Salivary exosomal microRNA profile as biomonitoring tool for diagnosis and prognosis of patients with head and neck squamous cell carcinoma: a systematic review.

OBJECTIVE: Exosomes are extracellular vesicles found in saliva and other body fluids. These vesicles range in size from 30 to 150 nm and play a crucial role in intercellular communication, transporting different biomolecules, actively targeting cells. These vesicles regulate both physiological and pathological processes within recipient cells. MicroRNAs (miRs) are transported within exosomes and are delivered to target cells where they influence signaling pathways, taking on a crucial regulatory role in oncogenesis; for example, they are implicated in progression and infiltration of various cancers, such as head and neck squamous cell carcinoma (HNSCC). MATERIAL AND METHODS: A systematic literature search based on specific keywords, according to the PRISMA guidelines, was carried out on PubMed, Web of Science, Scopus, and Google Scholar. Only original articles were selected during this review. The risk of bias was assessed by QUADAS-2. RESULTS: At the end of the selection process 9 articles were included. In these studies, 41 miRs showed differential expression between healthy subjects and patient with HNSCC. The techniques varied among studies for the extraction and analysis of exosomal miRs. We presented also salivary exosomal miRs pathways, to give insights about pathogenetic mechanisms. CONCLUSIONS: Exosomal microRNA are promising biomarkers for HNSCC detection. MiR-10b-5p, miR-486-5p, miR-24-3p, miR-412-3p, and miR-512-3p are the most promising markers applicable to diagnostics, while miR-1307-5p and miR-519c-3p resulted overexpressed and correlated to worse survival outcomes.

Irisin prevents trabecular bone damage and tumor invasion in a mouse model of multiple myeloma.

Bone disease associated with multiple myeloma (MM) is characterized by osteolytic lesions and pathological fractures, which remain a therapeutic priority despite new drugs improving MM patient survival. Antiresorptive molecules represent the main option for the treatment of MM-associated bone disease (MMBD), whereas osteoanabolic molecules are under investigation. Among these latter, we here focused on the myokine irisin, which is able to enhance bone mass in healthy mice, prevent bone loss in osteoporotic mouse models, and accelerate fracture healing in mice. Therefore, we investigated irisin effect on MMBD in a mouse model of MM induced by intratibial injection of myeloma cells followed by weekly administration of 100 µg/kg of recombinant irisin for 5 wk. By micro-Ct analysis, we demonstrated that irisin improves MM-induced trabecular bone damage by partially preventing the reduction of femur Trabecular Bone Volume/Total Volume (P = .0028), Trabecular Number (P = .0076), Trabecular Fractal Dimension (P = .0044), and increasing Trabecular Separation (P = .0003) in MM mice. In cortical bone, irisin downregulates the expression of Sclerostin, a bone formation inhibitor, and RankL, a pro-osteoclastogenic molecule, while in BM it upregulates Opg, an anti-osteoclastogenic cytokine. We found that in the BM tibia of irisin-treated MM mice, the percentage of MM cells displays a reduction trend, while in the femur it decreases significantly. This is in line with the in vitro reduction of myeloma cell viability after 48 h of irisin stimulation at both 200 and 500 ng/mL and, after 72 h already at 100 ng/mL rec-irisin. These results could be due to irisin ability to downregulate the expression of Notch 3, which is important for cell-to-cell communication in the tumor niche, and Cyclin D1, supporting an inhibitory effect of irisin on MM cell proliferation. Overall, our findings suggest that irisin could be a new promising strategy to counteract MMBD and tumor burden in one shot.

Molecules Inducing Dental Stem Cells Differentiation and Bone Regeneration: State of the Art.

Teeth include mesenchymal stem cells (MSCs), which are multipotent cells that promote tooth growth and repair. Dental tissues, specifically the dental pulp and the dental bud, constitute a relevant source of multipotent stem cells, known as dental-derived stem cells (d-DSCs): dental pulp stem cells (DPSCs) and dental bud stem cells (DBSCs). Cell treatment with bone-associated factors and stimulation with small molecule compounds are, among the available methods, the ones who show excellent advantages promoting stem cell differentiation and osteogenesis. Recently, attention has been paid to studies on natural and non-natural compounds. Many fruits, vegetables, and some drugs contain molecules that can enhance MSC osteogenic differentiation and therefore bone formation. The purpose of this review is to examine research work over the past 10 years that has investigated two different types of MSCs from dental tissues that are attractive targets for bone tissue engineering: DPSCs and DBSCs. The reconstruction of bone defects, in fact, is still a challenge and therefore more research is needed; the articles reviewed are meant to identify compounds useful to stimulate d-DSC proliferation and osteogenic differentiation. We only consider the results of the research which is encouraging, assuming that the mentioned compounds are of some importance for bone regeneration.

Irisin Modulates Inflammatory, Angiogenic, and Osteogenic Factors during Fracture Healing.

Bone fractures are a widespread clinical event due to accidental falls and trauma or bone fragility; they also occur in association with various diseases and are common with aging. In the search for new therapeutic strategies, a crucial link between irisin and bone fractures has recently emerged. To explore this issue, we subjected 8-week-old C57BL/6 male mice to tibial fracture, and then we treated them with intra-peritoneal injection of r-Irisin (100 µg/kg/weekly) or vehicle as control. At day 10 post fracture, histological analysis showed a significant reduced expression of inflammatory cytokines as tumor necrosis factor-alpha (TNFα) (p = 0.004) and macrophage inflammatory protein-alpha (MIP-1α) (p = 0.015) in the cartilaginous callus of irisin-treated mice compared to controls, supporting irisin's anti-inflammatory role. We also found increased expressions of the pro-angiogenic molecule vascular endothelial growth factor (VEGF) (p = 0.002) and the metalloproteinase MMP-13 (p = 0.0006) in the irisin-treated mice compared to the vehicle ones, suggesting a myokine involvement in angiogenesis and cartilage matrix degradation processes. Moreover, the bone morphogenetic protein (BMP2) expression was also upregulated (p = 0.002). Taken together, our findings suggest that irisin can contribute to fracture repair by reducing inflammation and promoting vessel invasion, matrix degradation, and bone formation, supporting its possible role as a novel molecule for fracture treatment.

Myoglobin expression by alternative transcript in different mesenchymal stem cells compartments.

BACKGROUND: The metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not completely defined role in this context. In a previous study, we reported the ectopic expression of myoglobin (MB) in bone marrow-derived hematopoietic stem/progenitor cells. Here, we have extended the analysis to mesenchymal stem cells (MSCs) isolated from different tissues. METHODS: MSCs were isolated from human placental membrane, mammary adipose tissue and dental pulp and subjected to RT-PCR, Western blotting and mass spectrometry to investigate the expression of MB. A combination of metabolic flux analysis and cyto-imaging was used to profile the metabolic phenotype and the mitochondria dynamics in the different MSCs. RESULTS: As for the hematopoietic stem/progenitor cells, the expression of Mb was largely driven by an alternative transcript with the protein occurring both in the monomer and in the dimer forms as confirmed by mass spectrometry analysis. Comparing the metabolic fluxes between neonatal placental membrane-derived and adult mammary adipose tissue-derived MSCs, we showed a significantly more active bioenergetics profile in the former that correlated with a larger co-localization of myoglobin with the mitochondrial compartment. Differences in the structure of the mitochondrial network as well as in the expression of factors controlling the organelle dynamics were also observed between neonatal and adult mesenchymal stem cells. Finally, the expression of myoglobin was found to be strongly reduced following osteogenic differentiation of dental pulp-derived MSCs, while it was upregulated following reprogramming of human fibroblasts to induce pluripotent stem cells. CONCLUSIONS: Ectopic expression of myoglobin in tissues other than muscle raises the question of understanding its function therein. Properties in addition to the canonical oxygen storage/delivery have been uncovered. Finding of Mb expressed via an alternative gene transcript in the context of different stem cells with metabolic phenotypes, its loss during differentiation and recovery in iPSCs suggest a hitherto unappreciated role of Mb in controlling the balance between aerobic metabolism and pluripotency. Understanding how Mb contributes through modulation of the mitochondrial physiology to the stem cell biology paves the way to novel perspectives in regenerative medicine as well as in cancer stem cell therapy.

Retrospective Analysis of Clinical and Radiologic Data Regarding Zygomatic Implant Rehabilitation with a Long-Term Follow-Up.

BACKGROUND: Zygomatic implants have been introduced to rehabilitate edentulous patients with severely atrophic maxillae. Their use has been reported by several studies, describing high overall survival rates at medium-long follow-up. The aim of this study was to retrospectively analyze if a few patient-related and implant-related features are correlated with implant success or the onset of complications. MATERIALS AND METHODS: Data of patients treated with zygomatic implants between May 2005 and November 2012 at three private clinics were collected and retrospectively analyzed. For each implant, the following data were collected: implant length, insertion path, ridge atrophy and sinus characteristics (width, pneumatization, thickness of mucosae, patency of sinus ostium). General patient characteristics and health status data were also recorded. The outcomes evaluated were implant failure, infective complications, early neurologic complications and overall complications. RESULTS: A total of 33 patients (14 men, 17 women, mean age 59.1) that received 67 zygomatic implants were included in the study. The mean duration of the follow-up was of 141.6 months (min 109; max 198). In this period, a total of 16 (23.88%) implants in 8 (24.24%) patients were removed and 17 (51.51%) patients with 36 (53.73%) implants reported complications. Immediate loading resulted in a significantly lower risk of complications compared with the two-stage prosthetic rehabilitation (OR: 0.04, p = 0.002). A thickness of the sinus mucosa > 3 mm emerged to be correlated with a greater occurrence of infective complications (OR: 3.39, p = 0.019). Severe and extreme pneumatization of the sinus was significantly correlated with the incidence of overall complications (p = 0.037) and implant failure (p = 0.044). A large sinus width was predisposed to a higher risk of neurologic complications, infective complications and implant failure (p = 0.036, p = 0.032, p = 0.04, respectively). CONCLUSIONS: zygomatic implants are an alternative procedure for atrophic ridge rehabilitation when a conventional implant placement is not possible. Several clinical and anatomical factors can have a significant role in complication occurrence.

Osteogenic and Chondrogenic Potential of the Supramolecular Aggregate T-LysYal®.

Hard tissue regeneration represents a challenge for the Regenerative Medicine and Mesenchymal stem cells (MSCs) could be a successful therapeutic strategy. T-LysYal® (T-Lys), a new derivative of Hyaluronic Acid (HA) possessing a superior stability, has already been proved efficient in repairing corneal epithelial cells damaged by dry conditions in vitro. We investigated the regenerative potential of T-Lys in the hard tissues bone and cartilage. We have previously demonstrated that cells isolated from the tooth germ, Dental Bud Stem Cells (DBSCs), differentiate into osteoblast-like cells, representing a promising source of MSCs for bone regeneration. Herewith, we show that T-Lys treatment stimulates the expression of typical osteoblastic markers, such as Runx-2, Collagen I (Col1) and Alkaline Phosphatase (ALP), determining a higher production of mineralized matrix nodules. In addition, we found that T-Lys treatment positively affects αVß3 integrin expression, key integrin in the osteoblastic commitment, leading to the formation of focal adhesions (FAs). The efficacy of T-Lys was also tested on chondrogenic differentiation starting from human articular chondrocytes (HACs) resulting in an increase of differentiation markers and cell number.

LIGHT/TNFSF14 Promotes Osteolytic Bone Metastases in Non-small Cell Lung Cancer Patients.

Tumor necrosis factor superfamily member 14 (TNFSF14), LIGHT, is a component of the cytokine network that regulates innate and adaptive immune responses, which promote homeostasis of lymphoid organs, liver, and bone. Metastatic tumors often disrupt the tissue microenvironment, thus altering the homeostasis of the invaded organ; however, the underlying mechanisms required further studies. We investigated the role of LIGHT in osteolytic bone disease induced by metastatic non-small cell lung cancer (NSCLC). Patients diagnosed with NSCLC bone metastasis show significantly higher levels of LIGHT expressed in monocytes compared with non-bone metastatic tumors and healthy controls. Serum LIGHT levels were also higher in patients with bone metastases than in controls, suggesting a role for LIGHT in stimulating osteoclast precursors. In bone metastatic patients, we also detected increased RNA expression and serum RANKL levels, thus by adding anti-LIGHT or RANK-fragment crystallizable region (RANK-Fc) in PBMC cultures, a significant inhibition of osteoclastogenesis was observed. To model this observation in mice, we used the mouse lung cancer cell line LLC-1. After intratibial implantation, wild-type mice showed an increased number of osteoclasts but reduced numbers of osteoblasts and decreased osteoid formation. In contrast, Tnfsf14-/- mice showed no significant bone loss or other changes in bone homeostasis associated with this model. These data indicate LIGHT is a key control mechanism for regulating bone homeostasis during metastatic invasion. Thus, LIGHT may be a novel therapeutic target in osteolytic bone metastases. © 2019 American Society for Bone and Mineral Research.

Polydatin, Natural Precursor of Resveratrol, Promotes Osteogenic Differentiation of Mesenchymal Stem Cells.

Bone loss and fractures are consequences of aging, diseases or traumas. Furthermore the increased number of aged people, due to the rise of life expectancy, needs more strategies to limit the bone loss and regenerate the lost tissue, ameliorating the life quality of patients. A great interest for non-pharmacological therapies based on natural compounds is emerging and focusing on the oligostilbene Polydatin, present in many kinds of fruits and vegetables, when resveratrol particularly in red wines. These molecules have been extensively studied due to their antioxidant and anti-inflammatory effects, showing more recently Resveratrol the ability to enhance osteogenic differentiation and bone formation. However, the clinical applications of Resveratrol are limited due to its low bioavailability and rapid metabolism, while its natural glycosilated precursor Polydatin shows better metabolic stability and major abundance in fresh fruits and vegetables. Nevertheless the role of Polydatin on osteogenic differentiation is still unexplored. Mesenchymal stem cells (MSCs) from dental tissues, such as dental bud stem cells (DBSCs), are able to differentiate toward osteogenic lineage: thus we investigated how Resveratrol and Polydatin influence the differentiation of DBSCs, eventually affecting bone formation. Our results showed that Polydatin increases MSCs osteogenic differentiation sharing similar properties with Resveratrol. These results encourage to deepen the effects of this molecule on bone health and its associated mechanisms of action, wishing for the future a successful use in bone loss prevention and therapy.

High expression of TRAIL by osteoblastic differentiated dental pulp stem cells affects myeloma cell viability.

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 tumor­homing ability, are currently being used as cell­based delivery systems for cancer protein therapeutics, such as the TNF­related apoptosis­inducing 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 TRAIL­apoptotic effect was also associated with low levels of intracellular anti­apoptotic proteins, such as c­FLIP, XIAP and the activation of caspase­8 and ­3. DPSC­differentiated 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.

Impairment of Bone Remodeling in LIGHT/TNFSF14-Deficient Mice.

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.

Bone Regeneration Induced by Bone Porcine Block with Bone Marrow Stromal Stem Cells in a Minipig Model of Mandibular "Critical Size" Defect.

Introduction. Adding stem cells to biodegradable scaffolds to enhance bone regeneration is a valuable option. Different kinds of stem cells with osteoblastic activity were tested, such as bone marrow stromal stem cells (BMSSCs). Aim. To assess a correct protocol for osteogenic stem cell differentiation, so BMSSCs were seeded on a bone porcine block (BPB). Materials and Methods. Bone marrow from six minipigs was extracted from tibiae and humeri and treated to isolate BMSSCs. After seeding on BPB, critical-size defects were created on each mandible of the minipigs and implanted with BPB and BPB/BMSSCs. After three months, histomorphometric analysis was performed. Results. Histomorphometric analysis provided percentages of the three groups. Tissues present in control defects were 23 ± 2% lamellar bone, 28 ± 1% woven bone, and 56 ± 4% marrow spaces; in BPB defects were 20 ± 5% BPB, 32 ± 2% lamellar bone, 24 ± 1% woven bone, and 28 ± 2% marrow spaces; in BPB/BMSSCs defects were 17 ± 4% BPB/BMSSCs, 42 ± 2% lamellar bone, 12 ± 1% woven bone, and 22 ± 3% marrow spaces. Conclusion. BPB used as a scaffold to induce bone regeneration may benefit from the addition of BDPSCs in the tissue-engineered constructs.

The effects of bone pâté on human osteoblasts cell cultures.

The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone paté has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone paté taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone patè, with bone paté mixed with fibrin glue, with fibrin glue and untreated. After 24 h, the viability of the cells was evaluated; after 1 week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24 h osteoblasts showed increased viability when treated with bone paté (19 % increase) and bone pate mixed with fibrin glue (34 % increase). After 1 week, the number of alkaline phosphatase positive cells increased by 97 and 94 % in cultures treated with bone paté alone and bone pate mixed with fibrin glue. Treatment with bone patè upregulated transcription factors and components of the extracellular matrix. The present data show that bone paté has a high osteoinductive potential on human osteoblasts, enhancing their activity.

Osteogenic differentiation of mesenchymal stem cells from dental bud: Role of integrins and cadherins.

Several studies have reported the beneficial effects of mesenchymal stem cells (MSCs) in tissue repair and regeneration. New sources of stem cells in adult organisms are continuously emerging; dental tissues have been identified as a source of postnatal MSCs. Dental bud is the immature precursor of the tooth, is easy to access and we show in this study that it can yield a high number of cells with ≥95% expression of mesenchymal stemness makers and osteogenic capacity. Thus, these cells can be defined as Dental Bud Stem Cells (DBSCs) representing a promising source for bone regeneration of stomatognathic as well as other systems. Cell interactions with the extracellular matrix (ECM) and neighboring cells are critical for tissue morphogenesis and architecture; such interactions are mediated by integrins and cadherins respectively. We characterized DBSCs for the expression of these adhesion receptors and examined their pattern during osteogenic differentiation. Our data indicate that N-cadherin and cadherin-11 were expressed in undifferentiated DBSCs and their expression underwent changes during the osteogenic process (decreasing and increasing respectively), while expression of E-cadherin and P-cadherin was very low in DBSCs and did not change during the differentiation steps. Such expression pattern reflected the mesenchymal origin of DBSCs and confirmed their osteoblast-like features. On the other hand, osteogenic stimulation induced the upregulation of single subunits, αV, ß3, α5, and the formation of integrin receptors α5ß1 and αVß3. DBSCs differentiation toward osteoblastic lineage was enhanced when cells were grown on fibronectin (FN), vitronectin (VTN), and osteopontin (OPN), ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. In addition we established that integrin αVß3 plays a crucial role during the commitment of MSCs to osteoblast lineage, whereas integrin α5ß1 seems to be dispensable. These data suggest that functionalization of biomaterials with such ECM proteins would improve bone reconstruction therapies starting from dental stem cells.

Bone-immune cell crosstalk: bone diseases.

Bone diseases are associated with great morbidity; thus, the understanding of the mechanisms leading to their development represents a great challenge to improve bone health. Recent reports suggest that a large number of molecules produced by immune cells affect bone cell activity. However, the mechanisms are incompletely understood. This review aims to shed new lights into the mechanisms of bone diseases involving immune cells. In particular, we focused our attention on the major pathogenic mechanism underlying periodontal disease, psoriatic arthritis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, metastatic solid tumors, and multiple myeloma.

Human myeloma cell lines induce osteoblast downregulation of CD99 which is involved in osteoblast formation and activity.

CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs.

LIGHT/TNFSF14 increases osteoclastogenesis and decreases osteoblastogenesis in multiple myeloma-bone disease.

LIGHT, a TNF superfamily member, is involved in T-cell homeostasis and erosive bone disease associated with rheumatoid arthritis. Herein, we investigated whether LIGHT has a role in Multiple Myeloma (MM)-bone disease. We found that LIGHT was overproduced by CD14+ monocytes, CD8+ T-cells and neutrophils of peripheral blood and bone marrow (BM) from MM-bone disease patients. We also found that LIGHT induced osteoclastogenesis and inhibited osteoblastogenesis. In cultures from healthy-donors, LIGHT induced osteoclastogenesis in RANKL-dependent and -independent manners. In the presence of a sub-optimal RANKL concentration, LIGHT and RANKL synergically stimulated osteoclast formation, through the phosphorylation of Akt, NFκB and JNK pathways. In cultures of BM samples from patients with bone disease, LIGHT inhibited the formation of CFU-F and CFU-OB as well as the expression of osteoblastic markers including collagen-I, osteocalcin and bone sialoprotein-II. LIGHT indirectly inhibited osteoblastogenesis in part through sclerostin expressed by monocytes. In conclusion, our findings for the first time provide evidence for a role of LIGHT in MM-bone disease development.

Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand.

BACKGROUND/OBJECTIVES: Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2. METHODS: Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA. RESULTS: Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium. CONCLUSIONS: TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.

The Interplay between the bone and the immune system.

In the last two decades, numerous scientists have highlighted the interactions between bone and immune cells as well as their overlapping regulatory mechanisms. For example, osteoclasts, the bone-resorbing cells, are derived from the same myeloid precursor cells that give rise to macrophages and myeloid dendritic cells. On the other hand, osteoblasts, the bone-forming cells, regulate hematopoietic stem cell niches from which all blood and immune cells are derived. Furthermore, many of the soluble mediators of immune cells, including cytokines and growth factors, regulate the activities of osteoblasts and osteoclasts. This increased recognition of the complex interactions between the immune system and bone led to the development of the interdisciplinary osteoimmunology field. Research in this field has great potential to provide a better understanding of the pathogenesis of several diseases affecting both the bone and immune systems, thus providing the molecular basis for novel therapeutic strategies. In these review, we reported the latest findings about the reciprocal regulation of bone and immune cells.

Osteoblasts display different responsiveness to TRAIL-induced apoptosis during their differentiation process.

Apoptosis can occur throughout the life span of osteoblasts (OBs), beginning from the early stages of differentiation and continuing throughout all stages of their working life. Here, we investigated the effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal human OBs showing for the first time that the expression of TRAIL receptors is modulated during OB differentiation. In particular, the TRAIL receptor ratio was in favor of the deaths because of the low expression of DcR2 in undifferentiated OBs, differently it was shifted toward the decoys in differentiated ones. Undifferentiated OBs treated with TRAIL showed reduced cell viability, whereas differentiated OBs displayed TRAIL resistance. The OB sensitiveness to TRAIL was due to the up-regulation of DR5 and the down-regulation of DcR2. The main death receptor involved in TRAIL-reduced OB viability was DR5 as demonstrated by the rescue of cell viability observed in the presence of anti-DR5 neutralizing antibody. Besides the ratio of TRAIL receptors, the sensitivity of undifferentiated OBs to TRAIL-cytotoxic effect was also associated with low mRNA levels of intracellular anti-apoptotic proteins, such as cFLIP, the activation of caspase-8 and -3, as well as the DNA fragmentation. This study suggests that apoptotic effect exerted by TRAIL/TRAIL-receptor system on normal human OB is strictly dependent upon cell differentiation status.