Braz. j. . vol.82 número5

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www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

The

influence

of

growth

factors

on

skin

wound

healing

in

rats

夽

Elen

Carolina

David

João

De

Masi

a,∗

_,

_Antonio

_Carlos

_Ligocki

_Campos

a

_,

Flavia

David

João

De

Masi

b

_,

_Marco

_Aurelio

_Soatti

_Ratti

a

_,

_Isabela

_Shin

_Ike

a

_,

Roberta

David

João

De

Masi

c

a_Universidade_Federal_do_Paraná_(UFPR),_Curitiba,_PR,_Brazil

b_Pontifícia_Universidade_Católica_do_Paraná_(PUCPR),_Curitiba,_PR,_Brazil c_Universidade_da_Região_de_Joinville_(Univille),_Joinville,_SC,_Brazil

Received27August2015;accepted3September2015

Availableonline7January2016

KEYWORDS Woundsinrats; Growthfactor; Healing

Abstract

Introduction:Healingisaprocessthatrestoresthephysicalintegrityofbodystructures.Itisa dynamic,complex,multicellularprocessthatinvolvestheextracellularmatrix,cytokines,blood cells,andgrowthfactors.Growthfactorsareproteinsthatactivateandstimulatecell prolifer-ationthroughtheactivationofangiogenesis,mitogenesis,andgenetranscription,accelerating thehealingprocess.

Objective:Toassesstheinfluenceofgrowthfactorsonthehealingprocessofwoundsmadeon thebacksoffemaleratscomparedtothecontrolwound,throughmacroandmicroscopy.

Methods:Thisstudyused45femaleWistarrats,inwhichthreewoundsweremadeontheback. Thefirstwasthecontrolwound,thesecondreceivedepithelialgrowthfactorinjection,and thethirdreceivedacombination offactors. Macroscopicandmicroscopic assessmentswere performedon thethird, seventh,and 15thdays ofthe experiment.For microscopic analy-sis,hematoxylin---eosinstainingwasutilizedtoassesstheinflammatoryprocess;vimentin,for assessmentofbloodvesselsandfibroblasts,andSiriusRedforcollagenassessment.

Results:Inthemacroscopicassessment, theuseofgrowthfactorsresultedinfasterhealing anddecrease ofgranulationtissue ondays seven and15; (80.31%reduction inthecontrol woundvs.83.24%intheepithelialwoundvs.100%inthemixedwound).Utilizingmicroscopy, atthethreestagesoftheexperiment,therewerenosignificantdifferencesbetweenthethree wounds;however,whencomparingthedayofeuthanizationforeachtypeofwound,therewas afavorableoutcomeforepithelialandmixedwounds(betweenthethirdvs.15thday,p<0.001, andinthecomparisonoftheseventhvs.15thday;p=0.002andp=0.001forepithelialand

夽 _Please_cite_this_article_as:_João_De_Masi_EC,_Campos_AC,_João_De_Masi_FD,_Ratti_MA,_Ike_IS,_João_De_Masi_RD._The_influence_of_growth

factorsonskinwoundhealinginrats.BrazJOtorhinolaryngol.2016;82:512---21.

∗_{Corresponding}_author.

E-mails:[email protected],[email protected](E.C.JoãoDeMasi).

http://dx.doi.org/10.1016/j.bjorl.2015.09.011

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mixedwounds,respectively)withahighernumberoffibroblasts,angiogenesis,andcollagen typeI.

Conclusion: Theuseofgrowthfactorsaccelerateshealing,stimulatesgreaterangiogenic activ-ity,andacceleratesfibroplasiaandcollagenmaturation.

© 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

PALAVRAS-CHAVE Feridasemratas; Fatordecrescimento; Cicatrizac¸ão

Ainfluênciadefatoresdecrescimentonacicatrizac¸ãodeferidascutâneasderatas

Resumo

Introdução: Acicatrizaçãoéumprocessoderestauraçãodaintegridadefísicadasestruturas docorpo.Éumprocesso dinâmico,complexo,multicelular queenvolvematrizextracelular, citosinas,célulassanguíneasefatoresdecrescimento.Osfatoresdecrescimentosãoproteínas queestimulameativamaproliferaçãocelularmedianteaativaçãodaangiogênese,mitogênese, transcriçãogenética,acelerandooprocessodecicatrização.

Objetivo: Avaliarainfluência dosfatores de crescimento noprocesso cicatricial deferidas realizadasnodorsoderatasemcomparac¸ãocomaferida,controleatravésdamacroe micro-scopia.

Método: Foramutilizadas45 ratasWistar,submetidasàcriaçãodetrês feridas nodorso. A primeira controleasegundacominjeçãodefatordecrescimentoepitelialeaterceira com fatormisto.Asavaliaçõesmacroscópicasemicroscópicasforamrealizadasno3◦_,_no₇◦_e_no₁₅◦

diadoexperimento.Paraanálisemicroscópica,utilizou-secolorac¸ãodeHematoxilina-Eosina para avaliaroprocessoinflamatório;vimentina,paraaavaliac¸ãodosvasosefibroblastos,e

SiriusRed,paraavaliarocolágeno.

Resultados: Na avaliação macroscópica, o uso de fatores de crescimento proporcionou cicatrização maisrápida ediminuição dotecidode granulaçãono7◦ _e₁₅◦ _dia;_(80,31%_de

reduçãonaferidacontrolevs.83,24%naferidaepitelialvs.100%naferidamista).Na micro-scopia, nostrêsmomentosdoexperimento,não foramencontradasdiferenças significativas entreastrêsferidas;entretanto,quandocomparadososdiasdemorteemrelaçãoacadatipo deferida,observou-seresultadofavorávelparaasferidasepiteliaisemistas(entre3◦_×₁₅◦_dia

apresentoup<0,001enacomparac¸ãoentre7◦_×₁₅◦ _dias;_p₌_0,002_e_p₌_0,001_para_as_feridas

epiteliaisemistas)commaiornúmerodefibroblasto,angiogêneseecolágenotipo1.

Conclusão:autilizaçãodefatoresdecrescimentoaceleraacicatrização,estimulamaior ativi-dadeangiogênica,aceleraafibroplasiaematuraçãodocolágeno.

© 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Publicado por Elsevier Editora Ltda. Este é um artigo Open Access sob uma licença CC BY (http:// creativecommons.org/licenses/by/4.0/).

Introduction

Healing is a process that restores the internal and/or externalphysical integrityofbodystructuresandinvolves complexinteractionsbetweencellsandseveralother fac-tors.Itisadynamicandcomplexprocess,consistingofthree phases:tissueinflammation,proliferation,andremodeling.1 The healing process comprises the extracellular matrix, cytokines,blood cells,andgrowthfactors.Growthfactors areproteins that stimulateandactivate cell proliferation throughactivationofangiogenesis,myelogenesis,andgene transcription, amongother reactions, which activate and acceleratethehealingprocess.1,2

Among the growth factors, the most important ones forwound healinginclude:epithelial growthfactor(EGF), platelet-derivedgrowthfactor(PDGF),transforminggrowth factor(TGF-b),vascularendothelial growthfactor (VEGF),

fibroblast growth factor (FGF), and insulin growth factor (IGF);thelatterstimulatescellproliferation,tissue remod-eling, and collagen and elastin increase. VEGF acts on angiogenesis and tissue granulation at the early stage of healing.PDGFiscrucialfor inflammation,granulation, re-epithelialization, and remodeling in the three stages of woundhealing.3,4

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A great number of growth factors and cytokines are presentatthewoundsite.Theirdynamicexpression mani-feststemporalandspatialcharacteristicsintheregulation andchangesinthepatternofexpressionofgrowthfactors thatareassociatedwithimpairedwoundhealing.Important alterationsinthelevelsofonefactoreventuallyaffectthe productionofothergrowthfactorsandcytokines.Thus,it hasbeenshownthatpro-inflammatorycytokinesandgrowth factors are released in serum during the early phase of woundhealing,andactaspotentstimulatorsofthe expres-sionof several other growth factors. One example is the regulationofFGF7,agrowthfactorproducedbyfibroblasts at the wound site. Another example is the regulation of VEGF,amajorregulatorofangiogenesis,whichisproduced by keratinocytes and macrophages at the wound site. It wasfoundthatpro-inflammatorycytokinescaninduceVEGF expressioninbothcelltypes.Theseexampleshighlightthe complexinteractionsthatoccurduringwoundhealing.Such interactions should be considered when interpreting the resultsobtainedby theoverexpressionorelimination ofa singlegrowthfactoratthewoundsite.6

A study performed to compare the effectiveness of platelet-rich plasma (PRP) in the healing of wounds in rabbitscomparedtwogroups: onethat received chondro-cytes+PRP and the other that received only PRP. These components were subcutaneously injected on the dorsal regionoftherabbits;ascontrol,only PRPwasinjectedin four rabbits.After two months they underwent magnetic resonance imaging (MRI) assessment, histological analy-sis, and quantification of glycosaminoglycans. The MRI showed formation of new cartilage, indicating that PRP regenerates the cartilage, and demonstrating the poten-tialuse ofthismethod forthe reconstructionofcartilage defects.5,7

Shi8 _showed _that _the _conjunctival _growth _factor acti-vation and scar formation in corneal wounds in rabbits markedlyimprovedthearchitectureofthecornealstroma and reduced scar formation. However, the authors con-cluded thatthere is nomeasurable in vivoimpactof the cornealwoundscaranditshouldbeconsideredasaspecific targetofdrugtherapyforcornealscar.

Feng etal.9 _carried_out _a_study _in _diabetic_rats; _to_do so,theymadewoundsontheanimals’backsandinjected keratinocytegrowthfactorstoassesshealing.Twowounds measuring 2cm in diameter were made on each side of thespinalcolumn;growthfactorwasinjectedinoneside, whereassalinesolutionwasinjectedintheotherside.The studyperiodlastedfourweeksandphotographsweretaken dailyoveraperiodof28days.Theresultsshowedcell pro-liferationanda significanthealingstimulus inthewounds withgrowthfactor.

ThepresentstudyusedEGFalone,andVEGFgrowth fac-tor together with IGF and FGF --- which we called mixed factors(MF)---toquantifythecollagen,elastin,vessels,and cellproliferationintheskinhealinginrats,aswellasthe effectivenessofgrowthfactorsinthehealingprocessin rela-tiontothecontrolwound,thatdidnotreceivegrowthfactor orsalineinjection,tomimicnaturalhealing.

Theobjectiveofthisstudywastoassesstheinfluenceof growthfactorsonthehealingprocessthroughmacroscopic evolution,andmicroscopyofthewoundhealingprocesson thebacksoffemaleratsthathadbeeninjectedwithEGFor

0 h

6 h

3 h 9 h

Figure1 Schematicdrawingshowingthelocationofgrowth

factorinjectionintheepithelialandmixedwoundsinthefour

quadrants:three,six,nine,and12o’clock.

VEGFcombinedwithMF,andtocomparetheresultsto con-trolwoundsthatdidnotreceiveinjectionofgrowthfactors.

Methods

The animals werehandled according tothe Brazilian Col-lege of Animal Experimentation (Colégio Brasileiro de Experimentac¸ãoAnimal---COBEA)criteriaandthe require-ments established in Guide for the Care and Use of Experimental Animals(Canadian Council onAnimal Care). The study wasapproved bythe EthicsCommittee on Ani-mal Use (Comissão de Ética no Uso de Animais --- CEUA), Decree 787/03-BL of June 11, 2003, under Process No. 23075.013736/2012-11.ThestudywasconductedfromJuly 2012toJuly2015.

Forty-fivefemaleWistarratswereused(Rattus norvegi-cus albinus, Rodentia mammalia), agedbetween 115 and 130days,weighing200---253g.Aftertheywereweighed,the animalswererandomlydividedintothreegroupsof15 ani-mals.Healingwasassessedatdifferentstagesineachgroup. InGroup1,healingwasevaluatedonthethirdday;inGroup 2,ontheseventhday,andinGroup3,onthe15thday.

Threewoundsweremade:onemeasuring1cmin diame-ter andtwomeasuring0.6cm.The wounds weremade in different sizes in order to measure their tension. Due to non-availability of a tensiometer, wound tension was not performedandthestudywascarriedoutbyevaluatingthe macroscopicandthemicroscopic aspects.Ineachanimal, theexcisionextendedfromtheskintothemusclelayer.

The woundswere named 1---3 (F1,F2, F3). F1 wasthe controlwound(proximal),F2(centralormedial),received aninjectionofEGF,andF3(distal)receivedaninjectionof VEGF combinedwithFGFand IGF(whichwe calledmixed factorMF).

The growth factorswere injected only on the day the woundsweremade.Fourinjectionsof0.5mLweregivenfor eachwoundatfourpoints:three,six,nine,and12o’clock intothedermisandsubcutaneoustissue(Figs.1and2).The woundswereallowedtohealspontaneously.

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F1 F2 F3

Figure 2 Denomination ofthe wounds: wound 1--- control

(1.0cmindiameter);wound2---epithelial(0.6cmindiameter),

andwound3---mixed(0.6cmindiameter).

Aftertheeuthanizationoftheanimalsonthethird, sev-enth,and15thdaysoftheexperiment,theskin-aponeurotic flaps containingthewounds wereremoved. Subsequently, thematerialwassampledandsubmittedtoanautomated histologicalprocessandembeddedinparaffin.Histological

slidescontainingthesectionswerestainedbyhematoxylin and eosin (HE) and Sirius Red. Additional sections were preparedfortheimmunohistochemistryanalysis,usingthe antivimentinantibody.

Visualizing the sections stained with hematoxylin and eosin, the number of polymorphonuclear neutrophils, chronicinflammatorycells(lymphocytesandplasmacells), newly-formed capillaries, fibroblast proliferation, and amountofdepositedfiberswereassessed.Epithelial regen-erationwasconsideredaspartialorcomplete.

Sections stained in Sirius Red were used to assess the presenceandtypeofcollagen(matureorimmature)through polarized light microscopy, quantifying both types of col-lagen as percentages (immature collagen=green; mature collagen=red).

Histological sections, submitted to the immunohisto-chemical study and marked with antivimentin antibody, were used to identify fibroblasts and newly formed capillaries.

Afterassessingthemacroscopicandmicroscopicaspects ofF1,F2,andF3,theywerecomparedtotheHEstainingfor

Day 01 Day 02 Day 03

Day 06 Day 05

Day 04

Day 07

Day 10 Day 11 Day 12

Day 15 Day 14

Day 13

Day 08 Day 09

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100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

0 1 2 3 4 5 6 7 8 9 10

Day

Wound reduction

11 12 13 14 15 Wound 1 Wound 2 Wound 3

Figure4 Chartcomparingofthehealingofwounds1,2,and3

bymeasuringthewounds1,2,and3inpercentages,throughout

the15daysoftheexperiment.

allstudyvariables(neutrophils,lymphocytes,macrophages, fibroblasts,vessels)andaseparateanalysiswasperformed foreachmomentofassessment(three,seven,and15days) andsubmittedtostatisticalanalysis,considering the non-parametric Friedman test. Times of euthanization were compared using the nonparametric Kruskal---Wallis test. p-Values<0.05wereconsideredstatisticallysignificant.Data wereanalyzedusingStatisticav.8.0software.

Results

The macroscopic wound assessment was performed daily andrecordedinphotographs.TheF1,F2,andF3ofallthe ratsweremeasured usingaruler graduatedinmillimeters positionedatthelevel ofthelesion,andthemeansofall woundsweretakenoneachdayoftheexperiment(Fig.3). On the third day of the experiment, all wounds were open;in F1,therewaslittlewoundcontraction and gran-ulationtissue;inF2andF3,therewasaslightimprovement inrelationtoF1.

Ontheseventhdayoftheexperiment,thewoundswere stillopenwithaslightcontractionofallwounds,butF1had granulationtissue,showingdelayedwoundhealing.

Onthe15thdayoftheexperiment,thewoundscarswere asfollows:inF1therewasa reductionof 80%±0.311%of

Note: * p < 0.05 for the 3-7 days difference in the wound with epithelial growth factor.

# p < 0.05 for the 3-5 days difference in the wound with epithelial factor and in the wound with mixed growth.

32

30

28

26

24

22

20

18

16

14

12

10

8

6

Control

*

# #

Mixed growth factor Epithelial growth

factor

Hemato

xylin and eosin – L

ymphocytes

(Median;

25

%–

75%;

min - max)

4

3 days 7 days 15 days

Figure5 Hematoxylinandeosinevolution---lymphocytesin

eachwound,assessment.

thewounddiameter;inF2,thereductionwas83%±0.201%, andinF3itwas100%(Fig.4).

Whencomparingthethreetypesofwoundoneach day oftheanimals’euthanization,therewerenosignificant dif-ferencesinthenumberofneutrophils(Table1).However, whenevaluatingtheprogress ofeach woundonthethree daysoftheanimals’euthanization,asignificantdifference wasfoundforF1,whichshowedthehighestnumberof neu-trophils. Forthiswound, there weredifferences between thethirdandseventhdays(p=0.029)andbetweenthe sev-enthand15thdays(p=0.007).Betweenthethirdand15th daystherewasnosignificant difference(p=0.557).ForF2 andF3, theresultswerelower,withatendency to signif-icanceamongthe threedaysof assessment (p=0.058 and p=0.076,respectively).

Regarding the lymphocytes, when comparing F1, F2, and F3 in each of the euthanization moments, it was

Table1 Neutrophilassessmentwithhematoxylinandeosinonthethird,seventh,and15thdaysineachwound;assessment betweenthegroups.

Euthanizationday Wound n Mean±SD pa

Thirdday Control 15 24.2±4.5 0.482

Epithelialwound 15 21.7±5.7

Mixedwound 15 22.9±5.2

Seventhday Control 15 31.6±8.4 0.155

15thday Control 15 23.1±9.6 0.856

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Table2 Macrophagecountperfieldandcomparisonofwounds1,2,and3ateachmomentofeuthanization.

Euthanizationday Wound n Mean±SD pa_value_(F1_×_F2_×_F3)

Thirdday Control 15 28.2±10.5 0.247

Seventhday Control 15 45.5±9.9 0.344

15thday Control 15 33.9±5.5 0.005

a _{Nonparametric}_Friedman_test,_p_<_0.05.

observed that there was no significant difference among them(p=0.270).Asfortheseventhday,therewasalsono significantdifferencebetweenthethreewounds(p=0.759). Thesamewasobservedforthe15thday(p=0.451).

In F1, the results were similar regarding lymphocytes in the three days of comparison (p=0.774). As for F2, an increase in the number of lymphocytes was observed betweenthethirdandseventhdays(p=0.007)andbetween thethirdandthe15thdays(p=0.007).Whencomparingthe assessment days for F3, an increasednumber of lympho-cyteswerefound onlywhencomparingthethirdand15th days(Fig.5).

Regardingmacrophages,theresultsweresimilaramong thethreewoundsfor boththethirdandtheseventhdays, (p=0.247andp=0.344,respectively).Asforthe15thday, theresultsshowedasignificantdifferenceamongthethree wounds, with a lower number of macrophages in F2 and F3 (p=0.005). In addition, for the 15th day, when com-paringthewoundstwoby two,adifferencewasobserved betweenF1andF2(p=0.001),withfewermacrophagesin

120

100

80

60

40

20

Control

* * *

* #

Hemato

xylin and eosin – Fibrob

lasts

(Median;

25

%–

75%;

min - max)

Epithelial growth factor

Mixed growth factor 0

Figure 6 Hematoxylin and eosin evolution --- fibroblasts in

eachwound,betweenthetimesofeuthanization,assessment

betweengroups.

F2;andbetweenF1andF3,therewerefewermacrophages inF3(p=0.004).F2andF3showednosignificantdifference (p=0.660;Table2).

When comparing the days of euthanization regarding fibroblasts, significant differences were obtained among them,withthehighest numberoffibroblasts inF2andF3 (p=0.018forF1;p=0.003forF2,andp<0.001forF3).When comparingthedaysofeuthanizationtwobytwo,therewere differencesbetweenthethird andseventhdays(p=0.004 forF1andp<0.001forF2andF3,whichshowedhigher num-beroffibroblastsondayseven).Whencomparingbetween thethirdand15thdays,asignificantdifferencewasverified onlyforF2 (p=0.044)andinthecomparisonbetweenthe seventhand15thdays,onlyforF3(p=0.003;Fig.6).

Whenassessingtheevolutionofeachwoundinthethree days of euthanization, significant differences were found regardingtheincreaseinthenumberofvessels(p<0.001).

Note: * p < 0.05 for the 3-7 days difference in the three wounds and 7-15 days difference in control wounds and with epithelial growth. # p < 0.05 for the 3-15 days difference in the three wounds. 50

45

40

35

30

25

20

15

10

5

0

Control

# # #

* *

*

Hemato

xylin and eosin –V

essels

(Median;

25

%–

75%;

min - max)

Epithelial growth factor

Mixed growth factor

Figure7 Hematoxylinandeosinevolution---vesselsineach

wound,evaluationbetweenthetimesofeuthanization,

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Wound 1 Wound 2 Wound 3

Figure8 Photomicrograph ofvimentin ---inwhich macrophages, fibroblasts, andvessels areevaluated, onthe third dayof

evolution,inthewounds1,2,and3(VIM×200).

Figure9 Photomicrograph of vimentin---where macrophages, fibroblasts,andvessels areevaluated, onthe seventh dayof

evolution,inwounds1,2and3(VIM×200).

Figure10 Photomicrographofvimentin---wheremacrophages,fibroblasts,andvesselsareevaluated,onthe15thdayofevolution,

inwounds1,2,and3(VIM×200).

When comparing the days of euthanization in pairs, sig-nificantdifferences wereobserved betweenthe thirdand seventh days (p<0.001), and there were also significant differences between the third and 15th days (p<0.001). Betweentheseventhand15thdays,therewasnosignificant differencefor F1 and F2 (p=0.044 and p=0.035, respec-tively)(Fig.7).

Figs.8---10showthehistologicaldetailsofthe

immunohis-tochemicalassessmentofscarsinallthreeassessedperiods oftime.

WhenassessingtypeIcollagenonthedaysof euthaniza-tion,therewasnostatisticalsignificanceforF1(p=0.240), whereas there was a greater amount of type I collagen forF2andF3(p≤0.001),demonstratingadecreaseinthe amount of inflammatory infiltrate, increased angiogenesis andfibroplasia,witha faster and moreorganized healing (Figs.11---13).

RegardingcollagentypeIII,significantdifferenceswere foundinF2andF3,withaloweramountoftypeIIIcollagen (p≤0.001).

Discussion

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100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

72.1% 72.4% _70.4%

27.9% 27.6%

Wound 1

Collagen percentage

on the 3

th day

Wound 2 Wound 3 29.6%

Red Green

Figure11 DemonstrationofthepercentageoftypeIandtype

IIIcollagen onthe third dayof the experiment inthe three

wounds. 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Collagen percentage

on the 7

th day

Wound 1 41.9% 58.1% 36.9% 63.1% 41.8% 58.2%

Wound 2 Wound 3

Red Green

IIIcollagenontheseventhdayoftheexperimentinthethree

wounds. Red Green 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Collagen percentage

on the 15

th day

Wound 1 49.2% 50.8% 64.7% 35.3% 73.3% 26.7%

Wound 2 Wound 3

III collagenon the 15th day of the experiment in the three

wounds.

Wound healing is a complex series of reactions and interactions of inflammatory mediators and cell growth interactions.10---14_Many_intrinsic_and_extrinsic_factors_affect wound healing, and there is a great variety of commer-cialoptions that aim tocounteract negativeinterference or stimulate the healing process.14 _The _{interrelationship} between nutrition and the wound healing process acts together with the immune system and the immunomodu-latory function.15 _The _present _study _analyzed _the _healing process evaluating macroscopic and microscopic aspects, as well as the influence of growth factors on the heal-ing process. In the beginning of the healing process, thereisamigrationofneutrophilsstimulatedbyplatelets, and after that themacrophages, which contribute to the angiogenesisand fibroplasia.This occurred in thepresent

study, in which the wounds with EGF and MF infiltra-tion exhibited an increased number of neutrophils, with p=0.058andp=0.076;forthemacrophages,significant dif-ferences were found in the three wounds (p<0.01); but when comparing the wounds two by two, there was sig-nificant difference for the F2 and F3 (p<0.001). In the HE evaluation, regarding the vessels, there were signifi-cantdifferencesbetweenthethreewounds(p<0.001).The immunohistochemicalanalysisalsoshowedincreased fibro-blasts, macrophages, and vessels for wounds F2 and F3 (p<0.001). FortypeIcollagen, F2andF3 showedgreater depositionofthistypeofcollagen(p=0.001andp<0.001), demonstratinggreaterinflammatoryresponseandincreased angiogenesis,withfaster healing.The typeIcollagen per-centagesonthe 15thday of theexperiment were:for F1 (49.2%), for F2 (64.7%), and for F3 (73.3%). This demon-strates the improvement in wound healing where there wasinjectionof growth factors in relation tothe control wound.

Withregardtogrowthfactorsderivedfromplatelets,van den Dolder et al. carried out a study in rat bone matrix andperformedaninvitroculture,asampleofbonematrix coveredwith skin and PRP andanother sample withonly bone matrix with skin; they concluded that PRP stimu-lates the matrix and bone differentiation, as a positive correlationwasfoundbetween woundhealingandgrowth factor.16 _The_present _study_analyzed_the_use_of_VEGF,_EGF, FGF,andIGFtoacceleratewoundhealing;favorableresults were obtained when they were compared with the con-trolwound,withoutthepresenceofgrowthfactors.Wound areawasmacroscopicallyanalyzedandshowedthe follow-ing results: F1 showed an area of 0.031cm2 _at _the _end

oftheexperiment andthe F2andF3 areas were, respec-tively, 0.008cm2 _and _0, _{demonstrating} _a _more _effective

healing.

Thepercentageofcollageninthepresentstudy,observed throughthemicroscopyusingHE,washigherinwoundswith growthfactor,inadditiontoalowerinflammatoryresponse whencomparedwiththe control wound (p<0.05=0.016). Similarresults arefound inthe literature.Xieetal. used aratmodelandcomparedhealinginwoundstreatedwith growthfactorsandacontrolwound.Thewoundswere mon-itoreddailyandmeasuredwithacaliperondaysone,seven, 14,and28,wheneuthanasiaandexcisionalbiopsywere per-formed. The microscopic analysis wasperformed withHE andcollagenwasmeasuredinpercentages.3_The methodol-ogywassimilartothat usedin thisstudyfor thecreation ofthewounds,measurements,biopsy,andmicroscopy.The resultswerealsosimilar,astheyshowedahigherpercentage ofcollageninwoundswithgrowthfactor.

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showedthattopicaluseofgrowthfactorpromotedhealing improvement,asdemonstratedinthepresentstudy.17

Inanotherstudy,theauthorscomparedawoundhealing modelinWistarratsthroughtheexpressionofgrowth fac-tors---Group1:healthywithoutwound;Group2:excisional wound; Group 3: transcutaneous electrical nerve stimu-lation (TENS); Group 4: topical saline solution; Group 5: povidone iodine; Group 6: lavender oil. The experiment lastedfivedaysandthegrowthfactorsweremeasuredby immunoassayandimmunohistochemistryanalysis.The lev-elsofPDGFandEGFgrowthfactorsweresignificantlyhigher in theTENS group comparedtothe other groups andthe controlgroup (p<0.05).4 _This _study _showed _the_different treatments for wound healing, distinct from the present studyinwhichonlytheeffectofgrowthfactorsonwound healingwasassessed;therewasnoexternalstimulus,only growthfactorswereused.

Itisnoteworthythatalthoughthepresentstudyobtained favorableresultswiththeuseofEGF andMF(p<0.05)on thethree daysof assessment and a higherpercentage of maturecollagenforF2andF3,with64.7%and73.3%; respec-tively,whencomparedwithF1withonly49.2%,therewas astudythatevaluatedtheeffectsofPRPinhorsewounds withnegative results. Three wounds were created in the metacarpalregionof sixmares of differentbreeds, total-ing36wounds;of these36wounds,18weretreated with PRPandbandageand18werecontrolwounds,treatedonly withbandagewithoutmedication.Ananatomopathological studywasperformed one,two,andthreeweeksafterthe woundwasmade.Alargeamountofgranulationtissueand highconcentrationof transforminggrowth factorB1were observedinthewoundstreatedwithPRP, whencompared withwoundsthatwerenottreatedwithPRP.Histological, biochemicaldata,andgeneexpressiondidnotsignificantly differbetweenwoundstreatedwithPRPandtheuntreated. Theauthorsconcludedthatthetopicalapplicationof autol-ogousPRPdoesnotaccelerateorimprovehealingofwounds withgranulationinhorselimbs.Thiskindoftreatmentcan improvewoundswithextensivetissuelossoralternatively, chronicwoundsthatwouldbenefitmediatorstoaccelerate thehealingprocess.18

ThecareandobjectivesofthestudybyPradeepconsisted incomparingtheclinicaleffectivenessoftworegenerative techniques in the treatment of bone defects in humans. PRPandPRPassociatedwithinorganicmatrixderivedfrom bovinetissueandpeptide-15(ABM/P-15)wereused. Analyz-ingtheclinicalandradiologicalparametersin14patients, theycreatedbonedefectsandinjectedPRP,andinanother group,PRPassociatedwithAMB/P-15.Allanalyzedclinical parameters(plaqueindex,groovebleedingindex,levelof insertion,gingival margin level, probing depth) showed a statisticallysignificant difference(p<0.001) for the graft groupassociated withPRP.The computerized tomography (CT)scanshowed that bone growthwassignificantly bet-terwhenPRP+AMB/P-15werecombined(p<0.001).Itwas concludedthatthecombinationof PRPandAMB/P-15was more effective than PRP alone in treating intraosseous defects.19_Similar_results_were_obtained_in_the_present_study, asthe wounds with MF showed slightly better healing in relation to wound with EGF, which leads to the conclu-sionthatacombinationofgrowthfactorsresultsinbetter healing.

Conclusion

The macroscopicresultsrevealed areduction oftheopen wound area, and the microscopic findings support the conclusionthattheuseofgrowthfactorsinjectedintothe bordersofskinwoundsinrats,bothepithelialgrowthanda combinationofvascularendothelialgrowthfactor,fibroblast growthfactorandinsulingrowthfactor,acceleratethe heal-ingprocesscomparedtothecontrolwound,astheyfoster greaterangiogenicactivity,andacceleratedfibroplasiaand thedepositionoftypeIcollagen,inadditiontoaccelerating thematurationofhealing.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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