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.

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.

Simultaneous assessment of mechanical and electrical function in Langendorff-perfused ex-vivo mouse hearts.

Background: The Langendorff-perfused ex-vivo isolated heart model has been extensively used to study cardiac function for many years. However, electrical and mechanical function are often studied separately-despite growing proof of a complex electro-mechanical interaction in cardiac physiology and pathology. Therefore, we developed an isolated mouse heart perfusion system that allows simultaneous recording of electrical and mechanical function. Methods: Isolated mouse hearts were mounted on a Langendorff setup and electrical function was assessed via a pseudo-ECG and an octapolar catheter inserted in the right atrium and ventricle. Mechanical function was simultaneously assessed via a balloon inserted into the left ventricle coupled with pressure determination. Hearts were then submitted to an ischemia-reperfusion protocol. Results: At baseline, heart rate, PR and QT intervals, intra-atrial and intra-ventricular conduction times, as well as ventricular effective refractory period, could be measured as parameters of cardiac electrical function. Left ventricular developed pressure (DP), left ventricular work (DP-heart rate product) and maximal velocities of contraction and relaxation were used to assess cardiac mechanical function. Cardiac arrhythmias were observed with episodes of bigeminy during which DP was significantly increased compared to that of sinus rhythm episodes. In addition, the extrasystole-triggered contraction was only 50% of that of sinus rhythm, recapitulating the "pulse deficit" phenomenon observed in bigeminy patients. After ischemia, the mechanical function significantly decreased and slowly recovered during reperfusion while most of the electrical parameters remained unchanged. Finally, the same electro-mechanical interaction during episodes of bigeminy at baseline was observed during reperfusion. Conclusion: Our modified Langendorff setup allows simultaneous recording of electrical and mechanical function on a beat-to-beat scale and can be used to study electro-mechanical interaction in isolated mouse hearts.

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.

3-Dimensional Analysis and Systematic Review of Root Canal Morphology and Physiological Foramen Geometry of 109 Mandibular First Premolars by Micro-computed Tomography in a Mixed Swiss-German Population.

INTRODUCTION: The aim of this study was to systematically revise the root canal configuration (RCC) literature and to investigate the root canal morphology of mandibular first premolars (Ma1Ps) of 2 populations by means of micro-computed tomographic imaging. METHODS: This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines including RCC randomized controlled trials and cross-sectional, cohort, comparative, validation, and evaluation studies. Furthermore, the RCC, physiological foramina, the frequency of accessory and connecting canals, and the physiological foramina morphology of 109 Ma1Ps were investigated by means of micro-computed tomographic imaging. The RCC results are described using a 4-digit system code. RESULTS: The most frequent RCCs observed were 1-1-1/1 (70.6%), 1-1-2/2 (7.3%), 1-2-2/2 (7.3%), and 1-2-1/1 (5.5%). Accessory canals were observed in 31.2%. Connecting canals were observed in 1-1-2/2 (4.6%), 1-2-2/2 (4.6%), 1-1-2/1 (1.8%), and 1-2-1/1 (1.8%) RCCs. Accessory foramina were observed in 52.3%; 30.3% of the Ma1Ps had 1 accessory foramen, 12.8% had 2, 2.8% had 3, 2.8% had 4, 2.8% had 5, and 0.92% had 6. The narrow and wide diameter mean of 136 physiological foramina was 0.28 mm (±0.9) and 0.37 mm (±0.11) when only 1 physiological foramen was present. CONCLUSIONS: This study provides detailed root canal morphology of Ma1Ps in a Swiss-German population. Within the limitations of the study, the authors recommend a final physiological foramen preparation size of instrument tip sizes 30-40; yet, such a decision should be carefully considered on an individual basis.

An Ex Vivo Study of Root Canal System Configuration and Morphology of 115 Maxillary First Premolars.

INTRODUCTION: The aim of this study was to investigate the root canal system morphology of maxillary first premolars by means of micro-computed tomographic imaging in a Swiss-German population. METHODS: The root canal configuration (RCC) of 115 maxillary first premolars (Mx1Ps) were investigated by means of micro-computed tomographic imaging and 3-dimensional imaging. The RCC and the physiological foramina results are described by a 4-digit system code. RESULTS: Twelve different RCCs were observed in 30 single-rooted Mx1Ps; 2-2-2/2 (30.0%), 1-2-2/2 (13.3%), 1-2-1/2 (10%), and 2-2-1/2 (10.0%) were the most frequent ones. Seven different RCCs were observed in 2-rooted Mx1Ps (n = 81) in which the 1-1-1/1 (56.8%), 1-1-1/2 (29.6%), and 1-1-2/2 (8.6%) in the buccal root and 1-1-1/1 (92.6%) and 1-1-1/2 (6.2%) in the palatal root RCCs appeared most frequently. Three-rooted Mx1Ps (n = 4) showed a 1-1-1/1 (100.0%) RCC in all roots. The buccal root canal in 2-rooted Mx1Ps had 1 physiological foramen in 59.3% and 2 in 40.7% and 1 to 6 accessory foramina in 38.2%. The palatal root canal showed 1 physiological foramen in 93.8% and 2 in 6.2% and 1 to 2 accessory foramina in 14.8%. Single-rooted Mx1Ps showed 1 physiological foramen in 10.0%, 2 in 70.0%, 3 in 13.3%, and 4 in 6.7% and 1 to 3 accessory foramina in 46.7%. CONCLUSIONS: The results of this study provide detailed morphologic RCC information of Mx1Ps in a Swiss-German population. Single-rooted Mx1Ps showed morphologic diversifications more frequently than 2- or 3-rooted Mx1Ps. Within 2-rooted Mx1Ps, the buccal root had higher RCC variety, accessory canals, and foramina number than the palatal root.

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.

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.

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.

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.

Sodium-dependent phosphate transporters in osteoclast differentiation and function.

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes.

Endochondral ossification is regulated by many factors, including mechanical stimuli, which can suppress or accelerate chondrocyte maturation. Mathematical models of endochondral ossification have suggested that tension (or shear stress) can accelerate the formation of endochondral bone, while hydrostatic stress preserves the cartilage phenotype. The goal of this study was to test this hypothesis by examining the expression of hypertrophic chondrocyte markers (transcription factor Cbfa1, MMP-13, type X collagen, VEGF, CTGF) and cartilage matrix proteins under cyclic tension and cyclic hydrostatic pressure. Chondrocyte-seeded alginate constructs were exposed to one of the two loading modes for a period of 3 h per day for 3 days. Gene expression was analyzed using real-time RT-PCR. Cyclic tension upregulated the expression of Cbfa1, MMP-13, CTGF, type X collagen and VEGF and downregulated the expression of TIMP-1. Cyclic tension also upregulated the expression of type 2 collagen, COMP and lubricin, but did not change the expression of SOX9 and aggrecan. Cyclic hydrostatic pressure downregulated the expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 compared to the unloaded controls. Hydrostatic pressure may slow chondrocyte differentiation and have a chondroprotective, anti-angiogenic influence on cartilage tissue. Our results suggest that cyclic tension activates the Cbfa1/MMP-13 pathway and increases the expression of terminal differentiation hypertrophic markers. Mammalian chondrocytes appear to have evolved complex mechanoresponsive mechanisms, the effects of which can be observed in the histomorphologic establishment of the cartilaginous skeleton during development and maturation.

Collagen fibrillogenesis by chondrocytes in alginate.

Collagen is the primary structural component in connective tissue. The poor mechanical properties of most cell-seeded cartilage grafts used for cartilage repair can be attributed to the low level of collagen synthesized compared with native cartilage. In this study, the synthesis and assembly of collagen by chondrocytes in hydrogels were investigated, with particular attention paid to the role of cross-link formation in this process. Primary bovine chondrocytes were seeded in alginate and collagen synthesis was assessed in the presence and absence of beta-aminopropronitrile (BAPN), a potent inhibitor of the enzyme lysyl oxidase and collagen cross-link formation. Cultures on days 21, 35, and 49 were evaluated by stereology, biochemistry, and real-time reverse transcriptase-polymerase chain reaction. All measures of collagen synthesis (except hydroxyproline) significantly increased in the presence of 0.25 mM BAPN. By 35 days of culture, the average collagen fibril diameter was 62 +/- 10 nm in control cultures and 109 +/- 20 nm with BAPN supplementation. The collagen volume density increased from 5 +/- 3% in control cultures to 17 +/- 1% in the presence of BAPN. Likewise, the expression of cartilage-specific collagens (type II and XI) and aggrecan increased significantly as a result of BAPN culture. These findings demonstrate the prominent role of collagen cross-linking in collagen fibrillogenesis and suggest approaches by which collagen synthesis and assembly could be controlled in tissue-engineered constructs.

The mechanosensitivity of cartilage oligomeric matrix protein (COMP).

Physical forces are known to influence the synthesis, assembly and degradation of the cartilage extracellular matrix. The expression of cartilage oligomeric matrix protein (COMP) was found to be sensitive to long term cyclic compression. Explants of calf articular cartilage as well as cylindrical alginate/chondrocyte constructs were subjected to uniaxial unconfined dynamic compression for 18 hours after which total mRNA was extracted from samples. COMP expression was assessed by means of semi-quantitative RT-PCR and Northern blot techniques. The COMP transcript was found to be significantly enriched upon compression in both experimental systems. Incubation with anti-beta1 integrin blocking antibodies abolished the mechanosensitivity of COMP expression. In addition, the presence of a fully developed pericellular matrix was shown to be a prerequisite for enhanced COMP expression with cyclic loading. Cell/matrix interactions are therefore one of the key events in mechanotransduction in chondrocytes.

Expression of antagonists of WNT and BMP signaling after non-rigid fixation of osteotomies.

Delayed fracture healing and non-unions represent rare but severe complications in orthopedic surgery. Further knowledge on the mechanisms of the bone repair process and of the development of a pseudoarthrosis is essential to predict and prevent impaired healing of fractures. The present study aimed at elucidating differences in gene expression during the repair of rigidly and non-rigidly fixed osteotomies. For this purpose, the MouseFix™ and the FlexiPlate™ systems (AO Development Institute, Davos, CH), allowing the creation of well defined osteotomies in mouse femora, were employed. A time course following the healing process of the osteotomy was performed and bones and periimplant tissues were analyzed by high-resolution X-ray, MicroCT and by histology. For the assessment of gene expression, Low Density Arrays (LDA) were done. In animals with rigid fixation, X-ray and MicroCT revealed healing of the osteotomy within 3 weeks. Using the FlexiPlate™ system, the osteotomy was still visible by X-ray after 3 weeks and a stabilizing cartilaginous callus was formed. After 4.5 weeks, the callus was remodeled and the osteotomy was, on a histological level, healed. Gene expression studies revealed levels of transcripts encoding proteins associated with inflammatory processes not to be altered in tissues from bones with rigid and non-rigid fixation, respectively. Levels of transcripts encoding proteins of the extracellular matrix and essential for bone cell functions were not increased in the rigidly fixed group when compared to controls without osteotomy. In the FlexiPlate™ group, levels of transcripts encoding the same set of genes were significantly increased 3 weeks after surgery. Expression of transcripts encoding BMPs and BMP antagonists was increased after 3 weeks in repair tissues from bones fixed with FlexiPlate™, as were inhibitors of the WNT signaling pathways. Little changes only were detected in transcript levels of tissues from rigidly fixed bones. The data of the present study suggest that rigid fixation enables accelerated healing of an experimental osteotomy as compared to non-rigid fixation. The changes in the healing process after non-rigid fixation are accompanied by an increase in the levels of transcripts encoding inhibitors of osteogenic pathways and, probably as a consequence, by temporal changes in bone matrix synthesis.

Mitogen-activated protein kinase 2 regulates physiological and pathological bone turnover.

The objective of this study was to investigate the role of the serine-threonine kinase mitogen-activated protein kinase 2 (MK2) in bone homeostasis. Primary bone cell cultures from MK2(+/+) and MK2(-/-) mice were assessed for osteoclast and osteoblast differentiation, bone resorption, and gene expression. Bone architecture of MK2(+/+) and MK2(-/-) mice was investigated by micro-computed tomography and histomorphometry. Ovariectomy was performed in MK2(+/+) and MK2(-/-) mice to assess the role of MK2 in postmenopausal bone loss. Osteoclastogenesis, bone resorption, and osteoclast gene expression were significantly impaired in monocytes from MK2(-/-) compared to MK2(+/+) mice. Mechanistically, loss of MK2 causes impaired DNA binding of c-fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) to tartrate-resistant acid phosphatase (TRAP) and the calcitonin receptor gene promoter. In addition, MK2(-/-) mice showed an age-dependent increase in trabecular bone mass and cortical thickness, fewer osteoclasts, and lower markers of bone resorption than MK2(+/+) mice. Furthermore, MK2(-/-) mice were protected from ovariectomy-induced bone loss. Osteoblastogenesis and bone formation were unchanged in MK2(-/-) mice, whereas osteoblast expression of osteoprotegerin (OPG) and serum levels of OPG were higher in MK2(-/-) than in MK2(+/+) mice. Loss of MK2 effectively blocks bone resorption and prevents the development of postmenopausal bone loss. Small-molecule inhibitors of MK2 could thus emerge as highly effective tools to block bone resorption and to treat postmenopausal bone loss.