Assessment of subchondral bone microdamage quantification using contrast-enhanced imaging techniques.

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.

Effect of a protease-activated receptor-2 antagonist (GB88) on inflammation-related loss of alveolar bone in periodontal disease.

BACKGROUND AND OBJECTIVE: Protease-activated receptor-2 (PAR2 ), a pro-inflammatory G-protein coupled receptor, has been associated with pathogenesis of periodontitis and the resulting bone loss caused by oral pathogens, including the keystone pathogen Porphyromonas gingivalis (P. gingivalis). We hypothesised that administration of a PAR2 antagonist, GB88, might prevent inflammation and subsequent alveolar bone resorption in a mouse model of periodontal disease. METHODS: Periodontitis was induced in mice by oral inoculations with P. gingivalis for a total of eight times over 24 days. The infected mice were treated with either GB88 or vehicle for the duration of the trial. Following euthanasia on day 56, serum was collected and used for the detection of mast cell tryptase. The right maxillae were defleshed and stained with methylene blue to measure the exposed cementum in molar teeth. The left maxillae were prepared for cryosections followed by staining for tartrate-resistant acid phosphatase to identify osteoclasts or with toluidine blue to identify mast cells. Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of inflammatory cytokines in the gingival tissue. Supernatants of T-lymphocyte cultures isolated from the regional lymph nodes were assayed using a cytometric bead array to measure the Th1/Th2/Th17 cytokine levels. RESULTS: Measurement of the exposed cementum showed that GB88 reduced P. gingivalis-induced alveolar bone loss by up to 69%. GB88 also prevented the increase in osteoclast numbers observed in the infected mice. Serum tryptase levels were significantly elevated in both the infected groups, and not altered by treatment. RT-qPCR showed that GB88 prevented the upregulation of Il1b, Il6, Ifng and Cd11b. In T-lymphocyte supernatants, only IFNγ and IL-17A levels were increased in response to infection, but this was prevented by GB88 treatment. CONCLUSIONS: GB88 significantly reduced osteoclastic alveolar bone loss in mice infected with P. gingivalis, seemingly by preventing the upregulation of several inflammatory cytokines. PAR2 antagonism may be an effective treatment strategy for periodontal disease.

Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease.

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-1ß, 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.

Differences in bone turnover markers and injury risks between local and international horses: A Victorian Spring Racing Carnival study.

BACKGROUND: Musculoskeletal injuries (MSI) are common in racehorses and have been of increasing concern in horses travelling internationally to compete. Understanding the differences in bone turnover between local horses and international horses following long-distance air transportation may inform MSI prevention strategies. OBJECTIVES: To understand the differences in bone turnover markers and risk of MSI between local horses and international horses following long-distance air transportation. STUDY DESIGN: Prospective cohort. METHODS: The concentrations of bone turnover markers (OCN and CTXI), markers of stress (cortisol), inflammation (serum amyloid A) and circadian rhythm (melatonin), and bisphosphonates were determined in blood samples collected twice (14-17 days apart), from horses following international travel (n = 69), and from local horses (n = 79). The associations between markers, long-distance travel and MSI were determined using multivariable generalised linear regression models. RESULTS: Within 3-5 days post-transport, concentrations of cortisol in international horses were higher than those of local horses (main effect, Coef. 0.39; 95% CI 0.24, 0.54; p < 0.001) but they decreased and were not different to those of local horses at the second timepoint (interaction effect, Coef. -0.27; 95% CI -0.46, -0.07; p = 0.007). After adjusting for age and sex, OCN and CTXI were not significantly different between international and local horses; however, OCN was lower in international horses at timepoint 2 (interaction effect, Coef. -0.16; 95% CI -0.31, -0.01; p = 0.043). The prevalence of MSI was higher in the international (26%; 95% CI 16, 38%) compared with local horses (8%; 95% CI 3, 16%; p < 0.001), with all severe MSI sustained by the international horses. At the second timepoint compared with the first timepoint post-transport, cortisol remained high or increased (interaction effect, Coef. 0.43; 95% CI 0.24, 0.61; p < 0.001) and OCN increased (interaction effect, Coef. 0.26; 95% CI 0.08, 0.44; p = 0.006) in the horses that sustained severe MSI. MAIN LIMITATIONS: Horse population and racing career parameters differed between groups. Bone turnover markers have low sensitivity to detect local bone changes. CONCLUSIONS: Most horses showed minimal effects of long-distance air transport within 2 weeks relative to local horses as assessed by stress and bone turnover markers. Screening for persistent high cortisol and evidence of net bone formation after long-distance air transportation may help to identify racehorses at high risk of catastrophic MSI.

CONTEXTE: Les blessures musculosquelettiques (MSI) sont communes chez les chevaux de course et demeurent une source d'inquiétude pour les chevaux voyageant à l'international. Comprendre les différences de remodelage osseux entre les chevaux locaux et ceux voyageant suivant un trajet aérien longue distance pourrait aider au développement de stratégies de prévention des dommages musculosquelettiques. OBJECTIFS: Comprendre les différences de marqueurs de remodelage osseux et de risques de MSI entre les chevaux locaux et ceux voyageant à l'international suivant un transport aérien de longue distance. TYPE D'ÉTUDE: Étude de cohorte prospective. MÉTHODES: Les concentrations des marqueurs de remodelage osseux (OCN et CTXI), de stress (cortisol), d'inflammation (serum amyloid A), de rythme circadien (melatonin) et les bisphosphonates ont été mesurés dans des échantillons sanguins à deux reprises (14­17 jours à part) chez des chevaux ayant été à l'international (n = 69) et étant restés localement (n = 79). L'association entre les marqueurs, le transport longue distance et les MSI a été déterminée par modèles de régression linéaire multivarié généralisé. RÉSULTATS: Entre 3 à 5 jours suivant le transport, les concentrations de cortisol chez les chevaux internationaux étaient supérieures aux chevaux locaux (effet primaire, Coef. 0.39; 95% CI 0.24, 0.54; P < 0.001), mais ont diminué par la suite jusqu'à ne plus être différent de ceux des chevaux locaux à la deuxième mesure (effet interaction, Coef. −0.27; 95% CI −0.46, −0.07; P = 0.007). Après ajustement pour l'âge et le sexe, OCN et CTXI n'étaient pas significativement différents entre les chevaux internationaux et locaux. Cependant, OCN était inférieur chez les chevaux internationaux à la deuxième mesure (effet interaction, Coef. −0.16; 95% CI −0.31, −0.01; P = 0.043). La prévalence de MSI était plus élevée chez les chevaux internationaux (26%; 95% CI 16, 38%) comparativement aux chevaux locaux (8%; 95% CI 3, 16%; p < 0.001), avec toutes les MSI sévères subi par les chevaux internationaux. Au moment de la deuxième mesure comparée à la première mesure après le transport, le cortisol est demeuré élevé ou a augmenté (effet interaction, Coef. 0.43; 95% CI 0.24, 0.61; P < 0.001) et l'OCN a augmenté (effet interaction, Coef. 0.26; 95% CI 0.08, 0.44; P = 0.006) chez les chevaux ayant subi une MSI sévère. LIMITES PRINCIPALES: La population équine et leurs paramètres de course diffèrent entre les groupes. Les marqueurs de remodelage osseux ont une faible sensibilité pour la détection de changements osseux localisés. CONCLUSION: En deux semaines, les effets de transport aérien longue distance ont été minimaux pour la majorité des chevaux comparativement aux chevaux locaux, tel que démontré par les marqueurs de stress et de remodelage osseux. La détection de niveau élevé de cortisol de façon persistante et d'évidence d'os néoformé suivant un transport aérien de longue distance pourrait aider à détecter les chevaux de course à haut risque de MSI.

Reducing the prosthesis modulus by inclusion of an open space lattice improves osteogenic response in a sheep model of extraarticular defect.

Introduction: Stress shielding is a common complication following endoprosthetic reconstruction surgery. The resulting periprosthetic osteopenia often manifests as catastrophic fractures and can significantly limit future treatment options. It has been long known that bone plates with lower elastic moduli are key to reducing the risk of stress shielding in orthopedics. Inclusion of open space lattices in metal endoprostheses is believed to reduce the prosthesis modulus potentially improving stress shielding. However, no in vivo data is currently available to support this assumption in long bone reconstruction. This manuscript aims to address this hypothesis using a sheep model of extraarticular bone defect. Methods: Initially, CT was used to create a virtual resection plan of the distal femoral metaphyses and to custom design endoprostheses specific to each femur. The endoprostheses comprised additively manufactured Ti6Al4V-ELI modules that either had a solid core with a modulus of ∼120 GPa (solid implant group) or an open space lattice core with unit cells that had a modulus of 3-6 GPa (lattice implant group). Osteotomies were performed using computer-assisted navigation followed by implantations. The periprosthetic, interfacial and interstitial regions of interest were evaluated by a combination of micro-CT, back-scattered scanning electron microscopy (BSEM), as well as epifluorescence and brightfield microscopy. Results: In the periprosthetic region, mean pixel intensity (a proxy for tissue mineral density in BSEM) in the caudal cortex was found to be higher in the lattice implant group. This was complemented by BSEM derived porosity being lower in the lattice implant group in both caudal and cranial cortices. In the interfacial and interstitial regions, most pronounced differences were observed in the axial interfacial perimeter where the solid implant group had greater bone coverage. In contrast, the lattice group had a greater coverage in the cranial interfacial region. Conclusion: Our findings suggest that reducing the prosthesis modulus by inclusion of an open-space lattice in its design has a positive effect on bone material and morphological parameters particularly within the periprosthetic regions. Improved mechanics appears to also have a measurable effect on the interfacial osteogenic response and osteointegration.

Microstructural properties of the proximal sesamoid bones of Thoroughbred racehorses in training.

BACKGROUND: Proximal sesamoid bone fractures are common catastrophic injuries in racehorses. Understanding the response of proximal sesamoid bones to race training can inform fracture prevention strategies. OBJECTIVES: To describe proximal sesamoid bone microstructure of racehorses and to investigate the associations between microstructure and racing histories. STUDY DESIGN: Cross-sectional. METHODS: Proximal sesamoid bones from 63 Thoroughbred racehorses were imaged using micro-computed tomography. Bone volume fraction (BVTV) and bone material density (BMD) of the whole bone and four regions (apical, midbody dorsal, midbody palmar and basilar) were determined. Generalised linear regression models were used to identify the associations between bone parameters and race histories of the horses. RESULTS: The mean sesamoid BVTV was 0.79 ± 0.08 and BMD was 806.02 ± 24.66 mg HA/ccm. BVTV was greater in medial sesamoids compared with lateral sesamoids (0.80 ± 0.07 vs 0.79 ± 0.08; P < .001) predominantly due to differences in the apical region (medial-0.76 ± 0.08 vs lateral-0.72 ± 0.07; P < .001). BVTV in the midbody dorsal region (0.86 ± 0.06) was greater than other regions (midbody palmar-0.79 ± 0.07, basilar-0.78 ± 0.06 and apical-0.74 ± 0.08; P < .001). BVTV was greater in sesamoids with more microcracks on their articular surface (Coef. 0.005; 95% CI 0.001, 0.009; P = .01), greater extent of bone resorption on their abaxial surface (Grade 2-0.82 ± 0.05 vs Grade 1-0.80 ± 0.05 or Grade 0-0.79 ± 0.06; P = .006), in horses with a low (0.82 ± 0.07) or mid handicap rating (0.78 ± 0.08) compared with high rating (0.76 ± 0.07; P < .001), in 2- to 5-year-old horses (0.81 ± 0.07) compared with younger (0.68 ± 0.08) or older horses (0.77 ± 0.08; P < .001) and in horses that commenced their racing career at less than 4 years of age (0.79 ± 0.08 vs 0.77 ± 0.77; P < .001). BMD was greater in the midbody dorsal (828.6 ± 19.6 mg HA/ccm) compared with other regions (apical-805.8 ± 21.8, midbody palmar-804.7 ± 18.4 and basilar-785.0 ± 17.1; P < .001), in horses with a handicap rating (low-812.1 ± 20.0, mid-821.8 ± 21.3 and high-814.6 ± 19.4) compared with those with no rating (791.08 ± 24.4, P < .001), in females (806.7 ± 22.0) and geldings (812.2 ± 22.4) compared with entires (792.7 ± 26.2; P = .02) and in older horses (<2-year-old-763.7 ± 24.8 vs 2- to 5-year-old-802.7 ± 23.4, and 6- to 12-year-old-817.8 ± 20.0; P = .002). MAIN LIMITATIONS: Data were cross-sectional. CONCLUSIONS: Densification of the proximal sesamoid bones is associated with the commencement of racing in younger horses and the presence of bone fatigue-related pathology. Lower sesamoid BVTV was associated with longevity and better performance.

Associations between the radiographic appearance of vascular channels in proximal sesamoid bones, their microstructural characteristics and past racing performance in Thoroughbreds.

BACKGROUND: Abnormalities in vascular channel appearance within the proximal sesamoid bone (PSB) are the most common findings in Thoroughbred yearling presale radiographs and are often evaluated on radiographs of adult racehorses. Despite this, their pathogenesis and clinical significance are poorly understood, and associations with racing performance are inconsistent. OBJECTIVES: To determine microstructural characteristics of the PSBs associated with the radiographic appearance of vascular channels using microcomputed tomography (µCT) and to determine associations with past racing performance in mature horses. STUDY DESIGN: Cross-sectional. METHODS: One pair of PSBs were isolated from a forelimb of 59 Thoroughbred racehorses undergoing post-mortem examination. Each PSB (n = 118) was radiographed, assigned a vascular channel grade using previously published and novel grading systems, then imaged using µCT. Associations between radiographic, µCT and performance variables were investigated with uni- and multivariable generalised linear models. RESULTS: All PSBs had at least one vascular channel (mean 3.6 ± 0.89) observed on µCT originating from the abaxial border, yet in only 63.6% (75/118) were channels observed radiographically. Proximal sesamoid bones with a higher bone volume fraction (odds ratio [OR] 1.08; 95% confidence interval [CI] 1.01-1.15; P = .03) and wider channel diameter (mm) on µCT (OR 20.67; 95% CI 3.29-130.00; P = .001) were more likely to have vascular channels identified on radiographs. Greater radiographic channel number (OR 0.96; 95% CI 0.92-1.00; P = .04) and channel diameter (mm; OR 0.96; 95% CI 0.92-1.00; P = .04) were associated with fewer career placings. MAIN LIMITATIONS: Radiographs of isolated bones avoided the normal superimposition of tissue encountered in the live horse. CONCLUSIONS: The ability to identify vascular channels radiographically indicates widening of channels and densification of the PSB. More radiographic channels and greater channel diameter were associated with similar or poorer measures of past performance, suggesting that these changes are not desirable.

The vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation.

Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase) V0 subunit d2 (ATP6V0D2), was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium). In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.

A T cell-specific knockout reveals an important role for protease-activated receptor 2 in lymphocyte development.

Activation of protease-activated receptor-2 (PAR2) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR2 conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR2-null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR2 in thymocytes isolated from T cell-specific PAR2-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 PAR2-null mice compared to Cre expressing PAR2 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 PAR2-null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR2-null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR2-null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR2 plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR2 conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR2.

Identification of novel osteochondrosis--Associated genes.

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 ß4 (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.