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Bone loss induced by ovariectomy is due to the direct activity on bone cells and mesenchymal cells and to the dysregulated activity of bone marrow cells, including immune cells and stromal cells, but the underlying mechanisms are not completely known. Here, we demonstrate that ovariectomy induces the T-cell co-stimulatory cytokine LIGHT, which stimulates both osteoblastogenesis and osteoclastogenesis by modulating osteoclastogenic cytokine expression, including TNF, osteoprotegerin, and the receptor activator of nuclear factor-κB ligand (RANKL). Predictably, LIGHT-deficient (Tnfsf14-/- ) mice are protected from ovariectomy-dependent bone loss, whereas trabecular bone mass increases in mice deficient in both LIGHT and T and B lymphocytes (Rag -/- Tnfsf14 -/- ) and is associated with an inversion of the TNF and RANKL/OPG ratio. Furthermore, women with postmenopausal osteoporosis display high levels of LIGHT in circulating T cells and monocytes. Taken together, these results indicate that LIGHT mediates bone loss induced by ovariectomy, suggesting that patients with postmenopausal osteoporosis may benefit from LIGHT antagonism. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Peroxisome proliferator-activated receptor-gamma coactivator (PGC1α) is a transcription coactivator that interacts with a broad range of transcription factors involved in several biological responses. Here, we show that PGC1α plays a role in skeletal homeostasis since aged PGC1α-deficient mice (PGC1α−/−) display impaired bone structure. Micro-CT of the tibial mid-shaft showed a marked decrease of cortical thickness in PGC1α−/− (− 11.9%, p

Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT-deficient mice (Tnfsf14-/- ) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti-osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro-osteoblastogenic Wnt10b in CD8+ T cells in Tnfsf14-/- mice. LIGHT stimulation increases OPG levels in B, CD8+ T, and osteoblastic cells, as well as Wnt10b expression in CD8+ T cells. The high bone mass in Light and T- and B-cell-deficient mice (Rag- /Tnfsf14- ) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease. © 2017 American Society for Bone and Mineral Research.

Previous results showed that intermittently administered irisin improves bone mass in normal mice and prevents the development of disuse-induced osteoporosis and muscular atrophy in hindlimb-suspended mice, a murine model able to mimic the absence of mechanical loading. A recent study showed that irisin increases survival of osteocytes acting through integrin αV/β5 receptors. To better understand the action of irisin on these cells, we investigated the downstream signaling cascades in osteocyte-like cells (MLO-Y4) treated with recombinant irisin (rec-irisin) in vitro and we analyzed survival of osteocytes and caspase activation in cortical bone of osteoporotic mice treated with rec-irisin in vivo. Our results revealed that rec-irisin activated the MAP kinases Erk1 and Erk2 and increased the expression of the transcription factor Atf4 (2.5-fold, p < .05) through an Erk-dependent pathway in osteocytes. Some key genes expressed by MLO-Y4 cells were modulated by long-term irisin treatment, either continuously administered or given with intermittent short pulses. Interestingly, Sost mRNA was severely downregulated only upon intermittent irisin administration (10-fold, p < .001). Furthermore, rec-irisin upregulated Tfam mRNA (fourfold, p < .05) and Bcl2/Bax ratio (twofold, p < .05) in MLO-Y4 cells. By detecting caspase-9 and caspase-3, we also found that rec-irisin inhibited apoptosis induced by hydrogen peroxide and dexamethasone, respectively. In cortical bone of unloading C57BL6 mice treated with vehicle (unload-veh), irisin prevented disuse-induced reduction of viable osteocytes (+30% versus unload-veh, p < .05) and increase of empty lacunae (+110% versus unload-veh, p < .05), as well as caspase-9 (threefold, p < .05) and caspase-3 (twofold, p < .05) activations. Our findings revealed underlying mechanisms of irisin action on osteocytes, which increases their functions and exerts anti-apoptotic effects, confirming that mechanosensor cells of bone are sensitive to the exercise-mimetic myokine irisin. © 2019 American Society for Bone and Mineral Research.

Cells from dental tissues have a mesenchymal stem cell (MSC) phenotype, are multipotent and can differentiate into osteoblastic cells, as we have previously found. MSCs, due to their tumor‑homing ability, are currently being used as cell‑based delivery systems for cancer protein therapeutics, such as the TNF‑related apoptosis‑inducing ligand (TRAIL). In the present study we revealed that dental pulp stem cells (DPSCs) expressed TRAIL to a greater extent when they were differentiated into the osteoblastic lineage. TRAIL affected the viability of undifferentiated DPSCs, while osteoblastic differentiated DPSCs were not sensitive to TRAIL. The expression trend of TRAIL receptors underwent changes during the osteoblastic differentiation of DPSCs exhibiting low DcR2 and high DR5 levels in the undifferentiated DPSCs and an opposite scenario was presented in the differentiated cells. The sensitivity of the undifferentiated DPSCs to the TRAIL‑apoptotic effect was also associated with low levels of intracellular anti‑apoptotic proteins, such as c‑FLIP, XIAP and the activation of caspase‑8 and ‑3. DPSC‑differentiated osteoblasts expressing high TRAIL levels were capable to affect the cell viability of the human myeloma cell line H929, thus representing an effective anticancer therapeutic method.

We previously showed that Irisin, a myokine released from skeletal muscle after physical exercise, plays a central role in the control of bone mass. Here we report that treatment with recombinant Irisin prevented bone loss in hind-limb suspended mice when administered during suspension (preventive protocol) and induced recovery of bone mass when mice were injected after bone loss due to a suspension period of 4 weeks (curative protocol). MicroCT analysis of femurs showed that r-Irisin preserved both cortical and trabecular bone mineral density, and prevented a dramatic decrease of the trabecular bone volume fraction. Moreover, r-Irisin protected against muscle mass decline in the hind-limb suspended mice, and maintained the fiber cross-sectional area. Notably, the decrease of myosin type II expression in unloaded mice was completely prevented by r-Irisin administration. Our data reveal for the first time that Irisin retrieves disuse‐induced bone loss and muscle atrophy. These findings may lead to development of an Irisin-based therapy for elderly immobile osteoporotic and physically disable patients, and might represent a countermeasure for astronauts subjected to microgravity-induced bone and muscle losses.

Vitamin D deficiency is common in the general population and may impair skeletal muscle function. Very few data are available regarding this condition in professional athletes.To evaluate some skeletal parameters and in particular serum 25-hydroxyvitamin D status in professional rugby players during two different sunlight exposure times (October and early April) and to assess its impact on bone metabolism.Twenty-one male healthy professional rugby players living in northern Italy at latitude of 44°55'N (age 24.6 ± 4.3 years; height 182.0 ± 0.05 cm; mass 96.3 ± 14.6 kg; BMI 28.9 ± 3.7 kg/m(2)) participated in this observational study. During 2012/2013 Italian rugby season, 25-hydroxyvitamin D, PTH and other related biochemical parameters were monitored. Dietary calcium intake and body composition by DXA were also evaluated.Significant changes were observed between October and April data for 25-hydroxyvitamin D concentration (22.8 ± 5.8 vs. 19.1 ± 5.3 ng/ml; p = 0.001) whereas serum PTH, calcium and phosphorus plasma levels did not change. They presented with an appropriate daily intake of calcium (1,304.8 ± 477.9 mg; max 1,939 mg; min 228 mg).Professional rugby athletes practicing a sport characterized by intense outdoor training and with good calcium intake are at higher risk of hypovitaminosis D that worsens significantly during times of low cutaneous vitamin D production. Further studies are warranted to evaluate whether an appropriate supplementation with cholecalciferol in professional athletes is needed.

Periostin is a matricellular protein highly expressed in periodontal ligament and periostium and has been shown to be required for tissue development and maintenance. We showed that the adhesion of murine osteoblastic MC3T3 cells to thiolated hyaluronic acid/polyethyleneglycol hydrogels was greatly improved by enrichment with periostin. Polished or sand-blasted/acid-etched (SLA) commercially pure titanium surfaces were also coated with this protein and periostin ameliorated cell adhesion and dramatically affected cell morphology on both surfaces, as assessed at fluorescence microscopy, scanning electron microscopy, and chemiluminescence-based viability assay. Moreover, periostin increased the expression of alkaline phosphatase, osteoprotegerin, connective tissue growth factor, collagen 1a1, osteocalcin, Runx2, and osterix transcription factors on smooth surfaces. However, it did not affect, or even decreased, the expression of these genes on SLA discs. Transcript levels for connexin 43 were greatly increased on both surfaces in the presence of periostin. Taken together, these results show that periostin coatings can be a viable approach to improve cell adhesion and differentiation on implantable biomaterials.

The aim of the present work was to investigate the morphology and activity of the murine osteoblastic cell line MC3T3 on control smooth (Machined), commercially available rough (ZT) titanium discs, and on titanium samples obtained by modifying the ZT treatment protocol, and herein labelled as ZTF, ZTM and ZTFM. Cells were evaluated at SEM and immunofluorescence for morphology and cell-to-cell interactions and by MTT assay and real time PCR for cell growth and function. Microscopy showed that ZT modified protocols could differently affect cell shape and distribution. All the tested surfaces showed good biocompatibility by viability assay. However, cells on smoother surfaces appeared to express higher levels of transcript for Collagen 1a1, the main component of extracellular matrix, by real time PCR. Expression of the early differentiation marker Alkaline Phosphatase was higher on ZTF surfaces and ZTM enhanced the expression of later osteoblastic markers Osteoprotegerin and Osteocalcin. Noteworthy, the expression of Connexin 43, a component of cell-to-cell contacts and hemichannels, followed a similar pattern to differentiation marker genes and was higher in cells on ZTM surfaces, consistently with the microscopic observation of cell clusters. Taken together, this data showed that ZTF and ZTM treatment protocols appeared to improve the basal sand-blasting/acid-etching ZT procedure with ZTM surfaces promoting the most mature stage of differentiation.

Surface topography affects cell function and differentiation. It has been previously shown that rough surfaces can enhance the activation of canonical Wnt signaling, an important pathway for osteoblast differentiation and bone maintenance, but the underlying mechanisms are still poorly understood. The present paper investigates whether cytoskeletal organization contributes to regulating this pathway. Rho-associated protein kinase (ROCK), an important controller of actin microfilaments, was inhibited with 2mM specific antagonist Y-27632 in mesenchymal and osteoblastic cells growing on titanium discs with a polished or acid-etched, sand-blasted (SLA) surface. Y-27632 subverted the morphology of the cytoskeleton on polished and, to a lesser extent, on SLA surfaces, as evidenced by fluorescence microscopy. Although ROCK inhibition did not affect cell viability, it increased activation of Wnt signaling in uncommitted C2C12 mesenchymal cells on polished surfaces but not on SLA discs upon reporter assay. Consistently with this, real-time polymerase chain reaction analysis showed that MC3T3 cells on polished surfaces expressed higher mRNA levels for β-catenin and alkaline phosphatase, a known Wnt target gene, and for the osteoblastic differentiation marker osteocalcin after ROCK inhibition. Taken together, these data demonstrate that cytoskeletal organization mediates activation of Wnt canonical signaling in cells on titanium surfaces with different topographies.

Endosseous implants are important tools to replace missing teeth or damaged tissue segments. Their clinical success depends on their integration in bone and, thus, on the response of bone cells to material and surface characteristics. Recent evidence has shown that surface topography and chemistry affect WNT signalling, a pivotal pathway for the commitment of mesenchymal progenitors to the osteoblast lineage and for bone homeostasis. WNT signalling comprises several cascades that, acting through different effectors, modulate several aspects of cell behaviour. It has been shown that cells growing on rough titanium surfaces display a different expression profile for WNT factors, and that surface features can alter the response of bone cells to WNT factors. Although the underlying mechanisms to this regulation are still poorly understood, the present review reports intriguing evidence that that cell cytoskeletal signalling is involved in activating WNT signalling in cells growing on rough implant surfaces.

Aims Promoting bone formation at the tissue interface is an important step to improve implant success. This study investigated whether stimulation of Wnt signaling by GSK3b inhibitor lithium chloride (LiCl) could affect the response of mesenchymal or osteoblastic cells growing on titanium surfaces with different topography and wettability, and improve their differentiation along the osteoblastic lineage. Material and methods Murine mesenchymal C2C12 cells were plated on Pickled, acid-etched/sand-blasted (SLA), and hydrophilic SLA titanium disks (modSLA) and stimulated with increasing doses of LiCl. Cell viability was measured using chemiluminescence-based ATP quantitation and activation of Wnt canonical signaling was measured using a Luciferase-based reporter assay. Gene expression was measured using real time PCR in C2C12 cells, murine osteoblastic MC3T3 cells or murine primary bone marrow cells. Results LiCl stimulated Wnt activation and expression of Wnt markers in C2C12 cells on modSLA. Addition of 1 mM LiCl increased levels for bone marker Osteocalcin in MC3T3 cells on modSLA surfaces. Similarly, LiCl potently enhanced Osteoprotegetrin levels in MC3T3 cells on modSLA. When primary bone marrow cells were stimulated with LiCl, the expression of Wnttarget genes and osteoblastic differentiation markers was increased on modSLA surfaces. Conclusions Stimulation of the canonical Wnt pathway promoted osteoblast differentiation on hydrophilic modSLA surfaces. Taken together, these results demonstrate that Wnt activators such as LiCl should be further tested as a possible approach to improve implant osseointegration.

Laser light has been proposed as a tool to decontaminate the surface of endosseous implants. The effects of this maneuver on the interactions between cells and surface, however, are poorly known. The goal of the present study is to investigate osteoblast growth and differentiation on three commercially available surfaces untreated or after irradiation by erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser at two levels: 150 and 200 mJ/pulse at 10 Hz.Human osteoblastic Saos-2 cells were plated on machined, sandblasted and acid-etched titanium, or titanium plasma-sprayed disks. The effects of lasing were observed with a scanning electronic microscope, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Moreover, we measured the production of the osteoblast-specific protein osteocalcin and of osteoprotegerin in the supernatants by immunoenzymatic assays.Although no visible changes were observed on machined or titanium plasma-sprayed disk samples at the tested levels, titanium peaks on sandblasted and acid-etched titanium disks appeared fused as a consequence of laser irradiation. Interestingly, cell proliferation was slower on irradiated titanium at both intensities on all the surfaces. Cell differentiation, as assessed by osteocalcin production, was generally unaffected by laser treatment, whereas the production of osteoprotegerin was decreased on all the surfaces irradiated at the intensity of 200 mJ/10Hz.These results indicate that Er:YAG laser at energy levels used in this study can alter the surface profile of titanium implants and these changes may negatively affect the viability and the activity of osteoblastic cells. Therefore, Er:YAG lasers should be used with caution on titanium surfaces.

Little is known about how surface topography can modulate mesenchymal cell responses to oxygen-related stress occurring with age, or during the early phases of wound healing or inflammation. To antagonize Reactive Oxygen Species (ROS), cells resort to defense mechanisms, relying on β-catenin, a molecular switch between a TCF-mediated pathway, which promotes cells proliferation and commitment, and an alternative one controlled by FoxO, which induces quiescence and defenses against ROS. In the present study, we show that mesenchymal C2C12 cells are protected from H2O2-induced oxidative stress when they grow on rough (SLA) titanium surfaces. The expression of anti-ROS genes and FoxO/β-catenin signaling, as measured by a reporter assay, were increased on SLA surfaces. We also show that TCF-mediated transcription was inhibited by ROS in cells growing on either smooth or SLA titanium. Our results demonstrate that surface topography modulates cell resistance to ROS and the balance between the molecular pathways regulating cell growth and cell defense against oxidative stress.

Mechanical loading is of pivotal importance in the maintenance of skeletal homeostasis, but the players involved in the transduction of mechanical stimuli to promote bone maintenance have long remained elusive. Osteocytes, the most abundant cells in bone, possess mechanosensing appendices stretching through a system of bone canaliculi. Mechanical stimulation plays an important role in osteocyte survival and hence in the preservation of bone mechanical properties, through the maintenance of bone hydratation. Osteocytes can also control the osteoblastic differentiation of mesenchymal precursors in response to mechanical loading by modulating WNT signaling pathways, essential regulators of cell fate and commitment, through the protein sclerostin. Mutations of Sost, the sclerostin-encoding gene, have dramatic effects on the skeleton, indicating that osteocytes may act as master regulators of bone formation and localized bone remodeling. Moreover, the development of sclerostin inhibitors is opening new possibilities for bone regeneration in orthopedics and the dental field.

Background and Aim Sparse evidence suggests a possible link between exposure to airborne nanoparticles (NPs) and cardiovascular (CV) risk, perhaps through mechanisms involving oxidative stress and inflammation. We assessed the effects of TiO2 and Co3O4 NPs in human circulating angiogenic cells (CACs), which take part in vascular endothelium repair/replacement. Methods CACs were isolated from healthy donors’ buffy coats after culturing lymphomonocytes on fibronectin-coated dishes in endothelial medium for 7 days. CACs were pre-incubated with increasing concentration of TiO2 and Co3O4 (from 1 to 100 μg/ml) to test the effects of NP – characterized by Transmission Electron Microscopy – on CAC viability, apoptosis (caspase 3/7 activation), function (fibronectin adhesion assay), oxidative stress and inflammatory cytokine gene expression. Results Neither oxidative stress nor cell death were associated with exposure to TiO2 NP (except at the highest concentration tested), which, however, induced a higher pro-inflammatory effect compared to Co3O4 NPs (p

Bone regeneration techniques increasingly rely on the use of exogenous molecules able to enhance tissue formation in pathologic and traumatic defects. An enamel matrix derivative (EMD) has been largely used to promote tooth ligament regeneration within periodontal pockets. Recent evidence suggests that EMD may contribute to inducing osteoblast growth and differentiation. We investigated the effects of EMD on growth and osteogenic marker modulation in human mandibular osteoblasts.We focused our attention on cell growth by 3-(4,5-dimethyl[thiazol-2-yl]-3,5-diphery)tetradium bromide (MTT) assay, cell differentiation, mineralized nodule formation, and, in particular, the expression of receptor activator of nuclear factor-kappa B ligand (RANKL), the main osteoclast differentiation factor, and its decoy receptor, osteoprotegerin (OPG), by enzyme-linked immunosorbent assay.Cell growth was significantly increased by EMD. Similarly, a significantly higher quantity of OPG and a lower amount of RANKL were detectable in groups treated with 50 and 100 microg/ml at weeks 1, 2, and 3, and alkaline phosphatase activity and osteocalcin production were enhanced in cultures treated with 50 and 100 microg/ml at weeks 2 and 3. Mineralized nodules appeared bigger and more numerous in cultures treated with 50 and 100 microg/ml EMD.EMD was able to enhance osteoblast cell growth and the expression of markers of osteoblastic phenotype and differentiation. EMD also seemed able to create a favorable osteogenic microenvironment by reducing RANKL release and enhancing osteoblastic OPG production.

Aging is accompanied by a complex dynamics of CD8+ T cell subsets whose origin is unclear. To evaluate the impact of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) chronic infections on CD8+ T cells in far advanced age, we studied CD8+ T cells frequencies and phenotype in nonagenarians and centenarians by HLA-A*0201- and HLA-B*0702-tetramers incorporating epitopes specific of both viruses along with viral replication. The results demonstrate that EBV and CMV infections induce quantitatively and qualitatively different CD8+ T-cell responses in advanced aging. The frequency and absolute number of CD8+ T cells specific for one lytic and two latent EBV-epitopes, were relatively low and mostly included within CD8+ CD28+ cells. By contrast, CMV infection was characterized by highly variable numbers of CD8+ T cells specific for two differently restricted CMV-epitopes that, in some subjects, were strikingly expanded. Moreover, the great majority of anti-CMV CD8+ T cells did not bear CD28 antigen. Notwithstanding the expansion of CMV-specific CD8+ lymphocytes, CMV-DNA detection in blood samples was invariably negative. Altogether, we suggest that CMV, but not EBV, can sustain chronic activation of the HLA-class I restricted effector arm in elderly that might have detrimental effects on age-associated diseases.

OBJECTIVE Interleukin-6 (IL-6) seems to be a key mediator of the increased bone loss that follows loss of ovarian function. Based on this and on evidence that oestrogen deficiency may also increase cell sensitivity to IL-6, we studied the effects of ovariectomy and of oestrogen replacement therapy on the serum levels of IL-6 and of soluble IL-6 receptor (sIL-6R) in vivo. DESIGN AND PATIENTS Thirty-seven fertile women undergoing surgery for benign uterine diseases were divided into 3 groups and monitored for 12 months: hysterectomized women (n = 9), ovariectomized untreated women (n = 12) and ovariectomized women starting treatment with transdermal estradiol (E2, 50 μg/d) 1 month after surgery (n = 16). RESULTS Hysterectomy alone caused no significant changes of sIL6R whereas serum levels of sIL-6R rose progressively after ovariectomy (mean ± SEM: 31 ± 9% and 38 ± 7% over baseline, at 6 and 12 months, respectively; P

Alcohol abuse is commonly associated with reduced bone mass and osteoporosis as a consequence of both systemic and direct cellular effects. To clarify some of the pathways by which alcohol exerts its actions directly on bone cells, we investigated the formation of early osteoblast progenitors (colony-forming units for fibroblasts; CFU-F) in long-term murine and human bone marrow cultures exposed to ethanol and to its main metabolite, acetaldehyde. In murine bone marrow cultures, obtained from Swiss female mice, ethanol inhibited CFU-F formation (maximal reduction +/- SEM: 50 +/- 2%; p0.01) at concentrations ranging from 0.04% to 0.6% that are similar to those reached in vivo in alcoholics. Acetaldehyde strongly reduced CFU-F formation at concentrations of 0.004% and 0.02%, and completely abolished it at the dose of 0.06%. Similarly, ethanol (at concentrationsor =0.02%) and acetaldehyde (from 0.004% to 0.06%) significantly decreased the number of CFU-F in human bone marrow cultures; the mean reduction observed with ethanol was 63 +/- 12% (p0.05), whereas acetaldehyde completely prevented CFU-F formation at the concentration of 0.06%. These in vitro observations were confirmed by the in vivo findings that the CFU-F formation in bone marrow cultures from nine young, chronic, noncirrhotic alcoholics was significantly reduced (70 +/- 15%), compared with seven age-matched normal subjects (p0.01). In addition, acetaldehyde inhibited cell proliferation in human osteoblastic cells (MG-63 and HOBIT cell lines), whereas ethanol reduced proliferation only in MG-63 cells. Our results indicate that ethanol and acetaldehyde may directly inhibit the osteoblastogenic potential of the bone marrow, and this effect may contribute to the decreased bone formation observed in alcoholics.

As recent studies highlight the importance of alternative mechanisms in the control of bone turnover, new therapeutic approaches can be envisaged for bone diseases and periodontitis-induced bone loss. Recently, it has been shown that Fluoxetine and Venlafaxine, serotonin re-uptake inhibitors commonly used as antidepressants, can positively or negatively affect bone loss in rat models of induced periodontitis. Serotonin is a neurotransmitter that can be found within specific nuclei of the central nervous system, but can also be produced in the gut and be sequestered inside platelet granules. Although it is known to be mainly involved in the control of mood, sleep, and intestinal physiology, recent evidence has pointed at far reaching effects on bone metabolism, as a mediator of the effects of Lrp5, a membrane receptor commonly associated with Wnt canonical signaling and osteoblast differentiation. Deletion of Lrp5 in mice lead to increased expression of Tryptophan Hydroxylase 1, the gut isoform of the enzyme required for serotonin synthesis, thus increasing serum levels of serotonin. Serotonin, in turn, could bind to HTR1B receptors on osteoblasts and stop their proliferation by activating PKA and CREB. Although different groups have reported controversial results on the existence of an Lrp5-serotonin axis and the action of serotonin in bone remodeling, there is convincing evidence that serotonin modulators such as SSRIs can affect bone turnover. Consequently, the effects of this drug family on periodontal physiology should be thoroughly explored.

A 64-year-old man with chronic liver disease presented with dyspnoea, and a chest contrast computed tomography (CT) was performed to evaluate the suspect of pulmonary embolism ( Panel A ). A mass occupying the right atrium, of the same density of the myocardium at …

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.

Stromal cells of the bone marrow control the development of osteoclasts through the production of cytokines capable of promoting the proliferation and differentiation of hematopoietic progenitors. Moreover, the deregulated production of the cytokine IL-6 in the bone marrow mediates an increase in osteoclastogenesis after estrogen loss. IL-6, however, does not influence osteoclastogenesis in the estrogen-replete state, suggesting that other cytokines might be responsible for osteoclast development under physiologic circumstances. We report here that IL-11, a newly discovered cytokine that is produced by marrow stromal cells, induced the formation of osteoclasts exhibiting an unusually high degree of ploidy in cocultures of murine bone marrow and calvarial cells. Osteoclasts formed in the presence of IL-11 were capable of bone resorption, as evidenced by the formation of resorption pits, as well as the release of 45Ca from prelabeled murine calvaria. Further, an antibody neutralizing IL-11 suppressed osteoclast development induced by either 1,25-dihydroxyvitamin D3, parathyroid hormone, interleukin-1, or tumor necrosis factor; whereas inhibitors of IL-1 or TNF had no effect on IL-11-stimulated osteoclast formation. The effects of IL-11 on osteoclast development were blocked by indomethacin; more important, however, they were independent of the estrogen status of the marrow donors.

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

The contribution of the immune system to healthy aging and longevity is still an open question. For this reason, several immune parameters (T, B, and natural killer [NK] cell subsets; non-major histocompatibility complex [MHC]-restricted cytotoxic activities, ie, natural and redirected killing [RDK] activities) were studied in a total of 138 healthy subjects of different ages, from 4 to 106 years of age, including 26 centenarians. The major age-related modifications were the following: (1) a decrease in the absolute number of T lymphocytes (CD3+), involving both CD4+ and CD8+ subsets, accompanied by a marked concomitant increase in the number of activated T cells (CD3+, HLA- DR+); (2) a marked decrease in the number of B lymphocytes (CD19+); and (3) an increase in the number of cells with markers of NK activity and of T lymphocytes able to mediate non-MHC-restricted cytotoxicity. These modifications linearly progressed with age and centenarians followed the trend, suggesting that their immune system did not escape the aging process. However, other immunohematologic parameters (number of red blood cells, platelets, and leukocytes) and important immune functions, such as cytotoxic activities (NK and RDK cell activities), were well preserved throughout life until the last decades of life. Unexpectedly, in apparently healthy middle-aged subjects, a decrease of cytotoxic activities was observed in comparison with those of both young controls and centenarians. In conclusions, our data suggest that in centenarians some immune responses are kept at a high level of efficiency, likely contributing to their successful aging. However, this selected group of people does not escape the aging process, as shown by the progressive derangement of a variety of immune parameters.

We have previously shown that cytokine-induced production of interleukin-6 (IL-6) by cultured bone marrow-derived stromal and osteoblastic cells is inhibited by 17 beta-estradiol, and that estrogen withdrawal (ovariectomy) in mice causes an up-regulation of osteoclast development which can be prevented by a neutralizing antibody against IL-6 or estrogen replacement. To directly establish the link between estrogen loss and altered IL-6 production, implied by our earlier studies, we have now compared IL-6 production in ex vivo cultures of bone marrow cells from mice that were sham operated, ovariectomized, or ovariectomized and treated with 17 beta-estradiol. In addition, we have examined the effect of the in vitro withdrawal of estrogens from primary cell cultures of neonatal murine calvaria on IL-6 production. IL-6 production in ex vivo cultures of bone marrow cells maintained in the presence of 1,25-dihydroxyvitamin D3 or PTH was greater in marrow cells from ovariectomized mice than in those from sham-operated animals or ovariectomized animals receiving estrogen replacement. In line with this finding, addition of 17 beta-estradiol to calvaria cell cultures followed by withdrawal of the steroid caused an increase in the amount of IL-6 produced in response to the subsequent stimulation of these cultures with IL-1 or PTH compared to that in cultures that had never been treated with estradiol; when the inactive isomer 17 alpha-estradiol was used, no change in IL-6 production was observed. These results establish that estrogen loss causes an up-regulation of IL-6 production by bone marrow cells and that a similar phenomenon can be elicited in vitro by withdrawal of 17 beta-estradiol from primary cultures of bone cells.

Osteoclasts, the cells that resorb bone, develop from hematopoietic precursors of the bone marrow under the control of factors produced in their microenvironment. The cytokine interleukin-6 can promote hematopoiesis and osteoclastogenesis. Interleukin-6 production by bone and marrow stromal cells is suppressed by 17 beta-estradiol in vitro. In mice, estrogen loss (ovariectomy) increased the number of colony-forming units for granulocytes and macrophages, enhanced osteoclast development in ex vivo cultures of marrow, and increased the number of osteoclasts in trabecular bone. These changes were prevented by 17 beta-estradiol or an antibody to interleukin-6. Thus, estrogen loss results in an interleukin-6-mediated stimulation of osteoclastogenesis, which suggests a mechanism for the increased bone resorption in postmenopausal osteoporosis.

The erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser has been shown to be a promising tool for root treatment in periodontitis, but little information is available regarding the surface characteristics after this treatment, mainly because it is difficult to obtain standardized dentin samples for in vitro studies.Commercially available standardized dentin disks were treated with an Er:YAG laser at different settings and used as a substrate for human primary osteoblastic cells (hOBs) and periodontal ligament fibroblasts (PLFs). Cell proliferation on untreated dentin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 3, 6, 12, 24, and 48 hours of culture. The effects of the laser on dentin and cell morphology on treated and untreated samples were investigated by scanning electron microscopy after 3, 6, 24, and 48 hours of culture.Dentin samples supported proliferation for both cell types, although growth kinetics were different. The laser dramatically affected the dentin profile, creating a rough and irregular surface. Cells grew easily on untreated dentin, but fewer cells were present on treated areas, often displaying long filopodes. hOBs showed poorer adhesion to treated dentin than PLFs.The dentin disks provide a standardized and useful tool to study dentin surface modifications in vitro. PLFs behaved differently from hOBs on dentin, possibly because of their different affinity to this tissue and/or their differentiation state. The changes induced by the laser produced a less favorable environment for cell adhesion or growth, and treated dentin seemed to be more suitable for PLF adhesion compared to hOB adhesion.

Purpose: Nowadays fiber-reinforced composites (FRCs) are widely used in dentistry to substitutemetallic restoration. The fiber reinforcement provides better mechanical properties of the restoration (Alla et al. J Biomater Nanobiotechnol 2013:95). Various types of fibers are available commercially. Previous studies showed that some components of dental composites can be released into the oral cavity (Gupta et al. Toxicol Int 2012:225) and caused several adverse effects (Jan et al. J Formoson Med Assoc 2014:349). Little information exists regarding to the effect of different fibers reinforcement on FRCs biocompatibility. The purpose of this study was to determine the influence of fiber types on cytotoxicity properties of FRCs. Methods and materials: The materials used were ultrahigh molecular weight polyethylene (UHMWPE) fibers (CONSTRUCT Kerr TM, USA), E-glass fibers (Ahlstrom Fiberglass, Karhula, Finland), composite resin (MASTERFLOW® Biodinamica, Brazil), and Vero fibroblast cell line (Universitas Gadjah Mada, Indonesia). Fifteen specimens of FRCs (2× 2× 25)mm were prepared and divided into 3 groups. The experimental groups were FRCs by UHMWPE fiber reinforcement (UHMWPE-group) and E-glass fiber reinforcement (E-glass group) whilst the control-group was composite resin without fiber reinforcement. Specimens were milled and diluted in culture medium (0.1mg powder/1mL medium). Specimen solution of 100 L was added into 96-well plate containing fibroblast cells of 2× 104 cells/100 L and incubated for 24h. MTT of 10 L was added to the well, incubated for 4h, then 100 L stop solution was added. The optical density (OD) of the cells viability was determined by ELISA reader with a wavelength of 550nm. Cells viability were calculated in percentage and analyzed by ANOVA and LSD. Results: The average of fibroblast cells viability in percentage (%) showed for the UHMWPE-group was 88.59± 1.42, E-glass-group was 97.05± 2.24, and control-group was 80.97± 1.70. Analysis by one way ANOVA indicated significant difference of cells viability among the groups (p

To investigate the prevalence of thyroid autoantibodies in very old subjects, we assayed sera from 34 healthy centenarians (7 men, 27 women; age range 100-108 years) for these antibodies. There was a clear age-dependent increase in prevalence of thyroid autoantibodies in sera from 549 control subjects aged 7-85 years, prevalence in 40 subjects aged 70-85 being significantly greater (p less than 0.001, chi 2) than that in 436 subjects aged less than 50. By contrast, prevalence of thyroid autoantibodies in centenarians was not significantly different from that in controls aged less than 50. Cytofluorimetric analysis of peripheral blood lymphocytes showed a striking age-dependent decrease in total and CD5+B cells (without changes in their ratio), which reached its nadir in centenarians. The age-dependent increase in prevalence of thyroid autoantibodies in the elderly is not seen after the ninth decade of life. What relation this characteristic has to derangement of circulating B cells is unknown.

Recent in vitro findings suggest that bisphosphonates, potent inhibitors of osteoclastic bone resorption, may also have a direct action on osteoblasts. The purpose of this study was to search for potential effects of etidronate and alendronate on the formation of early and late osteoblastic cell precursors by measuring the number of colony-forming units for fibroblasts (CFU-F) and colony-forming units for osteoblasts (CFU-OB) in murine and human bone marrow cultures. In murine marrow cultures, etidronate (10(-5) to 10(-9) mol/L) significantly stimulated the formation of CFU-F with a maximal effect at 10(-5) mol/L (mean increase over control values+/-SD: 106+/-17%;p0.001), whereas alendronate had a biphasic effect, being stimulatory at concentrations below 10(-7) mol/L (78+/-5%; p0.001), and inhibitory at higher doses. The formation of CFU-OB was also inhibited by both bisphosphonates at the highest concentrations (10(-5) mol/L and 10(-6) mol/L), but it was significantly stimulated at lower concentrations (from 10(-7) to 10(-9) mol/L for etidronate and 10(-7) to 10(-10) mol/I, for alendronate; p0.001). In human bone marrow cultures, alendronate (10(-8) to 10-(12) mol/L) increased CFU-F formation with a maximal effect at 10(-10) mol/L (161+/-12 %; p0.01). CFU-OB formation, observed only in the presence of dexamethasone (10(-8) mol/L), was markedly stimulated by alendronate at the above concentrations with a maximal increase at 10(-10) mol/L (133+/-34%; p0.001). The in vivo short-term effects of bisphosphonates on the formation of early osteoblast precursors were also studied in bone marrow cultures from young female mice treated with weekly subcutaneous injections of etidronate (0.3, 3, and 30 mg/kg) or alendronate (0.3, 3, and 30 microg/kg) and from aging female mice treated with the two lowest doses of both drugs. After 1 month of treatment, etidronate (0.3 and 3 mg/kg) and alendronate (0.3 and 3 microg/kg) significantly increased the number of CFU-F colonies in the bone marrow from young and old animals, whereas the highest dose of both drugs had no effect in young mice. Our results, together with previously reported observations of bone-forming effects in osteoporosis, suggest that bisphosphonates may have, in vivo, a potentially relevant influence on cells of the osteoblastic lineage, distinct from their inhibitory action on osteoclasts.

Since bisphosphonates prevent bone loss in osteoporosis and rheumatoid arthritis, diseases in which the osteoclastogenic and inflammatory cytokine interleukin-6 plays a pathophysiologic role, we studied whether these drugs regulate the production of this cytokine by osteoblasts. Spontaneous and IL-1 + TNF-alpha stimulated IL-6 release was measured in supernatants of cultures of human osteoblastic osteosarcoma cells MG-63, pretreated for 4 hours with different doses of etidronate, clodronate or alendronate using a specific bioassay. Etidronate [from 10(-4) to 10(-8) M] or alendronate [from 10(-6) to 10(-11) M] inhibited in a dose-dependent manner the cytokine-induced IL-6 secretion [60+/-9.5% at 10(-5) M and 65+/-12% at 10(-7) M, respectively; p < 0.01]. Though significant, the inhibitory effect of clodronate was less [35+/-7% at 10(-5) M, p < 0.05]. These in vitro observations might have in vivo relevance in explaining at least in part the mechanisms by which bisphosphonates inhibit systemic and periarticular bone resorption.

We have shown earlier that 17 beta-estradiol inhibits cytokine-induced interleukin-6 (IL-6) production by bone marrow-derived stromal cells as well as osteoblasts, two types of cells with a critical influence on osteoclast development, and that ovariectomy causes an IL-6-mediated up-regulation of osteoclastogenesis in mice. Prompted by this, we have searched here for the presence of estrogen receptors (ERs) in two murine bone marrow-derived stromal cell lines, +/+ LDA11 and MBA 13.2, and the osteoblast-like cell line MC3T3-E1. All three cell lines exhibited high affinity saturable binding for [125I]17 beta-estradiol with a dissociation constant of approximately 10(-10) M and concentration of binding sites of 260 +/- 30, 170 +/- 10, and 90 +/- 10 sites per cell, respectively. In addition, we amplified complementary DNA from the stromal cell lines by polymerase chain reaction using oligonucleotide primers flanking the DNA binding domain of the murine uterine ER. The amplified product showed an identical nucleotide sequence to the DNA binding domain of the murine uterine receptor. Consistent with the functionality of the ER in stromal cells, and specifically its role in the regulation of IL-6 by 17 beta-estradiol, we found that the pure estrogen antagonist ICI 164,384 completely prevented the effect of 17 beta-estradiol on IL-6. All three cell lines also expressed receptors for 1,25-dihydroxyvitamin-D3 [1,25(OH)2D3] (dissociation constant, approximately 10(-10) M), with a concentration of binding sites of 490 +/- 20, 920 +/- 20, and 1110 +/- 70 sites per cell, respectively. 1,25(OH)2D3 treatment of the stromal cells caused a 2-fold increase in the concentration of ERs and a decrease in cell proliferation. These data establish that bone marrow-derived stromal cells express functional estrogen as well as vitamin D receptors, which serve to mediate actions of their respective ligands on the biosynthetic activity of these cells and presumably the effects of these two steroid hormones on osteoclastogenesis.

Abstract 2961 Reduced osteoblast activity contributes to the development of multiple myeloma-bone disease. Wingless-type (Wnt) signalling pathway is critical in osteoblastogenesis, and it is negatively regulated by molecules such as frizzled-related proteins (sFRPs), Dickkopf proteins (DKKs) and sclerostin. Myeloma cells are known to induce inhibition of osteoblastogenesis through Wnt antagonists such as DKK-1 and sFRP-2 and -3 whereas the role of sclerostin, an osteocyte-expressed negative regulator of bone formation, has not been yet investigated. We provide novel evidence showing sclerostin expression by myeloma cells from patients with multiple myeloma-bone disease and human myeloma cell lines (HMCLs). By means of a co-culture system of bone marrow stromal cells (BMSCs) and HMCLs, we demonstrated that sclerostin expression by myeloma cells and HMCLs is responsible for reduced expression of major osteoblastic specific proteins namely bone-specific alkaline phosphatase, collagen-type I, bone sialoprotein II and osteocalcin as well as decreased mineralized nodule formation and attenuated expression of member of the AP-1 transcription factor family (i.e. Fra-1, Fra-2 and Jun-D). The addition of a neutralizing anti-sclerostin antibody to our co-culture system can restore the above parameters, through the intranuclear accumulation of β-catenin in BMSCs. On the other hand, we demonstrated that sclerostin is also involved in inducing increased receptor activator of nuclear factor-k B ligand (RANKL) and decreased osteoprotegerin (OPG) expression in osteoblasts, contributing to the enhanced osteoclast activity occurring in patients with multiple myeloma-bone disease. Our data suggest that myeloma cells contribute to the suppression of bone formation through sclerostin secretion. Disclosures: No relevant conflicts of interest to declare.

The clinical, morphological, immunological, and molecular features of a case of expansion of large granular lymphocytes (LGL) are reported. Surface marker analysis of peripheral blood and spleen mononuclear cells showed that the majority of these cells were CD3 − , CD2 + , CD16 + , and Leu 7 − . Ultrastructural characteristics of CD16 + cells revealed a low nuclear/cytoplasmatic ratio, irregularly shaped nucleus, and numerous cytoplasmatic granules. Functional studies showed reduced proliferative responses to mitogens (PHA, Con A, PWM) and high levels of natural killer (NK) activity as well as antibody-dependent cell cytotoxicity (ADCC) and lymphokine-activated killer (LAK) activities. Molecular analysis of the T cell receptor genes revealed a germline configuration of the β, γ, and δ genes; however, as for normal NK cells, δ-related mRNA transcripts were found. Three months from diagnosis, the patient developed profound thrombocytopenia and splenectomy was carried out with complete normalization of the platelet counts and of hematological values while LGL lymphocytosis persisted. Although no tools are available for studying the monoclonality of CD3 − lymphoproliferative disease, the clinical course, the absence of chromosomal abnormalities, and a liver histology indicative of chronic active hepatitis suggest that LGL expansion in this patient could be part of a benign, possibly reactive, process.