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Introduction To investigate the role of the low-density lipoprotein receptor-related protein 5 (Lrp5) in bones' responses to loading, we analysed changes in multiple measures of bone architecture in tibias subjected to loading or disuse in male and female mice with the Lrp5 loss of function mutation (Lrp5−/−) or heterozygous for the Lrp5 G171V High Bone Mass (HBM) mutation (Lrp5HBM+). Materials and methods The right tibias of these 17 week old male and female mice and their Wild Type (WT) littermates were subjected to short periods of loading three days a week for two weeks. Each tibia was loaded for 40 cycles, to produce peak strains at the midshaft within the low, medium or high physiological range (~ 1500, 2400 and 3000 microstrain, respectively). In similar groups of mice the right sciatic nerve was severed causing disuse of the right tibia for 3 weeks. Data from microCT of loaded, neurectomised and contra-lateral control tibias were analysed to quantify changes in the cortical and cancellous regions of the bone in the absence of functional strains and in response to graded strains in addition to those derived from function. Results and conclusion Male WT+/+ controls showed significant strain:response curves for cortical area and trabecular thickness, but Lrp5−/− mice showed no detectable strain:response in those same outcomes. Female mice of either WT+/+ or Lrp5−/− genotype did not show significant strain:response curves for cortical or trabecular parameters, the one exception being Tb.Th in Lrp5−/− mice. Since female WT+/+ mice did not respond to loading in a significant dose:responsive manner, the similar lack of responsiveness of the Lrp5−/− females could not be ascribed to their Lrp5 status. Cortical bone loss associated with disuse showed no differences between Lrp5−/− mice and WT+/+ controls, but in cancellous bone of both male and females of these mice, there was a greater loss than in WT+/+ controls. In contrast, the tibias of male and female mice heterozygous for the Lrp5 G171V HBM mutation showed greater osteogenic responsiveness to loading and less bone loss associated with disuse than their WTHBM− controls. These data indicate that the presence of the Lrp5 G171V HBM mutation is associated with an increased osteogenic response to loading but support only a marginal gender-related role for normal Lrp5 function in this loading-related response., Research highlights ► Lrp5−/− increased cancellous but not cortical bone loss with disuse. ► Lrp5−/− decreases load-induced new bone formation in males but not in females. ► Lrp5 G171V High Bone Mass mutation protected against bone loss with disuse but increased bone response to loading.

To establish whether the combination of anti-resorptive therapy with mechanical loading has a negative, additive or synergistic effect on bone structure, we assessed the separate and combined effects of risedronate and non-invasive dynamic loading on trabecular and cortical bone. Seventeen-week-old female C57BL/6 mice were given daily subcutaneous injections of vehicle (n = 20) or risedronate at a dose of 0.15, 1.5, 15 or 150 μg/kg/day (n = 10 in each) for 17 days. From the fourth day of treatment, the right tibiae were subjected to a single period of axial loading (40 cycles/day) for three alternate days per week for two weeks. The left tibiae were used as internal controls. Trabecular and cortical sites in the tibiae were analyzed by high-resolution micro-computed tomography and imaging of fluorochrome labels. In the non-loaded tibiae, treatment with the higher doses of risedronate at 15 or 150 μg/kg/day resulted in higher trabecular bone volume and trabecular number than in vehicle-treated controls, whereas such treatment was associated with no differences in cortical bone volume at any dose. In the loaded tibiae, loading induced increases in trabecular and cortical bone volume compared with contra-lateral controls primarily through increased trabecular thickness and periosteal expansion, respectively, independently of risedronate treatment. In conclusion, the response to mechanical loading in both trabecular and cortical bone in mice is therefore not impaired by short-term treatment with risedronate, even over a 1000-fold dose range. In considering the optimization of treatments for osteoporosis, it is reassuring that anti-resorptive therapy and mechanical loading can exert independent beneficial effects. This article is part of a Special Issue entitled Bisphosphonates., Research highlights ► We assessed the combined effect of a bisphosphonate and loading on bone structure. ► Non-invasive tibia loading and a 1000-fold dose range of risedronate were used. ► Loading significantly increased both cortical and trabecular bone mass. ► Risedronate does not reduce the osteogenic effect of loading.

Summary Reasons for performing study: No large scale epidemiological studies have previously quantified the occurrence of carpal, metacarpo- and metatarsophalangeal (MCP/MTP) joint injuries in Thoroughbred racehorses. Objectives: To develop an objective classification system for carpal and MCP/MTP joint injuries and estimate the incidence of these injuries in young Thoroughbreds in flat race training. Methods: In a prospective cohort study, data on daily exercise and veterinary-diagnosed carpal and MCP/MTP joint injuries were collected from Thoroughbreds monitored since starting training as yearlings, for up to 2 years. Cases were classified in one of 4 categories: 1) localised to a carpal or MCP/MTP joint based on clinical examination and/or diagnostic analgesia; no diagnostic imaging performed; 2) localised to a carpal or MCP/MTP joint based on clinical examination and/or diagnostic analgesia; radiographs taken but no abnormalities detected; 3) evidence of abnormality of subchondral bone and/or articular margin(s) on diagnostic imaging and 4) evidence of discontinuity of the articular surface on diagnostic imaging. Incidence rates and rate ratios were estimated using Poisson regression, adjusting for trainer-level clustering. Results: A total of 647 horses from 13 trainers throughout England contributed 7785 months at risk of joint injury. One-hundred-and-eighty-four cases of carpal (n = 82) or MCP/MTP (n = 102) joint injury were reported in 165 horses and classified in Category 1 (n = 21), Category 2 (n = 21), Category 3 (n = 72) or Category 4 (n = 70). The overall joint injury rate was 1.8 per 100 horse months (95% CI = 1.2, 2.8); rates did not differ significantly between 2- and 3-year-olds but females sustained Category 1 injuries at triple the rate of males (P = 0.03). Joint injury rates differed significantly between trainers (P

Summary 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 12.365 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.

Summary Reasons for performing study: Injuries to the skeleton are a major cause of morbidity and mortality in racehorses and age, gender and season have all been shown to influence risk of injury. Objectives: To use biochemical markers of bone cell activity to establish to whether cellularprocesses in bone underlie these described effects. Methods: Blood samples were collected monthly from 2-year-old horses in race training between November 1998 and September 1999. Mean age at the start of the study was 20 months (range 18–23 months), with no significant difference in average age between colts (n = 84) and fillies (n = 63). Three markers were measured; osteocalcin (OC, bone formation), the carboxyte rminal cross-linked telopeptide of type I collagen (ICTP, bone resorption) and the carboxyterminal propeptide of type I collagen (PICP), which is less ‘bone-specific’ than the other 2 markers. Results: Colts had, on average, 3.62 ng/ml higher O C concentrations (P = 0.044) and 0.68 mg/l higher ICTP concentrations (P = 0.01) than fillies. The effect of gender was not statistically significant for PICP. However, in May, PI CP concentrations were on average 157 mg/l higher in fillies than colts. There was no effect of age or season on marker concentrations. Conclusions: This study has shown that there are gender differences in bone turnover markers in 2-year-old Thoroughbreds; however, age, within the limited range studied, did not have a significant effect on bone cell activity. Lower bone marker concentrations may reflect smaller bone size and/or earlier skeletal maturation in fillies. An increase in concentrations of PICP in fillies in spring and early summer may relect an influence of sex hormones on collagen turnover. Potential relevance: Gender differences in bone cell activity in 2-year-old colts and fillies may influence bone's adaptive responses to training and risk of injury.

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.

Objective—To investigate the relationship between stage of estrous cycle and bone cell activity in Thoroughbreds. Sample Population—Blood samples collected from forty-seven 2-year-old Thoroughbred mares in training for racing. Procedures—Blood samples were collected monthly (in April through September) from the mares. Stage of estrus was determined by assessing serum progesterone concentration. Bone cell activity was determined by measuring concentrations of 2 markers of bone formation (osteocalcin and the carboxy-terminal propeptide of type I collagen [PICP]) and a marker of bone resorption (the cross-linked carboxy-terminal telopeptide of type I collagen [ICTP]) in sera. Results—When the relationship between stage of the estrous cycle and markers of bone cell activity was examined, serum concentrations of both osteocalcin and ICTP were significantly higher in mares that were in the luteal phase, compared with mares that were at other stages of the estrous cycle. Stage of estrus did not affect serum PICP concentration. Conclusions and Clinical Relevance—Results indicate that bone cell activity in Thoroughbred mares fluctuates during the estrous cycle; serum concentrations of markers of bone formation and bone resorption are increased during the luteal phase. Further studies are required to determine whether these changes are of clinical importance and increase the risk of injury for mares in training during the breeding season. As in humans, stage of estrus must be considered as a source of uncontrollable variability in serum bone marker concentrations in horses.

Biochemical markers of bone cell activity have recently been shown to be useful for monitoring skeletal health in domestic animals, including dogs and horses. The aim of this study was to evaluate a number of biochemical assays, originally developed for use in humans, for their ability to measure indicators of bone cell activity in serum and urine of normal cats over a range of ages. Bone alkaline phosphatase (BAP), a marker of bone formation, was measured in serum using wheatgerm lectin precipitation (WGL) and by ELISA. The curve derived from serial dilution of feline serum was parallel with the ELISA standard curve, indicating species cross-reactivity, and there was a significant relationship between assays (rs = 0.97, P0.001). Deoxypyridinoline (DPD), a marker of bone resorption, was measured in its total form in urine by HPLC and ELISA, and in its free form in serum and urine by ELISA. The dilution curve for free DPD in urine showed parallelism with the assay standard curve; however, the curves for total DPD in urine and serum did not. A significant relationship was established between total urinary DPD (HPLC) with total serum DPD (rs = 0.69, P0.001), and with free urinary DPD (rs = 0.95, P0.001) concentrations. Carboxy-terminal telopeptide of type I collagen (CTX) concentration, another marker of bone resorption, was measured in serum and urine by ELISA, and there was a significant relationship between assays (rs = 0.82, P0.001). CTX could not be measured reliably using an auto-analysis method. A significant relationship was established between total urinary DPD (HPLC) with serum CTX (rs = 0.59, P0.05), and urinary CTX (rs = 0.65, P0.001) concentrations. BAP (ELISA and WGL), total urinary DPD (HPLC), urinary CTX (ELISA), and serum CTX (ELISA) concentrations were significantly inversely correlated with age (rs = -0.66, -0.88, -0.61, -0.70, and -0.51, P0.05 respectively). Cats under two years of age had significantly higher BAP, total urinary DPD (HPLC), and urinary CTX concentrations compared to older cats. In conclusion, this study has shown that a number of commercially available assays provide reliable methods for non-invasively monitoring bone cell activity in cats and has shown that bone turnover decreases within the first two years of life, until complete skeletal maturity is attained. Future studies can now be directed at evaluating the potential clinical application of these methods.

Studies in humans have found circadian changes to be one of the most important sources of controllable preanalytical variability when evaluating bone cell activity using biochemical markers. It remains unclear whether similar circadian changes influence bone marker concentrations in the horse. The aim of this study was to characterize changes in serum concentrations of three biochemical markers of bone cell activity over a 24-h period in six 2-yr-old Thoroughbred mares, and to determine circadian variability in IGF-I, which regulates bone turnover. Three bone markers were measured in serum: osteocalcin, a marker of bone formation, the carboxy-terminal propeptide of type-I collagen (a marker of bone formation), and the carboxy-terminal telopeptide of type-I collagen (a marker of bone resorption). Data were analyzed using the cosinor technique, which fits a 24-h cycle to each dataset. A significant circadian rhythm was observed for osteocalcin (P = 0.028), with an estimated amplitude of 7.6% of the mean (95% confidence interval 1.3% to 16.3%), and an estimated peak time of 0900. However, the observed rhythm for the carboxy-terminal telopeptide of type-I collagen (amplitude = 7.4%) was not significant (P = 0.067), and there were no significant changes in concentrations of the carboxy-terminal propeptide of type-I collagen over the 24-h study period (P = 0.44). There was a small but significant circadian rhythm for IGF-I (P = 0.04), with an estimated amplitude of 3.4% (95% confidence interval 0.2 to 7.1%) and peak at 1730. Further studies are now required to determine the potential association between circadian changes in IGF-I and osteocalcin in the horse. Although no significant circadian variation was found in concentrations of the car-boxy-terminal propeptide of type-I collagen and the carboxy-terminal telopeptide of type-I collagen, this may in part be a result of the age of the animals that were still skeletally immature. Future studies should aim to determine whether these markers develop a circadian rhythm at a later age when growth is complete. In the meantime, consistency in time of sampling should continue to be considered best practice when measuring biochemical markers of bone turnover in the horse.

Periprosthetic bone loss is an important factor that limits implant survival after total hip arthroplasty (THA). In a randomized trial we previously reported that pamidronate therapy prevented periprosthetic bone loss and decreased urinary excretion of N-telopeptide collagen cross-links over the first 6 months after THA, but had no apparent effect on free deoxypyridinoline excretion (J Bone Miner Res 2001; 16:556–564). In this study we investigated this discrepant observation that pamidronate reduced conjugated cross-link excretion but had no effect on free cross-links. Free and total deoxypyridinoline (DPD) were assayed by reverse-phase high-performance liquid chromatography (HPLC) and by immunosorbent assay (ELISA) at preoperative baseline and at week 6 after surgery in 46 subjects who had taken part in the trial. Randomly selected, 22 subjects received a single 90 mg intravenous infusion of pamidronate and 24 received placebo. Acute rises in free and total DPD occurred in both study groups at week 6 (P < 0.05). Total DPD excretion was lower in the pamidronate group than in the placebo group when measured by both HPLC and ELISA (P < 0.05). No difference in free DPD was found between groups. A rise in the ratio of free to total DPD occurred in the pamidronate group at week 6 (P = 0.03), but not in the placebo group. Pamidronate treatment suppresses excretion of total DPD. This is consistent with the effect of pamidronate on other turnover markers and periprosthetic bone loss after THA. Urinary-free DPD is a poor marker of response to treatment as the ratio of free-to-total cross-links is affected by amino-bisphosphonate therapy.

This study describes longitudinal changes in serum levels of biochemical markers of bone cell activity in a group of 24 thoroughbred foals from birth to 18 months of age. The markers of bone formation included the type I collagen carboxy-terminal propeptide (PICP), the bone-specific isoenzyme of alkaline phosphatase (BAP), and osteocalcin (OC). Levels of the cross-linked telopeptide of type I collagen (ICTP), a marker of bone resorption, and the N-terminal propeptide of type III collagen (PNIIIP), a marker of soft tissue turnover, were also measured. Levels of all markers fell significantly between birth and 18 months of age (70-80 per cent); this decrease being most marked between 0 and 6 months. However, a transient increase in levels of the markers then occurred between 6 and 14 months of age. The timing of this increase was specific for each parameter. ICTP and OC concentrations increased between October and December. PICP concentrations increased between December and April whereas the increase in PIIINP was coincident with the peak in weight gain between April and June. Changes in BAP concentration were less distinct at this time. Season was shown to have significant effects on the biochemical markers independent from the effect of age. Concentrations of all markers decreased with increasing body weight and at any given age heavier horses had lower marker levels. These results show that biochemical markers of bone cell activity and soft tissue turnover follow characteristic patterns of change in growing thoroughbreds influenced by age, season and bodyweight. The demonstration that the reference ranges for the biochemical markers change from month to month means that single samples from individuals are of little value for monitoring bone cell activity in growing thoroughbreds.

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 carboxyterminal 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. In summary, the results of these assays for bone ALP and metabolites of type I collagen indicate that the treadmill exercise regimen used for this study resulted in a general increase in bone turnover in 2-year-old thoroughbreds. These findings indicate that biochemical marker determinations may provide a sensitive, noninvasive method of monitoring skeletal turnover during athletic training.

Biochemical markers of bone metabolism were analysed in serum samples obtained from 60 horses with no history of orthopaedic disease (age 3 months-20 years). Serum levels of the carboxyterminal propeptide of type I procollagen (PICP), a marker of bone formation and the pyridinoline cross linked telopeptide domain of type I collagen (ICTP), a putative marker of bone resorption, were measured by radioimmunoassay (RIA). Serum levels of the bone specific isoenzyme of alkaline phosphatase (BALP), another marker of bone formation, were measured by a wheatgerm agglutinin affinity (WGA) method. Total alkaline phosphatase levels were also determined. Serum levels of PICP were significantly correlated with bone ALP (r = 0.78, P < 0.0001) and ICTP (r = 0.87, P < 0.0001). ICTP levels also correlated significantly with bone ALP (r = 0.81, P < 0.0001). However, total alkaline phosphatase did not correlate significantly with PICP, ICTP and BALP in horses over 1 year of age. There was an inverse correlation between serum levels of all biochemical markers and age of animals, with the most significant changes seen over the first 2 years. In animals less than 1 year of age, the reference ranges (mean +/- s.d. 1.96) were as follows: PICP 1216-2666 micrograms/l, ICTP 13.8-26.7 micrograms/l, bone ALP 134-288 u/l and total ALP 223-498 u/l. In 2-year-olds, the equivalent reference ranges were: PICP 550-1472 micrograms/l, ICTP 7.96-22.8 micrograms/l, bone ALP 32.7-125 u/l and total ALP 134-238 u/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Bisphosphonate treatment of Paget's disease results in a large decrease in urinary peptide-bound pyridinolines but a smaller decrease in urinary free pyridinolines. This discrepancy could be explained by changes in renal handling of pyridinoline forms. We studied eight patients with Paget's disease treated with pamidronate. We collected blood and urine at baseline and at 3 and 14 days after treatment. We measured free and total deoxypyridinoline (DPD) in serum (S) and urine (U) by high-performance liquid chromatography (HPLC). The ratio of free to total DPD at baseline was (mean +/- SE) 13 +/- 1% in serum and 37 +/- 3% in urine; at 3 days, this had increased to 25 +/- 3% in serum and 62 +/- 7% in urine. Peptide-bound (pb) DPD decreased significantly 3 days after treatment: UpbDPD -63 +/- 11%, p < 0.001; SpbDPD -51 +/- 8%, p < 0.01. Free DPD decreased in the urine after 14 days: UfDPD -48 +/- 5%, p < 0.01; there was no significant change in SfDPD. The fractional excretion of pbDPD relative to creatinine was less than one at all time-points; however, the fractional excretion of fDPD was significantly greater than one throughout the study. As a consequence, the proportion of free DPD in the urine increased as bone turnover decreased. This resulted in a smaller decrease in urine free compared with peptide-bound DPD in response to bisphosphonate therapy. Thus, the conversion of peptide-bound to free DPD in the kidney may become more efficient as bone turnover decreases as a consequence of pamidronate treatment.