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

ArticleinJournal of Cellular Physiology 228(1):172-81 · January 2013with 171 Reads 
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DOI: 10.1002/jcp.24119 · Source: PubMed
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Abstract
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. J. Cell. Physiol. 228: 172-181, 2013. © 2012 Wiley Periodicals, Inc.

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  • ... UC-MSCs irradiated with laser were injected i.v. to the mice of groups II-b, IV-a and IV-c in the dose of 1 Â 10 4 cells/cm 2 [34,35]. ...
    ... The actual power is calculated to know the exact power that the cells received from laser and to eliminate any absorption taking place through the plastic walls of the container. The laser beam expanded to 20.72 cm 2 area by a concave lens [35,63,64]. Mvula et al., [63] studied irradiation of human Adipose derived stem cells hADSCs and they stated that is dependant on the wavelength as well as a fluence. ...
    ... Giannelli et al., [35] studied that diode laser, operating at a wavelength of 635 nm stimulates the proliferative potential of MSCs. The enhancement of cell growth after irradiation is dependent on the activation of physiological processes including membrane ion channel modulation. ...
    Article
    Full-text available
    Background and study aims: The therapeutic effects of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) exposed to diode laser and/or hepatocyte growth factor (HGF) were compared in mice with experimental liver fibrosis induced by carbon tetra chloride (CCl4). Material and methods: Animal model of liver cirrhosis was induced by intraperitoneal injection of CCl4 in a dose of 0.4ml/kg, twice a week for 6weeks. UC-MSCs were obtained from normal full term placentas and were exposed to diode laser and/or HGF. Before treatment, UC-MSCs were labelled with red fluorescent PKH26. Fifty four male mice weighing 25-35g were randomly divided into four groups control, stem cells, CCl4, and treated groups. After the experimental period, body and liver weights were recorded, and the liver specimens were processed for histological examination using haematoxylin and eosin, Periodic Acid-Schiff (PAS), and Masson's Trichrome staining (MT). Results: Results showed that administration of UC-MSCs stimulated by diode laser and/or HGF improved body and liver weights, reduced vascular dilatation and congestion, reduced mononuclear cellular infiltration, reduced hepatocyte vacuolation, eosinophilia, and pyknosis. Furthermore, periportal fibrosis was minimized and PAS reaction was increased. These effects were maximum when UC-MSCs were exposed to both diode laser and HGF. Conclusion: UC-MSCs stimulated by both diode laser and HGF proved to be an effective therapeutic option in experimental liver fibrosis induced by CCl4 in mice.
  • ... The authors suggest that PBMT could be a preliminary stage in the in vitro preparation of MSCs prior to cell transplantation under clinical conditions [33,34]. Khandra et al. [33] showed there was increased cellular attachment, proliferation, differentiation, osteocalcin synthesis, and transforming growth factor β1 production in human osteoblast-like cells after low level laser irradiation. ...
    ... Nurković et al. [21] demonstrated that PBMT accelerates MSC proliferation [21]. The results obtained were confirmed by the Gianellis team [34]. From a systematic review, Farahabi [35] indicated that there was limited evidence for laser irradiation increasing MSCs proliferation due to the limited number of experiments and the heterogeneity within their methods, making further research necessary. ...
    ... [Color figure can be viewed at wileyonlinelibrary.com] for 7 days) there were more MSCs than in untreated samples, with the biggest increase resulting from concurrent application of EMF and PBMT. [21] Gianelli et al. [34] indicated that laser irradiation (power, 0.89 W; wavelength, 635 nm; continuous emission) stimulates MSC proliferation associated with increased membrane conductance. Reviewing the literature, Farahani [35] concluded that low intensity laser irradiation at 660 nm (two irradiations: energy density, 3 J/cm 2 and power, 20 mW; energy density, 0.5 and 1 J/cm 2 and power, 30 mW), 810 nm (seven irradiations: energy density, 0.1 and 0.2 J/cm 2 and power, 60 mW), and 980 nm (a single irradiation: energy density, 3 J/cm 2 and power 100 mW) increased MSC proliferation. ...
    Article
    Background and objectives: We have investigated how low intensity laser irradiation emitted by a multiwave-locked system (MLS M1) affects the viability and proliferation of human bone marrow mesenchymal stem cells (MSCs) depending on the parameters of the irradiation. Study design/materials and methods: Cells isolated surgically from the femoral bone during surgery were identified by flow cytometry and cell differentiation assays. For irradiation, two wavelengths (808 and 905 nm) with the following parameters were used: power density 195, 230, and 318 mW/cm 2 , doses of energy 3, 10, and 20 J (energy density 0.93-6.27 J/cm 2 ), and in continuous (CW) or pulsed emission (PE) (frequencies 1,000 and 2,000 Hz). Results: There were statistically significant increases of cell viability and proliferation after irradiation at 3 J (CW; 1,000 Hz), 10 J (1,000 Hz), and 20 J (2,000 Hz). Conclusions: Irradiation with the MLS M1 system can be used in vitro to modulate MSCs in preparation for therapeutic applications. This will assist in designing further studies to optimize the radiation parameters and elucidate the molecular mechanisms of action of the radiation. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.
  • ... The effectiveness of PBM to encourage proliferation and/or differentiation of osteogenic cells has been described in many in vitro studies [10][11][12][13][14][15][16][17][18][19][20][21]. In addition, several pieces of in vivo research addressed the ability of PBM to accelerate the process of bone healing after oral and orthopedic surgery, by inducing an increase in bone density, neoformation, regeneration and mineralization [22][23][24][25][26][27][28] and some clinical trials are currently ongoing in different centers all over the world (Available online: https://clinicaltrials.gov/). ...
    ... We tested these wavelengths on the basis of previous reports showing the ability of 635 nm to modulate different cell type behavior [11,32], and the in vitro efficacy of photodynamic treatment with methylene blue photoactivated with 635 nm or with 405 nm wavelengths [33] as well as of 808 nm laser phototreatment [34] to selectively inactivate different bacteria playing a central role in the pathogenesis of periodontal and peri-implant diseases. In addition, the bactericidal effect of 405 nm has also been demonstrated on a variety of pathogenic bacteria related to the infections after orthopedic surgery [35]. ...
    ... The viability of human bone marrow-derived MSCs (hMSCs) subjected to the three different red, NIR and violet-blue PBM treatments was comparable to that of untreated cells, as shown by the results of nuclear PI/Syto16 staining test ( Figure 1C) and MTS assay ( Figure 1D). A slight but significant increase in the percentage of Ki67 positive cells was found when the cells were treated with 635 nm as compared to control cells, in accordance with our previous observations on murine bone marrow-derived MSCs [11] ( Figure 4A,B,E). Conversely, 808 nm and 405 nm did not alter the proliferative ability of the cells as compared to controls ( Figure 4A,C-E). ...
    Article
    Full-text available
    Photobiomodulation (PBM) has been used for bone regenerative purposes in different fields of medicine and dentistry, but contradictory results demand a skeptical look for its potential benefits. This in vitro study compared PBM potentiality by red (635 ± 5 nm) or near-infrared (NIR, 808 ± 10 nm) diode lasers and violet-blue (405 ± 5 nm) light-emitting diode operating in a continuous wave with a 0.4 J/cm2 energy density, on human osteoblast and mesenchymal stromal cell (hMSC) viability, proliferation, adhesion and osteogenic differentiation. PBM treatments did not alter viability (PI/Syto16 and MTS assays). Confocal immunofluorescence and RT-PCR analyses indicated that red PBM (i) on both cell types increased vinculin-rich clusters, osteogenic markers expression (Runx-2, alkaline phosphatase, osteopontin) and mineralized bone-like nodule structure deposition and (ii) on hMSCs induced stress fiber formation and upregulated the expression of proliferation marker Ki67. Interestingly, osteoblast responses to red light were mediated by Akt signaling activation, which seems to positively modulate reactive oxygen species levels. Violet-blue light-irradiated cells behaved essentially as untreated ones and NIR irradiated ones displayed modifications of cytoskeleton assembly, Runx-2 expression and mineralization pattern. Although within the limitations of an in vitro experimentation, this study may suggest PBM with 635 nm laser as potential effective option for promoting/improving bone regeneration.
  • ... Accordingly, MSCs have the ability of self-renewal and are able to differentiate into at least two cell types [8]. LPT has already shown improvement in the MSCs proliferative rate and differentiation [9][10][11]. Current results stress that the association of laser and MSCs may be of particular relevance in the dentistry field. ...
    ... Although we cannot mechanistically explain our current results, some previous studies corroborate to elucidate the positive effect of LPT observed here. Laser irradiation can upregulate levels of mRNA of Notch-1, which play an important role in MSCs self-renewal [9]. Accordingly, the modulation of channel gating by laser light may be a critical step in the upregulation of Notch-1 signaling in MSCs, thus stimulating their proliferation [9]. ...
    ... Laser irradiation can upregulate levels of mRNA of Notch-1, which play an important role in MSCs self-renewal [9]. Accordingly, the modulation of channel gating by laser light may be a critical step in the upregulation of Notch-1 signaling in MSCs, thus stimulating their proliferation [9]. Another study reported that laser irradiation is able to inhibit NF-B nuclear translocation due to LPS stimulation through an increase in the intracellular level of cyclic AMP (cAMP), suppressing, and, therefore, the release of important proinflammatory cytokines (COX-2, IL-1B, IL-6, and IL-8) [27]. ...
    Article
    Full-text available
    Background: We investigated the influence of laser phototherapy (LPT) on the survival of human mesenchymal stem cells (MSCs) submitted to substances leached from dental adhesives. Method: MSCs were isolated and characterized. Oral mucosa fibroblasts and osteoblast-like cells were used as comparative controls. Cultured medium conditioned with two adhesive systems was applied to the cultures. Cell monolayers were exposed or not to LPT. Laser irradiations were performed using a red laser (GaAlAs, 780 nm, 0.04 cm(2), 40 mW, 1 W/cm(2), 0.4 J, 10 seconds, 1 point, 10 J/cm(2)). After 24 h, cell viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction assay. Data were statistically compared by ANOVA followed by Tukey's test (P < 0.05). Results: Different cell types showed different viabilities in response to the same materials. Substances leached from adhesives were less cytotoxic to MSCs than to other cell types. Substances leached from Clearfil SE Bond were highly cytotoxic to all cell types tested, except to the MSCs when applied polymerized and in association with LPT. LPT was unable to significantly increase the cell viability of fibroblasts and osteoblast-like cells submitted to the dental adhesives. Conclusion: LPT enhances mesenchymal stem cells survival in response to substances leached from dental adhesives.
  • ... Несмотря на значительный разброс энергетических параметров лазерного воздействия в исследованиях по изучению влияния НИЛИ на пролиферацию МСК, данные литературы в отношении оптимальной ЭП для непрерывного НИЛИ с длиной волны 633-635 нм находятся в достаточно узком диапазоне -0,3-0,5 Дж/см 2 , при этом ПМ варьирует в значительно более широких пределах, от 3-6,61 мВт/см 2 [18][19][20][21][22][23] до 32,6 мВт/см 2 [20], а экспозиция -соответственно от 10 до 90 с (см. табл. ...
    ... Несмотря на значительный разброс энергетических параметров лазерного воздействия в исследованиях по изучению влияния НИЛИ на пролиферацию МСК, данные литературы в отношении оптимальной ЭП для непрерывного НИЛИ с длиной волны 633-635 нм находятся в достаточно узком диапазоне -0,3-0,5 Дж/см 2 , при этом ПМ варьирует в значительно более широких пределах, от 3-6,61 мВт/см 2 [18][19][20][21][22][23] до 32,6 мВт/см 2 [20], а экспозиция -соответственно от 10 до 90 с (см. табл. ...
    ... При этом M. Giannelli и соавт. [20] получили увели-а б в г Рис. 3. Микрофотография контрольной культуры, выращенной на среде с 10% сыворотки без освечивания НИЛИ в проходящем свете и окрашенной флюоресцентными красителями. ...
    Article
    Low-intensity laser radiation can be used as one of the methods for the non-specific regulation of the human mesenchymal stem cell (MSC) activity at the preliminary stage of their in vitro cultivation. The objective of the present study was to estimate the influence of the limiting regimes of continuous low-intensity laser radiation (CLIR) of red (635 nm) and green (525 nm) spectra. MATERIAL AND METHODS: The adhesive culture of human mesenchymal stem cells obtained from a donor's umbilical cord tissue was used in the experiments (following 4 passages). They were irradiated using a Lazmik-VLOK laser therapeutic device equipped with the KLO-635-40 (635 nm, 4,9 mW/cm(2)) and KLO-525-50 (525 nm, 5,4 mW/cm(2)) laser diode emitting heads operating in a continuous mode. A special nozzle (jar) for laser and vacuum massage (KB-5, 35 cm in diameter) was employed to fix the heads. The exposure time in all the irradiation regimes was 5 minutes. CONCLUSION: The study has demonstrated that neither the morphological features nor the viability of mesenchymal stem cells was altered under the influence of laser irradiation at the aforementioned energy and time parameters. The data obtained indicate that laser irradiation with the limiting levels of the chosen energy parameters produces no positive effect on the cell proliferative activity; more than that, it may cause its inhibition.
  • ... 19,20 Other authors report that biostimulation via the diode laser also has a positive affect on the bone cells and can be stimulatory to the bone cells at the crest around the implant. 21,22 When compared to conventional methods tissue healing as well as postoperative sensitivity was less with the diode laser then with other methods. 23 ...
    ... 19,20 Otros autores informan que la bioestimulación mediante el láser de diodo también tiene un efecto positivo en las células óseas y puede ser estimulante para éstas en la cresta alrededor del implante. 21,22 En comparación con los métodos convencionales, la cicatrización de los tejidos, así como la sensibilidad posoperatoria, fue menor con el láser de diodo que con otros métodos. 23 ...
    Article
    Full-text available
    Diode lasers have become more widely used in dental clinical practice and have demonstrated a safe effective method for treatment in and around implants that require soft tissue modification to either expose the implant for the restorative phase or reshape the gingival margins for esthetics. The article will discuss the methods that have been employed for soft tissue modification around implants traditionally and compare these to use of the diode laser. RESUMEN Los láseres de diodo se han vuelto más ampliamente utilizados en la práctica clínica dental y han demostrado ser un método seguro y eficaz para el tratamiento en y alrededor de los implantes que requieren modificación de los tejidos blandos, ya sea para exponer el implante para la fase de restauración o para cambiar la forma de los márgenes gingivales por estética. El artículo discutirá los métodos que se han empleado para la modificación de los tejidos blandos alrededor de los implantes de forma tradicional y los comparará con el uso del láser de diodo.
  • ... This approach consists in the direct application of a nonionizing light, usually delivered via a low-power coherent light (laser) or non-coherent light sources such as filtered lamps or light-emitting diode (LED), typically emitting in the 600-1000 nm spectrum range (red to near infrared), with an energy density <100 J/cm 2 . Given the low-energy density and wavelengths used in this therapy, light is able to easily enter the tissues in a non-destructive and nonthermal mode resulting in the biomodulation of various cellular processes [23][24][25][26][27]. ...
    ... Cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) assay (Promega Corp., Madison, WI), essentially as previously reported [25]. Briefly, the cells were cultured in 96-well plates (6 Â 10 3 cells/well) in DMEM supplemented with FBS 10% (control) and, after 24 hours, were transferred in low serum (FBS 2%) phenol red-free culture medium in the presence of 2 ng/ml human TGF-b1, diode laser irradiated for 10 seconds as indicated in Table 1 (treated surface diameter: 18 mm) or not, and cultured for 24 hours. ...
    Article
    Background and objective: Low-level laser therapy (LLLT) or photobiomodulation therapy is emerging as a promising new therapeutic option for fibrosis in different damaged and/or diseased organs. However, the anti-fibrotic potential of this treatment needs to be elucidated and the cellular and molecular targets of the laser clarified. Here, we investigated the effects of a low intensity 635 ± 5 nm diode laser irradiation on fibroblast-myofibroblast transition, a key event in the onset of fibrosis, and elucidated some of the underlying molecular mechanisms. Materials and methods: NIH/3T3 fibroblasts were cultured in a low serum medium in the presence of transforming growth factor (TGF)-β1 and irradiated with a 635 ± 5 nm diode laser (continuous wave, 89 mW, 0.3 J/cm(2) ). Fibroblast-myofibroblast differentiation was assayed by morphological, biochemical, and electrophysiological approaches. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 and of Tissue inhibitor of MMPs, namely TIMP-1 and TIMP-2, after laser exposure was also evaluated by confocal immunofluorescence analyses. Moreover, the effect of the diode laser on transient receptor potential canonical channel (TRPC) 1/stretch-activated channel (SAC) expression and activity and on TGF-β1/Smad3 signaling was investigated. Results: Diode laser treatment inhibited TGF-β1-induced fibroblast-myofibroblast transition as judged by reduction of stress fibers formation, α-smooth muscle actin (sma) and type-1 collagen expression and by changes in electrophysiological properties such as resting membrane potential, cell capacitance and inwardly rectifying K(+) currents. In addition, the irradiation up-regulated the expression of MMP-2 and MMP-9 and downregulated that of TIMP-1 and TIMP-2 in TGF-β1-treated cells. This laser effect was shown to involve TRPC1/SAC channel functionality. Finally, diode laser stimulation and TRPC1 functionality negatively affected fibroblast-myofibroblast transition by interfering with TGF-β1 signaling, namely reducing the expression of Smad3, the TGF-β1 downstream signaling molecule. Conclusion: Low intensity irradiation with 635 ± 5 nm diode laser inhibited TGF-β1/Smad3-mediated fibroblast-myofibroblast transition and this effect involved the modulation of TRPC1 ion channels. These data contribute to support the potential anti-fibrotic effect of LLLT and may offer further informations for considering this therapy as a promising therapeutic tool for the treatment of tissue fibrosis.
  • ... A wide variety of methods and techniques was used in the experiments to assess cell proliferation. With respect to the intervals of analysis of cell proliferation, the minimum time point used was 0 h [1,8,9,25,26,28] after irradiation and the maximum was 4 weeks after irradiation [4]. ...
    ... Soares et al. [25] Human periodontal ligament 0, 24, 48, and 72 h after irradiation The dose of 1.0 J/cm 2 promoted a significant increase in cell proliferation at 48 and 72 h when compared to the control and the group that received a dose of 0.5 J/cm 2 Giannelli et al. [26] Murine bone marrow 0, 24, 48, and 72 h after irradiation Cell proliferation was significantly higher at 72 h after irradiation compared to the control group Pereira et al. [27] Healthy and inflamed human dental pulp 24, 48, 72, and 96 h after irradiation ...
    Article
    Full-text available
    Low-level laser therapy (LLLT) has been used in several in vitro experiments in order to stimulate cell proliferation. Cells such as fibroblasts, keratinocytes, lymphocytes, and osteoblasts have shown increased proliferation when submitted to laser irradiation, although little is known about the effects of LLLT on stem cells. This study aims to assess, through a systematic literature review, the effects of LLLT on the in vitro proliferation of mesenchymal stem cells. Using six different terms, we conducted an electronic search in PubMed/Medline database for articles published in the last twelve years. From 463 references obtained, only 19 papers met the search criteria and were included in this review. The analysis of the papers showed a concentration of experiments using LLLT on stem cells derived from bone marrow, dental pulp, periodontal ligament, and adipose tissue. Several protocols were used to irradiate the cells, with variations on wavelength, power density, radiation time, and state of light polarization. Most studies demonstrated an increase in the proliferation rate of the irradiated cells. It can be concluded that the laser therapy positively influences the in vitro proliferation of stem cells studied, being necessary to carry out further experiments on other cell types and to uniform the methodological designs.
  • ... A previous study demonstrated that 90% of BMSCs undergo apoptosis in the first 3 days after injection; most become trapped in the lungs [22] because the pulmonary capillary diameter is smaller than the diameter of a stem cell [23]. However, in vivo fluorescence experiments have shown that MSCs can arrive at a fracture site via the lung 3 days after injection [15,24]. In a chemotactic chamber assay to examine the in vitro chemotactic effect of SDF-1 on mouse BMSCs, stem cells with a 30-μm diameter could pass through an aperture with a diameter of 8 μm [25]. ...
    ... At day 14 post-fracture, mice that received BMSCs on day 7 demonstrated marked differences in terms of the size and morphological features of their new mineralized calluses [24] compared to mice injected on day 1 post-fracture (Fig. 2). The injection of BMSCs on day 7 post-fracture resulted in an increased BMD and bone volume fraction. ...
    Article
    Full-text available
    Background Bone marrow is an important source of stem cells, which can promote bone fracture healing. Methods We investigated the optimal time to inject bone marrow mesenchymal stem cells (BMSCs) in a C57 murine unilateral, transverse, femur fracture model. BMSCs transfected with red fluorescent protein (RFP-BMSCs) were injected via the tail vein on day 1, 7, or 14 post-fracture. AMD3100 (inhibitor of stromal cell-derived factor 1 [SDF-1]) was also injected before RFP-BMSCs in one group for comparison; a control group received saline injections. RFP-BMSC migration and fracture healing were evaluated by in vivo fluorescence assay. Micro-CT was performed and mechanical testing and histological analysis. Chemokine levels were evaluated by quantitative real-time PCR and western blotting. Results Following injection on day 7 post-fracture, RFP-BMSCs more frequently homed to the fracture site and remained for a longer duration. Bone volume and bone mineral density were increased when BMSCs were injected on day 7 post-fracture (P < 0.05). The mechanical properties of fractured femurs were improved following day-7 BMSC injection. Histology confirmed that BMSC injection improved the formation of new bones. Conclusions Chemokines that induce BMSC migration were highly expressed, and protein levels of osteogenesis-related factors were increased. Seven days after fracture may be the optimal time for injection of BMSCs to promote fracture healing. Additionally, the SDF-1/CXCR4 pathway may play an important role in fracture healing following BMSC injection.
  • ... This approach consists in the direct application of a nonionizing light, usually delivered via a low-power coherent light (laser) or non-coherent light sources such as filtered lamps or light-emitting diode (LED), typically emitting in the 600-1000 nm spectrum range (red to near infrared), with an energy density <100 J/cm 2 . Given the low-energy density and wavelengths used in this therapy, light is able to easily enter the tissues in a non-destructive and nonthermal mode resulting in the biomodulation of various cellular processes [23][24][25][26][27]. ...
    ... Cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) assay (Promega Corp., Madison, WI), essentially as previously reported [25]. Briefly, the cells were cultured in 96-well plates (6 Â 10 3 cells/well) in DMEM supplemented with FBS 10% (control) and, after 24 hours, were transferred in low serum (FBS 2%) phenol red-free culture medium in the presence of 2 ng/ml human TGF-b1, diode laser irradiated for 10 seconds as indicated in Table 1 (treated surface diameter: 18 mm) or not, and cultured for 24 hours. ...
    Article
    Fibrosis consists in a excessive and persistent formation of fibrous connective tissue that occurs frequently in different organs or tissues after an injury. The cells principally involved in the onset and progression of fibrosis, are the activated form of the fibroblasts, namely myofibroblasts. Although required for the wound healing and the reparative response to organ/tissue damage, myofibroblast persistence contributes to the increased synthesis and deposition of extracellular matrix proteins, which replace the necrotic or damaged tissue with a scar. In such perspective, identification of treatments capable of preventing myofibroblast generation and defining their molecular targets appears a key step for the design of therapeutic strategies aimed at counteracting fibrosis. On these bases, in the present study, by a morphological, biochemical and electrophysiological approach, we evaluated the effects of a diode laser treatment (635 nm) on the differentiation of NIH3T3 fibroblasts into myofibroblasts. We found that the laser stimulation was able to inhibit TGF-β1-induced fibroblast-myofibroblast transition and modify the myofibroblast resting membrane potential and inwardly rectifying K+ currents. We also found that the treatment up-regulated metalloprotease (MMP)-2 and -9 expression and downregulated the tissue inhibitors of metalloproteinases (TIMP)1 and -2 in TGF-β1-treated cells. Interestingly, the effects of the laser on fibroblasts involved the Transient Receptor Potential Channel 1 (TRPC-1) functionality. In conclusion, the present study besides offering novel experimental evidence on the mechanisms of action of the diode laser, may provide a promising therapeutic perspective for the treatment of tissue fibrosis extending the potential clinical application of the low level laser therapy.
  • ... Ballini et al. presented that LLLT stimulates the proliferation of dental pulp SCs and raises the expression of important proteins for bone formation in culture (Ballini et al., 2015). Giannelli et al. proposed that LLLT with a diode laser might be an effective attitude for the preconditioning of MSCs in culture before cell transplantation (Giannelli et al., 2013). Leonida et al. concluded that LLLT might lead to a reduction in healing times and potentially reduces the risks of failure (Leonida et al., 2013). ...
    ... Other in vitro studies have shown that LLLT can enhance the viability and proliferation rate of healthy, and in particular, osteoporotic autologous BMMSCs, which could be very useful in regenerative medicine (Fallahnezhad et al., 2016): LED irradiation downregulates osteoclastogenesis by reducing ROS production (Sohn et al., 2015), and LLLT enhances the differentiation of BMMSCs into osteoblasts and increases their proliferation (Soleimani et al., 2012). So, the application of LLLT and diode laser could be effective methods for the preconditioning of MSCs in culture prior cell transplantation (Fallahnezhad et al., 2016;Giannelli et al., 2013;Soleimani et al., 2012;Wang et al., 2012;Li et al., 2010). The United States Food and Drug Administration approved the use of LIPUS for accelerating conservatively-managed fresh fracture healing in 1994 and for treatment of established nonunions in 2000 (Rubin et al., 2001). ...
    Article
    To compare the in vitro effectiveness of Low-Level Laser Therapy (LLLT) and Low Intensity Pulsed Ultrasound (LIPUS) on bony cells and related stem cells. In this study, we aim to systematically review the published scientific literature which explores the use of LLLT and LIPUS to biostimulate the activity or the proliferation of bony cells or stem cells in vitro. We searched the database PubMed for LLLT or LIPUS, with/without bone, osteoblast, osteocyte, stem cells, the human osteosarcoma cell line (MG63), bone-forming cells, and cell culture (or in vitro). These studies were subdivided into categories exploring the effect of LLLT or LIPUS on bony cells, stem cells, and other related cells. 75 articles were found between 1987 and 2016; these included: 50 full paper articles on LLLT and 25 full papers on LIPUS. These articles met the eligibility criteria and were included in our review. A detailed and concise description of the LLLT and the LIPUS protocols and their individual effects on bony cells or stem cells and their results are presented in five tables. Based on the main results and the conclusions of the reviewed articles in the current work, both, LLLT and LIPUS, apply a biostimulatory effect on osteoblasts, osteocytes, and enhance osteoblast proliferation and differentiation on different bony cell lines used in in vitro studies, and therefore, these may be useful tools for bone regeneration therapy. Moreover, in consideration of future cell therapy protocols, both, LLLT and LIPUS (especially LLLT), enhnce a significant increase in the initial number of SCs before differentiation, thus increasing the number of differentiated cells for tissue engineering, regenerative medicine, and healing. Further studies are necessary to determine the LLLT or the LIPUS parameters, which are optimal for biostimsulating bony cells and SCs for bone healing and regenerative medicine.
  • ... In general, FIR radiation transfers energy to the human body and manifests a wide variety of biological effects, including improvement of ischemic lesions 9,10 . Although the biological activities of FIR in the preconditioning of BMSCs by in vitro manipulation are not fully understood, our findings corroborate previous studies of wavelength in the red range [11][12][13][14][15][16][17] . For example, red (630 nm) and near infrared (850 nm) light-emitting diodes enhance the migration of MSCs derived from rat bone marrow [11][12][13] . ...
    ... Although the biological activities of FIR in the preconditioning of BMSCs by in vitro manipulation are not fully understood, our findings corroborate previous studies of wavelength in the red range [11][12][13][14][15][16][17] . For example, red (630 nm) and near infrared (850 nm) light-emitting diodes enhance the migration of MSCs derived from rat bone marrow [11][12][13] . Other studies have demonstrated that a diode (Ga-As) laser of wavelength 804 nm promots the proliferation of BMSCs and cardiac stem cells [14][15][16] . ...
    Article
    Full-text available
    Far-infrared radiation (FIR) has been shown to exert positive effects on the cardiovascular system. However, the biological effects of FIR on bone marrow-derived stem cells (BMSCs) are not understood. In the present study, BMSCs were isolated from rat femur bone marrow and cultured in vitro. To investigate the effects of an FIR generator with an energy flux of 0.13 mW/cm² on rat BMSCs, survival of BMSCs was measured by crystal violet staining, and cell proliferation was additionally measured using Ez-Cytox cell viability, EdU, and Brd U assays. FIR preconditioning was found to significantly increase BMSC proliferation and survival against H2O2. The scratch and transwell migration assays showed that FIR preconditioning resulted in an increase in BMSC migration. qRT-PCR and Western blot analyses demonstrated that FIR upregulated Nanog, Sox2, c-Kit, Nkx2.5, and CXCR4 at both the mRNA and protein levels. Consistent with these observations, PD98059 (an ERK inhibitor) and AMD3100 (a CXCR4 inhibitor) prevented the activation of CXCR4/ERK and blocked the cell proliferation and migration induced by FIR. Overall, these findings provide the first evidence that FIR confers a real and significant benefit on the preconditioning of BMSCs, and might lead to novel strategies for improving BMSC therapy for cardiac ischemia.
  • ... Many reports have shown that LPT can increase cell survival and proliferation in vitro, in particular on mesenchymal stem cells. [61][62][63] LPT photomodulatory effects are reported to be dependent on the delivery of reactive oxygen species (ROS). ROS, in turn, are capable of activating signaling molecules, such as growth factors, which elicit many of the microscopic and macroscopic effects observed after LPT. 15 Besides ROS production, LPT also promotes a change in the electrical potential of the cell membrane, triggering the sodium (Na 11 ) and potassium (K 11 ) pumps, which leads to the increased synthesis of adenosine triphosphate (ATP). ...
    Treatments for dentine hypersensitivity (DH) may produce positive effects, though do not have lasting results. We investigated the reparative potential of stem cells derived from deciduous teeth (SHEDs) in response to components delivered from substances used in the treatment of the DH, associated or not to laser phototherapy (LPT), to stimulate dentine formation. SHEDs were submitted to substances delivered from a laboratorial P-rich bioactive glass [57SiO2 -26CaO-17P2 O5 (wt %)] or a commercially available desensitizer (Gluma® Desensitizer), associated (or not) to LPT (InGAlP diode laser, 660 nm, 0.028 cm(2) , 20 mW, 5 J/cm(2) , 7 s, contact mode). Biomaterial characterization was performed by X-ray diffraction, scanning electron microscopy and the particle size was evaluated by dynamic light scattering. SHEDs proliferation and differentiation were analyzed by MTT and Alizarin Red staining, respectively. The conditioned media used in these tests were evaluated regarding their pH and the ionic concentration changes due to ions leached from the bioactive glass (BG). BG majority presented a non-crystalline solid structure and mixed particle sizes characterized by the agglomeration of nanoparticles. Cultures treated with BG alone or in association to LPT showed improved cell growth in relation to Gluma(®) (p < 0.05). Gluma(®) was cytotoxic in all tested conditions, regardless irradiated or not. BG associated to LPT induced intense mineral matrix formation. In conclusion, BG releases ionic dissolution products able to promote SHEDs differentiation. BG associated to LPT improves SHEDs proliferation and differentiation in vitro, and may be a promise therapeutic approach for the DH treatment. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015.
  • ... 14,15 Other authors report that biostimulation via the diode laser also has a positive affect on the bone cells and can be stimulatory to the bone cells at the crest around the implant. 16,17 When compared to conventional methods tissue healing as well as postoperative sensitivity was less with the diode laser than with other methods. 18 ...
  • ... 18 Accordingly, these barriers could be easier bypassed by using PBM therapy, which is able to optimize the environmental conditions to the newly implanted cells, such as increasing blood supply, preventing cell death, inducting cell proliferation and migration, and inducing growth factor synthesis or its activation. [19][20][21][22][23][24][25][26][27] In light of future cell therapy protocols, PBM therapy would allow significant increase of the primary number of stem cells before differentiation, thus increasing the number of viable differentiated cells for tissue engineering and the regenerative process. Also, it has already been demonstrated that PBM therapy is able to recruit stem cells from the host itself to collaborate in the regenerative process. ...
    Article
    Objective: This study aimed to conduct a systematic review of the literature published from 2000 to August 2015, to investigate the effect of photobiomodulation (PBM) therapy on dentoalveolar-derived mesenchymal stem cells (ddMSCs), assessing whether a clear conclusion can be reached from the data presented. Background data: Systematic reviews provide the best evidence on the effectiveness of a procedure and permit investigation of factors that may influence the performance of a method. To the best of our knowledge, no previous systematic review has evaluated the effects of PBM only on ddMSCs. Methods: The search was conducted in PubMed /MEDLINE(®), Scopus and Web of Science databases, and reported according to the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA Statement). Original research articles investigating the effects of PBM therapy on ddMSCs, published from 2000 to August 2015, were retrieved and used for this review according to the following eligibility criteria: evaluating PBM therapy, assessing stem cells of dentoalveolar origin, published in English, dealing with cells characterized as stem cells, and using light that did not need external chromophores. Results: From the initial 3467 potentially relevant articles identified, 6 were excluded because they were duplicates, and 3453 were considered ineligible based on the inclusion criteria. Therefore, eight articles remained, and these were fully analyzed in order to closely check exclusion criteria items. Only one of them was excluded because the cultured cells studied were not characterized as stem cells. Finally, seven articles served as the basis for this systematic review. Conclusions: PBM therapy has no deleterious effects on ddMSCs. Although no other clear conclusion was obtained because of the scarce number of publications, the results of these studies are pointing to an important tendency of PBM therapy to improve ddMSCs' viability and proliferation.
  • ... Исследование М. Гианелли и соавт. [34] на МСККМ не было включено в графический анализ. Из статьи непонятно, какому значению плотности мощности и времени экспозиции соответствует заявленная прибавка скорости пролиферации (в табл. 1 данные параметры помечены знаком «?»). ...
  • ... Low pulse energy neodymium: yttrium-aluminum-garnet (Nd:YAG) laser light increases intracellular Ca 2+ concentration in osteoblasts through the activation of TRPC1 ion channels (38). Membrane conductance through voltage-gated K + , BK, and Kir channels and T-and L-type Ca 2+ channels is increased following red laser exposure at low fluence and power (39). Also, infrared lasers at 810 nm raise mitochondrial membrane potential and reduce intracellular Ca 2+ concentration (40). ...
    Article
    Full-text available
    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4 endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4 endonuclease V. Low-intensity lasers: i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells, ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, and iv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.
  • ... Since LLLT has been scientifically proven as a beneficial therapeutic modality for numerous diseases and diseased conditions, it was applied to enhance MSCs proliferation and differentiation. The recent 3-year reports regarding LLLT application to increase MSCs proliferative and differentiation potential were summarized in Table 1 [126][127][128][129][130][131][132][133][134][135][138][139][140][141][142][143]. Abrahamse's group published some literatures for LLLT application to stem cells. ...
    Article
    Full-text available
    Low reactive level laser therapy (LLLT) is mainly focused on the activation of intracellular or extracellular chromophore and the initiation of cellular signaling by using low power lasers. Over the past forty years, it was realized that the laser therapy had the potential to improve wound healing and reduce pain and inflammation. In recent years, the term LLLT has become widely recognized in the field of regenerative medicine. In this review, we will describe the mechanisms of action of LLLT at a cellular level and introduce the application to mesenchymal stem cells and mesenchymal stromal cells (MSCs) therapies. Finally, our recent research results that LLLT enhanced the MSCs differentiation to osteoblast will also be described.
  • ... It was also revealed that using LLL not only activated Kir channels and stimulate Notch-1 signaling pathways but also coordinate upstream and downstream signaling of cell cycle regulators and induce mitogenic effects in the laser-irradiated cells (Table 1). 54 Mesenchymal Stem Cells Derived From Dental Pulp Dental pulp stem cells have fibroblastic morphology and self-renewal and are able to differentiate to different types of cells. 55 Encapsulating the pulp stem cells and loading rhbmp4 in them and then low-power laser irradiation significantly increased osteogenic and odontogenic differentiation. ...
    Article
    Full-text available
    Introduction: The purpose of this study is to investigate the effect of a low-power laser on the proliferation, migration, differentiation of different types of mesenchymal stem cells (MSCs) in different studies. Methods: The relevant articles that were published from 2004 to 2019 were collected from the sources of PubMed, Scopus, and only the articles specifically examining the effect of a lowpower laser on the proliferation, differentiation, and migration of the MSCs were investigated. Results: After reviewing the literature, only 42 articles were found relevant. Generally, most of the studies demonstrated that different laser parameters increased the proliferation, migration, and differentiation of the MSCs, except the results of two studies which were contradictory. In fact, changing the parameters of a low-power laser would affect the results. On the other hand, the source of the stem cells was reported as a key factor. In addition, the combination of lasers with other therapeutic approaches was found to be more effective. Conclusion: The different parameters of lasers has been found to be effective in the proliferation, differentiation, and migration of the MSCs and in general, a low-power laser has a positive effect on the MSCs, helping to improve different disease models.
  • ... This opens new modalities of therapeutics by adopting light therapies (PBM). Furthermore, Smith et al. [9] supported that the infrared irradiation activates ion channels at the plasma membrane transuding signals into biological responses, initiating the cascade of metabolic events, and affecting cytoplasmic membrane permeability and Na + -K + flux and Ca +2 concentrations [28]. Besides, Cerdeira et al. [29] have already shown that LLLT on human neutrophils (PMN) caused a superior production of reactive oxygen species (ROS), such as hydroxyl radicals (HO•) and hypochlorite anions (ClO−), leading finally to a higher functional profile. ...
    Article
    This study aimed to investigate the potential of low-level laser irradiation (LLLI) to promote odontogenic differentiation and biomineralization by dental pulp stem cells (DPSCs) seeded inside bioceramic scaffolds. Mg-based, Zn-doped bioceramic scaffolds, synthesized by the sol-gel technique, were spotted with DPSCs and exposed to LLLI at 660 nm with maximum output power of 140 mw at fluencies (a) 2 and 4 J/cm(2) to evaluate cell viability/proliferation by the MTT assay and (b) 4 J/cm(2) to evaluate cell differentiation, using real-time PCR (expression of odontogenic markers) and a p-nitrophenylphosphate (pNPP)-based assay for alkaline phosphatase (ALP) activity measurement. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis were used for structural/chemical characterization of the regenerated tissues. Exposure of the DPSCs/scaffold complexes to the proposed LLLI scheme was associated with statistically significant increase of odontogenesis-related markers (bone morphogenetic protein 2 (BMP-2): 22.4-fold, dentin sialophosphoprotein (DSPP): 28.4-fold, Osterix: 18.5-fold, and Runt-related transcription factor 2 (Runx2): 3.4-fold). ALP activity was significantly increased at 3 and 7 days inside the irradiated compared to that in the non-irradiated SC/DPSC complexes, but gradually decreased until 14 days. Newly formed Ca-P tissue was formed on the SC/DPSC complexes after 28 days of culture that attained the characteristics of bioapatite. Overall, LLLI treatment proved to be beneficial for odontogenic differentiation and biomineralization of DPSCs inside the bioceramic scaffolds, making this therapeutic modality promising for targeted dentin engineering.
  • ... In most studies, the visible spectrum (600-700nm) was effective for a cellular response (proliferation or differentiation) of stem cells [19,[72][73][74][75]. De Villiers et al., found an increase in cellular viability and proliferation on human adipose-derived MSCs (hADSCs) using a diode laser [76] (Table 1). Giannelli M et al., reported that the proliferation of mouse MSC increased after irradiation with the 635 nm diode laser [77]. Mvula et al., suggested that the proliferation of ADSCs significantly increased after exposure to a diode laser at 636 nm wavelength (5 J/cm 2 ) [78]. ...
    Background Stem cells have attracted the researchers increasing interest, due to their applications in regenerative medicine. Their self-renewal, capacity for multipotent differentiation, and immunomodulatory properties make them unique to significantly contribute in tissue repair and regeneration applications. Recently, cells have shown increased proliferation when irradiated with low level laser therapy or Photobiomodulation therapy (PBMT), which induces the activation of intracellular and extracellular chromophores and the initiation of cellular signaling. The purpose of this study was to evaluate this phenomenon in the literature. Method The literature investigated the articles written in English in four electronic data bases of PubMed, Scopus, Google scholar and Cochrane up to April 2019. Stem cell was searched by combining the search keyword of "low level laser therapy" OR "low power laser therapy" OR "low intensity laser therapy" OR "photobiomodulation therapy" OR "photo biostimulation therapy" OR "LED". In total, 46 articles had eligibility to be evaluated. Results Studies demonstrated that red to near-infrared light is absorbed by the mitochondrial respiratory chain. Mitochondria are the significant sources of reactive oxygen species (ROS). Mitochondria play an important role in metabolism, energy generation, and are also involved in mediating the effects induced by PBMT. PBMT may result in the increased production of (ROS), nitric oxide (NO), adenosine triphosphate (ATP), and cyclic adenosine monophosphate (cAMP). These changes in turn initiate cell proliferation and induce the signal cascade effect. Conclusion The findings of this review suggest that PBMT -based regenerative medicine could be a useful tool for future advances in tissue engineering and cell therapy.
  • ... In stationary growth phase, the low-intensity red and infrared lasers did not modify the surface areas of wild-type E. coli AB1157, JW1625-1 and JW2146-1 cells (Table 7). Some authors have reported that low-intensity lasers alter the function of ion channels in the plasmatic membrane (27,28) and in the mitochondrial membrane (29). The results of our morphological analyses can be explained by the effects of the low-intensity lasers on such membrane ion channels. ...
    Article
    Full-text available
    Semiconductor laser devices are readily available and practical radiation sources providing wavelength tenability and high monochromaticity. Low-intensity red and near-infrared lasers are considered safe for use in clinical applications. However, adverse effects can occur via free radical generation, and the biological effects of these lasers from unusually high fluences or high doses have not yet been evaluated. Here, we evaluated the survival, filamentation induction and morphology of Escherichia coli cells deficient in repair of oxidative DNA lesions when exposed to low-intensity red and infrared lasers at unusually high fluences. Cultures of wild-type (AB1157), endonuclease III-deficient (JW1625-1), and endonuclease IV-deficient (JW2146-1) E. coli, in exponential and stationary growth phases, were exposed to red and infrared lasers (0, 250, 500, and 1000 J/cm2) to evaluate their survival rates, filamentation phenotype induction and cell morphologies. The results showed that low-intensity red and infrared lasers at high fluences are lethal, induce a filamentation phenotype, and alter the morphology of the E. coli cells. Low-intensity red and infrared lasers have potential to induce adverse effects on cells, whether used at unusually high fluences, or at high doses. Hence, there is a need to reinforce the importance of accurate dosimetry in therapeutic protocols.
  • ... These findings suggest that biological ef- fects of laser irradiation on cells depend on not only on the wavelength and irradiation dose, but also many other things. The diversity of manifestations of the effect of laser irradiation on cells can result from the involvement of various molecular mechanisms [2,7,8,18], which deserve special study. This apparently explains contradictory conclusions of published reports [15]. ...
    Article
    We studied the effect of low-level laser irradiation on proliferative activity of cultured human Wharton's jelly mesenchymal stromal sells. Cells were irradiated with a solid-state laser emitting at 650 nm; irradiation doses were 0.04, 0.4, or 4 J/cm2. Laser irradiation was performed once at the start of the cell proliferation experiment or daily throughout the experiment. Cells were cultured for 7 days. The number of viable cells was assessed using the MTT test. An increase in cell proliferative activity was detected after daily laser irradiations; the maximum stimulating effect was achieved at a dose of 0.04 J/cm2. These results substantiate medical use of lasers for expansion of cells intended for transplantation.
  • ... Studies on the effects of PBM on cells and tissues have been showing the ability of PBM in increasing survival, proliferation, migration and finally cell differentiation depending on the parameters used ( Arany et al., 2007Arany et al., , 2014Choi et al., 2013;Eduardo et al., 2008;Giannelli et al., 2013;Moreira et al., 2017). In this sense, recent studies have pointed out to the possibility of this therapy be applied in tissue engineering, improving stem cells responses to promote tissue regeneration. ...
    Article
    Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering. This article is protected by copyright. All rights reserved
  • ... Area and perimeter are increased in endonuclease deficient E. coli cells exposed to laser. Low intensity red laser was demonstrated to alter slow potassium currents [32] and membrane conductance through K + and Ca 2+ channels is increased after red laser exposure [33]. In addition, infrared laser (810 nm) raises mitochondrial membrane potential and reduces intracellular calcium concentrations [34]. ...
    Article
    Full-text available
    Clinical applications of low intensity lasers are based on the biostimulation effect and considered to occur mainly at cells under stressful conditions. Also, although the cytochrome is a chromophore to red and near infrared radiations, there are doubts whether indirect effects of these radiations could occur on the DNA molecule by oxidative mechanisms. Thus, this work evaluated the survival, filamentation and morphology of Escherichia coli cultures proficient and deficient in oxidative DNA damage repair exposed to low intensity red laser under stress conditions. Wild type and endonuclease III deficient E. coli cells were exposed to laser (658 nm, 1 and 8 J cm−2) under hyposmotic stress and bacterial survival, filamentation and cell morphology were evaluated. Laser exposure: (i) does not alter the bacterial survival in 0.9% NaCl, but increases the survival of wild type and decreases the survival of endonuclease III deficient cells under hyposmotic stress; (ii) increases filamentation in 0.9% NaCl but decreases in wild type and increases in endonuclease III deficient cells under hyposmotic stress; (iii) decreases the area and perimeter of wild type, does not alter these parameters in endonuclease III deficient cells under hyposmotic stress but increases the area of these in 0.9% NaCl. Low intensity red laser exposure has different effects on survival, filamentation phenotype and morphology of wild type and endonuclease III deficient cells under hyposmotic stress. Thus, our results suggest that therapies based on low intensity red lasers could take into account physiologic conditions and genetic characteristics of cells.
  • ... This assumption has been supported by our previous results which have shown that laser therapy has positive effects on the protozoa and mammalian mitochondria activities (Amaroli et al., 2015b(Amaroli et al., ,c, 2016b and stimulates the cell proliferation (Amaroli et al., 2015a) without inducing cellular damage (Amaroli et al., 2017). Furthermore, the literature has supported the fact that MSCs proliferation can be induced after laser irradiation (Abramovitch et al., 2005;Hou et al., 2008;Soleimani et al., 2012;Wu et al., 2012;Giannelli et al., 2013). In addition, it has been demonstrated that elevated levels of TGF-β1 play an important role in downregulating the release of the proinflammatory cytokine such as IL-6 and IL-17, thus promoting favorable conditions for bone regeneration (Zhou et al., 2008). ...
    Article
    Full-text available
    The literature has supported the concept of mesenchymal stromal cells (MSCs) in bone regeneration as one of the most important applications in oro-maxillofacial reconstructions. However, the fate of the transplanted cells and their effects on the clinical outcome is still uncertain. Photobiomodulation (PBM) plays an important role in the acceleration of tissue regeneration and potential repair. The aim of this in vitro study is to evaluate the effectiveness of PBM with 808 nm diode laser therapy, using a flat-top hand-piece delivery system at a higher-fluence (64 J/cm2) irradiation (1 W, continuous-wave) on bone marrow stromal cells (BMSCs). The BMSCs of 3 old female Balb-c mice were analyzed. The cells were divided into two groups: irradiated group and control group. In the former the cells were irradiated every 24 h during 0 day (T0), 5 (T1), 10 (T2), and 15 (T3) days, whereas the control group was non-irradiated. The results have shown that the 64 J/cm2 laser irradiation has increased the Runt-related transcription factor 2 (Runx2). Runx2 is the most important early marker of osteoblast differentiation. The higher-fluence suppressed the synthesis of adipogenic transcription factor (PPARγ), the pivotal transcription factor in adipogenic differentiation. Also, the osteogenic markers such as Osterix (Osx) and alkaline phosphatase (ALP) were upregulated with an increase in the matrix mineralization. Furthermore, western blotting data demonstrated that the laser therapy has induced a statistically valid increase in the synthesis of transforming growth factor β1 (TGF-β1) but had no effects on the tumor necrosis factor α (TNFα) production. The data has statistically validated the down-regulation of the important pro-inflammatory cytokines such as interleukin IL-6, and IL-17 after 808 nm PBM exposition. An increase in anti-inflammatory cytokines such as IL-1rα and IL-10 was observed. These in vitro studies provide for first time the initial proof that the PBM of the 808 nm diode laser therapy with flat-top hand-piece delivery system at a higher-fluence irradiation of 64 J/cm2 (1 W/cm2) can modulate BMSCs differentiation in enhancing osteogenesis.
  • ... Studies have shown that preconditioning by exposure to reduced levels of oxygen, incubation with nitric oxide, hydrogen peroxide or diazoxide, and treatment with pharmacological drugs, including phosphodiesterase inhibitors, angiotensin II receptor blocker and neuropeptide Y, may greatly enhance the therapeutic promise of BM-MSCs [44]. Moreover, it has been recently shown by our group that the treatment with platelet-derived rich plasma (PRP) [139], as well as irradiation with low level lasers [140], represent promising preconditioning approaches for stimulating BM-MSC proliferation, encompassing senescence during cell expansion and influencing stemness gene expression. Of interest, the genetic manipulation of MSC to overexpress cytokines and growth factors, such as HGF, VEGF and SDF-1, have also been proposed to improve neo-angiogenesis and the endogenous mechanisms of tissue repair/regeneration [44, 141,142]. ...
    Article
    Full-text available
    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) represent the leading candidate cell in tissue engineering and regenerative medicine. These cells can be easily isolated, expanded in vitro and are capable of providing significant functional benefits after implantation in the damaged muscle tissues. Despite their plasticity, the participation of BM-MSCs to new muscle fiber formation is controversial; in fact, emerging evidence indicates that their therapeutic effects occur without signs of long-term tissue engraftment and involve the paracrine secretion of cytokines and growth factors with multiple effects on the injured tissue, including modulation of inflammation and immune reaction, positive extracellular matrix (ECM) remodeling, angiogenesis and protection from apoptosis. Recently, a new role for BM-MSCs in the stimulation of muscle progenitor cells proliferation has been demonstrated, suggesting the potential ability of these cells to influence the fate of local stem cells and augment the endogenous mechanisms of repair/regeneration in the damaged tissues.
  • ... Another study that uses diode laser to photo activate stem cells. In the present study the effects of 635 nm diode laser on mouse MSC proliferation (20) 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 signifi-cantly enhanced after laser irradiation, as judged by time lapse video microscopy and Edu incorporation. ...
  • ... Previously, researchers demonstrated that low-level laser irradiation could stimulate the proliferation of many different types of cultured cells, including stem cells [33]. For example, laser irradiation at 635 nm and 0.5 J/cm 2 significantly stimulated the proliferation of bone marrow derived MSCs from rats and mice [34,35]. The laser energy density seemed a critical factor to stimulate the MSCs. ...
    Article
    Full-text available
    A method was developed to pattern the surface of perfluorinated materials with graphene oxide thin film, and various biological applications of the patterned perfluorinated surface were illustrated. Perfluorinated surfaces such as Teflon, Cytop, and other perfluorinated materials are known to be both hydrophobic and oleophobic, with low adhesion for most materials. Modifying the perfluorinated surfaces has been difficult due to the extraordinary chemical inertness, which limits the applications of perfluorinated materials as anti-fouling substrates. Herein we successfully patterned Cytop surfaces with graphene oxide. Patterns of the graphene oxide thin film with feature dimension down to 40 microns were formed and remained stable on the Cytop surface against washing with water, ethanol and acetone. The graphene oxide thin film on the Cytop surface allowed non-specific protein adsorption. To illustrate the applications of the patterned Cytop surface, we used the patterned Cytop surface as the substrate to study the protein-protein interactions, stem cell culture, and stem cell proliferation.
  • ... In general, in vitro studies evaluating the effect of LLLI on stem cells demonstrated that LLLI increased the proliferation of stem cells [24][25][26]. Laser irradiation at the power and energy densities used in the current study has not been shown to induce any adverse effects on cells in culture. The results of the current study clearly demonstrated that LLLI (0.1 and 0.2 J cm −2 ) significantly enhanced the growth of DPSCs. ...
    Article
    Full-text available
    This study assessed the effect of low-level laser irradiation (LLLI) on the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were exposed to 810 nm laser light (0.1, 0.2, or 0.3 J cm−2) for 7 d (60 s daily). The negative control group (cells in regular medium) and positive control group (cells in osteogenic medium (OM)) were not lased. One group of cells in OM was irradiated with laser operated at 0.2 J cm−2. Cell viability was evaluated at 24 h and one week after the last day of laser irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteogenic differentiation was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and alizarin Red S staining. Cell proliferation was not affected by laser irradiation at 24 h except in one group (cells in OM exposed to laser at 0.2 J cm−2). However, one week after the last day of laser irradiation, it was significantly increased in groups exposed to laser at 0.1 or 0.2 J cm−2 and decreased in groups containing OM (P < 0.05). Osteoblast marker expression was observed in groups containing OM. LLLI at 0.2 J cm−2 dramatically enhanced cell differentiation. Laser at 0.3 J cm−2 increased bone sialoprotein (BSP) and decreased alkaline phosphatase (ALP). Mineralized nodules were only observed in groups containing OM. Considering these findings, LLLI may be used as a novel approach for preconditioning of DPSCs in vitro prior to bone tissue engineering.
  • ... A number of studies, on various cell types, have shown positive effects of photoirradiation. Studies have been conducted on stem cells [35][36][37][38], keratinocytes [39,40], mast cells [41,42], fibroblasts [43][44][45][46][47][48], smooth muscle cells [49], osteoblasts [50,51], and schwann cells [52] to name but a few. ...
    Article
    Full-text available
    Impaired wound healing is a common complication associated with diabetes with complex pathophysiological underlying mechanisms and often necessitates amputation. With the advancement in laser technology, irradiation of these wounds with low-intensity laser irradiation (LILI) or phototherapy, has shown a vast improvement in wound healing. At the correct laser parameters, LILI has shown to increase migration, viability, and proliferation of diabetic cells in vitro; there is a stimulatory effect on the mitochondria with a resulting increase in adenosine triphosphate (ATP). In addition, LILI also has an anti-inflammatory and protective effect on these cells. In light of the ever present threat of diabetic foot ulcers, infection, and amputation, new improved therapies and the fortification of wound healing research deserves better prioritization. In this review we look at the complications associated with diabetic wound healing and the effect of laser irradiation both in vitro and in vivo in diabetic wound healing.
  • ... of LLLI in MSCs [7][8][9]16], in which the irradiation has been carried out under normal conditions. To our knowledge, the cytoprotective role of LLLI against DOX has not been evaluated in MSCs. ...
    Article
    Full-text available
    Cancer is a leading cause of death worldwide, and doxorubicin (DOX) has become one of the most commonly prescribed drugs. Stem cell (SC) therapy is proving to be a promising strategy to alleviate DOX adverse effects on non-cancerous cells. However, the drug also has a toxic action on SCs, reducing the efficiency of cell therapy from a preventive view. The present study shows that the DOX toxicity in mesenchymal SCs (MSCs) can be partially overcome by low-level laser irradiation (LLLI). To achieve this, we applied the low-level red laser (wavelength: 660 nm; output power: 30 mW; laser beam: 0.028 cm ² ; irradiation: 1.07 mW/cm ² ; Ga-Al-As Photon Laser III, DMC, São Paulo, Brazil) in rat adipose tissue-derived MSCs before their exposure to different DOX concentrations. Results revealed that the DOX reduced the viability and adenosine triphosphate level of MSCs. These findings were followed by significantly increased apoptosis as well as oxidative stress in the MSCs. Interestingly, LLLI at the dose of 0.2 J alleviated the effects of DOX on cell viability and apoptosis, and inhibited oxidative stress in the MSCs. In summary, this study provides a crucial step toward the future application of LLLI as a protective approach against DOX-induced toxicity in MSCs, particularly cell death. This study also lays the groundwork for further investigation into the role of oxidative stress and inflammation as an instructive milieu for cell protection.
  • ... These mechanisms include increasing the level of ATP [17], inducing nitric oxide synthesis, suppressing inflammatory cytokines, and elevating expression levels of some growth factors, including PDGF, IGF-1, NGF, and FGFs [18]. Following this cellular effects, proliferation and differentiation were affected after laser irradiation [19,20]. On the other hand, it is well shown that long-term treatment with PBM lead to mobilization, migration, and homing of other tissue resident stem cells into damaged areas [21]. ...
    Article
    Pathophysiologic conditions associated with diabetes mellitus affect mesenchymal stem cells (MSCs), and this phenomenon may lead to some diabetic secondary complications. The present study was conducted to evaluate the impact of photobiomodulation (PBM) on rat diabetic MSC (DMSC) behavior in vitro. For the purpose of PBM, we used helium-neon laser with a wavelength of 632.8 nm at three different energy densities (0.5, 1, 2 J/cm²) and radiation periodicity of once, twice, and thrice. The survival, proliferation, and apoptosis in the normal MSCs (NMSCs), DMSCs, and diabetic MSCs, which were laser irradiated (DMSCs+L), were assessed using MTT assay, Ki67 immunofluorescence staining, and TUNEL assay, respectively. Our results demonstrated that DMSCs have significantly lower survival (P < 0.05) and proliferation rates (P < 0.001), and dramatically higher population doubling time (PDT, P < 0.001) and apoptosis rates (P < 0.001) as compared to NMSCs. Moreover, PBM with energy density of 1 J/cm² and the periodicity of 1 or 2 times could improve diabetic MSC capabilities in the term of survival, proliferation, and apoptosis. Considering these findings, it is suggested that PBM could improve the ability of diabetic MSCs in vitro prior to transplantation or may rise their capabilities in their native niche in vivo.
  • ... 12 On the other hand, a low-level red laser modifies slow potassium currents, 21 and the ion flux through K + and Ca 2+ channels is high following irradiation with a red laser. 22 Moreover, an infrared laser increases transmembrane potential in mitochondria and decreases Ca +2 levels into cells. 23 Alterations of area and perimeter could be related to modifications in the membrane ion channels of the E. coli cells. ...
    Article
    Introduction: Low-level lasers are successfully used to prevent and treat diseases in soft oral and bone tissues, particularly diseases in oral cavity caused by chemotherapy and radiotherapy in oncology. However, controversy exists as to whether these lasers induce molecular side effects, mainly on DNA. The aim of this work was to assess the effects of low-power lasers on mutant Escherichia coli cells in DNA repair. Methods: Escherichia coli wild type cultures as well as those lacking recombination DNA repair (recA- ) and la SOS responses (lexA- ) irradiated with lasers at different energy densities, powers, and emission modes for cell viability and morphology assessment were used in this study. Results: Laser irradiation: (i) did not affect cell viability of non-mutant and lexA- cells but decreased viability in recA- cultures; (ii) altered morphology of wild type and lexA, depending on the energy density, power, emission mode, and wavelength. Conclusion: Results show that low-level lasers have lethal effects on both recombination DNA repair and SOS response bacterial cells but do not induce morphological modifications in these cells.
  • ... Low pulse energy neodymium: yttrium-aluminum-garnet (Nd:YAG) laser light increases intracellular Ca 2+ concentration in osteoblasts through the activation of TRPC1 ion channels (38). Membrane conductance through voltage-gated K + , BK, and Kir channels and T-and L-type Ca 2+ channels is increased following red laser exposure at low fluence and power (39). Also, infrared lasers at 810 nm raise mitochondrial membrane potential and reduce intracellular Ca 2+ concentration (40). ...
    Article
    Full-text available
    Biostimulative effect of low intensity laser in tissues has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases. The aim of this work was to evaluate effects of laser exposure on the survival of Escherichia coli cultures and plasmid topological forms. Escherichia coli cultures and plasmids were exposed to infrared laser to study bacterial survival and electrophoretic profile, respectively. Data indicate low intensity infrared laser: (i) had no effect on E. coli wild type, endonuclease IV, exonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cultures, but decreased the survival of E. coli UvrA protein deficient cultures; (ii) there was no alteration in the electrophoretic profile of plasmids. Exposure to low intensity infrared laser decreases survival of Escherichia coli cultures deficient in nucleotide excision repair of DNA and this effect could depend on fluences, wavelength and tissues conditions.
  • Article
    Objective: The aim of this article is to review experimental studies of visible and infrared light irradiation of human and animal stem cells (SCs) in vitro and in vivo to assess photobiomodulation effects on their proliferation and differentiation. Background data: The clinical application of light irradiation remains controversial, primarily because of the complexity of the rational choice of irradiation parameters. In laboratories, the theoretical justification underlying the choice of irradiation parameters also remains a challenge. Methods: A systematic review was completed of original research articles that investigated the effects of light irradiation on human and animal SCs in vitro and in vivo (to June 2014). Relevant articles were sourced from PubMed and MEDLINE(®). The search terms were laser (light) therapy (irradiation), stem cells, and phototherapy, stem cells. Results: The analysis revealed the importance of cell type when choosing the cell irradiation parameters. The influence of wavelength on the SC proliferation rate seemed to be nonsignificant. The high values of increased proliferation or differentiation were obtained using high power density, low energy density, and short exposure time. SC exposure to light without inducers did not lead to their differentiation. The maximum differentiation was achieved using irradiation parameters different from the ones needed to achieve the maximum proliferation of the same cells. Conclusions: Increased power density and reduced energy density were needed to increase the SC response. Based on the analysis, we have presented a graph of the cell response to generalized photostimulus, and introduced the concepts of "photostress" and "photoshock" to describe the stages of this response.
  • Article
    The aim of this study was to evaluate the effects of low-level laser therapy (LLLT) on the bone repair of critical size defects (CSDs) filled with autogenous bone in the calvaria of immunosuppressed rats. A 5 mm-diameter CSD was created in the calvaria of 30 rats. The animals were divided into 5 groups (n=6): Control (C) – the defect was filled with a blood clot; Dexamethasone (D) – dexamethasone treatment, and the defect was filled with a blood clot; Autogenous bone (AB) – dexamethasone treatment, and the defect was filled with autogenous bone; LLLT – dexamethasone treatment, and the defect received LLLT (660 nm; 35 mW; 24.7 J/cm2); and AB+LLLT – dexamethasone treatment, and the defect was filled with autogenous bone and received LLLT. All animals were euthanized at 30 postoperative days. Histometric and histological analyses were performed. The new bone area (NBA) was calculated as the percentage of the total area of the original defect. Data were analysed statistically (an analysis of variance and Tukey’s test; P < 0.05). The AB+LLLT group showed the largest NBA of all groups (P < 0.05). The use of LLLT with AB effectively stimulated bone formation in CSDs in the calvaria of immunosuppressed rats.
  • Article
    Dichromatic and consecutive laser radiations have attracted increased attention for clinical applications as offering new tools for the treatment of dysfunctional tissues in situations where monochromatic radiation is not effective. This work evaluated the survival, filamentation and morphology of Escherichia coli cells, and the induction of DNA lesions, in plasmid DNA exposed to low-intensity consecutive dichromatic laser radiation. Exponential and stationary wild type and formamidopyrimidine DNA glycosylase/MutM protein deficient E. coli cultures were exposed to consecutive low-intensity dichromatic laser radiation (infrared laser immediately after red laser) to study the survival, filamentation and morphology of bacterial cells. Plasmid DNA samples were exposed to dichromatic radiation to study DNA lesions by electrophoretic profile. Dichromatic laser radiation affects the survival, filamentation and morphology of E. coli cultures depending on the growth phase and the functional repair mechanism of oxidizing lesions in DNA, but does not induce single/double strands breaks or alkali-labile DNA lesions. Results show that low-intensity consecutive dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation, suggesting that other therapeutic effects could be obtained using dichromatic radiation.
  • Article
    Objective: The purpose of this article was to review the molecular mechanisms of low-level laser irradiation (LLLI) preconditioning for heart cell therapy. Background data: Stem cell transplantation appears to offer a better alternative to cardiac regenerative therapy. Previous studies have confirmed that the application of LLLI plays a positive role in regulating stem cell proliferation and in remodeling the hostile milieu of infarcted myocardium. Greater understanding of LLLI's underlying mechanisms would be helpful in translating cell transplantation therapy into the clinic. Methods: Studies investigating LLLI preconditioning for cardiac regenerative therapy published up to 2015 were retrieved from library sources and Pubmed databases. Results: LLLI preconditioning stimulates proliferation and differentiation of stem cells through activation of cell proliferation signaling pathways and alteration of microRNA expression. It also could stimulate paracrine secretion of stem cells and alter cardiac cytokine expression in infarcted myocardium. Conclusions: LLLI preconditioning provides a promising approach to maximize the efficacy of cardiac cell-based therapy. Although many studies have reported possible molecular mechanisms involved in LLLI preconditioning, the exact mechanisms are still not clearly understood.
  • Article
    Physical characteristics and practical properties have made lasers of interest for biomedical applications. Effects of low-level lasers on biological tissues could occur or be measurable depending on cell type, presence of a pathologic process or whether the cells are in an adverse environment. The objective of this work was to evaluate the survival, morphology and filamentation of E. coli cells proficient and deficient in the repair of oxidative DNA lesions exposed low-level red and infrared lasers submitted to hyperosmotic stress. Wild type and endonuclease VIII deficient E. coli cells in exponential and stationary growth phase were exposed to red and infrared lasers and submitted to hyperosmotic stress. Cell viability, filamentation phenotype and cell morphology were evaluated. Cell viability was not significantly altered but previous laser exposure induced filamentation and an altered area of stressed cells depending on physiologic condition and presence of the DNA repair. Results suggest that previous exposure to low-level red and infrared lasers could not affect viability but induced morphologic changes in cells submitted to hyperosmotic stress depending on physiologic conditions and repair of oxidative DNA lesions.
  • Article
    Objective: The purpose of this study was to review available literature about the effect of photobiomodulation (PBM) on mesenchymal stem cells (MSCs). Background data: The effects of coherent and noncoherent light sources such as low-level lasers and light-emitting diodes (LEDs) on cells and tissues, known as PBM, form the basis of photomedicine. This treatment technique effects cell function, proliferation, and migration, and plays an important role in tissue regeneration. Stem cells have been found to be helpful elements in tissue regeneration, and the combination of stem cell therapy and laser therapy appears to positively affect treatment results. Materials and methods: An electronic search in PubMed was conducted of publications from the previous 12 years. English language articles related to the subject were found using selected key words. The full texts of potentially suitable articles were assessed according to inclusion and exclusion criteria. Results: After evaluation, 30 articles were deemed relevant according to the inclusion criteria. The energy density of the laser was 0.7-9 J/cm(2). The power used for visible light was 30-110 mW and that used for infrared light was 50-800 mW. Nearly all studies showed that low-level laser therapy had a positive effect on cell proliferation. Similar outcomes were found for LED; however, some studies suggest that the laser alone is not effective, and should be used as an adjunct tool. Conclusions: PBM has positive effects on MSCs. This review concluded that doses of 0.7-4 J/cm(2) and wavelengths of 600-700 nm are appropriate for light therapy. The results were dependent upon different parameters; therefore, optimization of parameters used in light therapy to obtain favorable results is required to provide more accurate comparison.
  • Article
    Objective: To histomorphometrically analyze the effect of low-level laser therapy (LLLT) on bone formation process in surgically created critical-size defects (CSDs) treated with bovine bone graft (BBG) and its influence over particles' resorption of BBG. Methods: A 10-mm diameter CSD was surgically created in the calvaria of 64 male rats, which were distributed into 4 experimental groups: the C group (control), only blood clot; the LLLT group, LLLT (GaAlAs, 660nm) and blood clot; the BBG group, CSD filled with BBG; the BBG/LLLT group, LLLT and CSD filled with BBG. Animals were euthanized at either 30 or 60days post-operation. A histological analysis was performed. Additionally, the percentage of newly formed bone area (NFBA) and remaining particles areas (RPA) of BBG were histometrically evaluated and data statistically analyzed. Results: The LLLT (5.82±2.05; 7.34±1.01) group presented significantly greater NFBA when compared to the C group (1.61±0.30; 5.59±0.94) at 30 and 60days post-operation (p<0.05). The BBG/LLLT group (7.39±1.45; 9.44±2.36) presented significantly greater NFBA than the BBG group (3.85±1.56; 8.02±0.63) at 30 and 60days postoperation (p<0.05). There was no significant difference in the mean percentage of implanted material RPA between the BBG and the BBG/LLLT groups. Conclusions: LLLT can improve bone formation process in CSD filled or not with BBG in rat calvaria, but it is not able to accelerate particles resorption of this material in the interior of bone defect.
  • Chapter
    Periodontal disease is a current public health problem compromising heavily patients’ quality of life. Scientific evidence on treatment of periodontal disease is associated with a high degree of variability in reported outcomes and potential efficacy. Thus, regeneration of periodontal tissues through safe and efficient treatment protocols remains an important challenge. Moreover, conventional therapies result habitually in repair rather than regeneration of deteriorated tissues. Recent development in tissue engineering and regenerative medicine has paved the path for new treatment approaches. Mesenchymal stem cells are an outstanding candidate for tissue regeneration. They are the key element of combined tissue engineering therapies for periodontal regeneration. Related scientific evidence has highlighted exciting potential therapeutic utilization accompanied by numerous limitations and future challenges. The present chapter has been assembled considering these aspects and with the hope of providing investigators with solid bases for the state of art and potential future directions of periodontal regenerative therapies.
  • Chapter
    Multipotent mesenchymal stromal cells (commonly referred as Mesenchymal stem cells; MSCs) are the most frequently used cell population in tissue engineering because of its multilineage potential, multiple sources and ability to self-renew [1].
  • Article
    Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors’ fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.
  • Article
    Tissue engineering aims to take advantage of the ability of undifferentiated stem cells to differentiate into multiple cell types to repair damaged tissue. Photobiomodulation uses either lasers or light-emitting diodes to promote stem cell proliferation and differentiation. The present study aimed to investigate single and dual combinations of laser wavelengths on mesenchymal stem cells (MSCs). MSCs were derived from rabbit iliac bone marrow. One control and eight laser irradiated groups were designated as Infrared (IR, 810 nm), Red (R, 660 nm), Green (G, 532 nm), Blue (B, 485 nm), IR–R, IR–B, R–G, and B–G. Irradiation was repeated daily for 21 days and cell proliferation, osseous, or cartilaginous differentiation was then measured. RT-PCR biomarkers were SOX9, aggrecan, COL 2, and COL 10 expression for cartilage and ALP, COL 1, and osteocalcin expression for bone. Cellular proliferation was increased in all irradiated groups except G. All cartilage markers were significantly increased by IR and IR–B except COL 10 which was suppressed by IR–B combination. ALP expression was highest in R and IR groups during osseous differentiation. ALP was decreased by combinations of IR with B and with R, and also by G alone. R and B–G groups showed stimulated COL 1 expression; however, COL 1 was suppressed in IR–B, IR–R, and G groups. IR significantly increased osteocalcin expression, but in B, B–G, and G groups it was reduced. Cartilage differentiation was stimulated by IR and IR–B laser irradiation. The effects of single or combined laser irradiation were not clear-cut on osseous differentiation. Stimulatory effects on osteogenesis were seen for R and IR lasers, while G laser had inhibitory effects.
  • Article
    Full-text available
    In the originally published article, the name of the 3rd and 4th authors were labeled incorrectly. The correct names are Mohammadreza Baghaban Eslaminejad and Leila Taghiyar. Also, affiliation 4 has been corrected.
  • Article
    Since the 1960’s, basic and clinical research has been conducted regarding the biostimulative effects of photobiomodulation (PBM) by means of low-reactive level laser therapy (LLLT). A number of studies have used low-level diode lasers in near-infrared to infrared wavelengths because of their high degree of tissue penetration. In the dental field, it has been shown that LLLT enhances proliferation, migration, and differentiation of gingival fibroblasts, osteoblasts, periodontal ligament cells, as well as mesenchymal stem cells in vitro. Furthermore, LLLT promoted wound healing and bone formation in vivo. Currently, less invasive and safer procedures are desired for dental treatment, especially for regenerative therapy. It is expected that PBM by means of LLLT will contribute to attaining that goal. Consequently, we have reviewed basic and clinical studies using the diode laser for LLLT, and we discuss the potential of LLLT in promoting periodontal tissue healing and regeneration.
  • Thesis
    Full-text available
    Regenerative medicine is a promising growing area worldwide, with the aim of restore and regenerate tissues and whole organs through the use of cells, proliferation factors and biomaterials. One branch of regenerative medicine is cell therapy, that uses stem cells, aiming at the substitution of functionally or structurally damaged tissues, presenting therapeutic fature. LASERs and LEDs are available as therapeutic tools, showing biostimulating ability. The photo-stimulation technique uses light to activate molecules and cellular functions, presenting potential to affect proliferation, cell differentiation and metabolism, stimulating oxidative phosphorylation and reducing the local inflammatory response. Data shows the importance of selecting an ideal wavelength, such as the use of an inappropriate choice, can lead to undisered results, such as bioinhibition. In the present work, we evaluated the action of LED 850nm, pulsatile, at the doses of 3, 5 and 10J/cm² in mesenchymal stem cells (CTM) with Bovine Fetal Serum (FBS) and with derived platelets – Hormones (HDP) and B-cell lymphoblastic cell culture, 10J /cm2, RAJI cells. In all light exposure experiments, wavelength of 850 nm inhibited cell proliferation. CTM culture, LED had a low proliferation rate, resulting in a decrease in cellular confluence, especially at 5 and 10J/cm2. Lymphoblastic lymphoma type B cells, in only one week of exposure presente the same behavior of bioinhibition at 10J/cm2. The control group had 7.0 x 105 cells on per vial, while cells subjected to irradiation underwent the unfolding time at a concentration of 4.2 x 105 on average per vial.
  • Article
    Full-text available
    Osteoarthritis (OA) resulting from injury or disease is associated with increased levels of several matrix metalloproteinases (MMPs), which degrade all components of the complex extracellular matrix in the cartilage. The objective of this study is to investigate the effect of low-level laser therapy (LLLT) on papain-induced joint damage in rats by histopathology and analysis of metalloproteinase 2 and 9 production. Sixty male Wistar rats were randomly distributed into four groups of 15 animals: (1) non-injury negative control, (2) injury positive control, (3) treated with LLLT at 50 mW, and (4) treated with LLLT at 100 mW. OA was induced in animals using papain (4 % solution) followed by treatment with LLLT. After 7, 14, and 21 days, the animals were euthanized. The articular lavage was collected and centrifuged; then, the supernatant was stored prior to protein analysis by western blot. The material was stained with hematoxylin and eosin for histopathological analysis, and Picrosirius Red was used to estimate the percentage of collagen fibers. To determine normal distribution, ANOVA and Tukey's post hoc test were used for comparison between and within each group at each time period. All data are expressed as mean and standard deviation values, with the null hypothesis considered as p < 0.05. Both laser groups (50 and 100 mW) were effective in tissue repair, decreasing collagen type III expression and increasing type I expression in all experimental periods; however, LLLT at 50 mW reduced metalloproteinase 9 more than at 100 mW in 21 days. LLLT at 50 mW was more efficient in the modulation of matrix MMPs and tissue repair.
  • HES and HERP families: Multiple effectors of the Notch signaling pathway
    • T Iso
    • L Kedes
    • Y Hamamori
    Iso T, Kedes L, Hamamori Y. 2003. HES and HERP families: Multiple effectors of the Notch signaling pathway. J Cell Physiol 194:237–255.
  • Low pulse energy Nd:YAG laser irradiation exerts a biostimulative effect on different cells of the oral microenvironment: ''An in vitro study
    • F Chellini
    • C Sassoli
    • D Nosi
    • C Deledda
    • P Tonally
    • S Zecchi-Orlandini
    • L Formigli
    • M Giannelli
    Chellini F, Sassoli C, Nosi D, Deledda C, Tonally P, Zecchi-Orlandini S, Formigli L, Giannelli M. 2010. Low pulse energy Nd:YAG laser irradiation exerts a biostimulative effect on different cells of the oral microenvironment: ''An in vitro study.'' Lasers Surg Med 42:527-539.
  • Stem cell proliferation under low intensity laser irradiation: A preliminary study
    • Eduardo Fde
    • P Bueno
    • D F De Freitas
    • P M Marques
    • M M Passos-Bueno
    • Eduardo Cde
    • P Zatz
    Eduardo Fde P, Bueno DF, de Freitas PM, Marques MM, Passos-Bueno MR, Eduardo Cde P, Zatz M. 2008. Stem cell proliferation under low intensity laser irradiation: A preliminary study. Lasers Surg Med 40:433-438.
  • Article
    Full-text available
    Both Notch signaling and Akt-mTOR signaling pathway are involved in glioma cell proliferation and survival. Previous studies have shown that Notch-1 is overexpressed in many glioma cell lines and primary human gliomas. Blocking of Notch signaling pathway can induces glioma cell apoptosis and growth suppression. However, the underlying molecular mechanism is not clear. We report that activation of the Notch pathway by intracellular domain of human Notch- I (NIC-1) strongly activates Akt and promotes U251 glioma cell proliferation. Knockdown of Notch-1 by RNA interference suppresses Akt activation, reduces glioma cell growth rate and induce cell apoptosis. Following Notch-1 suppression, phosphorylated Akt and its downstream effector mTOR were reduced. Knockdown of Notch-1 also involves down-regulation of anti-apoptotic protein MCL-1, in parallel with activation of apoptotic associate proteins PARP, caspase-9 and caspase-3. Our data demonstrate that Notch-1 can positively regulate Akt-mTOR pathways, which is associated with glioma cell proliferation and apoptosis. This also suggests a molecular mechanism for the inhibitory effect of Notch-1 RNA interference on glioma cell proliferation through Akt-mTOR signaling pathway.
  • Article
    Human mesenchymal stem cells (hMSC) have gained considerable interest due to their potential use for cell replacement therapy and tissue engineering. One strategy is to differentiate these bone marrow stem cells in vitro into cardiomyocytes prior to implantation. In this context ion channels can be important functional markers of cardiac differentiation. At present there is little information about the electrophysiological behaviour of the undifferentiated hMSC. We therefore investigated mRNA expression of 26 ion channel subunits using semiquantitative RT-PCR and recorded transmembrane ion currents with the whole-cell voltage clamp technique. Bone marrow hMSC were obtained from healthy donors. The cells revealed a distinct pattern of ion channel mRNA with high expression levels for some channel subunits (e.g. Kv4.2, Kv4.3, MaxiK, HCN2, and alpha1C of the L-type calcium channel). Outward currents were recorded in almost all cells. The most abundant outward current rapidly activated at potentials positive to +20 mV. This current was identified as a large-conductance voltage- and Ca(2+)-activated K(+) current, conducted by MaxiK channels, due to its high sensitivity to tetraethylammonium (IC(50)= 340 microm) and its inhibition by 100 nm iberiotoxin. A large fraction of cells also demonstrated a more slowly activating current at potentials positive to -30 mV. This current was selectively inhibited by clofilium (IC(50)= 0.8 microm). Ba(2+) inward currents, stimulated by 1 microm BayK 8644 were found in a few cells, indicating the expression of functional L-type Ca(2+) channels. Other inward currents such as sodium currents or inward rectifier currents were absent. We conclude that undifferentiated hMSC express a distinct pattern of ion channel mRNA and functional ion channels that might contribute to physiological cell function.
  • Article
    Full-text available
    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.
  • Article
    Background and Objectives Bone marrow derived mesenchymal stem cells (BMSCs) have shown to be an appealing source for cell therapy and tissue engineering. Previous studies have confirmed that the application of low-level laser irradiation (LLLI) could affect the cellular process. However, little is known about the effects of LLLI on BMSCs. The aim of this study was designed to investigate the influence of LLLI at different energy densities on BMSCs proliferation, secretion and myogenic differentiation.Study Design/Materials and MethodsBMSCs were harvested from rat fresh bone marrow and exposed to a 635 nm diode laser (60 mW; 0, 0.5, 1.0, 2.0, or 5.0 J/cm2). The lactate dehydrogenase (LDH) release was used to assess the cytotoxicity of LLLI at different energy densities. Cell proliferation was evaluated by using 3-(4, 5-dimethylithiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and 5-bromo-2′-deoxyuridine (BrdU) assay. Production of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA). Myogenic differentiation, induced by 5-azacytidine (5-aza), was assessed by using immunocytochemical staining for the expression of sarcomeric α-actin and desmin.ResultsCytotoxicity assay showed no significant difference between the non-irradiated group and irradiated groups. LLLI significantly stimulated BMSCs proliferation and 0.5 J/cm2 was found to be an optimal energy density. VEGF and NGF were identified and LLLI at 5.0 J/cm2 significantly stimulated the secretion. After 5-aza induction, myogenic differentiation was observed in all groups and LLLI at 5.0 J/cm2 dramatically facilitated the differentiation.ConclusionsLLLI stimulates proliferation, increases growth factors secretion and facilitates myogenic differentiation of BMSCs. Therefore, LLLI may provide a novel approach for the preconditioning of BMSCs in vitro prior to transplantation. Lasers Surg. Med. 40:726–733, 2008. © 2008 Wiley-Liss, Inc.
  • Article
    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 Ca²⁺ 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.
  • Article
    The bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells (MSCs), with pluripotent differentiation capacity, present an ideal source for cell transplantation or tissue engineering therapies, but exact understanding of regulating mechanism underling MSC proliferation and differentiation remains a critical issue in securing their safe and efficient clinical application. This review outlines current knowledge regarding MSC cell surface biomarkers and molecular mechanisms of MSC differentiation and proliferation with emphasis on Wnt/β-catenin signaling, Notch signaling pathway, bone morphogenesis proteins and various growth factors functioning in regulation of differentiation and proliferation of MSCs. Possible relation of oncogene and immunosuppressive activities of MSCs with tumorigenicity or tumor generation is also addressed for safe translational clinical application. Fast increase of MSC knowledge and techniques has led to some successful clinical trials and helped devising new tissue engineering therapies for bone and cartilage diseases that severely afflict human health. Production of adult MSC-derived functional neurons can further extend their therapeutic application in nerve injury and neurodegenerative diseases. It is promising that MSCs shall overcome ethical and immunorejection problems appeared in human embryonic stem cells, and specific molecular targeting manipulation may result in practical MSC therapy for personalized treatment of various diseases in the regeneration medicine.
  • Article
    Full-text available
    The Notch receptor is part of a core signalling pathway which is highly conserved in all metazoan species. It is required for various cell fate decisions at multiple stages of development and in the adult organism, with dysregulation of the pathway associated with genetic and acquired diseases including cancer. Although cellular and in vivo studies have provided considerable insight into the downstream consequences of Notch signalling, relatively little is known about the molecular basis of the receptor/ligand interaction and initial stages of activation. Recent advances in structure determination of the extracellular regions of human Notch-1 and one of its ligands Jagged-1 have given new insights into docking events occurring at the cell surface which may facilitate the development of new highly specific therapies. We review the structural data available for receptor and ligands and identify the challenges ahead.
  • Article
    Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm(2) energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm(2). The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm(2). No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm(2), but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm(2) promoted the most significant biostimulatory effects on cultured keratinocytes.
  • Article
    Significant progress in the molecular investigation of endogenous bioelectric signals during pattern formation in growing tissues has been enabled by recently developed techniques. Ion flows and voltage gradients produced by ion channels and pumps are key regulators of cell proliferation, migration, and differentiation. Now, instructive roles for bioelectrical gradients in embryogenesis, regeneration, and neoplasm are being revealed through the use of fluorescent voltage reporters and functional experiments using well-characterized channel mutants. Transmembrane voltage gradients (V(mem) ) determine anatomical polarity and function as master regulators during appendage regeneration and embryonic left-right patterning. A state-of-the-art recent study reveals that they can also serve as prepatterns for gene expression domains during craniofacial patterning. Continued development of novel tools and better ways to think about physical controls of cell-cell interactions will lead to mastery of the morphogenetic information stored in physiological networks. This will enable fundamental advances in basic understanding of growth and form, as well as transformative biomedical applications in regenerative medicine.
  • Article
    Endostatin (ES) is a c-terminal proteolytic fragment of collagen XVIII with promising antitumour properties in several tumour models, including human glioblastoma. We hypothesized that this peptide could interact with plasma membrane ion channels and modulate their functions. Using cell proliferation and migration assays, patch clamp and Western blot analysis, we studied the effects of ES on the proliferation and migration of human glioblastoma U87 cells, mediated by T-type Ca²⁺ channels. Extracellular application of ES reversibly inhibited T-type Ca²⁺ channel currents (T-currents) in U87 cells, whereas L-type Ca²⁺ currents were not affected. This inhibitory effect was associated with a hyperpolarizing shift in the voltage-dependence of inactivation but was independent of G-protein and protein tyrosine kinase-mediated pathways. All three α₁ subunits of T-type Ca²⁺ channels (Ca(V) 3), α(1G) (Ca(V) 3.1), α(1H) (Ca(V) 3.2) and α(1I) (Ca(V) 3.3), were endogenously expressed in U87 cells. Using transfected HEK293 or CHO cells, we showed that only Ca(V) 3.1 and Ca(V) 3.2, but not Ca(V) 3.3 or Ca(V) 1.2 (L-type), channel currents were significantly inhibited. More interestingly, ES inhibited the proliferation and migration of U87 cells in a dose-dependent manner. Pretreatment of the cells with the specific T-type Ca²⁺ channel blocker mibefradil occluded these inhibitory effects of ES. This study provides the first evidence that the antitumour effects of ES on glioblastoma cells is through direct inhibition of T-type Ca²⁺ channels and gives new insights into the future development of a new class of antiglioblastoma agents that target the proliferation and migration of these cells.
  • Article
    Full-text available
    Notch signaling plays a pivotal role in the regulation of many fundamental cellular processes, such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. At the molecular level, ligand binding induces the proteolytic cleavage of the Notch receptor. The intracellular domain of Notch translocates subsequently into the nucleus, associates with the central transcription factor RBP-J and activates transcription. Although, this pathway is remarkably short, with no second messenger involved, it regulates expression of more than hundred target genes in a tissue-specific manner. This review summarizes recent studies on transcriptional and chromatin control mechanisms, which set the stage for specific expression of Notch target genes. Furthermore, we review how the canonical (RBP-J dependent) Notch pathway is fine-tuned by downstream effectors and feedback loops in mammals.
  • Article
    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. 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. 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. 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.
  • Article
    Regenerative medicine is an emerging interdisciplinary field of research that uses several technological approaches including stem cells to repair tissues. Mesenchymal stem cells (MSCs), a type of adult stem cell, have generated a great amount of interest over the past decade in this field. Numerous studies have explored the role of MSCs in tissue repair and modulation of allogeneic immune responses. The mechanisms through which MSCs exert their therapeutic potential rely on some key properties of the cells as follows: the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, hepatocytes, endothelial, and neuronal cells; the ability to secrete multiple bioactive molecules capable of stimulating the recovery of injured cells and inhibiting inflammation; the lack of immunogenicity; and the ability to perform immunomodulatory functions. In the present review, we focus on these three aspects upon which the therapeutic effects of MSCs are mainly based. Furthermore, some pathological conditions under which the application of MSCs should be done with caution are also mentioned.
  • Article
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    Among the various types of cell-to-cell signaling, paracrine signaling comprises those signals that are transmitted over short distances between different cell types. In the human body, secreted growth factors and cytokines instruct, among others, proliferation, differentiation, and migration. In the hematopoietic stem cell (HSC) niche, stromal cells provide instructive cues to stem cells via paracrine signaling and one of these cell types, known to secrete a broad panel of growth factors and cytokines, is mesenchymal stromal cells (MSCs). The factors secreted by MSCs have trophic, immunomodulatory, antiapoptotic, and proangiogenic properties, and their paracrine profile varies according to their initial activation by various stimuli. MSCs are currently studied as treatment for inflammatory diseases such as graft-versus-host disease and Crohn's disease, but also as treatment for myocardial infarct and solid organ transplantation. In addition, MSCs are investigated for their use in tissue engineering applications, in which their differentiation plays an important role, but as we have recently demonstrated, their trophic factors may also be involved. Furthermore, a functional improvement of MSCs might be obtained after preconditioning or tailoring the cells themselves. Also, the way the cells are clinically administered may be specialized for specific therapeutic scenarios. In this review we will first discuss the HSC niche, in which MSCs were recently identified and are thought to play an instructive and supportive role. We will then evaluate therapeutic applications that currently try to utilize the trophic and/or immunomodulatory properties of MSCs, and we will also discuss new options to enhance their therapeutic effects.
  • Article
    The Notch signaling pathway regulates metazoan development, in part, by directly controlling the transcription of target genes. For a given cellular context, however, only subsets of the known target genes are transcribed when the pathway is activated. Thus, there are context-dependent mechanisms that selectively maintain repression of target gene transcription when the Notch pathway is activated. This review focuses on molecular mechanisms that have been recently reported to mediate selective repression of Notch pathway target gene transcription. These mechanisms are essential for generating the complex spatial and temporal expression patterns of Notch target genes during development.
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    TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba2+ blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba2+ at the assumed K+ binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba2+ was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K+-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K+ and proton.
  • Article
    Increased proliferation after low-level laser irradiation (LLLI) has been well demonstrated in many cell types including mesenchymal stem cells (MSCs), but the exact molecular mechanisms involved remain poorly understood. The aim of this study was to investigate the change in mRNA expression in rat MSCs after LLLI and to reveal the associated molecular mechanisms. MSCs were exposed to a diode laser (635 nm) as the irradiated group. Cells undergoing the same procedure without LLLI served as the control group. Proliferation was evaluated using the MTS assay. Differences in the gene expression profiles between irradiated and control MSCs at 4 days after LLLI were analyzed using a cDNA microarray. Gene ontology and pathway analysis were used to find the key regulating genes followed by real-time PCR to validate seven representative genes from the microarray assays. This procedure identified 119 differentially expressed genes. Real-time PCR confirmed that the expression levels of v-akt murine thymoma viral oncogene homolog 1 (Akt1), the cyclin D1 gene (Ccnd1) and the phosphatidylinositol 3-kinase, catalytic alpha polypeptide gene (Pik3ca) were upregulated after LLLI, whereas those of protein tyrosine phosphatase non-receptor type 6 (Ptpn6) and serine/threonine kinase 17b (Stk17b) were downregulated. cDNA microarray analysis revealed that after LLLI the expression levels of various genes involved in cell proliferation, apoptosis and the cell cycle were affected. Five genes, including Akt1, Ptpn6, Stk17b, Ccnd1 and Pik3ca, were confirmed and the PI3K/Akt/mTOR/eIF4E pathway was identified as possibly playing an important role in mediating the effects of LLLI on the proliferation of MSCs.
  • Article
    The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies.
  • Article
    Phototherapy with low intensity laser irradiation has shown to be effective in promoting the proliferation of different cells. The aim of this in vitro study was to evaluate the potential effect of laser phototherapy (660 nm) on human dental pulp stem cell (hDPSC) proliferation. The hDPSC cell strain was used. Cells cultured under nutritional deficit (10% FBS) were either irradiated or not (control) using two different power settings (20 mW/6 seconds to 40 mW/3 seconds), with an InGaAIP diode laser. The cell growth was indirectly assessed by measuring the cell mitochondrial activity through the MTT reduction-based cytotoxicity assay. The group irradiated with the 20 mW setting presented significantly higher MTT activity at 72 hours than the other two groups (negative control--10% FBS--and lased 40 mW with 3 seconds exposure time). After 24 hours of the first irradiation, cultures grown under nutritional deficit (10% FBS) and irradiated presented significantly higher viable cells than the non-irradiated cultures grown under the same nutritional conditions. Under the conditions of this study it was possible to conclude that the cell strain hDPSC responds positively to laser phototherapy by improving the cell growth when cultured under nutritional deficit conditions. Thus, the association of laser phototherapy and hDPSC cells could be of importance for future tissue engineering and regenerative medicine. Moreover, it opens the possibility of using laser phototherapy for improving the cell growth of other types of stem cells.
  • Article
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    Because of their immunomodulatory and engraftment-promoting properties, mesenchymal stromal cells (MSCs) have been tested in the clinical setting both to facilitate haematopoietic recovery and to treat steroid-resistant acute GVHD. More recently, experimental findings and clinical trials have focused on the ability of MSCs to home to damaged tissue and to produce paracrine factors with anti-inflammatory properties, resulting in functional recovery of the damaged tissue. The mechanisms through which MSCs exert their therapeutic potential rely on some key properties of the cells: the ability to secrete soluble factors capable of stimulating survival and recovery of injured cells; the capacity to home to sites of damage and the ability to blunt exaggerated immune responses. These fundamental properties are being tested within a novel therapeutic field defined as Regenerative Medicine. This review deals with recent research on the anti-inflammatory/reparative properties of MSCs and considers the possible mechanisms of function responsible for these effects. Moreover, current and potential clinical applications of MSC-based treatment strategies in the context of Regenerative Medicine are being discussed. Key issues such as optimal timing of MSC administration, cell dose and schedule of administration, advantages and disadvantages of using autologous or allogeneic cells are still open. Nonetheless, MSCs promise to represent a revolution for many severe or presently untreatable disorders.
  • Progress through the cell mitotic cycle requires precise timing of the intrinsic molecular steps and tight coordination with the environmental signals that maintain a cell into the proper physiological context. Because of their great functional flexibility, ion channels coordinate the upstream and downstream signals that converge on the cell cycle machinery. Both voltage- and ligand-gated channels have been implicated in the control of different cell cycle checkpoints in normal as well as neoplastic cells. Ion channels mediate the calcium signals that punctuate the mitotic process, the cell volume oscillations typical of cycling cells, and the exocytosis of autocrine or angiogenetic factors. Other functions of ion channels in proliferation are still matter of debate. These may or may not depend on ion transport, as the channel proteins can form macromolecular complexes with growth factor and cell adhesion receptors. Direct conformational coupling with the cytoplasmic regulatory proteins is also possible. Derangement or relaxed control of the above processes can promote neoplasia. Specific types of ion channels have turned out to participate in the different stages of the tumor progression, in which cell heterogeneity is increased by the selection of malignant cell clones expressing the ion channel types that better support unrestrained growth. However, a comprehensive mechanistic picture of the functional relations between ion channels and cell proliferation is yet not available, partly because of the considerable experimental challenges offered by studying these processes in living mammalian cells. No doubt, such studies will constitute one of the most fruitful research fields for the next generation of cell physiologists.
  • Article
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    Human adipose derived stem cells (hADSCs), with their impressive differentiation potential, may be used in autologous cell therapy or grafting to replace damaged tissues. Low intensity laser irradiation (LILI) has been shown to influence the behaviour of various cells, including stem cells. This study aimed to investigate the effect of LILI on hADSCs 24, 48 or 72 h post-irradiation and their differentiation potential into smooth muscle cells (SMCs). hADSCs were exposed to a 636 nm diode laser at a fluence of 5 J/cm(2). hADSCs were differentiated into SMCs using retinoic acid (RA). Morphology was assessed by inverted light and differential interference contrast (DIC) microscopy. Proliferation and viability of hADSCs was assessed by optical density (OD), Trypan blue staining and adenosine triphosphate (ATP) luminescence. Expression of stem cell markers, β1-integrin and Thy-1, and SMC markers, smooth muscle alpha actin (SM-αa), desmin, smooth muscle myosin heavy chain (SM-MHC) and smoothelin, was assessed by immunofluorescent staining and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Morphologically, hADSCs did not show any differences and there was an increase in viability and proliferation post-irradiation. Immunofluorescent staining showed expression of β1-integrin and Thy-1 72 h post-irradiation. RT-PCR results showed a down regulation of Thy-1 48 h post-irradiation. Differentiated SMCs were confirmed by morphology and expression of SMC markers. LILI at a wavelength of 636 nm and a fluence of 5 J/cm(2) does not induce differentiation of isolated hADSCs over a 72 h period, and increases cellular viability and proliferation. hADSCs can be differentiated into SMCs within 14 days using RA.
  • Article
    The purpose of this study was to investigate the efficacy of Integra, an artificial dermal matrix used as a dermal template for skin regeneration, to form a multifunctional scaffold with human bone marrow-derived mesenchymal stem cells (hMSCs) and platelet-rich plasma (PRP) for tissue engineering and regenerative technology. First, we showed that PRP, used as a supplement for growth medium, represented an optimal substitute for animal serum as well as a source of multiple growth factors, was able to satisfactorily support cell viability and cell proliferation and influence stemness gene expression in hMSCs. Moreover, Integra appeared to be a suitable substrate for hMSCs colonization, as judged by two-photon microscopy combined with fluorescence lifetime imaging (FLIM) and confocal analysis. The cells were then seeded on Integra + PRP for 24 and 48 h. Notably, in these conditions, the seeded cells exhibited a greater aptitude to colonize the scaffold, showed improved cell adhesion and spreading as compared with those cultured on Integra alone, and acquired a fibroblast-like phenotype, indicating that the bioengineered scaffold provided an appropriate environment for cellular growth and differentiation. In conclusion, these results, although preliminary, provide clues for the design of new therapeutic strategies for skin regeneration, consisting in the combination of mesenchymal stem cells with engineered biomaterials.
  • Article
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    The aim of this study was to measure the temporal pattern of the expression of osteogenic genes after low-level laser therapy during the process of bone healing. We used quantitative real-time polymerase chain reaction (qPCR) along with histology to assess gene expression following laser irradiation on created bone defects in tibias of rats. The animals were randomly distributed into two groups: control or laser-irradiated group. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation started 24 h post-surgery and was performed for 3, 6, and 12 sessions, with an interval of 48 h. A 830 nm laser, 50 J/cm(2), 30 mW, was used. On days 7, 13, and 25 post-injury, rats were sacrificed individually by carbon dioxide asphyxia. The tibias were removed for analysis. The histological results revealed intense new bone formation surrounded by highly vascularized connective tissue presenting slight osteogenic activity, with primary bone deposition in the group exposed to laser in the intermediary (13 days) and late stages of repair (25 days). The quantitative real-time PCR showed that laser irradiation produced an upregulation of BMP-4 at day 13 post-surgery and an upregulation of BMP4, ALP, and Runx 2 at day 25 after surgery. Our results indicate that laser therapy improves bone repair in rats as depicted by differential histopathological and osteogenic genes expression, mainly at the late stages of recovery.
  • Article
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    The aim of this work is to review the available literature on the details of low-level laser therapy (LLLT) use for the enhancement of the proliferation of various cultured cell lines including stem cells. A cell culture is one of the most useful techniques in science, particularly in the production of viral vaccines and hybrid cell lines. However, the growth rate of some of the much-needed mammalian cells is slow. LLLT can enhance the proliferation rate of various cell lines. Literature review from 1923 to 2010. By investigating the outcome of LLLT on cell cultures, many articles report that it produces higher rates of ATP, RNA, and DNA synthesis in stem cells and other cell lines. Thus, LLLT improves the proliferation of the cells without causing any cytotoxic effects. Mainly, helium neon and gallium-aluminum-arsenide (Ga-Al-As) lasers are used for LLLT on cultured cells. The results of LLLT also vary according to the applied energy density and wavelengths to which the target cells are subjected. This review suggests that an energy density value of 0.5 to 4.0 J/cm(2) and a visible spectrum ranging from 600 to 700 nm of LLLT are very helpful in enhancing the proliferation rate of various cell lines. With the appropriate use of LLLT, the proliferation rate of cultured cells, including stem cells, can be increased, which would be very useful in tissue engineering and regenerative medicine.
  • Article
    K+ channels are key molecules in the progression of several cancer types and are considered to be potential targets for cancer therapy. In this study, we investigated the intermediate- conductance Ca2+-activated K+ channels (hKCa3.1) expression in both breast carcinoma (BC) specimens and human breast cancer epithelial primary cell cultures (hBCE) using immuno-histochemistry (60 samples), quantitative Real-Time RT-PCR (30 samples) and Western blot assay (30 samples). We also looked at whether or not the expression of these channels is correlated with breast carcinomas grade tumours and metastasis status. Furthermore, we characterized the hKCa3.1 channel activity in hBCE cells by using the Whole Cell Patch Clamp Technique. We found that hKCa3.1 transcripts and proteins were expressed in both BC samples and hBCE cells. Clinicopathologic evaluation indicated a significant correlation between hKCa3.1-expression and tumour grade. hKCa3.1 mRNA and protein were more highly expressed in grade III tumours than in both grades I and II. However, the hKCa3.1 expression-increase according to grade was only observed in tumours with negative metastasis status. Moreover, the hKCa3.1 channels expressed in hBCE cells are functional. This was attested by patch-clamp recordings showing typical hKCa3.1-mediated currents in these cells. In conclusion, these data suggest that hKCa3.1 might contribute to breast tumour-progression and can serve as a useful prognostic marker for breast cancer.
  • Article
    Dental lasers represent a promising therapeutic tool in the treatment of periodontal and peri-implant diseases. However, their clinical application remains still limited. Here, we investigated the potential biostimulatory effect of low pulse energy neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation on different cells representative of the oral microenvironment and elucidated the underlying molecular mechanisms. Saos-2 osteoblasts, H-end endothelial cells, and NIH/3T3 fibroblasts pre-treated or not with photosensitizing dye methylene blue (MB), were irradiated with low pulse energy (20 mJ) and high repetition rate (50-70 Hz) Nd:YAG laser, and evaluated for cell viability and proliferation as well as for the expression of specific differentiation markers by confocal immunofluorescence and real-time RT-PCR. Changes in intracellular Ca(2+) levels after laser exposure were also evaluated in living osteoblasts. Nd:YAG laser irradiation did not affect cell viability in all the tested cell types, even when combined with pre-treatment with MB, and efficiently stimulated cell growth in the non-sensitized osteoblasts. Moreover, a significant induction in the expression of osteopontin, ALP, and Runx2 in osteoblasts, type I collagen in fibroblasts, and vinculin in endothelial cells could be observed in the irradiated cells. Pre-treatment with MB negatively affected cell differentiation in the unstimulated and laser-stimulated cells. Notably, laser irradiation also caused an increase in the intracellular Ca(2+) in osteoblasts through the activation of TRPC1 ion channels. Moreover, the pharmacologic or genetic inhibition of these channels strongly attenuated laser-induced osteopontin expression, suggesting a role for the laser-mediated Ca(2+) influx in regulating osteoblast differentiation. Low pulse energy and high repetition rate Nd:YAG laser irradiation may exert a biostimulative effect on different cells representative of the oral microenvironment, particularly osteoblasts. Pre-treatment with MB prior to irradiation hampers this effect and limits the potential clinical application of photosensitizing dyes in dental practice.
  • Article
    Both Notch signaling and Akt-mTOR signaling pathway are involved in glioma cell proliferation and survival. Previous studies have shown that Notch-1 is overexpressed in many glioma cell lines and primary human gliomas. Blocking of Notch signaling pathway can induces glioma cell apoptosis and growth suppression. However, the underlying molecular mechanism is not clear. We report that activation of the Notch pathway by intracellular domain of human Notch-1 (NIC-1) strongly activates Akt and promotes U251 glioma cell proliferation. Knockdown of Notch-1 by RNA interference suppresses Akt activation, reduces glioma cell growth rate and induce cell apoptosis. Following Notch-1 suppression, phosphorylated Akt and its downstream effector mTOR were reduced. Knockdown of Notch-1 also involves down-regulation of anti-apoptotic protein MCL-1, in parallel with activation of apoptotic associate proteins PARP, caspase-9 and caspase-3. Our data demonstrate that Notch-1 can positively regulate Akt-mTOR pathways, which is associated with glioma cell proliferation and apoptosis. This also suggests a molecular mechanism for the inhibitory effect of Notch-1 RNA interference on glioma cell proliferation through Akt-mTOR signaling pathway.
  • Article
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    Peripheral nerves are structures that, when damaged, can result in significant motor and sensory disabilities. Several studies have used therapeutic resources with the aim of promoting early nerve regeneration, such as the use of low-power laser. However, this laser therapy does not represent a consensus regarding the methodology, thus yielding controversial conclusions. The objective of our study was to investigate, by functional evaluation, the comparative effects of low-power laser (660 nm and 830 nm) on sciatic nerve regeneration following crushing injuries. Twenty-seven Wistar rats subjected to sciatic nerve injury were divided into three groups: group sham, consisting of rats undergoing simulated irradiation; a group consisting of rats subjected to gallium-aluminum-arsenide (GaAlAs) laser at 660 nm (10 J/cm(2), 30 mW and 0.06 cm(2) beam), and another one consisting of rats subjected to GaAlAs laser at 830 nm (10 J/cm(2), 30 mW and 0.116 cm(2)). Laser was applied to the lesion for 21 days. A sciatic functional index (SFI) was used for functional evaluation prior to surgery and on days 7, 14, and 21 after surgery. Differences in SFI were found between group 660 nm and the other ones at the 14th day. One can observe that laser application at 660 nm with the parameters and methods utilised was effective in promoting early functional recovery, as indicated by the SFI, over the period evaluated.
  • Article
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    Understanding stem cell commitment and differentiation is a critical step towards clinical translation of cell therapies. In past few years, several cell types have been characterized and transplanted in animal models for different diseased tissues, eligible for a cell-mediated regeneration. Skeletal muscle damage is a challenge for cell- and gene-based therapeutical approaches, given the unique architecture of the tissue and the clinical relevance of acute damages or dystrophies. In this review, we will consider the regenerative potential of embryonic and somatic stem cells and the outcomes achieved on their transplantation into animal models for muscular dystrophy or acute muscle impairment.
  • Article
    The Notch signalling pathway is repeatedly employed during embryonic development and adult homeostasis of a variety of tissues. In particular, its frequent involvement in the regulation of stem and progenitor cell maintenance and proliferation, as well as its role in binary fate decisions in cells that are destined to differentiate, is remarkable. Here, we review its role in the development of haematopoietic stem cells during vertebrate embryogenesis and put it into the context of Notch's functions in arterial specification, angiogenic vessel sprouting and vessel maintenance. We further discuss interactions with other signalling cascades, and pinpoint open questions and some of the challenges that lie ahead.
  • Article
    Full-text available
    The Notch gene encodes a transmembrane receptor that gave the name to the evolutionary highly conserved Notch signaling cascade. It plays a pivotal role in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. After specific ligand binding, the intracellular part of the Notch receptor is cleaved off and translocates to the nucleus, where it binds to the transcription factor RBP-J. In the absence of activated Notch, RBP-J represses Notch target genes by recruiting a corepressor complex. Here, we review Notch signaling with a focus on gene regulatory events at Notch target genes. This is of utmost importance to understand Notch signaling since certain RBP-J associated cofactors and particular epigenetic marks determine the specificity of Notch target gene expression in different cell types. We subsequently summarize the current knowledge about Notch target genes and the physiological significance of Notch signaling in development and cancer.
  • Article
    Targeted therapy is considerably changing the treatment and prognosis of cancer. Progressive understanding of the molecular mechanisms that regulate the establishment and progression of different tumors is leading to ever more specific and efficacious pharmacological approaches. In this picture, ion channels represent an unexpected, but very promising, player. The expression and activity of different channel types mark and regulate specific stages of cancer progression. Their contribution to the neoplastic phenotype ranges from control of cell proliferation and apoptosis, to regulation of invasiveness and metastatic spread. As is being increasingly recognized, some of these roles can be attributed to signaling mechanisms independent of ion flow. Evidence is particularly extensive for K(+) channels. Their expression is altered in many primary human cancers, especially in early stages, and they frequently exert pleiotropic effects on the neoplastic cell physiology. For instance, by regulating membrane potential they can control Ca(2+) fluxes and thus the cell cycle machinery. Their effects on mitosis can also depend on regulation of cell volume, usually in cooperation with chloride channels. However, ion channels are also implicated in late neoplastic stages, by stimulating angiogenesis, mediating the cell-matrix interaction and regulating cell motility. Not surprisingly, the mechanisms of these effects are manifold. For example, intracellular signaling cascades can be triggered when ion channels form protein complexes with other membrane proteins such as integrins or growth factor receptors. Altered channel expression can be exploited for diagnostic purposes or for addressing traceable or cytotoxic compounds to specific neoplastic tissue. What is more, recent evidence indicates that blocking channel activity impairs the growth of some tumors, both in vitro and in vivo. This opens a new field for medicinal chemistry studies, which can avail of the many available tools, such as blocking antibodies, antisense oligonucleotides, small interfering RNAs, peptide toxins and a large variety of small organic compounds. The major drawback of this approach is that some ion channel blockers produce serious side effects, such as cardiac arrhythmias. Therefore, drug developing efforts aimed at producing less harmful compounds are needed and we discuss possible approaches toward this goal. Finally, we propose that a novel therapeutic tactic could be developed by unlocking ion channels from multiprotein membrane signaling complexes.
  • Article
    The high variation often observed in the ex vivo fibroblastic-colony forming unit (CFU-f) assay is likely to be due to both biological and experimental variation. To determine whether we could improve experimental methods we developed an alternative method of bone marrow cell (BMC) isolation employing a centrifugation step. The osteogenic capacity of centrifugally isolated BMC was compared to that of BMC that were isolated using the standard "flushing" technique using the CFU-f assay. The centrifugation method was found to be both quick and simple to perform and allowed simultaneous preparation of all samples. Centrifugally isolated BMC gave rise to approximately 100% more cfu-ap and cfu-f in cultures from both tibiae and femurae. The proportion of alkaline phosphatase positive colonies remained the same and colony morphologies were similar for both isolation methods. Histological comparison of the flushed and spun bones showed that after the flushing procedure many cells remained in the marrow cavity especially in the trabecular area. In contrast, centrifugation completely emptied the marrow space of all cells except bone lining cells and osteoblasts. Thus the osteogenic capacity of the bone marrow can be expressed as the number of CFU-f per bone instead of the frequency as is the norm. Using these methods to isolate BMC for ex vivo investigations should lead to a reduction in CFU-f number variation due to the isolation method. http://link.springer-ny. com/link/service/journals/00223/bibs/65n5p411.html</++ +hea
  • Article
    Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologues in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, we have analysed the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes and made comparisons to E(spl)m8. We find that although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. We conclude that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators and, since the activation of target genes is critical in determining the developmental consequences of Notch activity, provide a framework for understanding Notch function in different developmental contexts.
  • Article
    Corneal wound repair was investigated in rabbits following excimer laser ablation of a 6 mm diameter and 90 microm deep disc. In the healing process particular attention was focused on the epithelium where gap junction expression and the rearrangement of desmosomes and hemidesmosomes were correlated with cell proliferation and epidermal growth factor receptor expression. Immunofluorescence-based confocal laser scanning microscopy, semithin resin section morphology and electron microscopy were utilized. In resting cornea two isotypes of gap junctions, confined to different regions in the same basal epithelial cells, were detected. Particulate connexin43 (alpha1) immunostaining was concentrated on the apical while the connexin26 type (beta2) in the baso-lateral cell membranes. This is the first report of connexin26 in the cornea. Connexin43 was found also in corneal keratocytes and endothelial cell. Since the two connexins do not form functioning heteromeric channels and have selective permeabilities they may serve alternative pathways for direct cell-cell communication in the basal cell layer. During regeneration both connexins were expressed throughout the corneal epithelium including the migrating cells. They also showed transient up-regulation 24 hr after wounding in the form of overlapping relocation to the upper cell layers. At this time, basal epithelial cells at the limbal region, adjacent to the wound and those migrating over the wounded area all expressed membrane bound epidermal growth factor receptor and they were highly proliferating. In conclusion, like in other stratified epithelia connexin26 is also expressed in the cornea. Transient up-regulation and relocation of connexins within the regenerating epithelium may reflect the involvement of direct cell-cell communication in corneal wound healing. Mitotic activity in the migrating corneal epithelial cells is also a novel finding which is probably the sign of the excessive demand for new epithelial cells in larger wounds not met alone by the proliferating limbal stock.