Mariasevera Di Comite

The myokine irisin increases cortical bone mass

Significance Although exercise is a well known and potent stimulus for new bone formation, and weightlessness or muscle loss characteristically cause bone loss, it has remained unclear how muscle talks to bone, despite their close proximity. Here, we show that a molecule irisin derived from skeletal muscle in response to exercise has profound effects in enhancing mass and improving the geometry and strength specifically of cortical bone, the key function of which is to resist bending and torsion. Trabecular bone, which is a reservoir for bodily calcium, is remarkably spared. Irisin may therefore not only be the molecule responsible for muscle–bone connectivity, but could also become a therapy for sarcopenia and osteoporosis, which occur in tandem in the elderly. It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg−1. We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg−1 per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin–injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle–bone connectivity.

Leptin–leptin receptor are involved in angiogenesis in human hepatocellular carcinoma

Recent evidence in vitro and in vivo indicates that leptin, an adipose tissue-secreted hormone which is involved in the regulation of satiety, metabolic rate and thermogenesis, is implicated in angiogenesis. However, the role of leptin-mediated angiogenesis in hepatic carcinogenesis has not yet been completely elucidated. In this study, we have correlated microvascular density and leptin/leptin receptor (Ob-R) expression in endothelial and tumor cells with the histopathological type in human hepatocellular carcinoma (HCC). For this purpose, specimens of 40 primary HCC were submitted to immunohistochemical investigation using anti-CD31, anti-leptin and anti-Ob-R antibodies. Poorly-differentiated HCC had a higher degree of vascularization than other stages and leptin/Ob-R expression in both tumor and endothelial cells increased in parallel with the grade of malignancy and was highly correlated with the degree of angiogenesis. In the chick embryo chorioallantoic membrane in vivo assay, HCC biopsy specimens induced a strong angiogenic response, which was counteracted by an anti-leptin antibody. Taken together, these findings indicate that leptin/Ob-R correlate with angiogenesis and tumor progression in patients with HCC and that an anti-leptin antibody exerts an angiostatic activity in HCC.

Osteogenic differentiation of dental follicle stem cells.

Background: Stem cells are defined as clonogenic cells capable of self-renewal and multi-lineage differentiation. A population of these cells has been identified in human Dental Follicle (DF). Dental Follicle Stem Cells (DFSCs) were found in pediatric unerupted wisdom teeth and have been shown to differentiate, under particular conditions, into various cell types of the mesenchymal tissues. Aim: The aim of this study was to investigate if cells isolated from DF show stem features, differentiate toward osteoblastic phenotype and express osteoblastic markers. Methods: We studied the immunophenotype of DFSCs by flow cytometric analysis, the osteoblastic markers of differentiated DFSCs were assayed by histochemical methods and real-time PCR. Results: We demonstrated that DFSCs expressed a heterogeneous assortment of makers associated with stemness. Moreover DFSCs differentiated into osteoblast-like cells, producing mineralized matrix nodules and expressed the typical osteoblastic markers, Alkaline Phosphatase (ALP) and Collagen I (Coll I). Conclusion: This study suggests that DFSCs may provide a cell source for tissue engineering of bone.

Dens invaginatus: a qualitative-quantitative analysis. Case report of an upper second molar.

Dens invaginatus (D.I.) is a developmental anomaly caused by the infolding of the surface of a tooth crown before calcification has occurred. Its aetiology is controversial and remains unclear. It occurs in all dentitions with a prevalence that ranges from 0.25% to 7.74% and is mostly seen in the maxillary permanent incisors, particularly in the lateral incisors. Posterior teeth are infrequently involved. The purpose of this study was to investigate the morpho‐structure of a second upper molar dens invaginatus compared with a control tooth. Ground and decalcified sections were prepared and histo‐morphological evaluation of dental tissues was performed by using light microscopy, microradiography, and confocal laser scanning microscopy analysis (CLSM). The mechanical behaviour was tested by means of microhardness (HV) test. The results of our investigation showed structural anomalies of hard tissues, such as a difference in enamel prism diameter, in number and diameter of peripulpal dentinal tubules and in surface and diameter of cementocyte lacunae between D.I. and control tooth.

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

Orofacial Manifestations and Temporomandibular Disorders of Sjögren Syndrome: An Observational Study

AIMS: Sjӧgren Syndrome is a disorder involving oral tissues, with xerostomia, dysgeusia, dysphagia, tooth decay, gingivitis, angular cheilitis and glossitis. Temporomandibular disorders are a generic term referred to clinical conditions involving the jaw muscles and temporomandibular joint. The aim of this study was to investigate the prevalence of oral manifestations and temporomandibular disorders (TMD) in Sjӧgren Syndrome (SS) patients compared with healthy people. METHODS: The study group included 72 SS patients (2 men, 70 women) diagnosed according to the American-European Consensus Group (AECG) Criteria. A randomly selected group of 72 patients, matched by sex and age, served as control group. The examination for TMD signs and symptoms was based on the standardized Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) through a questionnaire and clinical examination. RESULTS: SS patients complained more frequently (95.8%) of oral symptoms (xerostomia, dysgeusia, dysphagia) than controls (22.2%) (χ2= 80.66 p< 0.001). TMD symptoms (muscle pain on chewing, difficulty in mouth opening, arthralgia, headaches, tinnitus) were complained by 91.7% of SS patients and by 84.7% of controls (χ2= 1,667 p= 0,196). At the clinical examination, 91,7% of SS had at least one oral sign respect to 75 % of controls. The salivary flow measurements showed high statistical significance between the two groups (Unpaired test, p< 0,0001). Myofascial pain (caused by muscular contracture) was significantly higher in the study group than in the control one (p≤ 0,05). Furthermore 18,05% of SS patients showed deflection versus 5,5% of controls (χ2=5,402 p=0,020). CONCLUSIONS: Sjӧgrens Syndrome seems to play a role in temporomandibular joint disorders.

Micro-anatomical structure of the first spine of the dorsal fin of Atlantic bluefin tuna, Thunnus thynnus (Osteichthyes: Scombridae)

The first spine of the first dorsal fin (FS) of the Atlantic bluefin tuna (ABFT), Thunnus thynnus, is customarily used in age determination research because its transverse sections display well-defined growth marks. In this paper the FS structure was studied to explain its known dramatic age- and season-related morphological modifications, which are evidently caused by bone remodeling. Cross sections of samples from six adult ABFT were in part decalcified to be stained with histological, histochemical and immunohistochemical methods, and in part embedded in methyl-methacrylate to be either observed under a linear polarized light or microradiographed. FS showed an external compact bone zone and an inner trabecular bone zone. The compact bone zone consisted of an outer non-osteonic primary bone layer (C1) and an inner osteonic bone layer (C2). C1 was in turn characterized by alternate translucent and opaque bands. Evidence of spine bone remodeling was shown by the presence of osteoclasts and osteoblasts as well as by tartrate-resistant acid phosphatase (TRAP) positive bands at the boundary between old and newly formed bone. The examination of plain, i.e. not-fixed and not-decalcified, FS from 28 ABFT showed that the average thickness of C1 remained fairly constant during fish growth, whereas C2 increased significantly, indicating that the periosteal primary bone apposition is counterbalanced by the parallel bone remodeling occurring inside the compact bone zone. The present study revealed the structure of the ABFT FS and the pattern of its bone remodeling. Both of them underlay phenomena, never examined in detail before, such as the appearance followed by the progressive disappearance of growth bands.

LIGHT/TNFSF14 affects basal bone remodeling

LIGHT (TNFSF14), expressed by different cells of the immune system, binds two trans-membrane receptors: HVEM and LTβR. It is over-expressed in erosive rheumatoid arthritis and lytic myeloma-bone disease and controversial data have been published on its role in osteoclast (OC) formation in vitro. Here, we investigated the role of LIGHT on in vitro murine osteoclastogenesis model and bone phenotype in LIGHT-/- mice. Firstly, we showed that murine macrophages stimulated with LIGHT alone did not differentiate into OCs. Interestingly, the presence of LIGHT and sub-optimal RANKL concentration displayed synergic effects on OC formation through the early and sustained activation of Akt, NFκB and JNK pathways. Secondly, by microCT we found that the femurs of LIGHT-KO mice exhibited a 30% (p<0.01) decrease in trabecular BV/TV due to a significant reduction in trabecular thickness and number as well as the increase in trabecular spaces respect to wild-type (WT) mice. Furthermore, a five fold increase of OC number/bone surface was found in femora from KO mice compared to WT (p<0.008). To investigate the possible molecular mechanism/s responsible for this bone phenotype in LIGHT-/- mice we studied OPG levels in whole bone marrow (BM) extracts from the femurs of these mice and demonstrated a significant reduction in OPG mRNA transcript respect to WT. Further investigations showed that BM CD8+ T cells and B cell subpopulations from KO mice expressed lower levels of OPG compared to those from WT mice. Consistently, LIGHT treatment in a dose dependent manner increase OPG expression in BM CD8+ T cells and B-cells. In conclusion, our results identified LIGHT as a new important regulator of bone remodeling and highlighted a new modulator of OPG expression.

Quantitative Analysis of Defects at the Dentin-Post Space in Endodontically Treated Teeth

The objective of this study was to assess frequency and extension of the defects affecting the dentin-post interface after using different combinations of irrigants and sealers. The experimental work was conducted on single-rooted teeth extracted for orthodontic reasons. The specimens were divided into different groups, according to irrigant and endodontic cement utilized, and endodontically instrumented. After fiberglass posts cementation, cross sections were obtained at apical, middle and coronal level of the root and submitted to quantitative analyses. Different types of defects were found: bubbles, bonding defects, polymerization defect, and cement residues. The percent extension of each defect and its frequency were related to the specific irrigant/sealer combination and to the root level. Detachments of the material from dentin were found only at apical and middle levels. Chlorhexidine digluconate seems to have more beneficial effects if compared to sodium hypochlorite: samples prepared with chlorhexidine digluconate showed a higher performance, with roots including null to few defects. In detail, samples treated with chlorhexidine digluconate and Pulp Canal Sealer showed the lowest frequency and the smallest dimension of defects.

Non invasive moderate loading in vivo and osteoarthritis development

Mechanical loading is known to modify joint structure through a mechano-adaptative response and to increase proinflammatory cytokines, as IL-1β, modulating VEGF secretion by condrocytes1. VEGF is a potent angiogenic factor, also detectable in later stages of OA, able to increase matrix MMPs playing an important role in the development of OA2. The aim of this study is to evaluate “in vivo” changes of bone and cartilage leading to OA by means of nonsurgical intermittent moderate loading. Forty day-old mice was randomly divided into two groups of six animals each: sedentary and exercised. The exercised group was subjected to a treadmill running at 12m/min, two times a week for four weeks. The sedentary group did not undergo any physical training and was left free to walk inside the cages. After the sacrifice the femur heads were removed and processed for paraffin embedding. On 5μm coronal sections, safranine-O staining and immunostaining for IL-1 β, MMP-13 and VEGF was performed to evaluate: articular cartilage and subchondral bone trabeculae thickness; chondrocytes number/mm2; chondrocytes volume; % cell number expressing VEGF, IL-1β and MMP-13 in articular cartilage and bone. Significant increase of chondrocytes volume, VEGF and MMP-13 expression in cartilage was detected. These data seem indicate that our non-invasive experimental model could induce early alterations in articular cartilage only, confirming chondrocytes to play a central role in the pathogenesis of OA. Other alterations, such as articular cartilage cracking and thickening and sub-chondral bone sclerosis, would appear later.