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.

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.

TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1(-/-) mice are viable, fertile and show no altered hematopoietic compartment. In CD4(+) T cell- and dextran sodium sulfate-induced models of colitis, Trem1(-/-) mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1(-/-) mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1(-/-) mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1(-/-) mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1(+/+) controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.

Glutamate Receptor Agonists and Glutamate Transporter Antagonists Regulate Differentiation of Osteoblast Lineage Cells.

Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell-cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response in the skeletal system. Known antagonists of glutamate transporters will serve as lead compounds in developing new and specific bioactive molecules.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

Interleukin-17A stimulates granulocyte-macrophage colony-stimulating factor release by murine osteoblasts in the presence of 1,25-dihydroxyvitamin D(3) and inhibits murine osteoclast development in vitro.

OBJECTIVE: To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. METHODS: Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte-macrophage colony-stimulating factor-knockout (GM-CSF(-/-)) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D(3)(1,25[OH](2)D(3)), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5-6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. RESULTS: In both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)(2)D(3) to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF(-/-) mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. CONCLUSION: These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.

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 Gd 3+ (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: Gd 3+ 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 Gd 3+ 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 Gd 3+ , whereas differentiation was decreased by 45.3% ± 9.8%-48.5% ± 15.8% at 100 µM Gd 3+ 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.

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.

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.

Blockade of the hedgehog pathway inhibits osteophyte formation in arthritis.

BACKGROUND: Osteophyte formation is a common phenomenon in arthritis. Bone formation by endochondral ossification is considered a key pathophysiological process in the formation of osteophytes. OBJECTIVE: To examine the hypothesis that inhibition of smoothened (Smo), a key component of the hedgehog pathway inhibits osteophyte formation as the hedgehog pathway mediates endochondral ossification. METHODS: Arthritis was induced in 8-week-old C57/BL6 mice by serum transfer (K/BxN model). Mice were then treated by daily administration of either vehicle or LDE223, a specific small molecule inhibitor for Smo, over 2 weeks starting at the onset of disease. Clinical course of arthritis, histological and molecular changes of bone in the affected joints as well as systemic bone changes were assessed. RESULTS: Serum transfer-induced arthritis led to severe osteophyte formation within 2 weeks of onset. Blockade of Smo inhibited hedgehog signalling in vivo and also significantly inhibited osteophyte formation, whereas the clinical and histopathological signs of arthritis were not affected. Also, systemic bone mass did not change. Smo inhibitor particularly blocked the formation of hypertrophic chondrocytes and collagen type X expression. CONCLUSIONS: The data indicate that blockade of hedgehog signalling by targeting Smo specifically inhibits osteophyte formation in arthritis without affecting inflammation and without eliciting bone destruction at the local and systemic level. Blockade of Smo may thus be considered as a strategy to specifically influence the periosteal bone response in arthritis associated with bone apposition.

Association between cam-type deformities and magnetic resonance imaging-detected structural hip damage: a cross-sectional study in young men.

OBJECTIVE: Femoroacetabular impingement may be a risk factor for hip osteoarthritis in men. An underlying hip deformity of the cam type is common in asymptomatic men with nondysplastic hips. This study was undertaken to examine whether hip deformities of the cam type are associated with signs of hip abnormality, including labral lesions and articular cartilage damage, detectable on magnetic resonance imaging (MRI). METHODS: In this cross-sectional, population-based study in asymptomatic young men, 1,080 subjects underwent clinical examination and completed a self-report questionnaire. Of these subjects, 244 asymptomatic men with a mean age of 19.9 years underwent MRI. All MRIs were read for cam-type deformities, labral lesions, cartilage thickness, and impingement pits. The relationship between cam-type deformities and signs of joint damage were examined using logistic regression models adjusted for age and body mass index. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined. RESULTS: Sixty-seven definite cam-type deformities were detected. These deformities were associated with labral lesions (adjusted OR 2.77 [95% CI 1.31, 5.87]), impingement pits (adjusted OR 2.9 [95% CI 1.43, 5.93]), and labral deformities (adjusted OR 2.45 [95% CI 1.06, 5.66]). The adjusted mean difference in combined anterosuperior femoral and acetabular cartilage thickness was -0.19 mm (95% CI -0.41, 0.02) lower in those with cam-type deformities compared to those without. CONCLUSION: Our findings indicate that the presence of a cam-type deformity is associated with MRI-detected hip damage in asymptomatic young men.

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.

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.

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.

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.

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.

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.

Time course of biological activity in fresh murine osteochondral allografts paralleled to the recipient's immune response.

The use of fresh osteochondral allografts is a popular approach to treat articular cartilage lesions. Immunological reactions of the recipient elicited by the allograft's osseous portion, however, frequently result in their deterioration. So far, little emphasis has been put on describing morphology and biological activity in fresh allografts and paralleling these to the immunological processes triggered in the host. Therefore, in the present study murine neonatal femora, serving as osteochondral grafts, were transplanted as fresh isografts (controls) or allografts (the latter in non- or presensitized mice) and retrieved after 2, 5, 10, and 20 days. It was shown that (1) in isografts active bone cells (osteoblasts, osteoclasts) were present, the bone marrow was repopulated with hematopoietic cells, the diaphysis increased in length, and no specific immunological reaction by the recipient was evoked. (2) Allografts transplanted into nonsensitized hosts initially appeared similar as isografts, but activated T lymphocytes at the transplantation site preceded loss of active bone cells within the graft and development of fibrosis within the marrow cavity. (3) In allografts transplanted into presensitized recipients, severe deterioration of the graft was observed with very few active bone cells, accompanied by an invasion of T lymphocytes and fibrosis in the marrow cavity already in early stages. Similar to vital organ transplantation, the function of cells within osteochondral allografts is severely impaired after being recognized by the immune system. Therefore, emphasis has to be placed on the development of procedures preserving cartilage biology while reducing the antigenicity of the allograft's osseous portion.