SIRT1 haplo-insufficiency results in reduced cortical bone thickness, increased porosity and decreased estrogen receptor alpha in bone in adult 129/Sv female mice.

Introduction: Sirtuin 1 (SIRT1) is a key player in aging and metabolism and regulates bone mass and architecture. Sexual dimorphism in skeletal effects of SIRT1 has been reported, with an unfavorable phenotype primarily in female mice. Methods: To investigate the mechanisms of gender differences in SIRT1 skeletal effect, we investigated femoral and vertebral cortical and cancellous bone in global Sirt1 haplo-insufficient 129/Sv mice aged 2,7,12 months lacking Sirt1 exons 5,6,7 (Sirt1+/Δ ) and their wild type (WT) counterparts. Results: In females, femoral bone mineral content, peak cortical thickness, and trabecular bone volume (BV/TV%), number and thickness were significantly lower in Sirt1+/Δ compared to WT mice. Increased femoral cortical porosity was observed in 7-month-old Sirt1+/Δ compared to WT female mice, accompanied by reduced biomechanical strength. No difference in vertebral indices was detected between Sirt1+/Δ and WT female mice. SIRT1 decreased with aging in WT female mice and was lower in vertebrae and femur in 18- and 30- versus 3-month-old 129/Sv and C57BL/6J female mice, respectively. Decreased bone estrogen receptor alpha (ERα) was observed in Sirt1+/Δ compared to WT female mice and was significantly higher in Sirt1 over-expressing C3HT101/2 murine mesenchymal stem cells. In males no difference in femoral indices was detected in Sirt1+/Δ versus WT mice, however vertebral BV/TV%, trabecular number and thickness were higher in Sirt1+/Δ vs. WT mice. No difference in androgen receptor (AR) was detected in bone in Sirt1+/Δ vs. WT male mice. Bone SIRT1 was significantly lower in male compared to female WT mice, suggesting that SIRT1 maybe more significant in female than male skeleton. Discussion: These findings demonstrate that 50% reduction in SIRT1 is sufficient to induce the hallmarks of skeletal aging namely, decreased cortical thickness and increased porosity in female mice, highlighting the role of SIRT1 as a regulator of cortical bone quantity and quality. The effects of SIRT1 in cortical bone are likely mediated in part by its regulation of ERα. The age-associated decline in bone SIRT1 positions SIRT1 as a potential therapeutic target to ameliorate age-related cortical bone deterioration in females. The crosstalk between ERα, AR and SIRT1 in the bone microenvironment remains to be further investigated.

Skeletal ligament healing using the recombinant human amelogenin protein.

Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM(+)) resulted in enhanced healing of the tooth-supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM(+), dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 µg/µl rHAM(+) were similar to the normal un-transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 µg/µl rHAM(+) treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 µg/µl rHAM(+) increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM(+) dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers.

The Sirtuin1 activator SRT3025 down-regulates sclerostin and rescues ovariectomy-induced bone loss and biomechanical deterioration in female mice.

Estrogen deficiency leads to rapid bone loss and skeletal fragility. Sclerostin, encoded by the sost gene, and a product of the osteocyte, is a negative regulator of bone formation. Blocking sclerostin increases bone mass and strength in animals and humans. Sirtuin1 (Sirt1), a player in aging and metabolism, regulates bone mass and inhibits sost expression by deacetylating histone 3 at its promoter. We asked whether a Sirt1-activating compound could rescue ovariectomy (OVX)-induced bone loss and biomechanical deterioration in 9-week-old C57BL/6 mice. OVX resulted in a substantial decrease in skeletal Sirt1 expression accompanied by an increase in sclerostin. Oral administration of SRT3025, a Sirt1 activator, at 50 and 100 mg/kg·d for 6 weeks starting 6 weeks after OVX fully reversed the deleterious effects of OVX on vertebral bone mass, microarchitecture, and femoral biomechanical properties. Treatment with SRT3025 decreased bone sclerostin expression and increased cortical periosteal mineralizing surface and serum propeptide of type I procollagen, a bone formation marker. In vitro, in the murine long bone osteocyte-Y4 osteocyte-like cell line SRT3025 down-regulated sclerostin and inactive ß-catenin, whereas a reciprocal effect was observed with EX-527, a Sirt1 inhibitor. Sirt1 activation by Sirt1-activating compounds is a potential novel pathway to down-regulate sclerostin and design anabolic therapies for osteoporosis concurrently ameliorating other metabolic and age-associated conditions.

Exposure to omega-3 fatty acids at early age accelerate bone growth and improve bone quality.

Omega-3 fatty acids (FAs) are essential nutritional components that must be obtained from foods. Increasing evidence validate that omega-3 FAs are beneficial for bone health, and several mechanisms have been suggested to mediate their effects on bone, including alterations in calcium absorption and urinary calcium loss, prostaglandin synthesis, lipid oxidation, osteoblast formation and inhibition of osteoclastogenesis. However, to date, there is scant information regarding the effect of omega-3 FAs on the developing skeleton during the rapid growth phase. In this study we aim to evaluate the effect of exposure to high levels of omega-3 FAs on bone development and quality during prenatal and early postnatal period. For this purpose, we used the fat-1 transgenic mice that have the ability to convert omega-6 to omega-3 fatty acids and the ATDC5 chondrogenic cell line as models. We show that exposure to high concentrations of omega-3 FAs at a young age accelerates bone growth through alterations of the growth plate, associated with increased chondrocyte proliferation and differentiation. We further propose that those effects are mediated by the receptors G-protein coupled receptor 120 (GPR120) and hepatic nuclear factor 4α, which are expressed by chondrocytes in culture. Additionally, using a combined study on the structural and mechanical bone parameters, we show that high omega-3 levels contribute to superior trabecular and cortical structure, as well as to stiffer bones and improved bone quality. Most interestingly, the fat-1 model allowed us to demonstrate the role of maternal high omega-3 concentration on bone growth during the gestation and postnatal period.

Acidic peptide hydrogel scaffolds enhance calcium phosphate mineral turnover into bone tissue.

Designed peptides may generate molecular scaffolds in the form of hydrogels to support tissue regeneration. We studied the effect of hydrogels comprising ß-sheet-forming peptides rich in aspartic amino acids and of tricalcium phosphate (ß-TCP)-loaded hydrogels on calcium adsorption and cell culture in vitro, and on bone regeneration in vivo. The hydrogels were found to act as efficient depots for calcium ions, and to induce osteoblast differentiation in vitro. In vivo studies on bone defect healing in rat distal femurs analyzed by microcomputerized tomography showed that the peptide hydrogel itself induced better bone regeneration in comparison to non-treated defects. A stronger regeneration capacity was obtained in bone defects treated with ß-TCP-loaded hydrogels, indicating that the peptide hydrogels and the mineral act synergistically to enhance bone regeneration. In vivo regeneration was found to be better with hydrogels loaded with porous ß-TCP than with hydrogels loaded with non-porous mineral. It is concluded that biocompatible and biodegradable matrices, rich in anionic moieties that efficiently adsorb calcium ions while supporting cellular osteogenic activity, may efficiently promote ß-TCP turnover into bone mineral.

Bone loss in adult offspring induced by low-dose exposure to teratogens.

Maternal malnutrition during pregnancy was shown by numerous studies to result in the birth of offspring exhibiting altered bone characteristics, which are indicative of bone loss. We hypothesized that not only maternal malnutrition but also some developmental toxicants (teratogens) given at a dose inducing neither structural anomalies nor growth retardation can detrimentally affect skeletal health in adult offspring. To check this hypothesis, pregnant mice were exposed to a single injection of 5-aza-2-deoxycytidine (5-AZA) (a teratogen capable of inducing phocomelia of the hind limbs) at a sub-threshold teratogenic dose. Micro-computed tomography scanning revealed that femora of 5-month-old male offspring exposed in uterus to 5-AZA had trabecular microarchitecture indicative of bone loss. Furthermore, exposure to 5-AZA increased the susceptibility of offspring to postnatal chronic mild stress, which has been shown to induce bone loss in mice. While exploring possible mechanisms underlying this phenomenon, we observed that the expression of some microRNAs, which have been demonstrated as regulators of key osteoblastogenic genes, was altered in hind limb buds of embryos exposed to 5-AZA. Furthermore, the expression of receptor activator of nuclear factor kappa B ligand (RANKL) in femoral stromal/osteoblastic cells of 5-month-old offspring of 5-AZA-treated females was found to be increased. Collectively, this study implies for the first time that single low-dose exposure to a teratogen can induce bone loss in adult offspring, possibly via alteration of embryonic microRNAs and RANKL expression.

Lateral thoracic artery axial pattern flap in cats.

OBJECTIVE: To describe the location of the lateral thoracic artery (LTA), determine dimensions of an axial pattern flap based on this artery, and report use of this flap in 2 cats. STUDY DESIGN: Ex vivo study and case reports. ANIMALS: Cat cadavers (n=8); cats (n=2) with thoracic limb skin defects. METHODS: Dissection of the LTA was carried out on 1 side of each cadaver and the contralateral side was used for injection studies. In 4 specimens, the LTA was cannulated and injected with positive contrast material and the flap was raised and radiographed. In 4 specimens, the flap was injected with methylene blue. Adequacy of flap injection was subjectively evaluated and leakage of methylene blue from the cut edge was noted. RESULTS: The cutaneous location of the LTA caudal to the triceps muscle was confirmed. Mean flap size was 8.7 cm x 15.5 cm for a mature, average-sized cat. Perfusion of the entire flap was demonstrated and viability of the flap was confirmed in 2 clinical cases. CONCLUSION: The LTA flap is useful for repair of skin defects of the brachium and antebrachium in cats. CLINICAL RELEVANCE: The LTA flap is an alternative technique for repair of skin defects involving the thoracic limb of cats.

In vitro study of the ilial anatomic landmarks for safe implant insertion in the first sacral vertebra of the intact canine sacroiliac joint.

OBJECTIVE: To define landmarks on the canine ilial wing for accurate, consistent insertion of implants into the 1st sacral (S1) vertebral body when the sacroiliac joint is intact. STUDY DESIGN: Anatomic study. ANIMALS: Intact, cadaveric canine pelves and sacra (n=25). METHODS: Median sections (5 specimens) were drilled from the center of S1 in a lateral direction, exiting on the ilial wing. Landmarks on the ilial wing and shaft used to define this exit point were then used to locate this point on both wings of 20 articulated specimens, positioned and rigidly held so that the dorsal plane of the pelvis was aligned with a plumb line and the median plane of the pelvis was horizontal. A 2 mm hole was drilled from the marked point, parallel to the plumb line, until it exited the contralateral ilial wing. Distance of drill hole position from the geometric center (GC) of S1 was located on median and paramedian plane images derived from plane, computed tomographic (CT) scans. RESULTS: The entire drill hole was located within S1 in 18 specimens. Mean deviation of the hole from GC (ratio of the distance of GC from the closest S1 body border) in median section was 0.40 +/- 0.29 (craniocaudal direction) and 0.29 +/- 0.23 (dorsoventral). CONCLUSIONS: Use of ilial wing landmarks and drilling perpendicular to the median plane will improve accuracy for insertion of implants into S1 when the sacroiliac joint is intact. CLINICAL RELEVANCE: Ilial wing landmarks should be used to improve accuracy of implant insertion into S1.

Biomechanics of tibial plateau leveling of the canine cruciate-deficient stifle joint: a theoretical model.

OBJECTIVE: To evaluate the effect of tibial plateau leveling on the biomechanics of the canine stifle. STUDY DESIGN: Analysis of a 3-dimensional (3-D) anatomically accurate theoretical model of the canine stifle. METHODS: A 3-D, 3-segment mathematical model of the normal canine stifle was modified to simulate the effect of rotation of the tibial plateau during tibial plateau leveling osteotomy (TPLO). The model examined the normal stifle, the stifle with a tibial plateau angle (TPA) of 0 degrees, and the stifle with a TPA of 5 degrees. Analysis of the models at 10 consecutive equally spaced positions during the stance phase yielded data such as ligament forces and joint reaction forces at each position. RESULTS: Rotation of the tibial plateau to a TPA of 0 degrees almost eliminates forces in the cranial cruciate ligament (CCL) throughout the stance phase. Rotation to a TPA of 5 degrees did not, however, substantially decrease the load in the CCL. Both procedures increased the load in the caudal cruciate ligament (CaCL). CONCLUSIONS: Cranial tibial thrust (CTT) is converted into caudal tibial thrust when the TPA is 0 degrees ; however, rotating the plateau to a TPA of 5 degrees does not eliminate the CTT. CLINICAL RELEVANCE: The TPLO procedure performed as currently recommended (rotating the tibial plateau to a TPA of 5 degrees) may not eliminate the CTT, but only reduce it. Both TPLO procedures evaluated here were found to increase the load in the CaCL.

The use of the Objective Structured Clinical Examination (OSCE) in small-animal internal medicine and surgery.

The Objective Structured Clinical Examination (OSCE) has been used extensively to evaluate clinical skill of medical students. This article reports on its adaptation to veterinary medicine.

Expression of relaxin receptor LRG7, canine relaxin, and relaxin-like factor in the pelvic diaphragm musculature of dogs with and without perineal hernia.

OBJECTIVES: To compare the expression of canine relaxin, relaxin-like factor (RLF), and relaxin receptors within the muscles of the pelvic diaphragm of dogs with perineal hernia (PH) and clinically normal dogs. STUDY DESIGN: In vivo comparative study. ANIMALS: Fifteen client-owned intact male dogs with PH were studied. Four mature intact male dogs with no evidence of perineal pathology served as controls. METHODS: Biopsy samples from the levator ani, coccygeus, and internal obturator muscles were obtained. RNA samples were reverse transcribed and analyzed by real-time PCR for the expression of canine relaxin receptor LRG7, relaxin, and RLF. RESULTS: Significantly higher expression levels of canine relaxin receptors occurred in the musculature of the pelvic diaphragm and internal obturator muscle in dogs with PH compared with normal dogs. Expression of canine RLF revealed no significant difference between dogs with PH and controls. The difference in the expression of canine relaxin between groups was not statistically significant. CONCLUSIONS: Relaxin receptor up-regulation occurs in the coccygeus, levator ani, and internal obturator muscles of dogs with PH. CLINICAL RELEVANCE: The higher expression of relaxin receptors within the muscles of the pelvic diaphragm in dogs with PH suggests that relaxin might play a role in the pathogenesis of PH. Atrophy of these muscles, which predisposes to PH, may be attributable to increased relaxin activity.

Partial esophagectomy with single layer closure for treatment of esophageal sarcomas in 6 dogs.

OBJECTIVE: To report partial esophagectomy (PE) as a treatment for esophageal sarcoma in dogs. STUDY DESIGN: Retrospective study (2000-2002). ANIMALS: Six dogs with caudal thoracic esophageal tumors. METHODS: Medical records of 6 dogs that had surgical removal of esophageal tumors were reviewed. Signalment, medical history, physical examination results, complete blood count, surgical procedure, tumor classification, postoperative treatment, and complications were retrieved. RESULTS: Esophageal masses were approached by thoracotomy and esophagotomy on the side opposite the mass, removed with 1 cm margins by full thickness excision, and the defects closed with a single layer of interrupted sutures. All dogs recovered rapidly without major complications. Tumors were fibrosarcoma (3 dogs), undifferentiated sarcoma (1), and osteosarcoma (2). Five dogs were administered doxorubicin chemotherapy after surgery. Good quality of life was observed postoperatively in 5 dogs until deterioration necessitated euthanasia; survival ranged from 2-16 months. The remaining dog was alive, 20 months after surgery. CONCLUSIONS: Partial esophagectomy and closure using 1 suture layer, was an effective, simple, and safe technique for removal of sarcomas of the distal thoracic esophagus. CLINICAL RELEVANCE: Removal of esophageal masses by partial esophagectomy can be used reliably as a method of esophageal surgery.