Effect of curcumin on bone healing: An experimental study in a rat model of femur fracture

Injury, Int. J. Care Injured 50 (2019) 1915–1920

Effect

of

curcumin

on

bone

healing:

An

experimental

study

in

a

rat

model

of

femur

fracture

Selim

Safali

_,

_Bahattin

_Kerem

_Aydin

_*

_,

_Alaaddin

_Nayman

_,

_Ceyhan

_Ugurluoglu

b_{SelcukUniversity,DepartmentofRadiology,Turkey} c_{SelcukUniversity,DepartmentofPathology,Turkey}

ARTICLE INFO Articlehistory: Accepted1September2019 Keywords: Rat Femur Fracturehealing Curcumin ABSTRACT

Objective:Todeterminetheradiologic,histologicandbiomechanicaleffectsofcurcuminonbonehealing

usingatotalratfemurfractureinjurymodel.

Materialsandmethods:SixtyfourmaleWistar–Albinoratsweighing170–210gwereusedinthisstudy.

Theanimalswererandomlydividedintoeightgroupsand5or6animalswereplacedineachcage.A

transversefemurshaftfracturemodelused.Theanimalsinstudygroupsreceivedoralcurcuminatadose

of200mg/kgfor14daysor28days.Remaininganimalsreceivedonlysalinesolutionbyoralgavagefora

periodof14daysand28daysascontrolgroups.Aftersacrificationtheleftfemursusedforradiological,

histologicalandbiomechanicalevaluation.

Results: The groupstreated withcurcuminshowed nosignificant differencein termsof radiological,

histologicalandbiomechanicalevaluationsin14daysgroups.Alsotherewasnosignificantdifferencebetween

curcuminandcontrolgroupsfor28daysaccordingtoradiological,histologicalandbiomechanicaltests.

Conclusions:Accordingtoourresults,curcuminhasnopositiveeffectonfracturehealingnotonly

histologicallybutalsoradiologicallyandbiomechanically.Curcumin’santioxidanteffectmaybemore

noticeablewithlongtermfollowupinvestigationasitmayhaveapositiveeffectinremodellingphase.

Longtermfollowupdesignedstudiesmaybeplannedtoinvestigateitseffectonremodellingphaseof

fracturehealing.

©2019ElsevierLtd.Allrightsreserved.

Introduction

Fracture healing continues to be a significant challenge for orthopaedic surgeons. It leads to significant health care costs which resultin restrictedactivityand individualmorbidity [1]. Boneisauniquetissuewithastronghealingcapabilityintermsof restoringoriginaltissuebutnotintermsoffibrosisrepair.Healing isacomplexandorderlyregulatedprocessthatcanbeaffectedby multiplefactors.However,previouspublicationshaveshownthat in5%10%ofbonefractures,healingcannotbeachievedleadingto impairedqualityoflife[1,2].

Curcumin, a natural compound extracted from turmeric (Curcuma longa), has strongantioxidant and anti-inflammatory activities.Morethan10,000articleshavediscussedthemolecular

basisofcurcumin’spotentialantioxidant,anti-inflammatory,and anti-infectious activities during the last decade [3–7]. Many studieshaveshowncurcumin’spositiveeffectsontissueinjuries ofthedermis,nervefibres,andburnsaremediatedviaactivationof cellmigrationandwoundhealing[8–10].Ithasbeenreportedthat antioxidanttherapyandinhibitinginflammatoryprocessescould havepositiveeffectsonthehealingofsofttissueinjuries[1,11–13]. Manyfactorshavebeenstudiedfortheirroleinimprovingbone healing processes such as stem cells, growth factors, and haemostaticagents[14–17]. However,untilnow,therehasbeen only one reported study in the English literature assessing curcumin’seffectonbonehealingviahistologicandradiological evaluationbutnotbiomechanicalevaluation[18].

In thepresentstudy,weaimedtoinvestigatetheradiologic, histologic,andbiomechanicaleffectsofcurcuminonbonehealing usingaratfemurtotalfractureinjurymodel.

Materialsandmethods

Sixtyfour maleWistar albinoratsweighing 170–210gwere used.Theexperimentaldesignandallprocedureswereapproved

* Correspondingauthorat:SelcukUniversity,FacultyofMedicine,Departmentof Orthopaedicsand Traumatology, AlaeddinKeykubat Campus,42100, Selcuklu, Konya,Turkey.

E-mailaddresses:selimsafali@gmail.com(S.Safali),bkaydin@yahoo.com

(B.K.Aydin),naymanalaaddin@hotmail.com(A.Nayman),drceyhan@gmail.com

(C.Ugurluoglu).

https://doi.org/10.1016/j.injury.2019.09.002

0020-1383/©2019ElsevierLtd.Allrightsreserved.

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JINJ8304No.ofPages6

ContentslistsavailableatScienceDirect

Injury

1916 S. Safali et al. / Injury, Int. J. Care Injured 50 (2019) 1915–1920

byXXXAnimalResearchandEthicsCommittee(protocolnumber 2015/38).Alltheanimalsforthisstudywerepurchasedfromthe LaboratoryofExperimentalAnimals,XXXUniversity,BBcity,CCC Country.

Theanimalswererandomlydividedintoeightgroupsandfive orsixanimalswereplacedineachcage. Theeightgroupswere designatedas A,B, C, D,E,F,G,and H.Theproperties andthe numbersoftheanimalsofeachgroupareshowninTable1.

A well-known rat fracture model was used [18]. Surgical procedures were conducted under general anaesthesia with intraperitoneal injection of 50mg/kg ketamine (Ketalar; Eczacıbaşı, _Istanbul,Turkey)and10mg/kgxylazinehydrochloride (Rompun;Bayer,Leverkusen,Germany).Theleftlowerlimbsinthe rats were shaved and disinfected using povidone-iodine, and surgerywas performedundersterile conditions.A 3-cmlateral longitidinal incisions were made to expose the femurs. A transverse femur shaft fracture was made using an ossilating microsaw.A1-mmKirschnerwire(Hipokrat, _Izmir,Turkey)was usedforintramedullaryfixation.Thesubcutaneoustissueandthe skinwereclosedfollowingstandardprocedures.

Nobandageorcastingwasplacedafterthesurgeries.Allrats wereallowedtomovefreelyandwerefedwithstandardlaboratory feedandtapwater.

TheanimalsingroupsAandCreceivedcurcumin(curcumin, Sigma-Aldrich,Germanysuspendedinsalineatadoseof200mg/ kgorally)viaoralgavagefor14daysandtheanimalsinGroupE andFreceivedcurcuminforfourweeksstartingfromthedayof surgery.Theremaininganimalsreceivedonlysalinesolutionvia oralgavageforaperiodof14days(ingroupsBandD)or28days(in groupsEandF).Thedosageofcurcuminwasbasedonprevious similarstudies[13,19].

High-doseethervapourwasusedforeuthanasia.Aftertherats werekilled,theirleftfemursweredisarticulatedfromtheirhipand kneejoints.Softtissuesonthefemoralbonewerepeeledoffgently fromthebonewithoutanyharmtothecallustissue.Allofthe left femurs were studied addressing clinical, radiological, and histologicalaspects.

Alltheleftfemurswereremovedandtakenimmediatelyfor radiologicanalysis.ThefemursobtainedfromgroupsA,B,E,andF

were used for histological analysis. The femurs obtained from groupsC,D,G,andHwereusedforbiomechanicalanalysis. Radiologicanalysis

Sixtyfourfemurswereusedforradiologicalanalysis.Computed tomography (CT) imaging was performed with a 1282-slice dual-sourceCT (Somatom Definition Flash, Siemens, Germany). ThesampleswereplacedintheCTforobtainingmicro–CTimages byscanningalong thelongaxisof thefemoralsamplesandthe regionofinterest,10mmaroundthefracturesite,witha0.6mm collimation.TheKwireswerenotremovedbecausetheydidnot causeanyartefactsinradiologicalanalysis.

The software Radiant Dicom Wiever 4.6.9 was used for radiologicalmeasurements.Totalcallusdiameter,low-radiodensity bonemeasurements,highdensitybonemeasurements,lengthof callustissue,andfemoraldiameterparametersweremeasured.For measuringcallusdiameter,low-radiodensitybonemeasurements, high density bone measurements, and femoral diameter, axial sectionswereused.Analysisaftertwoweekswasdoneusingthe ratiooftotalcallusdiameter/femoraldiameter,andanalysisafter fourweekswas doneusingtheratio oflowdensitybone/highdensity bonemeasurements[Fig.1].

Histologicalanalysis

Atotal 32 femursfromgroups A,B, D,and Ewereusedfor histologicalanalysis. A bufferedformalin solutionwas usedfor fixationovertwodays.Next,10%aceticacidsolutionwasusedfor decalcificationoverfourdays.Next,thespecimenswere embed-dedinparaffinand3mm-thickserialsectionsweretakenfromthe callus site of each femur. Hematoxylin and eosin staining was performed.Histologicalclassificationofthehealingwasdoneper thehistologicalhealingscalepublishedbyHuoetal.[20]. Biomechanicalanalysis

Atotal32leftfemursoftheratsfromgroupsC,D,G,andHwere used for biomechanical analysis. Biomechanical testing were

Table1

Thedistrubutionofgroups,contentsandsacrificationtimes.

GroupName GroupFeature NumberofRats SacrificationTime

A 14daysHistologicalAssesmentCurcuminGroup 10 14days

B 14daysHistologicalAssesmentControlGroup 6 14dayd

C 14daysBiomechanicalAssesmentCurcuminGroup 10 14days

D 14daysBiomechanicalAssesmentControlGroup 6 14days

E 28daysHistologicalAssesmentCurcuminGroup 10 28days

F 28daysHistologicalAssesmentControlGroup 6 28days

G 28daysBiomechanicalAssesmentCurcuminGroup 10 28days

H 28daysBiomechanicalAssesmentControlGroup 6 28days

Fig.1.Schematicdrawingsofmeasurementsusedforradiologicalevaluation.A,longitudinalsection.B,Transversesection.

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S. Safali et al. / Injury, Int. J. Care Injured 50 (2019) 1915–1920 1917

byXXXAnimalResearchandEthicsCommittee(protocolnumber 2015/38).Alltheanimalsforthisstudywerepurchasedfromthe LaboratoryofExperimentalAnimals,XXXUniversity,BBcity,CCC Country.

Theanimalswererandomlydividedintoeightgroupsandfive orsixanimalswereplacedineachcage. Theeightgroupswere designatedas A,B, C, D,E,F,G,and H.The propertiesand the numbersoftheanimalsofeachgroupareshowninTable1.

A well-known rat fracture model was used [18]. Surgical procedures were conducted under general anaesthesia with intraperitoneal injection of 50mg/kg ketamine (Ketalar; Eczacıbaşı, _Istanbul,Turkey)and10mg/kgxylazinehydrochloride (Rompun;Bayer,Leverkusen,Germany).Theleftlowerlimbsinthe rats were shaved and disinfected using povidone-iodine, and surgerywas performedunder sterileconditions.A 3-cmlateral longitidinal incisions were made to expose the femurs. A transverse femur shaft fracture was made using an ossilating microsaw.A1-mmKirschnerwire(Hipokrat, _Izmir,Turkey)was usedforintramedullaryfixation.Thesubcutaneoustissueandthe skinwereclosedfollowingstandardprocedures.

Nobandageorcastingwasplacedafterthesurgeries.Allrats wereallowedtomovefreelyandwerefedwithstandardlaboratory feedandtapwater.

TheanimalsingroupsAandCreceivedcurcumin(curcumin, Sigma-Aldrich,Germanysuspendedinsalineatadoseof200mg/ kgorally)viaoralgavagefor14daysandtheanimalsinGroupE andFreceivedcurcuminforfourweeksstartingfromthedayof surgery.Theremaininganimalsreceivedonlysalinesolutionvia oralgavageforaperiodof14days(ingroupsBandD)or28days(in groupsEandF).Thedosageofcurcuminwasbasedonprevious similarstudies[13,19].

High-doseethervapourwasusedforeuthanasia.Aftertherats werekilled,theirleftfemursweredisarticulatedfromtheirhipand kneejoints.Softtissuesonthefemoralbonewerepeeledoffgently fromthebonewithoutanyharmtothecallustissue. Allof the left femurs were studied addressing clinical, radiological, and histologicalaspects.

Alltheleftfemurswereremovedandtakenimmediatelyfor radiologicanalysis.ThefemursobtainedfromgroupsA,B,E,andF

were used for histological analysis. The femurs obtained from groupsC,D,G,andHwereusedforbiomechanicalanalysis. Radiologicanalysis

Sixtyfourfemurswereusedforradiologicalanalysis.Computed tomography (CT) imaging was performed with a 1282-slice dual-source CT (Somatom Definition Flash, Siemens, Germany). ThesampleswereplacedintheCTforobtainingmicro–CTimages byscanningalongthelongaxisofthefemoralsamplesandthe regionofinterest,10mmaroundthefracturesite,witha0.6mm collimation.TheKwireswerenotremovedbecausetheydidnot causeanyartefactsinradiologicalanalysis.

The software Radiant Dicom Wiever 4.6.9 was used for radiologicalmeasurements.Totalcallusdiameter,low-radiodensity bonemeasurements,highdensitybonemeasurements,lengthof callustissue,andfemoraldiameterparametersweremeasured.For measuringcallusdiameter,low-radiodensitybonemeasurements, high density bone measurements, and femoral diameter, axial sectionswereused.Analysisaftertwoweekswasdoneusingthe ratiooftotalcallusdiameter/femoraldiameter,andanalysisafter fourweekswas doneusingtheratio oflowdensitybone/highdensity bonemeasurements[Fig.1].

Histologicalanalysis

Atotal 32 femursfromgroups A,B, D,and Ewereusedfor histologicalanalysis. A bufferedformalin solutionwas usedfor fixationovertwodays.Next,10%aceticacidsolutionwasusedfor decalcificationoverfourdays.Next,thespecimenswere embed-dedinparaffinand3mm-thickserialsectionsweretakenfromthe callus site of each femur. Hematoxylinand eosin staining was performed.Histologicalclassificationofthehealingwasdoneper thehistologicalhealingscalepublishedbyHuoetal.[20]. Biomechanicalanalysis

Atotal32leftfemursoftheratsfromgroupsC,D,G,andHwere used for biomechanical analysis. Biomechanical testing were

Table1

Thedistrubutionofgroups,contentsandsacrificationtimes.

GroupName GroupFeature NumberofRats SacrificationTime

A 14daysHistologicalAssesmentCurcuminGroup 10 14days

B 14daysHistologicalAssesmentControlGroup 6 14dayd

C 14daysBiomechanicalAssesmentCurcuminGroup 10 14days

D 14daysBiomechanicalAssesmentControlGroup 6 14days

E 28daysHistologicalAssesmentCurcuminGroup 10 28days

F 28daysHistologicalAssesmentControlGroup 6 28days

G 28daysBiomechanicalAssesmentCurcuminGroup 10 28days

H 28daysBiomechanicalAssesmentControlGroup 6 28days

Fig.1.Schematicdrawingsofmeasurementsusedforradiologicalevaluation.A,longitudinalsection.B,Transversesection.

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performedusingaElistaTST2500materialtestingmachine(Elista, Istanbul,Turkey).Thefemursweremountedwithminiclampson thetestingdevice.Theclampsgrippedeachspecimenatthedistal and proximal metaphyseal parts of the femurs for measuring resistance to longitudinal distraction forces (in Newtons). A distractionforcewasappliedat3mm/min.Thedistractionforced appliedwasincreaseduntilfailureofthehealingregion.Loadto failureparameterswererecordedforeachspecimen.Athree-point bendingtestwasplannedbutcouldnotbeusedbecausetwo-week specimenswerenotusableduetoonlysoftcallusformation.The recordedmeasurements(inNewtons)wereusedtocomparethe resultsofbiomechanicalevaluation.

Statisticalanalysis

Mann–Whitney U test was used for analysing radiological, histological, and biomechanical results to evaluate differences betweenthetwogroups. Alltheanalyseswereperformedwith IBMSPSSversion 20.0(IBMCorp., Armonk,NY,USA.A p value <0.05wasconsideredtobestatisticallysignificant).

Results

RadiologicalresultsbasedonCTimagesareshowninTable2. There was no significant difference in radiological evaluation resultsbetweenthecurcumin2nd weekgroupandthecontrol group (Mann–Whitney test; U=117.00; p>0.05) [Fig. 2]. For radiologicalevaluationaftertwoweeks,onlytotalcallusdiameter/ femoraldiameter measurement wereused becausehard callus formationwasinsufficient.Comparisonofradiologicalevaluation resultsfromtheratskilledinthesecondweekshowedthatthe

diameterofthecallusinthecurcumingroupwassmallerthanthat inthecontrolgroup.Atfourweeks,theratiooflowdensitybone/ highdensitybonewasusedtorepresenthealing.Lowdensitybone is a predictor of new bone formation and high density bone indicates the cortex of the intact (before fracture) bone. Radiological evaluation results of the rats killed in the fourth weekshowed thatthelow-radiodensity bone/highradiodensity bone ratiowas higherinthecurcumingroup[Fig.3].However, statisticalcomparisonofradiologicalevaluationresultsattwoor fourweeksdidnotshowanystatisticalsignificance.

Histological scores per the Huo classification are shown in

Table 3. There was no significant difference in histological examinationresultsbetweenthe2ndweekcurcumingroupand the 2ndweekcontrol group(Mann–WhitneyU test; U=30.00; p>0.05)[Fig.4].Therewasnosignificantdifferenceinhistological examinationresultsbetweenthe4thweek curcumingroupand 4th week control group (Mann–Whitney U test; U=21.00; p>0.05) [Fig. 5]. The mean scores of histological examination were4.8inthecontrolgroupand4.2inthecurcumingroupattwo weeks.Themeanscoreswere5.5inthecontrolgroupand5.9inthe curcumingroupatfourweeks.Thescoresattwoweekswerebetter inthecontrolgroup,andthoseatfourweekswerebetterinthe curcumingroup;however,thesedifferenceswerenotstatistically significant.

The results of biomechanical analysisare shown inTable 4. Therewasnosignificantdifferenceinbiomechanicalexamination resultsbetweenthe2ndweekcurcumingroupandthe2ndweek controlgroup(Mann–WhitneyUtest;U=29.00;p>0.05).There was nosignificantdifference inthebiomechanical examination resultsbetweenthe4thweekcurcumingroupandthe4thweek control group (Mann–Whitney U test; U=27.50; p>0.05). The meanloadtofailurewas8.3Ninthecontrolgroupand8.6Ninthe curcumingroupaftertwoweeks;itwas8.9inthecontrolgroup and8.2inthecurcumingroupafterfourweeks.

Discussion

There are limited studies on curcumin’s effect on fracture healing.Curcumin’spositiveeffectonfracturehealinghasbeen reported in a recent study, which found that curcumin has potentialinpromotingbonehealingbyactivatingautophagyina

Table2

Radiologicalresultsofcurcuminandcontrolgroups. Groups Numberofrats Averageradiological

assesmentscore Pvalue A+C 20 2,514 P>0.05 B+D 12 2,509 E+G 20 1,314 P>0.05 F+H 12 1,281

Fig.2.A,Sampleoftransverseradiologicalimaging(micro-CT)afterscarificationat14daysfromCurcumingroup.B,Sampleoftransverseradiologicalimagingafter scarificationat14daysfromcontrolgroup.

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ratmodel [18]. Histologicaland radiologicalevaluation showed positiveeffectsof curcuminonbonehealingbut biomechanical tests were not used in the above study. To the best of our knowledge, the present study is the first animal model study investigatingcurcumin’seffectonfracturehealingusing radiolog-ical,histological,andbiomechanicaltests.

Inthepresentstudy,wecreatedafemurfractureinjurymodel andevaluatedtheeffectsoforalcurcumin.Thehistologicalresults werebetterin thecurcumingroupattwo andfourweeks, but the differences were not statistically significant. This may be secondarytocurcumin’santi-inflammatoryeffect.Thefirstphase

of bone healing is theinflammation phase and curcumin may inhibit this phase. A similarsituation mayexistat fourweeks, representingthehealingorcallusphaseinfracturehealing.Thisis quitedifferentfrompreviouslyreportedfindings[18].Theirstudy designisquitedifferentfromourstudy.Firstly,theyusedalongK wirefixationtechniquepenetratingthekneejointandendsoutof theskin.Thistypeoffixationcanblockthemotionofthekneejoint andloadoftheextremitywhichmaybepositivefactorforfracture healing.InourstudyweusedshortKwireswhichwerecompletely buriedintramedullaryfornotlimitingtheearlyloadandrangeof motionofthejoints.Secondlythetimeperiodisforsacrifyingthe animalsisdifferentastheyusedtwoandsixweeksresults.Thirdly, Lietal.designedtheirstudyonblockingtheautophagyoftherats byusing3-methyladenineandcurcuminbuttheydidnotstudythe only effect of 3-methyladenine on fracture healing. Also the histologicalevaluationsysteminourstudywasdifferentfromthat usedintheabovestudy.Inourstudy,weevaluatedhealingusing Huo’s classification, which has 10parameters, using only light microscopywithhaematoxylinandeosinstaining.Theabovestudy usedosteoblastcellcounting,immunohistochemicaltissue stain-ing,andwesternblotting.However,thereisnoreportedobjective

Fig.3.A,Sampleoflongitudinalradiologicalimaging(micro-CT)afterscarificationat28daysfromcurcumingroup.B,Sampleoflongitudinalradiologicalimagingafter scarificationat28daysfromcontrolgroup.

Table3

Histologicalexaminationresultsofcontrolgroupandcurcumingroup. GroupName NumberofRats Averagehistological

assesmentscore Pvalue A 10 4,6 P>0.05 B 6 4,8 E 10 5,9 P>0.05 F 6 5,5

Fig.4.A,Histologicalimagesofhealingaftertwoweeks(H&EstainingX100).A,Curcumingroupshowingmainlychondroidtissueandsomewovenbone.B,Controlgroup showingequalamountofchondroidtissueandwovenbone.

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ratmodel [18]. Histologicaland radiologicalevaluation showed positiveeffectsof curcuminonbonehealingbut biomechanical tests were not used in the above study. To the best of our knowledge, the present study is the first animal model study investigatingcurcumin’seffectonfracturehealingusing radiolog-ical,histological,andbiomechanicaltests.

Inthepresentstudy,wecreatedafemurfractureinjurymodel andevaluatedtheeffectsoforalcurcumin.Thehistologicalresults werebetterin thecurcumingroupattwo andfourweeks, but the differences were not statistically significant. This may be secondarytocurcumin’santi-inflammatoryeffect.Thefirstphase

of bone healing is the inflammationphase and curcumin may inhibit this phase. A similarsituation mayexistat four weeks, representingthehealingorcallusphaseinfracturehealing.Thisis quitedifferentfrompreviouslyreportedfindings[18].Theirstudy designisquitedifferentfromourstudy.Firstly,theyusedalongK wirefixationtechniquepenetratingthekneejointandendsoutof theskin.Thistypeoffixationcanblockthemotionofthekneejoint andloadoftheextremitywhichmaybepositivefactorforfracture healing.InourstudyweusedshortKwireswhichwerecompletely buriedintramedullaryfornotlimitingtheearlyloadandrangeof motionofthejoints.Secondlythetimeperiodisforsacrifyingthe animalsisdifferentastheyusedtwoandsixweeksresults.Thirdly, Lietal.designedtheirstudyonblockingtheautophagyoftherats byusing3-methyladenineandcurcuminbuttheydidnotstudythe only effect of 3-methyladenine on fracture healing. Also the histologicalevaluationsysteminourstudywasdifferentfromthat usedintheabovestudy.Inourstudy,weevaluatedhealingusing Huo’s classification, which has 10parameters, using only light microscopywithhaematoxylinandeosinstaining.Theabovestudy usedosteoblastcellcounting,immunohistochemicaltissue stain-ing,andwesternblotting.However,thereisnoreportedobjective

Fig.3.A,Sampleoflongitudinalradiologicalimaging(micro-CT)afterscarificationat28daysfromcurcumingroup.B,Sampleoflongitudinalradiologicalimagingafter scarificationat28daysfromcontrolgroup.

Table3

Histologicalexaminationresultsofcontrolgroupandcurcumingroup. GroupName NumberofRats Averagehistological

assesmentscore Pvalue A 10 4,6 P>0.05 B 6 4,8 E 10 5,9 P>0.05 F 6 5,5

Fig.4.A,Histologicalimagesofhealingaftertwoweeks(H&EstainingX100).A,Curcumingroupshowingmainlychondroidtissueandsomewovenbone.B,Controlgroup showingequalamountofchondroidtissueandwovenbone.

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classificationsystemforthishistologicexamination.Indeed,the number ofosteoblasts and otherimmunohistochemical param-etersareimportantinfracturehealing,butwecouldnotfindany reportscomparingHuo’shistologicclassificationmethodandthe osteoblastcountingmethod.Itisknowninsomecases,although thenumberofblasticcellsishigh,itmaynotbefunctional(suchas hypertrophicpseudoarthrosis).Therefore,thedifferencein histo-logicalresultsbetweenthesesimilarstudiesmaybesecondaryto theusageofdifferenthistologicalevaluationmethods.

Theradiologicalevaluationresultswerebetterinthecurcumin group,whichalsodidnotshowstatisticallysignificantdifferences. CTevaluationwas used for investigatingbony union in therat femurs.Thediameterofcallusformationanditsratiowithintact bonediameterwasusedforradiologicexaminations.Radiological evaluationresultsfrom similarreports haveshown statistically betterresultswithcurcuminuseinX-rayvisualisation;onlythe callusdiameterwasusedasaparameterintheirCTevaluation.In ourstudy,weusedcallusdiameterpluscallusdiametertointact bone ratio. Thedifference in ourCT evaluation results may be secondarytothissituation.

Wealsoperformedanalysisofbiomechanical evaluation;the maximum load to failure parameter was measured in all rats. Three-pointbendingtestscouldnotbeappliedbecausethecallus tissuesweretoosoft.Allthefailureswereatthefracturehealing site;thisissimilartothatseeninhumanre-fractures.The two-weekresultswerebetterinthecurcumingroupandthefour-week results were better in the control group. However, when we comparedtheresults,therewasnostatisticallydifferencebetween thecontrolandcurcumingroups.Therehasbeenonlyonereport onthebiomechanicaleffectsofcurcuminonbonetissue[21];in theirstudy,theauthorsevaluatedtheeffectofcurcuminonintact bonetissuebyusinganovariectomisedratmodel.Theyconcluded thatcurcuminhadnostatisticallypositiveeffectonbonetissuevia evaluationofbiomechanicaltests.Ourresultsaresimilartothatof theabovestudy,althoughweusedafemurfracturemodel.

Therearealsosomelimitationsinourstudy.First,wecouldnot applythethree-pointbendingtestforbiomechanicalevaluation.

Althoughfracture-healedtissueswerenotapplicableforthistest, longer follow-up analysis can be performed to address this. Secondly,thenumberof ratsusedwould needtobehigher for moreaccurateresults.

Curcuminhasbeenused,especiallyinAsiancountries,totreat differentkindsofdiseasesduetoitsantioxidant, anti-inflamma-tory,immunomodulatory,andantimicrobialeffectsformanyyears [3–10]. In ourstudy, we found that curcumindidnot improve fracture healing based on biomechanical, radiological, and histologicalevaluation.Weinferthatcurcumin’s anti-inflamma-torypropertymayberesponsibleforitsineffectivenessinfracture healing.

Conclusion

Our resultsshowed thatcurcumin hasnopositive effecton fracturehealing,notonlyhistologicallybutalsoradiologicallyand biomechanically. Curcumin’s effect may be more noticeable in long-term follow-up investigations because of its potential positive effects in the remodelling phase. Long termfollow up designed studies may be planned to investigate its effect on remodellingphaseoffracturehealing.

Statementofwelfareofanimals

Allapplicableinternational,national andinstitutional guide-linesforthecareanduseofanimalswerefollowed.Allprocedures performed were in accordance with ethical standards of the institution (Selcuk University Experimental Research Centre, Konya,Turkey)wherethestudywasconducted.

Acknowledgement

ThisstudywasfundedbySelcukUniversityScientificResearch Office.

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Fig.5.A,Histologicalimagesofhealingafterfourweeks(H&EstainingX100).A,Curcumingroupshowingmainlywovenboneandsomechondroidtissue.B,Controlgroup showingmainlychondroidtissueandsomewovenbone.

Table4

Biomechanicalexaminationresultsofcurcuminandcontrolgroups.

Groups Numberofrats Averageloadtofailure Pvalue

C 10 37,7 P>0.05

D 6 31,9

G 10 49,2 P>0.05

H 6 48,6

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