Effect of LLLT on autogenous bone grafts in the repair of critical size defects in the calvaria of immunosuppressed rats

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Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7

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Journal of Cranio-Maxillo-Facial Surgery

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Effect of LLLT on autogenous bone grafts in the repair of critical sizedefects in the calvaria of immunosuppressed rats

Valdir Gouveia Garcia a,b, Angelita Strazzi Sahyon a, Mariéllen Longo a,Leandro Araújo Fernandes c, Erivan Clementino Gualberto Junior a,Vivian Cristina Noronha Novaes a, Edilson Ervolino d, Juliano Milanezi de Almeida a,Letícia Helena Theodoro a,*

aGroup of Research and Study on Laser in Dentistry (GEPLO), Department of Surgery and Integrated Clinic, Division of Periodontics, São Paulo StateUniversity, UNESP (“Univ. Estadual Paulista”), Araçatuba, SP, BrazilbDepartment of Periodontics, University Center of the Educational Foundation of Barretos (UNIFEB), Barretos, SP, BrazilcDepartment of Clinic and Surgery, Federal of University Alfenas, Alfenas, MG, BrazildDepartment of Basic Science, São Paulo State University, UNESP (“Univ. Estadual Paulista”), Araçatuba, SP, Brazil

a r t i c l e i n f o

Article history:Paper received 20 September 2013Accepted 13 February 2014

Keywords:Bone transplantationGlucocorticoidsImmunocompromised hostLaser therapy

* Corresponding author. Faculdade de Odontologia dBonifácio 1193, Centro. CEP: 16050-300 Araçatub36362860.

E-mail addresses: [email protected], letheodoro

http://dx.doi.org/10.1016/j.jcms.2014.02.0081010-5182/� 2014 European Association for Cranio-M

Please cite this article in press as: Garcia VG,immunosuppressed rats, Journal of Cranio-M

a b s t r a c t

The aim of this study was to evaluate the effects of low-level laser therapy (LLLT) on the bone repair ofcritical size defects (CSDs) filled with autogenous bone in the calvaria of immunosuppressed rats. A5 mm-diameter CSD was created in the calvaria of 30 rats. The animals were divided into 5 groups(n ¼ 6): Control (C) e the defect was filled with a blood clot; Dexamethasone (D) e dexamethasonetreatment, and the defect was filled with a blood clot; Autogenous bone (AB) e dexamethasone treat-ment, and the defect was filled with autogenous bone; LLLT e dexamethasone treatment, and the defectreceived LLLT (660 nm; 35 mW; 24.7 J/cm2); and AB þ LLLT e dexamethasone treatment, and the defectwas filled with autogenous bone and received LLLT. All animals were euthanized at 30 postoperativedays. Histometric and histological analyses were performed. The new bone area (NBA) was calculated asthe percentage of the total area of the original defect. Data were analysed statistically (an analysis ofvariance and Tukey’s test; P < 0.05). The AB þ 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 immunosup-pressed rats.

� 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rightsreserved.

1. Introduction orthopaedic and oral and maxillofacial surgery (De Long et al.,

Therapeutic strategies to promote bone repair represent a majorchallenge for many health professionals. To reduce the functionalincapacity and high socioeconomic costs associated with bonedefects, several interventions in the bone healing process havebeen investigated, including the use of low-level laser therapy(LLLT) (Liu et al., 2007; Pinheiro et al., 2009), bone substitutes(Dinopoulos et al., 2012) and specific biomaterials (Notodihardjoet al., 2012; Zanchetta et al., 2012).

Bone grafting is a frequently performed procedure to enhancebone regeneration under various conditions in the fields of

e Araçatuba-UNESP, Rua Joséa, SP, Brazil. Tel.: þ55 18

@foa.unesp.br (L.H. Theodoro).

axillo-Facial Surgery. Published by

et al., Effect of LLLT on autogeaxillo-Facial Surgery (2014)

2007). An increasing number of procedures that require boneaugmentation have recently been introduced, such as spinal fusionprocedures, revision arthroplasties, and limb salvage proceduressecondary to trauma, tumour, or skeletal abnormalities(Dinopoulos et al., 2012). Bone autografts are considered to be thegold standard for bone grafting (Streckbein et al., 2013) due to theirunique characteristics. They exhibit complete histocompatibility,thereby generating minimal immunological reactions. Bone auto-grafts usually contain viable osteogenic cells and bone matrixproteins that support bone growth (Bauer and Muschler, 2000).

Glucocorticoids (GCs) are potent anti-inflammatory andimmunosuppressive drugs that have been widely used for manydecades in the treatment of various autoimmune, pulmonary andgastrointestinal disorders (Kim, 2010). In recent years, organtransplant has become an accepted treatment for many acquiredand congenital disorders. GCs link to receptors inside the cell,

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nous bone grafts in the repair of critical size defects in the calvaria of, http://dx.doi.org/10.1016/j.jcms.2014.02.008

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V.G. Garcia et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e72

causing the redistribution of lymphocytes. GCs reduce T-cell pro-liferation, decreasing interleukin (IL)-2 and downregulating IL-1and IL-6, thereby reducing curtailing inflammation (Vasanthanand Dallal, 2007). However, prolonged therapy with glucocorti-coids may induce osteoporosis (Seymour, 2006), inhibit fibroblastactivity and cause collagen and connective tissue loss withdecreased re-epithelization and angiogenesis (Pessoa et al., 2004).CGs suppress the number, differentiation and function of osteo-blasts (Kim, 2010). The maintenance of bone mass requires thatbone formation equals bone resorption (Smith et al., 2002). GCsimpair this balance primarily by inhibiting osteoblastic bone for-mation (Smith et al., 2002).

Experimental studies using stem cells (Wang et al., 2011; deVilliers et al., 2011; AlGhamdi et al., 2012) and animal models(Pinheiro et al., 2009; Garcia et al., 2013) as well as clinical studies(Makhlouf et al., 2012; Cepera et al., 2012) have been conducted toevaluate the biostimulation effects of LLLT on bone repair. However,it is unclear whether the low-level laser biomodulation of boneformation is a consequence of the stimulation of mesenchymal cells(Giannelli et al., 2013) or the direct stimulation of osteoblasts (Xuet al., 2009). It may be that biostimulation results from anincreased release of fibroblast growth factor, which is found in bonetissue and acts on differentiated cells, increasing cell proliferationand the secretion of matrix components (Obradovi�c et al., 2009).

Although LLLT is increasingly used for the biostimulation ofbone repair and several studies have demonstrated its positive ef-fects on the healing of bone tissue (Luger et al., 1998; Garavello-Freitas et al., 2003; Liu et al., 2007; Pinheiro et al., 2009; Garciaet al., 2013), there are only a few previous reports on the associationof LLLT with autogenous bone (da Silva and Camilli, 2006; Weberet al., 2006). In particular, LLLT has been shown to stimulate therepair of bone defects implanted with biomaterials, such as inor-ganic bone, organic lyophilized bone, hydroxyapatite implants,inorganic bovine material (associated or not with biologicalmembrane), bone morphogenic proteins (BMPs), and organicbovine grafts (Obradovi�c et al., 2009). Moreover, few studies haveaddressed the mechanism of action of LLLT on the implantetissueinteraction. It has been shown that LLLT promotes bone healing andbone mineralization (Khadra et al., 2004). In the search for theoptimal implantetissue interaction, the effect of LLLT on these cellsis an important field of investigation.

The utility of LLLT in biomaterial osseointegration, therefore,remains unclear, and studies have not addressed the effect of LLLTon bone autografts under the conditions of immunosuppression.The aim of this study was to evaluate the effects of LLLT on the bonerepair of critical size defects (CSDs) filled with autogenous bone inthe calvaria of immunosuppressed rats.

2. Materials and methods

2.1. Experimental model

This study was conducted on 30 adult male Wistar rats (250e300 g) aged 3 months. Animals were kept in plastic cages, withaccess to food and water ad libitum, in a room with a 12-h light/dark cycle and temperature between 22 and 24 �C. The experi-mental protocol was approved by the Institutional Review Board ofAraçatuba Dental School, UNESP (Process 2008-008465).

Animals were randomly assigned to 5 experimental groups of 6animals each, according to the following systemic and local treat-ments: Control (C) e saline solution treatment, and the defect wasfilled with a blood clot; Dexamethasone (D) e dexamethasonetreatment, and the defect was filled with a blood clot; Autogenousbone (AB) e dexamethasone treatment, and the defect was filledwith AB; Low-level laser therapy (LLLT) e dexamethasone

Please cite this article in press as: Garcia VG, et al., Effect of LLLT on autogeimmunosuppressed rats, Journal of Cranio-Maxillo-Facial Surgery (2014)

treatment, and the defect received LLLT (660 nm; 35 mW; 24.7 J/cm2), autogenous bone and low-level laser therapy (AB þ LLLT) edexamethasone treatment, and the defect was filled with autoge-nous bone and received laser irradiation.

2.2. Protocol of drug administration

Animals of groups D, AB, LLLT, and AB þ LLLT received injectionsof 2 mg per kilogram of body weight of dexamethasone (Pessoaet al., 2004) (1 ml; DECADRON, 2 mg/ml, Prodome, Aché Pharma-ceutical Laboratories SA, Campinas, SP, Brazil). Animals of group Creceived injections of 1 ml of 0.9% saline solution. The subcutane-ous injections were initiated at 24 h and maintained every 3 daysuntil the euthanasia of the animals (Fernandes et al., 2009). Theadministration site was in the backs of the animals, next the ce-phalic region, and injections were always scheduled in the morn-ing. Animals were weighed weekly for dose maintenancethroughout the experimental period.

2.3. Surgical procedure

Rats were anaesthetized by an intramuscular injection of xyla-zine (6 mg/kg; Xilazin, Syntec do Brasil Ltd., Cotia, SP, Brazil) andketamine (70 mg/kg; Cetamin, Syntec do Brasil Ltd., Cotia, SP,Brazil). After aseptic preparation, a semilunar incision was made inthe scalp in the anterior region of the calvarium, allowing for thereflection of a full-thickness flap in a posterior direction. A CSD5 mm in diameter (Calixto et al., 2011; Nagata et al., 2013) wasmade with a trephine bur used in a low-speed handpiece undercontinuous sterile saline irrigation. The defect included a portion ofthe sagittal suture.

One L-shaped mark was made 2 mm anterior and one mark wasmade 2 mm posterior to the margins of the surgical defect by usinga small tapered carbide fissure bur and a surgical stent. The longaxes of the L-shaped marks were located on the longitudinal axisbisecting the surgical defect (Messora et al., 2008). The marks werefilled with amalgam (Messora et al., 2008). Their purpose was toallow identification of the centre line of the original defect duringlaboratory processing. The marks were also used as references tolocate the original bone margins of the surgical defect during his-tometric analysis (Messora et al., 2008; Nagata et al., 2009).

In groups AB and ABþ LLLT, autogenous bonewas obtained fromthe calvarium during the creation of the surgical defect. The boneremoved from the defect was particulated (172e210 mm) using aBone Grinder type pestle manual (Kopp, dental implant system,Curitiba, PR, Brazil) and placed in the defect.

The soft tissues were repositioned and sutured to achieve pri-mary closure (4-0 Silk, Ethicon, Johnson & Johnson, São Paulo, SP,Brazil). After surgery, each animal received an intramuscular in-jection of 24,000 IU of penicillin G-benzathine (Pentabiótico*Veterinário Pequeno Porte, Fort Dodge, Saúde Animal Ltd., Campi-nas, SP, Brazil).

2.4. LLLT treatment

The LLLT used in this study was indium-gallium-aluminium-phosphorus (InGaAlP; Theralase, DMC Equipments Ltd., São Carlos,SP, Brazil), with a wavelength of 660 nm, power of 35 mW, andbeam area of 0.0283 cm2. In the LLLT and AB þ LLLT groups, LLLTwas applied at 5 points on the spherical surgical wound, such thatall of the injuries received uniform treatment. 4 application pointswere distributed along the edge of the wound (at the 3, 6, 9, and 12o’clock positions, respectively), and 1 application point was locatedin the central region of the wound (axis of the clock; Fig. 1). Laserirradiation was released for 4 s/point (full exposure time of 20 s),


Fig. 1. Illustration of laser application points on CSD.

Table 1The mean and standard deviation (M � SD) of body weight (g) in each group andperiod.

Period/group Baseline (g) 30 days (g)

C 245.85 � 4.18a 306.00 � 0.81a

D 247.42 � 5.88a 177.14 � 1.34a,b

AB 247.28 � 5.31a 178.28 � 1.49a,b

LLLT 246.85 � 5.6a 178.28 � 1.11a,b

AB þ LLLT 248.85 � 6.64a 179.30 � 2.10a,b

N 30 30

a Significant difference between experimental periods in the same group (ANOVAand Tukey’s test; P < 0.05).

b Significant differences with the C group in the same period (ANOVA and Tukey’stest; P < 0.05).

V.G. Garcia et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2014) 1e7 3

with an energy density of 4.9 J/cm2/point, total energy density of24.7 J/cm2, and total energy of 0.70 J.

2.5. Tissue processing

All animals were euthanized at 30 days postoperative. The areaof the original surgical defect and the surrounding tissues wereremoved onto a block. The blocks were fixed in 4% formaldehyde,rinsed with water, and decalcified in 18% ethylenediaminotetra-acetic acid (EDTA) solution. After initial decalcification, each spec-imen was divided longitudinally into 2 blocks along the centre lineof the original surgical defect, using the long axis of both L marks asreferences (Fig. 1). Transverse cuts were made using the short axisof both L marks as references. Each specimen measured 9 mm inlength along the longitudinal axis running through the centre of thedefect. This consistency among specimen sizes allowed for theprecise identification of the original surgical defect margins duringthe histological and histometric evaluations (Messora et al., 2008).

After the samples were completely decalcified, they were pro-cessed and embedded in paraffin. Serial sections (6-mm thick) werecut in a longitudinal direction, beginning at the centre of theoriginal surgical defect. The sections were stained with haema-toxylin and eosin (HE) for analysis under light microscopy.

2.6. Histometric analysis

Four histological equidistant sections, representing the centre ofthe original surgical defect, were selected for the histological andhistometric analyses to increase the reliability of the data used(Messora et al., 2008; Garcia et al., 2013). Image analysis was per-formed by a calibrated examiner blinded to the treatment (LAF).Images of the histological sections were captured by a digitalcamera connected to a light microscope (Axiovision; 4.8.2, CarlZeiss MicroImaging GmbH, Jena, Germany; originalmagnification �5) and saved on a computer. Because it was notpossible to capture the entire defect in one image at the level ofmagnification that was used, a composite digital image was createdby combining 8 smaller images. The composite image was createdon the basis of anatomic reference structures (e.g., blood vesselsand bone trabeculae) within each of the histological sections.ImageLab 2000 software (Diracon Bio Informática Ltd., VargemGrande do Sul, SP, Brazil) was used for image analysis.

The following criteria, based on the work of Messora et al., wereused to standardize the analyses of the digital images. The total area(in mm2) corresponded to the entire area of the original surgicaldefect. This area was determined by identifying the external andinternal surfaces of the original calvarium at the right and left


margins of the surgical defect and connecting them with linesdrawn according to their respective curvatures. Given the totallength of the histological specimen, a length of 2mmwasmeasuredfrom the right and left edges of the specimen towards the centre todetermine themargins of the original surgical defect. The new bonearea (NBA, in mm2) and the remaining bone graft particle areaswere delineated within the confines of the total area. The total areawas considered to be 100% of the area to be analysed. The NBAwascalculated as a percentage of the total area.

2.7. Statistical analysis

For each animal, the NBA value was calculated as the meanpercentage of four histological sections. The significance of differ-ences between groups relative to NBA was determined by ananalysis of variance (ANOVA), followed by a post-hoc Turkey’s testwhen the ANOVA suggested a significant difference between thegroups (P < 0.05).

3. Results

3.1. Clinical analysis

All of the animals in group C showed no clinical differences interms of general health, and the weight gain was within the pre-dicted range for healthy rats (Table 1). Animals of groups D, AB,LLLT, and AB þ LLLT presented significant progressive weight losscompared to animals of group C (Table 1) and showed trends ofimmunosuppression and systemic alterations.

3.2. Qualitative analysis histological

In groups C (Fig. 2a and b) and D (Fig. 3a and b), a narrow band ofnewly formed bone tissue that was contained within the edges ofthe surgical wound was observed. Although the patterns of newbone present in these two groups were similar, the amount of boneobserved in group D was lower than that in group C. Almost all ofthe bone defects in these groups were filled with dense connectivetissue, inwhich therewas a large amount of collagen fibres orientedparallel to the wound surface, containing a scarce amount of in-flammatory cells, fibroblasts, and some blood vessels.

In group AB, bone tissue was present at the edges of the wound,resulting in the closure of more than two-thirds of the bone defectsin most specimens (Fig. 4a). The centre of the bone defect appearedto be occupied by connective tissue-containing residual bone graftparticles, and a thin layer of immature bone was found to be affixedto the surface of some remaining bone grafts. However, in theremaining group AB grafts that had no bone tissue, there was oftenresorption lacunae occupied by osteoclasts. Fibrous connectivetissue covered the surfaces of the bone defects and circumscribed


Fig. 2. a e Panoramic view of the defects at 30 days in group C (HE staining; originalmagnification �1.15); b e The bone defects were filled with connective tissue (ct).Newly formed bone (nb) was restricted to areas close to the borders of the surgicaldefects (HE staining; original magnification �160).

Fig. 3. a e Panoramic view of the defects at 30 days in group D (HE staining; originalmagnification �1.15); b e The bone defects were filled with connective tissue. Newlyformed bone (nb) was restricted to areas close to the borders of the surgical defects(HE staining; original magnification �160).

Fig. 4. a e Panoramic view of the defects at 30 days in group AB (HE staining; originalmagnification �1.15); b e The centre of the bone defect was filled with connectivetissue (ct) and remaining bone grafts (*). Newly formed bone (nb) was present in theborders of the surgical defects and a thin layer in the surface of some remaining bonegrafts. Osteoclasts (green arrowheads) were observed on the surface of some of theremaining bone grafts (HE staining; original magnification �160).

Fig. 5. a e Panoramic view of the defects at 30 days in group LLLT (HE staining;original magnification �1.15); b e Newly formed bone (nb) progressed from the pe-riphery to the centre of the bone defect. In this newly formed bone, osteoblasts (blackarrowheads) were observed that exhibited the morphological characteristics of intensebone matrix synthesis activity. The centre of the remaining defect was filled withconnective tissue (ct) (HE staining; original magnification �160).


residual bone graft particles (Fig. 4b). This tissue contained a largeamount of collagen fibres with a moderate amount of fibroblasts,numerous blood vessels, and absent or rare isolated foci of in-flammatory cells, especially in regions where the bone resorption ofthe graft particles was actively occurring.

In the LLLT group, newly formed immature bone occupied morethan two-thirds of the surgical wound (Fig. 5a). Bone strands,


which were full of osteoblasts that exhibited the morphologicalcharacteristics of intense bonematrix synthesis activity, progressedfrom the periphery to the centre of the bone defect (Fig. 5b). In thecentre of the defect, many fibroblasts and collagen fibres, both


Fig. 7. A graph showing the mean and standard deviation (M � SD) of new bone area(expressed as a percentage of the total defect area) in each group. *Statistically sig-nificant difference (ANOVA and Tukey’s test; P < 0.05).


oriented parallel to the defect surface, were observed together withnumerous blood vessels, the quantities of which were significantlygreater than the quantities observed in groups C and D.

The AB þ LLLT group exhibited two patterns of bone formationthat contributed to newly formed bone tissue, which occupied agreat extent of the surgical wound. The new bone formation thatprogressed from the edges of the wound toward the defect centrein the AB þ LLLT group was significantly higher than that observedin the groups described above. There was substantial immaturebone tissue on most of the remaining graft (Fig. 6a and b). The fociof active bone resorption on autogenous bone graft particles wererarely observed in AB þ LLLT group wounds. Intensely vascularizedtissue surfaces laced the wound and the adjacent remaining bonegraft particles. This tissue was composed of large amounts ofcollagen fibres, between which a moderate amount of fibroblastscould be observed.

3.3. Histometric and statistical analyses

The normality and homogeneity of the data variances wereverified. Fig. 7 shows the means and standard deviations of NBA foreach group and the results of statistical comparisons among thegroups. The histometric results at 30 postoperative days revealedsignificantly increased NBA for group AB þ LLLT compared togroups C, D, AB, and LLLT; for group AB compared to groups C, D andLLLT; and for group LLLT compared to groups C and D. Animals ingroup D showed significantly less bone formation than animals inall other groups (p < 0.05).

4. Discussion

The aim of this study was to evaluate the effects of LLLT on thebone repair of CSDs filled with autogenous bone in the calvaria ofimmunosuppressed rats treated with dexamethasone. Animals

Fig. 6. a e Panoramic view of the defects at 30 days in group AB þ LLLT (HE staining;original magnification �1.15): b e The centre of the bone defect was occupied byconnective tissue (ct) and remaining bone grafts (*). Newly formed bone (nb) waspresent in the borders of the surgical defects and as a thick layer on most of theremaining bone graft (HE staining; original magnification �160).


treated with this drug exhibited lethargy, haematoma, and alopeciawhen they were euthanized. The animals also underwent a sig-nificant weight reduction, which probably resulted from thedecreased gastrointestinal nutrient absorption caused by the drug(Metzger et al., 2002).

Group D showed less bone formation compared to the othergroups. This result may be explained by the use of high doses ofcorticoid, which are associated with reduced osteoblast numberand activity (Kim, 2010). They also potently inhibit osteoclastfunction, which, in the context of bone remodelling, leads to theadditional suppression of osteoblast function (Kim, 2010). Inaddition, GCs decrease the number of osteoblasts by inhibiting thepool of cells available for differentiation into osteoblasts (Kim,2010) and induce osteocyte autophagy (Yao et al., 2013).

We observed that group AB showed greater bone formationcompared to groups C, D, and LLLT. In group AB, bone tissue waspresent at the edges of the wound. The centre of the bone defectappeared to be occupied by connective tissue-containing residualbone graft particles, and a thin layer of immature bonewas found tobe affixed to the surface of some remaining bone grafts. Otherstudies in rabbit calvaria defects showed that the autogenous bonegraft was completely bridged by mineralized tissue (Pripatnanontet al., 2013). Calvarial bone is a cancellous and cortical bone.Dinopoulos et al. (2012) reported that the main advantage ofautologous cancellous grafts is their potential to transfer osteo-progenitor cells to the recipient site for osteogenesis. According tothese derived from the cellular population, only the osteoblasts andendosteal cells on the surface of the graft survived the trans-plantation. On the other hand a cortical graft acts mainly as anosteoconductive substrate by supporting the ingrowth of newblood vessels and the infiltration of new osteoblasts and osteoblastprecursors. Both grafts also contain osteoinductive agents, such asBMPs, which induce the differentiation of mesenchymal stem cells(MSCs) towards osteoblasts (Dinopoulos et al., 2012). Furthermoreosteoinductive factors released from the graft during the resorptiveprocess and cytokines released during the inflammatory phasemayalso contribute to bone healing (Bauer and Muschler, 2000; Khanet al., 2005).

The inflammatory process triggered by the inorganic matrixhinders graft integration. A drug capable of reducing or abolishingthe inflammatory process would be of great value in the study ofinorganic grafts (Ogston et al., 2002), but there is a lack of knowl-edge about the effects of anti-inflammatory agents on autogenousbone graft integration, primarily with regard to the grafteboneinterface. Silva et al., 2008 studied the effects of anti-inflammatoryagents (i.e., diclofenac sodium, dexamethasone, and meloxicam,



with isotonic saline solution as a control) on the integration ofautogenous bone grafts and bovine bone devitalized matrix in rats.Anti-inflammatory drugs were administered during the immediatepostoperative period and for 6 consecutive days. Diclofenac sodiumand meloxicam delayed bone graft repair, whereas dexamethasonedid not interfere with it. However, the drug was applied for only 6days; whereas in the present study, the drug was injected untileuthanasia (30 days).

The histometric results at 30 days postoperative indicated thatgroup AB þ LLLT showed significantly more bone formation (interms of the NBA) than groups C, D, AB, and LLLT. In addition to theproperties of autogenous bone described above, a possible expla-nation for these results might include the action of the LLLT onosteoinductive factors from the autogenous graft, such as growthfactors and mesenchymal stem cells. MSCs are a promising sourceof adult stem cells for cell transplantation and tissue engineering(Quattrocelli et al., 2010; Wang et al., 2011). Recent evidence sug-gests that the proliferation of various cultured stem cells, includingMSCs, may be enhanced by LLLT (de Villiers et al., 2011; AlGhamdiet al., 2012; Giannelli et al., 2013). However, there is a generalconsensus that the beneficial effects of MSCs on tissue repair andregeneration do not require their differentiation at the target sites.Rather, these effects mostly depend on the ability of the cells tosecrete a broad panel of growth factors and cytokines, whichinstruct the neighbouring cells and provide cues to stimulate neo-angiogenesis and extracellular matrix remodelling and to assist theendogenous regenerative response (Caplan, 2007).

Osteogenesis is the actual process of new bone formation fromMSCs or osteoprogenitor cells, which may originate from livingcells in the graft or from cells of host origin. MSCs clearly have anindirect role because they act as a source of progenitors for osteo-blasts, which are responsible for the anabolic half of homeostaticbalance and also regulate osteoclastogenesis via the expression ofRANKL and OPG (Bielby et al., 2007). Similar to MSCs, LLLT may alsoregulate the expression of RANKL and OPG. Xu et al. investigatedthe regulatory effect of LLLT on RANKL and OPG mRNA expressionin cultured calvarial cells, reporting that LLLT significantly down-regulated the RANKL: OPG mRNA ratio in osteoblasts. This findingindicates that LLLT may indirectly inhibit the differentiation andfunction of osteoclasts. Although the detailed mechanisms remainunknown, Giannelli et al. suggest that the stimulation of bonerepair by laser treatment may depend, at least in part, on theaugmented release of growth factors by MSCs.

The mechanism of action of laser irradiation in organisms is animportant area of research. The precise roles of laser irradiation inbone remodelling and repair are still not fully understood. Severalauthors have suggested that the application of a laser beam directlyover the bone lesion has a biostimulating effect on bone remodel-ling (Dortbudak et al., 2002; Pretel et al., 2007). In the presentstudy, the LLLT group showed more new bone formation comparedto the C and D groups (not irradiated). This finding suggests thatLLLT may be able to neutralize the harmful effects of GCs on healingbone.

Many variables may affect the biostimulatory effects of LLLT(e.g., laser wavelength, energy, exposition time, power, and thebiologic state of the cell). In this study, we reported the influence ofLLLT with a wavelength of 660 nm and a dose of 24.7 J/cm2. Moststudies have evaluated the effect of infrared laser light on bonehealing (Pinheiro et al., 2003; Gerbi et al., 2005; Weber et al., 2006;Lirani-Galvão et al., 2006; Pinheiro et al., 2009) because of itsdeeper penetration of tissues. One study found that the use of aninfrared laser at a dose of 10 J/cm2 during surgery resulted in apositive biomodulatory effect on the healing of bone defects on thefemur of normal rats submitted to autologous bone grafting (Weberet al., 2006). Another study showed that the use of a 637-nm


GaAlAs low-power laser (4 J/cm2 for 7 days) influenced the heal-ing speed of bone defects in the femur of rats (Markovic et al.,2005). The histopathological results suggested that bone regener-ation was faster under the influence of the laser, especially in theearly stages of wound healing (Markovic et al., 2005).

We only performed irradiation of the CSD during surgery. Usingthe infrared laser, a previous study demonstrated that the positivebiomodulatory effect on the healing of bone defects on the femursof rats submitted to autologous bone grafts was more evident whenlaser irradiation was performed on the surgical bed trans-operatively, prior to the placement of the bone graft, as well aspostoperatively (Weber et al., 2006). According to a recent study,the use of a 660-nm GaAlAs (57.14 J/cm2) low-power laser waseffective in stimulating bone formation in CSDs in the calvaria ofrats submitted to ovariectomy (Garcia et al., 2013).

In specimens treated with LLLT and autogenous bone, the de-fects were occupied by few remaining bone graft particles and byan organized connective tissue, with collagen fibres parallel to thewound surface and multiple fibroblasts. Large amounts of collagenfibres have been observed in animals irradiated with lasers inseveral studies (Pinheiro et al., 2003; Gerbi et al., 2005). Becausecollagen is an important component of the extracellular matrix ofthe bone, this result may indicate a positive effect of LLLT on thebone healing (Pinheiro et al., 2003). The effects of the laser may bedue to the increased levels of growth factors that act on differen-tiated cells, increasing the proliferation rate and stimulating thesecretion of bone tissue (Gerbi et al., 2005).

5. Conclusion

Within the limits of this study, it can be concluded that thecombination of autogenous bone grafts and LLLT (660 nm)improved bone healing in CSDs that were surgically created in thecalvaria of immunosuppressed rats. The laser promoted a bio-stimulatory effect on the bone graft that surpassed the inhibitoryeffects of dexamethasone. However, given themethodology used inthis study, it was not possible to clarify the exact mechanisms bywhich the laser compensated for this inhibitory effect on the bonematrix. These encouraging results suggest that further experi-mental and clinical studies should be performed to determine theeffective parameters of irradiation for clinical applications on bonerepair in immunosuppressed patients.

Sources of support in the form of grantsAngelita Strazzi Sahyon received a scholarship from the São

Paulo Research Foundation-FAPESP, São Paulo, SP, Brazil (Processno. 2008/10868-0).

Conflict of interest statementNone declared.

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Effect of LLLT on autogenous bone grafts in the repair of critical size defects in the calvaria of immunosuppressed rats