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Bone microdamage is common at subchondral bone (SCB) sites subjected to repeated high rate and magnitude of loading in the limbs of athletic animals and humans. Microdamage can affect the biomechanical behaviour of bone under physiological loading conditions. To understand the effects of microdamage on the mechanical properties of SCB, it is important to be able to quantify it. The extent of SCB microdamage had been previously estimated qualitatively using plain microcomputed tomography (μCT) and a radiocontrast quantification method has been used for trabecular bone but this method may not be directly applicable to SCB due to differences in bone structure. In the current study, SCB microdamage detection using lead uranyl acetate (LUA) and quantification by contrast‐enhanced μCT and backscattered scanning electron microscopy (SEM) imaging techniques were assessed to determine the specificity of the labels to microdamage and the accuracy of damaged bone volume metrices. SCB specimens from the metacarpus of racehorses, with the hyaline articular cartilage (HAC) removed, were grouped into two with one group subjected to ex vivo uniaxial compression loading to create experimental bone damage. The other group was not loaded to preserve the pre‐existing in vivo propagated bone microdamage. A subset of each group was stained with LUA using an established or a modified protocol to determine label penetration into SCB. The μCT and SEM images of stained specimens showed that penetration of LUA into the SCB was better using the modified protocol, and this protocol was repeated in SCB specimens with intact hyaline articular cartilage. The percentage of total label localised to bone microdamage was determined on SEM images, and the estimated labelled bone volume determined by μCT in SCB groups was compared. Label was present around diffuse and linear microdamage as well as oblique linear microcracks present at the articular surface, except in microcracks with high‐density mineral infills. Bone surfaces lining pores with recent mineralisation were also labelled. Labelled bone volume fraction (LV/BV) estimated by μCT was higher in the absence of HAC. At least 50% of total labels were localised to bone microdamage when the bone area fraction (B.Ar/T.Ar) of the SCB was greater than 0.85 but less than 30% when B.Ar/T.Ar of the SCB was less than 0.85. To adjust for LUA labels on bone surfaces, a measure of the LV/BV corrected for bone surface area (LV/BV BS−1) was used to quantify damaged SCB. In conclusion, removal of HAC and using a modified labelling protocol effectively stained damaged SCB of the metacarpus of racehorses and represents a technique useful for quantifying microdamage in SCB. This method can facilitate future investigations of the effects of microdamage on joint physiology. [ABSTRACT FROM AUTHOR]

ABSTRACT During the early stages of articular osteochondrosis, cartilage is retained in subchondral bone, but the pathophysiology of this condition of growing humans and domestic animals is poorly understood. A subtractive hybridization study was undertaken to compare gene expression between the cartilage of early experimentally induced equine osteochondrosis lesions and control cartilage. Of the many putative differentially expressed genes identified, eight were confirmed by quantitative PCR analysis as differentially expressed, in addition to those already known to be associated with early lesions. Genes encoding vacuolar H+-ATPase V0 subunit d2 (ATP6V0D2), cathepsin K, integrin-binding sialoprotein, integrin αV, low density lipoprotein receptor-related protein 4, lumican, osteopontin, and thymosin β4 (TMSB4) were expressed at higher levels in lesions than in control cartilage. These genes included 34 genes not previously identified in cartilage. Some genes identified as associated with early lesions are known chondrocyte hypertrophy-associated genes, and in transmission electron microscopy studies normal hypertrophic chondrocytes were observed in lesions. Differential expression of ATP6V0D2 and TMSB4 in the cartilage of early naturally occurring osteochondrosis lesions was confirmed by immunohistochemistry. These results identify novel osteochondrosis-associated genes and provide evidence that articular osteochondrosis does not necessarily result from failure of chondrocytes to undergo hypertrophy. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:404-411, 2016. [ABSTRACT FROM AUTHOR]

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease‐activated receptor 2 (PAR2). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL‐6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte‐specific PAR2‐null mice were generated using K14‐Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14‐Cre:F2rl1wt/wt), but not littermate keratinocyte‐specific PAR2‐null (K14‐Cre:F2rl1fl/fl) mice. Keratinocyte‐specific ablation of PAR2 prevented the significant Lig + Inf‐induced increase (2.8‐fold) in the number of osteoclasts in alveolar bone and the significant up‐regulation (2.4–4‐fold) of the inflammatory markers IL‐6, IL‐1β, interferon‐γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis‐induced bone loss and will help in designing novel therapies with which to treat the disease. [ABSTRACT FROM AUTHOR]

Activation of protease-activated receptor-2 (PAR 2 ) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR 2 conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR 2 -null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR 2 in thymocytes isolated from T cell-specific PAR 2 -null mice resulted in thymic and splenic hypoplasia and a reduction in the cells of the cortex and a loss of distinction between the cortex and the medulla of the thymus. FACS analysis confirmed significant reductions in CD4 and CD8 double negative (DN3 and DN4) sub-populations, as well as double positive and single positive T cells, in T cell-specific PAR 2 -null mice compared to Cre expressing PAR 2 wild-type mice. The proportion of annexin V positive and propidium iodide negative cells was increased in CD4 and CD8 double negative, double positive and single positive T cells from T cell-specific PAR 2 -null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR 2 -null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR 2 -null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR 2 plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR 2 conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR 2 . [ABSTRACT FROM AUTHOR]