Canine Brief Pain Inventory scores for dogs with osteoarthritis before and after administration of a monoclonal antibody against nerve growth factor.

OBJECTIVE: To determine changes in Canine Brief Pain Inventory scores for dogs with osteoarthritis after administration of a monoclonal antibody (mAb) against nerve growth factor (NGF) that was modified by use of a proprietary process for administration to dogs. ANIMALS: 11 adult dogs. PROCEDURES: Dogs received the anti-NGF mAb (0.2 mg/kg, IV) at various evaluation times during the study period; at other evaluation times, dogs received an equivalent volume of PBS solution IV. Owners determined Canine Brief Pain Inventory pain severity (PS) and pain interference (PI) scores immediately before (baseline) and 2, 4, and 6 weeks after administration of the anti-NGF mAb; owners were unaware of the evaluation time at which the mAb had been administered. RESULTS: Compared with baseline PS scores (median, 4.75; range, 0.75 to 8.5), dogs had significantly lower PS scores 2 weeks (median, 3; range, 1 to 5.5) and 4 weeks (median, 2.25; range, 0.25 to 7.25) after administration of anti-NGF mAb. Compared with baseline PI scores (median, 5.33; range, 1.17 to 9.33), dogs had significantly lower PI scores 2 weeks (median, 3; range, 0.67 to 6.83) and 4 weeks (median, 3.33; range, 0.67 to 6.67) after administration of anti-NGF mAb. The PS and PI scores 6 weeks after mAb administration were lower than baseline scores, although values were not significantly different. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study suggested the evaluated anti-NGF mAb decreased PS and PI scores for 4 weeks after administration. This treatment may be effective for alleviation of signs of pain in dogs with osteoarthritis for up to 4 weeks.

Widespread differential maternal and paternal genome effects on fetal bone phenotype at mid-gestation.

Parent-of-origin-dependent (epi)genetic factors are important determinants of prenatal development that program adult phenotype. However, data on magnitude and specificity of maternal and paternal genome effects on fetal bone are lacking. We used an outbred bovine model to dissect and quantify effects of parental genomes, fetal sex, and nongenetic maternal effects on the fetal skeleton and analyzed phenotypic and molecular relationships between fetal muscle and bone. Analysis of 51 bone morphometric and weight parameters from 72 fetuses recovered at day 153 gestation (54% term) identified six principal components (PC1-6) that explained 80% of the variation in skeletal parameters. Parental genomes accounted for most of the variation in bone wet weight (PC1, 72.1%), limb ossification (PC2, 99.8%), flat bone size (PC4, 99.7%), and axial skeletal growth (PC5, 96.9%). Limb length showed lesser effects of parental genomes (PC3, 40.8%) and a significant nongenetic maternal effect (gestational weight gain, 29%). Fetal sex affected bone wet weight (PC1, p < 0.0001) and limb length (PC3, p < 0.05). Partitioning of variation explained by parental genomes revealed strong maternal genome effects on bone wet weight (74.1%, p < 0.0001) and axial skeletal growth (93.5%, p < 0.001), whereas paternal genome controlled limb ossification (95.1%, p < 0.0001). Histomorphometric data revealed strong maternal genome effects on growth plate height (98.6%, p < 0.0001) and trabecular thickness (85.5%, p < 0.0001) in distal femur. Parental genome effects on fetal bone were mirrored by maternal genome effects on fetal serum 25-hydroxyvitamin D (96.9%, p < 0.001) and paternal genome effects on alkaline phosphatase (90.0%, p < 0.001) and their correlations with maternally controlled bone wet weight and paternally controlled limb ossification, respectively. Bone wet weight and flat bone size correlated positively with muscle weight (r = 0.84 and 0.77, p < 0.0001) and negatively with muscle H19 expression (r = -0.34 and -0.31, p < 0.01). Because imprinted maternally expressed H19 regulates growth factors by miRNA interference, this suggests muscle-bone interaction via epigenetic factors.

A policy at the University of Adelaide for student objections to the use of animals in teaching.

In veterinary medical education, the use of animals or cadaveric tissue as a component of teaching practice is common. Teachers are required, during the process of ethical review, to apply the 3 Rs principle (replacement, refinement, reduction) whenever they consider using animals during a teaching exercise. This often involves use of replacement strategies, such as utilization of video footage or simulation-based training. However, aside from legislative or ethical requirements imposed by a country's regulatory framework on the institution, students are often the key advocates for using alternative teaching practices that do not make use of animals. This has prompted many institutions with veterinary and other life sciences teaching programs to develop student-conscientious objection policies to the use of animals in teaching. In this article, we discuss the procedures implemented to make provision for student-conscientious objectors at a new Australian Veterinary School, at the University of Adelaide. We also describe the processes to provide information to students and faculty on this issue and to facilitate information gathering on alternatives.

Maternal and paternal genomes differentially affect myofibre characteristics and muscle weights of bovine fetuses at midgestation.

Postnatal myofibre characteristics and muscle mass are largely determined during fetal development and may be significantly affected by epigenetic parent-of-origin effects. However, data on such effects in prenatal muscle development that could help understand unexplained variation in postnatal muscle traits are lacking. In a bovine model we studied effects of distinct maternal and paternal genomes, fetal sex, and non-genetic maternal effects on fetal myofibre characteristics and muscle mass. Data from 73 fetuses (Day153, 54% term) of four genetic groups with purebred and reciprocal cross Angus and Brahman genetics were analyzed using general linear models. Parental genomes explained the greatest proportion of variation in myofibre size of Musculus semitendinosus (80-96%) and in absolute and relative weights of M. supraspinatus, M. longissimus dorsi, M. quadriceps femoris and M. semimembranosus (82-89% and 56-93%, respectively). Paternal genome in interaction with maternal genome (P<0.05) explained most genetic variation in cross sectional area (CSA) of fast myotubes (68%), while maternal genome alone explained most genetic variation in CSA of fast myofibres (93%, P<0.01). Furthermore, maternal genome independently (M. semimembranosus, 88%, P<0.0001) or in combination (M. supraspinatus, 82%; M. longissimus dorsi, 93%; M. quadriceps femoris, 86%) with nested maternal weight effect (5-6%, P<0.05), was the predominant source of variation for absolute muscle weights. Effects of paternal genome on muscle mass decreased from thoracic to pelvic limb and accounted for all (M. supraspinatus, 97%, P<0.0001) or most (M. longissimus dorsi, 69%, P<0.0001; M. quadriceps femoris, 54%, P<0.001) genetic variation in relative weights. An interaction between maternal and paternal genomes (P<0.01) and effects of maternal weight (P<0.05) on expression of H19, a master regulator of an imprinted gene network, and negative correlations between H19 expression and fetal muscle mass (P<0.001), suggested imprinted genes and miRNA interference as mechanisms for differential effects of maternal and paternal genomes on fetal muscle.

Sphene ceramics for orthopedic coating applications: an in vitro and in vivo study.

The host response to titanium alloy (Ti-6Al-4V) is not always favorable as a fibrous layer may form at the skeletal tissue-device interface, causing aseptic loosening. Recently, sphene (CaTiSiO(5)) ceramics were developed by incorporating Ti in the Ca-Si system, and found to exhibit improved chemical stability. The aim of this study is to evaluate the in vitro response of human osteoblast-like cells, human osteoclasts and human microvascular endothelial cells to sphene ceramics and determine whether coating Ti-6Al-4V implants with sphene enhances anchorage to surrounding bone. The study showed that sphene ceramics support human osteoblast-like cell attachment with organized cytoskeleton structure and express increased mRNA levels of osteoblast-related genes. Sphene ceramics were able to induce the differentiation of monocytes to form functional osteoclasts with the characteristic features of f-actin and alpha(v)beta(3) integrin, and express osteoclast-related genes. Human endothelial cells were also able to attach and express the endothelial cell markers ZO-1 and VE-Cadherin when cultured on sphene ceramics. Histological staining, enzyme histochemistry and immunolabelling were used for identification of mineralized bone and bone remodelling around the coated implants. Ti-6Al-4V implants coated with sphene showed new bone formation and filled the gap between the implants and existing bone in a manner comparable to that of the hydroxyapatite coatings used as control. The new bone was in direct contact with the implants, whereas fibrous tissue formed between the bone and implant with uncoated Ti-6Al-4V. The in vivo assessment of sphene-coated implants supports our in vitro observation and suggests that they have the ability to recruit osteogenic cells, and thus support bone formation around the implants and enhance osseointegration.

Bone growth is enhanced by novel bioceramic coatings on Ti alloy implants.

Calcium phosphate ceramics are widely used as coating materials to orthopedic implants and are found to enhance initial bony ingrowth by stimulating osseous apposition to the implant surface. In this study, two novel calcium orthophosphate materials were selected for coating onto the commonly used orthopedic implant material Ti-6Al- 4V. One was calcium alkali orthophosphate with the crystalline phase Ca10[K/Na](PO4)7 with a small addition of SiO2 (AW-Si) and the other was calcium orthophosphate composed of 70 mol % fluorapatite, Ca10(PO4)6F2 and 30 mol % CaZr4(PO4)6 (FA7Z). The coated implants were placed in cortical and cortico-cancellous bone of sheep femur for six weeks. Retrieved samples were tested for osseointegration and mechanical strength. It was found that both coatings produced enhanced bone/implant contact rate compared to the control when implanted in cortico-cancellous bone. This study demonstrates that the two coatings have the capability of encouraging bone growth, and hence the potential for being used as coating materials on Ti implants.

In vitro heterogeneity of osteogenic cell populations at various equine skeletal sites.

Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted. The numbers of nodules produced from osteogenic cells harvested from different skeletal sites were compared with the use of a mixed linear model. On average, cortical bone sites yielded significantly greater numbers of nodules than did cancellous bone sites. Between cortical bone sites, there was no significant difference in nodule numbers. Among cancellous sites, the radial cancellous bone yielded significantly more nodules than did the tibial cancellous bone. Among appendicular skeletal sites, tibial metaphyseal bone yielded significantly fewer nodules than did all other long bone sites. This study detected evidence of heterogeneity of equine osteogenic cell populations at various skeletal sites. Further characterization of the dissimilarities is warranted to determine the potential role heterogeneity plays in differential rates of fracture healing between skeletal sites.

Adsorption of bisphosphonate onto hydroxyapatite using a novel co-precipitation technique for bone growth enhancement.

Premature bone resorption and remodeling by osteoclasts can limit the longevity of implant fixation and recovery time. Orally administered bisphosphonates (BPs) have been used to inhibit osteoclast action at the implant/bone interface. Ideally, these should be delivered at the interface with the osteoblast-active hydroxyapatite (HA) for maximum effect. This investigation introduces a novel BP loading technique to achieve improved BP release from a simulated body fluid-grown HA (SBF-HA) with the aim of improving implant fixation. A solution co-precipitation technique incorporates the BP (pamidronate) into a thin SBF-HA coating. Surface analysis, using X-ray photoelectron spectroscopy (XPS), of the resultant coating was employed to confirm the presence of the adsorbed BP on the surface of SBF-HA. XPS analysis was also used to determine the optimal adsorption process. Osteoclast cell culture experiments confirmed the biological effectiveness of BP adsorption and proved that the pamidronate was biologically active, causing both decreased osteoclast numbers and decreased resorption.

Mechanical loading modulates glutamate receptor subunit expression in bone.

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-d-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GluR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity.

Wear-particle-induced osteoclast osteolysis: the role of particulates and mechanical strain.

Periprosthetic osteolysis involves osteoclast activation by wear particulates and their exposure to mechanical perturbation through exposure to shear forces generated by periprosthetic fluid as well as interface micromotion. This study aimed to determine the interactions between wear particulates, mechanical stimulation, and osteoclasts. In static cultures, wear particulates increased osteoclast differentiation. Addition of neutralizing antibodies to RANKL (receptor activator of nuclear factor kappa ligand) inhibited the particle-induced increase in osteoclast numbers. Cyclic 5000 microstrains were applied with the use of a custom-built device to marrow-derived cultures to assess the effect on osteoclast differentiation. Mechanical strain application alone decreased osteoclast differentiation, which was further decreased by the addition of particles despite increases in the soluble RANKL to osteoprotegerin (OPG) ratio. Mechanical strain alone induced mature osteoclast apoptosis in a dose-dependent manner. In contrast, in the mature osteoclast model, the addition of nonmetal particulates protected the osteoclasts from becoming apoptopic. Titanium (Ti) and cobalt chromium (CoCr) particles, however, induced osteoclast apoptosis, whereas polyethylene (PE) and polymethylmethacrylate (PMMA) did not. Wear particulates and mechanical stimulation interact via an eicosanoid-dependent pathway to alter osteoclast function and survival. The addition of mechanical perturbation to a particle-laden system thus appears to enhance the potential for osteolytic activity by enhancing osteoclast survival.

The effect of Gu-Sui-Bu (Drynaria fortunei) on bone cell activity.

We investigated the effects of Gu-Sui-Bu using in vitro bone cell cultures. Primary rabbit and mouse marrow cells were cultured with or without five different concentrations of Gu-Sui-Bu extract. Osteoclast numbers were assessed using tartrate-resistant acid phosphatase (TRAP) positive cell counts and for function, osteoclast resorption pits on bovine bone slices were performed. Alkaline phosphatase (AP) positive cell counts and mineralized nodule formation were examined to assess osteoblast function with Gu-Sui-Bu. TRAP+ osteoclast numbers increased, as did the number and size of resorption pits with 0.001 mg/ml of extract. Low doses of extract did not alter AP+ colony number or mineralized nodule formation, but both were inhibited by doses of 0.1 mg/ml or higher. The highest dose of extract (10 mg/ml) inhibited proliferation of all cell types. At 0.01 and 0.001 mg/ml doses, RANKL increased over time; however, osteoprotegerin levels only increased at doses > or = 0.1 mg/ml. Resorption pit formation was decreased without alteration in mature multinucleated (TRAP+) cell counts only at the highest dose of the putative active ingredient of Gu-Sui-Bu. In summary, lower concentrations of Gu-Sui-Bu extract had positive effects on osteoclast proliferation, survival and resorptive activity that may be mediated through enhanced prostaglandin secretion. However, high doses of extract proved detrimental to osteoclast and osteoblast survival. No effect of low doses of Gu-Sui-Bu extract was seen in osteoblast cultures. High doses of the putative active ingredient of Gu-Sui-Bu showed mild inhibition of mouse osteoclast function.

In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors.

OBJECTIVE: To compare the osteogenic potential of cancellous bone of conventional graft sites with that of one nonconventional site (fourth coccygeal vertebra) and to investigate the tibial periosteum as a donor site with respect to osteogenic potential. STUDY DESIGN: In vitro osteogenic cell culture system. SAMPLE POPULATION: Eight adult horses. METHODS: Cancellous bone or tibial periosteum was aseptically collected and cut into bone chips or periosteal strips of 1 to 2 mm(3) for primary explant cultures. After 2 weeks, primary tissue cultures that yielded a population of osteogenic cells were counted and subcultured at 1 x 10(5) cells/35-mm dish in osteogenic media. After 7 to 10 days, subcultures were stained with Von Kossa (VK) to assess mineralized bone nodule formation. VK-positive bone nodules were counted as osteoprogenitors and compared among 3 donor sites, which provided consistent primary osteogenic cells (tuber coxae, fourth coccygeal vertebra, periosteum) using ANOVA (P <.05). RESULTS: Sternal and tibial bone yielded viable osteogenic cells from 25% and 50% of horses, respectively, whereas yields from tuber coxae, coccygeal vertebra, and periosteum were 75%, 100%, and 100%, respectively. Tuber coxae and periosteum had significantly greater numbers of osteoprogenitors compared with fourth coccygeal vertebra. CONCLUSIONS: Among the conventional donor sites, tuber coxae most consistently yielded viable osteogenic cells with an acceptable percentage of osteoprogenitors. Sternal and tibial sites were unreliable in providing osteogenic cells. Two new donor sites, the fourth coccygeal vertebra and tibial periosteum, were tissues with good osteogenic potential. CLINICAL RELEVANCE: When a source of transplantable viable osteoprogenitor cells is desired, use of the tuber coxae as a conventional donor site is warranted. Use of tibial periosteum or fourth coccygeal vertebra as reliable sources of transplantable osteoprogenitors should be considered.

In vitro osteoclast resorption of bone substitute biomaterials used for implant site augmentation: a pilot study.

PURPOSE: This observational study examined the resorptive behavior of normal neonatal rabbit osteoclasts grown on slices of bovine cortical bone as compared to samples of commercially available bone substitute biomaterials. It also examined the surface characteristics of these materials. MATERIALS AND METHODS: The 11 materials tested fell into 3 groups: (1) bone-derived, including freeze-dried human rib block, human demineralized freeze-dried bone, and deproteinated bovine bone; (2) synthetic hydroxyapatites (HA); and (3) synthetic non-HA, including coated methacrylates and coated silica glass. After 4 days in culture, 1 group of samples of each material underwent scanning electron microscopy (SEM) to evaluate resorptive pitting versus controls, while another group underwent tartrate-resistant acid phosphatase staining and light microscopy to examine osteoclast numbers and morphology. The 2 bovine-derived HA materials also underwent immunohistochemical staining and surface chemistry analysis. RESULTS: While most of these materials supported osteoclast attachment, some spreading, and survival in culture, only the bone-derived materials, with the exception of sintered deproteinated bovine bone, showed large scalloped-edged resorption pits with trails and exposed collagen when examined by SEM, although not to the same extent as unprocessed natural bone material. The HA materials and the sintered deproteinated bovine bone showed evidence of etching with smaller pits but no evidence of resorptive trail formation. The non-HA materials showed no evidence of pit formation or trails. Under immunohistochemical staining, Bio-Oss appeared to be positive for type I collagen after osteoclast activity on its surface, while Osteograf/N showed no positive staining. Surface chemistry analysis revealed nitrogen present in Bio-Oss specimens (0.17% to 0.47%), while there was no nitrogen detected in the Osteograf/N (0.00%); the percent nitrogen observed in normal bovine bone controls was 6.01% to 9.25%. DISCUSSION: The bone-derived materials supported osteoclast activity on the material surface in a way that facilitated formation of the more complex resorption pits in vitro. Assuming the rate of pit formation observed in vitro mimics that observed in vivo, the quantity and type of osteoclastic remodeling seen on non-bone-derived materials--and perhaps sintered bone-derived materials--would be extremely slow to negligible. Physiologic removal of non-bone-derived bone substitutes in vivo may occur by methods other than osteoclast resorption. CONCLUSIONS: Allogenous and xenogenous bone-derived materials that undergo delayed physiologic resorption may be more appropriately used with a staged surgical approach when used in sites intended to support osseointegrated dental implants. The combination of collagen staining and the presence of nitrogen suggest that there may be residual protein in Bio-Oss.

Mechanical characterization of a novel cell stimulating system (CSS) to apply dynamic, uniform and isotropic biaxial strains to cells in vitro.

Mechanical loading alters cellular responses. While in vitro mechanical stimulation is a powerful tool for exploration of mechanotransduction, very little has been published documenting techniques for validation of such devices. We have developed an in vitro experimental system that imposes well-defined temporal and spatial strain profiles using a pressure-actuated, tethered diaphragm substrate (Bioflex dishes). More importantly we have accurately characterized the strain and strain rate performance of this system and herein describe that methodology. The prototype CSS deflected cell substrates over cylindrical platens, producing dynamic biaxial strains. Dynamic studies at 1 Hz were conducted at 8.0, 9.0, 10.0 and 13.0 kPa peak transmural pressures for a total of 1000 loading cycles. To study the effects of frequency, experiments were also run at 0.5 and 1.5 Hz at 8 and 13 kPa. A series of 33 dots were placed collinearly in rings on the membrane. Dot motions were monitored via a CCD video camera and acquisition was performed using an 8-bit gray-scale video board and N1H Image software. Strain fields and rates were subsequently calculated using Mathematica software. Results confirmed that the strains were biaxially uniform over the frequencies and pressures examined: e.g., at 9.0 KPa, max radial & circumferential strain = 0.009 +/- 0.001. It was also shown that, as transmural pressure was increased, both membrane strains and strain rates increased; however biaxial strain isotropy was preserved. While we cannot measure out-of-plane deflections, video-based image analysis is a very useful technique for validation of dynamic planar biaxial strains in cell stimulation systems.