The effect of flushing of the common bile duct on hepatobiliary markers and short-term outcomes in dogs undergoing cholecystectomy for the management of gall bladder mucocele: A randomized controlled prospective study.

OBJECTIVE: To determine the effect of flushing of the common bile duct (CBD) on hepatobiliary markers and short-term outcome in dogs undergoing cholecystectomy for the management of gallbladder mucocele (GBM). STUDY DESIGN: Randomized, controlled, prospective study. ANIMALS: Thirty-two client-owned dogs. METHODS: Dogs were allocated randomly to either a "flush" group or a "non-flush group." Flushing was performed in a normograde fashion, followed by a routine cholecystectomy. Data collected included presenting clinical signs, preoperative and 3-day postoperative hepatobiliary markers (alkaline phosphatase, ALP; alanine aminotransferase, ALT; gamma glumatyl-transferase, GGT; bilirubin; cholesterol; triglycerides), duration of hospitalization, and complications. These data were compared between groups. RESULTS: Sixteen dogs were enrolled in each group. One dog (in the flush group) was excluded following diagnosis of hepatic lymphoma. Border terriers were overrepresented (20/31). Overall, there were marked reductions from preoperative to 3 days postoperative in serum bilirubin (p = .004), ALP (p = .020), ALT (p < .001), GGT (p = .025), and cholesterol (p < .001) values. There was no difference in any marker between groups. Survival to discharge was 90.3% (28/31 dogs). CONCLUSION: Cholestatic markers decreased significantly 3 days postcholecystectomy. No short-term clinical or clinico-pathological benefits were identified when flushing the CBD in dogs undergoing cholecystectomy for GBM. CLINICAL SIGNIFICANCE: The findings of the study do not support routine flushing of the CBD during cholecystectomy for GBM in dogs.

Long-term outcome of female dogs treated for intramural ectopic ureters with cystoscopic-guided laser ablation.

OBJECTIVE: To report the complications and long-term outcome of female dogs with intramural ectopic ureter(s) (iEU) undergoing cystoscopic-guided laser ablation (CLA) and determine the effect of post-CLA neutering on urinary continence. STUDY DESIGN: Retrospective clinical study. ANIMALS OR SAMPLE POPULATION: Thirty-four client-owned dogs. METHODS: Medical records of female dogs that had iEU-CLA were reviewed. A 10-point continence score was assigned before, immediately after, and at a minimum of 12 months postprocedure via owner telephone contact. Neutering status prior to and postprocedure was recorded. RESULTS: Continence scores increased in all dogs after CLA (p < .0001, mean duration of follow-up: 63.9 ± 5.7 months) with an increase of the median score from 2 (preprocedure) to 10 (postprocedure). A urethral tear occurred in 2/34 dogs immediately after the procedure, successfully managed conservatively. Mild hematuria was present in 2/34, lasting less than 48 h. Postoperative urinary tract infections were documented in 6/34 dogs. Two dogs died of urinary-related issues at 1 and 5 months after CLA. Complete and near-complete urinary continence (scores 9 and 10/10) was achieved in 26/32 dogs including 3 dogs requiring medical (2) or surgical interventions (1). Post-CLA neutering did not affect continence scores (p = .44). CONCLUSION: A large proportion of dogs regained and maintained full continence after CLA alone. Subsequent medical or surgical therapy allowed further improvements when needed. Post-CLA neutering did not negatively impact urinary continence score. CLINICAL SIGNIFICANCE: The beneficial effect of iEU-CLA in female dogs is long standing and not affected by postprocedural neutering.

Influence of muscle-sparing lateral thoracotomy on postoperative pain and lameness: A randomized clinical trial.

OBJECTIVE: To assess and compare the magnitude of lameness and level of pain after muscle-sparing lateral thoracotomy (MSLT) and standard lateral thoracotomy (SLT) in dogs. STUDY DESIGN: Randomized, blinded, prospective clinical study. ANIMALS: Twenty-eight client-owned dogs. METHODS: The latissimus dorsi muscle was retracted in the MSLT group and was transected in the SLT group. Gait was analyzed with a force plate, and the peak vertical force symmetry index (SI) was calculated within 24 hours before surgery, 3 days postoperatively, and 8 to 12 weeks postoperatively. Symmetry index and pain scores as measured by the Glasgow Composite Measure Pain Scale - Short Form were assessed as primary outcome measures. RESULTS: The SI 3 days postoperatively was lower compared with the preoperative SI value in all dogs, consistent with lameness of the ipsilateral thoracic limb (P < .001). The absolute differences in preoperative and 3-day-postoperative SI provided evidence that this change was 3.1-fold greater after SLT compared with after MSLT (P = .009). Pain scores 1 day after surgery were lower after MSLT (1) compared with after SLT (2.5, P < .001). CONCLUSION: Lateral thoracotomies caused postoperative pain and ipsilateral forelimb lameness, and both were reduced by sparing the latissimus dorsi. CLINICAL SIGNIFICANCE: Sparing the latissimus dorsi should be considered to decrease immediate postoperative morbidity in dogs undergoing lateral thoracotomy.

Outcomes of dogs treated for extrahepatic congenital portosystemic shunts with thin film banding or ameroid ring constrictor.

OBJECTIVE: To compare the outcomes of dogs treated at a single institution for single extrahepatic congenital portosystemic shunts (CPSS) by thin film banding (TFB) or by placement of an ameroid constrictor (AC). STUDY DESIGN: Retrospective case series. ANIMALS: Seventy-six client-owned dogs with CPSS treated with TFB (n = 53) or AC (n = 23). METHODS: Records were reviewed for signalment, preoperative, intraoperative, and postoperative management and short-term outcomes. Data on second surgeries were reviewed. Long-term outcomes were obtained via an owner-directed health-related quality of life questionnaire. The rates of complications, mortality, and revision surgery were compared between the treatment groups. RESULTS: Postoperative complications occurred in 15 (28%) dogs with TFB (9% mortality, n = 5) and 8 (35%) dogs with an AC (4% mortality, n = 1). Long-term follow-up was available in 41 of 56 dogs at a median of 55 months (range, 15-89). Revision surgery for persistent shunting was performed in 14 (29%) dogs treated initially with TFB and in no dogs treated initially with AC (P = .007). Median long-term outcome scores were good in both groups; nine of 14 revision surgeries led to favorable outcomes. CONCLUSION: Persistent shunting requiring revision surgery was more common when CPSS were treated with TFB than with an AC, but both treatments achieved favorable long-term outcomes. CLINICAL SIGNIFICANCE: Treatment of CPPS by placement of an AC rather than TFB seems more reliable for shunt attenuation and prevention of revision surgeries.

Mechanical properties of 6 finger-trap suture techniques.

OBJECTIVE: To identify the most common methods used by surgeons to place finger-trap sutures (FTS), and determine their influence on the biomechanical properties of constructs. STUDY DESIGN: Questionnaire and experimental study. METHODS: Six commonly used FTS methods (A-F) were identified from literature review and questionnaire. Constructs made with 3-metric nylon suture and 18-French polyurethane esophagostomy tubing were tested in axial loading to failure. Two patterns (B and D) selected based on common use and biomechanical performance were further tested, with 2, 4, and 8 repeats along the tube. Displacement, load, and energy at failure were compared between constructs, and failure mode was video recorded. RESULTS: Patterns E and F were susceptible to slipping (P < .001). Patterns A and D were stiffer than pattern E, and patterns A-D were stiffer than pattern F (P = .012). Patterns A and B had less extension than pattern E and F, and patterns A-D had less extension than pattern F (P = .002). 87.5% of FTS failed by breaking at the first suture knot. The number of repeats had no effect on FTS performance, but catastrophic failure occurred in 2 constructs with 2 repeats. CONCLUSION: The mechanical behavior of suture-tube constructs and failure mode is affected by the FTS pattern. Patterns E and F are not advocated due to suture slippage. The number of repeats may not affect the FTS performance, but a minimum of 4 repeats is recommended. Overall, patterns B, C, and D performed the best in axial loading.

Electrosurgery reduces blood loss and immediate postoperative inflammation compared to cold instruments for midline celiotomy in dogs: A randomized controlled trial.

OBJECTIVES: To compare the use of an electrosurgical device with traditional cold instruments (scalpel and scissors) for midline celiotomy incision. STUDY DESIGN: Prospective randomized controlled clinical trial. SAMPLE POPULATION: One hundred and twenty client-owned dogs undergoing abdominal surgery. METHODS: Dogs were prospectively recruited and randomized to receive electroincision or cold instrument incision. For cold incision, surgeons used basic surgical instruments including scalpel and scissors. For electroincision, surgeons only used the electrosurgical device in cutting mode. Time for the approach, blood loss, and the incision length were recorded. A blinded observer assessed pain and incision redness, swelling, and discharge at 24 and 48 hours postoperative (graded 0-3). Owner assessment of incision healing was recorded by telephone interview. RESULTS: Blood loss during surgery was significantly lower for electroincision (mean 0.7, SD 1.7 mL) than cold incision (mean 3.0, SD 4.3 mL, P < .0001) with no significant difference in incision length or time for approach. Electroincision was associated with significantly less incision redness (cold median 1, range 0-3; electroincision median 0, range 0-2, P = .02) and less incision discharge (cold median 0.5 range 0-3; electroincision median 0, range 0-1, P = .006) at 24 hours postoperative. There was no significant difference in pain scores or incision healing in dogs receiving the two techniques. No incisional hernias were reported. A surgical site infection occurred in 1 dog (cold incision). CONCLUSIONS: Electroincision for a celiotomy approach in the dog reduces blood loss, and incision redness and discharge in the immediate postoperative period without affecting the occurrence of wound complications such as infection and dehiscence (including linea alba).

Colored Indicator Undergloves Increase the Detection of Glove Perforations by Surgeons During Small Animal Orthopedic Surgery: A Randomized Controlled Trial.

OBJECTIVE: To determine whether use of colored indicator gloves affects perforation detection rate and to identify risk factors for glove perforation during veterinary orthopedic surgery. STUDY DESIGN: Prospective randomized controlled trial. SAMPLE POPULATION: 574 double pairs of gloves worn during 300 orthopedic surgical procedures (2,296 gloves). METHODS: Primary and assistant surgeons double-gloved for all orthopedic surgical procedures. Type of inner glove (standard or colored indicator) was randomized for the first 360 double pairs of gloves worn by surgeons during 180 procedures. Perforations detected by surgeons were recorded and gloves changed if requested. For a further 120 procedures, indicator gloves were used exclusively. All gloves were leak-tested after surgery to identify perforations. Association between potential risk factors and perforation was explored using multivariate logistical regression analysis. RESULTS: Glove perforations occurred during 43% of surgeries with a mean of 2.3 holes/surgery. Inner gloves were intact in 63% of glove pairs where an outer perforation occurred. Intraoperative perforation detection was improved when colored indicator gloves were worn (83% sensitivity) vs. standard gloves (34% sensitivity; P<.001). Independent risk factors for perforation were placement of plates and/or screws (P=.001; OR=2.4; 95% CI, 1.4-4.0), placement of an external skeletal fixator (P=.002; OR=7.0; 95% CI, 2.1-23.8), use of orthopedic wire (P=.011; OR=2.4; 95% CI, 1.2-4.7), and primary surgeon being board-certified (P=.016; OR=1.9; 95% CI, 1.1-3.1). CONCLUSION: Increased surgeon recognition of glove perforations through use of colored indicator gloves enables prompt change of gloves if perforation occurs and may reduce potential contamination of the surgical site.

Protein kinase Cα (PKCα) regulates bone architecture and osteoblast activity.

Bones' strength is achieved and maintained through adaptation to load bearing. The role of the protein kinase PKCα in this process has not been previously reported. However, we observed a phenotype in the long bones of Prkca(-/-) female but not male mice, in which bone tissue progressively invades the medullary cavity in the mid-diaphysis. This bone deposition progresses with age and is prevented by disuse but unaffected by ovariectomy. Castration of male Prkca(-/-) but not WT mice results in the formation of small amounts of intramedullary bone. Osteoblast differentiation markers and Wnt target gene expression were up-regulated in osteoblast-like cells derived from cortical bone of female Prkca(-/-) mice compared with WT. Additionally, although osteoblastic cells derived from WT proliferate following exposure to estradiol or mechanical strain, those from Prkca(-/-) mice do not. Female Prkca(-/-) mice develop splenomegaly and reduced marrow GBA1 expression reminiscent of Gaucher disease, in which PKC involvement has been suggested previously. From these data, we infer that in female mice, PKCα normally serves to prevent endosteal bone formation stimulated by load bearing. This phenotype appears to be suppressed by testicular hormones in male Prkca(-/-) mice. Within osteoblastic cells, PKCα enhances proliferation and suppresses differentiation, and this regulation involves the Wnt pathway. These findings implicate PKCα as a target gene for therapeutic approaches in low bone mass conditions.

Estrogen receptor α mediates proliferation of osteoblastic cells stimulated by estrogen and mechanical strain, but their acute down-regulation of the Wnt antagonist Sost is mediated by estrogen receptor ß.

Mechanical strain and estrogens both stimulate osteoblast proliferation through estrogen receptor (ER)-mediated effects, and both down-regulate the Wnt antagonist Sost/sclerostin. Here, we investigate the differential effects of ERα and -ß in these processes in mouse long bone-derived osteoblastic cells and human Saos-2 cells. Recruitment to the cell cycle following strain or 17ß-estradiol occurs within 30 min, as determined by Ki-67 staining, and is prevented by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride. ERß inhibition with 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-ß]pyrimidin-3-yl] phenol (PTHPP) increases basal proliferation similarly to strain or estradiol. Both strain and estradiol down-regulate Sost expression, as does in vitro inhibition or in vivo deletion of ERα. The ERß agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and ERB041 also down-regulated Sost expression in vitro, whereas the ERα agonist 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl]tris-phenol or the ERß antagonist PTHPP has no effect. Tamoxifen, a nongenomic ERß agonist, down-regulates Sost expression in vitro and in bones in vivo. Inhibition of both ERs with fulvestrant or selective antagonism of ERß, but not ERα, prevents Sost down-regulation by strain or estradiol. Sost down-regulation by strain or ERß activation is prevented by MEK/ERK blockade. Exogenous sclerostin has no effect on estradiol-induced proliferation but prevents that following strain. Thus, in osteoblastic cells the acute proliferative effects of both estradiol and strain are ERα-mediated. Basal Sost down-regulation follows decreased activity of ERα and increased activity of ERß. Sost down-regulation by strain or increased estrogens is mediated by ERß, not ERα. ER-targeting therapy may facilitate structurally appropriate bone formation by enhancing the distinct ligand-independent, strain-related contributions to proliferation of both ERα and ERß.

Cortical thickness adaptation to combined mechanical loading and parathyroid hormone treatments is site specific and synergistic in the mouse tibia model.

In this study, we aimed to quantify the localised effects of mechanical loading (ML), low (20 µg/kg/day), moderate (40 µg/kg/day) or high (80 µg/kg/day) dosages of parathyroid hormone (PTH), and combined (PTHML) treatments on cortical bone adaptation in healthy 19-week old female C57BL/6 mice. To this end, we utilise a previously reported image analysis algorithm on µCT data of the mouse tibia published by Sugiyama et al. (2008) to measure changes in cortical area, marrow cavity area and local cortical thickness measures (ΔCt.Ar, ΔMa.Ar, ΔCt.Th respectively), evaluated at two cross-sections within the mouse tibia (proximal-middle (37 %) and middle (50 %)), and are compared to a superposed summation (P + M) of individual treatments to determine the effectiveness of combining treatments in vivo. ΔCt.Ar analysis revealed a non-linear, synergistic interactions between PTH and ML in the 37 % cross-section that saturates at higher PTH dosages, whereas the 50 % cross-section experiences an approximately linear, additive adaptation response. This coincided with an increase in ΔMa.Ar (indicating resorption of the endosteal surface), which was only counteracted by combined high dose PTH with ML in the middle cross-section. Regional analysis of ΔCt.Th changes reveal localised cortical thinning in response to low dose PTH treatment in the posteromedial region of the middle cross-section, signifying that PTH does not provide a homogeneous adaptation response around the cortical perimeter. We observe a synergistic response in the proximal-middle cross-section, with regions of compressive strain experiencing the greatest adaptation response to PTHML treatments, (peak ΔCt.Th of 189.32, 213.78 and 239.30 µm for low, moderate and high PTHML groups respectively). In contrast, PTHML treatments in the middle cross-section show a similar response to the superposed P + M group, with the exception of the combined high dose PTHML treatment which shows a synergistic interaction. These analyses suggest that, in mice, adding mechanical loading to PTH treatments leads to region specific bone responses; synergism of PTHML is only achieved in some regions experiencing high loading, while other regions respond additively to this combined treatment.

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.

Cortical Thickness Adaptive Response to Mechanical Loading Depends on Periosteal Position and Varies Linearly With Loading Magnitude.

The aim of the current study was to quantify the local effect of mechanical loading on cortical bone formation response at the periosteal surface using previously obtained µCT data from a mouse tibia mechanical loading study. A novel image analysis algorithm was developed to quantify local cortical thickness changes (ΔCt.Th) along the periosteal surface due to different peak loads (0N ≤ F ≤ 12N) applied to right-neurectomised mature female C57BL/6 mice. Furthermore, beam analysis was performed to analyse the local strain distribution including regions of tensile, compressive, and low strain magnitudes. Student's paired t-test showed that ΔCt.Th in the proximal (25%), proximal/middle (37%), and middle (50%) cross-sections (along the z-axis of tibia) is strongly associated with the peak applied loads. These changes are significant in a majority of periosteal positions, in particular those experiencing high compressive or tensile strains. No association between F and ΔCt.Th was found in regions around the neutral axis. For the most distal cross-section (75%), the association of loading magnitude and ΔCt.Th was not as pronounced as the more proximal cross-sections. Also, bone formation responses along the periosteum did not occur in regions of highest compressive and tensile strains predicted by beam theory. This could be due to complex experimental loading conditions which were not explicitly accounted for in the mechanical analysis. Our results show that the bone formation response depends on the load magnitude and the periosteal position. Bone resorption due to the neurectomy of the loaded tibia occurs throughout the entire cross-sectional region for all investigated cortical sections 25, 37, 50, and 75%. For peak applied loads higher than 4 N, compressive and tensile regions show bone formation; however, regions around the neutral axis show constant resorption. The 50% cross-section showed the most regular ΔCt.Th response with increased loading when compared to 25 and 37% cross-sections. Relative thickness gains of approximately 70, 60, and 55% were observed for F = 12 N in the 25, 37, and 50% cross-sections. ΔCt.Th at selected points of the periosteum follow a linear response with increased peak load; no lazy zone was observed at these positions.

Murine Axial Compression Tibial Loading Model to Study Bone Mechanobiology: Implementing the Model and Reporting Results.

In vivo, tibial loading in mice is increasingly used to study bone adaptation and mechanotransduction. To achieve standardized and defined experimental conditions, loading parameters and animal-related factors must be considered when performing in vivo loading studies. In this review, we discuss these loading and animal-related experimental conditions, present methods to assess bone adaptation, and suggest reporting guidelines. This review originated from presentations by each of the authors at the workshop "Developing Best Practices for Mouse Models of In Vivo Loading" during the Preclinical Models Section at the Orthopaedic Research Society Annual Meeting, San Diego, CA, March 2017. Following the meeting, the authors engaged in detailed discussions with consideration of relevant literature. The guidelines and recommendations in this review are provided to help researchers perform in vivo loading experiments in mice, and thus further our knowledge of bone adaptation and the mechanisms involved in mechanotransduction. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:233-252, 2020.

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.

Parathyroid hormone's enhancement of bones' osteogenic response to loading is affected by ageing in a dose- and time-dependent manner.

Decreased effectiveness of bones' adaptive response to mechanical loading contributes to age-related bone loss. In young mice, intermittent administration of parathyroid hormone (iPTH) at 20-80µg/kg/day interacts synergistically with artificially applied loading to increase bone mass. Here we report investigations on the effect of different doses and duration of iPTH treatment on mice whose osteogenic response to artificial loading is impaired by age. One group of aged, 19-month-old female C57BL/6 mice was given 0, 25, 50 or 100µg/kg/day iPTH for 4weeks. Histological and µCT analysis of their tibiae revealed potent iPTH dose-related increases in periosteally-enclosed area, cortical area and porosity with decreased cortical thickness. There was practically no effect on trabecular bone. Another group was given a submaximal dose of 50µg/kg/day iPTH or vehicle for 2 or 6weeks with loading of their right tibia three times per week for the final 2weeks. In the trabecular bone of these mice the loading-related increase in BV/TV was abrogated by iPTH primarily by reduction of the increase in trabecular number. In their cortical bone, iPTH treatment time-dependently increased cortical porosity. Loading partially reduced this effect. The osteogenic effects of iPTH and loading on periosteally-enclosed area and cortical area were additive but not synergistic. Thus in aged, unlike young mice, iPTH and loading appear to have separate effects. iPTH alone causes a marked increase in cortical porosity which loading reduces. Both iPTH and loading have positive effects on cortical periosteal bone formation but these are additive rather than synergistic.

Old age and the associated impairment of bones' adaptation to loading are associated with transcriptomic changes in cellular metabolism, cell-matrix interactions and the cell cycle.

In old animals, bone's ability to adapt its mass and architecture to functional load-bearing requirements is diminished, resulting in bone loss characteristic of osteoporosis. Here we investigate transcriptomic changes associated with this impaired adaptive response. Young adult (19-week-old) and aged (19-month-old) female mice were subjected to unilateral axial tibial loading and their cortical shells harvested for microarray analysis between 1h and 24h following loading (36 mice per age group, 6 mice per loading group at 6 time points). In non-loaded aged bones, down-regulated genes are enriched for MAPK, Wnt and cell cycle components, including E2F1. E2F1 is the transcription factor most closely associated with genes down-regulated by ageing and is down-regulated at the protein level in osteocytes. Genes up-regulated in aged bone are enriched for carbohydrate metabolism, TNFα and TGFß superfamily components. Loading stimulates rapid and sustained transcriptional responses in both age groups. However, genes related to proliferation are predominantly up-regulated in the young and down-regulated in the aged following loading, whereas those implicated in bioenergetics are down-regulated in the young and up-regulated in the aged. Networks of inter-related transcription factors regulated by E2F1 are loading-responsive in both age groups. Loading regulates genes involved in similar signalling cascades in both age groups, but these responses are more sustained in the young than aged. From this we conclude that cells in aged bone retain the capability to sense and transduce loading-related stimuli, but their ability to translate acute responses into functionally relevant outcomes is diminished.

Exercise does not enhance aged bone's impaired response to artificial loading in C57Bl/6 mice.

Bones adapt their structure to their loading environment and so ensure that they become, and are maintained, sufficiently strong to withstand the loads to which they are habituated. The effectiveness of this process declines with age and bones become fragile fracturing with less force. This effect in humans also occurs in mice which experience age-related bone loss and reduced adaptation to loading. Exercise engenders many systemic and local muscular physiological responses as well as engendering local bone strain. To investigate whether these physiological responses influence bones' adaptive responses to mechanical strain we examined whether a period of treadmill exercise influenced the adaptive response to an associated period of artificial loading in young adult (17-week) and old (19-month) mice. After treadmill acclimatization, mice were exercised for 30 min three times per week for two weeks. Three hours after each exercise period, right tibiae were subjected to 40 cycles of non-invasive axial loading engendering peak strain of 2250 µÎµ. In both young and aged mice exercise increased cross-sectional muscle area and serum sclerostin concentration. In young mice it also increased serum IGF1. Exercise did not affect bone's adaptation to loading in any measured parameter in young or aged bone. These data demonstrate that a level of exercise sufficient to cause systemic changes in serum, and adaptive changes in local musculature, has no effect on bone's response to loading 3h later. This study provides no support for the beneficial effects of exercise on bone in the elderly being mediated by systemic or local muscle-derived effects rather than local adaptation to altered mechanical strain.

Wnt16 Is Associated with Age-Related Bone Loss and Estrogen Withdrawal in Murine Bone.

Genome Wide Association Studies suggest that Wnt16 is an important contributor to the mechanisms controlling bone mineral density, cortical thickness, bone strength and ultimately fracture risk. Wnt16 acts on osteoblasts and osteoclasts and, in cortical bone, is predominantly derived from osteoblasts. This led us to hypothesize that low bone mass would be associated with low levels of Wnt16 expression and that Wnt16 expression would be increased by anabolic factors, including mechanical loading. We therefore investigated Wnt16 expression in the context of ageing, mechanical loading and unloading, estrogen deficiency and replacement, and estrogen receptor α (ERα) depletion. Quantitative real time PCR showed that Wnt16 mRNA expression was lower in cortical bone and marrow of aged compared to young female mice. Neither increased nor decreased (by disuse) mechanical loading altered Wnt16 expression in young female mice, although Wnt16 expression was decreased following ovariectomy. Both 17ß-estradiol and the Selective Estrogen Receptor Modulator Tamoxifen increased Wnt16 expression relative to ovariectomy. Wnt16 and ERß expression were increased in female ERα-/- mice when compared to Wild Type. We also addressed potential effects of gender on Wnt16 expression and while the expression was lower in the cortical bone of aged males as in females, it was higher in male bone marrow of aged mice compared to young. In the kidney, which we used as a non-bone reference tissue, Wnt16 expression was unaffected by age in either males or females. In summary, age, and its associated bone loss, is associated with low levels of Wnt16 expression whereas bone loss associated with disuse has no effect on Wnt16 expression. In the artificially loaded mouse tibia we observed no loading-related up-regulation of Wnt16 expression but provide evidence that its expression is influenced by estrogen receptor signaling. These findings suggest that while Wnt16 is not an obligatory contributor to regulation of bone mass per se, it potentially plays a role in influencing pathways associated with regulation of bone mass during ageing and estrogen withdrawal.

Quantification of Alterations in Cortical Bone Geometry Using Site Specificity Software in Mouse models of Aging and the Responses to Ovariectomy and Altered Loading.

Investigations into the effect of (re)modeling stimuli on cortical bone in rodents normally rely on analysis of changes in bone mass and architecture at a narrow cross-sectional site. However, it is well established that the effects of axial loading produce site-specific changes throughout bones' structure. Non-mechanical influences (e.g., hormones) can be additional to or oppose locally controlled adaptive responses and may have more generalized effects. Tools currently available to study site-specific cortical bone adaptation are limited. Here, we applied novel site specificity software to measure bone mass and architecture at each 1% site along the length of the mouse tibia from standard micro-computed tomography (µCT) images. Resulting measures are directly comparable to those obtained through µCT analysis (R (2) > 0.96). Site Specificity analysis was used to compare a number of parameters in tibiae from young adult (19-week-old) versus aged (19-month-old) mice; ovariectomized and entire mice; limbs subjected to short periods of axial loading or disuse induced by sciatic neurectomy. Age was associated with uniformly reduced cortical thickness and site-specific decreases in cortical area most apparent in the proximal tibia. Mechanical loading site-specifically increased cortical area and thickness in the proximal tibia. Disuse uniformly decreased cortical thickness and decreased cortical area in the proximal tibia. Ovariectomy uniformly reduced cortical area without altering cortical thickness. Differences in polar moment of inertia between experimental groups were only observed in the proximal tibia. Aging and ovariectomy also altered eccentricity in the distal tibia. In summary, site specificity analysis provides a valuable tool for measuring changes in cortical bone mass and architecture along the entire length of a bone. Changes in the (re)modeling response determined at a single site may not reflect the response at different locations within the same bone.