ORIGINAL ARTICLE

Digital photogrammetry and histomorphometric assessmentof the effect of non-coherent light (light-emitting diode) therapy(l640±20 nm) on the repair of third-degree burns in rats

Silvana Maria Véras Neves & Renata Amadei Nicolau &

Antônio Luiz Martins Maia Filho &

Lianna Martha Soares Mendes & Ana Maria Veloso

Received: 28 July 2012 /Accepted: 15 March 2013 /Published online: 13 April 2013# Springer-Verlag London 2013

Abstract Recent studies have demonstrated the efficacy ofcoherent light therapy from the red region of the electro-magnetic spectrum on the tissue-healing process. This studyanalysed the effect of non-coherent light therapy (light-emitting diode—LED) with or without silver sulfadiazine(sulpha) on the healing process of third-degree burns. In thisstudy, 72 rats with third-degree burns were randomly divid-ed into six groups (n=12): Gr1 (control), Gr2 (non-contactLED), Gr3 (contact LED), Gr4 (sulfadiazine), Gr5 (sulfadi-azine + non-contact LED) and Gr6 (sulfadiazine + contactLED). The groups treated with LED therapy received treat-ment every 48 h (l=640±20 nm, 110 mW, 16 J/cm2; 41 swith contact and 680 s without contact). The digital photo-metric and histomorphometric analyses were conducted

after the burn occurred. The combination of sulpha andLED (contact or non-contact) improved the healing of burnwounds. These results demonstrate that the combination ofsilver sulfadiazine with LED therapy (l=640±20 nm,4 J/cm2, without contact) improves healing of third-degreeburn wounds, significantly reduces the lesion area andincreases the granulation tissue, increases the number offibroblasts, promotes collagen synthesis and preventsburn infections by accelerating recovery.

Keywords Light-emitting diode . Healing . Third-degreeburns . Rats

Introduction

Burns are an important public health problem in Brazil andthroughout the world. The healing process for these lesionsis highly complex and can be divided into three phases:inflammation, proliferation and maturation [1].

Research using experimental models has suggestedthat phototherapy positively modulates cellular prolifer-ation and activity, angiogenesis and collagen synthesis[2–4]. The therapeutic effects of electromagnetic radia-tion (coherent and non-coherent) on biological tissue arefundamentally dependent on the measured parameters[5].

Some studies have demonstrated that skin lesions in non-diabetic and diabetic rats are repaired more quickly when

S. M. V. Neves (*) :A. L. M. M. FilhoFaculdade Integral Diferencial—FACID, Rua Veterinário BugyjaBrito, 1354—Bairro Horto Florestal,64052-410, Teresina, Piauí, Brazile-mail: silvanafisio7@yahoo.com.br

S. M. V. Neves : R. A. Nicolau :A. L. M. M. FilhoUniversidade do Vale do Paraíba, São José dos Campos,São Paulo, Brazil

L. M. S. MendesUniversidade Federal do Piauí, Teresina, Piaui, Brazil

A. M. VelosoPathology and Cytopathology Laboratory, São Marcos Hospital,Teresina, Piaui, Brazil

Lasers Med Sci (2014) 29:203–212DOI 10.1007/s10103-013-1312-7

light-emitting diode (LED) therapy is employed because itfacilitates the healing process, promotes collagen proteinsynthesis and aids wound recovery [6–9].

Several studies have employed coherent light to optimisethe process of tissue repair [10–15] because it has importantbiomodulatory effects. However, more research on non-coherent light therapy is needed to scientifically test itseffects on cutis burn lesions with and without medications.

The objective of this study was to test the biomodulatoryeffects of LED therapy with and without 1 % silver sulfadi-azine on healing of third-degree burns in rats.

Materials and methods

Ethical aspects

After the Research Ethics Committee of Faculdade IntegralDiferencial granted approval (under protocol number496/2008), 72 male Wistar rats weighing 250±25 g wereused in the study. The International Norms for BiomedicalResearch in Animals were followed.

Induction of experimental burn

Anaesthesia was administered intraperitoneally using 10 %ketamine and 2 % xylazine chloride (0.1 ml/100 g of bodyweight). A dorsal trichotomy was performed on the animals(6 cm from the skull to the tail and 4 cm from side to side). Toinduce the third-degree burns, a test tube with a 2.44-cmdiameter containing 50 ml of distilled water was heated to100ºC and then placed in contact with the skin for 20 s [16].After these procedures, the animals were placed in polypro-pylene cages during the experiment. They were kept underideal hygienic, light and temperature conditions and fed thestandard animal house diet and water ad libitum.

Experimental groups

The animals included in the study were randomly dividedinto six experimental groups. Each group was subdividedinto two groups (n=6) for euthanasia and analysed after 7 or14 days (Table 1).

Treatment parameters and equipment

The first irradiations occurred 24 h after the burns wereinduced at 48-h intervals in Gr2, Gr3, Gr5 and Gr6. (Table 2).The LED equipment was used [gallium–aluminium–arsenide(GaAlAs)] with 40 nm bandwidth (full width at half maxi-mum), centred at 640 nm, which was a prototype built for theexperiment. The optical output power of the light source wasfound before the experiments with the use of a broadband

power and energy metre: System 13 PEM 001/J (Melles GriotPhotonics Components Group, Carlsbad, CA, USA).

For the irradiations of Gr2 and Gr5, the LED was fixedon a tripod at a distance of 2.44 cm from the animal's dorsalso that the LED beam covered the entire affected area, butthe equipment did not make direct contact with the lesionregion. Thirty-six animals were euthanised 7 days after thelesion was induced. The animals' euthanised on the 7th dayreceived three therapy sessions, whereas the animals'euthanised on the 14th day received six sessions.

Silver sulfadiazine topical use

Applications of 0.5 g of 1 % silver sulfadiazine (Prati,Donaduzzi, Toledo, Paraná, Brazil) were performed in Gr4,Gr5 and Gr6 covering the entire lesion area every 48 h until theday before euthanasia.

Digital photogrammetry—macroscopic analysis

The digital photogrammetry was performed immediately afterthe burn and on the 7th and 14th days of treatment. The digitalimage was obtained using a Sony model DSC-S730 camera(Cyber-shot © 2008, Sony Corporation, São Paulo, Brazil)with a resolution of 7.2 megapixels. The camera was fixedon a tripod and kept at a constant distance of 12 cm from thebase. The diameter of the wounds was measured using ImageJ

Table 1 Distribution of the experimental groups

Group (n=12) Treatment

Gr1 Control

Gr2 Non-contact LED

Gr3 Contact LED

Gr4 Sulfadiazine only

Gr5 Sulfadiazine + non-contact LED

Gr6 Sulfadiazine + contact LED

Table 2 LED irradiation parameters used in the experiment

Parameters LED contact LED without contact

Wavelength (nm) 640±20 640±20

Power (W) 0.11 0.11

Irradiation area (cm2) 1.13 4.67

Irradiation time (seconds) 41 680

Energy density (J/cm2)/point 4 16

Energy (J)/point 4.51 74.8

Number of points 4 1

Energy density (J/cm2) total 16 16

Total energy (J) 74.8 74.8

204 Lasers Med Sci (2014) 29:203–212

software (version 1.39u), and the programme calibrationguidelines were followed to determine the area of the lesion.

Histological analysis

Longitudinal histological cuts with a thickness of 5 μm wereobtained for analysis. For each animal, eight cuts wereobtained: four cuts stained with haematoxylin and eosin andfour cuts stained with Masson's trichrome for specific collagencoloration.

The histological analysis of inflammatory reactions wasconducted using optical microscopy following the protocolof Meirelles et al. [16].

Histomorphometric analysis

The histological laminas were submitted to analysis usingcaptured images evaluated with a system of optical microsco-py. The image capture was taken with a Samsung Super VisionSVIII digital camera (1280×1024 pixels) attached to a NikonEclipse Microscope E 400 (Japan) and a Samsung Synmastercomputer 740n. The images were standardised according to themicroscope light intensity and magnification level (×40 objec-tive) and stored in tagged image file format for subsequent cellcounting. For quantitative analysis, inflammatory cells, fibro-blasts and blood vessels from the experimental periods (days 7and 14) were counted using the ImageJ programme. Thequalitative evaluation analysed the evolution of lesion repairby investigating the degree of collagen fibre remodelling usingthe same software.

Statistical analysis

The variables measured in the study were analysed with the aidof GraphPad Prism software 3.0 (Platform Windows, 3.0 ver-sion). A nonparametric Kruskal–Wallis test and Dunn's post-testwere used to analyse the differences between groups. P≤0.05was considered statistically significant.

Results

Macroscopic evolution of wound repair

Seven days after burning (DAB), thermal necrosis with thepresence of crusts that extended to the subcutaneous tissuewas still observed in all of the specimens in the six groups(Fig. 1).

Intragroup analysis The intragroup analysis showed that thelesion area was significantly smaller (p<0.001) in all groups 7and 14 DAB compared with the lesion area at baseline(0 DAB). It was observed that the injury area was significantly

smaller 14 DAB compared with the area 7 DAB (p<0.05) inall groups.

Intergroup analysis There was no significant difference in theburn area between the groups 7 DAB (p>0.05). Gr4 had asignificantly smaller lesion area (p<0.01) than Gr1, Gr3 andGr5 (Fig. 2). The area of the legions in Gr4 was not signifi-cantly different than the area in Gr2 andGr6. The lesion area inGr6 was significantly smaller than in Gr2. The lesion area inGr2 was significantly larger than in Gr1 (p=0.0206), whileGr4 had a significantly smaller legion area than Gr1 (p=0.0011). Fourteen DAB, Gr1 (p=0.0079), Gr2 (p=0.0111),Gr3 (p=0.0183) and Gr6 (p=0.0325) had significantly largerlesion areas than Gr4. However, 14 DAB, the lesion area wassimilar between Gr4 and Gr5.

Microscopic evolution of wound repair The inflammatorycells, blood vessels and fibroblasts were counted in the areadelimited by the ImageJ software. On day 7, acute inflam-mation and neovascularisation were intense in the majorityof analysed specimens, and chronic inflammation was gen-erally moderate. Re-epithelisation was absent in the majorityof analysed samples and was present in more than 50 % ofspecimens in only Gr1, Gr2 and Gr5. The presence of gran-ulation tissue was moderate in all specimens of the controlgroup and mild in the majority of animals in the other groups.The number of fibroblasts was low in the majority of animalsin all of the experimental groups. In all of the animals, thecollagen deposits were also mild in this phase. On day 14,granulation tissue was generally moderate in the sulpha groupand intense in the other groups. Neovascularisationwas intensein nearly all of the animals. The number of fibroblasts wasmoderate in Gr1 and Gr3, moderate to intense in Gr2 andintense in Gr4 and Gr5. Collagen deposition was mild in themajority of the animals. Re-epithelisation was predominantlymild in Gr2 and Gr5, varied from mild to moderate in Gr1 andGr3 and varied from moderate to intense in Gr4 and Gr6(Table 3, Fig. 3).

Intragroup analysis There was a significant reduction in thenumber of inflammatory cells from 7 to 14 DAB in Gr1 (p=0.0073), Gr4 (p=0.0297) and Gr6 (p=0.0159). There were nosignificant differences (p>0.05) between the two time points inGr2, Gr3 or Gr5. Gr2 and Gr5 showed similar results at bothexperimental time points (7 and 14DAB), but there were fewerinflammatory cells in Gr1 and Gr3 (Fig. 4). There were nosignificant differences in the number of vessels from 7 to 14DAB in any of the experimental groups. An increase in thenumber of fibroblasts from 7 to 14 DAB was observed in Gr2(p=0.0004) and Gr4 (p=0.0208).

Intergroup analysis The number of inflammatory cells 7DAB was greater in Gr1 than in Gr2 (p=0.0040), Gr4

Lasers Med Sci (2014) 29:203–212 205

(p=0.0060) and GR5 (p=0.0060). The number of inflam-matory cells 14 DAB was significantly lower in Gr4 com-pared with Gr1 (p=0.0079), Gr2 (p=0.0111), Gr3 (p=0.0183) and Gr6 (p=0.0325). There were no significantdifferences between Gr4 and Gr5 (p=0.2035) in the numberof inflammatory cells. The number of blood vessels 7 DABwas significantly lower in Gr5 than in Gr1 (p=0.0169). Thenumber of blood vessels 14 DAB was significantly higher inGr1 than in Gr3 (p=0.0153) and Gr4 (p=0.0352). Gr4 andGr3 had significantly fewer vessels than Gr2 (p=0.0175 andp=0.0098, respectively). The number of vessels in Gr3 wassignificantly lower than in Gr6 (p=0.0448). The number offibroblasts 7 DAB was significantly lower in Gr4 than inGr1 (p=0.0386) and Gr5 (p=0.033). Although differenceswere observed between Gr4 and Gr2 (p=0.1137), Gr3 (p=0.0534) and Gr6 (p=0.0996), these differences were not

significant. The number of fibroblasts 14 DAB in Gr4was significantly higher than in Gr1 (p=0.0357) andGr5 (p=0.0297).

Discussion

The beneficial effects of coherent and non-coherent light forhealing tissue burns in diabetic and non-diabetic rats and humanshave been reported in several studies [6, 11, 13, 16–19].However, some studies are contradictory, and some do not showpositive results for phototherapy [20–22]. Burns were chosen asthe woundmodel because there is a great need for more researchon the biomodulation effects of laser or LED therapy. Therewere no significant differences in burn area between thedifferent groups, indicating that the effects of third-degree

GroupsDays after burns

0 7 14

No sulfadiazine

Control

Non-contact LED

Contact LED

With sulfadiazine

Control

Non-contact LED

Contact LED

3 cm

Fig. 1 Burn injury during theexperimental periods (days 0, 7and 14) in animals treated withor without sulfadiazine and/orLED

206 Lasers Med Sci (2014) 29:203–212

burns were consistent, as previously described by Meirelleset al. [16]. There is evidence that coherent light therapydecreases the number of inflammatory cells [2, 3].Photobiomodulation helps increase the quantity of growthfactors and cytokines liberated by monocytes, which playan important role in the inflammatory phase of healing [23,24]. There have been no studies to date on LED therapywith silver sulfadiazine for burns, although some recentstudies have suggested an association of low-intensity coher-ent light with other medicines [25–28]. The effectivenessof phototherapy in dosages between 1 and 6 J/cm2 inaccelerating the healing process has been demonstratedpreviously [4, 10, 24, 29–32]. From this perspective, thebenefits of phototherapy were evident in this study; theLED energy employed at the cellular level acceleratedthe inflammatory response in the initial healing phase.Phototherapy in elevated dosages (above 16 J/cm2) mayinhibit the inflammatory response and harm the healingprocess [33–36].

The presence of crusts was observed in all the groups onday 7. Some authors say that crusts are necessary becausethey serve as a mechanical barrier between the externalenvironment and lesion tissue, facilitating cell migration tothe healing tissue and producing better conditions for cellu-lar repair [17]. All of the groups showed significant burnreduction in the first and second weeks after the burn oc-curred. We suggest that there is no negative change in thetissue repair process after LED therapy (contact or non-

contact) with or without silver sulfadiazine. Previous studieshave observed similar results for wounds treated with non-coherent light (LED) or coherent light (laser) as the radiationsource [2, 3, 34, 35]. The results of the lesion area analysis 7DAB suggest that non-contact LED had an effect that wassimilar to sulpha therapy but less pronounced than thecombination of sulpha and contact LED. Confirming thedata obtained in this study, Minatel et al. [37] observed theeffect of phototherapy on the healing of leg ulcers in twodiabetic patients. The combination of a laser (660/890 nm)with topical sulfadiazine was effective for surgical healingas a non-invasive, biostimulatory therapy and had an addi-tional analgesic effect. There is no significant differenceassociated with changes in the electromagnetic spectrum ofthe LED used in coherent light (laser) therapy in the resultsobtained in cultures or tissue when they are irradiated inhomeostasis [38, 39]. Diabetic and immunosuppressed ani-mals, for example, have no systemic homeostasis. Unlikeprevious studies, when phototherapy was used on diabeticanimals with damaged skin, it promoted an area reduction ofmore significant injuries [2, 3].

Seven DAB, the lesion area in Gr2 was larger than inthe control group (Gr1). This result suggests that non-contact LED therapy can promote effective control ofthe inflammatory cascade, resulting in less oedema andleading to a larger area of injury because of the swell-ing and oedema of the lower edges. Other studies haveoffered evidence of a significant reduction in cytokines

0

2

4

6

8

Gr1 Gr2 Gr3 Gr4 Gr5 Gr6

Groups

Les

ion

area

(cm

2 )

0 days

7 days

14 days

¥

§

¥

§#

¥ ¥¥§†

¥

Fig. 2 Lesion area during the experiment. Gr1 (control), Gr2 (non-contact LED), Gr3 (contact LED), Gr4 (sulfadiazine), Gr5 (sulfadia-zine + non-contact LED) and Gr6 (sulfadiazine + contact LED). Thevalues are presented as the mean ± SEM for the six groups. Intragroup

significant difference (p<0.05); (asterisk) vs. day 0, (yen) vs. day 7,Intergroup; (section sign) vs. Gr1, (black diamond) vs. Gr2, (numbersign) vs. Gr3, (dagger) vs. Gr6

Lasers Med Sci (2014) 29:203–212 207

after irradiation with red region light [40], and thus, wecould obtain the same energy density in the groups thatwere irradiated. The irradiation time employed for Gr2was higher than for Gr3, suggesting that the irradiationtime may have been an important factor in the positivephotobiomodulator effect and control of inflammation.This conclusion is strongly supported by the significant-ly lower number of inflammatory cells in Gr2 comparedwith the control and contact LED therapy groups. Thesimilarity of the results related to the inflammatory cellsin Gr1, Gr4 and Gr5 (in the parameters tested) maysuggest that non-contact LED therapy promotes localantimicrobial control and activates the immune system,as shown in a recent review by Piva et al. [40].

The presence of granulation tissue was intense and accom-panied by a large number of fibroblasts and low number ofcollagen fibres in Gr4, Gr5 and Gr6 14 DAB. These findings

confirm the results of a previous study [30] that used the sameperiod of observation, wavelengths from 660 and 780 nm andan energy density of 5 J/cm2 per point.

Intense neovascularisation was observed in 100 % of theanimals in Gr2, Gr3, Gr5 and Gr6 on day 7, which suggestpositive effects of photo induction on vascular formationbased on the parameters used. These data corroborate aprevious study [24] that found that blood vessel prolifera-tion on days 3, 7 and 14 was superior to the non-treatedgroup in all of the irradiated groups (p<0.05). For theseauthors, irradiation with 5 J/cm2 fluency stimulated moreintense angiogenesis than the 20 J/cm treatment. On day 14,the tissue therapy with a non-contact LED (Gr2) showed ahigher number of blood vessels compared with Gr4 (p<0.05).Thus, the LED therapy may have generated and increasedblood vessels, contributing to the re-establishment of localblood flow. These data corroborate the results of the authors

Table 3 Summary of the results of the histological analysis (%) on days 7 and 14

CriterionControl Non-contact LED Contact LED

I M Md I M Md I M Md

Day 7

No sulfadiazine Acute inflammation 83 17 – 83 17 – 100 – –

Chronic inflammation – 100 – – 83 17 – 100 –

Fibroblasts – – 100 – – 100 – – 100

Granulation tissue – 100 – – – 100 – – 100

Neoangiogenesis 100 – – 100 – – 100 – –

Collagen – – 100 – – 100 – – 100

With sulfadiazine Acute inflammation 50 33 17 50 33 17 66 17 17

Chronic inflammation – 83 17 – 50 50 – 66 33

Fibroblasts 17 17 66 – – 100 – – 100

Granulation tissue 17 33 50 – – 100 – – 100

Neoangiogenesis 83 17 – 100 – – 100 – –

Collagen – 17 83 – – 100 – – 100

Day 14

No sulfadiazine Acute inflammation – 67 33 33 50 17 17 67 17

Chronic inflammation – 83 17 – 33 67 – 33 67

Fibroblasts – 100 – 33 67 – 17 83 –

Granulation tissue – 100 – 50 50 – 83 17 –

Neoangiogenesis 100 – – 83 17 – 100 – –

Collagen – – 100 – – 100 – – 100

With sulfadiazine Acute inflammation – 67 33 – 100 – – 50 50

Chronic inflammation – 67 33 – 100 – – 17 83

Fibroblasts 100 – – 100 – – 100 – –

Granulation tissue 100 – – 100 – – 100 – –

Neoangiogenesis 67 33 – 100 – – 67 33 –

Collagen – 17 83 – – 100 – – 100

I Intense, M Moderate, Md Mild

208 Lasers Med Sci (2014) 29:203–212

that have employed a laser in repairing third-degreeburns [16, 17, 19].

Fibroblasts play a decisive role in healing. Three and 4DAB, fibroblasts originated from mesenchymal cells. Theyproliferated and became dominant around the 10th day, whenthey became involved in collagen production. The resultsindicated that non-coherent light had better stimulatory effectswhen combined with sulpha and was superior to it when usedalone. On the cellular level, coherent and non-coherent lightmodulates fibroblast proliferation, increases collagen bondand synthesis, promotes angiogenesis, improves energy me-tabolism and produces biological effects during the healing

phase [3, 33–36]. The collagen deposition varied according tothe period and according to the group analysed; it was minorin the great majority of animals in the six groups on the14th day of observation. Our results concur with the findingsof previous research that employed wavelengths of 660 and780 nm and a dosage of 5 J/cm2 on four points [16, 30] andfound a small and moderated number of immature and frag-mentary collagen fibres on the 14th day of treatment usingoptical microscopy. The studies indicated that the 660 nmlaser produced better results than the 780 nm laser in acceler-ating the healing process of third-degree burns in diabetic andnon-diabetic rats.

GroupsDay

7 14

No sulfadiazine

Control(Gr1)

Non-contact LED (Gr2)

Contact LED(Gr3)

With sulfadiazine

Control(Gr4)

Non-contact LED (Gr5)

Contact LED(Gr6)

Fig. 3 Microscopy showingwound healing on the 7th and14th day after the burn.Masson's trichrome (×400)

Lasers Med Sci (2014) 29:203–212 209

The results obtained in this study demonstrate thatthe majority of the treated groups (Gr2 to Gr5) had asmaller concentration of inflammatory cells at the lesionsite in the initial phase of repair, especially in the LEDgroups combined with sulpha compared with the controlgroup, which corroborated some studies that haveemployed laser therapy [2, 31]. Gr1, Gr2, Gr3 andGr6 had significantly larger lesion areas than Gr4. How-ever, the lesion area was similar 14 DAB for Gr4 andGr5. These results demonstrate the cumulative effects ofsulpha drug therapy and occurred in association with

non-contact LED and sulpha. The positive resultsobtained in this study regarding the reduction in thelesion area (7 and 14 DAB) corroborate previous studiesthat have found an effect of phototherapy using a doseof 4 J/cm2. To evaluate the interference of the laser(GaAlAs, 670 nm) on the elastic and collagen fibres,the researchers induced standardised skin wounds on theback of Wistar rats and then performed regular applica-tions of laser therapy with different energy densities.The animals were sacrificed after 5, 7 and 14 days.The results showed that there was a greater reduction

0

10

20

30

40

50

Infl

amm

ator

y ce

lls (

n) ..

7 days 14 days

0

2

4

6

8

Ves

sels

(n)

0

10

20

30

40

Gr1 Gr2 Gr3 Gr4 Gr5 Gr6Groups

Fib

robl

asts

(n)

‡

¥

*

¥*

‡

*† *†¥

¥*§

¥*

‡

*§†*§

*

Fig. 4 Number ofinflammatory cells, bloodvessels and fibroblasts presentin the repair area of the differentgroups 7 and 14 DAB. Gr1(control), Gr2 (non-contactLED), Gr3 (contact LED), Gr4(sulfadiazine), Gr5(sulfadiazine + non-contactLED) and Gr6 (sulfadiazine +contact LED). The values arepresented as the mean ± SEMfor the six groups. Intragroupsignificant difference (p<0.05);(yen) vs. day 7. Intergroupsignificant difference (p<0.05);(asterisk) vs. Gr1, (section sign)vs. Gr2, (double dagger) vs.Gr4, (dagger) vs. Gr6

210 Lasers Med Sci (2014) 29:203–212

in oedema and inflammatory infiltration and increasedexpression of collagen and elastic fibres in the experi-mental group, although the difference was not statisti-cally significant (p>0.05). Treatment with a dose of4 J/cm2 yielded better results than a dose of 8 J/cm2

[29].On 7 DAB of healing, there were significantly fewer in-

flammatory cells in the groups treated with contact LED (Gr3and Gr6) compared with the non-contact LED groups (Gr2and Gr4). This result suggests that the pressure exerted by theequipment in Gr3 and Gr6 may have generated a greaterinflammatory response in these groups. This information con-tributes to knowledge about treating wounds with photother-apy because it guides decisions about the type of lightapplication. The loss of radiation from reflection under non-contact treatment did not decrease the positive biomodulatoryeffects of phototherapy.

Conclusion

The combination of sulpha and LED (contact or non-contact) improves the healing of burn wounds. These resultsdemonstrate that the combination of silver sulfadiazine withLED therapy (l=640±20 nm, 4 J/cm2 without contact)improves the quality of the wound healing after third-degree burns, promotes a significant reduction in the lesionarea and granulation tissue, increases the number offibroblasts, improves collagen synthesis and preventsburn infections by encouraging wound repair.

Acknowledgments R. A. Nicolau thanks the National Council forScientific and Technological Development (CNPq, Brazil) for theproductivity fellowship (process no. 314455/2009-4).

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