Efficacy of Combined Photoablative-Photodynamic Diode Laser Therapy Adjunctive to Scaling and Root Planing in Periodontitis: Randomized Split-Mouth Trial with 4-Year Follow-Up.

OBJECTIVE: We previously showed that photoablative laser therapy followed by multiple photodynamic cycles (PAPD) after scaling/root planing (SRP) improved healing of periodontitis patients as compared with conventional SRP after 1-year follow-up. This study reports the favorable results of PAPD plus SRP in patients with chronic periodontitis at a 4-year follow-up. MATERIALS AND METHODS: Twenty-four patients were studied. Maxillary left or right quadrants were randomly assigned to PAPD laser treatment or sham-treatment and SRP. PAPD consisted of: (1) photoablative intra/extrapocket de-epithelization with diode laser (λ 810 nm, 1 W), and (2) photodynamic treatments (4-10 weekly) with diode laser (λ 635 nm, 100 mW) and 0.3% methylene blue as photoactive antiseptic, performed after SRP. Sham treatment was performed with switched- off laser. Probing depth (PD), clinical attachment level (CAL), and bleeding-on-probing (BOP) were evaluated. Additional disease markers, namely polymorphonuclear leukocytes (PMN), erythrocytes (RBC), damaged epithelial cells (DEC), and bacteria were assayed by cytofluorescence on gingival exfoliative samples. RESULTS: At 4-year follow-up, PAPD plus SRP significantly improved PD, CAL, and BOP, as well as bacterial contamination and PMN-RBC shedding in the exfoliative samples, compared with sham treatment plus SRP. This effect was greater than that observed at 1-year follow-up. CONCLUSIONS: PAPD plus SRP provided significant, durable improvement of chronic periodontitis over sham treatment plus SRP alone.

Melatonin affects voltage-dependent calcium and potassium currents in MCF-7 cell line cultured either in growth or differentiation medium.

Big efforts have been dedicated up to now to identify novel targets for cancer treatment. The peculiar biophysical profile and the atypical ionic channels activity shown by diverse types of human cancers suggest that ion channels may be possible targets in cancer therapy. Earlier studies have shown that melatonin exerts an oncostatic action on different tumors. In particular, it was shown that melatonin was able to inhibit growth/viability and proliferation, to reduce the invasiveness and metastatic properties of human estrogen-sensitive breast adenocarcinoma MCF-7 cell line cultured in growth medium, with substantial impairments of epidermal growth factor (EGF) and Notch-1-mediated signaling. The purpose of this work was to evaluate on MCF-7 cells the possible effects of melatonin on the biophysical features known to have a role in proliferation and differentiation, by using the patch-clamp technique. Our results show that in cells cultured in growth as well as in differentiation medium melatonin caused a hyperpolarization of resting membrane potential paralleled by significant changes of the inward Ca(2+) currents (T- and L-type), outward delayed rectifier K(+) currents and cell capacitance. All these effects are involved in MCF-7 growth and differentiation. These findings strongly suggest that melatonin, acting as a modulator of different voltage-dependent ion channels, might be considered a new promising tool for specifically disrupting cell viability and differentiation pathways in tumour cells with possible beneficial effects on cancer therapy.

Inhibitory effects of relaxin on cardiac fibroblast-to-myofibroblast transition: an electrophysiological study.

NEW FINDINGS: What is the central question of this study? Fibroblast-to-myofibroblast transition is a key mechanism in the reparative response to tissue damage, but myofibroblast persistence in the wound leads to fibrosis and organ failure. The role of relaxin as an antifibrotic agent capable of counteracting the acquisition of biophysical features of differentiated myofibroblasts deserves further investigation. What is the main finding and its importance? Electrophysiological analysis showed that relaxin, administered during profibrotic treatment, hyperpolarizes the membrane potential and attenuates delayed rectifier and inwardly rectifying K(+) currents, which usually increase in the transition to myofibroblasts. These findings provide further clues to the therapeutic potential of relaxin in fibrosis. The hormone relaxin (RLX) is produced by the heart and may be involved in endogenous mechanisms of cardiac protection against ischaemic injury and fibrosis. Recent findings in cultured cardiac stromal cells suggest that RLX can inhibit fibroblast-to-myofibroblast transition, thereby counteracting fibrosis. In order to explore its efficiency as an antifibrotic agent further, we designed the present study to investigate whether RLX may influence the electrophysiological events associated with differentiation of cardiac stromal cells to myofibroblasts. Primary cardiac proto-myofibroblasts and NIH/3T3 fibroblasts were induced to myofibroblasts by transforming growth factor-ß1, and the electrophysiological features of both cell populations were investigated by whole-cell patch clamp. We demonstrated that proto-myofibroblasts and myofibroblasts express different membrane passive properties and K(+) currents. Here, we have shown, for the first time, that RLX (100 ng ml(-1) ) significantly reduced both voltage- and Ca(2+) -dependent delayed-rectifier and inward-rectifying K(+) currents that are typically increased in myofibroblasts compared with proto-myofibroblasts, suggesting that this hormone can antagonize the biophysical effects of transforming growth factor-ß1 in inducing myofibroblast differentiation. These newly recognized effects of RLX on the electrical properties of cardiac stromal cell membrane correlate well with its well-known ability to suppress myofibroblast differentiation, further supporting the possibility that RLX may be used for the treatment of cardiac fibrosis.

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.

MSCs seeded on bioengineered scaffolds improve skin wound healing in rats.

Growing evidence has shown the promise of mesenchymal stromal cells (MSCs) for the treatment of cutaneous wound healing. We have previously demonstrated that MSCs seeded on an artificial dermal matrix, Integra (Integra Lifesciences Corp., Plainsboro, NJ) enriched with platelet-rich plasma (Ematrix) have enhanced proliferative potential in vitro as compared with those cultured on the scaffold alone. In this study, we extended the experimentation by evaluating the efficacy of the MSCs seeded scaffolds in the healing of skin wounds in an animal model in vivo. It was found that the presence of MSCs within the scaffolds greatly ameliorated the quality of regenerated skin, reduced collagen deposition, enhanced reepithelization, increased neo-angiogenesis, and promoted a greater return of hair follicles and sebaceous glands. The mechanisms involved in these beneficial effects were likely related to the ability of MSCs to release paracrine factors modulating the wound healing response. MSC-seeded scaffolds, in fact, up-regulated matrix metalloproteinase 9 expression in the extracellular matrix and enhanced the recruitment of endogenous progenitors during tissue repair. In conclusion, the results of this study provide evidence that the treatment with MSC-seeded scaffolds of cutaneous wounds contributes to the recreation of a suitable microenvironment for promoting tissue repair/regeneration at the implantation sites.

Relaxin protects cardiac muscle cells from hypoxia/reoxygenation injury: involvement of the Notch-1 pathway.

In animal models, the cardiotropic hormone relaxin has been shown to protect the heart against ischemia and reperfusion-induced damage, acting by multiple mechanisms that primarily involve the coronary vessels. This in vitro study evaluates whether relaxin also has a direct protective action on cardiac muscle cells. H9c2 rat cardiomyoblasts and primary mouse cardiomyocytes were subjected to hypoxia and reoxygenation. In some experiments, relaxin was added preventatively before hypoxia; in others, at reoxygenation. To elucidate its mechanisms of action, we focused on Notch-1, which is involved in heart pre- and postconditioning to ischemia. Inactivated RLX was used as negative control. Relaxin (17 nmol/L, EC50 4.7 nmol/L), added 24 h before hypoxia or at reoxygenation, protected against cardiomyocyte injury. In fact, relaxin significantly increased cell viability (assayed by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), decreased apoptosis (assayed by TUNEL and bax/bcl-2 ratio), and reduced nitroxidative damage (assayed by nitrotyrosine expression and 8-hydroxy-deoxyguanosine levels). These effects were partly attributable to the ability of relaxin to upregulate Notch-1 signaling; indeed, blockade of Notch-1 activation with the specific inhibitor DAPT reduced relaxin-induced cardioprotection during hypoxia and reoxygenation. This study adds new mechanistic insights on the cardioprotective role of relaxin on ischemic and oxidative damage.

Mesenchymal stromal cell secreted sphingosine 1-phosphate (S1P) exerts a stimulatory effect on skeletal myoblast proliferation.

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.

Minocycline but not tigecycline is neuroprotective and reduces the neuroinflammatory response induced by the superimposition of sepsis upon traumatic brain injury.

OBJECTIVE: The development of sepsis in patients with traumatic brain injury increases mortality, exacerbates morphological and functional cerebral damage, and causes persistent neuroinflammation, including microglial activation. The administration of antibiotics possessing both antimicrobial and immunomodulatory activity might attenuate both sepsis and posttraumatic cerebral inflammation. We compared the potential therapeutic efficacy of two tetracyclines, minocycline and the newer generation tigecycline, on functional neurobehavioral impairment and regional histopathological damage in an experimental model of combined traumatic brain injury and sepsis. DESIGN: Prospective, experimental animal study. SETTING: University Research Laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Controlled cortical impact was used to induce traumatic brain injury and cecal ligation and puncture for sepsis. Immediately following injury, animals were treated with minocycline (45 mg/kg intraperitoneal), tigecycline (7.5 mg/kg intraperitoneal), or saline every 12 hours for 3 days. MEASUREMENTS AND MAIN RESULTS: The development of sepsis and cerebral inflammatory response were evaluated, respectively, by 1) growth of peritoneal microorganisms and clinical variables and 2) tumor necrosis factor-α expression in the perilesional cortex. To assess posttraumatic outcome, vestibulomotor and cognitive function were evaluated at different time points for 14 days post injury whereupon animals were killed and cerebral tissue analyzed for lesion volume, regional hippocampal (CA1/CA3) cell death, and microglial activation in the perilesional cortex, lesion core zone, and choroid plexus. Treatment with both antibiotics reduced microorganism growth, body weight loss, and mortality but had no effect on vestibulomotor or cognitive function. Minocycline alone attenuated postinjury cortical lesion volume, hippocampal CA3 neuronal cell loss, tumor necrosis factor-α expression, and the extent of microglial activation and infiltration. CONCLUSIONS: The significantly heightened mortality caused by the superimposition of sepsis upon traumatic brain injury can be reduced by administration of both antibiotics but only minocycline can decrease the extent of cell death in selectively cortical and hippocampal brain regions, via, in part, a reduction in cerebral inflammation.

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells.

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7(+) satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.

Comparative evaluation of photoablative efficacy of erbium: yttrium-aluminium-garnet and diode laser for the treatment of gingival hyperpigmentation. A randomized split-mouth clinical trial.

BACKGROUND: The use of lasers in periodontology is a matter of debate, mainly because of the lack of consensual therapeutic protocols. In this randomized, split-mouth trial, the clinical efficacy of two different photoablative dental lasers, erbium:yttrium-aluminum-garnet (Er:YAG) and diode, for the treatment of gingival hyperpigmentation is compared. METHODS: Twenty-one patients requiring treatment for mild-to-severe gingival hyperpigmentation were enrolled. Maxillary or mandibular left or right quadrants were randomly subjected to photoablative deepithelialization with either Er:YAG or diode laser. Masked clinical assessments of each laser quadrant were made at admission and days 7, 30, and 180 postoperatively by an independent observer. Histologic examination was performed before and soon after treatment and 6 months after irradiation. Patients also compiled a subjective evaluation questionnaire. RESULTS: Both diode and Er:YAG lasers gave excellent results in gingival hyperpigmentation. However, Er:YAG laser induced deeper gingival tissue injury than diode laser, as judged by bleeding at surgery, delayed healing, and histopathologic analysis. The use of diode laser showed additional advantages compared to Er:YAG in terms of less postoperative discomfort and pain. CONCLUSIONS: This study highlights the efficacy of diode laser for photoablative deepithelialization of hyperpigmented gingiva. It is suggested that this laser can represent an effective and safe therapeutic option for gingival photoablation.

Relaxin prevents cardiac fibroblast-myofibroblast transition via notch-1-mediated inhibition of TGF-ß/Smad3 signaling.

The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-ß1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-ß1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-ß1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-ß1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-ß1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-ß1 signaling, since the addition of RLX to TGF-ß1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-ß1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-ß1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects.

A new thermographic and fluorescent method for tuning photoablative laser removal of the gingival epithelium in patients with chronic periodontitis and hyperpigmentation.

OBJECTIVE: The purpose of this study was to optimize gingival laser photoablation by thermographic and autofluorescent feedbacks. BACKGROUND DATA: Photoablative laser treatment is commonly used for gingival de-epithelization in patients with chronic periodontitis or hyperpigmentation. The reduction of collateral thermal damage of periodontal tissues is crucial for optimal treatment outcome. METHODS: Nineteen patients with chronic periodontitis, seven of whom showing gingival hyperpigmentation, were subjected to de-epithelization with an 810 nm diode laser used in continuous (1 W, 66.67 J/cm2) or pulsed wave mode (69 µJ, 18 µs, 8000 Hz, corresponding to peak/mean power of 3.8 W/0.6 W, 40 J/cm2), depending upon individual gingival features. Photoablation was controlled in real time with a 405 nm violet light probe, which stimulated a yellow autofluorescence of the laser-coagulated tissue. The temperature at the target tissue was controlled with an infrared thermographic probe. When appropriate, small biopsies were taken to evaluate epithelial ablation and thermal effects. RESULTS: The energy density transferred to the treated tissue surface was computed based on the irradiation modality of the target tissues. Laser photoablation performed under thermographic control yielded complete removal of the gingival epithelium with minimal injury to the underlying lamina propria. Irradiation-evoked autofluorescence, conceivably the result of epithelial keratins, allowed very sharp recognition of the borders between laser-ablated and intact epithelium, thus preventing repeated irradiation. CONCLUSIONS: This study further supports the favorable characteristics of photoablative diode laser for gingival de-epithelization. Concurrent thermographic and fluorescent analysis can provide substantial help to the setup of a safe and well-tolerated protocol.

A molecular imaging analysis of Cx43 association with Cdo during skeletal myoblast differentiation.

Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin 43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects is independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43.

Photoactivation of bone marrow mesenchymal stromal cells with diode laser: effects and mechanisms of action.

Mesenchymal stromal cells (MSCs) are a promising cell candidate in tissue engineering and regenerative medicine. Their proliferative potential can be increased by low-level laser irradiation (LLLI), but the mechanisms involved remain to be clarified. With the aim of expanding the therapeutic application of LLLI to MSC therapy, in the present study we investigated the effects of 635 nm diode laser on mouse MSC proliferation and investigated the underlying cellular and molecular mechanisms, focusing the attention on the effects of laser irradiation on Notch-1 signal activation and membrane ion channel modulation. It was found that MSC proliferation was significantly enhanced after laser irradiation, as judged by time lapse videomicroscopy and EdU incorporation. This phenomenon was associated with the up-regulation and activation of Notch-1 pathway, and with increased membrane conductance through voltage-gated K(+) , BK and Kir, channels and T- and L-type Ca(2+) channels. We also showed that MSC proliferation was mainly dependent on Kir channel activity, on the basis that the cell growth and Notch-1 up-regulation were severely decreased by the pre-treatment with the channel inhibitor Ba(2+) (0.5 mM). Interestingly, the channel inhibition was also able to attenuate the stimulatory effects of diode laser on MSCs, thus providing novel evidence to expand our knowledge on the mechanisms of biostimulation after LLLI. In conclusions, our findings suggest that diode laser may be a valid approach for the preconditioning of MSCs in vitro prior cell transplantation.

Relaxin, cardiac stem cells and heart regeneration.

The notion that the adult heart of mammals including humans contain a small population of resident cardiac progenitor/stem cells (CSCs) have questioned the traditional paradigm of the myocardium as a post-mitotic terminally differentiated tissue. These cells, however, are relatively scarce and unable to be recruited in large number at the site of tissue damage. This has sparkled novel interest in the identification of molecules capable of stimulating the regenerative potentials of CSCs in their microenvironment. In this context, the peptide hormone relaxin (RLX), recently validated as a cardiovascular hormone, deserves a key place. This article summarizes the most recent findings of our group on the ability of RLX to modulate growth and differentiation of mouse neonatal cardiomyocytes, suggesting that this hormone, for which the heart is both a source and target organ, may participate in the endogenous mechanisms of myocardial repair/regeneration. Moreover, we have recently observed that RLX, by a Notch-1-mediated mechanism, inhibits cardiac myofibroblast differentiation and function, suggesting that the known anti-fibrotic effects of RLX may be part of a complex network of actions whereby this hormone facilitates cardiac stem cell growth and improves myocardial regeneration.

Combined photoablative and photodynamic diode laser therapy as an adjunct to non-surgical periodontal treatment: a randomized split-mouth clinical trial.

AIM: Comparing the efficacy of photoablative and photodynamic diode laser in adjunct to scaling -root planing (SRP) and SRP alone for the treatment of chronic periodontitis. MATERIALS AND METHODS: Twenty-six patients were studied. Maxillary left or right quadrants were randomly assigned to sham-laser treatment + SRP or laser + SRP. This consisted of photoablative intra/extra-pocket de-epithelization with diode laser (λ = 810 nm), followed by single SRP and multiple photodynamic treatments (once weekly, 4-10 applications, mean ± SD: 3.7 ± 2.4) using diode laser (λ = 635 nm) and 0.3% methylene blue as photosensitizer. The patients were monitored at days 0 and 365 by clinical assessment (probing depth, PD; clinical attachment level, CAL; bleeding on probing, BOP) and at days 0, 15, 30, 45, 60, 75, 90, 365 by cytofluorescence analysis of gingival exfoliative samples taken in proximity of the teeth to be treated (polymorphonuclear leukocytes, PMN; red blood cells, RBC; damaged epithelial cells, DEC; bacteria). RESULTS: At day 365, compared with the control quadrants, the laser + SRP therapy yielded a significant (p < 0.001) reduction in PD (-1.9 mm), CAL (-1.7 mm) and BOP (-33.2% bleeding sites), as well as in bacterial contamination - especially spirochetes - and PMN and RBC shedding in the gingival samples (p < 0.001). CONCLUSIONS: Diode laser treatment (photoablation followed by multiple photodynamic cycles) adjunctive to conventional SRP improves healing in chronic periodontitis patients.

Bone marrow mesenchymal stromal cells stimulate skeletal myoblast proliferation through the paracrine release of VEGF.

Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies.

Combined effects of melatonin and all-trans retinoic acid and somatostatin on breast cancer cell proliferation and death: molecular basis for the anticancer effect of these molecules.

Melatonin has been shown to inhibit breast cancer cell growth in numerous studies. However, our understanding of the therapeutic effects of this hormone is still marginal and there is little information concerning its combination with other antitumor agents to achieve additional potential benefits. All-trans retinoic acids or somatostatin have been used in combination with melatonin in several pre-clinical and clinical trials, but they have never been combined altogether as an anti-breast cancer treatment. In the present study, we investigated whether the association of melatonin, all-trans retinoic acid and somatostatin leads to an enhanced anticancer activity in MCF-7 breast cancer cells. In such conditions, MCF-7 cells were investigated for cell growth/viability and proliferation, as well as for the expression of cyclin A, and components of the Notch and EGFR pathways, by Western blotting and confocal immunofluorescence. Electrophysiological, morphological, and biochemical analysis were also performed to reveal signs of cell damage and death. We found that melatonin in combination with all-trans retinoic acid and somatostatin potentiated the effects of melatonin alone on MCF-7 cell viability and growth inhibition; this phenomenon was associated with altered conductance through Ca²âº and voltage-activated K⁺ (BK) channels, and with substantial impairments of Notch-1 and epidermal growth factor (EGF)-mediated signaling. The combined treatment also caused a marked reduction in mitochondrial membrane potential and intracellular ATP production as well as induction of necrotic cell death. Taken together our results indicate that co-administration of melatonin with all-trans retinoic acid and somatostatin may be of significant therapeutic benefit in breast cancer.

Comparative evaluation of the effects of different photoablative laser irradiation protocols on the gingiva of periodontopathic patients.

OBJECTIVE: We aimed at quantifying the presence of periodontopathogens in gingival biopsies from periodontitis patients treated with different photoablative lasers (diode GaAs, Er:YAG, Nd:YAG, and CO(2) lasers) and histologically analyzing their effects on the gingiva. BACKGROUND DATA: Substantial evidence indicates that intracellular location of periodontal bacteria in the gingival epithelium may contribute to chronic periodontitis. METHODS: Sixteen adult subjects with chronic periodontitis were subjected to conventional scaling/root planing and topical chlorhexidine, and immediately laser-irradiated on the inner and outer free gingiva. Small gingival biopsies were subjected to real-time polymerase chain reaction and cytofluorescence to identify periodontopathogens; tissue damage and endothelial ICAM-1 expression were assessed by histological and immunofluorescence analyses. RESULTS: High DNA levels of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Treponema denticola, Prevotella intermedia, and Ekenella corrodens, were detected in all samples. Nd:YAG and diode lasers were capable of eradicating periodontopathogenic bacteria endowed within gingival epithelial cells outside periodontal pockets, without causing connective tissue damage and microvessel rupture. They also reduced ICAM-1 immunolabelling by the vascular endothelium. Conversely, Er:YAG lasers induced marked microvessel rupture and bleeding and failed to completely and selectively ablate the infected gingival epithelium, whereas CO(2) laser caused heat-induced coagulation of the lamina propria. CONCLUSIONS: This study indicates that periodontopathogens can persist within cells outside the pocket epithelium, despite conventional periodontal treatment. Nd:YAG and diode lasers are able to eradicate intra- and extracellular bacteria from these sites, suggesting that they can be considered suitable devices to improve the clinical outcome of periodontal disease.

Relaxin promotes growth and maturation of mouse neonatal cardiomyocytes in vitro: clues for cardiac regeneration.

The demonstration that the adult heart contains myocardial progenitor cells which can be recruited in an attempt to replace the injured myocardium has sparkled interest towards novel molecules capable of improving the differentiation of these cells. In this context, the peptide hormone relaxin (RLX), recently validated as a cardiovascular hormone, is a promising candidate. This study was designed to test the hypothesis that RLX may promote the growth and maturation of mouse neonatal immature cardiomyocytes in primary culture. The cultures were studied at 2, 12, 24 and 48 hrs after the addition of human recombinant H2 RLX (100 ng/ml), the main circulating form of the hormone, or plain medium by combining molecular biology, morphology and electrophysiology. RLX modulated cell proliferation, promoting it at 2 and 12 hrs and inhibiting it at 24 hrs; RLX also induced the expression of both cardiac-specific transcription factors (GATA-4 and Nkx2-5) and cardiac-specific structural genes (connexin 43, troponin T and HCN4 ion channel) at both the mRNA and protein level. Consistently, RLX induced the appearance of ultrastructural and electrophysiological signs of functionally competent, mature cardiomyocytes. In conclusion, this study provides novel circumstantial evidence that RLX specifically acts on immature cardiomyocytes by promoting their proliferation and maturation. This notion suggests that RLX, for which the heart is both a source and target organ, may be an endogenous regulator of cardiac morphogenesis during pre-natal life and could participate in heart regeneration and repair, both as endogenous myocardium-derived factor and exogenous cardiotropic drug, during adult life.