphometry descriptors in relation to inflammation and phenotypic outcome. Acute vs. persistent exposure to IL-1β and how IL-1β modulated dynamic changes in cell morphology in relation to the phenotype, donor and OA grade in healthy and osteoarthritis (OA) chondrocytes was investigated. A panel of quantitative cell morphometry descriptors was measured using an automated high-throughput method. Absolute quantification of gene expression was measured by ddPCR followed by correlation analyses. In OA chondrocytes, chronic IL-1β significantly decreased COL2A1, SOX9, and ACAN, increased IL-6 and IL-8 levels and caused chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype. In healthy chondrocytes, 3 days after acute (72 h) IL-1β exposure, COL1A2 and IL-6 significantly increased but had minor effects on cell morphology. However, in healthy chondrocytes, persistent IL-1β led to more profound effects in all cell morphology descriptors and chondrocytes expressed significantly less COL2A1 and more IL-6 and IL-8 vs. controls and acutely-stimulated chondrocytes. In both OA and healthy chronically-stimulated chondrocytes, area, width and circularity were sensitive to the persistent presence of the IL-1β cytokine. Moreover, there were many significant and strong correlations among the measured parameters, with several indications of an IL-1β-mediated mechanism. Cell morphology combined with gene expression analysis could guide researchers interested in understanding inflammatory effects in the complex domain of cartilage/chondrocyte biology. Use of quantitative cell morphometry could complement classical approaches by providing numerical data on a large number of cells, thereby providing a biological fingerprint for describing chondrocyte phenotype, which could help to understand how changes in cell morphology lead to disease progression

Objectives. This study aims to evaluate if micro-CT can work as a method for the 3D assessment and analysis of cancellous bone by comparing micro-CT with undecalcified histological sections in OVX rats. Methods. The mandible and tibia of sham, ovariectomised (OVX) and zoledronate-injected ovariectomised (OVX-ZOL) rats were assessed morphometrically. Specimens were scanned by micro-CT. Undecalcified histological sections were manufactured from the specimen scanned by micro-CT and stained with haematoxylin and eosin. Bivariate linear regressions and one-way analysis of variance were undertaken for statistics using SPSS 16.0.1 software. Results. There were highly significant correlations between undecalcified histological sections and micro-CT for all parameters (bone volume density (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp))in the mandible and tibia. Bone histomorphometric parameters analysed by both methods exhibited significant differences among sham, OVX, and OVX-ZOL groups. There were significant correlations between mandible and tibia in BV/TV, BS/BV, and Tb.Sp. Conclusions. Micro-CT is a complementary tool to histological sections in basic research that could improve our understanding of bone histomorphometry. The mandible can be used as an effective site to assess bone morphometry of OVX or metabolic bone disease rat models. Cite this article: H. Liu, W. Li, Y. S. Liu, Y. S. Zhou. Bone micro-architectural analysis of mandible and tibia in ovariectomised rats: A quantitative structural comparison between undecalcified histological sections and micro-CT. Bone Joint Res 2016;5:253–262

Introduction and Objective. Forced external rotation is hypothesized as the key mechanism of syndesmotic ankle injuries. This complex trauma pattern ruptures the syndesmotic ligaments and induces a three-dimensional deviation from the normal distal tibiofibular joint configuration. However, current diagnostic imaging modalities are impeded by a two-dimensional assessment, without taking into account ligamentous stabilizers. Therefore, our aim is two-fold: (1) to construct an articulated statistical shape model of the normal ankle with inclusion of ligamentous morphometry and (2) to apply this model in the assessment of a clinical cohort of patients with syndesmotic ankle injuries. Materials and Methods. Three-dimensional models of the distal tibiofibular joint were analyzed in asymptomatic controls (N= 76; Mean age 63 +/− 19 years), patients with syndesmotic ankle injury (N = 13; Mean age 35 +/− 15 years), and their healthy contralateral equivalent (N = 13). Subsequently, the statistical shape model was generated after aligning all ankles based on the distal tibia. The position of the syndesmotic ligaments was predicted based on previously validated iterative shortest path calculation methodology. Evaluation of the model was described by means of accuracy, compactness and generalization. Canonical Correlation Analysis was performed to assess the influence of syndesmotic lesions on the distal tibiofibular joint congruency. Results. Our presented model contained an accuracy of 0.23 +/− 0.028 mm. Mean prediction accuracy of ligament insertions was 0.53 +/− 12 mm. A statistically significant difference in anterior syndesmotic distance was found between ankles with syndesmotic lesions and healthy controls (95% CI [0.32, 3.29], p = 0.017). There was a significant correlation between presence of syndesmotic injury and the morphological distal tibiofibular configuration (r = 0.873, p <0,001). Conclusions. In this study, we constructed a bony and ligamentous statistical model representing the distal tibiofibular joint Furthermore, the presented model was able to detect an elongation injury of the anterior inferior tibiofibular ligament after traumatic syndesmotic lesions in a clinical patient cohort

Chronic glucocorticoid use causes osteogenesis loss, accelerating the progression of osteoporosis. Histone methylation is shown to epigenetically increase repressive transcription, altering lineage programming of mesenchymal stem cells (MSC). This study is undertaken to characterize the action of histone demethylase UTX to osteogenic lineage specification of bone-marrow MSC and bone integrity upon glucocorticoid treatment. Bone-marrow MSC were incubated in osteogenic medium containing supraphysiological dexamethasone. Osteogenic gene expression and mineralized nodule formation were probed using RT-PCR and von Kossa staining. The enrichment of trimethylated lysine 27 at histone 3 (H3K27me3) in Dkk1 promoter was quantified using chromatin immunoprecipitation-PCR. Bone mass and trabecular morphometry in methylprednisolone-treated skeletons were quantified using microCT analysis. Supraphysiological dexamethasone decreased osteogenic genes Runx2 and osteocalcin expression and mineralized matrix production along with reduced UTX expression in MSC. Forced UTX expression attenuated the glucocorticoid-mediated loss of osteogenic differentiation, whereas UTX knockdown provoked osteogenesis loss and cytoplasmic oil overproduction. UTX demethylated H3K27 and reduced the glucocorticoid-mediated the H3K27 enrichment in Dkk1 promoter, reversing beta-catenin signal, but downregulating Dkk1 production by MSC. In vivo, treatment with UTX inhibitor GSK-J4 significantly suppressed bone mineral density, trabecular volume, and thickness along with porous trabecular, fatty marrow and disturbed beta-catenin/Dkk1 histopathology comparable with glucocorticoid-induced osteoporosis condition. This study offers a productive insight into how UTX protects MSC from methylated histone-mediated osteogenesis repression in the development of glucocorticoid-induced osteoporosis

Introduction and Objective. In multiple trauma patients, as well as in the healing of isolated fractures (Fx) with heavy bleeding (trauma haemorrhage, TH), complications occur very often. This is particularly evident in elderly patients over 65 years of age. Since these accompanying circumstances strongly influence the clinical course of treatment, the influence of age on bone regeneration after femoral fracture and severe blood loss was investigated in this study. Materials and Methods. 12 young (17–26 weeks) and 12 old (64–72 weeks) male C57BL / 6J mice per group were examined. The fracture group Fx underwent an osteotomy after applying an external fixator. The THFx group also received blood pressure-controlled trauma hemorrhage (35 mmHg for 90 minutes) and reperfusion with Ringer's solution for 30 minutes. The Sham group received only the catheter and one external fixator. μCT scans of the femora were performed in vivo after 2 weeks and ex vivo after 3 weeks. Histological and biomechanical examinations were also carried out. The statistical significance was set at p ≤ 0.05. The non-normally distributed data were analyzed using the Mann-Whitney-U or Kruskal-Wallis test. Results. The histology showed less mineralized bone in the fracture gap in old animals of the Fx (25.41% [1.68%]) and THFx groups (25.50% [4.07%]) compared with the young ones (34.20% [6.36%], p = 0.003; 34.31% [5.12%], p=0.009). Moreover, a severe blood loss lead to more cartilage in both young (6.91% [5.08%]) and old animals (4.17% [1.42%]) compared to animals with only a fracture (2, 45% [1.04%], p=0.004; 2.95% [1.12%], p=0.032). In old animals (11.37 / nm. 2. [17.17 / nm. 2. ]) in contrast the young mice with an isolated fracture (33.6/nm. 2. [8.83/nm. 2. ]) fewer osteoclasts were present (p=0.009). Therefore, the severe blood loss further reduced the number of osteoclasts only in young animals (16.83/nm. 2. [6.07/nm. 2. ]) (p=0.004). In the in vivo μCT, after 2 weeks, a lower volume of bone, cortex and callus was found in old THFx animals (3.14 mm. 3. [0.64 mm. 3. ]); 1.01 mm. 3. [0.04 mm. 3. ]; 2.07 mm. 3. [0.57 mm. 3. ]) compared with the Fx animals (4.29 mm. 3. [0.74 mm. 3. ], p=0.008; 1.18 mm. 3. [0, 25 mm. 3. ], p=0.004; 3.02 mm. 3. [0.77 mm. 3. ], p=0.008) After 3 weeks, the ex vivo μCT scans also showed a reduced callus percentage in old THFx animals (61.18% [13.9 9%]), as well as a low number of trabeculae (1.81 mm. -1. [0.23 mm. -1. ]) compared to animals without blood loss (68.72% [15.71%], p = 0.030; 2.06mm. -1. [0.37mm. -1. ], p=0.041). In the biomechanical test, a reduced elasticity limit of the old THFx mice (7.75 N [3.33 N]) in contrast to the old Fx (10.24 N [3.32 N]) animals was shown (p=0.022). Conclusions. A severe blood loss has a higher negative effect on the healing, morphometry, and biomechanical properties of previously fractured femora in old compared to young individuals

Trochlear dysplasia is a specific morphotype of the knee, characterized by but not limited to a specific anatomy of the trochlea. The notch, posterior femur and tibial plateau also seem to be involved. In our study we conducted a semi-automated landmark-based 3D analysis on the distal femur, tibial plateau and patella. The knee morphology of a study population (n=20), diagnosed with trochlear dysplasia and a history of recurrent patellar dislocation was compared to a gender- and age-matched control group (n=20). The arthro-CT scan-based 3D-models were isotropically scaled and landmark-based reference planes were created for quantification of the morphometry. Statistical analysis was performed to detect shape differences between the femur, tibia and patella as individual bone models (Mann-Whitney U test) and to detect differences in size agreement between femur and tibia (Pearson's correlation test). The size of the femur did not differ significantly between the two groups, but the maximum size difference (scaling factor) over all cases was 35%. Significant differences were observed in the trochlear dysplasia (TD) versus control group for all conventional parameters. Morphometrical measurements showed also significant differences in the three directions (anteroposterior (AP), mediolateral (ML), proximodistal (PD)) for the distal femur, tibia and patella. Correlation tests between the width of the distal femur and the tibial plateau revealed that TD knees show less agreement between femur and tibia than the control knees; this was observed for the overall width (TD: r=0.172; p=0.494 - control group: r=0.636; p=0.003) and the medial compartment (TD: r=0.164; p=0.516 - control group: r=0.679; p=0.001), but not for the lateral compartment (TD: r=0.512; p=0.029 - control: r=0.683; p=0.001). In both groups the intercondylar eminence width was strongly correlated with the notch width (TD: r=0.791; p=0.001 - control: r=0.643; p=0.002). The morphology of the trochleodysplastic knee differs significantly from the normal knee by means of an increased ratio of AP/ML width for both femur and tibia, a smaller femoral notch and a lack of correspondence in mediolateral width between the femur and tibia. More specifically, the medial femoral condyle shows no correlation with the medial tibial plateau

Osteophyte deposition and subchondral bone damage are notable features of osteoarthritis (OA). Deregulated mineralization contributes to osteophyte and subchondral irregularity. The microRNA-29 (miR-29) family is associated with arthritic disorders. This study is aimed to investigate miR-29a function to OA osteophyte formation and subchondral integrity. Intact and damaged articular cartilage in patients with end-stage knee OA who required total knee arthroplasty were harvested to probe miR-29a, cartilage, and mineralized matrix expression using RT-PCR and in situ hybridization. Osteophyte volume and subchondral morphometry of collagenase-induced OA knees in mice were quantified using μCT and histomorphometry. Increased bone matrix expression (collagen I and bone alkaline phosphatase) and reduced cartilage matrix (collagen II and aggrecan) along with low miR-29a expression existed in human OA specimens. Aged miR-29a knockout mice showed spontaneous osteophyte formation and articular cartilage erosion. In primary articular chondrocytes, miR-29a deficiency significantly reduced cartilage matrix synthesis, whereas von Kossa staining-positive mineralized matrix production was increased. Of interest, the severity of collagenase-induced osteophyte accumulation and subchondral damage along with serum cartilage breakdown products CTX-II and COMP levels were significantly compromised in mice overexpressing miR-29a. Intra-articularly injecting miR-29a significantly reduced osteophyte volume and subchondral integrity and retained cartilage morphology in collagenase-injured knees. Reduced miR-29a signalling worsens osteophyte and subchondral destruction in OA through increasing mineralized matrix formation of chondrocytes. Restoring miR-29a shields joints from cartilage degradation, osteophyte and subchondral destruction. This study conveys new mechanistic underlying OA osteophyte pathogenesis and shines light on the remedial potential of miR-29a to OA

Introduction. Histology remains the gold standard in morphometric and pathological analyses of osteochondral tissues in human and experimental bone and joint disease. However, histological tissue processing is laborious, destructive and only provides a two-dimensional image in a single anatomical plane. Micro computed tomography (μCT) enables non-destructive three-dimensional visualization and morphometry of mineralized tissues and, with the aid of contrast agents, soft tissues. In this study, we evaluated phosphotungstic acid-enhanced (PTA) μCT to visualize joint pathology in spine osteoarthritis. Methods. Lumbar facet joint specimens were acquired from six patients (5 female, age range 31–78) undergoing decompression surgery. Fresh osteochondral specimens were immediately fixed in formalin and scanned in a benchtop μCT scanner (65 kV, 153 mA, 25 μm resolution). Subsequently, samples were completely decalcified in 5% formic acid, equilibrated in 70% ethanol and stained up to ten days in 1% PTA (w/v) in 70% ethanol. PTA-stained specimens were scanned at 70 kV, 140 mA, 15 μm resolution. Depth-dependent analysis of X-ray attenuation in cartilage tissues was performed using ImageJ. Bone structural parameters of undecalcified and PTA-stained specimens were determined using CT Analyser and methods were compared using correlation and Bland-Altman analysis. Results. The maximal penetration depth of PTA in decalcified facet joint was 5 mm. Bone tissue showed strong and uniformly distributed X-ray attenuation, while mild to moderate and differentially distributed attenuation was observed in articular cartilage and subchondral marrow spaces. Measurements of bone volume (r=0.90, p=0.01) and bone surface (r=0.95, p=0.004) were strongly correlated between undecalcified and PTA-stained samples. Compared with PTA-stained samples, measurements in undecalcified specimens were consistently higher (∼14%). PTA-enhanced μCT visualization of cartilage tissues enabled the identification of individual chondrocytes and their pericellular microenvironment (chondrons). Owing to loss of collagen lower X-ray attenuation was observed in the middle and deep cartilage layers at the central, but not peripheral, regions of the degenerated facet joint specimens. Depth-dependent analysis of PTA-staining intensity suggested that the extent of collagen loss in articular cartilage might correlate with the thickness of the subchondral cortical plate. Conclusion. PTA-enhanced μCT is a low-cost, non-toxic and highly feasible method for ex vivo 3D-visualization of osteochondral pathology in human osteoarthritis. The method enables bone morphometric analysis, as well as collagen distribution in all anatomical planes. Contrast enhanced μCT has several applications in bone and osteoarthritis research including 3D histopathological grading, tissue stratification, and imaging and analysis of aberrant collagen metabolism in osteochondral disease

The study of the chondrocyte maturation cycle and endochondral ossification showed that the developing vascular supply has appeared to play a key role in determining the cortical or trabecular structure of the long bones. The chondrocyte maturation cycle and endochondral ossification were studied in human, foetal cartilage anlagen and in postnatal meta-epiphyses. The relationship between the lacunar area, the inter territorial fibril network variations and CaP nucleation in primary and secondary ossification centres were assessed using light microscopy and SEM morphometry. The anlage topographic, zonal classification derived from the anatomical nomenclature of the completely developed long bone (diaphysis, metaphyses and epiphyses) allowed to follow the development of long bones cartilage model. A significant increase in chondrocyte lacunar area (p<0.001) was documented from the anlage epiphyseal zone 4 and 3 to zone 2 (metaphysis) and zone 1 (diaphysis), with the highest variation from zone 2 to zone 1. An inverse reduction in the intercellular matrix area (p<0.001) and matrix interfibrillar empty space (p<0.001) was also documented. These findings are consistent with the osmotic passage of free cartilage water from the interfibrillar space into the swelling chondrocytes, raising ion concentrations up to the critical threshold for mineral precipitation in the matrix. The mineralised cartilage served as a scaffold for osteoblasts apposition both in primary and secondary ossification centres and in the metaphyseal growth plate cartilage, but at different periods of bone anlage development and with distinct patterns for each zone. They all shared a common initial pathway, but it progressed with different times, modes and organisation in diaphysis, metaphysis and epiphysis. In the ossification phase the developing vascular supply has appeared to play a key role in determining the cortical or trabecular structure of the long bones

Summary. Macroscopic grading, histologic grading, morphometry, mineral analysis, and mechanical testing were performed to better understand the changes that occur in the cartilage, calcified cartilage, and subchondral bone in early osteoarthritis. Introduction. The earliest changes in osteoarthritis (OA) remain poorly understood due to the difficulty in detecting OA before patients feel pain. We have published details of the mature bovine patella model showing the pre-OA state where no gross macroscopic changes are visible yet microstructural changes indicate very early degeneration. In this new study, we proceed to investigate this model further by more comprehensively quantifying the changes in articular cartilage (AC), zone of calcified cartilage (ZCC), and subchondral bone (SB) in pre and early OA. Methods. Patellae from mature cow were studied. Gross examination with India ink was used to classify macroscopic cartilage degeneration. Two groups were selected in this study: one with no visible surface degeneration (pre-OA) and the other with mild to moderate macroscopically visible surface degeneration (early OA). Histologic staining with Safranin O and Fast Green was analysed with two osteoarthritic scoring systems: Mankin and OOCHAS. Differential Interference Contrast (DIC) microscopy was used to quantify morphometric changes. Degree of mineralisation was analysed with energy dispersive X-ray spectroscopy (EDS) to quantify the calcium and phosphorus content of the mineralised tissues. Material properties of calcified cartilage and subchondral bone were tested macroscopically using 3 point bending. Results. In the early OA group, cartilage was fissured and showed matrix loss. In its hydrated state, average cartilage thickness was significantly greater (p<0.05) in the early OA group by 24% compared to pre-OA group. The early OA group showed an 88% increase in ZCC thickness. Early OA tissue was graded significantly higher in OOCHAS grading and structure scores, cellularity, and staining scores of Mankin grading but not in the tidemark integrity score. Pre-OA and early OA tissues showed no significant differences in ZCC or SB mineralisation although all samples showed an increase in the degree of mineralisation going from the upper to the deeper ZCC and SB. Macroscopic mechanical testing showed no significant differences in mechanical properties between pre-OA and early OA groups. However within groups, the ZCC was an order of magnitude less stiff than the SB. Micromechanical testing showed that deeper ZCC and SB were stiffer than their regions closer to the joint surface. Conclusions. Early osteoarthritic changes in the joint tissues produce macro-level cartilage degeneration as well as microstructural changes. The combination of mineralisation and mechanical data show that though calcified cartilage and subchondral bone have similar mineralisation profiles, their material properties are drastically different, suggesting that stiffness is not purely the result of the mineral phase

We studied the morphometry of 35 femora from 31 female patients with developmental dysplasia of the hip (DDH) and another 15 from 15 age- and sex-matched control patients using CT and three-dimensional computer reconstruction models. According to the classification of Crowe et al 15 of the dysplastic hips were graded as class I (less than 50% subluxation), ten as class II/III (50% to 100% subluxation) and ten as class IV (more than 100% subluxation). The femora with DDH had 10 to 14° more anteversion than the control group independent of the degree of subluxation of the hip. In even the most mildly dysplastic joints, the femur had a smaller and more anteverted canal than the normal control. With increased subluxation, additional abnormalities were observed in the size and position of the femoral head. Femora from dislocated joints had a short, anteverted neck associated with a smaller, narrower, and straighter canal than femora of classes I and II/III or the normal control group. We suggest that when total hip replacement is performed in the patient with DDH, the femoral prosthesis should be chosen on the basis of the severity of the subluxation and the degree of anteversion of each individual femur

Objectives

Healing in cancellous metaphyseal bone might be different from midshaft fracture healing due to different access to mesenchymal stem cells, and because metaphyseal bone often heals without a cartilaginous phase. Inflammation plays an important role in the healing of a shaft fracture, but if metaphyseal injury is different, it is important to clarify if the role of inflammation is also different. The biology of fracture healing is also influenced by the degree of mechanical stability. It is unclear if inflammation interacts with stability-related factors.