The Effect of Photosensitizer Drugsand Light Stimulation on Osteoblast Growth

Daniela Cervelle Zancanela, M.S.,1 Fernando Lucas Primo, Ph.D.,1,2 Adalberto Luiz Rosa, Ph.D.,3

Pietro Ciancaglini, Ph.D.,1 and Antonio Claudio Tedesco, Ph.D.1

Abstract

Objective: A promising new treatment in dentistry involves the photodynamic process, which utilizes a com-bination of two therapeutic agents, namely a photosensitizer drug and a low dose of visible light. We investi-gated the in vitro effect of low intensity laser irradiation (visible light irradiation at 670 nm) using doses rangingbetween 0.5 and 3 J/cm2, combined with nanoemulsion (NE) of the photosensitizer drug aluminum phthalo-cyanine chloride (AlClPc), ranging from 0.5 to 5 lmol/L, on the growth and differentiation of osteoblastic cellsisolated from rat bone marrow. Background data: Treatments using laser radiation of low intensity in dentistryare of great interest, especially in bucco-maxillofacial surgery and dental implantology, where this approach iscurrently employed to stimulate osteogenesis. In the presence of oxygen, the combination of these agents couldinduce cellular biostimulation, via an efficient noninvasive method. Methods: We have done the colorimetricMTT assay, collagen content, total protein content, ALP activity and bone-like nodule formation. Results:We observed that an increased number of viable cells was evident upon application of a laser dosage equal to0.5 J/cm2 when combined with 0.5 lmol/L of AlClPc/NE, suggesting cellular biostimulation. Conclusions: Itwas possible to demonstrate that low intensity laser irradiation can play an important role in promotingbiostimulation of osteoblast cell cultures. Therefore, whether biostimulation of osteoblastic cell cultures byphotodynamic therapy or the cytotoxic effect of this therapy occurs only depends upon the light dose, and theresults can be completely reversed.

Introduction

Tissue repair is a dynamic interactive process in-volving several biochemical and cellular changes. Bone

remodeling via bone resorption and bone formation (or bio-logical calcification) happens throughout our lifetime. Os-teoblasts and osteoclasts are the primary cells involved inbone remodelling.1 The functional coupling between thesetwo types of cells is the basis of bone growth, metabolism, andrepair.2,3

Low-intensity laser therapy has become an accepted toolfor several clinical applications and has been successfullyestablished in regenerative medicine and dentistry becauseof its anti-inflammatory, analgesic, and regenerative effects.4

The latter effect, also called photostimulation, produces non-destructive events as well as increased cellular activity on

tissues at the cellular level, and it has been used in a varietyof medical therapies.5,6

In the last two decades photodynamic therapy (PDT) hasbeen successfully employed for clinical treatment of cancerusing different photosensitizer molecules. Photosensitizersare the active agents in the photodynamic process. PDT isbased on the photoactivation of photosensitizers when irra-diated by light in a specific wavelength window (generallythe maximum absorption band of the photosensitizer com-pound). The light-excited photosensitizer generates reactiveoxygen species (ROS) that induce reduction or destruction oftumors by multifactorial mechanisms.7

The explanation for the photobiological effects of laserlight is based on the light absorption by primary endogenouschromophores (mitochondrial enzymes, porphyrins, flavins,and cytochromes).8 Many in vivo and in vitro studies have

1Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto, Departamento de Quımica – Universidade de Sao Paulo, Ribeirao Preto, SaoPaulo, Brazil.

2Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Departamento de Ciencias Farmaceuticas, Universidade de Sao Paulo, RibeiraoPreto, Sao Paulo, Brazil.

3Departamento de Cirurgia Buco-Maxillofacial e Periodontologia, Faculdade de Odontologia de Ribeirao Preto, Universidade de SaoPaulo, Ribeirao Preto, Sao Paulo, Brazil.

Photomedicine and Laser SurgeryVolume 29, Number 10, 2011ª Mary Ann Liebert, Inc.Pp. 699–705DOI: 10.1089/pho.2010.2929

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reported on the influence of laser irradiation on the cellularfunctional state.9–11

The exact mechanism of action of the laser irradiation inliving cells is not yet understood, although the photo-stimulation and therapeutic effects of low-power visible ir-radiation of different wavelengths and light doses are wellknown.1,4,9–15 Experimental and clinical studies have sug-gested that low-intensity laser irradiation affects cellularmetabolic processes, leading to enhanced regeneration ofbiological tissues.16,17 The biological effects of irradiationdepend upon wavelength, power, time of exposure, and ir-radiation dose or fluence.5,18

Lubart et al.19 have suggested that low-intensity laser ir-radiation might promote changes in the cellular redox state,playing a pivotal role in sustaining cellular activities, thuspromoting photobiostimulative processes. Other studies,however, have emphasized that different biological re-sponses can be achieved, depending on the applied dose,wavelength, irradiation time, and the conditions of thetreated tissue.20–22 Laser light processes present some specialfeatures that allow them to selectively and precisely influ-ence some targets, including biological ones.11,12,18 Smalldoses induce stimuli, large doses depress physiologicalprocesses, while extreme ones cause destruction of biologicalstructures (for example, lipoperoxidation or heating, whichcauses protein denaturation).5,18,23–26

Some in vitro effects of photostimulation on the extracel-lular matrix, collagen production, macrophage stimulation,and fibroblast proliferation have been described.27,28

Some authors20,29 have suggested that the biologicalmechanism behind the effects of light used in therapy is re-lated to absorption of red and near-infrared light by chro-mophores contained in the protein components of therespiratory chain located in the mitochondria, particularly incytochrome c oxidase. Moreover, stimulation of the latterenzyme in isolated extracts has been recently demonstrated.8

Therefore, the purpose of this work is to associate thephotosensitive drug aluminum phthalocyanine chloride andlow intensity laser in order to study its efficiency in biocel-lular biostimulation and in vitro osteogenesis through thephotodynamic process for a possible acceleration of the bonedamage recovery. The main purpose of this study was toinvestigate the in vitro effects of visible light radiation at670 nm, as obtained from an Eagle diode laser (QuantumTech, Sao Carlos, SP, Brazil) at low doses varying between0.5 and 3 J/cm2, combined with the previous application ofan aluminum phthalocyanine chloride (AlClPc) dye as aphotosensitizer, on the growth and differentiation of osteo-blastic cells using a primary osteoblastic cell line isolatedfrom the bone marrow of rats. New evidence that low-intensity laser irradiation may play an important role inpromoting bone formation is presented on the basis of theMTT assay, collagen content, total protein content, alkalinephosphatase (ALP) activity, and bone-like nodule formation.

Methods

Materials

All the solutions were prepared using pure apyrogenicwater (Millipore Direct-Q, Millipore, Barueri, SP, Brazil). Allthe reagents were of the highest commercially available pu-rity. Epikuron, Poloxamer 188, Polomazer 170, and soybean

phospholipids were acquired from Lucas Meyer, France.Miglyol 812 N Hulls (Witten, Germany) was purchased fromPuteaux, France. Aluminum chloride phthalocyanine or(chlorine [29H, 31H-phthalocyaninato] aluminum) (AlClPc),3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-mide (MTT), b-glycerophosphate, sodium lauryl sulfate(SDS), Lowry solution, phenol reagent of Folin-Ciacalteau,bovine albumin, alizarin red, and dexamethasone were ob-tained from Sigma-Aldrich Co., St. Louis, MO. Hank’s buffer,a-MEM, trypsin, ascorbic acid, fetal bovine serum, gentami-cin, and fungizone were supplied by Gibco – InvitrogenTechnologies (Grand Island, NY). Acetic acid, chloramine T,and Ehrlich’s reagent were provided by Acros, Pittsburgh,PA. Glutharaldehyde and sodium cacodylate were obtainedfrom Electron Microscopy Sciences, Hatfield, PA. ALP ac-tivity was measured using a commercial kit (DiagnosticLabtest, Belo Horizonte, MG, Brazil).

In addition to these materials, different organic solventsand other inorganic salts commonly used in the laboratorywere employed. All the solvents were analytical grade.

Preparation of nanoemulsions

The nanoemulsions (NE) were obtained by the spontane-ous emulsification process described by Yu et al.30 Briefly,the organic phase (acetone) was prepared with oil, soyphospholipid, and AlClPc, at 55�C. The photosensitizer hadbeen previously dissolved in Miglyol 812N and was addedto the phospholipid organic solution at a concentration of0.05 mg/mL. The organic solution was slowly added to theaqueous phase containing Poloxamer 188, under magneticstirring. After total addition of the organic solution andspontaneous emulsification, the organic solvent was re-moved by evaporation under reduced pressure, at 65�C. Fi-nally, the formulations were concentrated to a final volumeof 10 mL. Formulations without the drug were preparedunder the same conditions, for use as reference compoundsin the spectroscopic analyses. All formulations were charac-terized by their mean size, polydispersity index (PdI), and fpotential, as described by Siqueira-Moura et al.31 The meansize and PdI of the colloidal dispersions were determined at25�C by laser light scattering at an angle of 173o, whereas thef potential was measured by electrophoretic mobility using aZetasizer� (Nano ZS, Malvern PCS Instruments, Worcester-shire, UK). Data are the mean ( – SD) of three different in-dependent preparations.

Rat bone marrow cell isolation and culture

Cells were obtained and cultured according to Maniato-poulos et al.,32 with modifications standardized by Simaoet al.33 Bone marrow was obtained from young adult malerats of the Wistar strain weighing 110–120 g. The femorawere aseptically excised, cleaned of soft tissues, and washedthree times (15 min each) in culture medium containing 10times the usual concentration of antibiotics.

The femoral epiphyses were cut off, and the marrow wasflushed out with 20 mL culture medium. Released bonemarrow cells were collected in a 75 cm2 plastic culture flaskcontaining 10 mL osteogenic culture medium, which allowsfor the osteoblastic differentiation and is composed by a-MEMsupplemented with 15% fetal bovine serum, 50 lg/mL gen-tamicin, 0.3 lg/mL fungizone, 10- 7 M dexamethasone, 5 lg/

700 ZANCANELA ET AL.

mL ascorbic acid, and 2.16 mg/mL b-glycerophosphate.33

Cells were cultured until subconfluence enzymatically-re-leased and first-passage cells were cultured in the same me-dium, at a concentration of 2 · 104 cells per well, in 24-wellmicroplates (Falcon, Franklin Lakes, NJ). Cells were culturedup to 21 days at 37�C in a humidified atmosphere of 5% CO2

and 95% air, and the medium was changed every 48 h.

Cytotoxicity assays

The methodology used to evaluate cell viability wasthe classical MTT assay. The tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)produced the highly coloured formazan dye upon NADHreduction, which reflects a living cellular dehydrogenase.34

To investigate the toxicity of the photosensitizer, a suspen-sion of 2 · 104 osteoblastic cells in 1000 lL medium was ad-ded into each well in the 24-well microplate. After culturingin CO2 incubator for 24 h, monolayer cultures of osteoblastswere treated with AlClPc/NE at a final concentration of0.5 lmol/L (for the darkness toxicity and photobiologicaltoxicity assays). Three hours after incubation, the cells thatwere going to be used for the darkness toxicity experimentwere washed twice, and the volume was completed withaddition of 1000 lL a-MEM to each well, overnight.

The 1.0 mg/mL MTT solution (250 lL per well) was addedto the cells placed in the 24-well microplates, followed byincubation for 4 h, at 37�C. The crystals formed as a result ofinteraction between the mitochondrial dehydrogenases andthe MTT reagent were dissolved with 2-propanol. The sam-ples were shaken until complete dissolution of the formedproduct. After the reaction was finished, the absorbance at560 and 690 nm of each well was measured by means of theSafire II multiplate reader from TECAN (Tecan Trading AG,Mannedorf, Switzerland), for determination of mitochon-drial dehydrogenases and MTT, respectively. Cell viability isexpressed as the percentage of cells incubated withoutAlClPc/NE.

Photobiological assays

For the photobiological tests, the cells were washed twiceand the volume was completed by addition of 1000 lL phos-phate buffer to each well of the microplate. Then, the wellswere irradiated with the following light dosages: 0.5, 1.0, and3.0 J/ cm2, using the Eagle diode laser (Quantum Tech, SanCarlos, SP, Brazil). The light source was a continuous-wavelaser operating at 670 nm, with maximum power of 50–300 mW in the end of the optical fibers. The Eagle diode lasermodel is a laser system that operates in a set wavelength of670 nm of the visible electromagnetic spectra. The system isattached to an optical fiber system (200 lm) for light delivery.For the red laser, the active material is a diode emitter oper-ating with a 50–300 mW potency through an optical fiber,continuous operation.

The laser equipment used presents all the internationallyrequired specifications and a calibration point that guaranteesthat the programmed dosage be the same as that dischargedby the outlet spot of the laser. The description of laser irra-diation parameters used in this article are shown in Table 1.

After irradiation, the phosphate buffer was removed, thevolume was completed by addition of 1000lL osteogenic me-dium to each well, and the solution was incubated overnight.

The percentage of cell viability was determined using theMMT assay and is expressed as the percentage of cultureswithout either photosensitizer or laser irradiation.

Total protein content

Total protein content was calculated at 7 days using amodification of the Lowry method.35 Culture medium wasremoved, and the wells were washed three times with PBS at37�C and filled with 2 mL 0.1% sodium lauryl sulfate. After30 min, 1 mL of this solution (from each well) was mixedwith 1 mL Lowry solution and left for 20 min at room tem-perature. After this period, 0.5 mL of the solution of phenolreagent of Folin and Ciocalteau was added, and the solutionwas left to stand for 30 min at room temperature, to allowcolor development. Absorbance was then spectrophotomet-rically measured at 680 nm, and the total protein content(lg/mL) was calculated from a standard curve.

Collagen content

The collagen content was calculated at 7 days, accordingto the method of Reddy and Enwemeka.36 Samples of thesame solutions used for determination of the total proteincontent were assayed for collagen content. Aliquots con-taining 0.5 mL of this solution were lyophilized and re-suspended in 50 lL acetic acid 6 N. Then, the samples werehydrolyzed by autoclaving at 120�C for 30 min. After thisprocedure, 450 lL chloramine-T was added to the hydroly-sate, which was then mixed gently, and the oxidation wasallowed to proceed for 25 min at room temperature. Next,0.5 mL Ehrlich’s aldehyde reagent was added to each sample,followed by gentle mixing, and the color was developed byincubating the samples at 65�C for 20 min. The absorbancewas measured at 550 nm in a spectrophotometer, and thecollagen content was calculated from a standard curve andexpressed as lg/mL.

ALP activity

The ALP activity was assayed at 7 days as the release oftymolphtaleine from tymolphtaleine monophosphate using acommercial kit, and the specific activity was calculated.Aliquots of the same solutions used for calculating totalprotein content were assayed for the ALP activity, accordingto the instructions on the kit. The absorbance was thenspectrophotometrically measured at 590 nm, and the ALPactivity was calculated (lmol tymophtaleine/h/mg) from astandard curve.

Bone-like nodule formation

Bone-like nodule formation was assessed at 21 days byalizarin red S method. The culture medium was removed,

Table 1. Description of Laser Irradiation Parameters

0.5 J/cm2 1.0 J/cm2 3.0 J/cm2

Power (mW) 150 150 150Beam diameter

at the surface (cm)1 1 1

Irradiation time (s) 3 6 18

DRUGS AND LIGHT STIMULATION EFFECT ON OSTEOBLAST GROWTH 701

cells were washed three times with PBS at 37�C, and theattached cells were fixed in 10% formalin for 24 h, at roomtemperature. After fixation, the specimens were dehydratedthrough a graded series of alcohol and processed for stainingwith alizarin red, which stains calcium-rich bone-like nod-ules. Images were obtained for documentation; the fieldswere randomly selected from different wells using a micro-scope Axiovert 40 CFL Ph 1 with objective 10x / 0.25 coupledwith camera 7.2 (Zeiss, Thornwood, NY). Quantification ofstaining was assessed by a colorimetric method described byGregori et al.37 Then, 360 lL 10% acetic acid was added toeach well previously stained with alizarin red, and the platewas shaken for 30 min at room temperature. The contents ofeach well were transferred to Eppendorf tubes (EppendorfManufacturing Corp., Enfield, CT), which were centrifugedat 20,000g for 15 min. Next, 100 lL supernatant from eachtube were transferred to new tubes. Finally, 40 lL ammo-nium hydroxide 10% were added, to neutralize the acid, andthe absorbance was measured at a wavelength of 405 nm.

Statistical analysis

Data presented in this work are the result of a singleculture with n = 3 for each surface treatment, for each ex-periment. All the data were submitted to an analysis ofvariance (One-Way ANOVA) followed by Tukey’s test whenappropriate. Differences at p < 0.05 were considered statisti-cally significant.

Results

Cytotoxicity assays

Cytotoxicity studies performed in the absence of light andusing osteoblasts as a biologicical model and a photosensi-tizer (AlClPc) combined in NE (AlClPc/NE) are described inTable 2. As observed, cell viability in the presence of the NEalone or in the presence of AlClPc/NE remained unaffectedwith up to 3.0 lmol/L photosensitizer. For AlClPc/NE con-centrations > 3.0 lmol/L, there was a reduction in totalprotein content and ALP activity.

Because the cytotoxic effect was observed from 1 lmol/LAlClPc/NE thereafter, further biostimulation studies wereconducted using the 0.5 lmol/L concentration only, whichwould guarantee a good safety margin.

Photobiological assays

The photostimulation results obtained by using low-powerlaser and AlClPc/NE (0.5 lmol/L) during the in vitro assay of

osteoblasts cells are described in Fig. 1. Irradiation of smalldoses, *0.5 J/cm2, resulted in significant increase in cell via-bility, total protein, ALP activity, and collagen, suggestingstimulation of cell growth. On the other hand, this stimulationprocess wasn’t observed when irradiation of about 1 J/cm2 wasemployed. An irradiation of 3 J/cm2 led to considerable re-duction in cell viability and, consequently, loss of total protein,and collagen content as well as ALP activity, indicating pho-totoxicity. The use of light from low-power laser, in the absenceof AlClPc/NE, did not affect any of the parameters studiedhere up to irradiations at 3 J/cm2 (results not shown).

Bone-like nodule formation

The experiments concerning bone-like nodule formationevidenced formation of calcified nodules, quantified bymeans of three parameters, namely, control (ct), treatmentwith AlClPc/NE at a light dose of 0.5 J/cm2, and treatmentwith AlClPc/NE at a light dose of 3.0 J/cm2. As observed inFig. 2B, irradiation at 0.5 J/cm2, elicited increased minerali-zation of bone-like nodules, compared with ct (Fig. 2A).However, Fig. 2C reveals that radiation at 3.0 J/cm2 led to alarge reduction in the number of nodules, thus reinforcingthe cytotoxic condition.

Discussion

As far as laser therapy is concerned, it is known that thistype of treatment has been successfully employed over thepast years and its applications are countless. Its effect has

Table 2. In vitro Toxicity of AlClPc/NE in Cultured Osteoblasts. Tested in the Absence

of Light After 3 H of Incubation with the Investigated AlClPc/NE Concentrations

[AlClPc] Cell viability Total protein ALP activityOsteoblasts cultures (lmol/L) (%) (lg/mL) (lmol thymolphthalein / h/ mg)

Control - 100 21.1 – 1.2 616.11 – 52.1NE - 99.9 – 1.2 20.9 – 0.8 660.29 – 23.9AlClPc/NE 0.5 100.6 – 3.5 20.4 – 0.9 656.86 – 9.8AlClPc/NE 1.0 96.6 – 1.0 19.9 – 1.3 623.12 – 30.2AlClPc/NE 3.0 98.1 – 5.6 19.6 – 0.6 *326.53 – 30.6AlClPc/NE 5.0 *38.4 – 2.6 *15.0 – 0.4 *326.67 – 30.3

*Represents results considered statistically significant.

FIG. 1. In vitro with AlClPc / NE in culture of osteoblasts inthe presence of light. Laser dosage of 0 (control), 0.5, 1.0, and3.0 J/cm2.

702 ZANCANELA ET AL.

already been proved in the case of orthodontic treatment,38

bone defects,39,40 and bone fractures,41 and after implantplacement.42

The biostimulation assays conducted here demonstratedincrease in the number of viable cells with high levels of totalprotein and collagen content as well as ALP activity at a laserdose equal to 0.5 J/cm2. This suggests that cellular biosti-mulation occurred because of the combination of a photo-sensitive drug (AlClPc/NE) with a laser energy dose.Because AlClPc is hydrophobic, only the formulation incor-porated into the NE was studied here.39

Indeed, experimental studies have shown that lower doseshave no effect and that very high doses have an inhibitorytoxic effect on cell cultures. An example of such studies is thework of Van Breughel et al.,43 who tested the effect of laserlight output (HeNe laser; wavelength 633 nm) on humanfibroblast cultures.

Using laser stimulation, researchers have observed thedifferentiation of mesenchymal stem cells into osteoblasts ina three-dimensional biomatrix.44 Another study has shownthat low-intensity laser therapy has a positive effect on thebone healing of diabetic rats.45

Finally, a recent work has described that the use of low-intensity laser (830 nm) at 1.91 J/cm2, without associationwith a photosensitizer, stimulates in vitro mineralizationthrough increased insulin-like growth factor (IGF-I) andbone morphogenetic protein production, which occurs as aresult of Runx2 expression and ERK phosphorylation inmouse osteoblastic MC3T3-E1 cells, associated with an in-crease in the calcium content of the cell culture.46

Currently, there are not many studies using laser therapyassociated with a photosensitizer for the stimulation of bonecell growth.

As described by other authors,14 a possible mechanism forthe biostimulation of healing by low-level laser light is the

absorption of light energy by mitochondria, which increasesthe energy of the osteoblast, thereby stimulating release ofchemical mediators. In addition, biostimulation (in the visibleregion) could be associated with the irradiated light, leading tothe generation of minimum levels of ROS. The latter species, inturn, play an important role in the triggering of many cellularprocesses. On the other hand, high concentrations of ROS causecell death, probably by ATP depletion and lipid peroxidation.

Conclusions

Unlike biostimulation, this technique seeks to increase cellgrowth and this is obtained only by using a low light dose( < 1.0 J/cm2). Therefore, whether biostimulation of osteo-blastic cell cultures by PDT or the cytotoxic effect of thistherapy occurs only depends upon the light dose, and theresults can be completely reversed.

Acknowledgments

This work was partly supported by Conselho Nacional deDesenvolvimento Cientifico e Tecnologico (CNPq), Fundacaode Amparo a Pesquisa do Estado de Sao Paulo (FAPESP),and Coordenacao de Aperfeicoamento de Pessoal de NivelSuperior (CAPES). F.L.P. and D.C.Z. received Ph.D. and M.S.scholarships from FAPESP and CNPq, respectively. Wethank Cynthia Maria de Campos Prado Manso and PriscilaCerviglieri for linguistic advice.

Author Disclosure Statement

No conflicting financial interests exist.

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Address correspondence to:Antonio Claudio Tedesco

Departamento de QuımicaFaculdade de Filosofia Ciencias e Letras de Ribeirao Preto

FFCLRP - USP, 14040-901Ribeirao Preto, SP

Brazil

E-mail: atedesco@usp.br

DRUGS AND LIGHT STIMULATION EFFECT ON OSTEOBLAST GROWTH 705