Adriana Di Benedetto

Periodontal disease: linking the primary inflammation to bone loss.

Periodontal disease (PD), or periodontitis, is defined as a bacterially induced disease of the tooth-supporting (periodontal) tissues. It is characterized by inflammation and bone loss; therefore understanding how they are linked would help to address the most efficacious therapeutic approach. Bacterial infection is the primary etiology but is not sufficient to induce the disease initiation or progression. Indeed, bacteria-derived factors stimulate a local inflammatory reaction and activation of the innate immune system. The innate response involves the recognition of microbial components by host cells, and this event is mediated by toll-like receptors (TLRs) expressed by resident cells and leukocytes. Activation of these cells leads to the release of proinflammatory cytokines and recruitment of phagocytes and lymphocytes. Activation of T and B cells initiates the adaptive immunity with Th1 Th2 Th17 Treg response and antibodies production respectively. In this inflammatory scenario, cytokines involved in bone regulation and maintenance have considerable relevance because tissue destruction is believed to be the consequence of host inflammatory response to the bacterial challenge. In the present review, we summarize host factors including cell populations, cytokines, and mechanisms involved in the destruction of the supporting tissues of the tooth and discuss treatment perspectives based on this knowledge.

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.

Microgravity during spaceflight directly affects in vitro osteoclastogenesis and bone resorption

During space flight, severe losses of bone mass are observed. Both bone formation and resorption are probably involved, but their relative importance remains unclear. The purpose of this research is to understand the role of osteoclasts and their precursors in microgravity‐induced bone loss. Three experiments on isolated osteoclasts (OCs) and on their precursors, OSTEO, OCLAST, and PITS, were launched in the FOTON‐M3 mission. The OSTEO experiment was conducted for 10 d in microgravity within bioreactors with a perfusion system, where the differentiation of precursors, cultured on a synthetic 3‐dimensional bonelike biomaterial, skelite, toward mature OCs was assessed. In OCLAST and in PITS experiments, differentiated OCs were cultured on devitalized bovine bone slices for 4 d in microgravity. All of the experiments were replicated on ground in the same bioreactors, and OCLAST also had an inflight centrifuge as a control. Gene expression in microgravity, compared with ground controls, demonstrated a severalfold increase in genes involved in osteoclast maturation and activity. Increased bone resorption, proved by an increased amount of collagen telopeptides released VS ground and centrifuge control, was also found. These results indicate for the first time osteoclasts and their precursors as direct targets for microgravity and mechanical forces.— Tamma, R.,Colaianni, G., Camerino, C., Di Benedetto, A., Greco, G., Strippoli, M., Vergari, R., Grano, A., Mancini, L., Mori, G., Colucci, S., Grano, M., Zallone, A. Microgravity during spaceflight directly affects in vitro osteoclastogenesis and bone resorption. FASEB J. 23, 2549–2554 (2009)

Regulation of bone remodeling by vasopressin explains the bone loss in hyponatremia

Significance In this study, we show that a primitive brain hormone, arginine vasopressin (AVP), which has hitherto been implicated in regulating water balance in mammals, has a function in skeletal homeostasis. Using genetically modified mice that are lacking one of the AVP receptors as well as pharmacologic inhibitors, we show that AVP negatively regulates osteoblasts (cells that form new bone) and stimulates osteoclasts (cells that remove old bone). Our findings explain the bone loss that is known to accompany low blood sodium levels in patients when AVP levels are high. Although hyponatremia is known to be associated with osteoporosis and a high fracture risk, the mechanism through which bone loss ensues has remained unclear. As hyponatremic patients have elevated circulating arginine-vasopressin (AVP) levels, we examined whether AVP can affect the skeleton directly as yet another component of the pituitary-bone axis. Here, we report that the two Avp receptors, Avpr1α and Avpr2, coupled to Erk activation, are expressed in osteoblasts and osteoclasts. AVP injected into wild-type mice enhanced and reduced, respectively, the formation of bone-resorbing osteoclasts and bone-forming osteoblasts. Conversely, the exposure of osteoblast precursors to Avpr1α or Avpr2 antagonists, namely SR49059 or ADAM, increased osteoblastogenesis, as did the genetic deletion of Avpr1α. In contrast, osteoclast formation and bone resorption were both reduced in Avpr1α−/− cultures. This process increased bone formation and reduced resorption resulted in a profound enhancement of bone mass in Avpr1α−/− mice and in wild-type mice injected with SR49059. Collectively, the data not only establish a primary role for Avp signaling in bone mass regulation, but also call for further studies on the skeletal actions of Avpr inhibitors used commonly in hyponatremic patients.

Bone marrow oxytocin mediates the anabolic action of estrogen on the skeleton

Background: The mechanism underlying the anabolic effect of estrogen on the skeleton is unclear. Results: We report that estrogen-induced bone formation in mice occurs through oxytocin (OT) produced by osteoblasts in bone marrow. Conclusion: Feed-forward OT release in bone marrow by a rising estrogen level may facilitate rapid skeletal recovery after lactation. Significance: The study highlights a novel mechanism for estrogen action on bone. Estrogen uses two mechanisms to exert its effect on the skeleton: it inhibits bone resorption by osteoclasts and, at higher doses, can stimulate bone formation. Although the antiresorptive action of estrogen arises from the inhibition of the MAPK JNK, the mechanism of its effect on the osteoblast remains unclear. Here, we report that the anabolic action of estrogen in mice occurs, at least in part, through oxytocin (OT) produced by osteoblasts in bone marrow. We show that the absence of OT receptors (OTRs) in OTR−/− osteoblasts or attenuation of OTR expression in silenced cells inhibits estrogen-induced osteoblast differentiation, transcription factor up-regulation, and/or OT production in vitro. In vivo, OTR−/− mice, known to have a bone formation defect, fail to display increases in trabecular bone volume, cortical thickness, and bone formation in response to estrogen. Furthermore, osteoblast-specific Col2.3-Cre+/OTRfl/fl mice, but not TRAP-Cre+/OTRfl/fl mice, mimic the OTR−/− phenotype and also fail to respond to estrogen. These data attribute the phenotype of OTR deficiency to an osteoblastic rather than an osteoclastic defect. Physiologically, feed-forward OT release in bone marrow by a rising estrogen concentration may facilitate rapid skeletal recovery during the latter phases of lactation.

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.

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.

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, β3, α5, and the formation of integrin receptors α5β1 and αVβ3. 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 αVβ3 plays a crucial role during the commitment of MSCs to osteoblast lineage, whereas integrin α5β1 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.

Regulated production of the pituitary hormone oxytocin from murine and human osteoblasts

Oxytocin (OT) is a primitive neurohypophyseal hormone that plays a primary and indispensible role in mammalian lactation. We have shown recently that OT also regulates bone remodeling, mainly bone formation, with remarkable sensitivity. We now show that OT, apart from its neurohypophyseal origin, is produced in abundance by both human and murine osteoblasts. Production of osteoblast OT is under the control of estrogen, which acts by activating the MAP kinase Erk. This non-genomic mechanism of estrogen action is in stark contrast to its genomic control of OT receptor (OTR) expression. We surmise that there is a local feed-forward loop in bone marrow through which the OT so produced from osteoblasts in response to estrogen acts upon its receptor to exert a potent anabolic action.

Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor

Significance We have shown previously that oxytocin (Oxt), other than regulating lactation and social bonding, is a potent stimulator of bone formation by the osteoblast. Here, we present evidence that this action is exerted through the nuclear localization of the Oxt receptor (Oxtr). Our findings prompt additional studies into the contribution of nuclear Oxtr signaling in regulating lactation and social bonding. We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr–EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.