Joanna S. Price

Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha.

The Wnt/β-catenin pathway has been implicated in bone cell response to their mechanical environment. This response is the origin of the mechanism by which bone cells adjust bone architecture to maintain bone strength. Osteoporosis is the most widespread failure of this mechanism. The degree of osteoporotic bone loss in men and women is related to bio-available estrogen. Here we report that in osteoblastic ROS 17/2.8 cells and primary osteoblast cultures, a single short period of dynamic mechanical strain, as well as the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl, increased nuclear accumulation of activated β-catenin and stimulated TCF/LEF reporter activity. This effect was blocked by the estrogen receptor (ER) modulators ICI 182,780 and tamoxifen and was absent in primary osteoblast cultures from mice lacking ERα. Microarray expression data for 25,000 genes from total RNA extracted from tibiae of wild-type mice within 24 h of being loaded in vivo showed differential gene regulation between loaded and contralateral non-loaded bones of 10 genes established to be involved in the Wnt pathway. Only 2 genes were involved in loaded tibiae from mice lacking ERα (ERα-/-). Together these data suggest that Wnt/β-catenin signaling contributes to bone cell early responses to mechanical strain and that its effectiveness requires ERα. Reduced effectiveness of bone cell responses to bone loading, associated with estrogen-related decline in ERα, may contribute to the failure to maintain structurally appropriate bone mass in osteoporosis in both men and women.

Osteocytes Use Estrogen Receptor α to Respond to Strain but Their ERα Content Is Regulated by Estrogen

The role of mechanical strain and estrogen status in regulating ERα levels in bone cells was studied in female rats. OVX is associated with decreased ERα protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulnas medial surface, where artificial loading stimulates reversal of resorption to formation.

Bones' adaptive response to mechanical loading is essentially linear between the low strains associated with disuse and the high strains associated with the lamellar/woven bone transition

There is a widely held view that the relationship between mechanical loading history and adult bone mass/strength includes an adapted state or “lazy zone” where the bone mass/strength remains constant over a wide range of strain magnitudes. Evidence to support this theory is circumstantial. We investigated the possibility that the “lazy zone” is an artifact and that, across the range of normal strain experience, features of bone architecture associated with strength are linearly related in size to their strain experience. Skeletally mature female C57BL/6 mice were right sciatic neurectomized to minimize natural loading in their right tibiae. From the fifth day, these tibiae were subjected to a single period of external axial loading (40, 10‐second rest interrupted cycles) on alternate days for 2 weeks, with a peak dynamic load magnitude ranging from 0 to 14 N (peak strain magnitude: 0–5000 µε) and a constant loading rate of 500 N/s (maximum strain rate: 75,000 µε/s). The left tibiae were used as internal controls. Multilevel regression analyses suggest no evidence of any discontinuity in the progression of the relationships between peak dynamic load and three‐dimensional measures of bone mass/strength in both cortical and cancellous regions. These are essentially linear between the low‐peak locomotor strains associated with disuse (∼300 µε) and the high‐peak strains derived from artificial loading and associated with the lamellar/woven bone transition (∼5000 µε). The strain:response relationship and minimum effective strain are site‐specific, probably related to differences in the mismatch in strain distribution between normal and artificial loading at the locations investigated.

Mechano-transduction in Osteoblastic Cells Involves Strain-regulated Estrogen Receptor α-mediated Control of Insulin-like Growth Factor (IGF) I Receptor Sensitivity to Ambient IGF, Leading to Phosphatidylinositol 3-Kinase/AKT-dependent Wnt/LRP5 Receptor-independent Activation of β-Catenin Signaling

The capacity of bones to adjust their mass and architecture to withstand the loads of everyday activity derives from the ability of their resident cells to respond appropriately to the strains engendered. To elucidate the mechanisms of strain responsiveness in bone cells, we investigated in vitro the responses of primary mouse osteoblasts and UMR-106 osteoblast-like cells to a single period of dynamic strain. This stimulates a cascade of events, including activation of insulin-like growth factor I receptor (IGF-IR), phosphatidylinositol 3-kinase-mediated phosphorylation of AKT, inhibition of GSK-3β, increased activation of β-catenin, and associated lymphoid-enhancing factor/T cell factor-mediated transcription. Initiation of this pathway does not involve the Wnt/LRP5/Frizzled receptor and does not culminate in increased IGF transcription. The effect of strain on IGF-IR is mimicked by exogenous des-(1–3)IGF-I and is blocked by the IGF-IR inhibitor H1356. Inhibition of strain-related prostanoid and nitric oxide production inhibits strain-related (and basal) AKT activity, but their separate ectopic administration does not mimic it. Strain-related IGF-IR activation of AKT requires estrogen receptor α (ERα) with which IGF-1R physically associates. The ER blocker ICI 182,780 increases the concentration of des-(1–3)IGF-I necessary to activate this cascade, whereas estrogen inhibits both basal AKT activity and its activation by des-(1–3)IGF-I. These data suggest an initial cascade of strain-related events in osteoblasts in which strain activates IGF-IR, in association with ERα, so initiating phosphatidylinositol 3-kinase/AKT-dependent activation of β-catenin and altered lymphoid-enhancing factor/T cell factor transcription. This cascade requires prostanoid/nitric oxide production and is independent of Wnt/LRP5.

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.

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.

Deer antlers : a zoological curiosity or the key to understanding organ regeneration in mammals?

Many organisms are able to regenerate lost or damaged body parts that are structural and functional replicates of the original. Eventually these become fully integrated into pre‐existing tissues. However, with the exception of deer, mammals have lost this ability. Each spring deer shed antlers that were used for fighting and display during the previous mating season. Their loss is triggered by a fall in circulating testosterone levels, a hormonal change that is linked to an increase in day length. A complex ‘blastema‐like’ structure or ‘antler‐bud’ then forms; however, unlike the regenerative process in the newt, most evidence (albeit indirect) suggests that this does not involve reversal of the differentiated state but is stem cell based. The subsequent re‐growth of antlers during the spring and summer months is spectacular and represents one of the fastest rates of organogenesis in the animal kingdom. Longitudinal growth involves endochondral ossification in the tip of each antler branch and bone growth around the antler shaft is by intramembranous ossification. As androgen concentrations rise in late summer, longitudinal growth stops, the skin (velvet) covering the antler is lost and antlers are ‘polished’ in preparation for the mating season. Although the timing of the antler growth cycle is clearly closely linked to circulating testosterone, oestrogen may be a key cellular regulator, as it is in the skeleton of other male mammals. We still know very little about the molecular machinery required for antler regeneration, although there is evidence that developmental signalling pathways with pleiotropic functions are important and that novel ‘antler‐specific’ molecules may not exist. Identifying these pathways and factors, deciphering their interactions and how they are regulated by environmental cues could have an important impact on human health if this knowledge is applied to the engineering of new human tissues and organs.

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.

Role of endocrine and paracrine factors in the adaptation of bone to mechanical loading

There appears to be no unique mechanically sensitive pathway by which changes in bone loading regulate bone mass and architecture to ensure adequate structural strength. Rather, strain-derived changes in bone cells activate a number of nonspecific strain-sensitive pathways (including calcium fluxes, prostanoids, nitric oxide, extracellular signal-regulated kinase, and sclerostin), the activities of which are modified by a number of factors (including estrogen receptors) for which this contribution is subsidiary to other purposes. The strain-sensitive pathways modified by these factors interact with a number of other pathways, some of which appear to have specific osteoregulatory potential (eg, the parathyroid hormone pathway), whereas others such as the Wnt pathway appear to be associated primarily with the response mechanisms of proliferation, differentiation, and apoptosis. The outcome of these multiple interactions are stimuli for local bone formation, resorption, or maintenance of the status quo, to maintain existing bone architecture or adapt it to a new mechanical regimen.

Loading-related regulation of gene expression in bone in the contexts of estrogen deficiency, lack of estrogen receptor α and disuse

Loading-related changes in gene expression in resident cells in the tibia of female mice in the contexts of normality (WT), estrogen deficiency (WT-OVX), absence of estrogen receptor α (ERα−/−) and disuse due to sciatic neurectomy (WT-SN) were established by microarray. Total RNA was extracted from loaded and contra-lateral non-loaded tibiae at selected time points after a single, short period of dynamic loading sufficient to engender an osteogenic response. There were marked changes in the expression of many genes according to context as well as in response to loading within those contexts. In WT mice at 3, 8, 12 and 24 h after loading the expression of 642, 341, 171 and 24 genes, respectively, were differentially regulated compared with contra-lateral bones which were not loaded. Only a few of the genes differentially regulated by loading in the tibiae of WT mice have recognized roles in bone metabolism or have been linked previously to osteogenesis (Opn, Sost, Esr1, Tgfb1, Lrp1, Ostn, Timp, Mmp, Ctgf, Postn and Irs1, BMP and DLX5). The canonical pathways showing the greatest loading-related regulation were those involving pyruvate metabolism, mitochondrial dysfunction, calcium-induced apoptosis, glycolysis/gluconeogenesis, aryl hydrocarbon receptor and oxidative phosphorylation. In the tibiae from WT-OVX, ERα−/− and WT-SN mice, 440, 439 and 987 genes respectively were differentially regulated by context alone compared to WT. The early response to loading in tibiae of WT-OVX mice involved differential regulation compared to their contra-lateral non-loaded pair of fewer genes than in WT, more down-regulation than up-regulation and a later response. This was shared by WT-SN. In tibiae of ERα−/− mice, the number of genes differentially regulated by loading was markedly reduced at all time points. These data indicate that in resident bone cells, both basal and loading-related gene expression is substantially modified by context. Many of the genes differentially regulated by the earliest loading-related response were primarily involved in energy metabolism and were not specific to bone.