Photomedicine and Laser SurgeryVolume 26, Number 4, 2008© Mary Ann Liebert, Inc.Pp. 401–404DOI: 10.1089/pho.2007.2101

Evaluation of Low-Level Laser Therapy of Osteoblastic Cells

Deise A.A. Pires Oliveira, M.D.,1 Rodrigo Franco de Oliveira, M.D.,1 Renato Amaro Zangaro, Ph.D.,2

and Cristina Pacheco Soares, Ph.D.1

Abstract

Objective: The purpose of the present study was to evaluate the effect of biomodulation on osteoblastic cellsusing a gallium-aluminium-arsenide diode laser.Background Data: Low-level laser therapy (LLLT) is a non-pharmacological therapeutic resource to which bi-ological tissues respond well, producing such effects as the acceleration of bone formation and bone repair.Materials and Methods: Osteoblastic cell cultures (OFCOL II) were irradiated with a gallium-aluminium-ar-senide diode laser (GaAlAs � � 830 nm; 50 mW; 3 J/cm2; 600-�m-diameter optical fiber) and divided into twogroups: group 1—irradiated cells, and group 2—non-irradiated cells. Irradiation occurred at 24-h intervals fora total of 3 d. After each interval, the cells were marked with Mito Tracker Orange dye to assess the biostim-ulatory effect on mitochondrial activity and cell proliferation using an MTT assay.Results: Intense grouping of mitochondria in the perinuclear region was observed at 24 h and 48 h followingirradiation. Changes from a filamentous to a granular appearance in mitochondrial morphology and mito-chondria distributed throughout the cytoplasm were observed 72 h following proliferation. Such changes ledto an in vitro proliferation process, as confirmed by the MTT assay.Conclusion: LLLT has shown itself capable of altering mitochondrial activity and the population of OFCOL IIcells.

401

Introduction

LOW-LEVEL LASER THERAPY (LLLT) using the far-red to near-infrared spectral range (630–1000 nm) or light-emitting

diode arrays has been found to modulate various biologicalprocesses in cell cultures and animal models.1 At the cellu-lar level, LLLT generates significant biological effects, in-cluding cell proliferation, collagen synthesis, and the releaseof growth factors.2

According to Carnevalli et al.,3 laser energy in the redspectral range favors the formation of giant mitochondria, aswell as an increase in the number of mitochondria.4–6 Irra-diation induces an increase in mitochondrial membrane po-tential, causing alterations in the ADP:ATP ratio, an increasein the DNA:RNA ratio, activation of nucleotides and chro-matin rearrangements, as well as an increase in mitochon-drial protein synthesis.7

The mitochondria play an important role in many phys-iopathological conditions, such as calcium homeostasis, oxy-

gen generation, and the control of apoptosis. The evaluationof mitochondrial activity in cells submitted to irradiation isa topic of ongoing interest.4 Yamada8 studied the effects ofhelium-neon laser irradiation (� � 632.8 nm, power output8.5 mW, energy densities of 0.01–1.0 J/cm2) on culturedclonal osteoblastic cells at different stages of the culturephase. Photobiomodulation was found to accelerate cell pro-liferation only in the growing phase, when cells were con-sidered undifferentiated osteoprogenitor cells.

LLLT has a positive effect on the proliferation and differ-entiation of osteoblastic cells in vitro.8 Osteoblast prolifera-tion in particular is of great clinical interest in the regenera-tion of lost bone. As the mechanisms acting on the bone havenot yet been fully elucidated, the exact mechanism of laser-induced cell biostimulation remains unclear. It is, however,the subject of a number of studies.9

The purpose of the present study was to evaluate the ef-fect of biomodulation using a Ga-Al-As diode laser on os-teoblast cells.

1Laboratório de Dinâmica de Compartimentos Celulares, Instituto de Pesquisa e Desenvolvimento (UNIVAP), and 2Laboratório de Flu-orescência, Instituto de Pesquisa e Desenvolvimento (UNIVAP), São José dos Campos, São Paulo, Brazil.

Materials and Methods

Cell culture

A mouse OFCOL II cell line derived from bone marrow(ATCC CCL-61; American Type Culture Collection,Rockville, MD, USA) obtained from the Paul Ehrlich Tech-nical Scientific Association (UFRJ Rio de Janiero, Brazil)was routinely cultivated using minimum essentialmedium (MEM) (GibcoBRL, Grand lsland, NY, USA), con-taining 5% (vol:vol) fetal bovine serum (FBS) (GibcoBRL),100 U/mL penicillin, 100 mM/mL streptomycin, and 0.25�g/mL Fungizone® (GibcoBRL). The cells were culturedin an incubator at 37°C in a humidified atmosphere con-taining 5% CO2. The medium was changed every 2 d.When the cells became confluent, the medium was re-moved and the cell layer was washed with phosphate-buffered saline; 0.25% trypsin (GibcoBRL) in bufferedEDTA (Carlos Erba, ABC Lab, São Paulo, Brazil) was thenadded and incubation continued for 2–4 min. A solutionwith a concentration of approximately 1.5 � 104 cells/mLwas prepared and poured into each well of 24-well tissueculture plates (Nunc, Denmark).

Irradiation

Cultures were irradiated with a low-level GaAlAs laser(� � 830 nm, 50 mW, 3.0 J/cm2, 600-�m-diameter opticalfiber) (Thera Laser; DMC Equipment Ltd., São Carlos,Brazil). Laser energy was delivered to the culture in a con-tinuous mode, with the laser positioned vertically above eachwell at a distance of 2 cm from the plate. Irradiation time foreach well was 36 sec. Cell cultures were divided into twogroups: group 1 was irradiated at 24-h intervals, with 24-,48-, and 72-h incubation times following irradiation; andgroup 2 was the non-irradiated control. For the MTT assay,irradiated cells were sub-cultured on 96-well culture plates;irradiation time for each well was 36 sec. Cells in the controlgroup were kept under the same conditions as cells in theirradiated group. The experiment was carried out in tripli-cate. The plates were positioned in a black mask, with onlythe area to be irradiated exposed.

Procedures for fluorescence staining

At each time point (24, 48, and 72 h), OFCOL II cellswere stained with Mito Tracker Orange Dye® (CMTMros;CITY?, STATE?, COUNTRY?) molecular probes10 (150nM), incubated for 20 min in the dark, immediately washedwith PHEM buffer (60 nM PIPES, 20 nM HEPES, 10 nMEGTA, and 5 mM MgCl2), and fixed in 3% paraformalde-hyde (Sigma-Aldrich Chemie GmBh, Steinheim, Germany)in 0.1 M PHEM for 10 min. After washing away the excessdye, cover slips were mounted on slides with n-propyl gallate. Observations and photographs were made using aLeica microscope. Specific filters were used for the following wavelengths: excitation (554 nm) and emission(576 nm).

Cell viability using the MTT technique (cytotoxicity)

Cytotoxicity experiments evaluated the irradiated culturesat 24-, 48-, and 72-h intervals using the MTT method [3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide].11

The colorimetric MTT assay was used to assess cell survivaland proliferation. For analysis, the culture medium was re-moved and each well received 20 �L of MTT, for a final con-centration of 0.5 mg/mL of MTT (Sigma-Aldrich ChemieGmbh). The cells were then incubated for 1 h at 37°C in a5% CO2 atmosphere; 100 �L of dimethyl sulfoxide (DMSO)was then added to each well. The plate was kept in agita-tion for 30 min for solubilization of the formazan crystals.Readings were performed using a 570-nm filter in an ELISASpectracount Reader (Packard Instrument Company Inc.,Meriden, CT, USA).

Statistical analysis

Results are expressed as mean values � SEM. Compar-isons between groups were performed with one-way analy-sis of variance (ANOVA). The post hoc Tukey-HSD analysiswas used to determine significant differences betweengroups. Values of p � 0.05 were considered statistically sig-nificant. Values were analyzed using the GraphPad Prism(version 4.0) statistical package (GraphPad Software, Inc., LaJolla, CA, USA).

Results

Results from the fluorescence microscopy reveal that con-trol cells had a small number of mitochondria with a fila-mentous appearance. The low intensities of fluorescence sug-gest low mitochondrial activity (Fig. 1A, C, and E). Inirradiated cells, intense grouping of mitochondria was ob-

PIRES OLIVEIRA ET AL.402

FIG. 1. OFCOL II cells marked with Mito Tracker Orangedye. (A) Control group (not irradiated) marked after 24 h.(B) Irradiated group marked 24 h after biomodulation. (C)Control group (not irradiated) marked after 48 h. (D) Irra-diated group marked 48 h after biomodulation. (E) Controlgroup (not irradiated) marked after 72 h. (F) Irradiated groupmarked 72 h after biomodulation. Arrows indicate mito-chondria.

served in the perinuclear region 24 h following irradiation(arrow in Fig. 1B), as well as at 48 h following irradiation(Fig. 1D). Alterations in mitochondria from a filamentous toa granular appearance were observed 72 h after prolifera-tion, and mitochondria were also distributed throughout thecytoplasm more so than in the control cells (Fig. 1F).

The results reveal that irradiation with MTT reflected thenumber of living cells in the culture. Significant increases of30% and 50% were observed in comparison to controls after9.6 min (3 J/cm2) of laser emission (p � 0.05), primarily 48and 72 h following the first irradiation (Fig. 2).

Discussion

Mito Tracker CMTMros dyes are fluorescent markers thathave been widely used to measure mitochondrial membranepotential (��m), mitochondrial mass, and photosensitivisa-tion.12 Perrin-Tamietti et al.13 found that Mito TrackerCMTMros demonstrated mitochondrial migration to the per-inuclear region, suggesting that intact mitochondria were so-licited for possible cell division.

The results obtained in the present study reveal that OF-COL II cells submitted to proliferation showed alterations inmitochondrial morphology. Cells in the control group ex-hibited a filamentous appearance and a small number of thisorganelle, which is an indication of low mitochondrial ac-tivity, as described by Bortoleto et al.4 In the irradiated cells,there was a grouping of mitochondria with a granular ap-pearance concentrated in the perinuclear region and intensefluorescence, which indicates high mitochondrial activity.The concentration of mitochondria in the perinuclear regionindicates the need for energy3 for protein synthesis and theduplication of genetic material. This corroborates resultsfrom other studies.3–6 According to Carnevalli et al.,3 thesedifferences must be the result of mitochondrial sensitivity to

various visible wavelengths. Bortoleto et al.4 also observedthat cells from the control group exhibited mitochondriawith a filamentous appearance and low membrane poten-tial, whereas irradiated cells exhibited mitochondria with agranular appearance. These observations suggest that alter-ations in mitochondrial activity, along with alterations in cellmetabolism, play a key role in the function of osteoblasts.5,6

The short-term effect of LLLT improves mitochondrialstimulation, which is in agreement with the data from thepresent study. However, long-term results may include stim-ulation or transcription inhibition and DNA replication.14

Short-term LLLT application can promote significant prolif-eration and differentiation of human osteoblasts in vitrowhen compared to non-irradiated cells.15 Pinheiro andGerbi16 report that non-differentiated mesenchymal cellscould be positively biomodulated to become osteoblasts,which would provoke a faster change in the osteocytes. Onthe other hand, LLLT seems ineffective when used on nor-mal tissues. LLLT may act as an inducing factor, improvingbone matrix production due to improved vascularizationand anti-inflammatory effects. These aspects would result inan increased release of mediators and microvascularization,which would subsequently accelerate bone healing.

When LLLT is performed with light in the visible regionof the electromagnetic spectrum, there is an initial photo-biostimulation of the cell mitochondria. When irradiation isin the infrared region, it stimulates the plasmatic membrane.After photoreception in both cases, there is transduction andamplification of signals and a subsequent photoresponse, atwhich point there may be proliferation, differentiation, orsynthesis of proteins, including cell growth factors.6

The stimulation of cell proliferation results from an in-crease in mitochondrial respiration and ATP synthesis. Mi-tochondrial activity involved in ATP synthesis undergoescontinuous ��m dissipation, which should be reflected by anaverage low-polarized condition. The presence of mitochon-dria in the perinuclear region may be associated with theregulation of the proton gradient along the mitochondrialmembrane.17

Stimulation of the plasmatic membrane and mitochondriais an effect of LLLT observed in the cell structure. The increasein mitochondrial ATP observed following irradiation withsome types of laser light induces further reactions that inter-fere with cell metabolism, as the absorbed energy stimulatescell functions. The biomodulating effect of LLLT occursthrough the activation of ATP production and an increase inthe mitotic process due to the excitation of cell respiration andendogenous porphyrins. The difficulty in measuring variablessuch as pain and tissue repair in both animal and human mod-els increases the importance of studies of the photobiologicaleffects of LLLT on cells.3 The biological response of cells irra-diated with LLLT reveals an alteration in the mitochondrial ac-tivity of oxidation reduction, resulting in a cascade of bio-chemical reactions. The irradiation of isolated mitochondriaalso induces changes in mitochondrial transcription and trans-lation, increasing the cascade reactions and number of respi-ratory chain components.18

Ueda and Shimizu19 also demonstrated that even a lowincrease in frequency (from 0–1 Hz) affects the stimulatoryaction of the GaAlAs diode laser (830 nm, 50 mW) on theproliferation of osteoblasts cultured in vitro. Similar resultshave been found using infrared laser irradiation of different

LLLT OF OSTEOBLASTIC CELLS 403

FIG. 2. MTT colorimetric test on OFCOL II cells irradiatedwith the 830-nm laser. Cell growth due to LLLT and culti-vation time; cell viability of control populations (p � 0.05).

cell models, which suggests that the administration modemay be a more important factor than wavelength with re-gard to treatment results. Castro et al.20 assessed the prolif-eration process of KB cells subjected to two wavelengths (830nm and 685 nm LLLT; 4 J/cm2) using an MTT assay. The au-thors found that the two wavelengths had different effectson the cells; the higher wavelength (830 nm) had a greaterproliferative effect, resulting in a photochemical effect di-rectly on the mitochondria, whereas the 685-nm wavelengthhad a photophysical effect on the cell membrane. These find-ings corroborate the results of the present study, demon-strating that the increase in proliferation may be a result ofthe positive biomodulatory effect of LLLT at 830 nm on thecells.

Therapeutic progress resulting from technological ad-vances in recent decades has led to a significant increase inlife expectancy, with a subsequent growth of the populationaged 65 years and over. This has created the need for re-search and studies on the physiopathology, prevention, andtreatment of alterations and diseases related to aging. Al-though the findings from such studies have been positive,not all results are clear regarding the interactions betweenlaser irradiation and biological systems. This calls for furtherstudies to establish the benefits and parameters of this in-creasingly widespread therapeutic resource used in the re-habilitation clinic.

Conclusion

In the present study, we concluded that LLLT proved ca-pable of altering mitochondrial activity and populations ofOFCOL II cells.

Acknowledgments

This work was supported by a grant from the Fundaçãode Amparo à Pesquisa do Estado de São Paulo (FAPESP) no.01/07380-6, CAPES (scholarship). The authors are also grate-ful to Professor Dr. Drauzio Eduardo Naretto Rangel.

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Address reprint requests to:Dr. Cristina Pacheco Soares, Ph.D.

Laboratório de Dinâmica de Compartimento CelularInstituto de Pesquisa e Desenvolvimento, UNIVAP

Av. Shishima Hifumi2911 Urbanova

São José dos CamposSão Paulo, CEP 12244-000, Brazil.

E-mail: cpsoares@univap.br

PIRES OLIVEIRA ET AL.404

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