The Large Zinc Finger Protein ZAS3 Is a Critical Modulator of Osteoclastogenesis

Mice deficient in the large zinc finger protein, ZAS3, show postnatal increase in bone mass suggesting that ZAS3 is critical in the regulation of bone homeostasis. Although ZAS3 has been shown to inhibit osteoblast differentiation, its role on osteoclastogenesis has not been determined. In this report we demonstrated the role of ZAS3 in bone resorption by examining the signaling mechanisms involved in osteoclastogenesis.Comparison of adult wild-type and ZAS3 knockout (ZAS3-/-) mice showed that ZAS3 deficiency led to thicker bones that are more resistant to mechanical fracture. Additionally, ZAS3-/- bones showed fewer osteoclasts and inefficient M-CSF/sRANKL-mediated osteoclastogenesis ex vivo. Utilizing RAW 264.7 pre-osteoclasts, we demonstrated that overexpression of ZAS3 promoted osteoclastogenesis and the expression of crucial osteoclastic molecules, including phospho-p38, c-Jun, NFATc1, TRAP and CTSK. Contrarily, ZAS3 silencing by siRNA inhibited osteoclastogenesis. Co-immunoprecipitation experiments demonstrated that ZAS3 associated with TRAF6, the major receptor associated molecule in RANK signaling. Furthermore, EMSA suggested that nuclear ZAS3 could regulate transcription by binding to gene regulatory elements.Collectively, the data suggested a novel role of ZAS3 as a positive regulator of osteoclast differentiation. ZAS3 deficiency caused increased bone mass, at least in part due to decreased osteoclast formation and bone resorption. These functions of ZAS3 were mediated via activation of multiple intracellular targets. In the cytoplasmic compartment, ZAS3 associated with TRAF6 to control NF-kB and MAP kinase signaling cascades. Nuclear ZAS3 acted as a transcriptional regulator for osteoclast-associated genes. Additionally, ZAS3 activated NFATc1 required for the integration of RANK signaling in the terminal differentiation of osteoclasts. Thus, ZAS3 was a crucial molecule in osteoclast differentiation, which might potentially serve as a target in the design of therapeutic interventions for the treatment of bone diseases related to increased osteoclast activity such as postmenopausal osteoporosis, Paget's disease, and rheumatoid arthritis

Biomechanical Comparison of 2 Veterinary Locking Plates to Monocortical Screw/Polymethylmethacrylate Fixation in Canine Cadaveric Cervical Vertebral Column

OBJECTIVE: To compare the biomechanical properties of 2 veterinary locking plates and monocortical screws/polymethylmethacrylate (PMMA) fixation in canine cadaveric cervical vertebral columns. STUDY DESIGN: Biomechanical cadaveric study. MATERIALS: Nineteen cervical vertebral columns (C2-C7) from large breed, skeletally mature, canine cadavers were used. A cortical ring was placed as a disk spacer at C4-C5 in all specimens. Seven vertebral columns were plated at C4-C5 with two 4-hole, 3.5 mm string of pearls plates (SOP) and 6 vertebral columns were plated with two 6-hole, 2.4 mm titanium locking reconstruction plates (Ti recon plate). All screws were placed monocortically. Six vertebral columns had monocortical titanium screws and PMMA (Ti screws/PMMA) placed, tested as part of a prior study. METHODS: Stiffness testing in 3 directions was performed of the unaltered C4-C5 vertebral motion unit and repeated after placement of the disk spacer and implants. Data were compared using a linear mixed model that incorporated data from previously tested spines (Ti screw/PMMA). RESULTS: The mean (95% CI) stiffness (N/m) in extension for SOP was 407 N/mm (330-503), for Ti recon plate was 284 N/mm (198-407) and for Ti screws/PMMA was 365 N/mm (314-428); in flexion for SOP was 250 N/mm (178-354), for Ti recon plate was 147 N/mm (106-204) and for Ti screws/PMMA was 311 (235-416); in lateral bending for SOP was 528 N/mm (441-633), for Ti recon plate was 633 N/mm (545-735) and for Ti screws/PMMA was 327 N/mm (257-412). There were no significant differences in stiffness between the 3 fixations for any outcome. CONCLUSION: Monocortical fixation with two 3.5 mm SOP or two 2.4 mm Ti recon plates may be an alternate fixation to monocortical screws and PMMA

Measurement of shoulder abduction angles in dogs: an ex vivo study of accuracy and repeatability

OBJECTIVE: The aim of this study was to determine the accuracy and repeatability of the shoulder abduction test and to assess the effect of transection of the medial shoulder support structures in canine cadavers. MATERIALS AND METHODS: The shoulder abduction angle was measured by three separate observers, both with the shoulder extended and at a neutral angle. Shoulder abduction was then measured, using craniocaudal fluoroscopic images. Arthroscopy was performed in all shoulder joints, with the medial support structures transected in one shoulder of each dog. The three observers again measured shoulder abduction angles in all dogs. Shoulder abduction was measured again using fluoroscopy. Accuracy and repeatability of the abduction test were assessed using linear mixed models. RESULTS: All three observers had different measured abduction angles when compared with fluoroscopy ( < 0.01); however, the experienced surgeon had an error of only 2.9°. Inter-observer repeatability was poor, with all three observers having different abduction measurements ( < 0.001). Intra-observer repeatability, however, indicated no differences on repeated measurements ( = 0.26). Placing the shoulder at a neutral standing angle, and transection of support structures caused an average increase in abduction by 8.2° ( < 0.001) and 4.4° respectively. CONCLUSION: Significant variation exists between observers performing this test, increased accuracy seen in the more experienced observer. Shoulder flexion angle can significantly affect measured abduction angles

Biomechanical comparison between bicortical pin and monocortical screw/polymethylmethacrylate constructs in the cadaveric canine cervical vertebral column.

OBJECTIVE To compare biomechanical stiffness of cadaveric canine cervical spine constructs stabilized with bicortical stainless steel pins and polymethylmethacrylate (PMMA), monocortical stainless steel screws with PMMA, or monocortical titanium screws with PMMA. STUDY DESIGN Biomechanical cadaver study. ANIMALS Eighteen canine cervical vertebral columns (C2-C7) were collected from skeletally mature dogs (weighing 22-32 kg). METHODS Specimens were radiographed and examined by dual energy X-ray absorptiometry. Stiffness of the unaltered C4-C5 intervertebral motion unit was measured in extension, flexion and lateral bending using non-destructive 4-point bend testing. Specimens were then stabilized by (1) bicortical stainless steel pins/PMMA, (2) monocortical stainless steel screws/PMMA, or (3) monocortical titanium screws/PMMA. Mechanical testing was repeated and stiffness data from unaltered specimens and the 3 treatment groups were compared. RESULTS All 3 surgical methods significantly increased stiffness of the C4-C5 motion unit compared with the unaltered specimen (P < .001 for all treatments), but stiffness was not significantly different among the 3 fixation groups (P = .578). CONCLUSIONS In this model, monocortical screw fixation (with stainless steel or titanium screws) was biomechanically equivalent to bicortical fixation

Evaluating Stiffness of Fibreglass and Thermoplastic Splint Materials and Inter-fragmentary Motion in a Canine Tibial Fracture Model.

OBJECTIVES:  Various materials are used to construct splints for mid-diaphyseal tibial fracture stabilization. The objective of this study was to compare construct stiffness and inter-fragmentary bone motion when fibreglass (FG) or thermoplastic (TP) splints are applied to either the lateral or cranial aspect of the tibia in a mid-diaphyseal fracture model. METHODS:  A coaptation bandage was applied to eight cadaveric canine pelvic limbs, with a custom-formed splint made of either FG or TP material applied to either the lateral or cranial aspect of the osteotomized tibia. Four-point bending tests were performed to evaluate construct stiffness and inter-fragmentary motion in both frontal and sagittal planes. RESULTS:  For a given material, FG or TP, construct stiffness was not affected by splint location. Construct stiffness was significantly greater with cranial FG splints than with cranial TP splints (p < 0.05), but this difference was not significant when comparing splints applied laterally (p = 0.15). Inter-fragmentary motions in the sagittal and frontal planes were similar across splint types for cranial splints, but for lateral splints there was a 64% reduction in frontal plane motion when FG was used as the splint material (p = 0.03). CLINICAL SIGNIFICANCE:  FG produces a stiffer construct, but the difference is not reflected in a reduction in inter-fragmentary motion. For lateral splints, FG splints are associated with reduced inter-fragmentary motion as compared with TP and may therefore have slight superiority for this application

Evaluation of Three Human Cervical Fusion Implants for Use in the Canine Cervical Vertebral Column.

OBJECTIVE: To assess technical feasibility and mechanical properties of 3 locking plate designs (Zero-P, Zero-P VA, and Uniplate 2) for use in the canine cervical spine. STUDY DESIGN: Prospective ex vivo study. ANIMALS: Cadaver cervical spines from skeletally mature large breed dogs (n = 18). METHODS: Specimens were screened using radiography and allocated into balanced groups based on bone density. Stiffness of intact C4-C5 vertebral motion units was measured in extension, flexion, and lateral bending using nondestructive 4-point bend testing. Uniplate 2 was then implanted at C4-C5 and mechanical testing was repeated. Mechanical test data were compared against those from 6 spines implanted with monocortical screws, an allograft ring spacer, and PMMA. RESULTS: The Zero-P and Zero-P VA systems could not be surgically implanted due to anatomical constraints in the vertebral column sizes of the canine cervical spines used in this study. Fixation with Uniplate 2 or with screws/PMMA significantly increased stiffness of the C4-C5 vertebral motion units compared to unaltered specimens (P < .001) in extension. Stiffness of the titanium screw/PMMA fixation was significantly greater than the Uniplate 2 construct in extension. Flexion and lateral bending could not be evaluated in 3 of 6 specimens in the Uniplate 2 group due to failure at the bone/implant interface during extension testing. CONCLUSION: Fixation with Uniplate 2 was biomechanically inferior to screws/PMMA. Particularly concerning was the incidence of vertebral fracture after several testing cycles. Based on our results, Zero-P, Zero-P VA, and Uniplate 2 cannot be recommended for use in dogs requiring cervical fusion.College of Veterinary Medicine, The Ohio State University (intramural award)This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1111/vsu.1253