Physico-chemical characterization collagen scaffolds of for tissueengineering Technology Journal of Applied Researchand

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Availableonlineatwww.sciencedirect.com

Journal of Applied Research and Technology

www.jart.ccadet.unam.mx JournalofAppliedResearchandTechnology14(2016)77–85

Original

Physico-chemical characterization of collagen scaffolds for tissue engineering

B.H. León-Mancilla

^a,∗

, M.A. Araiza-Téllez

^b

, J.O. Flores-Flores

^c

, M.C. Pi˜na-Barba

^d,∗

aDepartamentodeCirugía,FacultaddeMedicina,UniversidadNacionalAutónomadeMéxico,CircuitoEscolars/n,CiudadUniversitaria,Del, Coyoacán,MéxicoD.F.CP.04510,Mexico

bLaboratoriodeBiomaterialesDentales,FacultaddeOdontología,UniversidadNacionalAutónomadeMéxico,MéxicoD.F.,Mexico

cCentrodeCienciasAplicadasyDesarrolloTecnológico,UniversidadNacionalAutónomadeMéxico,Mexico

dLaboratoriodeBiomateriales,InstitutodeInvestigacionesenMateriales,UniversidadNacionalAutónomadeMéxico,Mexico Received24August2015;accepted2September2015

Availableonline10March2016

Abstract

Theobjectivewastoresearchthephysicalandchemicalpropertiesofcollagenscaffolds(CS)obtainedfrombonematrixNukbone^®subjectto ademineralizationprocessusinghydrochloricacid.TheCSsampleswerecharacterizedbyopticalandscanningelectronmicroscopy,elemental chemicalanalysis,X-raydiffraction,spectroscopy Infrared,thermalanalysis,differential scanningcalorimetry andnitrogenadsorption. The microanalysiswereusedtosetthemacro-andmicrostructuresofCS.Theyshowedthatthe CSretainedthe morphologyofNukbone^® with interconnectedporesandtheirsizebetween100and500␮m,anditiscomposedof20%byweightofHAandtherestiscollagentypeI.Byinfrared spectroscopythefunctionalgroupsofcollagentypeI(amideA–3285,B–2917,I–1633,II–1553andIII–1239cm⁻¹)wereidentified.By thermalanalysisitwasdeterminedthatthephenomenonofdenaturationofthecollagentypeIbeganintherangeof75–85^◦Candburnedabove 200^◦C.

Keywords:Tissueengineering;Collagenscaffolds;Bonematrix;Biomaterials

1. Introduction

In the mid-1980s came the so-called tissue engineering, whichhascontinued toevolveas an excitingand multidisci- plinaryfield,aimingtodevelopbiologicalsubstitutestorestore, replaceor regeneratedefectivetissues(Chan&Leong,2008;

Lanza,Langer,&Vacanti,2013)Tissueengineeringmainlyuses threefactors:scaffoldsmadeofbiomaterialsmustmeetcertain requirements,cellsandgrowth-stimulating signals,whichare knownasthetriadoftissueengineering.Thescaffoldsaretyp- icallymadeofpolymericbiomaterials,mustbebiocompatibles andbiodegradableuponimplantation,witharatematchingthat

∗Correspondingauthors.

E-mailaddresses:[email protected](B.H.León-Mancilla), [email protected](M.C.Pi˜na-Barba).

PeerReviewundertheresponsibilityofUniversidadNacionalAutónomade México.

of the new matrix production by the developing tissue; they should be absorbable bythe humanorganism and should be porouswithinterconnectedporesofadequatesizetoallowcell adhesionandcellproliferation,givingtheopportunitytosubse- quenttissuesdevelopment,andtheymustalsohavetheability to bind to surrounding tissues and be clinically manageable (Langer&Vacanti,1993).

Thereare agreatvariety of choicestoselectscaffoldsfor tissueengineering;ingeneral,thebiomaterialsusedformaking porousscaffoldscanbeclassifiedintotwocategories accord- ing to their sources, natural and synthetic (Meyer, Meyer, Handschel,&Wiesmann,2009).Thenaturalbiomaterialsgener- allyhaveexcellentbiocompatibilitysothatthecellscanadhere andgrow withexcellent viability.In thissense,the scaffolds can be obtained from different animal species: pigs, bovine andhorses,usingintestine,pericardium,skin,tendon,boneand demineralizedbone(Carletti,Motta,&Migliaresi,2011;Chen, Torian,Price,&McKittrick,2011).

http://dx.doi.org/10.1016/j.jart.2016.01.001

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Themainobjectiveofthisworkwastodeterminethephys- icochemicalpropertiesofcollagenscaffolds(CS),producedby immersion of bone matrix (BM) blocks inhydrochloric acid (HCl)at0.5M, throughdifferent characterizationtechniques:

the histological studies were made with optical microscopy (OM) using the following stainings: hematoxylin & eosin (H&E)(MontuengaBadía&CalvoGonzáles,2009),vonKossa (Penney, Powers, Frank, Willis, & Churukian, 2002), Mas- son’sTrichrome(Sampedro,2014)andpolarizedlight(Narváez, 2014).Scanningelectronicmicroscopy(SEM)andenergydis- persive spectrometry (EDS) (Vázquez & Echeverría, 2000);

thermogravimetric analysis (TGA) and differential scanning calorimetry(DSC) (Skoog,Holler, &Crouch,2008); Fourier transforminfraredspectrometry(FTIR)(Coman,Grecu,Baciut, Prodan & Simon, 2007; Griffiths & De Haseth, 2007) the totalareawas measuredusing N2 adsorption(BET) (Nishad, Dhathathreyan & Ramasami, 2002) was measured. For each techniqueatleast5sampleswereused.

The collagen scaffolds characterized in this work were obtainedfromNukbone^®,bonematrixwhoseporesareinter- connectedin arange of size from100 to500␮mthat allow the cellular growth, and were already used without cells in patientswithbonedefects,successfully(Aguilar,Piña,López, Rodríguez,&Driessens,2005;Piña,Munguía,Palma&Lima, 2006;Cueva-Del Castilloetal., 2008;León, Araiza,&Piña, 2012;Rodríguezetal.,2013).

2. Materialandmethods 2.1. Samplepreparation

BlocksofNukbone^®(BM)of2.5cm×2.5cm×0.5cmwere employed,andeachonewasimmersedfor10mininstirringin 50mLof chloridricacid(HCl)(TJBaker,Germany)solution withbidistilledwaterat0.5M.Thentheywererinsedwithbidis- tilledwateranddried,thuscollagenscaffoldswereobtained,as showninFigure1.

Fig.1.Collagenscaffolds(CS).

2.2. Opticalmicroscopy(OM)

To performhistologicalstudiescollagenscaffoldscubesof 1cm×1cm×0.5cmwereused;theyweredehydratedinalco- holsatdifferentconcentrations:50,60,70,80,96and100%,in anhistoquinet during2h ineachconcentrationandthenwere included inwaxcubesand3␮mthickslices werecut witha microtome.

To determinethe internalstructure of the CS, bright-field microscopywasused.ThesamplesofCSwerecutintoslices of3␮mthickness.Thesampleswerestainedusinghematoxylin

&eosin(H&E), Masson’s Trichrome(MT),von Kossa(vK), andtheywerealsoobservedusingpolarizedlightmicroscopy (PLM).

2.3. Scanningelectronmicroscopy(SEM)andelemental chemicalanalysis(EDS)

The microstructure of sampleswas analyzedwithaJEOL electron microscope JSM7600F, using the technique of sec- ondaryelectronswithavoltageof5kV.Thesizeofthesamples used in SEM was 1cm×1cm×0.5cm, andno sample was covered with conductive material; they were placed directly in the sample holder. EDS technique was also employed for determiningthemaincompoundsofdifferentsuperficialzones.

2.4. X-raydiffraction(XRD)

ThepowdersofBMandCSweretestedbyanX-raydiffrac- tometer D8 Advance of Bruker AXS, withCu K␣ radiation (λ=0.154nm) and 2θ from 18^◦ to 80^◦, to obtain structural informationonanatomicscalefrombothmaterials(BMand CS).

2.5. Fouriertransforminfrared(FTIR)

ToidentifythefunctionalgroupsofcollagenusedtheFourier transforminfrared(FTIR)analysisofCSwasdonewithaSci- entificNicolet^TM 6700FT-IRspectrometer,inawavenumber rangeof500–3500cm⁻¹;thesamplesof0.2cmthicknesswere placeddirectlyinthespectrometer.

2.6. Thermogravimetricanalysis(TGA)–differential scanningcalorimetry(DSC)

The behaviorofthe sampleswithtemperature wasstudied usingTAInstrumentsSDTQ600innitrogenatmosphere.TGA andDSCcurvesweretakenintherange25–700^◦Cataheating rateof10^◦C/min.

2.7. Nitrogenadsorption(BET)

ThetotalareaofthesampleswasdeterminedusingaQuanta- chromeInstrumentsAutosorbequipment,employingcubesof 20mm×20mm×20mmofBMandCSwereused.Theanal- ysis was carried out using liquid nitrogen, and the samples

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werecleanedpriortotheanalysisat77K.Theareawasdeter- minedbeforeandafterthe demineralizationprocess(Chen&

McKittrick,2011)

3. Resultsanddiscussion 3.1. Preparationofsamples

Actually, HCl is used for demineralization of hard tissue with allograft purposes, as reported by Gruskin, Doll, Futrell,Schmitz,andHollinger(2012),orforobtainingmatrices fromdemineralizedhumanandbovine bone,for useintissue engineering; Murugan et al. reported 3 days of treatment to

demineralize the samples of 10mm³ at environment temperature(Murugan, Ramakrishna,&Rao,2008);meanwhileour treatmentrequiresonly10min.

Theresultsshowedthatthedemineralizationofbonematrix usingHCldidnotalteritsstructureandholdtheorganiccom- poundthatwasidentifiedastypeIcollagen.

3.2. Opticalmicroscopy(OM)

ThesamplesofCScutinto3␮mthicksliceswereusedin bright-fieldopticalmicroscopy,withseveralstainingtechniques mentionedabove.

T

P

T

T T

P

a

d

g h

T

P

T

50 µm 100 µm

P

e f

b c

Ca

Coll

L

Fig.2.Morphologicalcharacterizationwithopticalmicroscopy.(a)CSstainedwithH&E,pores(P)andtrabecules(T)withmultipleslacunae(L)canbeseen.(b) CSstainedwithMasson’sTrichrome,identifyingthefiberofcollagen(Coll)withabluecolorandresidueofcalcium(Ca)identifiedbyredcolor.(c)Elsewherein theCSinwhichahigherconcentrationofCaisobserved.a–c:100×.(d–f)CSstainedwithvonKossatechnique.Thedarkareasinvolvingahigherconcentration ofcalciumremainingafterdemineralization.Pdenotesapore,TdenotesatrabeculaandCadenotescalcium.d:10×,e:40×,f:100×.(gandh)Photographsofthe CStakenwithpolarizedlight;thecollagenfibersorientationgivesthearchitectureofbonetrabeculae(T)andthepores(P).g:10×,h:40×.

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P

R

S

R

S

R

10 µm 100 µm

100 µm X250 X500

X100

S

a b c

Fig.3.(a)Bonematrix(BM)observedbySEM,poresareobserved(P)andasmoothzonecorrespondingtothesurfaceofHA(S),andarougharea(R)lacking ofHA,correspondingtocollagenfibers.SEMimagesofCS(bandc)showedtwoareaswellidentified;thesmooth(S)surfacecorrespondedwiththeareaofHA whichwasremovedbythedemineralizationprocess.Theotherzonewasobservedasrough(R)correspondingtocollagenfibers.a:100×,b:250×,c:500×.

Figure2ashowsacutof CSstainedwithH&E;Pdenotes the spaceof aporewhileTdenotesthe spaceoccupied bya trabecula,nowwithouttheceramiccomponent.Themorphol- ogyofthistissueisobservedwitheosinophilicpigment(red), thusexpressing itsaffinityby the organicstructure, whichin thiscaseisthecollagen.Thebonegapswerespaces(L)occu- piedbyosteocytes,preservedtheirarchitecture(sizeandform), andtheywerebasophilicpigmented(blue),whichmatcheswith descriptionreportedbyDomínguezandTorres(2006).

MeanwhileFigure2bandcshowsa3␮mthicknesshisto- logicalsection of CSstained withMasson’s techniquewhere thestructurewasoccupiedbyatrabeculameshwork,nowwith- outceramiccompound.Here,theredcolordenotesthecalcium presence(Ca)andthebluecolordenotescollagen(Coll);itis possibletoseethatthecalciumisfoundmostlyontheedges, andit isnot distributeduniformlyinthe volumeas couldbe expected.

VonKossastaining,showninFigure2d–f,isemployedto determineCaconcentrationinnewformationtissue(ZongMing, Jian Yu, Rui Xin, Hao,& Yong, 2013). Thisstain allows to observe the concentration Caplaces; these places are related withthesiteswheretheHAisassociatedwiththecollagenin Masson’sstain,observedinFigure2c(inredcolor).InFigure2e onlytwodarkzonescorrespondtohighconcentrationofCa,and intherestofthebodyofCS,smallsitesofCawereobserved.The architectureoftrabeculaeisconserved;inFigure2fthedetails ofthedepositionofCaincludingacirclewayofthetrabeculae areshown.

Figure2gandharecutsof3␮mthickness;theseimageswere obtainedwithapolarizedlightmicroscope,anditcorresponds tothecollagenofatrabeculae.Thistechniqueshowedhowthe collagenfibersarearrangedinbundlesthatdeterminethefinal morphologyofthebonematrixandthescaffolds;theporesare preservedandtheirsizesvariedintherangeof100–500␮m.

3.3. Scanningelectronmicroscopy(SEM)andelemental chemicalanalysis(EDS)

Figure3a(X100)showsthemicrostructureoftheBM;three zonesmaybeseen;oneofthemisrough(R)andcorrespondsto acut fromtrabecula; another zone is smooth(S) which cor- responds to the extern surface of the trabeculae constituted byhydroxyapatite(ceramiccomponent ofthebones),andthe lastzoneisaporouszone(P),thatcorrespondstoopenedand

interconnectedpores,whichhavearangesizeof100–200␮m, andafracture(circle)duetoexcessivechargeofcompressionis alsoobserved.

Figure3bshowsaphotomicrographofCS(X250);itisinter- estingtonotetwodifferentsurfaces;oneofthemisasmooth zonefeaturetohydroxyapatite(HA,maincompoundofbone) andwhichmaintainsitsmorphology(S)andiscoveringtorough volumes (R) that correspond to the trabecula conformed by collagenfibrous;thisfactspeaks ofthe kindnessof thedem- ineralizationmethod.

Figure 3c(X500) also showsa microstructure of CS,and it can beseenthat the exterior surface(S) retainsits smooth morphologywhilethevolumeismadeoflongfibersasexpected.

ThiswasalsoreportedbyChen&McKittrick,(2011).

3.4. MacroporesmeasuredbySEM

Through SEMthe poresizebeforeandafterdemineraliza- tionwascompared.Figure4aandbareSEMimages(X50),and theyallowedmeasurementstothebonematrix,firstmineralized (BM)andafterdemineralized(CS).Theporesweremeasured and therewas no difference inthe measurements beforeand after demineralization.The poresizesremainedinarange of 180–350␮m. Murphyreportedthat the optimumporesizeto promote cellproliferation andmigration inbone tissueengi- neeringis325microns(Murphy,Haugh,&O’Brien,2010).The poresgenerallyhaveanovalshapethatthelargestdiameterwas inarangebetween100and500␮m,andwasidealforuseasa cellscaffold.

3.5. EnergydispersiveX-rayanalysis(EDS)

ElementalanalysiswasmadeusingEDSofthesmoothand roughtareasidentifiedbySEMinFigure4aandb,tobepunc- tualisasemiquantitativeanalysis.Onthesurface,theidentified elements of higher percentage in weight were: O (41.34%);

Ca (29.36%); C (16.08%); P (12.16%); Mg (0.66%)and Na (0.39%)thatcanbeassociatedtocalciumphosphateslikeHA, as observed inFigure 3a. In the fibrous zone under the surface,theidentifiedelementswereC(43.92%),N(23.87%)and O (23.87%); these elements are part of the amidefunctional groupsofthecollagen(NH,C O)betweenothers,asobserved inFigure 3c. InTable 1,the elementsfoundonCS andtheir proportionarereported.

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X 50 X 50 230 µm

100 µm

230 µm 300 µm 300 µm

350 µm 350 µm

130 µm 130 µm

180 µm b

a

100 µm 100 µm

Fig.4.SEMimagesshowingtheeffectofdemineralizationprocessinthemorphologyandstructureofBM(a).Therewerenosignificantchangesasaresultof demineralizationindistributionofpores,neitherinshapenorsize,thuspreservingthethreedimensionalstructureintheCS(b).

Table1

ElementalanalysisbyEDSofcollagenscaffolds.

Element Inorganicarea Organicarea

Weight% Weight%

C 16.08 43.92

O 41.34 31.41

P 12.16 –

Na 0.39 –

Mg 0.66 –

Ca 20.36 –

N – 23.87

3.5.1. X-raydiffraction(XRD)

The XRD of BM and CS is shown in Figure 5 and was comparedwiththedatasheet9-432fromJointCommitteePow- derDiffraction(JCDP),ofPDF-2(PowderDiffractionField)of InternationalCenterforDiffractionData(ICDD,2006),thatcor- respondstohydroxyapatite,mineralcompoundofbone,whichis acrystallineceramic.ThediffractogramoftheCSappearsasan amorphousmaterial;however,themainpeaksofHA:2θ=25.8

A.U. (002) (121) (112)(300)

Bone matrix

Collagen scaffolds

30 60

2θ Degree (°)

Fig.5.X-raydiffractogramsforbonematrix(BM)andcollagenscaffold(CS).

ThereferencepeakscorrespondtoJCPD9-432file.

(002),2θ=30.4(121),2θ=30.6(112),2θ=32.9(300)aredis- tinguished,whichindicatesthatacertainamountofitispresent inthesample.

3.6. Infraredspectroscopy(FTIR)

Theresultsobtainedthroughthistechniqueallowustoiden- tify the functional groups of the organic part of the CS, see Figure6.However,thereisaphosphategroupPO4(1076cm⁻¹), correspondingtoremainderhydroxyapatite(Xiao,Cai,&Liu, 2007).FunctionalgroupsidentifiedintheIRspectrawereamide A,B,I,IIandIIIcorrespondingtocollagen(Xiaoetal.,2007).

The sample of CS shows the following bands:

1650–1665cm⁻¹ (C O) corresponding to Amide I;

1530–1550cm⁻¹ corresponding to Amide II ␦(NH)+␯(CN);

3325–3330 for Amide A (N H); the peaks at 2956cm⁻¹ corresponding to functional group CH3; 2924cm⁻¹ toCH2; 1239cm⁻¹ to Amide III ␦(NH)+␯(CN); and 3080cm⁻¹ to Amide B (CH2) can be seen. Those functional groups are corresponding tocollagen(Sionkowska&Kozłowska,2010).

Table2summarizesthedataobtainedfortheCSIRspectra.

110

100

Collagen scaffolds

90

80

Amide A 3285

Amide B 2917

Amide I 1633

Amide III 1239

PO₄ 1076

Amide II 1535

500 1000 1500

3500 3000 2500 2000

Wavenumbers (cm^-1)

Transmitance

70

60

50

Fig.6.InfraredspectraofCSwherefunctionalgroupsforamides(A,B,I,II, III)andphosphate(PO4)areidentified.

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Table2

Infraredspectrum.

Functionalgroups IR(cm⁻¹) IRofCS

CH3 2956 –

CH2 2924 –

AmideI(C O) 1650–1665 1633

AmideII␦(NH)+␯(C-N) 1530–1550 1535

AmideIII␦(NH)+␯(C-N) 1239 1239

AmideA(NH) 3325–3330 3285

AmideB(CH2) 3080 2917

3.7. Thermogravimetricanalysis(TGA)

TheTGAprovidedinformationaboutthebehaviorwiththe temperaturepresentedbytheBM(Fig.7a)ascomparedwithCS

(Fig.7b).Themainlossispresentedinthreedifferenttemper- aturerangesgivenbyA:(50–200^◦C);B:(200–450^◦C)andC:

(450–700^◦C),asshowninFigure7aandb.ThecurveinAcorre- spondedtothelossofthephysisorbedandchemicalwaterinthe material, whichrepresentedthe 6.07%wtof the bonematrix, whichoccurredat79^◦C.Meanwhile forCStheloss ofwater correspondedto9.6%wtandoccurredat114.74^◦C.Thesefind- ingsaresimilartothosereportedbyLozano,Pe˜na&Heredia, (2003).

The followingloss,occurringintherangeof temperatures Binthethermogram,forBMwasobservedbetween341and 493^◦Ccorrespondingto30.34wt%losscomparedto67.76%

corresponding to 311.63^◦C for CS. These losses are related tothecombustion ofcollageninthetwo samples.The differ- ences in mass loss in the BM could be dueto the structure

A

a

B C

A B C

0.20

0.15

0.10

Deriv. Weight (%°C)

0.05

0.00 100

90 ^79.69°C

93.93% 341.07°C

81.91%

493.26°C 69.08%

0 100 200 300 400

Temperature (°C)

500 600 700

Universal v4.5A TA instruments 63.59%

(3.212mg) Residue 80

70 60

50

Weight, %

40 30 20 10 0

100

80

60

Weight, %

40

20

0 100 200 300

311.29°C 66.00%

25.57%

(1.504mg) 77.71°C

92.85%

400 500 600 700

Temperature (°C) Universal V4.5A TA instruments

0.004

0.003

0.002

0.001

0.000

Deriv. Log[Weight] (1%°C)

Resldue

b

+

Fig.7.(a)TGAgraphofbonematrix(BM),upperlinecorrespondstotheweightlossandlowerlineisthederivativecurve(ω/T).(b)TGAgraphofcollagen scaffold(CS).

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Table3

Thermogramsdataforbonematrixandcollagenscaffold.

Condition BM CS

Massloss(%) ^◦C Massloss(%) ^◦C

Dehydratedcollagendenaturalized 6.07 79.69 9.6 114.76

Collagencombustion 30.34 341/493 64.76 311.63

Remainder 63.59 700 25.64 700

4

2

0

–2

Heat flow (W/g)

–4

–60 100

Exo Up

Endothermic Endothermic Exothermic

200 300

Temperature (°C) 500

400 600 700

Universal V4.5A TA instruments

BM CS

Fig.8.ComparativeDSCanalysisofthebonematrix(dottedline)andofscaffold collagen(continuousline).

ofthetrabeculaedevoidfrommineralphase(HA),sothatthe combustionoccursatalowertemperature.Finally,theweight lossinC,correspondingto450–700^◦C,presentingaminimum changeinmassloss,leavingaresidueof63.59%for theBM and25.64%forCSsurelycorrespondstocalciumcompounds.

Theuseofderivativeofthermalanalysis(DTG)alloweddeter- miningthatthedegradationprocessofcollagenhadmaximum

rateat311.95^◦C.Thedataobtainedfromthethermogramsfor BMandCSaresummarizedinTable3.

3.8. Differentialscanningcalorimetry(DSC)

Thistechnique allowedtoidentify changes inthethermo- dynamic variables that occurred during the physic-chemical transformations induced by heating or cooling the BM and CS. Thefirst endothermicpeakobserved inBM andCS was between85and90^◦C,correspondingtodehydrationprocessof surfacewater,seeFigure8.Thisresultisalsopresentedinthe study by Lozanoetal.(2003).The second endothermicphe- nomenonobservedintheBM andCSwas inthetemperature range between275^◦Cand325^◦C.Thisisrelated tothe loss of hydrogen bonds, so thatthe phenomenon of protein dena- turation,couldbeinitiatedinthisintervalinwhichthetertiary structureislost.Thisphenomenonisreversibleiftheproteinis newlyhydrated.

Anexothermicprocesswasfoundinbothsamples(BMand CS)observedinatemperaturerangebetween350^◦Cand425^◦C, corresponding to the combustion of the collagen fibers. The resultsobtainedwiththeDSCtechniquearecomplementedby TGA,whichsuggeststhatBMandCSarebasicallycomposed ofthreecomponents,whicharewater,collagenandhydroxyap- atite.

38.48

19.24

Volume (cc/g)Volume (cc/g)

Isotherm

Isotherm Relative pressure, P/Po

Relative pressure, P/Po

a

b

0.00

16.52

8.26

0.00

0.000 0.2000 0.4000 0.6000 0.8000

0.000 0.2000 0.4000 0.6000 0.8000 1.00

1.00

Fig.9.N2physisorptioncurvesrecordedat77Kofbonematrix(a)andcollagenscaffold(b).Linered:adsorption,lineblue:desorption.

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Table4

TotalareaBETunderIUPACclassification.

Surfacearea(m²/g) Isotherm Poresize Poreform

BM 5.39 TypeII Macroporous Open

CS 7.34 TypeII Macroporous

Difference 36.18%

3.9. Nitrogenadsorption(BET)

The total area of the BM was determined by BET and wascomparedwiththeareaofCS.Theresultsobtained were 5.39m²/gforBMand7.34m²/gforCS.Thedifferenceofthe total surface areabetween the BM andCS was 36.18%,the resultcanbeusedforinvitrostudies,becauseitistheareathat willbeincontactwiththecells.Figure9ashowstheisotherm presentedbytheBM,anditshowsthattherewerenochanges (hysteresis)betweenadsorption(redline)anddesorption(blue line).Figure9bistheisothermfromCS,andthereisamini- mumdifferencebetweentheadsorptionanddesorptionofgas.

TheisothermsobservedareType2andcorrespondtoanopen macroporestype,accordingtotheIUPAC.Table4summarizes alldataobtained.

4. Conclusions

Thesescaffoldshavetheadvantagethattheycanbemolded to give them different shapes. The scaffolds preserved the morphologyfromtrabecularbone,theporesareopenandinter- connected, and the pore sizes are adequate for use as cell scaffolds(tissueregeneration).Thisbiomaterialisproposedfor useasascaffoldintissueregeneration.Thecollagenscaffolds obtainedarecompletelybiocompatible,biodegradableandeasy touse.

Acknowledgments

Thanks to E. Fregoso, V. Rodríguez, A. Pérez, O. Nov- elo, A. Tejeda, M.A. Canseco, M. Herrera, A. Zepeda, F.

PasosandV.Maturanofortheirtechnicalsupport.Alsothanks to DGAPA-UNAM for financial support through projects:

IG100114,IT114911,IT119111andIN113108.

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