αSMA Osteoprogenitor Cells Contribute to the Increase in Osteoblast Numbers in Response to Mechanical Loading.

Bone is a dynamic tissue that site-specifically adapts to the load that it experiences. In response to increasing load, the cortical bone area is increased, mainly through enhanced periosteal bone formation. This increase in area is associated with an increase in the number of bone-forming osteoblasts; however, the origin of the cells involved remains unclear. Alpha-smooth muscle actin (αSMA) is a marker of early osteoprogenitor cells in the periosteum, and we hypothesized that the new osteoblasts that are activated by loading could originate from αSMA-expressing cells. Therefore, we used an in vivo fate-mapping approach in an established axial loading model to investigate the role of αSMA-expressing cells in the load-induced increase in osteoblasts. Histomorphometric analysis was applied to measure the number of cells of different origin on the periosteal surface in the most load-responsive region of the mouse tibia. A single loading session failed to increase the number of periosteal αSMA-expressing cells and osteoblasts. However, in response to multiple episodes of loading, the caudal, but not the cranial, periosteal surface was lined with an increased number of osteoblasts originating from αSMA-expressing cells 5 days after the initial loading session. The proportion of osteoblasts derived from αSMA-labeled progenitors increased by 70% (p < 0.05), and the proportion of αSMA-labeled cells that had differentiated into osteoblasts was doubled. We conclude that αSMA-expressing osteoprogenitors can differentiate and contribute to the increase in periosteal osteoblasts induced by mechanical loading in a site-specific manner.

Disuse rescues the age-impaired adaptive response to external loading in mice.

UNLABELLED: We aimed to determine whether aged bone's diminished response to mechanical loading could be rescued by modulating habitual activity. By reducing background loading, aged bone's response to loading increased to a level no different to young mice. This suggests, given the right stimulus, that ageing bone can respond to mechanical loading. INTRODUCTION: Age-related decline in bone mass has been suggested to represent an impaired ability of bone to adapt to its mechanical environment. In young mice, the tibia's response to external mechanical loading has been shown to increase when habitual activity is reduced by sciatic neurectomy. Here we investigate if neurectomy can rescue bone's response to loading in old mice. METHODS: The effect of tibial disuse, induced by unilateral sciatic neurectomy (SN), on the adaptive response to a single peak magnitude of dynamic load-engendered mechanical strain was assessed in 19-month-old (aged) mice. In a second experiment, a range of peak loads was used to assess the load magnitude-related effects of loading on a background of disuse in young adult and aged mice. Bone architecture was analysed using micro-computed tomography (µCT) and dynamic histomorphometry. RESULTS: In the first experiment, SN in aged mice was associated with a significant periosteal osteogenic response to loading not observed in sham-operated mice (7.98 ± 1.7 vs 1.02 ± 2.2 % increase in periosteally enclosed area, p < 0.05). In the second experiment, SN abrogated the expected age-related difference in the bones' osteogenic response to peak strain magnitude (p > 0.05). CONCLUSIONS: These data suggest that bones' age-related decline in osteogenic responsiveness to loading does not originate in bone cells to either assess, or appropriately respond to strain, but rather is likely to be due to inhibitory "averaging" effects derived from the habitual strains to which the bone is already adapted. If such "strain averaging" is applicable to humans, it suggests that gentle exercise may degrade the beneficially osteogenic effects of short periods of more vigorous activity.

The cyclooxygenase-2 selective inhibitor NS-398 does not influence trabecular or cortical bone gain resulting from repeated mechanical loading in female mice.

UNLABELLED: A single injection of the cyclooxygenase-2 (COX-2) selective inhibitor NS-398 reduces bone's osteogenic response to a single period of mechanical loading in female rats, while women taking COX-2 selective inhibitors do not have lower bone mass. We show that daily NS-398 injection does not influence bone gain from repeated loading in female mice. INTRODUCTION: Prostaglandins are mediators of bone cells' early response to mechanical stimulation. COX-2 expression is up-regulated by exposure of these cells to mechanical strain or fluid flow, and the osteogenic response to a single loading period is reduced by COX-2 inhibition. This study determined, in female mice in vivo, the effect of longer term COX-2 inhibition on adaptive (re)modelling of cortical and trabecular bone in response to repeated loading. METHODS: Nineteen-week-old female C57BL/6 mice were injected with vehicle or NS-398 (5 mg/kg/day) 5 days a week for 2 weeks. On three alternate days each week, the right tibiae/fibulae were axially loaded [40 cycles (7 min)/day] three hours after injection. Left limbs acted as internal controls. Changes in three-dimensional bone architecture were analysed by high-resolution micro-computed tomography. RESULTS: In control limbs NS-398 was associated with reduced trabecular number but had no influence on cortical bone. In loaded limbs trabecular thickness and cortical periosteally enclosed volume increased. NS-398 showed no effect on this response. CONCLUSION: Pharmacological inhibition of COX-2 by NS-398 does not affect trabecular or cortical bone's response to repeated mechanical loading in female mice and thus would not be expected to impair the functional adaptation of bone to physical activity in women.

Mechanical loading-related changes in osteocyte sclerostin expression in mice are more closely associated with the subsequent osteogenic response than the peak strains engendered.

UNLABELLED: Osteocyte sclerostin is regulated by loading and disuse in mouse tibiae but is more closely related to subsequent local osteogenesis than the peak strains engendered. INTRODUCTION: The purpose of this study was to assess the relationship between loading-related change in osteocyte sclerostin expression, local strain magnitude, and local bone modeling/remodeling. METHODS: The right tibiae of 19-week-old female C57BL/6 mice were subjected to non-invasive, dynamic axial loading and/or to sciatic neurectomy-induced disuse. The sclerostin status of osteocytes was evaluated immunohistochemically, changes in bone mass by micro-computed tomography, new bone formation by histomorphometry, and loading-induced strain by strain gauges and finite element analysis. RESULTS: In cortical bone of the tibial shaft, loading engendered strains of similar peak magnitude proximally and distally. Proximally, sclerostin-positive osteocytes decreased and new bone formation increased. Distally, there was neither decrease in sclerostin-positive osteocytes nor increased osteogenesis. In trabecular bone of the proximal secondary spongiosa, loading decreased sclerostin-positive osteocytes and increased bone volume. Neither occurred in the primary spongiosa. Disuse increased sclerostin-positive osteocytes and decreased bone volume at all four sites. Loading reversed this sclerostin upregulation to a level below baseline in the proximal cortex and secondary spongiosa. CONCLUSION: Loading-related sclerostin downregulation in osteocytes of the mouse tibia is associated preferentially with regions where new bone formation is stimulated rather than where high peak strains are engendered. The mechanisms involved remain unclear, but could relate to peak surface strains not accurately reflecting the strain-related osteogenic stimulus or that sclerostin regulation occurs after sufficient signal processing to distinguish between local osteogenic and non-osteogenic responses.

Identifying early osteoclastic resorptive lesions in feline teeth: a model for understanding the origin of multiple idiopathic root resorption.

BACKGROUND AND OBJECTIVE: Domestic cats commonly suffer from external osteoclastic tooth resorption, a disease with many similarities to human multiple idiopathic root resorption. In both diseases, it is unclear whether anatomical features of the tooth surface are associated with a predisposition for resorptive lesions. The aim of the present study was to investigate the origin and progression of early feline osteoclastic resorptive lesions in teeth exhibiting no clinical signs of disease. MATERIAL AND METHODS: The entire surfaces of 138 teeth from 13 adult cats were analysed using back-scattered electron microscopy. The distribution of lesions was assessed by tooth type, location and between individuals. RESULTS: Seventy-three (53%) teeth showed at least one resorptive lesion. Eleven (85%) cats had lesions, and there was a significant association between increasing age and incidence of resorptive lesions. The highest frequency occurred in mandibular molars (82%). On average, there were 3.5 lesions per tooth. Fifty-two (38%) teeth featured resorptive lesions at the cemento-enamel junction. Twenty-three per cent of teeth with resorptive lesions showed evidence of repair of lesions that was limited to the root surface. There was no evidence of repair of resorptive lesions at the cemento-enamel junction. CONCLUSION: Resorption is prevalent without evidence of clinical disease, and occurred at younger ages than previously reported. It can initiate anywhere on the root surface, but lack of repair of lesions at the cemento-enamel junction indicates that mechanisms of replacement are absent or compromised in this region. Whereas resorption of the root may undergo repair, resorption at the cervix may progress to clinically evident lesions.

Bone biomarkers and risk of fracture in two- and three-year-old Thoroughbreds.

The aim of this study was to determine whether bone biomarkers (osteocalcin, PICP, ICTP and CTX-I) could be used to identify 2- and 3-year-olds at increased risk of fracture in the subsequent flat racing season. It was concluded that these bone biomarkers cannot be used to identify 2- and 3-year-olds that sustain a fracture. Whether bone biomarkers have better predictive value in older horses or when measured serially in the same animal remains to be determined.

Descriptive epidemiology of fracture, tendon and suspensory ligament injuries in National Hunt racehorses in training.

REASONS FOR PERFORMING STUDY: While fractures and tendon injuries are known to be important diseases in National Hunt (NH) racehorses during racing, there are no accurate estimates of their incidence in NH training yards. OBJECTIVES: To estimate the incidence of fractures and tendon and suspensory ligament injuries (TLIs) in NH racehorses in training; to describe the injuries incurred and to compare injury incidence rates by horse age, trainer, gender and background (ex-flat vs. ex-store horses). METHODS: Cohort data were collected from 14 UK NH training yards for 2 racing seasons. Daily exercise regimens and details of fractures and TLIs occurring in study horses were recorded. RESULTS: Data were gathered from 1223 horses that spent 9466 months at risk of injury. The fracture incidence rate was 1.1/100 horse months and varied significantly by trainer (P<0.001) but not by gender, age or background. The pelvis and third metacarpal bone (MCIII) were the most common fracture sites, although this varied between racing and training. The TLI incidence rate was 1.9/100 horse months and varied significantly by trainer (P = 0.05) and age (P<0.001) but not by gender or background. However, ex-store horses were significantly more likely to have a TLI on the racecourse than ex-flat horses (P = 0.01). Superficial digital flexor injuries accounted for 89% of all TLIs, the remainder being suspensory ligament injuries. CONCLUSIONS AND POTENTIAL RELEVANCE: Fractures and TLIs are important causes of morbidity and mortality in NH racehorses in training in England. This study provides accurate estimates of their incidence in this population and provides a baseline against which to monitor the effect of future interventions.

Ultrasonographic assessment of the superficial digital flexor tendons of National Hunt racehorses in training over two racing seasons.

REASONS FOR PERFORMING STUDY: It is important to ascertain the prevalence of superficial digital flexor tendon (SDFT) injuries and to improve methods of predicting injury in National Hunt (NH) racehorses. OBJECTIVES: To establish: 1) the prevalence of SDFT tendinopathy in NH horses; 2) whether routine ultrasonography can be used to predict SDFT injuries; 3) whether previous tendinopathy predisposes to reinjury; 4) a normal range for the SDFT cross-sectional area (CSA); and 5) the effects of gender, age, background (ex-flat or ex-store), limb, training and rest periods on SDFT CSA. METHODS: Routine ultrasound assessment of the palmar metacarpal soft tissues of 263 NH racehorses was performed on up to 6 occasions over 2 NH racing seasons. RESULTS: The prevalence of SDFT pathology detected using ultrasonography was 24% (n = 148), with a nonsignificant variation between yards of 10-40%. No changes in SDFT CSA or ultrasonographic appearance were detected prior to injury. Older horses had a significantly higher prevalence of SDFT pathology compared to younger horses, and horses with tendinopathy were more likely to suffer an acute injury compared to horses with no evidence of pathology. A reference range for normal CSA measurements was established as 77-139 mm2 at level 4, from 142 horses with no ultrasonographic evidence of SDFT pathology. The CSA of normal horses did not vary significantly with age, limb or over 2 racing seasons, but did with sex and background. CONCLUSIONS: The study confirms that SDFT tendinopathy is common in NH horses, with substantial variation between training yards. Ultrasonography at 3 month intervals did not seem to predict acute SDFT injuries. POTENTIAL RELEVANCE: Variation in the prevalence of tendinopathy between yards suggests that training methods may influence injury rate. It was not possible to predict injury using routine ultrasonography and therefore other methods must be identified. A normal reference range for SDFT CSA is provided.

Transport of chloride and deuterated water in peat: The role of anion exclusion, diffusion, and anion adsorption in a dual porosity organic media.

The dual-porosity structure of peat and the extremely high organic matter content give rise to a complex medium that typically generates prolonged tailing and early 50% concentration breakthrough in the breakthrough curves (BTCs) of chloride (Cl-) and other anions. Untangling whether these observations are due to rate-limited (physical) diffusion into inactive pores, (chemical) adsorption or anion exclusion remains a critical question in peat hydrogeochemistry. This study aimed to elucidate whether Cl- is truly conservative in peat, as usually assumed, and whether the prolonged tailing and early 50% concentration breakthrough of Cl- observed is due to diffusion, adsorption, anion exclusion or a combination of all three. The mobile-immobile (MiM) dual-porosity model was fit to BTCs of Cl- and deuterated water measured on undisturbed cores of the same peat soils, and equilibrium Cl- adsorption batch experiments were conducted. Adsorption of Cl- to undecomposed and decomposed peat samples in batch experiments followed Freundlich isotherms but did not exhibit any trends with the degree of peat decomposition and sorption became negligible below aqueous Cl- concentrations of ~310 mg L-1. The dispersivity determined by fitting the Cl- BTCs whether assuming adsorption or no adsorption were significantly different than determined by the deuterated water (p < .0001). However, no statistical differences in dispersivity (p = .27) or immobile water content (p = .97) was observed between deuterated water and Cl- when accounting for anion exclusion. A higher degree of decomposition significantly increased anion exclusion (p < .0001) but did not influence the diffusion of either tracer into the immobile porosity. Contrary to previous assumptions, Cl- is not truly conservative in peat due to anion exclusion, and adsorption at higher aqueous concentrations, but the overall effect of anion exclusion on transport is likely minimal.

PEAT-CLSM: A Specific Treatment of Peatland Hydrology in the NASA Catchment Land Surface Model.

Peatlands are poorly represented in global Earth system modeling frameworks. Here we add a peatland-specific land surface hydrology module (PEAT-CLSM) to the Catchment Land Surface Model (CLSM) of the NASA Goddard Earth Observing System (GEOS) framework. The amended TOPMODEL approach of the original CLSM that uses topography characteristics to model catchment processes is discarded, and a peatland-specific model concept is realized in its place. To facilitate its utilization in operational GEOS efforts, PEAT-CLSM uses the basic structure of CLSM and the same global input data. Parameters used in PEAT-CLSM are based on literature data. A suite of CLSM and PEAT-CLSM simulations for peatland areas between 40°N and 75°N is presented and evaluated against a newly compiled data set of groundwater table depth and eddy covariance observations of latent and sensible heat fluxes in natural and seminatural peatlands. CLSM's simulated groundwater tables are too deep and variable, whereas PEAT-CLSM simulates a mean groundwater table depth of -0.20 m (snow-free unfrozen period) with moderate temporal fluctuations (standard deviation of 0.10 m), in significantly better agreement with in situ observations. Relative to an operational CLSM version that simply includes peat as a soil class, the temporal correlation coefficient is increased on average by 0.16 and reaches 0.64 for bogs and 0.66 for fens when driven with global atmospheric forcing data. In PEAT-CLSM, runoff is increased on average by 38% and evapotranspiration is reduced by 19%. The evapotranspiration reduction constitutes a significant improvement relative to eddy covariance measurements.

Days lost from training by two- and three-year-old Thoroughbred horses: a survey of seven UK training yards.

REASONS FOR PERFORMING STUDY: The first major epidemiological study of injury incidence in the UK flat racing Thoroughbred (TB), published in 1985, found lameness to be the single largest reason for days when horses failed to train. It was considered advisable to ascertain if progress has been made in reducing the problem of musculoskeletal injuries in the intervening period. OBJECTIVE: To quantify injury incidence and days lost from training by 2- and 3-year-old TBs in UK training yards during 2002 and 2003. METHODS: One-hundred-and-eighty-two yearling TBs were recruited at the end of 2001 and daily training and injury records maintained over the following 2 training and racing seasons. Days were defined as lost from training when a horse failed to train at a slow canter speed or faster, and could be assigned to one of 4 categories: lameness, medical, traumatic and unknown. The incidence and number of days lost due to specific injuries and medical conditions was determined by further subdividing the lameness and medical categories. RESULTS: The study period provided a total of 52,601 2-year-old and 29,369 3-year-old days available for training, with 2-year-olds failing to train on a significantly greater proportion of days available than 3-year-olds. Lameness was the most important condition causing horses to miss training, with stress fractures being the most important cause of lameness. Medical conditions were a relatively minor cause of days lost from training, accounting for approximately 5% of untrained days in the 2 age groups. CONCLUSIONS: In UK flat racehorses there has been little change in the proportion of days lost from training due to lameness over the last 20 years. POTENTIAL RELEVANCE: This study highlights the need for further efforts to reduce the problem of lameness in the racing TB.

Nutrient and mercury transport in a sub-arctic ladder fen peatland subjected to simulated wastewater discharges.

Safely treating wastewater in remote communities and mining operations in sub-arctic Canada is critical to protecting the surrounding aquatic ecosystems. Undisturbed fen peatlands have been used to minimize the release of contaminants to the aquatic ecosystems; however, there is a limited understanding of wastewater transport or polishing in undisturbed fen peatlands. To elucidate these processes, a small (9800m2, ~250m long) ladder fen was continuously injected with a wastewater surrogate derived from a custom fertilizer blend and 38m3day-1 of water for 51days. The simulated wastewater included sulphate (27.2mgL-1), nitrate (7.6mgL-1), ammonium (9.1mgL-1), phosphate (7.4mgL-1), and chloride (47.2mgL-1). Major ion, total mercury (THg) and methylmercury (MeHg) pore water concentrations were measured throughout the study period. No wastewater contaminants were detected in the site outlet (~250m down-gradient) and most wastewater contaminants, except for SO42- and Cl-, remained relatively immobile. Within the SO42- plume, MeHg and THg concentrations became highly elevated relative to background (up to 10ngL-1, ~ three to five-fold increase) and MeHg comprised 60-100% of dissolved THg in the pore water. No MeHg or THg was exported at the outflow. The large increase in THg cannot be solely accounted for by the increase in MeHg and was likely due to enhanced decomposition of the peat substrate by increased microbial activity due to electron acceptor loading. Since the added nutrients were effectively transformed, sequestered or otherwise removed from pore waters in this experimental system, it appears that fen peatlands have a large capacity to safely treat residential wastewater nutrients; however, the inadvertent increases in THg and MeHg require further investigation and potential management.

Cost-consequence analysis of two different active flowable hemostatic matrices in spine surgery patients.

OBJECTIVES: A recently published retrospective analysis comparing two different active flowable hemostatic matrices (FLOSEAL and SURGIFLO Kit with Thrombin) showed significantly increased resource use and complications (surgery time, risk of blood product transfusion, and amount of matrix used) with SURGIFLO use compared to FLOSEAL in major spine surgery, and also significantly increased surgical time with SURGIFLO use in severe spine surgery. This analysis was developed as a follow-up to this prior analysis, to evaluate the cost-consequence of using FLOSEAL vs SURGIFLO in major and severe spine surgery. METHODS: A cost consequence model was constructed from a US hospital provider perspective. Model parameters combined clinical inputs from the published retrospective analysis with supplemental analyses on annual spine surgery volume using the 2012 National Inpatient Sample (NIS) database. Cost of hemostatic matrices, blood product transfusion, and operating room time were identified from published literature. Various one-way and probabilistic sensitivity analyses were performed. RESULTS: The base case for a medium volume hospital showed that, compared to SURGIFLO, patients receiving FLOSEAL required three fewer blood product transfusions and saved 27 h of OR time, resulting in annual savings of $151 per major and $574 per severe spine surgery. Additional scenarios for high and low volume hospitals supported cost savings in the base case. Probabilistic sensitivity analysis revealed FLOSEAL was cost-saving in 76% of simulations in major spine and 97% of iterations in severe spine surgery. CONCLUSIONS: This economic analysis indicates that use of FLOSEAL instead of SURGIFLO hemostatic matrices to induce hemostasis in both major and severe spine surgery could potentially lead to sizable cost savings in US hospitals, regardless of spinal surgery case-mix.

A case-control study of factors associated with pelvic and tibial stress fractures in Thoroughbred racehorses in training in the UK.

Few epidemiological studies have investigated risk factors for musculoskeletal injury occurring in Thoroughbred racehorses during training, although it is the major cause of wastage in the racing industry. We recently conducted a large-scale epidemiological study to estimate the incidence of fracture in racehorses in training in the UK and to identify associated risk factors. Thirteen racehorse trainers provided data on horses in their care, with daily recording of training information and provision of details on any fractures incurred. Data were collected for 2 years, including two consecutive flat racing seasons (1999 and 2000). This paper describes findings from a nested case-control study investigating factors associated with the occurrence of pelvic and tibial stress fractures in our study population. Cases were identified from the main study and defined as horses with a pelvic or tibial stress fracture, confirmed through routine diagnostic imaging. Randomly selected controls were matched on date of fracture in the case. Age and gender of the horse, its exercise history and training surfaces were examined as explanatory variables. Exercise was quantified as cumulative distances cantered and worked at high speed in 30- and 60-day periods prior to date of fracture in the case. Conditional logistic regression was used to construct multivariable models for the 30- and 60-day periods, respectively. We hypothesised that larger cumulative exercise distances would be associated with an increased risk of pelvic or tibial stress fracture and that different training surfaces would be associated with differences in fracture risk. In the 30-day period, when adjusting for trainer, the risk of pelvic or tibial stress fracture increased with increasing distance cantered, reaching a peak at around 50 km, after which the risk reduced. This trend was not obvious in the 60-day period, with no significant association between exercise distances and risk of stress fracture. Predominant use of one particular sand-based all-weather surface was related to an increased risk of pelvic or tibial stress injury, although this finding should be interpreted with caution. Surface maintenance and construction may play a role; variables that were not considered in the current analyses, which were based on a relatively small number of cases. Trainer was associated with differences in stress fracture risk after adjusting for exercise distances and surface but age and gender were not.

Training-related factors associated with dorsometacarpal disease in young Thoroughbred racehorses in the UK.

REASONS FOR PERFORMING STUDY: Dorsometacarpal disease (DMD) is common in young Thoroughbred (TB) racehorses, often interfering with their training programme. No reports exist on incidence rate or associated risk factors in the UK. OBJECTIVES: To estimate the incidence rate of DMD in TB horses in training for flat racing in the UK and identify associated risk factors, particularly in relation to training regimens. METHODS: Data were drawn from a large prospective study in which TB horses in 13 training yards were monitored for a period of up to 2 years, recording daily training information and details of musculoskeletal injury, including DMD. The incidence of DMD was calculated and survival analysis performed to determine features of the training regimen related to the risk of disease. RESULTS: A total of 335 horses provided 4235 months at risk of DMD. Seventy-nine first episodes were recorded, resulting in an incidence rate of 1.87/100 horse months (95% confidence interval 1.50-2.33). Increasing exercise distances at canter and high speed in short periods (up to 1 month) were associated with an increasing risk of DMD. However, increasing cumulative exercise distances since entering training were associated with a decreasing risk of the disease. CONCLUSIONS: The risk of DMD in young TBs decreases with accumulation of distances exercised at canter and high speed; this reflects the adaptive response of the third metacarpal bone to the loads placed upon it. However, increasing exercise distances in short periods (up to 1 month) increases the risk of DMD, probably as a consequence of microdamage and its associated remodelling response, and should therefore be avoided. POTENTIAL RELEVANCE: Training regimens for young TBs can be adjusted to prevent the occurrence of DMD. Early but gradual introduction of small amounts of high-speed exercise may be beneficial. Canter exercise should be kept minimal on high-speed work days and large amounts of both cantering and high-speed work per week, 2 weeks or per month should be avoided, particularly during the early stages of training, in order to minimise the risk of DMD.

Biochemical markers of bone metabolism and risk of dorsal metacarpal disease in 2-year-old Thoroughbreds.

REASONS FOR PERFORMING STUDY: Dorsal metacarpal disease (DMD) is a common problem in 2-year-old racehorses and results in loss of a significant number of days from training. Biochemical markers of bone cell activity measured early in the training season could have value for identifying 2-year-old Thoroughbred racehorses that develop DMD. OBJECTIVES: To determine the association between serum concentrations of osteocalcin, the carboxyterminal propeptide of type I collagen (PICP) and the carboxyterminal cross-linked telopeptide of type I collagen (ICTP) measured early in the training season and the risk of DMD. METHODS: Blood samples were collected from 165 two-year-old Thoroughbreds during late November/early December. Osteocalcin and PICP were measured as markers of bone formation, and ICTP as a marker of bone resorption. Training and veterinary records for each horse were monitored over the following training/racing season (10 months). Cases were defined as an episode where signs of DMD were sufficiently severe for a horse to miss at least 5 consecutive days of training. Classification tree and logistic regression analysis were used to identify the most important factors suitable for prediction of DMD risk. RESULTS: There were 24 cases of DMD during the season (14.6% cumulative incidence), with an average time to recognition of approximately 6 months (May). The earliest recognised case was in February and the latest in September. Osteocalcin and ICTP concentrations in the early stages of the training season were significantly higher in horses that subsequently developed DMD (P = 0.017 and 0.019, respectively). DMD cases were also significantly older compared to noncases (21.04 vs. 20.44 months, P = 0.023). Using a multivariable logistic regression model, it was possible to postulate a set of diagnostic rules to predict the likelihood of DMD injury during the season. This suggested that horses with ICTP concentrations above 12365 ug/l and older than 20.5 months are 2.6 times more likely to develop DMD. CONCLUSIONS: The measurement of the bone resorption marker ICTP could be useful for identification of 2-year-olds at increased risk of developing DMD. POTENTIAL RELEVANCE: These findings, together with other strategies such as modification of training regimens, e.g. early introduction of short distances of high-speed exercise into the training programme, could help reduce the days lost to training as a result of DMD.

Mechanical strain stimulates osteoblast proliferation through the estrogen receptor in males as well as females.

Mechanical strain, testosterone, and estrogen all stimulate proliferation of primary cultures of male rat long bone (LOB)-derived osteoblast-like cells as determined by [3H]thymidine incorporation. The maximum proliferative effect of a single period of mechanical strain (3400 microepsilon, 1 Hz, and 600 cycles) is additional to that of testosterone (10(-8) M) or estrogen (10(-8) M). The cells' proliferative response to strain is abolished both by concentrations of tamoxifen that cause proliferation (10(-8) M) and by those that have no effect (10(-6) M). Strain-related proliferation also is reduced by the estrogen antagonist ICI 182,780 (10(-8) M) but is unaffected by the androgen receptor antagonist hydroxyflutamide (10(-7) M). Tamoxifen, ICI 182,780, and the aromatase inhibitor 4-dihydroandrostenedione, at concentrations that have no effect on basal proliferation, significantly reduce the proliferative effect of the aromatizable androgen testosterone but not that of the nonaromatizable androgen 5alpha-dihydrotestosterone. Hydroxyflutamide, at a concentration that has no effect on basal proliferation (10(-7) M), eliminates the proliferative effect of 5alpha-dihydro-testosterone but had no significant effect on that caused by testosterone. Proliferation associated with strain is blocked by neutralizing antibody to insulin-like growth factor II (IGF-II) but not by antibody to IGF-I. Proliferation associated with testosterone is blocked by neutralizing antibody to IGF-I but is unaffected by antibody to IGF-II. These data suggest that in rat osteoblast-like cells from males, as from females, strain-related proliferation is mediated through the estrogen receptor (ER) in a manner that does not compete with estrogen but that can be blocked by ER modulators. Proliferation associated with testosterone appears to follow its aromatization to estrogen and is mediated through the ER, whereas proliferation associated with 5alpha-dihydrotestosterone is mediated by the androgen receptor. Strain-related proliferation in males, as in females, is mediated by IGF-II, whereas proliferation associated with estrogen and testosterone is mediated by IGF-I.

The estrogen receptor's involvement in osteoblasts' adaptive response to mechanical strain.

The estrogen receptor's role in bone cells' response to mechanical strain was investigated by studying the effect of the estrogen receptor modulators ICI 182, 780 and tamoxifen on the proliferation of primary cultures of rat long bone-derived osteoblasts stimulated by the independent and combined effects of 17beta-estradiol, mechanical strain, and the mitogens basic fibroblast growth factor (bFGF), truncated insulin-like growth factor (tIGF)-I and tIGF-II, and epidermal growth factor (EGF). 17Beta-estradiol (10(-10) M to 10(-8) M) increased [3H]thymidine incorporation equally in cells from males and females, as did a single period of cyclical strain in the plastic strips onto which the cells had been seeded (peak strain 3,400 microepsilon, 600 cycles, 1 Hz). At 10(-8) M, neither ICI 182,780 nor tamoxifen had any effect on basal [3H]thymidine incorporation in these cells, but both compounds prevented their proliferative responses to 10(-8) M 17beta-estradiol. Tamoxifen eliminated and ICI 182,780 substantially reduced the proliferation stimulated by strain. 17Beta-estradiol partially rescued the strain-related response from the effect of tamoxifen but not that of ICI 182,780. Both tamoxifen and ICI 182,780 reduced proliferation stimulated by 10(-8) M EGF but had no effect on that by 10(-7) M bFGF or tIGF-I and tIGF-II. That both ICI 182,780 and tamoxifen, which in other tissues act as estrogen antagonists, should reduce osteoblast proliferation stimulated by 17beta-estradiol and EGF, but not that by FGF or the IGFs, was expected since the mitogenic effects of estrogen and EGF involve the estrogen receptor, whereas those of FGF and the IGFs do not. That these compounds should prevent osteoblasts' proliferative response to strain suggests that strain also stimulates mitogenesis by a mechanism involving the estrogen receptor. If this is so, bones' reduced ability to maintain their structural strength after the menopause could be explained by less effective strain-related (re)modeling when estrogen is absent and, among other changes, the estrogen receptor could be down-regulated.

Nuclear localization of type I parathyroid hormone/parathyroid hormone-related protein receptors in deer antler osteoclasts: evidence for parathyroid hormone-related protein and receptor activator of NF-kappaB-dependent effects on osteoclast formation in regenerating mammalian bone.

Parathyroid hormone-related protein (PTHrP) is not required for osteoclastogenesis during embryonic development; however, after birth it has been shown to regulate osteoclast formation during tooth eruption. Our study explores the hypothesis that PTHrP also may regulate osteoclast differentiation in the regenerating skeletal tissues of deer antlers, bones capable of complete regeneration. Osteoclast-like multinucleated cells (MNCs) formed spontaneously in micromass cultures derived from antler cartilage and these cells had the phenotypic characteristics of osteoclasts. PTHrP and receptor activator of NF-kappaB ligand (RANKL) stimulated antler osteoclast formation although the effect of RANKL was less marked than that of PTHrP. The addition of osteoprotegerin (OPG) only partially decreased (by approximately 65%) the number of osteoclasts in PTHrP-treated cultures. To determine whether PTHrP also potentially could have direct effects on antler osteoclasts, we studied, by confocal microscopy, the expression of the type I PTH/PTHrP receptor (PTH1R) in MNCs cultured on glass and found the receptor protein to have a nuclear localization. In situ hybridization showed that antler MNCs also expressed PTH1R and PTHrP messenger RNAs (mRNAs). PTHrP was immunolocalized in MNCs cultured on glass but was undetectable in cells resorbing a dentine substrate. In tissue sections of antler cartilage, PTHrP and PTH1R were expressed in vitronectin receptor-positive (VNR+) osteoclast-like cells localized in the perivascular stroma. Thus, these data show that PTHrP plays a role in the regulation of osteoclast differentiation in regenerating skeletal tissues and that PTHrP can have effects on osteoclastogenesis that are independent of RANKL synthesis. Ours is the first study to describe the expression of the type I PTH/PTHrP receptor in mammalian osteoclasts at a protein and mRNA level, which indicates that PTHrP also may have a direct effect on osteoclasts. This also is the first study to show a nuclear localization of the PTHIR in cells of the osteoclast lineage, although the functional significance of this observation has yet to be established.

The response of the skeleton to physical training: a biochemical study in horses.

In this study we tested the hypothesis that exercise induces an adaptive response in the developing skeleton which may be monitored in vivo by measuring biochemical markers of bone metabolism. The effects of exercise on two biochemical markers of bone formation were determined; the carboxy-terminal propeptide of type I procollagen (PICP), and the bone-specific isoenzyme of alkaline phosphatase (BAP), and one putative marker of resorption, the pyridinoline crosslinked telopeptide domain of type I collagen (ICTP). All three markers were measured for a year in 2-year-old thoroughbred horses exercised three times a week on a treadmill, and values compared to a control group of age-matched animals. Levels of all three markers fell in both exercised and control groups over the 12-month period reflecting normal age changes. However, there were differences between groups in the pattern of this decrease. When expressed as a percentage of baseline values, BAP was higher (p < 0.05) at 2 months and both BAP and the PICP were higher at 4 months (p < 0.01 and p < 0.05, respectively) in the exercised group, reflecting an increase in bone turnover in this group in the early stages of training. PICP levels were also elevated in the exercised group at 10 months and this result indicates an increase in bone turnover at this time. The changes in ICTP were different; at 2 months, levels were higher in exercised animals than in controls, but there was no significant difference between the two groups at 4 and 6 months. After 8 months, ICTP levels in the exercised group increased returning to near baseline values at 10 months.(ABSTRACT TRUNCATED AT 250 WORDS)