Guido M. Macaluso

Osteocytes and WNT: the Mechanical Control of Bone Formation

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.

FoxOs, Wnts and oxidative stress-induced bone loss: new players in the periodontitis arena?

BACKGROUND AND OBJECTIVE Chronic periodontitis is a widespread disease affecting tooth-supporting structures that can lead to extensive loss of periodontal ligament and bone, ultimately resulting in tooth loss. Extensive evidence has demonstrated a strong association between age, metabolic disorders such as type II diabetes, oxidative stress and alveolar bone loss. The molecular players controlling bone maintenance and underlying age-related bone loss and its links to the general metabolism are currently the object of intense research. MATERIAL AND METHODS Recent findings are summarized to elucidate the molecular mechanisms linking oxidative stress, bone loss and metabolic factors. RESULTS It is well known that reactive oxygen species are an inevitable consequence of cellular respiration and that organisms have developed an efficient array of defenses against them. The core of this complex defense line is a family of transcription factors, known as FoxOs, which can bind to β-catenin and initiate a transcriptional programme regulating cell apoptosis, DNA repair and degradation of reactive oxygen species. An increase in reactive oxygen species due, for example, to age or insulin resistance, generates a situation in which bone formation is impaired by activation of FoxO, and a decrease in Wnt signaling and bone resorption are promoted. CONCLUSION The balance between FoxO and the Wnt pathway is finely tuned by systemic and local factors, creating a far-reaching mechanism that dictates the fate of mesenchymal progenitors and regulates the homeostasis of bone, providing a rationale for the impairment of systemic and alveolar bone maintenance clinically observed with age and metabolic diseases.

Rough surface topography enhances the activation of Wnt/β-catenin signaling in mesenchymal cells

It is known that the roughness of titanium surfaces affects cell proliferation and differentiation. However, the mechanisms mediating the cellular responses to surface topography are only partially understood. The present study investigated whether Wnt canonical signaling, an important pathway in determining cell fate, is modulated by surface roughness. This study analyzed the behavior of the murine C2C12 mesenchymal cell line on polished or acid-etched, sand-blasted (SLA) commercially pure titanium. When we transfected cells with Wnt3a or wild-type β-catenin and a reporter construct, we found that stimulation of Wnt canonical signaling was enhanced in cells on SLA surfaces. Moreover, more β-catenin translocated to the nucleus in cells on SLA surfaces after stimulation with Wnt3a as evidenced by immunofluorescence. However, when cells were transfected with constitutively active S33Y β-catenin mutant, no difference was observed between the groups. Higher levels of transcripts of Wnt target genes were detected in C2C12 cells cultured on SLA surfaces following transfection with Wnt3a, but the expression of a gene regulating β-catenin degradation, Axin 2, was reduced on SLA surfaces. Inhibition of β-catenin mediated transcription by dnTCF in murine osteoblastic MC3T3 cells, reversed the effects of topography on cell differentiation. Taken together, these results show that surface roughness modulates the responsiveness of mesenchymal cells to Wnt3a, that this requires the control of β-catenin degradation, and that the control of β-catenin signaling by surface topography is accountable for at least part of the effects of surface on cell differentiation.

Motor-evoked potentials in masseter muscle by electrical and magnetic stimulation in intact alert man

The electromyographic responses of the masseter after different types of transcranial stimulation were recorded with surface and needle electrodes. Magnetic stimulation at 4 cm lateral to the vertex on the biauricular line elicited MEPs in the contralateral masseter (latency 6.9 ms) due to activation of motor cortex or adjacent elements along the cortico-nuclear pathway. The ipsilateral responses to the same stimuli and to more lateral ones had shorter latencies and were ascribed to direct stimulation of the trigeminal nerve, probably its intracisternal portion. This was also the probable origin of the ipsilateral MEPs after both anodic and cathodic bipolar electrical stimulation at 7 and 11 cm lateral to the vertex on the biauricular line.

Actin cytoskeleton controls activation of Wnt/β-catenin signaling in mesenchymal cells on implant surfaces with different topographies

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.

H-reflexes in masseter and temporalis muscles in man

In contrast with limb muscles, studies on H-reflexes in the trigeminal system are scarce. The present report aimed at reevaluating the responses obtained in the masseter and temporalis muscles after electrical stimulation of their nerves. Twenty-four subjects participated in the experiments. The reflexes were elicited in the masseter and temporal muscles by monopolar stimulation and recorded using surface electrodes. Stimulation of the masseteric nerve evoked an M-response in the masseter and an H-reflex in both the masseter and the temporal muscles. In contrast with the masseter muscle, where the homonymous H-reflex disappeared at higher stimulation intensities, the heteronymous temporal H-reflex remained and reached a plateau. Simultaneous stimulation of the masseteric and deep temporal nerves resulted in an M-response and an H-reflex in both the masseter and temporal muscles. Increasing stimulus intensitites led to disappearance of the H-reflex in both muscles. The results were compared with those obtained by others on limb muscles. As in these muscles, the presence of heteronymous H-reflexes in the jaw muscles can be used in future studies of motoneuronal excitability.

Fresh‐Frozen Bone Blocks for Horizontal Ridge Augmentation in the Upper Maxilla: 6‐Month Outcomes of a Randomized Controlled Trial

PURPOSE This randomized controlled trial compared fresh-frozen versus autologous bone blocks for maxillary horizontal ridge augmentation in patients with Cawood and Howell class IV atrophies. MATERIALS AND METHODS Twenty-four patients were allocated to the autologous and fresh-frozen groups in a 1:1 ratio. Patients underwent computed tomography scans 1 week and 6 months after surgery for graft volume and density analysis. Doxycycline was administered at day 120 and day 150 to label new bone formation. Biopsy for histologic and histomorphometric analyses was performed at reentry for implant insertion, 6 months after grafting. RESULTS Fresh-frozen grafts had lower density than autologous bone. Autologous and fresh-frozen grafts lost, respectively, 25% and 52% of their initial volume (p = .0041). Histology revealed the presence of newly formed bone within both graft types, but clear signs of inflammation were present in fresh-frozen blocks. CONCLUSIONS According to these 6-month results, autologous bone blocks are preferable to fresh-frozen bone grafts.

The response of osteoblastic MC3T3-E1 cells to micro- and nano-textured, hydrophilic and bioactive titanium surfaces

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.

Biomimetic coating with phosphoserine‐tethered poly(epsilon‐lysine) dendrons on titanium surfaces enhances Wnt and osteoblastic differentiation

OBJECTIVES Phosphoserine-based functionalization has been proposed as a tool to improve integration of endosseous implants by promoting osteoblast adhesion and differentiation in vitro. The present work investigates whether phosphoserine-tethered poly(epsilon-lysine) dendrons, when applied as a film to titanium surfaces, enhance the differentiation of osteoblastic cells and the activation of Wnt/β-catenin signaling. MATERIALS AND METHODS These films were tested in a murine model of calvaria-derived MC3T3 osteoblastic cells, primary bone marrow cells and mesenchymal, undifferentiated C2C12 cells. Gene expression was assayed by Real Time PCR, and activation of Wnt signaling pathway was measured with a reporter assay. RESULTS Dendrons increased expression of alkaline phosphatase and osteocalcin, two osteoblastic markers, in both murine osteoblastic MC3T3 cells and primary bone marrow cells. The expression of osteoprotegerin, a protein opposing osteoclastogenesis was also significantly higher in cells growing on dendron-coated substrates both at 3 and 6 days of culture. Similarly, the mRNA levels of Wisp-2 and of β-catenin, two Wnt target genes, were also markedly increased in this group at day 6. The activation of this signaling pathway in cells growing on the dendron-coated surfaces was confirmed by use of a TCF/β-catenin reporter system in the C2C12 cell line. CONCLUSIONS The findings of the present study show that phosphoserine-tethered poly(epsilon-lysine) dendron films act as stimuli for the activation of specific signal cascades and promote the differentiation of adhering progenitor cells into an osteoblastic phenotype.

Serotonin: a novel bone mass controller may have implications for alveolar bone

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.