SHIP1 deficiency causes inflammation-dependent retardation in skeletal growth.

Inflammation and skeletal homeostasis are closely intertwined. Inflammatory diseases are associated with local and systemic bone loss, and post-menopausal osteoporosis is linked to low-level chronic inflammation. Phosphoinositide-3-kinase signalling is a pivotal pathway modulating immune responses and controlling skeletal health. Mice deficient in Src homology 2-containing inositol phosphatase 1 (SHIP1), a negative regulator of the phosphoinositide-3-kinase pathway, develop systemic inflammation associated with low body weight, reduced bone mass, and changes in bone microarchitecture. To elucidate the specific role of the immune system in skeletal development, a genetic approach was used to characterise the contribution of SHIP1-controlled systemic inflammation to SHIP1-dependent osteoclastogenesis. Lymphocyte deletion entirely rescued the skeletal phenotype in Rag2 -/- /Il2rg -/- /SHIP1 -/- mice. Rag2 -/- /Il2rg -/- /SHIP1 -/- osteoclasts, however, displayed an intermediate transcriptomic signature between control and Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts while exhibiting aberrant in vitro development and functions similar to Rag2 +/+ /Il2rg +/+ /SHIP1 -/- osteoclasts. These data establish a cell-intrinsic role for SHIP1 in osteoclasts, with inflammation as the key driver of the skeletal phenotype in SHIP1-deficient mice. Our findings demonstrate the central role of the immune system in steering physiological skeletal development.

Bisphosphonates do not affect healing of a critical-size defect in estrogen-deficient mice.

Bisphosphonates (BP) are anti-resorptive drugs that are widely used to prevent bone loss in osteoporosis. Since inhibition of bone resorption will cause a decrease in bone formation through a process called coupling, it is hypothesized that extended treatment protocols may impair bone healing. In this study, ß-tri­calcium-phosphate (ßTCP) ceramics were inserted into critical-size long bone defects in estrogen-deficient mice under BP therapy. The study assessed the benefits of coating the ceramics with Bone Morphogenetic Protein-2 (BMP2) and an engineered BMP2 analogue (L51P) that inactivates BMP antagonists on the healing process, implant resorption, and bone formation. Female NMRI mice (11-12 weeks of age) were ovariectomized (OVX) or sham operated. Eight weeks later, after the manifestation of ovariectomy-induced osteoporotic bone changes, BP therapy with Alendronate (ALN) was commenced. After another five weeks, a femoral critical-size defect was generated, rigidly fixed, and ßTCP-cylinders loaded with 0.25 µg or 2.5 µg BMP2, 2.5 µg L51P, and 0.25 µg BMP2/2.5 µg L51P, respectively, were inserted. Unloaded ßTCP-cylinders were used as controls. Femora were collected six and twelve weeks post-implantation. Histological and micro-computer tomography (MicroCT) evaluation revealed that insertion of cylinders coated with 2.5 µg BMP2 accelerated fracture repair and induced significant bone formation compared to controls (unloaded cylinders or coated with 2.5 µg L51P, 0.25 µg BMP2) already six weeks post-implantation, independent of estrogen-deficiency and BP therapy. The simultaneous administration of BMP2 and L51P (0.25 µg BMP2/2.5 µg L51P) did not promote fracture healing six and twelve weeks post-implantation. Moreover, new bone formation within the critical-size defect was directly linked to the removal of the ßTCP-implant in all experimental groups. No evidence was found that long-term therapy with ALN impaired the resorption of the implanted graft. However, osteoclast transcriptome signature was elevated in sham and OVX animals upon treatment with BP, with transcript levels being higher at six weeks than at twelve weeks post-surgery. Furthermore, the transcriptome profile of the developing repair tissue confirmed an accelerated repair process in animals treated with 2.5 µg BMP2 implants. L51P did not increase the bioefficacy of BMP2 in the applied defect model. The present study provides evidence that continuous administration of BP does not inhibit implant resorption and does not alter the kinetics of the healing process of critical-size long bone defects. Furthermore, the BMP2 variant L51P did not enhance the bioefficacy of BMP2 when applied simultaneously to the femoral critical-size defect in sham and OVX mice.

Gadolinium-Based Contrast Agents and Free Gadolinium Inhibit Differentiation and Activity of Bone Cell Lineages.

OBJECTIVES: Administration of gadolinium-based contrast agents (GBCA) in magnetic resonance imaging results in the long-term retention of gadolinium (Gd) in tissues and organs, including the bone, and may affect their function and metabolism. This study aims to investigate the effects of Gd and GBCA on the proliferation/survival, differentiation, and function of bone cell lineages. MATERIALS AND METHODS: Primary murine osteoblasts (OB) and osteoclast progenitor cells (OPC) isolated from C57BL/6J mice were used to test the effects of Gd3+ (12.5-100 µM) and GBCA (100-2000 µM). Cultures were supplemented with the nonionic linear Gd-DTPA-BMA (gadodiamide), ionic linear Gd-DTPA (gadopentetic acid), and macrocyclic Gd-DOTA (gadoteric acid). Cell viability and differentiation were analyzed on days 4-6 of the culture. To assess the resorptive activity of osteoclasts, the cells were grown in OPC cultures and were seeded onto layers of amorphous calcium phosphate with incorporated Gd. RESULTS: Gd3+ did not affect OB viability, but differentiation was reduced dose-dependently up to 72.4% ± 6.2%-73.0% ± 13.2% (average ± SD) at 100 µM Gd3+ on days 4-6 of culture as compared with unexposed controls (P < 0.001). Exposure to GBCA had minor effects on OB viability with a dose-dependent reduction up to 23.3% ± 10.2% for Gd-DTPA-BMA at 2000 µM on day 5 (P < 0.001). In contrast, all 3 GBCA caused a dose-dependent reduction of differentiation up to 88.3% ± 5.2% for Gd-DTPA-BMA, 49.8% ± 16.0% for Gd-DTPA, and 23.1% ± 8.7% for Gd-DOTA at 2000 µM on day 5 (P < 0.001). In cultures of OPC, cell viability was not affected by Gd3+, whereas differentiation was decreased by 45.3% ± 9.8%-48.5% ± 15.8% at 100 µM Gd3+ on days 4-6 (P < 0.05). Exposure of OPC to GBCA resulted in a dose-dependent increase in cell viability of up to 34.1% ± 11.4% at 2000 µM on day 5 of culture (P < 0.001). However, differentiation of OPC cultures was reduced on day 5 by 24.2% ± 9.4% for Gd-DTPA-BMA, 47.1% ± 14.0% for Gd-DTPA, and 38.2% ± 10.0% for Gd-DOTA (P < 0.001). The dissolution of amorphous calcium phosphate by mature osteoclasts was reduced by 36.3% ± 5.3% upon incorporation of 4.3% Gd/Ca wt/wt (P < 0.001). CONCLUSIONS: Gadolinium and GBCA inhibit differentiation and activity of bone cell lineages in vitro. Thus, Gd retention in bone tissue could potentially impair the physiological regulation of bone turnover on a cellular level, leading to pathological changes in bone metabolism.

Influence of the sintering atmosphere on the physico-chemical properties and the osteoclastic resorption of ß-tricalcium phosphate cylinders.

One of the most widely used materials for bone graft substitution is ß-Tricalcium phosphate (ß-TCP; ß-Ca3(PO4)2). ß-TCP is typically produced by sintering in air or vacuum. During this process, evaporation of phosphorus (P) species occurs, leading to the formation of a calcium-rich alkaline layer. It was recently shown that the evaporation of P species could be prevented by co-sintering ß-TCP with dicalcium phosphate (DCPA; CaHPO4; mineral name: monetite). The aim of this study was to see how a change of sintering atmosphere could affect the physico-chemical and biological properties of ß-TCP. For this purpose, three experimental groups were considered: ß-TCP cylinders sintered in air and subsequently polished to remove the surface layer (control group); the same polished cylinders after subsequent annealing at 500 °C in air to generate a calcium-rich alkaline layer (annealed group); and finally, ß-TCP cylinders sintered in a monetite-rich atmosphere and subsequently polished (monetite group). XPS analysis confirmed that cylinders from the annealed group had a significantly higher Ca/P molar ratio at their surface than that of the control group while this ratio was significantly lower for the cylinders from the monetite group. Sintering ß-TCP in the monetite-rich atmosphere significantly reduced the grain size and increased the density. Changes of surface composition affected the activity of osteoclasts seeded onto the surfaces, since annealed ß-TCP cylinders were significantly less resorbed than ß-TCP cylinders sintered in the monetite-rich atmosphere. This suggests that an increase of the surface Ca/P molar ratio leads to a decrease of osteoclastic resorption. STATEMENT OF SIGNIFICANCE: Minimal changes of surface and bulk (< 1%) composition have major effects on the ability of osteoclasts to resorb ß-tricalcium phosphate (ß-TCP), one of the most widely used ceramics for bone substitution. The results presented in this study are thus important for the calcium phosphate community because (i) ß-TCP may have up to 5% impurities according to ISO and ASTM standards and still be considered to be "pure ß-TCP", (ii) ß-TCP surface properties are generally not considered during biocompatibility assessment and (iii) a rationale can be proposed to explain the various inconsistencies reported in the literature on the biological properties of ß-TCP.

The Allelic Variant A391T of Metal Ion Transporter ZIP8 (SLC39A8) Leads to Hypotension and Enhanced Insulin Resistance.

The metal ion transporter ZIP8 (SLC39A8) mediates cellular uptake of vital divalent metal ions. Genome-wide association studies (GWAS) showed that the single-nucleotide polymorphism (SNP) variant A391T (rs13107325) is associated with numerous human traits, including reduced arterial blood pressure, increased body mass index and hyperlipidemia. We analyzed in vitro the transport properties of mutant ZIP8 A391T and investigated in vivo in mice the physiological effects of this polymorphism. In vitro, the intrinsic transport properties of mutant ZIP8 were similar to those of wild type ZIP8, but cellular uptake of zinc, cadmium and iron was attenuated due to reduced ZIP8 plasma membrane expression. We then generated the ZIP8 A393T mice (ZIP8KI) that carry the corresponding polymorphism and characterized their phenotype. We observed lower protein expression in lung and kidney membrane extracts in ZIP8KI mice. The ZIP8KI mice exhibited striking changes in metal ion composition of the tissues, including cobalt, palladium, mercury and platinum. In agreement with GWAS, ZIP8KI mice showed reduced arterial blood pressure. Body weight and plasma lipid composition remained unchanged, although these features were reported to be increased in GWAS. ZIP8KI mice also exhibited remarkable insulin resistance and were protected from elevated blood glucose when challenged by dietary sucrose supplementation. We showed that increased hepatic insulin receptor expression and decreased ZnT8 (slc30a8) metal ion transporter mRNA expression are associated with this phenotypic change. In conclusion, our data reveal that ZIP8 plays an important role in blood pressure regulation and glucose homeostasis.

Deletion of the sodium/hydrogen exchanger 6 causes low bone volume in adult mice.

The sodium/hydrogen exchanger 6 (NHE6) localizes to recycling endosomes, where it mediates endosomal alkalinization through K+/H+ exchange. Mutations in the SLC9A6 gene encoding NHE6 cause severe X-linked mental retardation, epilepsy, autism and corticobasal degeneration in humans. Patients with SLC9A6 mutations exhibit skeletal malformations, and a previous study suggested a key role of NHE6 in osteoblast-mediated mineralization. The goal of this study was to explore the role of NHE6 in bone homeostasis. To this end, we studied the bone phenotype of NHE6 knock-out mice by microcomputed tomography, quantitative histomorphometry and complementary ex vivo and in vitro studies. We detected NHE6 transcript and protein in both differentiated osteoclasts and mineralizing osteoblasts. In vitro studies with osteoclasts and osteoblasts derived from NHE6 knock-out mice demonstrated normal osteoclast differentiation and osteoblast proliferation without an impairment in mineralization capacity. Microcomputed tomography and bone histomorphometry studies showed a significantly reduced bone volume and trabecular number as well as an increased trabecular space at lumbar vertebrae of 6 months old NHE6 knock-out mice. The bone degradation marker c-terminal telopeptides of type I collagen was unaltered in NHE6 knock-out mice. However, we observed a reduction of the bone formation marker procollagen type 1 N-terminal propeptide, and increased circulating sclerostin levels in NHE6 knock-out mice. Subsequent studies revealed a significant upregulation of sclerostin transcript expression in both primary calvarial cultures and femora derived from NHE6 knock-out mice. Thus, loss of NHE6 in mice causes an increase of sclerostin expression associated with reduced bone formation and low bone volume.

Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age.

Adipose tissue eosinophils (ATEs) are important in the control of obesity-associated inflammation and metabolic disease. However, the way in which ageing impacts the regulatory role of ATEs remains unknown. Here, we show that ATEs undergo major age-related changes in distribution and function associated with impaired adipose tissue homeostasis and systemic low-grade inflammation in both humans and mice. We find that exposure to a young systemic environment partially restores ATE distribution in aged parabionts and reduces adipose tissue inflammation. Approaches to restore ATE distribution using adoptive transfer of eosinophils from young mice into aged recipients proved sufficient to dampen age-related local and systemic low-grade inflammation. Importantly, restoration of a youthful systemic milieu by means of eosinophil transfers resulted in systemic rejuvenation of the aged host, manifesting in improved physical and immune fitness that was partially mediated by eosinophil-derived IL-4. Together, these findings support a critical function of adipose tissue as a source of pro-ageing factors and uncover a new role of eosinophils in promoting healthy ageing by sustaining adipose tissue homeostasis.

Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype.

OBJECTIVE: Spinal fusion surgery is a common treatment modality for various pathologic conditions of the spine. The bone morphogenetic protein 2 (BMP2) analogue L51P acts as a general inhibitor of BMP antagonists, whereas it shows a weak affinity for BMP type I receptor. It is suggested that L51P applied in bone disorders might prevent side effects of highly concentrated BMP dosage applications in the order of milligrams. The objective of this study was to investigate the effects of L51P and BMP2 on intervertebral disc cells (IVDCs), i.e. on nucleus pulposus cells, on annulus fibrosus cells (AFCs), and on cartilaginous endplate cells (CEPCs), respectively, in 3-dimensional (3D) culture. METHODS: Low-passage primary IVDCs were cultured in 3D alginate bead culture and exposed to 100-ng/mL BMP2 and/or L51P for 21 days. Here, we analyzed glycosaminoglycan (GAG) and DNA content and further performed gene expression analysis for major matrix genes. RESULTS: AFCs and cartilaginous CEPCs stimulated with each 100-ng/mL L51P and BMP2, showed a significant upregulation in GAG (AFCs: p = 0.00347 and CEPCs: p = 0.0115) and DNA production (AFCs: p = 0.0182 and CEPCs: p = 0.0179) compared to control. CONCLUSION: These results allow first insights into the behavior of IVDCs upon L51P stimulation.

Effects of local application of alendronate on early healing of extraction socket in dogs.

OBJECTIVE: The aim of the present study was to assess the effects of alendronate (ALN) on bone remodeling following tooth extraction in a dog model. MATERIAL AND METHODS: For the study, fifteen male Beagles dogs of approximately 12 months of age were used. Mesial roots of four mandibular premolars were endodontically treated, and the distal roots were removed. ALN concentrations of 0.5, 1, and 2 mg/mL were topically applied for 15 min, while a sterile saline was used as a negative control. After the healing period of 1, 2, and 8 weeks, the samples were analyzed by micro-CT and histology. RESULTS: Treatment with ALN increased vertical distance between the lingual and the buccal crestal bones. While the ALN-treated sockets had preserved more lingual bone areas, control sockets showed better preservation of the buccal bone areas. ALN treatment resulted in more osteoid formation within the extraction sockets compared with the control. Higher bone volume was found in ALN groups than in the control at 2-week and 8-week healing periods, reaching the significant difference only for the extraction sockets pooled for the ALN treatment. CONCLUSIONS: Although ALN treatment could not prevent buccal bone resorption following tooth extraction in dogs, it proved beneficial for the preservation of the lingual bone and formation of new bone within the socket. There was no clear relation between the ALN dosages and the alterations within the extraction sockets. CLINICAL RELEVANCE: ALN affects bone remodeling of the extraction socket. The optimal concentration remains to be determined in future studies.

Application of Cytokines of the Bone Morphogenetic Protein (BMP) Family in Spinal Fusion - Effects on the Bone, Intervertebral Disc and Mesenchymal Stromal Cells.

Low back pain is a prevalent socio-economic burden and is often associated with damaged or degenerated intervertebral discs (IVDs). When conservative therapy fails, removal of the IVD (discectomy), followed by intersomatic spinal fusion, is currently the standard practice in clinics. The remaining space is filled with an intersomatic device (cage) and with bone substitutes to achieve disc height compensation and bone fusion. As a complication, in up to 30% of cases, spinal non-fusions result in a painful pseudoarthrosis. Bone morphogenetic proteins (BMPs) have been clinically applied with varied outcomes. Several members of the BMP family, such as BMP2, BMP4, BMP6, BMP7, and BMP9, are known to induce osteogenesis. Questions remain on why hyper-physiological doses of BMPs do not show beneficial effects in certain patients. In this respect, BMP antagonists secreted by mesenchymal cells, which might interfere with or block the action of BMPs, have drawn research attention as possible targets for the enhancement of spinal fusion or the prevention of non-unions. Examples of these antagonists are noggin, gremlin1 and 2, chordin, follistatin, BMP3, and twisted gastrulation. In this review, we discuss current evidence of the osteogenic effects of several members of the BMP family on osteoblasts, IVD cells, and mesenchymal stromal cells. We consider in vitro and in vivo studies performed in human, mouse, rat, and rabbit related to BMP and BMP antagonists in the last two decades. We give insights into the effects that BMP have on the ossification of the spine. Furthermore, the benefits, pitfalls, and possible safety concerns using these cytokines for the improvement of spinal fusion are discussed.

Rapid evaluation of bioactive Ti-based surfaces using an in vitro titration method.

The prediction of implant behavior in vivo by the use of easy-to-perform in vitro methods is of great interest in biomaterials research. Simulated body fluids (SBFs) have been proposed and widely used to evaluate the bone-bonding ability of implant materials. In view of its limitations, we report here a rapid in vitro method based on calcium titration for the evaluation of in vivo bioactivity. Using four different titanium surfaces, this method identifies that alkaline treatment is the key process to confer bioactivity to titanium whereas no significant effect from heat treatment is observed. The presence of bioactive titanium surfaces in the solution during calcium titration induces an earlier nucleation of crystalline calcium phosphates and changes the crystallization pathway. The conclusions from this method are also supported by the standard SBF test (ISO 23317), in vitro cell culture tests using osteoblasts and in vivo animal experiments employing a pelvic sheep model.

Effect of grain orientation and magnesium doping on ß-tricalcium phosphate resorption behavior.

The efficiency of calcium phosphate (CaP) bone substitutes can be improved by tuning their resorption rate. The influence of both crystal orientation and ion doping on resorption is here investigated for beta-tricalcium phosphate (ß-TCP). Non-doped and Mg-doped (1 and 6 mol%) sintered ß-TCP samples were immersed in acidic solution (pH 4.4) to mimic the environmental conditions found underneath active osteoclasts. The surfaces of ß-TCP samples were observed after acid-etching and compared to surfaces after osteoclastic resorption assays. ß-TCP grains exhibited similar patterns with characteristic intra-crystalline pillars after acid-etching and after cell-mediated resorption. Electron BackScatter Diffraction analyses, coupled with Scanning Electron Microscopy, Inductively Coupled Plasma-Mass Spectrometry and X-Ray Diffraction, demonstrated the influence of both grain orientation and doping on the process and kinetics of resorption. Grains with c-axis nearly perpendicular to the surface were preferentially etched in non-doped ß-TCP samples, whereas all grains with simple axis (a, b or c) nearly normal to the surface were etched in 6 mol% Mg-doped samples. In addition, both the dissolution rate and the percentage of etched surface were lower in Mg-doped specimens. Finally, the alignment direction of the intra-crystalline pillars was correlated with the preferential direction for dissolution. STATEMENT OF SIGNIFICANCE: The present work focuses on the resorption behavior of calcium phosphate bioceramics. A simple and cost-effective alternative to osteoclast culture was implemented to identify which material features drive resorption. For the first time, it was demonstrated that crystal orientation, measured by Electron Backscatter Diffraction, is the discriminating factor between grains, which resorbed first, and grains, which resorbed slower. It also elucidated how resorption kinetics can be tuned by doping ß-tricalcium phosphate with ions of interest. Doping with magnesium impacted lattice parameters. Therefore, the crystal orientations, which preferentially resorbed, changed, explaining the solubility decrease. These important findings pave the way for the design of optimized bone graft substitutes with tailored resorption kinetics.

Bisphosphonates reduce biomaterial turnover in healing of critical-size rat femoral defects.

Treatment of osteoporotic patients with bisphosphonates (BPs) preserves bone mass and microarchitecture. The high prescription rate of the drugs brings about increases in the numbers of fractures and bone defects requiring surgical interventions in these patients. Currently, critical-size defects are filled with biomaterials and healing is supported with bone morphogenetic proteins (BMP). It is hypothesized that BPs interfere with biomaterial turnover during BMP-supported repair of defects filled with ß-tricalcium phosphate (ßTCP) ceramics. To test this hypothesis, retired breeder rats were ovariectomized ( OVX). After 8 weeks, treatment with alendronate (ALN) commenced. Five weeks later, 6 mm diaphyseal femoral defects were applied and stabilized with locking plates. ßTCP cylinders loaded with 1 µg and 10 µg BMP2, 10 µg L51P, an inhibitor of BMP antagonists and 1 µg BMP2/10 µg L51P were fitted into the defects. Femora were collected 16 weeks post-implantation. In groups receiving calcium phosphate implants loaded with 10 µg BMP2 and 1 µg BMP2/10 µg L51P, the volume of bone was increased and ßTCP was decreased compared to groups receiving implants with 1 µg BMP2 and 10 µg L51P. Treatment of animals with ALN caused a decrease in ßTCP turnover. The results corroborate the synergistic effects of BMP2 and L51P on bone augmentation. Administration of ALN caused a reduction in implant turnover, demonstrating the dependence of ßTCP removal on osteoclast activity, rather than on chemical solubility. Based on these data, it is suggested that in patients treated with BPs, healing of biomaterial-filled bone defects may be impaired because of the failure to remove the implant and its replacement by authentic bone.

Redox-Dependent Bone Alkaline Phosphatase Dysfunction Drives Part of the Complex Bone Phenotype in Mice Deficient for Memo1.

Mediator of ErbB2-driven cell Motility 1 (MEMO1) is an intracellular redox protein that integrates growth factors signaling with the intracellular redox state. We have previously reported that mice lacking Memo1 displayed higher plasma calcium levels and other alterations of mineral metabolism, but the underlying mechanism was unresolved and the bone phenotype was not described. Here, we show that Cre/lox-mediated MEMO1 deletion in the whole body of C57Bl/6 mice (Memo cKO) leads to severely altered trabecular bone and lower mineralization, with preserved osteoblast and osteoclast number and activity, but altered osteoblast response to epidermal growth factor (EGF) and FGF2. More strikingly, Memo cKO mice display decreased alkaline phosphatase (ALP) activity in serum and in bone, while ALPL expression level is unchanged. Bone intracellular redox state is significantly altered in Memo cKO mice and we inferred that ALP dimerization was reduced in Memo cKO mice. Indeed, despite similar ALP oxidation, we found increased ALP sensitivity to detergent in Memo cKO bone leading to lower ALP dimerization capability. Thus, we report a severe bone phenotype and dysfunctional bone ALP with local alteration of the redox state in Memo cKO mice that partially mimics hypophosphatasia, independent of ALPL mutations. These findings reveal Memo as a key player in bone homeostasis and underline a role of bone redox state in controlling ALP activity.

Healing of fractures in osteoporotic bones in mice treated with bisphosphonates - A transcriptome analysis.

Bisphosphonates (BP) are inhibitors of bone resorption and are used to treat postmenopausal osteoporosis. Long-term treatment with BP attenuates bone remodeling, possibly leading to detrimental consequences for the bones' ability to repair defects. To test this hypothesis, an animal model was established. Twelve week old mice were ovariectomized (OVX). Following confirmation of bone loss 8 weeks after OVX, the animals were treated with Alendronate (ALN) until sacrifice. After 5 weeks of ALN injections, the femoral bones were osteotomized and the osteotomies were either rigidly or non-rigidly stabilized. In rigidly fixed defects, no callus developed between 1 and 5 weeks after osteotomy, whereas after non-rigid fixation, callus development occurred. The administration of ALN resulted in an increase in newly formed bone at the defect site 5 weeks after osteotomy, irrespective of the estrogen status or fixation system. Transcriptome analysis demonstrated that both rigid and non-rigid fixation affected gene expression primarily during the middle phase of bone repair. Furthermore, the number of differentially expressed genes in tissues from non-rigidly fixed defect sites increased in animals treated with ALN over the course of bone repair. This indicates that ALN-dependent repair processes become increasingly dominant in the late phases of the healing process. Ranking of the factors affecting the composition of the transcriptome and their impact on the healing process revealed fixation at the defect site to be the strongest causative factor, followed by bisphosphonate treatment and estrogen deficiency. The present study suggests that the continuous administration of ALN is detrimental to bone repair, eventually causing a delay in healing in mechanically compromised situations. Consequently, rigid fixation may prove essential for a successful intervention.

Osteosynthesis of partial rib osteotomy in a miniature pig model using human standard-sized magnesium plate/screw systems: Effect of cyclic deformation on implant integrity and bone healing.

Magnesium alloys are candidates for resorbable material in bone fixation. However, the degradation and performance of osteosynthesis plate/screw systems in vivo, under cyclic deformation, is unknown. We evaluated the outcomes of human standard-sized magnesium plate/screw systems with or without plasma-electrolytic surface modifications in a miniature pig rib model. Of a total of 14 minipigs, six were implanted with coated magnesium WE43 six-hole plates/screws, six received magnesium uncoated plates/screws, and two received titanium osteosynthesis systems. The performance of the plate/screw fixation system on partially osteotomized 7th ribs was compared with that on intact 9th ribs. Radiological examinations were performed in vivo at 1, 4 and 8 weeks and after euthanasia at 12 and 24 weeks. After euthanasia the bone blocks were analyzed by computed tomography (CT), microfocus computed tomography (micro-CT), histology and histomorphometry. Follow-up post-surgery showed no trouble with wound healing. In vivo radiological examinations showed higher amounts of gas formation above the uncoated magnesium plates fixed on the partially osteotomized and intact ribs. CT scans showed no broken plates or implant displacement. The micro-CT examination demonstrated better surrounding bone properties around the coated than the uncoated magnesium implants 12 weeks after surgery. No negative influence of magnesium degradation on bone healing was observed with histological examinations. Plastic deformation during surgery and cyclic deformation did not affect the integrity of the used magnesium plates. This study showed promising results for the further development of coated magnesium plate/screw systems for bone fixation.

Granulocyte-macrophage colony-stimulating factor-dependent CD11c-positive cells differentiate into active osteoclasts.

Levels of circulating cytokines are elevated in inflammatory diseases. Previously, it was shown that interleukin (IL-)17A, in synergism with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and tumor necrosis factor α (TNFα), induces the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) by murine osteoblasts in vitro. In this study, we further analyzed the effects of GM-CSF on osteoclast development in vitro. The effects of IL-17A, TNFα, and 1,25(OH)2D3 on the regulation of osteoclast development were investigated in cocultures of bone marrow-derived osteoclast progenitor cells (OPC) and mouse calvarial osteoblasts. Additionally, OPC were grown for 3days in media containing macrophage colony-stimulating factor (M-CSF), GM-CSF, or M-CSF/GM-CSF. Subsequently, the osteoclastogenic potential and the capacity to dissolve amorphous calcium phosphate were assessed in each of the three populations of OPC. IL-17A, in synergism with TNFα and 1,25(OH)2D3, inhibited the development of osteoclasts in cocultures by stimulating the osteoblast lineage cells to release GM-CSF. GM-CSF-treated OPC expressed traits characteristic of dendritic cells. Upon removal of GM-CSF and supplementation of the culture media with M-CSF/RANKL, the cells lost their dendritic cell characteristics and differentiated into osteoclasts. OPC pretreated with GM-CSF and M-CSF/GM-CSF exhibited delayed development to osteoclasts and an extended proliferation phase. Elevated levels of GM-CSF in systemic inflammatory diseases may cause an expansion of the OPC pools in the bone, bone marrow, and blood. Upon homing to the bone, this may lead to an increase in the number of osteoclasts and in bone resorption.

Role of the plasma cascade systems in ischemia/reperfusion injury of bone.

Ischemia/reperfusion (I/R) injury has been extensively studied in organs such as heart, brain, liver, kidney, and lung. As a vascularized organ, bone is known to be susceptible to I/R injury too, but the respective mechanisms are not well understood to date. We therefore hypothesized that, similar to other organs, plasma cascade-induced inflammation also plays a role in bone I/R injury. Reperfusion injury in rat tibia was induced by unilateral clamping of the femoral artery and additional use of a tourniquet, while keeping the femoral vein patent to prevent venous congestion. Rats were subjected to 4h ischemia and 24h reperfusion. Deposition of complement fragment C3b/c and fibrin as well as expression of tissue factor (TF), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and E-selectin was detected by immunohistochemistry. In plasma, the levels of high mobility group box1 (HMGB1) were measured by ELISA. The total level of complement in serum was assessed by the CH50 test. Our results show that deposition of C3b/c was significantly increased with respect to healthy controls in cortical bone as well as in marrow of reperfused limbs. C3b/c deposition was also increased in cortical bone, but not in bone marrow, of contralateral limbs. Deposition of fibrin, as well as expression of PAI-1, was significantly increased in bone after ischemia and reperfusion, whereas expression of tPA was reduced. These differences were most prominent in vessels of bone, both in marrow and cortical bone, and both in reperfused and contralateral limbs. However, PAI-1, was only increased in vessels of reperfused cortical bone and there were no significant changes in expression of E-selectin. With respect to solid bone tissue, a significant increase of C3b/c and fibrin deposition was shown in osteocytes, and for fibrin also in the bone matrix, in both contralateral and reperfused cortical bone compared with normal healthy controls. A slight expression of TF was visible in osteocytes of the normal healthy control group, while TF was not present in the experimental groups. Moreover, CH50 values in serum decreased over time and HMGB1 was significantly increased in plasma of animals at the end of reperfusion. We conclude that ischemia and reperfusion of bone leads to activation of the complement and coagulation systems and a downregulation of the fibrinolytic cascade. In the acute phase, a vascular inflammation induced by activation of the plasma cascade systems also occurs in the bone. This is similar to I/R injury of other vascularized organs and tissues.

Increased bone resorption by osteoclast-specific deletion of the sodium/calcium exchanger isoform 1 (NCX1).

Calcium is a key component of the bone mineral hydroxyapatite. During osteoclast-mediated bone resorption, hydroxyapatite is dissolved and significant quantities of calcium are released. Several calcium transport systems have previously been identified in osteoclasts, including members of the sodium/calcium exchanger (NCX) family. Expression pattern and physiological role of NCX isoforms in osteoclasts, however, remain largely unknown at the moment. Our data indicate that all three NCX isoforms (NCX1, NCX2, and NCX3) are present in murine osteoclasts. RANKL-induced differentiation of murine osteoclast precursors into mature osteoclasts significantly attenuated the expression of NCX1, while NCX2 and NCX3 expressions were largely unaffected. To study the role of NCX1 during osteoclast differentiation and bone resorption, we crossed mice with exon 11 of the NCX1 gene flanked by loxP sites with cathepsin K-Cre transgenic mice. Mature osteoclasts derived from transgenic mice exhibited an 80-90% reduction of NCX1 protein. In vitro studies indicate that NCX1 is dispensable for osteoclast differentiation, but NCX1-deficient osteoclasts exhibited increased resorptive activity. In line with these in vitro findings, mice with an osteoclast-targeted deletion of the NCX1 gene locus displayed an age-dependent loss of bone mass. Thus, in summary, our data reveal NCX1 as a regulator of osteoclast-mediated bone resorption.

In vivo degradation of a new concept of magnesium-based rivet-screws in the minipig mandibular bone.

Self-tapping of magnesium screws in hard bone may be a challenge due to the limited torsional strength of magnesium alloys in comparison with titanium. To avoid screw failure upon implantation, the new concept of a rivet-screw was applied to a WE43 magnesium alloy. Hollow cylinders with threads on the outside were expanded inside drill holes of minipig mandibles. During the expansion with a hexagonal mandrel, the threads engaged the surrounding bone and the inside of the screw transformed into a hexagonal screw drive to allow further screwing in or out of the implant. The in vivo degradation of the magnesium implants and the performance of the used coating were studied in a human standard-sized animal model. Four magnesium alloy rivet-screws were implanted in each mandible of 12 minipigs. Six animals received the plasmaelectrolytically coated magnesium alloy implants; another six received the uncoated magnesium alloy rivet-screws. Two further animals received one titanium rivet-screw each as control. In vivo radiologic examination was performed at one, four, and eight weeks. Euthanasia was performed for one group of seven animals (three animals with coated, three with uncoated magnesium alloy implants and one with titanium implant) at 12weeks and for the remaining seven animals at 24weeks. After euthanasia, micro-computed tomography and histological examination with histomorphometry were performed. Significantly less void formation as well as higher bone volume density (BV/TV) and bone-implant contact area (BIC) were measured around the coated implants compared to the uncoated ones. The surface coating was effective in delaying degradation despite plastic deformation. The results showed potential for further development of magnesium hollow coated screws for bone fixation.