NELL-1 in the treatment of osteoporotic bone loss.

NELL-1 is a secreted, osteoinductive protein whose expression rheostatically controls skeletal ossification. Overexpression of NELL-1 results in craniosynostosis in humans and mice, whereas lack of Nell-1 expression is associated with skeletal undermineralization. Here we show that Nell-1-haploinsufficient mice have normal skeletal development but undergo age-related osteoporosis, characterized by a reduction in osteoblast:osteoclast (OB:OC) ratio and increased bone fragility. Recombinant NELL-1 binds to integrin ß1 and consequently induces Wnt/ß-catenin signalling, associated with increased OB differentiation and inhibition of OC-directed bone resorption. Systemic delivery of NELL-1 to mice with gonadectomy-induced osteoporosis results in improved bone mineral density. When extended to a large animal model, local delivery of NELL-1 to osteoporotic sheep spine leads to significant increase in bone formation. Altogether, these findings suggest that NELL-1 deficiency plays a role in osteoporosis and demonstrate the potential utility of NELL-1 as a combination anabolic/antiosteoclastic therapeutic for bone loss.

Allogeneic mesenchymal progenitor cells for posterolateral lumbar spine fusion in sheep.

BACKGROUND CONTEXT: Osteoconductive porous ceramic bone graft materials supplemented with mesenchymal precursor cells (MPC) derived from autologous bone marrow aspirates have been shown to stimulate successful interbody and posterolateral spine fusion in preclinical models. Recent advances in immunomagnetic cell sorting have enabled purification and isolation of pluripotent stem cells from marrow aspirates and have expanded stem cell technology to allogeneic cell sources. Allogeneic MPC technology combined with appropriate synthetic biomaterial carriers could provide both the osteogenic and osteoconductive components needed for successful posterolateral spine fusion without the need for autologous bone harvest or expensive recombinant protein technology. PURPOSE: To determine the safety and efficacy of a hydroxyapatite:tricalcium phosphate graft material supplemented with allogeneic mesenchymal precursor cells in posterolateral lumbar spine fusion using an ovine model. STUDY DESIGN: Skeletally mature ewes underwent single-level instrumented posterolateral lumbar spine fusion using either autograft (AG), hydroxyapatite:tricalcium phosphate carrier (CP), or CP supplemented with allogeneic mesenchymal progenitor cells (MPCs). Three doses of MPCs were evaluated: 25 × 106 cells (low dose, LD), 75 × 106 cells (mid dose, MD), and 225 × 106 cell (high dose, HD). Animals survived for either 4 or 9 months. METHODS: Plain radiographs were acquired and scored for bridging bone at regular intervals during healing to monitor fusion development. Hematology, coagulation, and serum chemistry were monitored at regular intervals throughout the study to monitor animal health. After necropsy, computed tomography, high-resolution radiography, biomechanical testing, organ pathology, bone histopathology, and bone histomorphometry were conducted to monitor the safety and ascertain the efficacy of MPC treatment. RESULTS: MPC treatment in this spine fusion model resulted in no observed adverse systemic or local tissue responses. Radiographically, fusion scores for MPC-treated animals were uniformly higher compared with those treated with carrier alone (CP) after 3 months and continued the same trend throughout 9 month of healing. Quantitative computed tomography confirmed better connectivity of the fusion for MPC treatment groups compared with CP. Biomechanical analyses were not able to differentiate between treatment groups. Histomorphometry results confirmed radiographic and quantitative computed tomography results; cell-supplemented treatment groups and autograft had equivalent amounts of bone within the fusion mass and less bony fusion tissue was found within the fusion mass in specimens from the CP treatment group. No conclusive effects of cell dose of fusion efficacy were noted. CONCLUSIONS: Adult allogeneic mesenchymal precursor cells delivered via a hydroxyapatite:tricalcium phosphate carrier were both safe and efficacious in this ovine spine fusion model. Results from this preclinical study support that allogeneic mesenchymal precursor cells produced fusion efficacy similar to that achieved using iliac crest autograft, thereby providing a safe and viable option to achieve successful posterolateral spine fusion.

Evaluation of effects of sciatic and femoral nerve blocks in sheep undergoing stifle surgery.

The authors evaluated the effects of locally anesthetizing the sciatic and femoral nerves in sheep undergoing stifle (femorotibial) surgery (16 sheep received nerve blocks; 16 sheep underwent a nerve localization procedure but received no nerve blocks). Heart rate, mean arterial blood pressure and end-tidal isoflurane were recorded every 5 min while sheep were anesthetized. At some of the observed time points, the mean heart rate in the sheep that had received no nerve blocks was significantly higher than in the sheep that had received the nerve blocks. Postoperatively, each sheep was assigned scores for comfort and attitude, movement, flock behavior, feeding behavior and appetite and respiratory rate (based on predefined descriptions). Though the authors found no undesirable effects of this local anesthesia, beneficial effects of the nerve blocks were minimal or not readily apparent under the conditions of this study.

Evaluation of a silica-containing bone graft substitute in a vertebral defect model.

Orthopedic and spine surgeons are in need of supplements or replacements for autograft. We investigated the histological properties of three formulations of Calcium Sodium Phosphosilicate [calcium sodium phosphosilicate putty with or without autograft and NovaBone 45S5 Bioglass particulate (NovaBone, LLC, Jacksonville, FL)] using a sheep vertebral bone void model. Bone voids were surgically created in L3, L4, and L5 in each of 22 sheep, and the voids were filled with one of the tested biomaterials or left empty as a control. Histological evaluations were performed at either: 0, 6, or 12 weeks after surgery. Undecalcified sections were digitized, and the areas of the original defect and new bone were quantified. Decalcified sections were evaluated qualitatively. Histomorphometry showed a significant increase in the amount of bone between 6 and 12 weeks in all groups, but there was no significant difference in new bone formation among the formulations or between any formulation and the empty defects. The granules of all three formulations were associated with an inflammatory reaction. Many of the particles appear to have a hollow center, and the narrow tunnel through the center of the particles was sometimes associated with acute inflammation especially at 6 weeks. These particles were also associated with chronic inflammation at both 6 and 12 weeks, although the extent of inflammation decreased between 6 and 12 weeks. The search for the optimum bone graft substitute/extender will continue.

Efficacy of silicated calcium phosphate graft in posterolateral lumbar fusion in sheep.

BACKGROUND CONTEXT: Conditions requiring posterior lumbar spinal fusion remain a clinical challenge. Achieving arthrodesis using autogenous bone graft is inconsistent when rigid internal fixation such as transpedicular instrumentation is applied. Synthetic materials, particularly calcium phosphate-based ceramics, have shown promise for spine fusion applications, especially when combined with autograft. Silicate substitution has been shown to enhance the bioactivity of calcium phosphates and may obviate the need for autologous supplementation. PURPOSE: Determine efficacy of silicated calcium phosphate (Si-CaP) compared with autograft to generate solid lumbar fusion. STUDY DESIGN: Comparison of healing of instrumented posterolateral lumbar fusion in ewes at 2 and 6 months using Si-CaP or iliac crest autograft. METHODS: Eighteen skeletally mature ewes underwent implantation of either autograft or Si-CaP in the space spanning the L4-L5 transverse process. In vivo quantitative computed tomography (CT) scans were made at 2-month intervals and after euthanasia. Harvested spine segments were radiographed and biomechanically tested in bending at 6 months. Histological assessments were made at 2 and 6 months. RESULTS: Animals receiving Si-CaP graft were biomechanically and radiographically equivalent to those receiving autograft. Fusion mass density and volume were higher for the Si-CaP group throughout the healing period. Si-CaP regenerated normal bone tissue morphology, cellularity, and maturation with no inflammatory responses despite the fact that no autograft, bone marrow aspirate, or blood was mixed with the material. Histomorphometrically, fusion mass was higher for Si-CaP and bony bridging was equivalent when compared with autograft treatment. CONCLUSIONS: Si-CaP was biomechanically, radiographically, and histologically equivalent to autograft in generating a solid, bony, intertransverse process fusion in an ovine model. Both treatment groups achieved 100% bridging fusion after 6 months of healing.

Histological evaluation of an impacted bone graft substitute composed of a combination of mineralized and demineralized allograft in a sheep vertebral bone defect.

Demineralized bone matrix (DBMs) preparations are a potential alternative or supplement to autogenous bone graft, but many DBMs have not been adequately tested in clinically relevant animal models. The aim of current study was to compare the efficacy of a new bone graft substitute composed of a combination of mineralized and demineralized allograft, along with hyaluronic acid (AFT Bone Void Filler) with several other bone graft materials in a sheep vertebral bone void model. A drilled defect in the sheep vertebral body was filled with either the new DBM preparation, calcium sulfate (OsteoSet), autologous bone graft, or left empty. The sheep were euthanized after 6 or 12 weeks, and the defects were examined by histology and quantitative histomorphometry. The morphometry data were analyzed by one-way analysis of variance with the post hoc Tukey-Kramer test or the Student's t-test. All of the bone defects in the AFT DBM preparation group showed good new bone formation with variable amounts of residual DBM and mineralized bone graft. The DBM preparation group at 12 weeks contained significantly more new bone than the defects treated with calcium sulfate or left empty (respectively, p < 0.05, p < 0.01). There was no significant difference between the DBM and autograft groups. No adverse inflammatory reactions were associated with any of the three graft materials. The AFT preparation of a mixture of mineralized and demineralized allograft appears to be an effective autograft substitute as tested in this sheep vertebral bone void model.

Evaluation of the effects of dietary n-3 fatty acid supplementation on the pharmacokinetics of doxorubicin in dogs with lymphoma.

OBJECTIVE: To determine the effect of dietary n-3 fatty acids on the pharmacokinetics of doxorubicin in dogs with lymphoma. ANIMALS: 23 dogs with lymphoma in stages IIIa, IVa, and Va. PROCEDURE: Dogs receiving doxorubicin chemotherapy were randomly allocated to receive food with a high (test group) or low (control group) content of n-3 fatty acids. Serum doxorubicin and doxorubicinol concentrations were measured via high-performance liquid chromatography before and 6 to 9 weeks after initiation of the diets. Lymph node concentrations of doxorubicin were assessed 6 hours after the initial treatment. Dogs' body composition was assessed by means of dual-energy x-ray absorptiometry scans. RESULTS: No significant differences in doxorubicin pharmacokinetics were detected between treatment groups. Significant differences existed between the first and second sampling times among all dogs for area under the curve, maximum serum concentration, and clearance. Differences in body composition did not affect measured pharmacokinetic variables. The terminal elimination half-life was longer in dogs in which a long-term remission was achieved than in dogs that did not have remission. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary supplementation of n-3 fatty acids is common in veterinary patients with neoplasia, but supplementation did not affect doxorubicin pharmacokinetics in this population of dogs. Explanations for the beneficial effects of n-3 fatty acids other than alterations in the pharmacokinetics of chemotherapy drugs should be investigated. Dogs may metabolize drugs differently prior to remission of lymphoma than when in remission. The pharmacokinetics of doxorubicin at the time of the first administration may predict response to treatment.

Effect of dietary-induced metabolic acidosis and ovariectomy on bone mineral density and markers of bone turnover.

Dietary-induced metabolic acidosis (DIMA) has been implicated as a significant confounder in the development of osteoporosis. Twenty-four mature ewes were randomly assigned to four groups of six sheep. Group 1 consumed a control diet (ND); group 2 consumed a normal diet (ND) and had ovariectomy (OVX), group 3 consumed a diet that induced metabolic acidosis (MA), without OVX, and group 4 consumed a diet that induced MA, with OVX. The study was conducted over 180 days and the sheep were maintained on the assigned diet throughout. Sheep were weighed and bone mineral density (BMD) was measured, using dual-energy X-ray absorptiometry (DEXA), on days 0 and 180. Serum bone alkaline phosphatase (BAP), urine deoxypyridinoline (DPD), and fractional excretions (FE) of Ca and P were determined on days 0, 90, and 180. Arterial blood pH was determined on day 180. Analysis consisted of a two-way analysis of variance for repeated measures with significance set at P < or = 0.05. Body weights, serum BAP, and urine DPD were not influenced by either diet or OVX status. DIMA did significantly increase urinary FE of Ca and P and significantly decreased lumbar BMD and arterial pH. Arterial pH remained within physiologic normal limits. DIMA was a more potent cause of calcium wasting than OVX over the time frame of this study. Sheep appear to be sensitive to DIMA and will therefore be a useful animal model to study the influence of diet on the development of osteoporosis. The specific mechanisms through which DIMA exerts its influence are still unknown and are the subject of ongoing studies.