the effect of low-level laser therapy (810 nm) on root development of immature permanent teeth in...

ORIGINAL ARTICLE

The effect of low-level laser therapy (810 nm) on root developmentof immature permanent teeth in dogs

Reza Fekrazad & Bahman Seraj & Sara Ghadimi &Parvin Tamiz & Pouriya Mottahary &

Mohammad-Mehdi Dehghan

Received: 20 December 2013 /Accepted: 24 April 2014# Springer-Verlag London 2014

Abstract Traumatic injuries and dental caries can be a bigchallenge to immature teeth. In these cases, the main purposeof treatment is to maintain the pulp vitality. The purpose ofthis study was to investigate the effect of low-level lasertherapy on accelerating the rate of dentinogenesis inpulpotomy of immature permanent teeth (apexogenesis).Three dogs, 4–6 months old, were used in this study. Onejaw in each dog was randomly assigned to laser irradiationgroup. All selected teeth were pulpotomized with mineraltrioxide aggregate (MTA) and restored with amalgam. In thelaser group, the Ga-Al-As laser (810 nm, 0.3 W, 4 J/cm2, 9 s)was used on buccal and lingual gingiva of each tooth in 48 hintervals for 2 weeks. In order to observe the newly formeddentine, tetracycline was injected on the 1st, 3rd, 7th, and 14thday after the operation. Then, ground sections of teeth wereobserved under a fluorescence microscope. The data wasanalyzed with Generalized Estimating Equations (GEE) test.The mean distance between the lines of tetracycline formed onthe 1st and 14th day was significantly higher in the laser group

(P=0.005). Within the limitation of this study, irradiation ofGa-Al-As laser (810 nm) can accelerate the rate ofdentinogenesis in apexogenesis of immature permanent teethwith MTA in dogs.

Keywords Pulpotomy . Low-level laser therapy .Mineraltrioxide aggregate . Dog

Introduction

Traumatic injuries and dental caries are the greatest challengeto immature permanent teeth which can cause the arrest ofnormal tooth development. The primary goal of treatment inthese cases is to maintain the pulp vitality; therefore,apexogenesis is the treatment of choice [1, 2].

Apexogenesis is defined as the physiological rootdevelopment, not only restricted to the apical segmentbut also deposition of dentin throughout the length ofthe root [3]. The abundant vascular supply of the im-mature tooth after the process of apexogenesis providesa greater potential for recovery following injury andstronger roots to withstand fracture in the event offurther trauma [3, 4].

In traumatic injuries, the pulp–dentin complex reacts toexposure of dentin due to fracture and subsequent bacterialinvasion into dentinal tubules which give rise to differenthealing and defense responses of the pulp [5].

During the years, a variety of materials have beenused in vital pulp treatment like calcium hydroxide anddentine bonding agents. Mineral trioxide aggregate(MTA) is one of the materials which is used in differenttypes of vital pulp therapy [6–8]. Several in vitro andin vivo studies have reported MTA as a biocompatiblematerial. It has been reported that it provides a greatseal and has excellent marginal adaptation. In addition,

R. FekrazadLaser Research Center in Medical Sciences, AJA University ofMedical Sciences, Tehran, Islamic Republic of Iran

B. SerajDental Research Center, Department of Pediatric Dentistry, Facultyof Dentistry, Tehran University of Medical Sciences, Tehran,Islamic Republic of Iran

S. Ghadimi (*)Laser Research Center of Dentistry, Department of PediatricDentistry, Faculty of Dentistry, Tehran University of MedicalSciences, Tehran, Islamic Republic of Irane-mail: [email protected]

M.<M. DehghanDepartment of Veterinary Surgery and Radiology, Faculty ofVeterinary Medicine, University of Tehran, Tehran,Islamic Republic of Iran

Lasers Med SciDOI 10.1007/s10103-014-1588-2

its good physical characteristics and the ability to main-tain a high pH for a long period of time suggest MTAas a suitable material to be used in vital pulp therapy[8].

However, the apexogenesis process requires a long treat-ment time which can increase the risk of treatment failure.Considering this fact, using a method to accelerate the processis of increasing interest [9].

Dentinogenesis involves different mechanisms like celldifferentiation and interactions, synthesis of an organic matrix,and the eventual mineral crystals formation in this extracellu-lar matrix [10]. Accelerating the rate of dentinogenesis in theprocess of apexogenesis can occur via any of thesemechanisms.

Several studies have demonstrated the wide range of thebenefits of low-level laser therapy (LLLT) for biological tis-sues. It has been reported that LLLT can increase the func-tional activity of the cells, promote cell proliferation, andreduce inflammation. Moreover, it has analgesic effects as aresult of increased endorphin release [9, 11].

Also, previous studies have demonstrated that LLLT usedon the exposed pulp reduces the inflammation [12]. It hasbeen shown that low power laser irradiation can acceleratetissue repair by formation of a fibrous matrix and dentinbridge [12, 13], and increases the production of collagenous[12] and non-collagenous proteins [14] from the extracellularmatrix (ECM). These proteins have an important role inmineralized tissue formation and in differentiation, migration,and proliferation of cells during the different stages ofdentinogenesis [14].

However, we did not find any reports on the effects ofLLLT on root development of pulpotomized teeth. Consider-ing the positive effect of MTA and LLLT on the dental pulp,we hypothesized that the combination therapy of pulpotomywith MTA and LLLT could have positive effects on the rootdevelopment of the permanent teeth in dogs. The aim of thisstudy was to investigate the effect of combined therapy ofMTA and LLLT on the rate of dentinogenesis in immaturepermanent teeth in dogs.

Materials and methods

Animal models

Three healthy male dogs of Iranian mixed generation in mixeddentition, aged 4–6 months, were used for the present study.The ethics committee of Tehran University of Medical Sci-ences approved the experimental protocol (#89-04-97-12065-26589). A period of 2 weeks was considered in order tovaccinate the animals and standardize their diet and environ-mental condition. Prior to each treatment, the animals wereanesthetized with the injection of ketamine hydrochloride(5 mg/kg) and diazepam (1 mg/kg) and hydrated with ringerlactate solution [15]. All teeth were caries free and pre-operative radiographs were taken from anterior and premolarteeth to confirm the immaturity and rule out any pathosis(Fig. 1).

Experimental procedures

In each group, 20 teeth with appropriate distances fromthe second and third incisors, canines, and premolarswere assigned to the experiment. The quadrants of eachjaw were randomly divided into irradiation and non-irradiation groups. After fixing the mouth opener andtongue retractor, the teeth were isolated with cottonrolls. Then, the pulps of the selected teeth were exposedwith a diamond high speed bur with copious waterspray. The access cavities were completed using anappropriate round bur in a high speed handpiece. Thecoronal portion of the pulps was removed by the samebur. The pulp chambers were rinsed with sterile normalsaline solution and hemostasis was achieved by placinga small moistened sterile cotton pellet directly on thepulp with a light pressure for 5 min. Then, MTA pow-der (Angelus, Londrina, Brazil) was mixed with normalsaline in a 1:1 ratio (according to the manufacturer’sinstructions) and the pulps were covered with a thicklayer of MTA. After that, all the cavities were sealed

Fig. 1 a Occlusal radiograph ofmaxillary anterior teeth; bocclusal radiograph ofmandibular anterior teeth; and clateral radiograph of the dogs’ jawwere taken before operation

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with a layer of zonalin (Zonalin, Kemdent, Purton,Swindon, Wiltshire, UK) and restored with amalgam.

Low-level laser therapy

The teeth in the irradiation group received laser therapy onmid root areas of the buccal and lingual surfaces in 48 hintervals for 2 weeks (Fig. 2). The Gallium–Aluminum–Ar-senide laser (DenLase, China) with a wavelength of 810 nmand output power of 300 mWwas used in this study. The laserbeam was delivered with an optical fiber of 0.9 cm diameter.The teeth were irradiated with a continuous emission of thelaser for 9 s,E=2.7 J and the total dose of each application was4 J/cm2.

Histological evaluation

In order to observe newly formed dentin, tetracyclinehydrochloride was injected intravenously to each dog 1,3, 7, and 14 days after irradiation. Ten days after thelast irradiation, the teeth were examined clinically andradiologically which revealed no pathologic signs in

none of the teeth. Then, the dogs were sacrificed byvital perfusion. The jaws were separated from the ani-mals and kept in 10 % formalin solution. Radiographswere then taken from jaws to determine the approximateareas of the following cuts (Fig. 3). The teeth wereseparated with a high speed saw (Dorsa, Iran) andground sections were prepared using a low speed saw(Isomet, Beuhler, Lakebluff, IL, USA). Each tooth wasfirst sectioned horizontally in the apical third of the rootand then sectioned vertically parallel to the longitudinalaxis of the tooth. The slices were 100 μm thick andembedded in normal saline 0.9 % in a dark container.The samples were then observed under a fluorescencemicroscope (Olympus, Tokyo, Japan) using the U-MW2filter (Universal- Mirror unit Wide band2, Excitationfilter: BP 400–410 nm, Dichroic mirror: DM 455)(Figs. 4 and 5). An image was obtained from eachsample in×20 magnification using a software (analySISLS Professional, Germany), and the distances of tetra-cycline lines were measured by two calibrated observersusing the same software (Fig. 6). The observers wereblinded to the group classification. The SPSS 20 soft-ware (Statistical Package for the Social Science, Chica-go, IL, USA) was used for analysis using GeneralizedEstimating Equations (GEE) test. The significance levelwas set at 0.05.

Result

At the beginning of the study, there were 20 teeth in eachgroup. After the observation of samples under the fluores-cence microscope, the tetracycline lines were not detectable infive samples of each group so they were excluded from theanalysis.

Statistical analysis showed that the distance betweentetracycline lines during the intervention in vertical andhorizontal slices were in the same range, so the bestsection was selected for analysis in each sample(Fig. 7).

Fig. 2 The teeth in the irradiation group received laser therapy on midroot areas of the buccal and lingual surfaces in 48 h intervals for 2 weeks

Fig. 3 After cutting the jaws,parallel radiographs were takenfrom maxillary (a) andmandibular (b) sections of jawsto determine the approximateareas of the following cutsfrom teeth and to measuredistance from the borders ofbone to apex of the tooth

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As it is shown in Table 1, the mean distance betweentetracycline lines during the intervention (line 1 and 14)was 91.2 μm in the irradiation group and 80.12 μm inthe non-irradiation group (P=0.005). Statistical analysisrevealed significantly different results between the dis-tances of tetracycline lines on the 1st and 3rd(P<0.001), 3rd and 7th (P=0.005), 1st and 7th (P=0.013), 3rd and 14th (P=0.01), and 7th and 14th days(P=0.005). The findings showed that the rate ofdentinogenesis was significantly higher in the irradiationgroup versus the non-irradiation group in all the dis-tances of tetracycline lines.

Discussion

In an attempt to find a technique to accelerate bothdentinogenesis and apexogenesis, we used combined

therapy with MTA and Ga-Al-As laser (810 nm), tospeed up the treatment and improve the prognosis oftreated teeth. Statistical analysis of data obtained frompathologic analysis showed a greater amount ofdentinogenesis in apexogenesis of immature permanentteeth with MTA in dogs in the laser radiation groupthan the non-laser group.

Because of the modern method of treatment in thepresent study, it was impractical and sometimes unethi-cal to test it in human. Therefore, an animal model wasused to study various aspects of this procedure [16].Dogs have comparable dental tissue structures andhealthy and diseased states as observed in humans. So,they are one of the most frequently used animal speciesin regenerative endodontics [17].

Wilson in 1996 reported that all of the apices ofpermanent teeth in dogs younger than 6 months of ageare open [16]. Therefore, we used 4–6 months old dogsin this study.

Since 1956, several studies have reported that tetra-cyclines incorporate into tissues calcifying at the time oftheir administration, which results in the discoloration ofdeveloping teeth [18, 19]. Also, it has been reportedthat histopathologic examination of the ground sectionof such teeth under the ultraviolet light shows golden-yellow fluorescence bands in dentin. These bands cor-respond to the dose and administration pattern of tetra-cycline [20, 21]. The pattern of tetracycline lines indentin follows the growth increments of the tooth andcan be an excel lent indica tor of the ra te ofdentinogenesis [20]. Considering these facts, tetracyclinewas used as the indicator of root development in thepresent study.

The pulp shows a repair potential in accidental cor-onal exposure after traumatic dental injuries. The pulpwound healing process contains fibrinogen exudationunder capping material, replacement of inflammatoryinfiltration with granulation tissue along wound surfacewhich consist newly formed fibroblasts and capillary

Fig. 4 Tetracycline lines in a horizontal slice under fluorescencemicroscope

Fig. 5 Tetracycline lines in a vertical slice under fluorescencemicroscope

Fig. 6 The distances of tetracycline lines were measured by two cali-brated observers

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vessels, increase of fibroblast layers around the lesion,and synthesis of new collagen fibers. After several days,calcifying nodules are found in new extracellular andlater dentin bridge can be seen [5].

Retaining the vitality of an exposed dental pulp fol-lowing any trauma or decay in newly erupted permanentteeth would be the main goal of treatment. Previousstudies have reported the positive effect of MTA in vitalpulp therapy [9]. Moreover, the positive effect of LLLTin the process of dentinogenesis has been already doc-umented [12, 13, 22, 23]. Therefore, we hypothesizethat combination therapy with MTA and LLLT couldaccelerate the process of apexogenesis.

Since the laser beam had to pass the surroundingtissues of the teeth and reach the dental pulp cells, itwas needed to use a laser with an infrared wavelength(700–1,000 mm). The energy of such lasers has a lowabsorption coefficient in hemoglobin and water [9].Therefore, we used a diode laser with a 810-nm wave-length, in addition diode lasers are portable, inexpensivedevices, which have been used for LLLT.

Previous studies suggest 2 to 4 J/cm2 density ofirradiation when using directly on the teeth or indirectlyabove the apex [24]. In this study, 4 J/cm2 energydensity was used.

In order to prevent the accumulated dose of LLLT whichcan result in the bio-inhibition range, we chose 48 h intervalsfor the treatment.

Several studies have reported the positive effect ofLLLT on dental pulp tissues. In 1998, Utsunomiya showedthat Ga-Al-As laser irradiation (output power=300 mW,wavelength=830 nm, and power density=105.9 J/s/cm2)on exposed pulps could accelerate the dentin bridge for-mation and expression of collagens in dogs [12]. Theseresults confirm our findings.

In a study by Godoy et al. (2007), low-power laser irradi-ation (660 nm, 30 mW, and 2 J/cm2) was used in Class Icavities prepared in human teeth. Microscopic analysisshowed odontoblast processes in higher contact with the ex-tracellular matrix, and collagen fibrils were more aggregatedand organized than those of the control group [25]. Almost90 % of the dentin organic matrix consists of collagen [10]although acceleration of its expression and maturation canresult in the acceleration of dentin formation, as shown inour study.

Ohbayashi et al. and Matsui et al. reported enhance-ment of calcified nodule formation in human dentalpulp (HDP) cells irradiated with Ga-Al-As laser [13,23]. Matsui et al. demonstrated that the ability of LLLTto accelerate the dentin formation might be also attrib-uted to the stimulation of ·OH production in HDP cellswhich can promote their mineralization [23].

Tate et al. also reported that Ga-Al-As laser irradiation(810 nm and 0.5 to 1.5 W) on the mesial surface of the upperright first molar ofWistar rats induced the formation of tertiarydentin by influencing the secretary activity of odontoblasts

Distance between

lines 1 and 3 in ver�cal sec�on

Distance between

lines 1 and 3 in horizontal sec�on

D istance between

lines 3and 7

in ver�cal sec�on

D istance between

lines 3and 7

in horizontal sec�on

D istance between

lines 7and 14

in ver�cal sec�on

D istance between

lines 7and 14

in horizontal sec�on

Fig. 7 The distance betweentetracycline lines during theintervention in vertical andhorizontal slices were in thesame range

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[22] which is consistent with our results. None of these studiesreported the positive effect of LLLTon one of the mechanismswhich are involved in dentinogenesis; however, the rate ofdentinogenesis was measurable during the intervention in thepresent study.

In an animal study, Toomarian et al. showed thatlow-level laser irradiation (808 nm, 300 mW with thesame condition as to our study) accelerated rat molarroot development, and histological findings showed thatthe occurrence of secondary cement formation was sig-nificantly higher in laser groups [9]. The teeth wereintact and the acceleration of root development wasshown with mammography. However, in the presentstudy, the teeth were pulpotomized with MTA and the

rate of dentinogenesis was observed using histopatholo-gy evaluations.

In the present study, the positive effect of combinationtherapy of pulpotomy with MTA and LLLT was shown.However, further studies with larger sample sizes and long-term follow-ups are required.

Conclusion

Considering the limitations of this study, combined therapywithMTA and LLLTcan increase the rate of dentinogenesis inimmature permanent teeth in dogs.

Table 1 Distances between tetracycline lines in MTA and MTA + laser groups (μm)

MTA group MTA + laser group P value

Distance between the lines of the 1st and 3rd days Mean 9.83 11.67 <0.001Median 10.03 12.18

Standard deviation 5.78 7.00

Maximum 13.95 14.65

Minimum 2.37 2.70

Distance between the lines of the 3rd and 7th days Mean 28.26 32.89 0.005Median 28.47 33.22


Maximum 38.34 45.65

Minimum 4.96 5.79

Distance between the lines of the 1st and 7th days Mean 39.03 44.72 0.013Median 39.77 43.66


Maximum 48.98 58.71

Minimum 7.06 7.57

Distance between the lines of the 7th and 14th days Mean 41.08 46.56 0.005Median 42.57 47.40


Maximum 57.29 63.27

Minimum 6.92 8.45

Distance between the lines of the 1st and 14th days Mean 80.12 91.27 0.005Median 79.33 93.50


Maximum 106.27 121.98

Minimum 13.36 15.05

Distance between the lines of the 3rd and 14th days Mean 70.29 79.60 0.01Median 70.79 82.84


Maximum 96.24 107.33

Minimum 11.94 13.51

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Acknowledgments This project has been supported by Tehran Univer-sity of Medical Sciences & health Services grant number 89-04-97-12065.

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