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Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.

A major challenge in orthopedics is the repair of large non-union bone fractures. A promising therapy for this indication is the use of biodegradable bioinspired biomaterials that stabilize the fracture site, relieve pain and initiate bone formation and healing. This study uses a multidisciplinary evaluation strategy to assess immunogenicity, allergenicity, bone responses and physicochemical properties of a novel biomaterial scaffold. Two-photon stereolithography generated personalized custom-built scaffolds with a repeating 3D structure of Schwarz Primitive minimal surface unit cell with a specific pore size of ?400 ?m from three different methacrylated poly(D,L-lactide-co-?-caprolactone) copolymers with lactide to caprolactone monomer ratios of 16?:?4, 18?:?2 and 9?:?1. Using in vitro and in vivo assays for bone responses, immunological reactions and degradation dynamics, we found that copolymer composition influenced the scaffold physicochemical and biological properties. The scaffolds with the fastest degradation rate correlated with adverse cellular effects and mechanical stiffness correlated with in vitro osteoblast mineralization. The physicochemical properties also correlated with in vivo bone healing and immune responses. Overall these observations provide compelling support for these scaffolds for bone repair and illustrate the effectiveness of a promising multidisciplinary strategy with great potential for the preclinical evaluation of biomaterials.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of β-tricalcium phosphate (β-TCP) disks. We also tested a β-TCP/Collagen (β-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

Novel thermo-sensitive elastin-like recombinamers (ELRs) containing bioactive molecules were created for use as a biomimetic biomaterial for tissue regeneration. For effective use for in vivo applications, it is essential to ensure that they do not induce adverse inflammatory, immune, or allergic responses that inhibit tissue repair. Therefore, we sought to establish a pre-clinical approach to evaluate biocompatibility in experimental mice using ELRs as a prototype biomaterial. First, we measured in vitro proliferation and cytokine production from BALB/c and C57BL/6 mouse splenocytes incubated with ELRs. Second, we used a rapid, high throughput in vivo approach in which inflammatory cells and cytokines were measured following an intraperitoneal implantation. Lastly, a subchronic in vivo approach was used in which ELRs or positive controls were subcutaneously implanted and the implantation sites were assessed for inflammation and gene expression. We found that ELRs induced mild inflammation and minimal fibrosis compared to the intense response to Vitoss. Additionally, implantation increased antigen-specific antibody titers for both groups and gene expression profiling of the implantation sites revealed the upregulation of inflammation, fibrosis, and wound healing-related genes in ELR and positive control-implanted mice compared to sham controls. These data demonstrate that ELRs appear safe for use in tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 924-934, 2018.

Beauvericin (BEA) and enniatins (ENN) are emerging Fusarium mycotoxins that are known to contaminate food and feed. BEA- and ENN-mediated cytotoxicity towards various mammalian and cancer cell lines is only partly understood yet and engages several cellular targets and molecular mechanisms. Thus, the channel forming ability of BEA and ENN selectively directs a flux of cations – particularly calcium – into the cell. The resulting increased intracellular calcium levels might be at least in part responsible for their cytotoxicity. Additionally, BEA and ENN activate programmed cell death via the internal mitochondrial pathway (release of cytochrome c, activation of pro-apoptotic proteins such as Bax and activation of caspases). Several cellular signalling pathways and regulators are influenced by these fusariotoxins including MAPK, NF-κB and p53. The in vitro cytotoxicity implicates that these compounds could be potentially used as cancer therapeutics. However, considering their high prevalence in grains destined for consumption, also potential systemic toxicity towards humans and animals has to be considered. Interestingly, the few studies that have addressed this issue in animals so far predominantly reported minor effects at least as far as acute toxicity is concerned. However, consequences especially of chronic exposure but also at pharmacologically active doses in humans/animals have not been explored in detail. Nevertheless, both compounds exhibit interesting pharmacological characteristics (as they are cytotoxic especially to cancer cells, inhibit drug efflux pumps, are non-mutagenic, inhibit bone resorption) which suggest them as potential drug candidates to fight disseminated cancer. Thus, detailed studies on the consequences of chronic and bolus BEA and ENN exposure are eagerly needed.

The bone-resorbing activity of thyroid hormones was evaluated in neonatal mouse calvaria maintained in organ culture for 96 h. Thyroxine (T4) between 10(-8) and 10(-5) mol/liter and triiodothyronine (T3) between 10(-8) and 10(-7) mol/liter caused a dose-dependent release of calcium from cultured bone. The thyroid hormone effect was delayed in onset for at least 24 h, and after 96 h of culture amounted to 50-90% of the bone-resorbing activity of 10(-8) mol/liter parathyroid hormone (PTH). The bone-resorbing action of T4 as well as of T3 was completely blocked by 100 U/ml interferon-gamma (IF-gamma) or 20 mU/ml salmon calcitonin (CT). "Escape" from CT inhibition, which is a well-known phenomenon in the action of PTH, was not observed with thyroid hormone-mediated bone resorption. Thyroid hormone treatment of cultured calvaria resulted in a gradual increase between 48 and 96 h of medium concentrations of prostaglandin (PG) E2 and particularly of 6-keto-PGF1 alpha, the inactive metabolite of prostacyclin (PGI2). The release of PGF2 alpha in general was not significantly affected. Although the effect of thyroid hormones on PG release from cultured calvaria was completely abolished by 5 x 10(-7) mol/liter indomethacin, in some experiments indomethacin reduced thyroid hormone-mediated bone resorption by only 50%. This indicates that thyroid hormone action on bone is also mediated by a PG-independent mechanism.

Osteoporosis is a chronic and progressive disease that can result in severe morbidity and mortality once falls and fractures occur. The objective was to develop a medication assessment tool (MAT) for use in the evaluation of implementation of evidencebased practice in the treatment and prevention of osteoporosis.

Background Cardiovascular mortality is extraordinarily high in renal allograft recipients and accounts for almost half of all allograft losses. Whereas the immunosuppression with calcineurin inhibitors is associated with an increased cardiovascular risk, beneficial effects of mammalian target of rapamycin inhibitors on the vascular system are suspected. Methods In a randomized clinical trial, we evaluated the impact on pulse wave velocity (PWV) of a switch from cyclosporine A (CsA) to everolimus (EVR), 6 months after transplantation, in 27 stable de-novo renal allograft recipients. PWV was assessed before and after randomization to the different immunosuppressive protocols at 6 and 15 months post-transplantation, respectively. Seventeen out of 27 patients included in the analysis were switched to EVR; 10 out of 27 were kept on CsA. Results The switch of immunosuppressive therapy from CsA to EVR resulted in stable PWV (9.50+/-1.92 vs. 9.13+/-1.62 m/s, DeltaPWV= -0.37+/-1.14 m/s, P=0.16), whereas a significant increase of PWV (9.93+/-1.94 vs. 10.8+/-2.24 m/s, DeltaPWV=0.89+/-1.47 m/s, P=0.03) was observed in patients on continued CsA therapy. Conclusion In renal allograft recipients, the prolonged treatment with CsA was associated with a significant increase of PWV whereas no further deterioration of large vessel compliance was observed in patients that were switched to EVR 6 months post transplantation. The cardiovascular risk profile in stable de-novo renal allograft recipients might therefore be positively impacted by an early switch of the primary immunosuppressive therapy from CsA to EVR.

Although polyphosphonates (PPs) were introduced as bone imaging agents in nuclear medicine in the early 1970s, the mechanisms involved in their uptake still remain unclear. Suggested mechanisms range from mineral adsorption with disputed binding to the organic phase, over incorporation into the mineralisation process to a combination of both mechanisms. Thus, our investigations aimed to: (1) evaluate adsorption parameters of (99m)Tc-MDP, (153)Sm-EDTMP and (18)F-fluoride on mineralising osteoblast cultures, (2) correlate the radiotracer binding measured in the cell cultures with binding values from our previously presented mineral model and (3) compare binding with cell number.Primary osteoblasts were obtained by sequential digestion of foetal mice calvariae. The cells were incubated with 0.3 mumol of radiolabelled PPs or 25 MBq (18)F-fluoride for 120 min. Gamma signals from labelled samples were detected with a Millennium Hawkeye SPECT camera or with a dedicated Advance full-ring PET scanner and the binding percentages were calculated.From days 8 to 15 of culture, the percent binding of all evaluated tracers increased significantly, whereas the protein concentration showed insignificant changes. Additional comparisons of the binding values with our recently published pre-vivo model revealed remarkable agreement, suggesting solely bone-forming minerals to be responsible for radiotracer binding.This study provides evidence that binding of the evaluated radiotracers is not associated with osteoblast numbers but only with the concentration of bone-forming minerals. The presented correlations substantiate our recently presented pre-vivo model for the evaluation of bone-seekers: mechanisms associated with the uptake of bone-seekers are irreversible and mineral-associated processes.

The population of incident dialysis patients is progressively ageing and dialysis outcome is particularly poor in the elderly. There is little documentation whether late referral is more frequent in the very elderly (or =75 years) as compared with non-elderly patients and whether it contributes, at least in part, to their particularly poor outcome.In a retrospective single center study we assessed all consecutive patients (n = 254) who had been admitted to haemodialysis between 1998 and 2001. Outcome in relation to the interval between the time of referral and start of dialysis was compared in very elderly and non-elderly patients. According to a previous analysis in our center major adverse outcome is seen in patients referredor =8 weeks before the start of dialysis. For the present study this time interval was therefore operationally defined as 'late referral'.Expectedly 1 year after start of dialysis mortality was higher (31%) in the very elderly compared with younger patients (19%). The interval between referral and first dialysis was less in patientsor =75 years (median interval 3.5 weeks) compared with patients75 years (median 20.5 weeks; P = 0.007). The difference in 1 year mortality between timely (8 weeks) vs late (or =8 weeks) referral, however, was as high in the very elderly (42% vs 16%) as in the younger patients (34% vs 9%). The relative risk of death conferred by late referral was also not significantly different in the very elderly (RR 1.80) compared with the younger (RR 2.32) patient. Using multivariate analysis timing of referral proves to be an independent factor with regard to the outcome and time of survival.We conclude that late referral is more frequent in the very elderly. Although the relative risk of death conferred by late referral is similar in the very elderly and non-elderly, due to higher frequency of late referral it accounts for a large proportion of excess mortality in the very elderly.

The goal of this study was to determine whether human osteoblasts might harbor the hepatitis C virus. We tested for positive-strand and negative-strand (replicative) hepatitis C virus RNA by reverse transcriptase-polymerase chain reaction, by in situ reverse transcriptase-polymerase chain reaction for intracellular localization of the hepatitis C virus, and by amplicon sequencing in in vitro differentiated mature osteoblasts from STRO-1+ osteoprogenitor cells from patients with chronic hepatitis C and from healthy individuals. We only detected the hepatitis C virus genome in STRO-1+ cells and mature osteoblasts from carriers with chronic hepatitis C, and we found hepatitis C virus negative strands expressed sporadically in these patients. Using in situ hepatitis C virus reverse transcriptase-polymerase chain reaction, we determined that the percentage of infected carrier osteoblasts ranged from 8.0-15.3%. These data provide evidence of hepatitis C virus presence and replication in human osteoprogenitors and osteoblasts, which may have important implications for bone allograft processing.

Chronic inflammation is a major trigger of local and systemic bone loss. Disintegration of cell–matrix interaction is a prerequisite for the invasion of inflammatory tissue into bone. CD44 is a type I transmembrane glycoprotein that connects a variety of extracellular matrix proteins to the cell surface. Tumor necrosis factor (TNF) is a major inducer of chronic inflammation and its overexpression leads to chronic inflammatory arthritis. By generating CD44−/− human TNF-transgenic (hTNFtg) mice, we show that destruction of joints and progressive crippling is far more severe in hTNFtg mice lacking CD44, which also develop severe generalized osteopenia. Mutant mice exhibit an increased bone resorption due to enhanced number, size, and resorptive capacity of osteoclasts, whereas bone formation and osteoblast differentiation are not affected. Responsiveness of CD44-deficient osteoclasts toward TNF is enhanced and associated with increased activation of the p38 mitogen-activated protein kinase. These data identify CD44 as a critical inhibitor of TNF-driven joint destruction and inflammatory bone loss.

A major complication of cortical bone grafting is nonunion at the host-graft junction. Many factors are thought to be involved in successful engraftment including the quality of the graft and the host response to it. In particular, the recipient osteoclasts (OCs) play a critical role by resorbing the engrafted bone. Thus, effective engraftment may depend on the inherent biological properties of the bone graft, which subsequently correlates with early and effective OC resorption. Normally, bone grafts are stored and processed by freezing, freeze-drying, irradiation, and lipid extraction. We sought to determine whether processing and storage affected bone quality, as evaluated by OC bone resorption. Cortical bone specimens from six human donors were either fresh, frozen at -75 degrees C, or had undergone combinations of freezing at -75 degrees C, freeze-drying, lipid extraction, irradiation, and treatment with hydrogen peroxide. Bone slices of 0.5-mm thickness taken from the surface, beneath the surface, and at a depth of 7.5 mm were incubated with isolated rabbit OCs and resorption lacunae were measured. We observed highest OC activity with fresh bone followed by frozen, partially processed, and fully processed bone. When allografts were stored at -75 degrees C for 12 months, there was up to a 4.2-fold reduction in OC activity on the surface layer. Additionally, we found reduced OC activity upon the outer surface bone compared to the inner layers. Removal of more than 0.5 mm of frozen and processed bone significantly improved OC activity. These results imply that inner bone layers of stored and processed bone allografts are protected against degradation of bone matrix components, except when frozen for extended periods of time. Taken together, these data suggest that bone allografts should be stored for less than 1 year and require the removal of at least 0.5 mm from their surface prior to transplantation.

In mouse bone marrow cultures, the formation of osteoclast-like, that is, tartrate-resistant acid phosphatase-positive (TRAP+) and calcitonin (CT) receptor-positive multinucleated cells (MNCs), induced by 10(-10) to 10(-8) mol/L 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], could be augmented by triiodothyronine (T3), which alone had no effect on osteoclast-like cell formation. The permissive effect of T3 increased the response to 1alpha,25(OH)2D3 by approximately one order of magnitude. Linear concentration dependence was observed between 10(-11) and 10(-8) mol/L T3. Importantly, inhibition of prostaglandin synthesis by indomethacin significantly impeded osteoclast-like cell formation by 1alpha,25(OH)2D3 and abrogated the effect of T3 thereon. Basal interleukin-6 (IL-6) production by cultured marrow cells was significantly stimulated by 1alpha,25(OH)2D3. However, even at an exceedingly high concentration of 20 ng/mL, IL-6 was ineffective in inducing osteoclast-like cell formation. Therefore, any hormonally induced rise in IL-6 release from bone marrow cells could not account for the observed changes in TRAP+ MNC numbers. Nevertheless, the stimulatory effect of 1alpha,25(OH)2D3 on osteoclastogenesis was partially dependent on IL-6 because it could be significantly blocked by a neutralizing monoclonal anti-IL-6 antibody, and to the same extent by a monoclonal anti-IL-6 receptor antibody. Unimpaired signaling through the IL-6/IL-6R system is also a prerequisite for the auxiliary effect of T3 on induction of osteoclast-like cells by 1alpha,25(OH)2D3. Our data provide evidence that 1alpha,25(OH)2D3 induces osteoclast-like cell formation, at least in part, in an IL-6-dependent mode of action, which is also subject to modulation by T3. The mechanism of interaction of the two hormones apparently involves joint stimulation of prostaglandin synthesis.

This study investigated the influence of the surface microporosity of beta-tri-calcium phosphate (β-TCP) ceramics on the resorption capacity of osteoclasts. This was achieved by first compacting commercially available β-TCP powder into disks that were sintered at various temperatures, thereby yielding different surface microporosities. Scanning electron microscopy (SEM) and subsequent image processing verified different degrees of surface microporosity on the disks. Rabbit osteoclasts in a bone marrow derived cell suspension were then seeded onto these disks and incubated for 48 h. Tartrate resistant acid phosphatase (TRAP) staining confirmed the presence of osteoclasts on all disks. Actin ring staining that detected actively resorbing OCs showed an inverse linear correlation between the number of actively resorbing osteoclasts (percentage of total OCs on the surfaces) with surface microporosity. These findings should be taken into consideration for the design and/or production of new β-TCP bone graft substitutes.

Amylin is a secretory product of pancreatic beta-cells which shares major sequence homology with calcitonin gene-related peptide. In neonatal mouse calvaria maintained in organ culture for 72 h, amylin inhibited basal (i.e., unstimulated) resorption in medium concentrations above 2.5 x 10(-9) M. In addition, amylin (> or = 1.0 x 10(-7) M) in a calcitonin-like fashion transiently inhibited bone resorption induced by 1.0 x 10(-8) M PTH ("escape phenomenon"). Pretreatment of calvarial bones with amylin (1.0 x 10(-8) - 1.0 x 10(-6) M) for 72 h attenuated the subsequent response to 1.0 x 10(-8) M PTH. Changes in location and appearance of osteoclasts in amylin-treated bones, as visualized by light microscopy, suggest that amylin inhibits bone resorption by causing a loss of specialized contact zones to the mineralized matrix in resorbing osteoclasts, and in addition, by preventing retraction of osteoblasts from the mineralized surface which impedes attachment of osteoclasts thereon.

The aim of this study was to analyse the cerebral venous outflow in relation to the arterial inflow during a Valsalva manoeuvre (VM). In 19 healthy volunteers (mean age 24.1 +/- 2.6 years), the middle cerebral artery (MCA) and the straight sinus (SRS) were insonated by transcranial Doppler sonography. Simultaneously the arterial blood pressure was recorded using a photoplethysmographic method. Two VM of 10 s length were performed per participant. Tracings of the variables were then transformed to equidistantly re-sampled data. Phases of the VM were analysed regarding the increase of the flow velocities and the latency to the peak. The typical four phases of the VM were also found in the SRS signal. The relative flow velocity (FV) increase was significantly higher in the SRS than in the MCA for all phases, particularly that of phase IV (p < 0.01). Comparison of the time latency of the VM phases of the MCA and SRS only showed a significant difference for phase I (p < 0.01). In particular, there was no significant difference for phase IV (15.8 +/- 0.29 vs. 16.0 +/- 0.28 s). Alterations in venous outflow in phase I are best explained by a cross-sectional change of the lumen of the SRS, while phases II and III are compatible with a Starling resistor. However, the significantly lager venous than the arterial overshoot in phase IV may be explained by the active regulation of the venous tone.

Clinical, CT, Doppler, and angiographic findings of 404 consecutive patients with brainstem/cerebellar strokes were registered in a data bank. One hundred and sixty-nine of the patients (41.8%) had a

BACKGROUND Although there exists a large body of knowledge on the regulation of arterial cerebral hemodynamics, little is known about the cerebral venous outflow (CVO). METHODS In 19 healthy volunteers, the middle cerebral artery and the straight sinus were examined using transcranial Doppler sonography. Arterial and venous mean flow velocities (aFV(mean), vFV(mean), respectively) were registered continuously while applying lower body positive (LBPP) or negative (LBNP) pressure of 30 mmHg and performing head-down (-20 degrees , HDT) and -up (+30 degrees , HUT) tilt manoeuvres. The arterial blood pressure was registered simultaneously with a noninvasive finger blood pressure monitor. Relative changes in parameters compared to the proceeding no-pressure, no-tilt baseline were used for analysis. RESULTS While aFV(mean) did not change significantly, vFV(mean) inc reased during LBPP by 10.5 +/- 2.9% and decreased during LBNP by 15.1 +/- 3.5% (mean +/- standard error of mean [SEM], P < .01). HUT resulted in a decrease in vFV(mean) by 25.5 +/- 3.3% and HDT, in an increase by 7.8 +/- 3.2% (P < .01) without alteration in aFV(mean). This may imply a decrease of cerebral blood volume (CBV) during LBPP and HDT and an increase during LBNP and HUT. CONCLUSIONS CVO cannot be neglected when studying cerebral hemodynamics because it might affect the CBV.

Understanding the mechanisms of osteoclastogenesis is crucial for developing new drugs to treat diseases associated with bone loss, such as osteoporosis. Here we report that the C-C chemokine receptor-2 (CCR2) is crucially involved in balancing bone mass. CCR2-knockout mice have high bone mass owing to a decrease in number, size and function of osteoclasts. In normal mice, activation of CCR2 in osteoclast progenitor cells results in both nuclear factor-kappaB (NF-kappaB) and extracellular signal-related kinase 1 and 2 (ERK1/2) signaling but not that of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. The induction of NF-kappaB and ERK1/2 signaling in turn leads to increased surface expression of receptor activator of NF-kappaB (RANK, encoded by Tnfrsf11a), making the progenitor cells more susceptible to RANK ligand-induced osteoclastogenesis. In ovariectomized mice, a model of postmenopausal osteoporosis, CCR2 is upregulated on wild-type preosteoclasts, thus increasing the surface expression of RANK on these cells and their osteoclastogenic potential, whereas CCR2-knockout mice are resistant to ovariectomy-induced bone loss. These data reveal a previously undescribed pathway by which RANK, osteoclasts and bone homeostasis are regulated in health and disease.

New pyrazolo[3,4-d]pyrimidines were synthesized and found to inhibit Src phosphorylation in a cell-free assay. Some of them significantly reduced the growth of human osteogenic sarcoma (SaOS-2) cells. The best compound, in terms of inhibitory properties toward both Src and SaOS-2 cells, was further investigated and found to reduce bone resorption when used to treat mouse osteoclasts, without interfering with normal osteoblast growth. Moreover, its metabolic stability prompted its study on a human SaOS-2 xenograft tumor model in nude mice, where the compound reduced significantly both the volume and weight of the tumor. These experimental findings make the new compound an interesting hit in the field of bone-related diseases.

Contains fulltext : 52277.pdf (Publisher’s version ) (Closed access) Blocking TNF effectively inhibits inflammation and structural damage in human rheumatoid arthritis (RA). However, so far it is unclear whether the effect of TNF is a direct one or indirect on up-regulation of other mediators. IL-1 may be one of these candidates because it has a central role in animal models of arthritis, and inhibition of IL-1 is used as a therapy of human RA. We removed the effects of IL-1 from a TNF-mediated inflammatory joint disease by crossing IL-1alpha and beta-deficient mice (IL-1-/-) with arthritic human TNF-transgenic (hTNFtg) mice. Development of synovial inflammation was almost unaffected on IL-1 deficiency, but bone erosion and osteoclast formation were significantly reduced in IL-1-/-hTNFtg mice, compared with hTNFtg mice based on an intrinsic differentiation defect of IL-1-deficient monocytes. Most dramatically, however, cartilage damage was absent in IL-1-/-hTNFtg mice. Chimera studies revealed that protection of cartilage is based on the loss of IL-1 on hematopoietic, but not mesenchymal, cells, leading to decreased expression of ADAMTS-5 and MMP-3. These data show that TNF-mediated cartilage damage is completely and TNF-mediated bone damage is partially dependent on IL-1, suggesting that IL-1 is a crucial mediator for inflammatory cartilage and bone degradation.

Heme oxygenase 1 (HO-1) plays an important role in vascular disease, transplantation, and inflammation. In animal models of acute and chronic inflammation, induction of HO-1 has anti-inflammatory and cytoprotective properties. Since inflammation is an important trigger of osteoclastogenesis, we hypothesized that HO-1 might influence osteoclastogenesis. We investigated the effects of induction of HO-1 on osteoclast formation in vitro and in vivo. Furthermore, we addressed the role of HO-1 in inflammatory bone loss in humans. When HO-1 was induced by hemin in vitro, a significant dose-dependent inhibition of osteoclastogenesis was observed. Up-regulation of HO-1 was mediated by activation of MAPK and primarily prevented differentiation of osteoclast precursors to osteoclasts, whereas it did not affect mature osteoclasts. Anti-osteoclastogenic properties of hemin were based on a down-regulation of c-fms, RANK, TRAF-6, and c-fos. In addition, induction of HO-1 inhibited TNF-triggered osteoclast differentiation in vitro as well as LPS-driven inflammatory bone loss in vivo. Furthermore, HO-1 induction suppressed osteoclastogenesis and bone destruction in a TNF-mediated arthritis. In line, assessment of synovial tissue from rheumatoid arthritis patients revealed that osteoclasts are usually HO-1 negative. Moreover, serum levels of bilirubin, a metabolite of HO-1, were elevated in rheumatoid arthritis patients without bone damage, suggesting HO-1 affects bone loss in humans. In summary, these data indicate that HO-1 negatively regulates osteoclastogenesis, leading to a positive net balance of bone.

Inactivation of the growth factor-regulated S6 kinase RSK2 causes Coffin-Lowry syndrome in humans, an X-linked mental retardation condition associated with progressive skeletal abnormalities. Here we show that mice lacking RSK2 develop a progressive skeletal disease, osteopenia due to impaired osteoblast function and normal osteoclast differentiation. The phenotype is associated with decreased expression of Phex, an endopeptidase regulating bone mineralization. This defect is probably not mediated by RSK2-dependent phosphorylation of c-Fos on serine 362 in the C-terminus. However, in the absence of RSK2, c-Fos-dependent osteosarcoma formation is impaired. The lack of c-Fos phosphorylation leads to reduced c-Fos protein levels, which are thought to be responsible for decreased proliferation and increased apoptosis of transformed osteoblasts. Therefore, RSK2-dependent stabilization of c-Fos is essential for osteosarcoma formation in mice and may also be important for human osteosarcomas.

The Src tyrosine kinase has been implicated in a wide variety of signal transduction pathways, yet despite the nearly ubiquitous expression of c-src, src−/− mice show only one major phenotype—osteopetrosis caused by an intrinsic defect in osteoclasts, the cells responsible for resorbing bone. To explore further the role of Src both in osteoclasts and other cell types, we have generated transgenic mice that express the wild-type and mutated versions of the chicken c-src proto-oncogene from the promoter of tartrate resistant acid phosphatase (TRAP), a gene that is expressed highly in osteoclasts. We demonstrate here that expression of a wild-type transgene in only a limited number of tissues can fully rescue the src−/− phenotype. Surprisingly, expression of kinase-defective alleles of c-srcalso reduces osteopetrosis in src−/− animals and partially rescues a defect in cytoskeletal organization observed in src−/− osteoclasts. These results suggest that there are essential kinase-independent functions for Src in vivo. Biochemical examination of osteoclasts from these mice suggest that Src may function in part by recruiting or activating other tyrosine kinases.

Im Bereich der Medizin ist der Einsatz informationsverarbeitender Verfahren immer starker gefragt. Mitarbeiter auf diesem Gebiet sind mit einem breiten Aufgabenspektrum konfrontiert. Diese spezifischen Anforderungen pragen das Berufsbild des Medizinischen Informatikers.

Kurzbeschreibung: Einleitung: Die ausgepragten Beschwerden der Patienten und die damit verbundenen hohen Kosten fur das Gesundheitssystem nach traumatischen Knochenverletzungen, Osteoporose oder osteolytischen Knochenmetastasen, unterstreichen die Wichtigkeit der Entwicklung neuer Ansatze in der Knochenwiederherstellung. Bei groseren Knochenverlusten ist es notwendig Knochentransplantate beziehungsweise Knochenersatzmaterialien einzusetzen. Viele fuhren jedoch zu verschiedenen Komplikationen wie, unter anderem, Reaktionen des Immunsystems, Abstosung und auf diesem Weg ubertragbaren Infektionskrankheiten. Es ist notwendig, neue Strategien in der Knochenregeneration zu entwickeln. Um optimale Regeneration zu gewahrleisten, mussen die Eigenschaften der neuen Knochenersatzmaterialien vollstandig geklart sein. Ziel der Studie: Untersuchung der Faktoren, die in die Anhaftung an und die Reaktion von Osteoklasten (OC) auf mineralisierte Substrate beeinflussen, um die besten Bedingungen fur die fruhen Stadien der Knochenregeneration zu schaffen. Methoden: OC aus Kaninchen oder aus muriner Cokultur von OB mit OC-Vorstufen aus dem Knochenmark, wurden auf Knochen, Elfenbein, Koralle, Muschelschalen und Glas kultiviert. Die Organisation des Zytoskeletts wurde bei verschiedenen Temperaturbehandlungen der Substrate (auser Glas) bei 50°C, 200°C, 400° oder 600°C analysiert. Durch die Temperaturbehandlung wurden Modifikationen der Zusammensetzung der Substrate, wie Proteingehalt oder kristalliner Struktur, erreicht. Die Zellen wurden nach der Kultur auf den jeweiligen Substraten fixiert und das Aktin-haltige Zytoskelett wurde durch Rhodamin-Phalloidin mittels Fluoreszenzmikroskopie sichtbar gemacht. Resultat: Unabhangig von der Temperaturbehandlung, wurden keine schadigenden Effekte der Substrate (Aragonit, Calcit, Perlmutt, Knochen, Elfenbein) auf OC festgestellt. Interessanterweise, hafteten OC stark an Knochen, Elfenbein und den anderen kalzifizierten Substraten, unabhangig von geandertem Proteingehalt oder modifizierter kristalliner Struktur. OC entwickelten auf allen Substraten (ebenfalls unabhangig von der Temperaturbehandlung) Sealing zones, mit Ausnahme der Glasoberflachen, die als Kontrolle dienten. Bemerkenswerterweise wurden „podosomen belts“ auf allen Substraten, inklusive Knochen, entdeckt. Diese Entdeckung widerspricht fruheren Untersuchungen, nach denen „podosomen belts“ nicht auf Knochen erscheinen. Zusammengefasst zeigen die Ergebnisse, dass der Ausloseimpuls fur die Bildung von Sealing zones durch OC weder Apatit noch Kalziumphosphat ist. Kalziumkarbonat ist ausreichend um die Entstehung von Sealing zones zu ermoglichen. Schlussfolgerung: Die Substrate Aragonit, Calcit und Perlmutt sind in ahnlicher Weise wie Knochen von OC abbaubar, eine Erkenntnis die in die Entwicklung neuer Knochenersatzmaterialien einfliesen sollte. Offenlegung von Interessenskonflikten: Keine

Heme oxygenase 1 (HO-1) plays an important role in vascular disease, transplantation and inflammation. In animal models of acute and chronic inflammation, induction of HO-1 has anti-inflammatory and cytoprotective properties. Since inflammation is an important trigger of osteoclastogenesis, we hypothesized that HO-1 might play a role in osteoclastogenesis. When HO-1 was induced by hemin in vitro, a significant dose-dependent inhibitory effect on osteoclastogenesis was observed. Hemin primarily inhibited differentiation of mononuclear osteoclast precusors to osteoclasts. These effects were based on a downregulation of the expression of c-fms and RANK, the receptors for monocyte-colony stimulating factor and RANKL, whereas MAP kinase, NF-κB or Akt signalling were not affected. In vivo, HO-1 induction prevented endotoxin-induced calvarial bone resorption. Furthermore, assessment of synovial tissue from rheumatoid arthritis (RA) patients showed expression of HO-1 in monocytes and fibroblasts, whereas osteoclasts were rarely HO-1 positive. To further assess the role of HO-1 activity in RA patients, we correlated the presence of local bone erosions with serum bilirubin levels. We observed significant higher bilirubin levels in non-erosive than in erosive RA patients. Thus, an increase of HO-1 expression is anti-osteoclastogenic in vitro and might protect from increased bone resorption in vivo.

The adenylate cyclase activator forskolin (1–10 μmol L ) inhibited 45 Ca release from parathyroid hormone (PTH; 10 nmol L ) stimulated prelabeled neonatal mouse calvaria in short term culture (24 h). This effect of forskolin was potentiated by rolipram, Ro 20-1724, and isobutyl-methylxanthine, three structurally different inhibitors of cyclic AMP phosphodiesterase. Forskolin (10 μmol L ) and calcitonin (30 mU/mL) inhibited the mobilization of stable calcium and inorganic phosphate as well as the release of the lysomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase from PTH-stimulated unlabeled bones. Osteoclasts in PTH-stimulated calvaria showed active ruffled borders with numerous membrane infoldings. Treatment of PTH-stimulated bones with forskolin and calcitonin resulted in a rapid (2 h) loss of the active ruffled border. In addition, forskolin and calcitonin induced similar changes with respect to the number and size distribution of cytoplasmic vesicles in PTH-activated osteoclasts. After 24 h, all signs of osteoclast inactivation were still prominent, whereas after 48 h of treatment with forskolin or calcitonin, the reappearance of a ruffled border on a number of osteoclasts signaled an escape from the inhibitory action of both calcitonin or forskolin. These data indicate that forskolin inhibits bone resorption by a cyclic AMP dependent mechanism and that the effect of forskolin and calcitonin on bone resorption and osteoclast morphology are comparable. These observations lend further support to the view that cyclic AMP may be an intracellular mediator of the inhibitory action of calcitonin on multinucleated osteoclasts.

A role of γ-interferon in the bone remodeling process can be implicated from its interference with bone resorptive processes in cultured neonatal mouse calvaria. The immune interferon is an efficient inhibitor of endogenous prostaglandin synthesis, particularly after stimulation by thrombin or arachidonic acid, and, in addition, has a calcitonin-like inhibitory effect on PTH-induced osteoclastic bone resorption. γ-Interferon Bone resorption Neonatal mouse calvaria Prostaglandin synthesis Parathyroid hormone Calcitonin