Lasers in Surgery and Medicine 40:433–438 (2008)
Stem Cell Proliferation Under Low Intensity Laser Irradiation: A Preliminary Study Fernanda de P. Eduardo, DDS, PhD,1 Daniela F. Bueno, PhD,2 Patricia M. de Freitas, PhD,3** Ma´rcia Martins Marques, PhD,4{ Maria Rita Passos-Bueno, PhD,5{ Carlos de P. Eduardo, PhD,6{ and Mayana Zatz, PhD7*,{ 1 Hospital Israelita Albert Einstein, Unit of Bone Marrow Transplantation, Sa˜o Paulo 05651-901, SP, Brazil 2 Departamento de Biologia, Centro de Estudos do Genoma Humano, Universidade de Sa˜o Paulo, Sa˜o Paulo 05508-090, SP, Brazil 3 Laborato´rio Especial de Laser na Odontologia (LELO), Faculdade de Odontologia, Departamento de Dentı´stica, Universidade de Sa˜o Paulo, Sa˜o Paulo 05508-000, SP, Brazil 4 Faculdade de Odontologia, Departamento de Dentı´stica, Universidade de Sa˜o Paulo, Sa˜o Paulo 05508-000, SP, Brazil 5 Departamento de Biologia, Centro de Estudos do Genoma Humano, Universidade de Sa˜o Paulo, Sa˜o Paulo 05508-090, SP, Brazil 6 Laborato´rio Especial de Laser na Odontologia (LELO), Departamento de Dentı´stica, Faculdade de Odontologia, Universidade de Sa˜o Paulo, Sa˜o Paulo 05508-000, SP, Brazil 7 Centro de Estudos do Genoma Humano, Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, Sa˜o Paulo 05508-090, SP, Brazil
Background and Objectives: 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. Study Design/Materials and Methods: 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. Results: 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. Conclusions: 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. Lasers Surg. Med. 40:433–438, 2008. ß 2008 Wiley-Liss, Inc. Key words: low intensity laser; stem cells ß 2008 Wiley-Liss, Inc.
INTRODUCTION The use of mesenchymal stem cells (MSC) for future tissue engineering and regenerative medicine to replace conventional therapeutic modalities has been the subject of growing interest in different areas [1]. These cells have self-renewing properties and are able to differentiate into one or many different specialized cell types under controlled in vitro conditions. They can be obtained from many tissues such as bone marrow [2,3], dental pulp [4–8], adipose tissue [9–11] and umbilical cord [12] among others. Laser phototherapy has been used to treat pathological tissue conditions, to control inflammatory processes and also to promote tissue healing [13,14]. The mechanism by which low intensity lasers induce biomodulation of cell activity has been well described by Karu [15,16]. Laser irradiation is postulated to intensify the formation of a transmembrane electromechanical proton gradient in mitochondria [17]. Thus, the efficiency of the proton-motive force (pmf) is increased and more calcium is
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Assistant Professor. Professor. Full Professor. Contract grant sponsor: Sa˜o Paulo Research Foundation; Contract grant numbers: FAPESP CEPID/GENOMA # 98/ 14254-2, CEPID/CEPOF # 98/14270-8; Contract grant sponsor: National Council of Scientific and Technological Development; Contract grant numbers: CNPq # 552210/2005, # 303798/2005-0. *Correspondence to: Mayana Zatz, PhD, Centro de Estudos do Genoma Humano, Universidade de Sa˜o Paulo, Rua do Mata˜o, 277-Cidade Universita´ria, Sa˜o Paulo 05508-090, SP, Brazil. E-mail:
[email protected] Accepted 27 March 2008 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/lsm.20646 { Associate {
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released into the cytoplasm from the mitochondria. At low laser doses, this additional calcium transported into the cytoplasm triggers mitosis and enhances cell proliferation. In addition to the mitogenic action of calcium release into the cytoplasm by the pmf, a short-term rise in the intracellular pH by the creation of the electromechanical proton gradient triggers mitogenic signals in the cells. Moreover, it is well known that the pmf increases ATP production, which activates Na, K-ATPase, and other ion carriers. Thus, the intracellular Kþ level is increased and the Naþ concentration and membrane potential are decreased. These factors also influence cell proliferation [17,18]. It has been shown that exposure to low intensity laser can accelerate the growth of fibroblasts and osteoblasts [19–21]. However, laser phototherapy seems to act mainly in cells with committed functions [15,16,18–20] and several questions on the intracellular responses of stem cells to laser phototherapy are still open. The use of human dental pulp stem cells (hDPSCs) is of great interest in tissue engineering as they can easily be isolated and expanded in culture; moreover, they have shown multipotential plasticity in vitro and in vivo [4–7]. These cells seem to have immunosuppressive activity that could have potential clinical applications in allogeneic in vivo stem cell transplantation [8]. However, the effect of laser irradiation on hDPSC has not been reported before. Here, the effect of laser phototherapy under different culture conditions has been tested, since improvement in stem cell proliferation could be of importance in the future use of these cells for tissue regenerating therapy in dentistry and medicine. MATERIALS AND METHODS Cell Culture The strain of hDPSCs used was previously characterized [7]. The culture of hDPSC was maintained in DMEM/F12 (Dulbecco’s-modified Eagle’s medium/Ham’s F12, 1:1, Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS, HyClone, Logan, Utah, USA), 100 units/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 2 mM nonessential amino acids. The cells were maintained semi-confluent in order to prevent differentiation, and were passed every 4–5 days with the medium refreshed daily. The hDPSC were incubated at 378C in a 5% CO2 and high humidity environment.
supplementation that allows cell growth, but at a rate lower than that characteristic of cells grown on nutritional regular culture medium (15% FBS), 1103 cells/well were plated into four 96-well microtitration plates (12 wells/ plate). In 48 wells it was possible to analyze, in quadruplicate, the growth of cells cultured in medium supplement with four different FBS concentrations (15%, 12.5%, 10%, and 5%) at three different experimental times (0, 20, and 24 hours after plating). To infer the cell viability and to plot the cell growth curves, the cell mitochondrial activity was analyzed using the MTT-based cytotoxicity assay, which involves the conversion of the water soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to an insoluble formazan. The formazan is then solubilized, and the concentration determined by optical density at ffi570 nm. A MTT reduction analysis kit (Vybrant MTT, Molecular Probes, Eugene, OR) was used. Immediately after the end of the assay procedures the absorbance was read in a micro plate reader (Biotrak II, Biochrom Ltd., Eugendorf, Austria) using a 562 nm filter. The absorbance data was used to plot the cell growth curves. Laser Irradiation Laser irradiation was delivered with InGaAlP lasers (MM Optics Ltd., Sa˜o Carlos, SP, Brazil). Irradiations were performed in contact, using the punctual irradiation mode in a 3.6 mm2 area. The 660 nm laser was applied with output powers of 40 and 20 mW. The energy density was 3 J/cm2 in 40 mW with 3 seconds exposure time and 20 mW with 6 seconds exposure time. The experiments were set up to standardize the procedures as previously described [20,21]. Briefly, knowing that the distance between the laser source and the surface of application is critical, the laser application was performed through the bottom of the optically clear 96-well microtitration plates. Therefore, the laser beam did not transpose the culture medium, being applied straight onto the cell and the distance between the laser beam and the cell monolayer was held constant at 1 mm. Finally, the laser irradiation was carried out in partial darkness without light influence other than laser. The LaserCheck power meter (Coherent, Inc., Santa Clara, CA) was used to verify the output power of the laser equipment. The control groups were treated under identical conditions except that the laser equipment was kept off.
Determination of Serum Concentration
Effect of Different Laser Irradiation Power Settings on Cell Growth
Effects of laser phototherapy on cultured cell growth are noticeably observed when the cultures are grown in nutritional deficit [19–21]. This in vitro situation is comparable to in vivo stress conditions where the laser phototherapy has shown to be effective. In previous studies, it has been demonstrated that when the cells are grown in culture medium supplemented with reduced FBS concentrations (nutritional deficient culture media) the growth rate of the cell cultures is diminished [18–21]. In order to determine a FBS concentration for cell culture medium
For cell growth analysis, control (non-irradiated) and treated cultures (irradiated) were plated in 96-well microtitration plates and grown in nutritional deficit (10% FBS). For this part of the study the experimental groups were: negative control (10%); lased 40 mW with 3 seconds exposure time and 20 mW with 6 seconds exposure time (Table 1). The cultures were incubated in a humidified air-5% CO2 atmosphere for 6 hours before irradiation. Before irradiation, the culture medium was replaced by the nutritional
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TABLE 1. Parameters Used for Cells Irradiation in Order to Determine the Effect of Different Power Settings on Cell Growth Groups Negative control Lased 20 Lased 40
Power (mW)
Energy density (J/cm2)
20 40
Exposure time (seconds)
Without laser irradiation 3.0 6 3.0 3
Total energy (J) 0.12 0.12
Experimental groups (negative control, G1 and G2) were submitted to nutritional deficit (10% FBS).
deficient culture medium supplemented with 10% FBS. Then, two irradiations with 6 hours intervals were performed. Samples from each group were taken for mitochondrial activity analysis at 20, 24, 48, and 72 hours after the first irradiation. The cell mitochondrial activity analysis was also used to infer the cell viability and to plot the cell growth curves. All the experiments were done in four replicates. Experiment #1. For this experiment the hDPSC irradiated cells were plated at the density of 1103 cells/ well in 96-well microtitration culture plates, divided into four sub-groups: (a) Positive control (15% FBS)—cultures grown in regular nutritional conditions and non-irradiated; (b) negative control (10%)—cultures grown in nutritional deficit (10% FBS) and non-irradiated; (c) lased (15%)— cultures grown in regular nutritional conditions and irradiated; and (d) lased (10%)—cultures grown in nutritional deficit (10% FBS) and irradiated. For growth analysis, control (non-irradiated) and irradiated cultures (lased 660 nm, two irradiations, 6 hours intervals, 20 mW, 3 J/cm2, 6 seconds exposure time) were plated in 96-well plates. The cultures were incubated in a humidified air 5% CO2 atmosphere for 6 hours before irradiation. Four identical samples from each of the four groups (lased and control) were taken for mitochondrial activity analysis 0, 20, 24, and 48 hours after the first irradiation. The cell mitochondrial activity analysis was also used to infer the cell viability and to plot the cell growth curves. Experiment #2. This experiment was done to assess the cell growth of lased and non-lased cells grown under nutritional deficit. The experimental groups for this part of the study were: positive control (10% FBS)—cultures grown in regular nutritional conditions and non-irradiated; lased (10%)—cultures grown in nutritional deficit (10% FBS) and irradiated. This experiment was held under the same conditions as those in experiment #1 except for the plating cell number (5102 cells/well) and the shorter experimental intervals, which were: 0, 6, 12, 18, 24, 30, and 36 hours after the first irradiation. The cell mitochondrial activity analysis was also used to infer the cell viability and to plot the cell growth curves. All the experiments were done in four replicates.
Statistical Analysis The optical density data, corresponding to the cell viability, obtained in four replicates are presented as mean the standard error of the mean (SEM). The data were compared by ANOVA test followed by the Tukey test. The level of significance was 5% (P0.05). RESULTS Effect of Serum Concentration on Cell Growth The growth of hDPSC cells in culture medium supplemented with different FBS concentrations (15%, 12.5%, 10%, and 5%) was compared in order to determine the appropriate serum concentration for the proposed laser experiments. The growth curves are presented in Figure 1. A significant growth was observed in cultures grown in culture media supplemented with all FBS concentrations tested, except those grown in medium supplement with 5% FBS. The growth of cultures treated with culture medium supplemented with 12.5% and 10% FBS was similar and, significantly less than that of cultures grown in nutritional regular culture medium, which contains a FBS concentration of 15%. Effect of Different Laser Irradiation Power Settings on Cell Growth The effect of the laser irradiation was analyzed in hDPSC cell grown in nutritional deficient culture medium
Fig. 1. Mitochondrial activity under different serum concentration. Different letters indicate statistical differences at the same experimental time. (*) indicates statistical difference with the other group at the same experimental time.
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supplemented with 10% FBS. For each laser parameter tested comparative cell growth curves were plotted and are presented in Figure 2. When the cell viability of cultures from the different groups were compared at the same experimental times, the group irradiated with the 20 mW setting presented significantly higher MTT activity at 72 hours than the other two groups. Experiment #1. The growth of both cultures grown under regular nutritional conditions (15% FBS) was similar, irrespective of the irradiation. The growth of these cultures was significantly higher than those of all groups grown under nutritional deficit during the entire experimental time. Cultures grown under nutritional deficit and irradiated (lased—10% FBS) starting 24 hours after the first irradiation presented significantly higher numbers of viable cells than the non-irradiated cultures grown under the same nutritional conditions (control—10%), as shown in Figure 3. Experiment #2. Experiment #2 confirmed the results of experiment #1 (Fig. 4). The differences in cell growth were observed starting 12 hours after the first irradiation. Lased cultures presented significantly higher cell growth than non-irradiated cultures, when both were cultured under nutritional deficit (10% FBS).
Fig. 3. Mitochondrial activity of cell cultures grown under regular nutritional conditions (15% FBS) under nutritional deficit (10% FBS) and submitted or not to laser irradiation.
The role of mesenchymal stem cells in repairing tissues has been studied for decades [1–9,22,23]. Phenotypic and genetic evidences suggest that hDPSC [4–7] are immature cell types, being a useful model for cell biology studies against normal and disease backgrounds. Knowledge of therapies able to improve stem cell growth conditions are of utmost interest. Here, for the first time, the effect of laser phototherapy on hDPSC was investigated, showing that a previously characterized strain of hDPSC [7] responded positively to
laser phototherapy using a 660 nm low intensity laser. These cells presented a fibroblast-like morphology and did not differentiate spontaneously during culture expansion. Under nutritional deficit conditions, the laser therapy was able to improve cell growth, although this cell growth rate was lower than the rate observed when hDPSC are grown in nutritional regular condition. The choice of the laser irradiation parameters of this cell culture study (660 nm, 40 mW with 3 seconds exposure time or 20 mW with 6 seconds exposure time, 3 J/cm2) was based in the authors’ previous in vitro studies, in which energy densities of 1–4 J/cm2, using either the visible red (660 nm) or infrared lasers (780 nm), showed a positive biostimulation effect on fibroblast [18–21] and osteoblast [24] proliferation. Moreover, a recent study revealed that the energy density of 3 J/cm2 had a significant influence on epithelial cell proliferation [18]. Laser phototherapy is based on photochemical and photobiological effects on cells and tissues [25]. Previous studies have reported that the process by which laser phototherapy leads to normalization of cell functions begins when photons enter the tissue, are absorbed by biological chromophores, located either in the mitochondria or in the
Fig. 2. Mitochondrial activity under different laser irradiation power settings. (*) indicates statistical difference with the other group at the same experimental time.
Fig. 4. Mitochondrial activity of cell cultures grown under nutritional deficit (10% FBS) and submitted or not to laser irradiation. (*) indicates statistical difference within groups at the same experimental time.
DISCUSSION
STEM CELLS AND LASER PHOTOTHERAPY
cell membrane. These chromophores interact strongly with the laser wavelengths. The photonic energy is converted into chemical energy within the cell, in the form of ATP, which leads to normalization of cell function, pain relief and healing. Cell membrane permeability alters, and then physiological changes can occur. These physiological changes can affect macrophages, fibroblasts, endothelial cells, mast cells, inflammation mediator secretion, and nerve conduction rates [26]. The present study revealed that the 660 nm wavelength at 20 mW was more effective in stimulating hDPSC cell growth when compared with the 40 mW power setting. Although some authors have reported that higher power densities and lower exposure times are usually more beneficial than lower power densities for a longer period of time [27], others have shown that higher power densities can be related to an inhibiting effect of cell activities [28]. This can be related to the type of tissue, the condition of the irradiated tissue and also to experimental methods used in cell culture studies. According to an important review of the literature published by Tuner and Hode [25] biostimulation can be reached with doses ranging from as low as 0.001 to 10 J/cm2. The authors point out that relevant difference may occur between irradiating naked cells in in vitro studies and overlapping layer tissues in vivo. Furthermore, the different laser irradiation conditions in laboratory studies, including laser beam characteristics and spreading, output power, equipment calibration, among other factors should also be considered. Interestingly, the hDPSC cell growth was not significantly different within the experimental groups under normal conditions, and showed no significant hDPSC proliferation in nutritional regular culture medium (15% FBS). However a statistically significant difference in growth was observed when cells were grown in nutritional deficit. According to Karu [29] the irradiation effect is strongly dependent on the cell redox state at the moment of irradiation; that is, an alteration of the redox state towards oxidation is correlated with stimulation, whereas reduction correlates with inhibition. The cellular response is weak or absent when the original redox potential is optimal or near optimal and stronger when shifted; and cells with lower than normal pH are considered to be more sensitive to the stimulative action of the light than those with the respective parameters being optimal or near to it. The hDPSCs used in this study were undifferentiated cells capable of self-renewal, with high proliferative capacity. Several reports suggest that MSCs were able to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, myocytes and neurons [4–7]. Whenever a tissue is injured the functions of the stem cells around it can also be compromised. Therefore, the results of the present study are promising, since laser phototherapy can significantly influence stem cell proliferation, leading to improved tissue healing. However, in order to verify whether this therapy can contribute to optimal attachment and functional improvement of the cells following implantation, as well as reduce tissue healing time, future studies are needed to evaluate its potential in bone and muscle
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neoformation after stem cell implantation in injured tissues. In short, this in vitro study suggests that hDPSC responds positively to laser phototherapy using 660 nm low intensity laser. 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.
ACKNOWLEDGMENTS This study was supported by the state of Sa˜o Paulo Research Foundation (FAPESP CEPID/GENOMA # 98/ 14254-2 and CEPID/CEPOF # 98/14270-8) and the National Council of Scientific and Technological Development (CNPq # 552210/2005 and # 303798/2005-0). The authors also thank Prof. Jan Tuner for his critical comments on the study. REFERENCES 1. Tuby H, Maltz L, Oron U. Low-level laser irradiation (LLLI) promotes proliferation of mesenchymal and cardiac stem cells in culture. Lasers Surg Med 2007;39:373–378. 2. Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9: 641–650. 3. Kuznetsov SA, Krebsbach PH, Satomura K, Kerr J, Riminucci M, Benayahu D, Robey PG. Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo. J Bone Miner Res 1997;12:1335– 1347. 4. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 2000;97:13625–13630. 5. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003;18:696–704. 6. Kerkis I, Kerkis A, et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cells markers. Cell Tissues Organs 2006;184(3–4):105–116. 7. de Mendonc¸a Costa AM, Bueno DF, Kerkis I, et al. Reconstruction of large cranial defects in non-immunosupressed rats with human stem cells A preliminary report. J Craniofac Surg 2008 Jan;19(1);204–210. 8. Pierdomenico L, Bonsi L, Calvitti M, et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 2005;80:836–842. 9. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279– 4295. 10. Woodruff LD, Bounkeo JM, Brannon WM, et al. The efficacy of laser therapy in wound repair: A meta-analysis of the literature. Photomed Laser Surg 2004 Jun;22(3):241–247. 11. Secco M, Zucconi E, Vieira NM, Fogac¸a LLQ, Cerqueira A, Carvalho MDF, Jazedje T, Okamoto OK, Muotri AR, Zatz M. Multipotent stem cells from umbilical cord: Cord is richer than blood. Stem Cells 2008 Jan;26(1):146–150. 12. Vieira NM, Zucconi E, Brandalise V, Jazedje T, Nunes VA, Strauss BE, Vainzof M, Zatz M. Human multipotent adipose derived stem cells restore dystrophin expression of Duchenne skeletal muscle cells in vitro. Biol Cell 2008 Apr; 100(4):231– 241. 13. Mester E, Mester AF, Mester A. The biomedical effects of laser application. Laser Surg Med 1985;5:31–39. 14. Stein A, Benayahu D, Maltz L, Oron U. Low-level laser irradiation promotes proliferation and differentiation of human osteoblasts in vitro. Photomed Laser Surg 2005;23: 161–166.
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