JosipJurajStrossmayerUniversityofOsijek

RuđerBoškovićInstitute,Zagreb

UniversityofDubrovnik

UniversityPostgraduateInterdisciplinaryDoctoralStudy

ofMolecularBiosciences

LidijaMilković

Beneficialeffectsoflipidperoxidationinthebonecellgrowth

onbioactiveglass‐Newperspectivesintissueengineering

andregenerativemedicine

PhDTHESIS

Osijek,2014.

  

BASICDOCUMENTATIONCARD  

JosipJurajStrossmayerUniversityofOsijekPhDthesisUniversityofDubrovnikRuđerBoškovićInstituteUniversityPostgraduateInterdisciplinaryDoctoralStudyofMolecularbiosciences

ScientificArea:InterdisciplinarySciencesScientificField:BiologyandBasicMedicalSciences

Beneficialeffectsoflipidperoxidationinthebonecellgrowthonbioactiveglass‐New

perspectivesintissueengineeringandregenerativemedicine

LidijaMilković

Thesisperformedat:LaboratoryforOxidativeStressoftheRuđerBoškovićInstituteSupervisor/s:ProfNevenŽarković,PhD,MD,SeniorScientistShortabstract:This research has revealed the role of lipid peroxidation, as well as its product 4‐hydroxynonenal in the process of stimulating osteogenesis assisted by using bioactive glass.Since the bioactive glass has found its important application in bone tissue engineering, newdiscoverieswillopennewpossibilitiesforcreatingimprovedbioactiveglassonwhichisbasedthefocusoftoday'sregenerativemedicine.

Numberofpages:101Numberoffigures:27Numberoftables:4Numberofreferences:121Originalin:EnglishKeywords:bonefracturehealing,lipidperoxidation,4‐hydroxynonenal,bioactiveglass,cooper,enhancedosteogenesisDateofthethesisdefence:22.07.2014.Reviewers:1.ProfJerkoBarbić,PhD,MD,Fullprofessor2.ProfNevenŽarković,PhD,MD,Seniorscientist(supervisor)3.ProfVeraCesar,PhD,Fullprofessor4.AnaČipakGašparović,PhD,Researchassociate(substitute)Thesisdepositedin:NationalandUniversityLibraryinZagreb,Ul.Hrvatskebratskezajednice4, Zagreb; City and University Library of Osijek, Europska avenija 24, Osijek; Josip JurajStrossmayerUniversityofOsijek,Trgsv.Trojstva3,Osijek

  

TEMELJNADOKUMENTACIJSKAKARTICA

SveučilišteJosipaJurjaStrossmayerauOsijekuDoktorskadisertacijaSveučilišteuDubrovnikuInstitutRuđerBoškovićSveučilišniposlijediplomskiinterdisciplinarnidoktorskistudijMolekularnebioznanostiZnanstvenopodručje:InterdisciplinarnopodručjeznanostiZnanstvenopolje:BiologijaiTemeljenemedicinskeznanostiPozitivniučincilipidneperoksidacijeurastustanicakostinabioaktivnomstaklu‐nove

mogućnostitkivnoginženjerstvairegenerativnemedicine

LidijaMilković

Radjeizrađenu:LaboratorijuzaoksidacijskistresInstitutaRuđerBoškovićMentor/i:Prof.dr.sc.NevenŽarković,Znanstvenisavjetnik,trajnozvanjeKratkisažetakdoktorskograda:Istraživanje je ukazalo na ulogu lipidne peroksidacije, kao i njezinog produkta 4‐hidroksinonenala u procesu poticanja osteogeneze potpomognute korištenjem bioaktivnihstakala. Obzirom da su bioaktivna stakla našla svoju značajnu primjenu u inženjerstvu tkivakosti,novespoznajećeotvoriti inovemogućnostikreiranjapoboljšanihbioaktivnihstakalanačemuseitemeljinaglasakdanašnjeregenerativnemedicine.

Brojstranica:101Brojslika:27Brojtablica:4Brojliteraturnihnavoda:121Jezikizvornika:EngleskijezikKljučne riječi: cijeljenje fraktura kosti, lipidna peroksidacija, 4‐hidroksinonenal, bioaktivnostaklo,bakar,potaknutaosteogenezaDatumobrane:22.07.2014.Stručnopovjerenstvozaobranu:1.Prof.dr.sc.JerkoBarbić,redovitiprofesor2.Profdr.sc.NevenŽarković,znanstvenisavjetnik,trajnozvanje(mentor)3.Prof.dr.sc.VeraCesar,redovitaprofesorica4.Dr.sc.AnaČipakGašparović,znanstvenasuradnica(zamjena)Radjepohranjenu:NacionalnojisveučilišnojknjižniciZagreb,Ul.Hrvatskebratskezajednice4,Zagreb;GradskojisveučilišnojknjižniciOsijek,Europskaavenija24,Osijek;SveučilištuJosipaJurjaStrossmayerauOsijeku,Trgsv.Trojstva3,Osijek

  

ResearchforthisPhDthesiswasperformedintheLaboratoryforOxidativeStress,atthe

Rudjer Bošković Institute, Zagreb, under the supervision of prof. dr. sc. Neven Žarković. This

workwassupportedbytheMinistryofScience,EducationandSportoftheRepublicofCroatia

[Project098‐0982464‐2519],bytheAustrianNationalBankJubilaeumsFundandbytheCOST

Action B35. Experiments with Real‐Time PCR were performed in the Centre for Medical

Research(ZMF),Graz,Austriaundersupervisionofdr.sc.NadiaDandachianddr.sc.MarijaBalić.

Experimentwith theCu‐doped45S5bioactiveglasswereperformed incollaborationwithour

colleagues from the Institute of Biomaterials, at the Department of Materials Science and

Engineering,UniversityofErlangen‐Nuremberg,Germanyduetothetwobilateralproject(one

in2012.andtheotherstillongoing,fortheperiod2013/2014.)

  

Dedication/Acknowledgement

Nothingisimpossiblewhenyourlovedonesbelieveinyou!

Thisisforallofus!

Iwould like to thankmymentor ProfN. Žarković for givingme the opportunity to pursuemy

dreamsandforhiskindsupport

IamverygratefultoProfR.Wildburgerforherkindsupportandourgoodcollaboration

IamthankfulforstartingcollaboratingwithProfBoccaccini,RainerandAlex.Iappreciateallyour

kindsupportandourfruitfulcollaboration.

IamalsosohappythatIhadanopportunitytostartworkingwithMarijaBalić,PhDandNadia

Dandachi,PhD.Thankyoubothforallthatyouhavedoneforme.Iwillcherishitalways.

Many thanks toallofmy colleagues (currentorpast) from theLaboratory forOxidativeStress:

Marina,Ana,Morana,Luka,Suzana,Nena,bothTeasandIva.Ilearnedfromyou,Ismiledwithyou

andIamsoprivilegedtoworkwithyou.Thankyouformakingmyworksowonderful!

Toallmyfriends…thankyouforbeingthereforme,cheeringmeandnotquestioning,evenjustfor

asecond,thatIwouldnotsucceed.

Tomymotherandmybrother...Therearenotenoughwordstoexpressmyappreciation.Thankyou

forbelievinginme.

TomyhusbandandourbestfriendMiki...thankyouforbeingyou!Yourunconditionallovewasmy

strength.

1  

Tableofcontents

1. INTRODUCTION....................................................................................................................................4

1.1. Bone.................................................................................................................................................5

1.1.1. Bonecomposition...............................................................................................................6

1.1.2. Bonecells...............................................................................................................................8

1.1.3. Boneremodellingandbonefracturehealing............................................................9

1.3. Biomaterials–bioactiveglasses...........................................................................................13

1.4. OxidativeStress.........................................................................................................................17

1.4. Lipidperoxidationand4‐hydroxynonenal.......................................................................21

1.5. HypothesisandAims................................................................................................................24

2. MATERIALSANDMETHODS............................................................................................................28

2.1. Bioactiveglasses........................................................................................................................28

2.1.1. Bioactiveglasses45S5and13‐93................................................................................28

2.1.2. Surfacecoatingtreatments............................................................................................28

2.1.3. Evaluation of the coating efficiency ‐ Immunochemical analysis of HNE

adsorptiontotheglasssurface......................................................................................................29

2.1.4. Preparationofbioactiveglasssamples.....................................................................30

2.2. Cellculture...................................................................................................................................31

2.3. Immunocytochemistry............................................................................................................32

2.4. Cellproliferation.......................................................................................................................32

2.5. Cellviability................................................................................................................................33

2.6. CellgrowthandapoptosisfortheBGstypes45S5and13‐93.....................................33

2.6.1. DAPIstaining......................................................................................................................33

2.7. CellgrowthandmaterialcharacterizationfortheBGtype45S5withCu...............34

2.7.1. DAPIstaining......................................................................................................................34

2.7.2. SEMandEDXanalysis......................................................................................................34

2.8. AnalysesofROSproduction...................................................................................................34

2  

2.9. Dot‐blotanalysis........................................................................................................................35

2.10. Celldifferentiation–Alkalinephosphatasestaining.................................................35

2.11. DetectionandquantificationofmineralizationwithAlizarinRed.......................36

2.12. RNAisolationandreversetranscription.......................................................................36

2.13. Primer.......................................................................................................................................37

2.14. RealtimePCR.........................................................................................................................38

2.15. Proteinisolation....................................................................................................................38

2.16. Westernblotanalyses.........................................................................................................38

2.17. Statisticalanalysis................................................................................................................39

3. RESULTS................................................................................................................................................41

3.1. Resultsobtainedwiththebioactiveglasses,types45S5and13‐93(MoSci).........42

3.1.1. ImmunochemicalanalysisofHNEadsorptiontotheglasssurface..................42

3.1.2. ImmunochemicalanalysisofcellsgrownonBGs...................................................43

3.1.3. AnalysesofROSproduction..........................................................................................45

3.1.4. Cellproliferation...............................................................................................................47

3.1.5. Cellviability........................................................................................................................49

3.1.6. Cellgrowthandapoptosis..............................................................................................51

3.1.7. Dot‐blotanalysis...............................................................................................................52

3.1.8. Celldifferentiation–Alkalinephosphatasestaining............................................54

3.1.9. Cellmineralization–AlizarinRedstaining..............................................................56

3.1.10. RealtimePCR.....................................................................................................................58

3.1.11. Western‐blotanalyses.....................................................................................................62

3.2. ResultsobtainedwithBG,type45S5,withorwithoutcopper....................................65

3.2.1. Cellviability........................................................................................................................65

3.2.2. Cellgrowthandmaterialcharacterization...............................................................66

3.2.3. Immunoblotanalysis.......................................................................................................68

4. DISCUSSION..........................................................................................................................................69

5. CONCLUSIONS......................................................................................................................................79

3  

6. REFERENCES........................................................................................................................................82

7. SUMMARY.............................................................................................................................................92

8. SAŽETAK...............................................................................................................................................94

9. CURRICULUMVITAE..........................................................................................................................97

4  

1. INTRODUCTION

INTRODUCTION

5  

1. INTRODUCTION

1.1. Bone

Humanskeletonofanadultiscomposedof206bonesanditsmainfunctionsareprovidinga

strong,supportivestructureforsofttissuesandmuscles,allowingbodymovement,protectionof

manyvitalorgansanditservesasaproductionsiteofbloodcellsaswellasastorageforcertain

mineralslikecalcium. Therefore,agreatvarietyofbonesthatbuildourbodycanbeclassified

accordingtotheirshape,position,sizeandstructure.

Allbones,whenlookedintransversesection,areformedoftwomaintypesofosseoustissue:

cancellousandcorticalbone.

Corticalbone,alsoknownascompactbone,presentsanouterlayerofabonewhichseems

very dense and compact but in reality it is highly porous and composed of closely packed

cylindrical parts called osteons or Haversian systems. The osteon consists of a central canal

calledtheHaversiancanal,whichissurroundedbyanumberoflamellae,bonematrixinaform

ofconcentric rings.TheHaversiancanalscontainbloodvessels thatareparallelwith the long

axis of the bone. These blood vessels interconnect, by way of perforating canals, called

Valksmann’s canals, with vessels on the surface of the bone. Between the lamellae, there are

spaces,calledlacunaewhichareoccupiedbythebonecells,osteocytes.Moreover,theselacunæ

are connected with each other and with the central Haversian canal by a number of small,

hairlikechannels,canaliculi.Theirmainroleofthecanaliculiistoprovidepassagewaysthrough

thehardmatrix(Fig.1).

Fig.1.Corticalboneandcancellousbone

(Sourceofillustration:http://training.seer.cancer.gov/anatomy/skeletal/tissue.html)

INTRODUCTION

6  

Thuseverypartof theHaversiansystem is suppliedwithnutrient fluidsderived from the

vesselsintheHaversiancanalanddistributedthroughthecanaliculiandlacunæ[1,2].

Ontheotherhand,cancellousbone,alsoknownasspongyortrabecularbone,hasalighter

andlessdensestructurethancompactbone.Cancellousboneconsistsofplates(trabeculae)and

bars of bone adjacent to small, irregular cavities which contain red bone marrow. Since the

cancellous bone does not contain the Haversian canal, its canaliculi are connected to the

adjacent cavities in order to receive their blood supply. Although it may appear that the

organisation of the trabeculae is poor and rather haphazardous, in reality they are organized

verypreciselyalongthelinesofstressinordertoprovidemaximumstrengthsimilartobraces

thatareusedtosupportabuilding[2].

Furthermore,basedonthepatternofcollagenformingtheosteoid(unmineralized,organic

portion of the bonematrix), bone can be classified into two types:woven bone and lamellar

bone[3].

Woven bone is characterized by a loosely organization of collagen fibres with no normal

lamellar pattern like observed in cancellous and cortical bone and therefore is weaker than

lamellarbone[3,4]. It isproducedbyosteoblasts incasesofneedforarapidboneproduction

which occurs in all foetal bones and in fracture healing. Thus formed newly bone is further

replacedbymoreresilientlamellarboneinaprocessofboneremodelling[3].

Lamellar bone is characterized by a regular parallel alignment of collagen into layers

(lamellae). As a result of the alternating orientations of collagen fibrils, lamellar bone has a

significantmechanicalstrength[3,5].

Virtuallyalltheboneinthehealthymatureadultislamellarbone.

1.1.1. Bonecomposition

Boneiscomposedof50to70%mineral,20to40%organicmatrix,5to10%water,and

<3% lipids [6]. Bone itself can be divided into the basic areas of matrix, cells and bioactive

factors. Bonematrixiscomposedoforganicandinorganicpart.Organicpartofthematrixare

bone proteins of which 85 to 90% are collagenous proteins and mostly type I collagen [7].

Noncollagenousproteinscompose10to15%oftotalboneprotein.Theyaregenerallydivided

into several categories, including proteoglycans, glycosylated proteins, glycosylated proteins

withpotentialcell‐attachmentactivities,andγ‐carboxylated(gla)proteins.Themostabundant

INTRODUCTION

7  

noncollagenous matrix bone proteins are osteonectin, osteocalcin, osteopontin, bone

sialoprotein, and alkaline phosphatase, as well as fibronectin, decorin and bioglycan [8]. The 

remaining noncollagenous proteins of bone matrix include bone morphogenetic proteins,

growthfactors,andavarietyofothermoleculesthatmayaffectbonecellactivity.Sincemanyof

the bone proteins seem to have multiple functions, including regulation of bone mineral

depositionandturnoverandregulationofbonecellactivity, therolesofeachofthemarestill

notwelldefined[6].

Osteonectinisthemostprevalentnoncollagenousprotein,accountingforapproximately

2% of total protein in developing bone. It is considered as a bone‐specific nucleator of

mineralization because of its strong affinity for both collagen and mineral. In vitro studies

indicate that osteonectin can bind collagen and regulate angiogenesis, metalloproteinase

expression, cell proliferation, and cell‐matrix interactions. Moreover, osteonectin‐null mice

developosteopeniawithdefectsinthefunctionofbothosteoblastsandosteoclasts[9].

Osteocalcin“synthesizedbyosteoblastswasinitiallythought to functionasapromoter

orinitiatorofcalciumdepositionatthenidusbetweentheendsofcollagenfibrilsandtherefore

regarded as amarker of bone formation” [6]. Recent results suggest that osteocalcin inhibits

bone formationbecauseofahighbonemassphenotypeobserved in theosteocalcinknockout

mice.Asideitsosteoblastsynthesis,osteocalcinisalsoderivedfrommatrixreleasebyosteoclast

activity,becauseofwhichcurrently it is ratherregardedasamarkerofbone turnover thana

specificmarkerofboneformation[6].

Osteopontin is abundant bone matrix protein functioning as an inhibitor of

mineralization and/or as a mediator of cell‐matrix and matrix‐matrix/mineral adhesion

(cohesion) during the formation, turnover, and repair of normal andpathologicalmineralized

tissues[10].

Alkalinephosphataseisthemainglycosylatedproteinpresentinbonewhereitisbound

to osteoblast cell surfaces via a phosphoinositol linkage but it can also be found free within

mineralizedmatrix. [11]. Although themechanismof its action is not completely understood,

alkalinephosphataseremainsanexcellentindicatorofbonedifferentiationandmineralization.

The mineral content of bone is mostly hydroxyapatite [Ca10(PO4)6 (OH)2], with small

amountsofcarbonate,magnesium,andacidphosphate,withmissinghydroxylgroupsthatare

normallypresent.Hydroxyapatite crystals inbonearevery small,when compared togeologic

hydroxyapatitecrystals,measuringonlyapproximately200Åintheirlargestdimension.These

INTRODUCTION

8  

small, poorly crystalline, carbonate‐substituted crystals are more soluble than geologic

hydroxyapatite crystals, thereby allowing them to support mineral metabolism [6]. Matrix

maturation is associatedwith expression of alkaline phosphatase and several noncollagenous

proteins, including osteocalcin, osteopontin, and bone sialoprotein. It is thought that these

calcium‐ and phosphate‐binding proteins help regulate ordered deposition of mineral by

regulatingtheamountandsizeofhydroxyapatitecrystalsformed[6].

1.1.2. Bonecells

Bonecellsareessentialfortheprocessofbonerenewalthatisoccurringinhumanbody

during the lifetime. Particularly interesting cells present in bone include osteoblasts that

synthesize andmineralizebonematrix, osteocytes responsible formaintenance of thematrix,

and,osteoclastsrequiredforboneresorption(Fig.2).

Osteoblasts are mononucleated “bone‐forming” cells found near the surface of bones.

They are derived from multipotent mesenchymal stem cells. Osteoblasts are responsible for

makingosteoid,whichconsistsmainlyofcollagen.Theosteoblastssecreteothermatrixproteins

including alkaline phosphatase to create sites for calcium and phosphate deposition, which

allowsthegrowthofbonemineralcrystalsandthusmineralisationoftheosteoid.Theyarealso

important in the regulation of osteoclastogenesis through secretion of receptor activator of

nuclear factor κ B ligand (RANKL) and osteoprotegerin (OPG) [12]. Moreover, osteoblasts

communicate with osteocytes to receive mechantransduction signals through gap junctional

connexions[12].

Fig2.Bonecells

(Sourceofillustration:Anatomy&Physiology,http://cnx.org/content/col11496/1.6/)  

INTRODUCTION

9  

Osteocytes represent terminally differentiated osteoblasts that are embedded into the

secretedbonematrix and thereforeare found in lacunaebetween the lamellae inbone.Their

main role is homeostasis – maintaining the correct oxygen and mineral levels in the bone,

achievedby their innercommunication throughconnexin‐mediatedgap junctions in fillipodial

extensionswithinthecanalicularnetwork.Osteocytesdonotcontainalkalinephosphatase,but

produceosteopontins,amongotherbonematrixproteins[12].

Osteoclastsarelargemultinucleatedcellswithauniqueabilitytoresorbbone.Theybind

tobonematrixviaintegrinreceptorsintheirmembranelinkingtobonematrixpeptidessuchas

osteopontin, bone sialoprotein, and others. Upon binding, osteoclasts polarize and develop

ruffled border with acidified vesicles. The sealing zone surrounds and isolates the acidified

resorption compartment from the surrounding bone surface. Osteoclasts secrete acid

phosphatase,whichunfixesthecalciuminmineralisedbonetobreakitdown[13].

1.1.3. Boneremodellingandbonefracturehealing

Boneremodellingoccursduringthelifetimetomaintaincalciumhomeostasisandtorepair

micro‐fractures that are result of mechanical stress. A highly complex interplay between

osteoblasts, osteocytes and osteoclasts is controlling this bone resorption and rebuilding

processviaOPG/RANKL/RANKsystem.Abnormalitiesinthissystemleadtoimbalanceinbone

formation/resorptionandtoincreasedordecreasedbonestrength[14].

Asadynamicandhighlyvascularised tissue,bonehasaunique internal repaircapacity to

healandremodelwithoutscarring[15].Skeletaldevelopmentandfracturerepair includesthe

coordinationofmultipleeventssuchasmigration,differentiation,andactivationofmultiplecell

typesandtissues[16].Themoleculareventsthatgovernfracturehealingareacomplexnetwork

of signals signifying tissue damage, cell death, cell recruitment, cell proliferation, cell

differentiation,andtissueformation[17].Thestagesoffracturehealingreiteratethesequential

stages of embryonic endochondral bone formation. Histological observations distinguish two

basictypesofbonehealing.Primaryhealingisrareandreferstoadirectattemptofthecellsin

cortical bone to re‐establish the disrupted continuity. It requires absolute contact of the

fragmentsandalmostcompletestabilityandminimisationoftheinter‐fragmentarystrains[18].

Secondarybonehealingoccurs in thevastmajorityofbonyinjuries.This typeofbonehealing

involves both intramembranous and endochondral ossification and leads to callus formation.

Likewise, committedosteoprogenitor cellsof theperiosteumandundifferentiatedmultipotent

mesenchymal stem cells (MSCs) are activated. Callus is a physiological reaction to inter‐

INTRODUCTION

10  

fragmentarymovementandrequires theexistenceof residual cellvitalityandadequateblood

flow [18]. Certain biologic prerequisites have been identified in this cascade of events. Many

local and systemic regulatory factors, cytokines and hormones, as well as extracellular

osteoconductivematrixareseentointeractwithseveralcelltypes.Avibrantcellpopulationisa

mandatoryfirstelementforanunimpededbonerepairprocess.Multipotentmesenchymalcells

arerecruitedatthefractureinjurysiteortransferredtoitwiththebloodcirculation.

Different set of phases can be observed during the fracture healing, such as hematoma

formation,inflammation,angiogenesis,cartilage/callusformation,andboneremodelling(Fig.3).

Therefore,thehealingprocesscanbedividedintothreeseparatebutoverlappingstages:1)the

earlyinflammatorystage;2)therepairstage;and3)thelateremodellingstage[19].

Intheinflammatorystage,hematomadevelopswithinthefractureduringfirstfewhoursand

days. The fracture hematoma has been proven to be a source of signalling molecules:

interleukins (IL‐1, IL‐6), tumour necrosis factor‐a (TNF‐a), fibroblast growth factor (FGF),

insulin‐like growth factor (IGF), platelet‐derived growth factor (PDGF), vascular endothelial

growthfactor(VEGF),andthetransforminggrowthfactorβ(TGFβ)superfamilymembers)that

mayinduceacascadeofcellulareventsthatinitiatehealing[18].

Figure3.Stagesinfracturerepair.PictureobtainedbytheServierMedicalArt

Since bone cells at the ends of the fracture are dead, this necrotic material elicits acute

inflammatory response observed as infiltration of inflammatory cells such as

polymorphonuclear leukocytes and macrophages. Cytokines and pro‐inflammatory factors

releasedbythesecellsstarthealingcascadebyattractionofundifferentiatedmesenchymalcells

and fibroblasts and support vascular ingrowth. This results in the formation of granulation

tissue[19].Inaddition,undifferentiatedmesenchymalcellsdifferentiateintochondrocytesand

osteoblasts.

INTRODUCTION

11  

In the reparative stage, fibroblasts and chondrocytes, which secrete collagen and

proteoglycans,createfibrocartilaginouscallus,knownassoftcallusthatreplacesthedeadbone

andprovidemechanicalsupport.Eventually,thesoftcallusisossifiedresultinginawovenbone

hardcallus,whichoccursviaacombinationofintramembranousandendochondralossification.

In the final stage of repair, the woven bone is slowly replaced by lamellar bone through the

remodelling stage in which the healing bone is restored to its original shape, structure, and

mechanicalstrength[19].

At the molecular level, different sets of molecules interact with both local cells and

circulatingcellstocoordinatethehealingcascade:effectorsofinflammation(IL‐1,IL‐6,COX‐2),

mitogens (transforming growth factorbeta (TGFβ), insulin‐like growth factor (IGF), fibroblast

growth factor (FGF) and platelet derived growth factor (PDGF)), morphogens (bone

morphogenetic proteins (BMPs)), and angiogenic factors (vascular endothelial growth factor

(VEGF)andangiopoietins)[16].

INTRODUCTION

12  

1.2. Bonegrafts

Bonefractures,especiallyof longbonesor large joints,representdifficultmedicaldisorder

andareoftennot followedby successful recovery inparticular in elderlypatients, in patients

that suffer from multifragmentary bone fractures or in polytraumatized patients. In a large

numberofcasestheresultingskeletaldeficienciesrequiresurgicalinterventionandrepair.

Currently there are three different grafts in usage for reconstructive surgery: autogenic

grafts,allogenicgraftsandbonesubstitutes.  

Autologousbonegraft,thatis,bonetakenfromanotherpartofthepatient’sownbody,has

beenthegoldstandardofbonereplacementformanyyearsbecauseitprovidesosteogeniccells

aswellasessentialosteoinductivefactorsneededforbonehealingandregeneration[20].It is

commonlytakenintheformoftrabecularbonefromthepatient’s iliaccrest,butcorticalbone

canbeusedaswell.However,andalthoughitpresentsrelativelygoodpercentagesofsuccess,

thespectrumofcasesinwhichitcanbeusedisrestricted,mainlyduetothelimitedamountof

theautograftthatcanbeobtainedandduetodonorsitemorbidity[20].

Allograft,bonetakenfromsomebodyelse’sbody,couldbeanalternative.However,therate

of graft incorporation is lower than with the autograft. Allograft bone also introduces the

possibilitiesofimmunerejectionandofpathogentransmissionfromdonortohost,andalthough

infrequent,infectionscouldoccurintherecipient’sbodyafterthetransplantation[20].

Althoughautologousbonegrafts are considered as a golden standard there arealso some

limitationspresent intheirusage likedonorsitemorbidityandlimiteddonorbonesupply.On

theotherhandallograftscanexhibitfurthernegativeeffectslikerejectionphenomenaandrisk

ofviralinfection.Therefore,attemptsoftheregenerativemedicinetodayistofind/createabone

substitute that will enhance normal bone healing/remodelling, will be easily accessible and

applicablewithoutunwantedside‐effects, thatwillbeableto formanormalbonestructureor

byintegrationintotheboneorbyitsresorbtionandreplacementwithanewlyformedbone.As

aconsequence,biomaterialsthatpossesmentionedpropertieshavefoundtheirplaceinthefield

oftissueengineeringandregenerativemedicine.

INTRODUCTION

13  

1.3. Biomaterials–bioactiveglasses

Thedrawbacksofautograftsandallograftsledtoaproductionofawiderangeofbioactive

materialscurrentlypresent in themarket.Someof themost interestingarerepresentativesof

calciumphosphateceramics(Ca‐P),bioactiveglasses(BG)andbioactiveglass‐ceramics.

Bioactive material is by definition “one that elicits a specific biological response at the

interface of thematerial that results in the formation of a bond between the tissues and the

material” [21]. Thesematerials generate a carbonated hydroxyapatite layer that is equivalent

chemically and structurally to the biological mineral of bone, what is known to be the

determining step for thebiointegration [22]. Furthermore, itwas found thatbioactive glasses

with composition of less than 55% SiO2exhibit not only osteoconductivity (able to support

abundant bone formation), but are also responsible for osteoproduction by stimulating

proliferationanddifferentiationofosteprogenitorcells[23].

Themajorbone‐bioactivematerial known todate is 45S5BG [21]. Thismaterial is highly

bioactiveanditisbothosteoinductive(abletoinduceboneformation)andosteoconductive.The

mechanisms of bioactivity and bone bonding of 45S5 BG have been widely studied, and

described[24].Basedonthosestudies,thebondingof45S5BGtobonehasbeenattributedto

theformationofacarbonate‐substitutedhydroxyapatite‐like(HCA)layerontheglasssurfacein

contactwiththebodyfluid.BecausethisHCAlayerissimilartothemineralconstituentofbone,

it bonds firmlywith livingboneand tissue.While somedetailsof thechemicalandstructural

changesarenotclear, theHCA layer isgenerallybelieved to formasa resultofa sequenceof

reactionsonthesurfaceoftheBGimplant,asdescribedbyHench[24]:

Stage1:Rapidionexchangereactionsbetweentheglassnetworkmodifiers(Na+andCa2+)

withH+(orH3O+)ionsfromthesolution,leadstohydrolysisofthesilicagroupsandthecreation

ofsilanol(Si−OH)groupsontheglasssurface:e.g.

Si−O−Na++H+→Si−OH+Na+(aq)

ThepHofthesolutionincreasesduetotheconsumptionofH+ions.

Stage2:TheincreaseinpH(orOH−concentration)leadstoattackoftheSiO2glassnetwork,

and the dissolution of silica, in the form of silicic acid, Si(OH)4, into the solution, and the

continuedformationofSi−OHgroupsontheglasssurface:

Si−O−Si+H2O→Si−OH+OH−Si

INTRODUCTION

14  

Whilethesolubilityofsilicaislow,theproductsof45S5BGandglass–ceramicdissolutionin

aqueoussolutionshaveshownanincreaseinSiconcentration[24]indicatingthatdissolutionof

silica is an important mechanism. However, other mechanisms could also contribute to the

increaseinSiconcentration.

Stage3:CondensationandpolymerizationofanamorphousSiO2‐richlayer(typically1–2µm

thick)onthesurfaceoftheglassdepletedinNa+andCa2+.

Stage 4: Further dissolution of the glass, coupledwithmigration of Ca2+ and (PO4)3− ions

fromtheglassthroughtheSiO2‐richlayerandfromthesolution,leadingtotheformationofan

amorphouscalciumphosphate(ACP)layeronthesurfaceoftheSiO2‐richlayer.

Stage 5: The glass continues to dissolve, as the ACP layer incorporates (OH)−and (CO3)2−

fromthesolutionandcrystallizesasanHCAlayer.

Figure4.Reactionstagesofbioactiveglassesincontactwithhumantissue/fluid[25]

WiththeinitialformationofanHCAlayer,thebiologicalmechanismsofbondingtoboneare

believed to involve adsorption of growth factors, followed by attachment, proliferation and

differentiation of osteoprogenitor cells [24]. Osteoblasts,which are bone‐forming cells, create

INTRODUCTION

15  

extracellularmatrix,mainlyofcollagen,whichmineralizestoformananocrystallinemineraland

collagenon thesurfaceof theglass implantwhile thedegradationandconversionof theglass

continuesovertime[24].

BGtype45S5remainsthegoldstandardforbioactiveglassbut,asascaffoldmaterial,ithas

several limitations. One limitation is the difficulty of processing 45S5 BG into porous 3‐D

scaffolds.Porousbioactiveglass scaffoldsare commonlypreparedbyheating (sintering)glass

particles,alreadyformedintothedesired3‐Dgeometry,tobondtheparticlesintoastrongglass

phasecontaininganinterpenetratingnetworkofpores.Becauseofthelimitedabilityof45S5BG

to sinterbyviscous flowabove its glass transition temperature (Tg), and thenarrowwindow

betweenTgandtheonsetofcrystallization,severedifficultiesareencounteredinsinteringthe

particles into a dense network. Consequently the scaffold often has low strength [26].

Commonly, theglassdevitrifiesduringsintering to formapredominantly combeitecrystalline

phase(Na2O−2CaO−3SiO2).Whiledevitrificationdoesnotinhibittheabilityof45S5BGtoform

anHA‐likesurfacelayer,ithastheeffectofreducingtherateofconversiontoHA[27].Another

limitationof45S5BG is its slowdegradation rateandconversion toanHA‐likematerial [28],

whichmakesitdifficulttomatchthedegradationrateofthescaffoldwiththerateofnewtissue

formation.TheconversionofthescaffoldtoanHA‐likematerialisoftenincomplete,sotherefore

aportionofunconvertedglasscontainingSiO2couldremaininthescaffold,raisinguncertainty

aboutthelong‐termeffectsofSiO2invivo.

Acomplicationwiththeuseof45S5BGandotherBGsandotherbiodegradablematerialsis

thatthelocalbiologicalmicroenvironmentcanbeinfluencedsignificantlybytheirdegradation.

Increases in theconcentrationof ions,suchasNa+andCa2+,andchanges in thepHoccurasa

resultofthedegradation,particularlyintheearlystageswhenthedegradationrateisfast[24].

The biological effects of these changes are difficult to predict from in vitro experiments.

Furthermore, thebiological rolesof these soluble species, their toxicity and their removal are

oftennotclearlyunderstood.

Moreover, it hasbeen shown that the ionicdissolutionproductsof 45S5BGmayenhance

new bone formation (osteogenesis) through direct control over genes that regulate cell

induction and proliferation [29]. Recent report demonstrates that the dissolution products of

45S5BGcreateanextracellularenvironmentthatiscapableofsupportingosteoblastphenotype

expression and extracellular matrix deposition and mineralization in vitro in human foetal

osteoblasts [30]. There are also indications from previous reports of invivostudies [31] that

bioactiveglass(45S5BG)enhancesvascularizationoftissueengineeringconstructs.

INTRODUCTION

16  

Anothersilicate‐basedbioactiveglassthatweexaminedinourresearchis13‐93BGwitha

slightlymodified45S5composition(higherSiO2contentandadditionalnetworkmodifiers,such

asK2OandMgO).Productsof13–93BGhavebeenapprovedforinvivouseinEurope.

Because 13–93 BG has better processing characteristics by viscous flow sintering (larger

windowbetweenTgandtheonsetofcrystallization),theglassphaseinporous3‐Dscaffoldscan

besinteredtohighdensitywithoutcrystallization.However,13–93BGdegrades(andconverts

toanHA‐likematerial)moreslowlythan45S5BG[24].

The13‐93BGsupportstheinvitrogrowthanddifferentiationofMC3T3‐E1preosteoblastic

cells [32] and quantitative measurement of DNA showed no significant difference in cell

proliferationbetweendensedisksof45S5and13–93BGs[33].

INTRODUCTION

17  

1.4. OxidativeStress

Themechanisms and pathways involved in oxidative stress are conserved inmammalian

cells.Oxidativestressresultsfromanimbalancebetweenpro‐oxidants(freeradicals)andanti‐

oxidantsinthefavouroftheformer[34].Freeradicalscanbedefinedasmoleculesormolecular

fragments containing one or more unpaired electrons. The presence of unpaired electrons

usuallyconfersaconsiderabledegreeofreactivityuponafreeradical.Radicalsthatarederived

fromoxygenrepresentthemostimportantclassofsuchspeciesgeneratedinlivingsystems[35].

Reactive oxygen species (ROS) comprise both free radical and non‐free radical oxygen

intermediatesofwhichthemostcommonarehydrogenperoxide(H2O2),superoxideanion(O2•‐)

and hydroxyl radical (•OH). Additionally, ROS can be produced from a variety of both

endogenousandexogenoussources.EndogenouslyROSaregeneratedasby‐productsofnormal

cellular metabolism from mitochondrial electron transport chain, nicotinamide adenine

dinucleotide phosphate (NADPH) oxidase (NOX), xanthine oxidase (XO), peroxisomes and

cytochromeP450[36,37].ExogenoussourcesofROSincludeultravioletlight,ionizingradiation,

environmental agents, pharmaceuticals, and industrial chemical. ROS, as highly reactive

molecules, may cause damage to the cellular macromolecules like DNA, proteins and lipids,

leading to changes in chromosome instability, genetic mutation, and/or modulation of cell

growth [37–39]. To avoid the damage, cells have evolved endogenous antioxidant defence

mechanisms including non‐enzymatic antioxidants such as glutathione, thioredoxin, ascorbic

acid, uric acid, alpha‐lipoic acid, coenzyme Q, ferritin, bilirubin, metallothionein, l‐carnitine,

melatonin and also enzymatic ones such as catalase (CAT), superoxide dismutase (SOD),

glutathionesignalling(GPX)andperoxiredoxins(PRXs)[40].

Superoxide anion, arising either through metabolic processes or following oxygen

“activation” by physical irradiation, is considered the “primary”ROS, and can further interact

with other molecules to generate “secondary” ROS, either directly or prevalently through

enzyme‐ or metal‐catalyzed processes [41]. It is a produced both enzymatically by NADPH

oxidase and xanthine oxidase, and non‐enzymatically, when a single electron is directly

transferred toO2 in electron transport chainwithinmitochondria. Superoxide anion does not

reactdirectlywithpolypeptides, sugars, ornucleic acids, and its ability to peroxidise lipids is

controversial[42].Thesuperoxideanioncanbeeasilyconvertedbysuperoxidedismutasesinto

H2O2.

INTRODUCTION

18  

H2O2is produced inmitochondria or by NOX enzymes upon their activation with various

growthfactorsandcytokines[43]. It isrelativelystable,membranepermeableandcandiffuse

within the cell making H2O2 considerably important ROS in redox signalling. If it is not

eliminated by the cell’s antioxidant system (CAT, GPX and thioredoxin signalling), in the

presence of metal ions (iron, copper, chromium, vanadium and cobalt), H2O2 can be further

transformedtothemostreactiveofROS,hydroxylradicalviaFentonreaction[44,45].

ThecurrentknowledgeisrecognizingtheJanus‐likerolesofROSandantioxidantsystemin

regulationof cellularprocesses,meaning that fluctuations in their levels could lead todiverse

effects. Alterations in ROS levels can modulate biologic activity through aberrant

stimulation/suppression of certain signalling pathways and through direct modifications of

biomolecules,especiallyproteins[46].ROS,onceperceivedasthemainactorsincell’sdamage,

are now recognized as important signalling molecules. To illuminate their role in redox

signalling,itshouldbementionedthatROSareoxidantsbynatureandhencecouldinfluencethe

redox status by inducing either a positive response (cell proliferation) or a negative cell

response(growtharrestorcelldeath),dependingontheirconcentration.

Therefore,redoxbalanceisplayingacriticalroleinmaintainingthebiologicprocessessince

theredoxsystemreliesmostlyondirectoxidativemodificationsoftheredox‐sensitivesignalling

proteinswhichreflectintheregulationoftheirexpression,posttranslationalmodificationsand

stabilities[46,47].

For that reason, to act as effective biological messengers, ROS have to bring about a

reversible change in the activity of a protein,which generally involvesmodification of a thiol

grouponacysteineresiduethatmediatesredoxsignalling[48,49].Forexample,whenH2O2acts

asaredoxsignalitoxidizesthethiolgrouponthetargetproteintoadisulphidegroup,thereby

changing the function of the protein. Once the level of H2O2 has returned to basal levels the

alterationisreversedandtheactivityoftheproteinrevertstoitsinitiallevel[49,50].

Moreover,ROS,actingassignallingmolecules,caninducecellularproliferationbyaffecting

several biochemical pathways from epidermal growth factor receptor (EGFR) to mTOR

(mammaliantargetofrapamycin)whichinvolvekeysignallingproteins,suchasnuclearfactor

erythroid 2‐related factor 2 (Nrf2), kelch‐like protein 19 (Keap1), Ras, Raf,mitogen activated

proteinkinases(MAPK)suchasERK1/2,MEK,p38α,c‐JunN‐terminalkinase(JNK),c‐myc,p53

and protein kinase C (PKC) [40,51,52]. Among them, Nrf2 is considered to be the master

regulatoroftheantioxidantresponse,butothersarealsoimportant.

INTRODUCTION

19  

Additionally,ROShasbeen implicated in regulationof the cytosolic calciumconcentration

(which itself regulates the above‐mentioned biological activities), regulation of protein

phosphorylation, and activation of certain transcription factors such as NF‐κB and the AP‐1

familyfactors[35,44].

DisruptioninredoxhomeostasisleadstoanoverallincreaseofintracellularROSlevel.Thus

generatedexcessiveROSconstantlyattackproteins,DNAandlipids,whichcouldleadtosevere

andirreversibleoxidativedamage.Dependingonthetargetandtheformofoxidativedamage,

protein oxidation can be reversible or irreversible with certain side chains (lysine, arginine,

histidine, proline and threonine) more susceptible to oxidation [46,53]. Processes involved

includeintramolecularorintermolecularproteincross‐linkage,glutathionylation,nitrosylation,

disulfide bond–mediated protein cross linkage or secondary oxidative modifications such as

adduct formation between oxidized proteins and lipid peroxides or glycation products. These

productscangenerateaggregationofbulkyproteincomplexes,whichmayinactivateboth26S

and20Sproteasome,leadingtoaccumulationofdamagedproteinsandcelldeath[54].

AlthoughDNAmay seem less susceptible tooxidativemodifications,becauseof itsdouble

helix structure together with the protective shield from histone and other coating proteins,

oxidativenuclearDNAdamageisdetectableundervariousconditions.Albeitmorethan20base

lesions have been identified, the most studied is 8‐oxo‐2’deoxyguanosine (8‐OH‐dG). The

consequenceofoxidativebaselesionspersistinginDNAismutationwhichisoneofthecrucial

stepsincarcinogenesis[55].

INTRODUCTION

20  

Figure5.Molecularmechanismofreactiveoxygenspecies(ROS)formation,involvementof

antioxidativedefenseandimpactofROSoncellularmacromolecules

ETC=electrontransportchain

 

INTRODUCTION

21  

1.4. Lipidperoxidationand4‐hydroxynonenal

The vastmajority consider lipids to be themost susceptiblemacromolecules to oxidative

modificationand, among them,polyunsaturated fattyacids (PUFAs) inparticular.Theprocess

initiatedbytheoxidationofPUFAs,knownaslipidperoxidation,isanautocatalyticprocessthat

resultsinaformationofhighlyreactivealdehydes(Fig.6)whicharemorestablethanROS,has

longerhalf‐life,candiffusefromtheirsiteoforiginandthusaffecttargetsdistantfromtheinitial

free radical attack. Increased lipid peroxidation has been implied with varying extents as

causative cofactor of various common age‐associated diseases (type 2 diabetes mellitus,

atherosclerosis), some formsof neurodegenerativedisorders and some types of cancer (liver,

kidney, brain, skin) [56,57]. On the otherhand, today it iswell‐known that lipid peroxidation

playsimportantroleincellphysiology,influencingthesignaltransductionpathways[58].

Figure6.Mechanismoflipidperoxidation

INTRODUCTION

22  

Oneofthemainproductsgeneratedduringlipidperoxidation,andthemostintensively

studiedone,istheα,β‐unsaturatedaldehyde4‐hydroxynonenal(HNE)whichisderivedfromω‐

6 PUFAs such as arachidonic acid and linoleic acid [59,60]. HNE has a dichotomous role

dependingonitsconcentration.Whereasifpresentinexcess,HNEexpressavarietyofcytotoxic

effectssuchastheinhibitionofDNA,RNAandproteinsynthesis,cellcyclearrest,mitochondrial

dysfunction,andthusplaysanimportantroleinpathologyofvariousdiseases[61].Ontheother

hand,atalow(physiological)level,HNEcaninduceproliferation,differentiationandapoptosis

affecting genome function and interacting with other growth regulating factors, in particular

cytokines[59,62–67].IntracellularHNEreactsrapidlywiththiolgroupsofGSHandcysteineand

with lysine and histidine residues of proteins leading to significant functional alterations

affectingsignallingpathways[65,68,69].

TheroleofHNEinsignallingprocessesisintriguingbecause,asalreadymentioned,ofits

double‐edged effects which are concentration dependent. For this reason, the regulation of

intracellularconcentrationsofHNEmightbecrucialforthenatureofcellcyclesignalling.Itwas

foundthatHNEcaninducecellgrowth,accompaniedbytheactivationofMAPKs(ERK,JNKand

p38)andinductionofc‐fos,c‐jungeneexpression,andAP‐1DNAbindingactivity[70].Studies

have also shown that HNE induces various enzymes including phospholipase C, adenylate

cyclase,caspase3,proteinkinaseCandotherkinasesinvolvedinsignaltransductioncascades,

stronglycomprisingaroleofHNEinsignallingcascades[71].Itisalsoabletoinfluencecellular

functionsbyregulatingthegenesencodingforothermoleculessuchasheat‐shockproteingenes

[72],c‐myc[73],c‐fos[74],c‐jun[75],c‐myb[76],cyclins[73],p53genefamily[77],procollagen

typeI[78],andTGF‐β1[79].Moreover,HNEisfoundtobeaphysiologicalconstituentofmany

human and animal tissues [80] however its physiological role has yet to be clarified, but it is

likelythatHNEcouldhaveanimportantroleintheregulationofcellulargrowth[66,71,81].

Relatively high steady state concentrations of HNE in cell membranes indicated the

primarysiteofitsorigin,fromwhereitcandiffuseactingasasecondmessengeroffreeradicals.

UnlikefreeradicalsortheotherROS,HNEhastheuniquefeaturetoremainstableforsometime

andbinds tomacromoleculeswithhigh reactivity, especially toproteins, causingalterationof

their tertiary structure and their function. These alterations are mainly conducted through

formationofcovalentbondwithcysteine,lysineandhistidineaminoacidresidues[65,67].HNE

mostlyreactswithSHgroupsofproteinsandpeptidesgeneratingHNE‐proteinconjugatesthat

can be detected by the use of monoclonal antibodies. On the other hand, such reaction with

glutathione(GSH)representsitsmostimportantdetoxificationmechanism[62].

INTRODUCTION

23  

Once formed, HNE is rapidly degraded by three major reactions: reduction to 1,4‐

dihydroxy‐2‐nonene by alcohol dehydrogenases, oxidation to 4‐hydroxy‐2‐nonenoic acid by

aldehyde dehydrogenase, or formation of the glutathione‐conjugate (GS‐HNE) catalyzed by

glutathione‐S‐transferase (GST). Thus formed GS‐HNE conjugate can easily be exported from

cells in an ATP‐dependent manner or, which is more often, by RLIP76, a GTPase‐activating

protein[82,83]andassuchisamajorfactorinthemechanismsofdrugresistance[83].

In addition, detoxificationmechanisms of HNE are important to define the amount of

cellular protein modified by HNE. GSH is the most important intracellular antioxidant that

protects cells from HNE. Consequently, after administration of HNE, the amount of HNE‐

proteinsadductscanbededucedby intracellular levelofGSH.This level is crucial for cellular

sensitivity to HNE toxicity [62]. Although GSH could act alone, detoxification reaction is

accelerated by GSH metabolizing enzymes such as glutathione S‐transferases (GSTs).

Noteworthy, Ramana et al. [84] reported that conjugates of GSHwith HNE activate PKC and

stimulateNF‐κBandAP‐1‐dependentgenetranscriptionleadingtoanincreaseincellgrowth.In

conclusion, the unique pathways and the primary molecular targets of intracellular signal

transductionmediatedbyHNEarestillunknown.However, itseemsthatauniquereceptor is

not necessary for HNE to achieve growth regulating effect. On the other hand, complexity of

possibleinteractionswithsignal‐transductionpathwaysincreasedenormously(Fig.7).

Figure7.PotentialsignallingbyHNEinpatophysiology.Figuretakenfrom[85]

INTRODUCTION

24  

1.5. HypothesisandAims

Ourpreviousresearchhaverevealedconnectionofstressresponsewiththephenomenonof

enhanced osteogenesis inpatientswith traumaticbrain injury that exert very shortperiodof

bonehealingfollowedbyheterotopicossificationorhypertrophiccallus formation.Thisstress

responseinvolveshormonalimbalanceassociatedwiththechangeofgrowthfactoractivities,in

particularbFGF[86]andIGF‐1[87]togetherwithoxidativestressandlipidperoxidation[88].

Thus, lipidperoxidationwasdefinedas importantparameterof systemic response inpatients

withtraumaticbraininjuriesandwithbonefractures.

LipidperoxidationoccurswhenROS,generatedduringoxidativestressdamagelipidsmostly

present inplasma,mitochondrial,andendoplasmicreticulummembranes.Recentresultshave

shown that the end product of lipid peroxidation HNE, acts as a growth regulating factor

interferingwiththeactivityofcytokinesandthusmayaffectthegrowthofculturedhumanbone

cells[71,89].HNEisknownasamajorbioactivemarkeroflipidperoxidation[67]thatactsasa

secondmessenger of free radicals and a signallingmolecule interferingwith the activities of

various signal kinases, such as protein kinase C (PKC) andmitogen‐activated protein kinases

(MAPKs),toregulatecellularprocessesfromproliferationtodifferentiationandapoptosis[85].

Because of its involvements, HNE is considered as particularly interesting biomolecule that

mightthereforeplayimportantroleregulatingregenerationofdamagedtissuesuchasbone.

Therefore, themainhypothesis of thiswork is that lipid peroxidation andmoreprecisely

HNEmaybe involved in theprocessof bone regeneration supportedby theBGs, and as such

couldpotentiallybeinterestinginthetissueengineeringanddesigningofthenewBGs.Reasons

for this lies in fact that ionicdissolutionproductsofBGsstimulatecertaingrowth factors that

couldgiverisetoendogenousROSlevels.ThusgeneratedROS,possibly,couldnotbeneutralised

by antioxidative system of the cell, especially during the initial phase, before the boosting of

antioxidative capacity, and therefore this excess of ROS can attack macromolecules, among

which are lipids. Consequently, lipid peroxidation is initiated leading to generation of HNE.

SinceBGssupportgrowthofbonecells,thelevelsofHNEshouldbeinphysiologicalrange,not

detrimentalforthecell.Moreover,bothHNEandgrowthfactorswerefoundtoregulatethecell

growthwiththeinvolvementofMAPKpathway(Fig.8).

INTRODUCTION

25  

Figure8.PossibleinvolvementoflipidperoxidationincellgrowthontheBGs

Previous research of our laboratory has elucidated HNE’s role in differentiation,

apoptosis and cell proliferation [89] in human osteoblast‐like cells as well as its ability to

increasecell growth inphysiological concentration [71].To test thehypothesis, first aim is to

findwhetherthereisapresenceofHNE‐proteinadductswhileincontactwithBGstypes45S5

and13‐93duringbonegrowthinvitro.

Subsequently,iflipidperoxidationhasitsfunctioninthegrowthofbonecellsonBGs,the

mechanismsinvolvedshouldberevealedaswellasifmanipulationoflipidperoxidation,inthis

case, coating with HNE could more benefit the growth of bone cells on BGs. Especially

implicationofMAPkinasepathwaywillbeinvestigated.

Furthermore,afterrevealingaroleoflipidperoxidationincellgrowthonBGs,thenext

stepistopreparenew,morefunctionalandpotentiallybetterBGs.Itiswellknownthatcertain

traceelementscanimprovepropertiesofBGs.Coopercouldbeaninterestingtraceelement,asit

is known both as an essential cofactor of several enzymes and for its proangiogenic and

INTRODUCTION

26  

antimicrobial properties that stimulate osteogenic differentiation [90]. Copper ions are also

well‐knowninducersofoxidativestressandconsequentlyoflipidperoxidation[91].Therefore,

designinganovelCu‐dopedBGcoupledwitha lipidperoxidationosteoinducingreadoutcould

providenewdirectionsinthefieldofbonetissueengineering.

Thisworkwill for the first time investigate theeffectsof aCu‐containingBG, i.e.45S5

Bioglass®on thegrowthof ahumanosteoblast‐like cell line (i.e.,HOS) and thecorrelationof

lipidperoxidation(i.e.,cellularproductionofHNE),withcellgrowth.

 

27  

2. MATERIALSANDMETHODS

MATERIALSANDMETHODS 

28  

2. MATERIALSANDMETHODS

 

2.1. Bioactiveglasses

2.1.1. Bioactiveglasses45S5and13‐93

 

Commercially available bioactive glasses (BGs), types 45S5 (GL‐0160 Bioactive Glass)

and13‐93(GL‐0811BioactiveGlass),werepurchasedas10cm longbars(MoSciCorporation,

HyPointNorthRolla,Missouri,USA).CompositionofbothbioactiveglassesisshowninTable1.

Forexperiments,thesebarswerecutintodiscswithfollowingdimensions:10mmindiameter×

2mm.Priortouseincellcultureexperiments,BGpelletsweresterilizedbydrysterilizationat

200°Cfor3hours.Sterilizeddiscswereputin24‐wellplate(TPP,Trasadingen,Switzerland)and

proceededforsurfacecoatingtreatments.

Table1.Compositionofbioactiveglasses45S5and13‐93

Typicalcompositions(wt%)

SiO2 Na2O K2O CaO P2O5 MgO

45S5 45 24.5 ‐ 24.5 6 ‐

13‐93 53 6 12 20 4 5

2.1.2. Surfacecoatingtreatments

 

SurfacesofBGs(MoSciCorporation),types45S5and13‐93werecoatedwith0,5mg/ml

bovine serum albumin (Sigma Aldrich Chemie GmbH, Steinheim, Germany), 2.5 µM 4‐

hydroxynonenal(HNE)andwithHNE‐BSAadductsobtainedbymixingHNEandBSA,thesame

concentrationsasfortheseparatetreatments.NecessarydissolutionsweremadewithddH2O.

HNE was prepared from (E)‐4‐hydroxynonenal‐dimethylacetal (HNE‐DA, Alexis

Biochemicals) inn‐hexane.Beforeuse,HNE‐DAwasactivatedaccording tothemanufacturer’s

protocol. Briefly, HNE‐DA was evaporated by nitrogen blow and 1mM HCl was added to

MATERIALSANDMETHODS 

29  

hydrolyzeHNEfor1h.HNEconcentrationwasdeterminedbyspectrophotometermeasuringthe

spectrafrom200to350nmandcalculatedvalueupontheabsorptionmaximumat223nm.HNE

concentrationwas calculated according to the formulae c(mol/l)= A×d/13750,where is c the

concentration of HNE in mol/l; A‐measured absorbance; d‐sample dilution; 13750‐molar

extinctioncoefficientofHNEinwater.

2.1.3. Evaluation of the coating efficiency ‐ Immunochemical analysis of HNE

adsorptiontotheglasssurface 

To evaluate whether the coating of the BG surfaces were efficient, normal glass (8‐

chamber glass slide; Thermo Scientific Nunc Lab‐Tek system) was treated with different

concentrationsofHNE(0;2.5;10;200μM)eithergivenaloneorincombinationwithBSA.

Higherconcentrations,thanusedinthecoatingtreatments,wereusedtobecertainthat

atleastsomeresultswillbeobtained.

NormalglasswasusedtobeabletoevaluateobtainedresultsproperlybecauseBGstend

toformcrystalswhenembeddedinculturemedium.Thesurfacecoatingwasmadeinthesame

manner like for BG samples and afterwards, complete medium was added to the chambers

(withoutthecells).After3days,mediawasreplacesandslidewaswashedtwiceinPhosphate

bufferedsaline(PBS)andfixedin10%bufferedformalin.

ImmunostainingfortheHNE‐modifiedproteinswascarriedwithmonoclonalantibodies

obtained from culture medium of the clone “HNE 1g4”, produced by a fusion of Sp2‐Ag8

myeloma cellswithB‐cells of aBALB‐cmouse immunizedwithHNEmodified keyhole limpet

hemocyanine.The antibody is specific for theHNE‐histidineepitope inHNE‐protein (peptide)

conjugatesandgivesonly5%crossreactivitywithHNE‐lysineand4%withHNE‐cysteine[92].

Immunostaining was performed as described before [93] using LSAB kit (DAKO, Glostrup,

Denmark). Briefly, a glass slidewas incubatedwith anti‐HNEmonoclonal antibodies (dilution

1:10) over night in humid chambers at room temperature,washed three times for 5minutes

withphosphate‐bufferedsaline(PBS)andthenincubatedwithbiotinylatedsecondaryantibody

during30min.Afterwashingwithtris‐buffered‐saline(TBS),threetimesfor5minutes,samples

wereincubatedwithstreptavidinperoxidaseduring30min.Finally,thereactionwasvisualized

byaDAB(3,3‐diaminobenzidinetetrahydrochlorideinorganicsolvent;DAKO,Denmark)giving

abrowncolourthatindicatedthepresenceofHNE.QualitativeevaluationofdifferencesinDAB

MATERIALSANDMETHODS 

30  

intensity was done by inverted microscopy (Krüss Optotronic, Germany) and quantitative

evaluationwasdonewithImageJsoftware.

2.1.4. Preparationofbioactiveglasssamples 

Cu‐containingmeltderivedBG(type45S5)wasproducedbymixinganalytical‐gradesof

siliconoxide(SiO2,Merck,Darmstadt,Germany),sodiumcarbonate(Na2CO3,Merck,Darmstadt,

Germany), calcium carbonate (CaCO3, Merck, Darmstadt, Germany), tricalcium phosphate

(Ca3(PO4)2, Sigma‐Aldrich, Germany) and copper carbonate ((Cu3(OH)2(CO3)2, Sigma‐Aldrich,

Germany). Table 2 shows the glass compositions used in this study. The rawmaterials were

homogenizedinaplanetarymillandmeltedinaplatinumcrucibleat1450°C for45min.The

glasswasfritted,pre‐milledinajawcrusherandmilledinaplanetarymilltothefinalparticles

sizeof~5µm.Thepowderwaspressedtopellets(diameter10mm,height2mm)andsintered

at1050°Cfor140min.Theheatingratewas2K/min.

Table2.Nominal45S5Bioglass®derivedglasscompositionswithdifferentcoppercontents.

Glass Compositionin%(w/w)

SiO2 Na2O P2O5 CaO CuO

45S5‐Ref 45 24.5 6 24.5 ‐

45S5‐0.1Cu 45 24.5 6 24.4 0.1

45S5‐1Cu 45 24.5 6 23.5 1

45S5‐2.5Cu 45 24.5 6 22 2.5

Priortouseincellcultureexperiments,BGsweresterilizedbydrysterilizationat200°C

for3hours.

MATERIALSANDMETHODS 

31  

2.2. Cellculture

Invitro cell culturestudieswereperformedusing thehumanosteosarcomacell line (HOS,

CRL 1543) as a cellular model of human osteoblastic functions. Cells were obtained from

AmericanTypeCultureCollection(ATCC)andculturedinaDulbecco'smodifiedEagle'smedium

(DMEM; Sigma‐Aldrich, St Louis,USA), supplementedwith 10% (v/v) foetal calf serum (FCS;

Sigma‐Aldrich, Steinheim, Germany), 1 % (v/v) penicillin and streptomycin solution and

incubatedat37°C in ahumidifiedatmosphere (5%CO2 in95%air),with themediumchange

everyotherday.Thesemi‐confluentculturesweredetachedfromBGsubstrateswitha0.25%

(w/v) trypsin‐EDTA solution for 5 minutes. Viable cells (upon Trypan Blue exclusion) were

countedinaBürker‐Türkhemocytometerandusedforexperiments.

In everyexperiment cellswerecultivated in the samemannerwith a smalldistinction

dependingwhether theBGs types 45S5 and 13‐93 fromMoSci Companywere used or newly

preparedBGtype45S5withacooper(Cu).SinceBGsfromMoSciCompanydidnotfit intothe

48‐well plates, these BG samples were cultivated in 24‐well plates (TPP, Trasadingen,

Switzerland)as follows: 2×104 cells in100µlofmediumwasseededon theupper sideofBG

samplesandleftfor4‐5hourstoallowcellattachmenttotheBGsurfacepriortoadditionofthe

restofthemedia,400μl,tocovertheBGsamples(Fig.9).

Figure9.ProtocolforcellcultivationontheBGsamples,types45S5and13‐93(MoSci

Company)

MATERIALSANDMETHODS 

32  

Ontheotherhand,newlypreparedBGsamplestype45S5withCuwereplacedin48‐well

plates(1BGsample/well)(Nunc/ThermoFisherScientific,Roskilde,Denmark)followedbycell

seedingatadensityof2×104cells/wellin400μloftheculturemedium(Fig.10).

Figure10.ProtocolforcellcultivationontheBGsamples,type45S5withcooper

2.3. Immunocytochemistry

Transparent BG samples, types 45S5 and 13‐93, at day 3 of cell culturing were used for

immunocytochemistry. Cells were seeded as described in previous section. Cell cultures

developedonthestandardhistologicalglassservedascontrols.

Immunostaining for theHNE‐modifiedproteinswascarriedon formalin‐fixedcell samples

asdescribedintheSection2.1.3.QualitativeevaluationofdifferencesinDABintensitywasdone

byinvertedmicroscopy(KrüssOptotronic,Germany).

ThesameprotocolwasrepeatedfortransparentBGsamples,types45S5and13‐93,with

orwithoutsurfacecoatingsperformedatdays3,7and14.

2.4. Cellproliferation

CellproliferationontheBGstypes45S5and13‐93fromMoSciCompanywasanalyzedwith

Trypan Blue exclusionmethod. Themethod is based on the fact that viable cells have intact

membranesandassuchtheyareabletoexcludetrypanbluedye,whiledeadcellarenotableto

dosoandassuchtheyareblue.CellswereseededasdescribedinSection3.2.andcultivatedfor

3,7and14dayswiththemediachangedeverysecondday.

MATERIALSANDMETHODS 

33  

Atspecified timepointscellsweretrypsinizedwitha0.25%(w/v) trypsin‐EDTAsolution

for 5 minutes and based on a colour appearance, counted as live or dead in a Bürker‐Türk

hemocytometer.

2.5. Cellviability

Cellviabilitywasmeasuredat3differenttimepoints:days3,7and14,withtheMTTassay

(EZ4U; Biomedica, Vienna, Austria) according to manufacturer’s instruction [94]. Briefly, the

method is based on the ability of living cells to reduce slightly coloured or uncoloured

tetrazoliumsaltsintointenselycolouredformazanderivatives.Afteradditionofthedyesolution,

thecellswereincubatedat37°Cfor4h.Theopticaldensitywasmeasuredat450and620nmas

areference,tocorrectthevaluesfornonspecificbackground.

2.6. CellgrowthandapoptosisfortheBGstypes45S5and13‐93

Cell growth on the BGs types 45S5 and 13‐93 was monitored by the invert microscope

(KrüssOptotronic,Germany).Detectionofapoptosis,micronucleusformationandnecrosiswas

performedatday3bytheDAPIstaining.

2.6.1. DAPIstaining

ForDAPI staining cells grownonBG sampleswere fixed in4% formaldehyde (w/v in

PBS), permeabilized for 2minutes in ice coldmethanol and stainedwith 1 µg/mlDAPI (4',6‐

diamidino‐2‐phenylindole;Sigma,St.Louis,USA)for30minutes.Beforeeachstep,sampleswere

washedtwiceinPBS,respectively.TheintercalationofDAPIwasvisualizedwithafluorescence

microscope(DM6000CFS,Leica,Wetzlar,Germany)andforeachsampleatleast500cellsfrom

3 different representative areas was counted and classified as normal, apoptotic, mitotic,

necroticorwithmicronucleus.

MATERIALSANDMETHODS 

34  

2.7. CellgrowthandmaterialcharacterizationfortheBGtype45S5withCu

Cellswere cultured andgrownon thebioactive glass surfaces, asdescribed in the section

3.2. The growth wasmonitored with DAPI staining and scanning electronmicroscopy (SEM)

analysis. DAPI stainingwas performed at day 14 and SEM analysiswas carried out at day 7,

respectively.MaterialcompositionwasdeterminedbyenergydispersiveX‐ray(EDX)analysis.

2.7.1. DAPIstaining

TheprocedurefortheDAPIstainingwasasdescribedinthesection2.6.1.Imageswere

obtainedwithafluorescencemicroscope(DM6000CFS,Leica,Wetzlar,Germany).

2.7.2. SEMandEDXanalysis

For SEM analysis, the cell culture medium was removed and the BG samples were

washed with PBS, fixed with a solution containing 3 % (v/v) glutaraldehyde (Sigma,

Taufkirchen,Germany)and3%(v/v)paraformaldehyde(Sigma,Taufkirchen,Germany)in0.2

Msodiumcacodylatebuffer(pH7.4)andfinallyrinsedthreetimeswithPBS.Allsampleswere

dehydrated in a graded ethanol series (30, 50, 75, 90, 95 and 99.8 % (v/v)). Samples were

maintained at 99.8 % (v/v) ethanol and critical‐point dried. Prior to SEM examination the

samples were sputteredwith gold and analyzed on an ESEM (Quanta 200, FEI, Netherlands)

equippedwithsecondaryelectronandbackscatteredelectrondetectors.Theoperatingvoltage

was 20 kV. Energy dispersive X‐ray (EDX) analysis was performed to determine materials

compositioninareasofinterest.

2.8. AnalysesofROSproduction

Cellswere cultivated on the BG samples in quadruplicates for 72 hours. Analyses of ROS

production were performed with addition of a nonfluorescent probe for intracellular ROS

detection2',7'‐dichlorofluorescindiacetate(DCFH‐DA,Fluka),finalconcentration10µM,tocell

mediumontheday3.Thiscell‐permeabledyeremainsnonfluorescent insidethecelluntil the

acetategroupsareremovedbyintracellularesterasesandoxidizedbyintracellularROStothe

MATERIALSANDMETHODS 

35  

fluorescent compound 2',7'‐dichlorofluorescein (DCF)which can be detected as ameasure of

intracellularROS.Fluorescenceintensitywasobservedwithamicroscope(ZeissAxiovert25).

For quantification of intracellular ROS, samples prepared on the sameway as previously

described,weretrypsinizedandresuspendedin500µlofHanks’BalancedSaltSolution(HBSS).

Technicalduplicatesof200µlwereplatedintowhite96‐wellplates(Nunc,Roskilde,Denmark)

and the fluorescence intensity was measured with a Cary Eclipse fluorescence

spectrophotometer(Varian,USA)withexcitationat500nmandemissionat529nm.

Thearbitraryunitswerebaseddirectlyonfluorescenceintensity.

2.9. Dot‐blotanalysis

Ondays:3,7and14,cellswerescrapedfromtheBGsurfaceinPBSandcollected.Celllysates

werepreparedbyafreeze/thawcycle.Priortotheimmunoblotanalysis,proteincontentineach

samplewasmeasuredbytheBradfordmethod[95].Adot‐blotanalysiswasperformed,incell

lysateswithadjustedproteincontentto100µg/ml,aspreviouslydescribed[96].Briefly,100μl

ofeachsamplewas loadedonanitrocellulosemembrane(Bio‐RadLaboratories,Hercules,CA,

USA)andwashedtwicewithPBSbeforeblockingofthemembranein2%(w/v)nonfatdrymilk

(Bio‐Rad Laboratories, Hercules, CA, USA) in PBS for 1 hour at room temperature (RT). The

membrane was then incubated overnight with mouse monoclonal antibody directed against

HNE‐histidineepitope.Nextday,afterblockingofendogenousperoxidases,themembranewas

incubatedwith a secondary antibody (EnVision; DakoNorth America, Carpinteria, USA) for 1

hour/RT and immune complexes were visualized using 3,3′‐diaminobenzidine (DAB; Dako

North America, Carpinteria, USA) and scanned for quantification of signals. Signals were

analyzedinImageJ,aJava‐basedimageprocessingprogram.

The resultswere recalculated from the standard curvepreparedwithHNE‐BSAstandards

(0;0.5;1;2.5;5;10;20)andexpressedasnmolofHNE‐proteinadducts/mgofproteins.

2.10. Celldifferentiation–Alkalinephosphatasestaining

At two time points, days 3 and 14, cells grown on BG samples (MoSci) were stained for

alkalinephosphatase(ALP)byNBT/BCIPassay(Roche,Switzerland).Thisassay isbasedona

chromogenic reaction initiated by the cleavage of the phosphate group of BCIP by alkaline

phosphatasepresentinthecells.Cellcultureswerewashedthreetimeswithtris‐bufferedsaline

MATERIALSANDMETHODS 

36  

(TBS),pH7.0andfixedwith4%bufferedparaformaldehydefor30minutesat4°C.Stainingwas

performedbyadding200µl/wellofstainandincubatedat37°Cfor60minutes.Imagesofpurple

precipitatesweretakenandanalysedwithImageJsoftware.

Forquantification,insolublepurpleprecipitatesweresolubilisedwith200µlofSDS10%HCl

andincubatedfor18h.Theopticaldensitymeasurementwasdoneat595nm.

2.11. DetectionandquantificationofmineralizationwithAlizarinRed

At day 14 the extent of matrix mineralization on the cell/BG samples (n = 3) was semi‐

quantifiedusingAlizarinredstaining,whichbindstotheCa2+ofcalciumdeposition.Briefly,the

BGsampleswerewashedtwicewithPBSandfixedwith10%neutralbufferedformalinfor30

minatroomtemperature.Thesampleswerewashedwithdistilledwater(dH2O)andincubated

with2%(w/v)AlizarinredS(Sigma‐Aldrich)indH2O,pH4.1,for1houratroomtemperature.

Afterwhich,unincorporateddyewaswashedawayusingseveral rinsesofdH2O. Imageswere

takenonlightmicroscope(ZeissAxiovert25).

Toquantifymineralization,Alizarinred–stainedcellsweresolubilisedin0.5NHCLand5%

SDS for 30min and detected at 405nmusing a LabSystemsMultiscanMSmicroplate reader.

Quantificationwillbedoneaccordingtothestandardcurvepreparedbythemodificationofthe

Osteogenesis assay kit (Millipore) protocol. Briefly, standard curve will be prepared with

differentconcentrationofAlizarinRedstain(0‐2mM)dilutedin0.5NHCLand5%SDS.

2.12. RNAisolationandreversetranscription

TotalRNAwasextractedfromcellsgrownonBGssamples, types45S5and13‐93,withor

withoutsurfacecoatingondays3and14.Toensureenoughsample,onday3octaplicateswere

pulledtogetherandforday14triplicateswerepulledtogether.RNAwasextractedusingTRIzol

Reagent (Invitrogen,Carlsbad,CA,USA)according to themanufacture’s recommendationwith

minor modifications.. Briefly, homogenized samples were incubated for 15 minutes at room

temperatureandtreatedwith0.2ml1‐Bromo‐3‐chloropropane(Sigma,St.Louis,MO,US)perml

TRIzol reagent.Aqueousphasewascollected innewtubeandproceededto theRNA isolation

with an RNeasy Mini kit (Qiagen, Hilden, Germany) while the organic phase was saved for

proteinisolation.Ontheaqueousphase0.5ml70%ethanolpermlofTRIzolreagentwasadded

MATERIALSANDMETHODS 

37  

andtransferredtoanRNeasyspincolumnplacedina2mlcollectiontubeandcentrifugedfor15

sat≥8000xg.Spincolumnwasreused forsuccessivealiquots.Thenthecolumnwaswashed

withRW1bufferandRPEbuffer,andRNAwaseluatedin30μlofRNase‐freewater.

RNAwasquantifiedandassessedforpuritybyNanodrop(ThermoScientific,Waltham,MA,

USA). One microgram of total RNA was reverse transcribed using the QuantiTect Reverse

TranscriptionKit (Qiagen,Hilden,Germany)according to themanufacturer’s instructions.The

finalvolumeofthereactionwas20μl.

2.13. Primer

Primers sequences were designed based on literature references and purchased from

Eurofins MWG Operon (Ebersberg, Germany). Glyceraldehyde‐3‐phosphate dehydrogenase

(GAPDH)wasusedasan„house‐keeping“gene.PCRproductsshowedasinglemeltingpeakin

themeltinganalysisandasinglebandonanagarosegel (2.5%agarose in1‐foldTBErunning

buffer). Sequence specificitywas verified by sequence analysis (3730 DNA Analyzer; Applied

Biosystems,FosterCity,CA,US).SequencesofprimersaresummarizedinTable3.

Table3.SequencesofprimersusedforRealTime‐PCR

Primer RefSeq Sequence Amplicon Ref.

Alkalinephosphatase

J04948Forward‐CCTACACGGTCCTCCTATAC

110 [97]Reverse‐TGACTGCTGCCGATACTC

CollagentypeI NM000088Forward‐GTGATGCTGGTCCTGTTGGT

123 [98]Reverse‐ CACCATCGTGAGCCTTCTCT

Osteocalcin NM199173Forward‐ AGCGAGGTAGTGAAGAGAC

142 [97]Reverse‐ GAAAGCCGATGTGGTCAG

Osteoprotegerin NM002546Forward‐ GCAGCGGCACATTGGACATG

135 [97]Reverse‐ AGGATCTGGTCACTGGGTTTGC

Osteopontin NM000582Forward‐ GCGAGGAGTTGAATGGTG

140 [99]Reverse‐ CTTGTGGCTGTGGGTTTC

Osteonectin J03040Forward‐ TCTTCCCTGTACACTGGCAGTTC

73 [97]Reverse‐ AGCTCGGTGTGGGAGAGGTA

Glyceraldehyde‐3‐phosphatedehydrogenase

BT006893Forward‐CCACTCCTCCACCTTTGAC

102 [100]Reverse‐ ACCCTGTTGCTGTAGCC

MATERIALSANDMETHODS 

38  

2.14. RealtimePCR

Real timePCRamplificationandmeltinganalysiswereperformedusingaLightCycler480

System (Roche Diagnostics GmbH, Mannheim, Germany). For the reaction, 20 ng cDNA was

added to a reactionmix containing LightCycler 480 SYBR Green IMaster (Roche Diagnostics

GmbH,Mannheim,Germany)and25μMofeachprimergivingafinalreactionvolumeof20μl.

The PCR reaction mixture was subjected to an initial denaturation at 95°C for 10 minutes,

followedby45cyclesofdenaturationat95°Cfor10seconds,annealingat60°Cat20seconds

andelongationat72°C for15seconds.Meltinganalysis startedwithan initialdenaturationat

95°C for 5 seconds followed by an increase in temperature from 65°C to 90°C with 10

acquisitions per degree. All qRT‐PCR reactions were conducted in duplicate and the

quantificationcyclevalueswereaveraged.Geneexpressionwascalculatedbythecomparative

Ctmethod.GAPDHwasusedasreferencegene.

2.15. Proteinisolation

OrganicphaseleftduringRNAisolationwithTRIzolreagentwasusedforprotein isolation

according to the manufacturer’s instructions. Protein concentration in each sample was

measuredaccordingtoBradfordmethod[95].

2.16. Westernblotanalyses

Samples,containingthesameamountofprotein,werepreparedwithLaemmlibuffer,heated

at95°Cfor10minandsubjectedtoSDS‐polyacrylamidegelelectrophoresis(SDS‐PAGE,10%).

After electrophoresis the separated proteins were transferred onto nitrocellulose membrane

andprobedforMAPKsusingspecificantibodies,accordingtothemanufacturer’sprotocol.

The primary antibodies, all obtained from Cell Signalling (Austria) include the following:

threerabbitantibodiesfromtheMAPKfamilyantibodysamplerkit(catalogueno.9926S),anti‐

p44/42MAPK(ERK1/2;clone137F5),anti‐SAPK/JNK(clone56G8),oranti‐p38MAPK; three

rabbit antibodies from thePhosphoMAPK family antibody samplerkit (catalogueno. 9910S),

anti‐phospho‐p38MAPK(Thr180/Tyr182;cloneD2F9),anti‐phospho‐p44/42MAPK(ERK1/2;

Thr202/Tyr294;cloneD13.14.4E),oranti‐phospho‐SAPK/JNK(Thr183/Tyr185;clone81E11);

rabbit anti. A secondary antibody against rabbit IgG, conjugatedwith horseradish peroxidase

MATERIALSANDMETHODS 

39  

(HRP; Cell Signalling) was used in all cases, and signal was detected using enzyme‐linked

chemiluminescencewithECLReagent(Calbiochem).Visualizationofboundsecondaryantibody

wasdonebyUvitec(Cambridge,UK).

Thechemiluminescentsignalwasquantifiedfromdensitometricreadingsofdigitalimages

usingImageJsoftware.

2.17. Statisticalanalysis

Allassayswereperformedinat leasttriplicatesandrepeatedin,at least, twoindependent

experiments. Values were expressed as means ± SD. Unless otherwise stated, comparisons

betweengroupswereassessedbyatwo‐tailedStudent’st‐testconsideringvaluesofp<0.05as

significant.

 

40  

3. RESULTS

RESULTS

41  

3. RESULTS

Formoreconvenienceandbetterfluencyofobtainedresults, thisSectionisseparatedinto

twoparts:

3.1.ResultsobtainedwiththecommerciallyavailableBGs,types45S5and13‐93and

3.2.ResultsobtainedwiththeBG,type45S5,withcooper,receivedfromourcolleaguesfrom

the Institute of Biomaterials, at the Department of Materials Science and Engineering,

UniversityofErlangen‐Nuremberg,Germany.

RESULTS

42  

3.1. Resultsobtainedwiththebioactiveglasses,types45S5and13‐93(MoSci)

3.1.1. ImmunochemicalanalysisofHNEadsorptiontotheglasssurface

ToevaluatewhetherthecoatingoftheBGsurfaceswereefficientnormalglassslidewas

treated with different concentrations of HNE (0; 2.5; 10; 200 μM) either given alone or in

combinationwithBSA.Resultshaveshownthateven2.5μMHNEcanbedetectedbymonoclonal

antibodyusedfor immunocytochemistry.What ismoreimportant, there isnodifference if the

coatingwasdonewithjustHNEincomparisontotheonedonewithHNE‐BSA,neverthelessthat

monoclonalantibodyrecognizesHNEboundedtothehistidineresiduesofproteins.Thereforeit

canbeconcludedthatcoatingtreatmentissuccessfulandobtainedresultsareduetotheeffect

ofHNE(Fig10).

0 2.5 10 2000

10000

20000

30000

HNE

BSA+HNE

HNE(M)

Arbitraryunits

A)

B)

Figure10.Efficiencyofacoatingtreatment

A)Pictureofaglassslideafterimmunochemicaldetectionwithanti‐HNE

monoclonalantibody

B)SemiquantificationoftheobtainedresultswithImageJ

RESULTS

43  

3.1.2. ImmunochemicalanalysisofcellsgrownonBGs

Immunochemicalanalysisrevealedthatduringincubationofhumanosteoblast‐likecells

invitroonBGstypes45S5and13‐93,HNEisgeneratedinthecells.Contrary,HNE‐protein

adductswerenotobservedincellsthatweregrownonthenormalhistologicalglass(Figure11.).

Figure11.ImmunochemicaldetectionoftheHNE‐histidineadductsdonebyspecific

monoclonalantibodiesafterthreedayscultivationofHOScellsonstandard

histologicalglassoronBGs.Scalebar:50µm.

(A) HOS cell cultured on normal standard histology glass ‐ negative control (without

addition of primary antibody while proceeding immunostaining) ‐ no brown coloured

immunostainingcanbeobserved;

(B) HOS cell cultured on normal standard histology glass ‐ no brown coloured

immunostainingcanbeobservedeither;

(C)HOScellculturedonBGtype45S5‐intensebrowncolourcanbeobservedshowing

abundantpresenceofHNE‐proteinadductswithinthecells

(D) HOS cell cultured on BG type 13‐93 with intense brown colour can be observed

showingabundantpresenceofHNE‐proteinadductswithinthecells

RESULTS

44  

Cellsgrownonstandardhistological glassweregrowingas typicalmonolayer cultures

anddidnotproduceendogenousHNE‐proteinadducts,whilethosegrownonBGweregrowing

overlapping in a tissue‐like form producing abundant HNE. Quadruplicates of cultures were

preparedfortypeofglass,whilerepresentativeimagesarepresentedontheFigure11.

The immunocytochemical analysis was repeated for the BG samples coatedwith BSA,

HNEandBSA‐HNEandaregiveninFigure12.

Figure12.ImmunochemicaldetectionoftheHNE‐histidineadductsafter3daysincellculture

ImmunochemicaldetectionofHNEpresentincellshasrevealedveryinterestingresults.

HNE was also detected in cells that were grown on control and BSA coated BG samples

implicatingitspossibleroleinosteogenesis.

RESULTS

45  

3.1.3. AnalysesofROSproduction

To furtherreveal if theHOScellsgrownonBGsundergophysiologicaloxidativestress

manifested by ROS andHNE production, presence of endogenousROS production during cell

growthontheBGswasdeterminedbyspecificfluorescencemethod(DCF,Figure13).

Figure13.DetectionofintracellularreactiveoxygenspeciesproducedbyHOScellscultured

for3daysonBGs.

Reactive oxygen species (ROS) were visualized by fluorescence using 2',7'

dichlorofluorescein(DCF).Scalebar:100µm.

A)HOScellsculturedfor3daysonBGtype45S5–severalcellsshowstrongpositivityof

theendogenousROSproduction,inparticularifgrowingintheupperlayerofthetissue‐

likeculture

B)HOScellsculturedfor3daysonBGtype13‐93–asincaseof45S5typeBG,several

cellsshowstrongpositivityoftheendogenousROSproduction, inparticularifgrowing

intheupperlayerofthetissue‐likeculture

QuadruplicatesofcultureswerepreparedforeachBG,whilerepresentativeimagesare

presentedontheFigure13.

ToquantifyROSlevels,analysisofROSproductionwascarriedoutincelllysatesgrownon

differentlycoatedsurfaces.Resultshaveshownthatinallsamplesthereisasignificantincrease

ofendogenousROSproductionincomparisontothecontrol(cellsgrownonpolystyreneplastic

in48‐wellplates)withthep<0.001,withexceptionforthesample13‐93(p<0.005).

Moreover, when samples were compared with its own control, BGs 45S5 and 13‐93,

significantincreaseinendogenousROSwasobservedinsamplesthatwerecoatedwithBSAfor

bothBGs(45S5Bp<0.05;13‐93Bp<0.001)whilecoatingwithBSA+HNEincreasedROSlevels

significantlyjustfor13‐93BG.

RESULTS

46  

ctrl(PS)

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0

50

100

150

200

**

**

** **** ** **

*

# ##

**%ofcontrol(PS)

Figure14.AnalysisofROSproductionmeasuredwithaCaryEclipsefluorescence

Spectrophotometer

Marks:H–HNE;B–BSA;BH‐BSA+HNE

#p<0.05;##p<0.01;*p<0.005;**p<0.001

Results support immunocytochemical detection ofHNE‐protein adducts, sinceROS should

beprimarilyformedtoinitiateaprocessoflipidperoxidationandthusgenerationofHNE.

RESULTS

47  

3.1.4. Cellproliferation

Results of cell proliferation are given in Figure 15. Stimulatory effect of the HNE

treatmentcouldbeseeninalltimepointsforBGtype45S5,withthesignificancep<0.05onday

7.BGtype13‐93isshowingthesameeffectwiththeexceptionofday3.

3rdday 7thday 14thday0

25000

50000

75000

100000

125000

150000

45S5

45S5B

45S5H

45S5BH

*Cellnumber

3rdday 7thday 14thday0

25000

50000

75000

100000

125000

150000

13‐93

13‐93B

13‐93H

13‐93BH

*

Cellnumber

A)

B)

Figure15. Proliferation ofhumanosteoblast‐like cells grownon the surfaceof theBGsA)

type45S5andB)13‐93.

Marks:H–HNE;B–BSA;BH‐BSA+HNE

*p<0.05

RESULTS

48  

While on the BG type 45S5 other coating treatments (BSA or BSA+HNE) excert mildly

stimulatory effect, in the case of BG type 13‐93, coating with the BSA‐HNE tends to show a

negative effect which is the most pronounced on day 3 (p<0.01). This negative effect is

decreasingwiththecultureperiodandonday14, there isapproximatelythesamenumberof

cellslikeonthecontrolsample(withoutcoating).

RESULTS

49  

3.1.5. Cellviability

Cell viabilitywasmeasured at 3 time points, days 3, 7 and 14. Stimulatory effectwas

seen for all coating treatments for BG 45S5 in comparison to the control but with slight

differencesregardingthetimepoints.

3rdday 7thday 14thday0

25

50

75

100

125

150

175

200

45S5

45S5B

45S5H

45S5BH

**

** **** **

**

%ofrespectivecontrol

3rdday 7thday 14thday0

25

50

75

100

125

150

13‐93

13‐93B

13‐93H

13‐93BH

*

** *

%ofrespectivecontrol

A)

B)

Figure16.CellviabilityofBGsamples:A)45S5andB)13‐93

Marks:H–HNE;B–BSA;BH‐BSA+HNE

*p<0.01;**p<0.001

RESULTS

50  

Sample45S5‐Bsignificantlyincreasedcellviabilityinalltimepoints(p<0.001).Samples

45S5‐Hand45S5‐BHdidnotincreaseviabilityinthefirstdaysofthecellculture,butlateron.In

caseof45S5‐H,viabilitywassignificantly increasedondays7and14(p<0.001)whilesample

45S5‐BHshowedenhancedviabilityjustafter14daysincellculture(p<0.001).

On the other hand,BG 13‐93 didnot express such stimulatory effect but rather just a

mildone,whichwassignificantlyincreasedinsamples13‐93‐Hand13‐93‐BH(p<0.01)ondays

3 and 14, respectively. In sample 13‐93‐B, on day 7, significant decrease in cell viabilitywas

observed(p<0.001).

RESULTS

51  

3.1.6. Cellgrowthandapoptosis

Genotoxycityanalyseshaveshownthattreatmentsarenotharmfulforcells(compared

tocontrolBGsthatarecurrentlyusedinreconstructivemedicine)andnosignificantdifference

betweenBGswasobserved(Fig17).

ctrl BSA HNE BSA+HNE0

2

4

6

8

10

45S513‐93

Apop

toticindex

(%)

ctrl BSA HNE BSA+HNE0

2

4

6

8

10

45S513‐93

Micronucleatedcells(%)

ctrl BSA HNE BSA+HNE0

2

4

6

8

10

45S513‐93

Mitoticin

dex(%)

ctrl BSA HNE BSA+HNE0

2

4

6

8

10

45S513‐93

Necroticindex(%)

A) B)

C) D)

Figure17.Analysesof:A)apoptotic,B)micronucleated,C)mitoticandD)necroticcellindex

after3rddayofhumanosteoblast‐likecellsgrowthonBGstypes45S5and13‐93

RESULTS

52  

3.1.7. Dot‐blotanalysis

ResultsforBGtype45S5haveshownagradualincreaseinconcentrationofHNE‐protein

adductsforallsurfacecoatingsandforalltimepoints,withtheexceptionforthesample45S5‐

BHinwhichadecreasecanbeseenonday14(Fig.18A).

From statistical point of view, if compared with the references sample, 45S5, for the

specific time point, then a significant increase can be seen: on day 3 in all samples (45S5‐B

(p<0.05);45S5‐Hand45S5‐BH(p<0.001));onday7justin45S5‐BH(p<0.05);andonday14in

samples,45S5‐Band45S5‐H(p<0.05).WhenallBGsamplestype45S5werecomparedwiththe

initial concentrationobtained for thereferencesample (45S5onday3),with theexceptionof

the sample 45S5 on day 7, all other samples are showing significant increase in the

concentrationofHNE‐proteinadductswithp<0.05,orp<0.005forsamples:45S5‐Band45S5‐H

(onday7)and45S5‐B(onday14);andevenp<0.001for45S5‐BH(onday3)and45S5(onday

14).

3rdday 7thday 14thday0.0

0.5

1.0

1.5

2.0

2.5

3.0

45S5

45S5B

45S5H

45S5BH

*****

*

**

nmolHNE‐proteinadducts/mgofprotein

A)

RESULTS

53  

3rdday 7thday 14thday0.0

0.5

1.0

1.5

2.0

2.5

3.0

13‐93

13‐93B

13‐93H

13‐93BH

* *

nmolHNE‐proteinadducts/mgofprotein

B)

Figure18.Dot‐blotanalysisofthecelllysatesgrownontheBGsamples:A)45S5andB)13‐93

Marks:H–HNE;B–BSA;BH‐BSA+HNE

*p<0.05;**p<0.001

Asseen forBG type45S5, resultsobtainedwith the type13‐93,are showing thesame

trend,gradual increaseofHNE‐proteinadduct inall samplesandagainwith theexception for

thesampletreatedwithBSA+HNE(Fig.18B).

Statistically, when compared with the references sample, 13‐93, for the specific time

point, significant increase inHNE‐protein adduct can be seen in 13‐93‐BH on day 3 (p<0.05)

while, on the other hand, significant decrease was observed in 13‐93‐BH on day 7 (p<0.05).

WhenallBGsamplestype13‐93werecomparedwiththeinitialconcentrationobtainedforthe

referencesample(13‐93onday3),increaseinconcentrationofHNE‐proteinadductswasseen

in:13‐93‐BH(day3)and13‐93‐B(day7)withp<0.05;13‐93(day14)withp<0.005;aswellas

in 13‐93‐B and 13‐93‐H (day 14)with p<0.001. In contrast, sample 13‐93‐BH on day 14 has

shownasignificantdecreasewithp<0.001.

When both types of BGswere compared one to another, the same time point and the

sametreatment,therewerefewdifferencesobserved, like inreferencesamples(45S5and13‐

93) on day 3, with higher levels measured in 13‐93 (p<0.01). Another example is coating

treatmentwithBSA+HNEonday7,whichshowedhigher levelsofHNE‐proteinadducts inBG

type45S5.Thelastdifferencewasobservedinreferencesamplesagainonday14,withhigher

levelsseeninBG13‐93.

RESULTS

54  

3.1.8. Celldifferentiation–Alkalinephosphatasestaining

Obtainedresultswereobservedunderthelightmicroscopeaswellassemiquantitatively

(Fig.19).

Staining for ALP, which is a marker of osteoblast differentiation, has revealed some

similaritiesanddifferencesdependingonthetypeoftheBGused.

BothtypesofBGshaveshownincreaseinALPonday14ifcomparedtothesamesample

onday3,whichwasstatisticallysignificantforsamples:45S5(p<0.05),45S5H(p<0.001),13‐93

H(p<0.01),13‐93BH(p<0.001)(Fig.19B).

While there were no differences between the surface coating treatments and control

sample forBGtype45S5onday3, incaseofBGtype13‐93asignificantdecrease inALPwas

observedincoatingswithHNE(p<0.01)andBSA+HNE(p<0.001)(Fig.19B).

Ontheotherhand,significantincreaseofALPwasobservedinthesampleoftheBGtype

45S5coatedwithHNE(p<0.005)whilefortheBGtype13‐93thereisatendencyofALPincrease

insamplescoatedwithHNEandBSA+HNE(Fig.19B).

A)  

RESULTS

55  

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0

50

100

150

200

250

300

350

3rdday

14thday

**

* ***

%ofrespectivecontrol

B)

 

Figure19.Observationofcelldifferentiationdetectedwithalkalinephosphatasestaining:

A) under a light microscope (magnification 200x; pictures in upper row for each

bioactiveglass)andonthesurfaceofbioactiveglassdiscs(belowrowforeachbioactive

glass);

B)quantificationofobserveddifferentiationwithVersaDoc

*p<0.01;**p<0.005;***p<0.001

RESULTS

56  

3.1.9. Cellmineralization–AlizarinRedstaining

ResultsofmineralizationofcellsgrownontheBGsamplesaregiveninFig20.

OnBG samples type13‐93, spherical cell structures are themost intensively stainedbut also

somesmallernodal formationscanbeseen inall samples.BGsamples type45S5haveshown

moreevenlydistributed stainingwith somenodal formation, especially observed in reference

sample(Fig.20A).

A)

Standardcurve

0.00 0.25 0.50 0.75 1.00 1.250.00

0.05

0.10

0.15

0.20

0.25

0.30

y=0.213x+0.0451R2=0.9995

AlizarinRed(mM)

ODat405nm

B)

 

RESULTS

57  

ctrl(PS)

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

AlizarinRedinmM

C)

 

Figure20.Detectionofcellmineralizationontheday14ofcellculture–stainingwithAlizarin

Red

A)Observedunderlightmicroscope

B)StandardcurvepreparedwithdifferentconcentrationsofAlizarinReddilutedin0.5N

HCland5%SDS

C)ConcentrationofAlizarinRedinmM,recalculatedfromthestandardcurve

InallBGsamplesthereisasignificantincreaseinmineralisationofextracellularmatrix

observed in comparison to the control sample, cells grown in 48‐well plate on polystyrene

plastic(p<0.005).Theonlyexceptionwassample45S5‐Bwhoshowedthesametendencybut

duetohighstandarddeviation,itwasnotstatisticallysignificant.

RESULTS

58  

3.1.10. RealtimePCR

 

Results of relative expression of genes for alkaline phosphatase (ALP), type I collagen

(COL1), osteocalcin (OC), osteoprotegerin (OPG), osteonectin (ON), osteopontin (OPN) were

showninFigure21.GAPDHwasusedasareferencegene.Geneexpressionwasdeterminedon

days3and14(Fig.21A‐F).

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

2.0 ALP3rdday

ALP14thday

relativemRNAexpression

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

2.0 COL3rdday

COL14thday

relativemRNAexpression

A)

B)

 

RESULTS

59  

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

2.0OC3rdday

OC14thdaydrelativemRNAexpression

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

2.0 ON3rdday

ON14thday

relativemRNAexpression

C)

D)

 

 

RESULTS

60  

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0

1

2

3

4

5

OPN3rdday

OPN14thdayrelativemRNAexpression

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

2.0

2.5 OPG3rdday

OPG14thday

relativemRNAexpression

E)

F)

 

Figure21.RealtimePCRanalysesof:A)ALP;B)COL1;C)OC;D)ON;E)OPN;andF)OPG

Statistical comparisons were done using two‐way analysis of variance (ANOVA) and

Bonferroni’spost‐test.Differenceswereconsideredstatisticallysignificantatp<0.05.

RESULTS

61  

Resultsindicatethatdifferentcoatingtreatmentaffectsosteogenicmarkersdifferentlyin

different time points. ALP, in the BG 45S5 was up regulated in the beginning (day 3) of cell

culture for samples treated with HNE and BSA+HNE while in the BG 13‐93, ALP was more

expressedonday14.COL1expressionwasthehighestinsample45S5‐BHonday14.OClevels

didnotsosignificantlydifferbetweenthetreatmentsandthedaysinculture.ONwasfoundto

beexpressedinsamples45S5‐H(day3)and45S5‐BH(day14).Thehighestfold‐changeingene

expressionwasobservedforOPNinsample45S5‐H(day14)witha4‐foldchangeobservedin

comparisontothereferencegene.Thisgenewasthemostexpressedincomparisontotheother

genes tested. According to the results obtained with this gene coating treatments seems to

stimulate the osteogenic potential of the cellwhat ismore pronounced for the BG type 45S5

eventhoughitwasalsoobservedfortheBGtype13‐93.Moreover,expressionofOPNwasup‐

regulated in the referencesample45S5onday14, incomparison today3,while in reference

sample13‐93therewasnochange.HigherexpressionofOPG, incomparisontodifferent time

points,wasobservedonday14inmostsamplesofBGtype45S5,especiallytheonecoatedwith

HNE and the reference sample, while, in case of the BG type 13‐93, higher expression was

observedonday3insamplescoatedwithBSAorBSA+HNE.

RESULTS

62  

3.1.11. Western‐blotanalyses

Results of the western blot analyses have shown that p44/42 (ERK 1/2) signalling

pathway is activated during cell growth on the surfaces of BGswhile p38 signalling pathway

remainedinactive,nophosphorilatedp38proteinsweredetected(datanotshown).

Analysis of SAPK/JNK was also carried out but no bands were detected with both,

phosphorilatedorunphosphorilatedantibodies(datanotshown).

ctrl(PS)

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

Arbitraryunits

A)

 

 

 

RESULTS

63  

ctrl(PS)

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

*

**

*

*

Arbitraryunits

B)

 

ctrl(PS)

45S5

45S5B

45S5H

45S5BH

13‐93

13‐93B

13‐93H

13‐93BH

0.0

0.5

1.0

1.5

Arbitraryunits

C)

Figure22.Western‐blotanalysisofp38(A),phosphorilatedp44/42(B),andp44/42(C)MAPK

signallingpathway

RESULTS

64  

Samplemarks:1‐45S5; 2‐45S5‐B; 3‐45S5‐H; 4‐45S5‐BH; 5‐13‐93;

6‐13‐93‐B; 7‐13‐93‐H; 8‐13‐93‐BH; 9‐ctrl(cellsgrownonplastic)

Treatmentmarks:B‐0.5mg/mlBSA;H‐2.5µMHNE;BH–0.5mg/mlBSA+2.5µMHNE

*p<0.05;**p<0.01

In comparison to the control sample (cells grown on plastic in 48‐well plate), significant

increaseinphosphorilatedp44/42wereobservedinsamples:45S5(p<0.05),45S5‐B(p<0.01)

and especially 45S5‐H (p<0.05). This results suggest that 45S5 BG itself activate the p44/42

MAPK pathway more than 13‐93 BG and that coatings, with BSA or with HNE, activate this

pathwayevenmore.ThecoatingmadewithcombinationofBSAandHNE,inbothBGs,didnot

increase theactivationofp44/42pathwaybutrather itstayed inthe levelsofcontrolsample,

grownon theplastic,whichwaseven lower than thecontrol sampleof45S5BG(without the

coating).

RESULTS

65  

3.2. ResultsobtainedwithBG,type45S5,withorwithoutcopper

3.2.1. Cellviability

Acytotoxiceffectofthe45S5‐1Cuand45S5‐2.5Cusampleswasobservedasadecrease

in cell viability at all time points. On day 3, cell viability of the 45S5‐1Cu sample was

approximately50%,whencompared to the45S5‐Ref (p<0.005),and itdecreased to~26%on

days7and14(p<0.001).Cellviabilityobservedon45S5‐2.5Cusampleswaslowerthan20%at

all 3 time points (p<0.001). In contrast, a trend of growth enhancement with a significant

increase inviability (p<0.05)wasobservedafter14days for45S5‐0.1Cu (Fig23).Resultsare

expressedaspercentagetotherespectivecontrol(45S5‐Ref).

3rdday 7thday 14thday0

50

100

150

20045S5‐Ref

45S5‐0.1Cu

45S5‐1Cu

45S5‐2.5Cu

*

**

*** ******

******

%ofrespectivecontrol

Figure23.CellviabilityoftheHOScellsgrownonthebioactiveglasssamplesmeasuredbyMTT

assayatdays:3,7and14.Theresultsaregivenasapercentageoftherespectivecontrol(45S5‐

Ref).Cellviabilitywasincreasedforthe45S5‐0.1Cuatday14(*p<0.05)whileitwasdecreased

forthe45S5‐1Cuand45S5‐2.5Cusampleatalltimepoints:

45S5‐1Cu–50%decreaseatday3 (**p<0.005) followedby the~75%atdays7 and14 (***

p<0.001).

45S5‐2.5Cu–>80%decreaseatalltimepoints(***p<0.001).

RESULTS

66  

3.2.2. Cellgrowthandmaterialcharacterization

3.2.2.1. DAPIstaining

DAPI stained images correlatedwith the cell viability results observed in Section 3.1.

Specifically, after 14 days, confluent cellswere observed on the 45S5‐Ref and 45S5‐0.1CuBG

surfaces.However,fewcellswerepresenton45S5‐1Cuand45S5‐2.5CuBGsurfaces(Fig24).

Figure24.DAPIstainingimagesobtainedattheday14:a)45S5‐Ref,b)45S5‐0.1Cu,c)45S5‐1Cu

andd)45S5‐2.5Cu.

RESULTS

67  

3.2.2.2. SEMandEDXanalysis

SEMimagesonday7ofcultureshowedanicelyspreadcellonthesurfaceofthe45S5‐

Refwhileonthe45S5‐1Cusurfacealotofprecipitatescouldbeseenwhichseemedtointerfere

withthecellgrowth(Fig25a&b).

Figure25.SEMfiguresofa)45S5‐Ref;b)45S5‐1Cuandc)45S5‐2.5Cusamplesafter7days in

cellculturemedium.

TheSEMimageoftheBG45S5‐2.5Cusamplesurface(Fig25c)indicatedthreedifferent

phases,i.e.,(i)glassmatrix(“BG”),(ii)calciumphosphateprecipitates(“HAp”)and(iii)copper‐

rich precipitations (“Cu”). The corresponding elemental concentrations derived from EDX

measurements of the precipitates are shown in Table 4. For the calcium rich area, the

determinedCa/Pratioof~1.61confirmedtheformationofhydroxyapatiteontheglasssurface.

However,theCu‐richareasseemedtocontainupto~13At.‐%Cu.

Table4.EDXanalysisof the45S5‐2.5Cu(atomic%).AreasofmeasurementcorrespondtoFig.

25c.

Sample Si P Ca Cu

45S5‐2.5Cu

Region“BG” 15.53 4.09 6.66 0.36

Region“HAp”

1.03 5.79 9.36 0.26

Region“Cu” 3.06 6.12 6.33 13.00

RESULTS

68  

3.2.3. Immunoblotanalysis

Thegrowthofthecontrolcells(45S5‐Ref) fromday3todays7and14wasassociated

withagradualincreaseoftheHNE‐proteinadducts(Fig26).Allcellsamplesthatweregrownon

Cu‐doped45S5BGshowedenhancedformationoftheHNE‐proteinadducts.Theenhancement

wasparticularlypronouncedondays3(p<0.001)and14(p<0.01),forthecellsgrownon45S5‐

2.5Cusamples.

3rdday 7thday 14thday0

1

2

3

4

5

6

7

8 45S5‐Ref

45S5‐0.1Cu

45S5‐1Cu

45S5‐2.5Cu

***

**

**

***

***

nmolHNE‐proteinadducts/mgofprotein

Figure26.Immunoblotting(dot‐blot)analysesoftheHNE‐proteinadductsformationatdays3,

7and14.ResultsareexpressedasnmolofHNE‐proteinadducts/mgofprotein.

Enhancementof the lipidperoxidationwas inparticularpronouncedondays3 (***,p<0.001)

and14(**,p<0.01),forthecellsgrownon45S5‐2.5Cusamples.*denotesp<0.05.

  

69  

4. DISCUSSION

DISCUSSION 

70  

4. DISCUSSION

Immunochemical analysis of the reference BGs types 45S5 and 13‐93 revealed that

during incubation of human bone cells invitro on their surface, HNE is generatedwithin the

cells,unlikewhilegrowingonthenormalhistologicalglass(Figure11.).Thissuggestspossible

physiological role of HNE in bone regeneration process which was not associated with

cytotoxicity because human osteoblast‐like cellswere proliferating (increasing their number)

during cultivation period. Moreover, since HNE is considered as "second messenger of free

radicals", this evidence gives additional support that oxidative stress and lipid peroxidation

should not only be considered as generators of biomolecular damage, and thus important

contributorstoagingaswellascausative factorsofdiseases,butshouldalsoberecognizedas

significant mediators of normal physiological functions [57]. That allowed consideration of

oxidative stress also as physiological process introducing new term ”oxidative homeostasis”,

whichimpliesinvolvementsofreactiveoxygenspecies(ROS)andlipidperoxidationinvarious

physiologicalprocesses[80].Infavourofthispossibilityisalsodifferenceofthegrowthpattern

observedwhenHOScellswerecultureonBGs.Namely,thecellsgrownonstandardhistological

glassweregrowingastypicalmonolayerculturesanddidnotproduceendogenousHNE‐protein

adducts,while thosegrownonBGsweregrowingoverlapping in a tissue‐like formproducing

abundantHNE(Figure11).

Since no HNE‐histidine adducts were detected in the cells cultured on normal

histological glass, but only if the cells were grown on BGs, conclusion arises that enhanced

growthof theHOS cells in tissue‐likepatternonBGswasassociatedwithproductionofHNE,

known toactas growth regulating factor and signallingmolecule consideredalsoas a second

messengeroffreeradicals[67].

To furtherreveal if theHOScellsgrownonBGsundergophysiologicaloxidativestress

manifested by ROS and HNE production, endogenous ROS production was determined to be

presentduring cell growthon theBGsby specific fluorescencemethod (DCF, Figure13).ROS

maytriggerlipidperoxidationandgenerationofHNE,whichisontheotherhandabletoinduce

ROSproduction[101],whichusuallyleadtotheself‐catalysedprocessoflipidperoxidationthat

coulddestroythecells.However,inthisstudyitappearedtoenhancethegrowthofHOScellson

BGs.

ThereisgrowingevidenceaboutthephysiologicalgenerationofROSthatcanoccurasa

byproductofotherbiologicalreactions.NADPHoxidaseisknowntobethemajorsourceofROS,

DISCUSSION 

71  

butROSgenerationalsooccurswithmitochondria,peroxisomes,cytochromeP‐450,andother

cellularelements [102].Severalgrowth factorsandcytokinesbinding todifferent typesofcell

membranereceptorscanelicitariseinintracellularROS.ItwasfoundthatgenerationofROSis

inducedwithTGF‐β inmouseosteoblastic cells and inhuman lung fibroblasts [103]andwith

PDGFinhumanlensepithelialcells[104].Furthermore,ithasbeenreportedthatROSactivate

MAPK pathways in several systems, including extracellular signal regulated kinase (ERK)1/2,

Jun‐NH2‐terminalkinaseandp38MAPKcascades[105].

Inthesecondstep,coatingofBGswithHNE,BSAortheircombinationhaveshownthat

the coating of the BG samples was efficient and that results obtained are due to the coating

treatmentandjoinedactivityoftheBGitself.ImmunochemicaldetectionofHNEpresentincells

wasincompliancewiththefirstobservations,thatHNE‐proteinadductswerepresentinallBG

samples,evenincontrolsamplesandsamplescoatedwithBSA.ResultsoftheROSproduction

wereconformingimmunocytochemicalresultssinceinallBGsamples,coatedorthereference

ones,theincreaseinendogenousROS,incomparisontothecellsgrowninacultureplateplastic,

wasobserved.Thisfindingindicatestherelevanceofoxidativestressandlipidperoxidationin

the process of enhanced growth of human osteoblast‐like cells on the surface of the BGs

[29,106].Moreover,when compared to their reference samples,bothBGs coatedwithBSAor

withBSA+HNEforBGtype13‐93,showedincreasedROSproduction.Recentfindingofalbumin‐

induced upregulation ofmatrixmetalloproteinase‐9 (MMP‐9) via increase of ROS production

andactivationofp38MAPKsignallingpathway[107],couldpotentiallylinkobservedincreaseof

ROSlevelsinBGsamplescoatedwithBSAwiththecoatingitself.

Analysis of the levels of HNE‐protein adducts in cell lysates grown on BG samples

showed gradual increase in endogenous concentration of HNE‐protein adducts with time in

cultureforallsampleswiththeexceptiontotheonestreatedwithBSA+HNEforthe45S5BG.In

thissamplethehighestlevelofHNE‐proteinadductswasobservedonday7anddecreasingon

day14.Thiscouldbedue to theboostingof theantioxidativecapacityof thecellsexposed to

higherconcentrationofHNE‐proteinadductsonday7.Moreover,resultsobtainedwiththeBG

type13‐93,areshowingthesametrend,gradualincreaseofHNE‐proteinadductinallsamples

withtheexceptionfortheBSA+HNEsample.Inthissamplethelowestlevelwasobservedonday

7andthehighestonday14.Since levelsofHNE‐proteinadductsonday3werehigher inthis

samplethaninallothers,thereisapossibilityofearlieractivationofantioxidativemechanisms.

Although, it was already reported that HNE is enhancing gene expression of antioxidant and

detoxifyingenzymesthroughNrf2andthatNrf2‐AREsignallingmechanisminthedetoxification

DISCUSSION 

72  

of HNE is very important [108] in order to confirm these claims additional experiments are

needed.

When present in cells, HNE forms stabile covalent‐bonded HNE‐protein adducts by

protein and peptidemodificationsmostly through reactionwith cysteine, lysine and histidine

aminoacidresidues[65,67].Theseprotein‐adductwerefoundinnormalskeletaldevelopment

duringembryogenesis[109]andinvarioustissuesofanimalandhumanorigin[80].Theimpact

ofHNEonthefateofthecellisdose‐dependent;atlowlevels,HNEpromotescellproliferation,

whereas at higher concentrations, it induces cell cycle arrest, differentiation, and finally

apoptosis[110].ExposuretoHNEinducesvascularsmoothmusclegrowth,accompaniedbythe

activationofMAPKs(ERK,c‐JunNH2‐terminalkinase,andp38),theinductionofc‐fosandc‐jun

geneexpression,andactivatorprotein1(AP‐1)DNAbindingactivity[70].Otherreportedthat

conjugatesof,themostimportantantioxidantincellsinvolvedinHNEdetoxification,GSHwith

HNE activate PKC and stimulate NF‐κB and AP‐1‐dependent gene transcription leading to an

increaseincellgrowth[84].Furthermore,recentobservationsshowthatstimulationwithHNE

at sublethal concentrations induces adaptive response and enhances cell tolerance, thereby

protectingcellsagainsttheforthcomingoxidativestress[58].

Inaddition,forthecoatingtreatmentwithHNEoritscombinationwithBSA,2.5μMHNE

was used, the concentration already shown to have stimulatory effect on the growth of the

humanosteoblast‐likecells[62].Furthermore,evaluationofapoptosis,micronucleusformation,

necrosisandmitoticindexcarriedoutbytheDAPIstainingmethod,hasshownthatthecoating

treatmentswerenotcytotoxicandgenotoxictothecells.Therewerenodifferencesbetweenthe

samplesandthereforethetreatmentswerenotnegativelyaffectingcellgrowth.

Onthecontrary,coatingtreatmentwithHNEseemstofurtherstimulatecellgrowthon

thesurfaceofbothBGwhileitismorepronouncedintheBGtype45S5.Thiswasobservedby

bothcellproliferation,analysedbythetrypanblueexclusiontest,andcellviability,analysedby

theMTTtestthatis,infact,ameasureofcellularmetabolicactivity.

Samples treatments with HNE and BSA+HNE on the 45S5 BG enhanced cell

differentiationandmatrixmineralizationwhatwasobservedbythegeneexpressionanalysisof

specificosteogenicmarkers,andalsostainingsforALPandwithAlizarinRed.Resultsofthegene

expression of the osteogenic markers indicate that different coating treatment affects them

differentlyindifferenttimepoints.IntheBGtype45S5,ALP,asanearlyosteogenicmarker,was

upregulatedinthebeginning(day3)ofcellcultureforsamplestreatedwithHNEandBSA+HNE

while in the BG 13‐93, ALPwasmore expressed in all samples on day 14. The highest fold‐

DISCUSSION 

73  

change in gene expression was observed for OPN in sample 45S5‐H (day 14) with a 4‐fold

change observed in comparison to the reference gene. This gene was the most expressed in

comparisontotheothergenestested.Accordingtotheresultsobtainedwiththisgene,coating

treatments seems to stimulate the osteogenic potential [111] of the cell what is more

pronouncedfortheBGtype45S5eventhoughitwasalsoobservedfortheBGtype13‐93.

ResultsofALPstainingandquantificationhaveshownthesametrend, increaseinALP

betweendays3and14 inbothBGs,whichwasstatisticallysignificantwhenBGswerecoated

withHNEorBSA+HNEforBGtype13‐93.WhileHNEcoatingnotablyincreasedALPonday14in

45S5BG,thesametreatmentdecreasedALPin13‐93typeBG.Thesameeffectwasobservedfor

13‐93typeBGcoatedwithBSA+HNE.Ontheotherhand,inbothBGsamplescoatedwithBSA,

detectedlevelsofalkalinephosphataseweresomewhereintherangeoftheresultsobtainedfor

thereferencesamples(withoutcoatings)forbothtimepoints.

Moreover,asignificantincreaseinmineralisationofextracellularmatrixwasobservedin

all BG samples when compared to the control sample, cells grown in 48‐well plate on

polystyreneplastic(p<0.005).Theonlyexceptionwassample45S5‐Bwherethesametendency

was shown, but due to high standard deviation, it was not statistically significant. Therefore,

abovementioned resultsgive evidenceof the stimulatoryeffectsof the treatments (especially

withHNE)uponcelldifferentiationandmineralization,andthussupportofbonegrowth.

The complexity of biological response that is involved in bone regeneration process

shouldbealsomentioned.ThereareseveralpossiblemechanismreportedlikeIndianhedgehog

(Ihh)pathway,bonemorphogeneticprotein(BMP)pathway,canonicalWnt‐β‐cateninpathway

andforourpointofviewthemostinterestingMAPKpathway.MAPKpathwaywasfoundtobe

obligatorytransducerandacrucialforpost‐fracturebonehealingbecauseittransducessignals

fromseveralgrowthfactors(FGF,IGF,EGF)[112].

BGs in contactwith thebody fluid,or in thiscaseculturemedium,behavespecifically,

that is, start to initiate a sequence of reactions in order to create carbonated hydroxyapatite

layer, similar to the mineral in bone, which is considered as indication of their bioactivity.

Consequently,ionicdissolutionproductsofBGarereleasedintothemediumandstimulategene

expressionofhumanosteoblastsandthusenhanceosteogenesisaswellasangiogenesis[113].

Furthermore, BG ionic dissolution products were found to stimulate few growth factors

includingIGF‐II,akeyregulatorofosteoblasthomeostasis,andVEGF,amemberofthefibroblast

growthfactorfamilywithosteogenicpotentialandalsoimportantinangiogenesis[25].

DISCUSSION 

74  

In addition, several intracellular signalling molecules are involved in osteoblast

activationduetoIGF‐IIandVEGF,includingERKandMAPKpathways.Fourkinaseswereshown

tobeinducedbythetreatment,MAPKAPkinase‐2,MAPK‐2,MAPKp38,andERK1[29].

The western blot analyses have revealed that p44/p42 (ERK 1/2) MAPK signalling

pathway is activated while p38 is not. In comparison to the control sample (cells grown on

plastic),significantincreaseinphosphorylatedp44/42wasobservedinsamples:45S5,45S5‐B

and especially 45S5‐H (p<0.05). This results suggest that 45S5 BG itself activate the p44/42

MAPK pathway more than 13‐93 BG and that coatings, with BSA or with HNE, activate this

pathwayevenmore.ThecoatingmadewithcombinationofBSAandHNE,inbothBGs,didnot

increase theactivationofp44/42pathwaybutrather itstayed inthe levelsofcontrolsample,

grownon theplastic,whichwaseven lower than thecontrol sampleof45S5BG(without the

coating).

Hence, taking all mentioned in consideration, the proposed mechanism of bone

regeneration during fracture healing supported by BGs, includes effect of BG dissolution

products that, on one hand, initiate oxidative stress and ROS formation, which triggers lipid

peroxidation and generationofHNE. On theotherhand, thesedissolutionproducts, together

with HNE, acting as a signalling molecule, enhance bone formation through MAPK pathway,

morepreciselyERK1/2MAPKsignallingpathway(Fig.27).

Therefore, the primary assumption that BG functionalized with physiological

concentrationofHNE,priortoitsimplantationindamagedbonetissue,couldtriggersignalling

reactionsand togetherwithpositiveeffectsofBGmight reflect inenhancedosteogenesis,was

shown to be true. These observations are supported by the report of successful use of

functionally activated carbonnanotubeswithHNE for stimulation of invitro neuronal growth

[114].

DISCUSSION 

75  

Fig. 27. Proposed mechanism of BG dissolution products without or with HNE that

through production of ROS, induction of lipid peroxidation enhance bone formation during

fracturehealing,whichismediatedthroughERK1/2MAPKpathway.

DISCUSSION 

76  

IntheotherpartofexperimentwiththeBGtype45S5preparedwithcertainamountof

cooper,resultshaverevealedfollowing.

EDXanalysis showed that theCu‐rich area seems to contain up to~13At.‐%Cu even

though the initial compositionof the45S5‐2.5Cusamplewasonly2.5%(w/w)Cu. It is likely

thatsuchapronouncedandrapidreleaseofCuintheinitialstageofthecellcultureincubation

results in focal saturation that might lead to precipitation of Cu‐containing mineral phases.

Whilemoredetailedsurfacespecificanalysiswouldbenecessaryfordeterminationoftheexact

originofthethreephasesdistinguishedonthe45S5‐2.5Cusample,thefindingofaCu‐richarea

explainsthecellviabilitydecreaseobservedforboth,45S5‐2.5Cuand45S5‐1Cu.Incontrast,the

45S5‐0.1Cusampleshoweddidnotshowdecreaseinviabilitybutanenhancedcellgrowth.

The observed results could be explained by the fact that an excess of Cu, an essential

traceelementvitalfortothehealthoflivingorganismsunderphysiologicalconditions,induces

cellular toxicity through the production of oxygen free radicals and direct oxidation of lipids,

proteinsandDNA[42,44].Thus,adelicatemechanismthatbalancestheconcentrationofCuis

needednotonlyatthecellularlevel,butalsoatthetissueandorganlevels[44].Oneofthemost

accepted explanations for Cu‐induced cytotoxicity presumes that Cu ions are prone to

participate in the formationof reactiveoxygenspecies (ROS)viaFentonreaction [115],while

themajorconsequenceofCuexcessisperoxidativedamagetomembranelipids[116].

In this respect, lipid peroxidation results in a formation of reactive aldehydes among

whichHNEisoneofthemostintensivelystudiedsinceitisahighlyelectrophilicmoleculethat

easily reacts with macromolecules (proteins, peptides, DNA and phospholipids) [59,60]. As

already mentioned, HNE is an important signalling molecule involved in various cellular

processes, from cell proliferation to differentiation as well as cell death in a concentration‐

dependentmanner [64,66,71,117].Moreover,HNE formsstablecovalentHNE‐proteinadducts

byreactionwithcysteine,lysineandhistidineresidues,withinthecell[65,69].

ResultsshowedagradualincreaseintheamountofHNE‐proteinadductsfromday3to

day14fortheBG45S5sampleswithoutCu(45S5‐Ref).Thesametrendwasobservedfor45S5‐

0.1Cusamples.Moreover,whencomparedto thecontrolsample,45S5‐Ref,all samplesofCu‐

doped 45S5 BG showed an enhanced formation of the HNE‐protein adducts, especially 45S5‐

2.5Cusamplesondays3and14.However,lowerHNEvalueswereobservedonthe7thdayfor

45S5‐1 Cu and 45S5‐2.5 Cu sampleswhich ismost likely due to the cell adaptation upon the

initial Cu‐induced generation of ROS and, consequently, HNE. This is inferred since the well‐

DISCUSSION 

77  

knownmechanismthatstimulatecellstoadaptationisthroughanincreaseoftheirintracellular

antioxidantmechanisms[42,68],althoughatthispointwehavenotexperimentallyconfirmedit.

Thepresentresultsareinagreementwithpreviousfindings,whichshowedthatgrowth

ofhumanbonecellsonBG isassociatedwith lipidperoxidation, i.e. formationofHNE‐protein

adducts[63].ThisprocessisenhancediftheBGcontainsCu,withaslightdifferenceinthat,on

theonehand,enhancedlipidperoxidationinducesacytotoxiceffect,asshownfor1%and2.5%,

respectively,whileat0.1%ofCuenhancedlipidperoxidationwasassociatedwithenhancedcell

growthandnotcytotoxycity.Theseresultsareincompliancewiththeconcentration‐dependent

role of HNE in regulation of cell growth and cell death [64,66,71,89]. Hence, it should be

mentionedthatcellsexposedtolevelsoflipidperoxidationabovephysiologicalarestillableto

adjusttothem[63,66],asobservedforthe45S5‐0.1Cusample.Themechanismsofsuchcellular

hormesis, an adaptive response of cells and organisms to a moderate (usually intermittent)

stress[118],arenotyetunderstood.ItiscertainthatinitiallyHNEactsthroughregulationofthe

c‐fosexpression,whichiscytokinedependentandleadstothecascadeofmultipleintracellular

andintercellulareventsaffectingproliferation,differentiationandapoptosisofthecellsexposed

tothedifferentlevelsofHNE[74].Furthermore,itwasalsoreportedthatHNEactivatesnuclear

factorerythroid2‐relatedfactor2(Nrf2)andantioxidantgeneexpressioninvascularcells[119]

aswellasotheradaptivestressresponsepathwaysthatpromotethesurvivalofcells[120,121].

Whileprinciplesof thebonegrowthenhancement remain tobeelucidated, it is clear that

HOS cells growing on BGs produce HNE, which makes stable protein adducts, indicating the

fundamental role of HNE in the bone cell growth enhancement by BG. The findings of

concentrationdependentCu‐induced,effectsonHOScellgrowthrelativetocellularproduction

levelsofHNEare relevant towardsabetterunderstandingof themechanisms involved in the

boneregenerationprocessaswellas theregenerationenhancementbyBG.Thecomplexityof

the bone regeneration process is also clear, with several signalling and metabolic pathways

possibly linked to HNE in potential cytotoxic or growth‐regulatory capacities. Thus, Cu‐

containingBGcouldofferapossiblesystemtocontroltheleveloflipidperoxidationresultingin

increasedosteogenesisandmayprovevaluableintissueengineeringapplications.

  

78  

5. CONCLUSIONS

CONCLUSIONS 

79  

5. CONCLUSIONS

CoatingofBGswithHNE,BSAorthecombinationwasefficientandresultsobtainedare

duetocombinedeffectofcoatingtreatmentandBGdissolutionproducts.

Coatingtreatmentsarenotgenotoxicnorinduceapoptosisornecrosisandthereforedo

notnegativelyaffectcellgrowth.

Immunochemical detection ofHNE present in cells has revealed its presence in all BG

samples,eventhereferenceones,implicatingitspossibleroleinosteogenesis.

Increase in endogenous ROS was observed in all BG samples in comparison to cells

growninaculture,whichisincompliancewithimmunocytochemistryresults.

CoatingtreatmentwithHNEseemstofurtherstimulatecellgrowthonthesurfaceofBGs,

morepronouncedforBGtype45S5.

Levels of HNE‐protein adducts in cell lysates show gradual increase in endogenous

concentrationofHNE‐proteinadductswith time in culture for all samplesexceptones coated

withBSA+HNE.Antioxidativecapacityshouldbeinvestigated.

Samples treatments with HNE and BSA+HNE on the 45S5 BG enhanced cell

differentiation and matrix mineralization what was observed as a gene expression and also

stainingsforALPandwithAlizarinRed.

The western blot analyses have revealed that p44/p42 (ERK 1/2) MAPK signalling

pathwayisactivatedwhilep38isnot.

CONCLUSIONS 

80  

For the experimentswith the 45S5 type BGswith different Cu contents (0, 0.1, 1 and

2.5%(w/w))resultshaveshownthatHOScellgrowthonall45S5BGsampleswasassociated

with lipid peroxidation, as measured through the formation of HNE‐protein adducts. This

processwasenhancedontheCucontainingBGinconcentration‐dependentmanner.

Cucontentwascytotoxicwhenpresentat1%andhigherconcentrations.

Onthecontrary, inthecaseof0.1%Cuanenhancedlipidperoxidationwasnotassociated

withcytotoxicitybutwithgrowthenhancement.

Therefore,Cucontaining45S5typeBGmightrepresentanovel,attractivebioactivematerial

forstudiesoftheroleoflipidperoxidationinvolvedintheboneregenerationprocess.

Overall conclusion is that lipid peroxidation is involved in a process of enhanced

osteogenesisofthecellsgrownontheBGsandthereforetheobtainedresultsareopeningnew

perspectivesintissueengineeringandinthefieldofregenerativemedicine.

 

81  

6. REFERENCES

REFERENCES

82  

6. REFERENCES

[1] Gray H. Anatomy of the Human Body. 20th ed. Philadelphia: Lea & Febiger, 1918;Bartleby.com,2000.;2000.

[2] Marieb EN, HoehnK. HumanAnatomy& Physiology. 7th editio. Pearson Prentice Hall;2007.

[3] Kini U, Nandeesh BN. Physiology of Bone Formation, Remodeling, andMetabolism. In:Fogelman I, Gnanasegaran G, Wall H, editors. Radionucl. Hybrid Bone Imaging, Berlin,Heidelberg:SpringerBerlinHeidelberg;2012,p.1046.

[4] Eriksen EF, Axelrod DW, Melsen F. Bone histomorphometry. New York: Raven Press;1994.

[5] Buckwalter JA, Glimcher MJ, Cooper RR, Recker R. Bone Biology. J Bone Jt Surgery1995;77:1276–89.

[6] Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol 2008;3 Suppl3:S131–9.

[7] Brodsky B, Persikov A V. Molecular structure of the collagen triple helix. Adv ProteinChem2005;70:301–39.

[8] Sroga GE, Karim L, ColónW, VashishthD. Biochemical characterization ofmajor bone‐matrixproteinsusingnanoscale‐sizebonesamplesandproteomicsmethodology.MolCellProteomics2011;10:M110.006718.

[9] DelanyA,AmlingM,PriemelM,HoweC,BaronR,CanalisE.Osteopeniaanddecreasedboneformationinosteonectin‐deficientmice.JClinInvest2000;105:1325.

[10] McKee MD, Nanci A. Osteopontin at mineralized tissue interfaces in bone, teeth, andosseointegrated implants: ultrastructural distribution and implications for mineralizedtissueformation,turnover,andrepair.MicroscResTech1996;33:141–64.

[11] GolubEE,HarrisonG,TayloraG,CamperS,ShapiroIM.Theroleofalkalinephosphataseincartilagemineralization.BoneMiner1992;17:273–8.

[12] ZaidiM.Skeletalremodelinginhealthanddisease.NatMed2007;13:791–801.

[13] TeitelbaumSL.BoneResorptionbyOsteoclasts.Science(80‐)2000;289:1504–8.

[14] HadjidakisDJ,AndroulakisII.Boneremodeling.AnnNYAcadSci2006;1092:385–96.

[15] Buckwalter JA, Glimcher MJ, Cooper RR, Recker R. Bone biology. I: Structure, bloodsupply,cells,matrix,andmineralization.InstrCourseLect1996;45:371–86.

[16] TsiridisE,UpadhyayN,GiannoudisP.Molecularaspectsoffracturehealing:whicharetheimportantmolecules?Injury2007;38Suppl1:S11–25.

REFERENCES

83  

[17] DimitriouR, Tsiridis E, Giannoudis PV. Current concepts ofmolecular aspects of bonehealing.Injury2005;36:1392–404.

[18] Giannoudis P V, Einhorn T a, Marsh D. Fracture healing: the diamond concept. Injury2007;38Suppl4:S3–6.

[19] KalfasIH.Principlesofbonehealing.NeurosurgFocus2001;10:E1.

[20] Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the art and futuretrends.MacromolBiosci2004;4:743–65.

[21] Hench LL, Splinter RJ, AllenWC, Greenlee TK. Bondingmechanisms at the interface ofceramicprostheticmaterials.JBiomedMaterRes1971;5:117–41.

[22] Kitsugi T, Nakamura T, Yamamura T, Kokubu T, Shibuya T, Takagi M. SEM‐EPMAobservation of three types of apatite‐containing glass‐ceramics implanted in bone: thevarianceofaCa‐P‐richlayer.JBiomedMaterRes1987;21:1255–71.

[23] Ohgushi H, Dohi Y, Yoshikawa T, Tamai S, Tabata S, Okunaga K, et al. Osteogenicdifferentiation of culturedmarrow stromal stem cells on the surface of bioactive glassceramics.JBiomedMaterRes1996;32:341–8.

[24] RahamanMN,DayDE,BalBS,FuQ,JungSB,BonewaldLF,etal.Bioactiveglassintissueengineering.ActaBiomater2011;7:2355–73.

[25] HenchLL,ThompsonI.Twenty‐firstcenturychallengesforbiomaterials.JRSocInterface2010;7Suppl4:S379–91.

[26] ChenQZ,ThompsonID,BoccacciniAR.45S5Bioglass‐derivedglass‐ceramicscaffoldsforbonetissueengineering.Biomaterials2006;27:2414–25.

[27] PeitlFilhoO,LaTorreGP,HenchLL.Effectofcrystallizationonapatite‐layerformationofbioactiveglass45S5.JBiomedMaterRes1996;30:509–14.

[28] HuangW, Day DE, Kittiratanapiboon K, RahamanMN. Kinetics andmechanisms of theconversionofsilicate(45S5),borate,andborosilicateglassestohydroxyapatiteindilutephosphatesolutions.JMaterSciMaterMed2006;17:583–96.

[29] Xynos ID, Hukkanen MVJ, Batten JJ, Buttery LD, Hench LL, Polak JM. Bioglass 45S5StimulatesOsteoblastTurnoverandEnhancesBoneFormationInVitro :ImplicationsandApplicationsforBoneTissueEngineering2000:321–9.

[30] Tsigkou O, Jones JR, Polak JM, Stevens MM. Differentiation of fetal osteoblasts andformation of mineralized bone nodules by 45S5 Bioglass conditioned medium in theabsenceofosteogenicsupplements.Biomaterials2009;30:3542–50.

[31] DayRM,BoccacciniAR, Shurey S, Roether JA, ForbesA,HenchLL, et al. Assessment ofpolyglycolic acid mesh and bioactive glass for soft‐tissue engineering scaffolds.Biomaterials2004;25:5857–66.

REFERENCES

84  

[32] FuQ,RahamanMN,BalBS,BrownRF,DayDE.Mechanicaland invitroperformanceof13‐93bioactive glass scaffoldspreparedby apolymer foam replication technique.ActaBiomater2008;4:1854–64.

[33] Brown RF, Day DE, Day TE, Jung S, RahamanMN, Fu Q. Growth and differentiation ofosteoblastic cells on 13‐93 bioactive glass fibers and scaffolds. Acta Biomater2008;4:387–96.

[34] SiesH.Oxidativestress:Introductoryremarks.In:SiesH,editor.OxidativeStress,London:AcademicPress;1985,p.1–7.

[35] ValkoM,RhodesCJ,MoncolJ,IzakovicM,MazurM.Freeradicals,metalsandantioxidantsinoxidativestress‐inducedcancer.ChemBiolInteract2006;160:1–40.

[36] Li X, Fang P,Mai J, Choi ET,WangH, Yang X. Targetingmitochondrial reactive oxygenspecies as novel therapy for inflammatory diseases and cancers. J Hematol Oncol2013;6:19.

[37] Klaunig JE, Kamendulis LM, Hocevar B a. Oxidative stress and oxidative damage incarcinogenesis.ToxicolPathol2010;38:96–109.

[38] VeskoukisAS,TsatsakisAM,KouretasD.Dietaryoxidativestressandantioxidantdefensewithanemphasisonplantextractadministration.CellStressChaperones2012;17:11–21.

[39] Nordberg J, Arnér ES. Reactive oxygen species, antioxidants, and the mammalianthioredoxinsystem.FreeRadicBiolMed2001;31:1287–312.

[40] Sosa V,Moliné T, Somoza R, Paciucci R, Kondoh H, LLeonart ME. Oxidative stress andcancer:anoverview.AgeingResRev2013;12:376–90.

[41] Fridovich I. Biological effects of the superoxide radical. Arch Biochem Biophys1986;247:1–11.

[42] Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals andantioxidantsinnormalphysiologicalfunctionsandhumandisease.IntJBiochemCellBiol2007;39:44–84.

[43] Gough DR, Cotter TG. Hydrogen peroxide: a Jekyll and Hyde signalling molecule. CellDeathDis2011;2:e213.

[44] Valko M, Morris H, Cronin MTD. Metals, toxicity and oxidative stress. Curr Med Chem2005;12:1161–208.

[45] LloydRV,HannaPM,MasonRP.TheoriginofthehydroxylradicaloxygenintheFentonreaction.FreeRadicBiolMed1997;22:885–8.

[46] Trachootham D, Lu W, Ogasawara M a, Nilsa R‐DV, Huang P. Redox regulation of cellsurvival.AntioxidRedoxSignal2008;10:1343–74.

[47] Ray PD, Huang B‐W, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redoxregulationincellularsignaling.CellSignal2012;24:981–90.

REFERENCES

85  

[48] EatonP.Proteinthioloxidationinhealthanddisease:techniquesformeasuringdisulfidesand related modifications in complex protein mixtures. Free Radic Biol Med2006;40:1889–99.

[49] CollinsY,ChouchaniET,JamesAM,MengerKE,CocheméHM,MurphyMP.Mitochondrialredoxsignallingataglance.JCellSci2012;125:801–6.

[50] D’Autréaux B, Toledano MB. ROS as signalling molecules: mechanisms that generatespecificityinROShomeostasis.NatRevMolCellBiol2007;8:813–24.

[51] MatsuzawaA, IchijoH. Redox control of cell fate byMAP kinase: physiological roles ofASK1‐MAP kinase pathway in stress signaling. BiochimBiophys Acta 2008;1780:1325–36.

[52] NguyenT,NioiP,PickettCB.TheNrf2‐antioxidantresponseelementsignalingpathwayanditsactivationbyoxidativestress.JBiolChem2009;284:13291–5.

[53] BaraibarMa,LiuL,AhmedEK,FriguetB.Proteinoxidativedamageatthecrossroadsofcellular senescence, aging, and age‐related diseases. Oxid Med Cell Longev2012;2012:919832.

[54] Poppek D, Grune T. Proteasomal defense of oxidative protein modifications. AntioxidRedoxSignaln.d.;8:173–84.

[55] Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms,mutation,anddisease.FASEBJ2003;17:1195–214.

[56] Gago‐DominguezM, Jiang X, Castelao JE. Lipid peroxidation, oxidative stress genes anddietaryfactorsinbreastcancerprotection:ahypothesis.BreastCancerRes2007;9:201.

[57] WildburgerR,MrakovcicL,StroserM,AndrisicL,BorovicSunjicS,ZarkovicK,etal.LipidPeroxidationandAge‐AssociatedDiseases‐CauseorConsequence?:Review.TurkiyeKlinJMedSci2009;29:189–93.

[58] NikiE.Lipidperoxidation:physiologicallevelsanddualbiologicaleffects.FreeRadicBiolMed2009;47:469–84.

[59] Guéraud F, Atalay M, Bresgen N, Cipak a, Eckl PM, Huc L, et al. Chemistry andbiochemistryoflipidperoxidationproducts.FreeRadicRes2010;44:1098–124.

[60] Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4‐hydroxynonenal,malonaldehydeandrelatedaldehydes.FreeRadicBiolMed1991;11:81–128.

[61] Negre‐SalvayreA,AugeN,AyalaV,BasagaH,BoadaJ,BrenkeR,etal.Pathologicalaspectsoflipidperoxidation.FreeRadicRes2010;44:1125–71.

[62] BorovicS,CipakA,MeinitzerA,KejlaZ,PerovicD,WaegG,etal.Differentialsensitivityto4‐hydroxynonenalfornormalandmalignantmesenchymalcells.RedoxRep2007;12:50–4.

REFERENCES

86  

[63] MrakovcicL,WildburgerR, JaganjacM,CindricM,CipakA,Borovic‐SunjicS,etal.Lipidperoxidation product 4‐hydroxynonenal as factor of oxidative homeostasis supportingboneregenerationwithbioactiveglasses.ActaBiochimPol2010;57:173–8.

[64] RudićM,MilkovićL,ZarkovićK,Borović‐ŠunjićS,SterkersO,WaegG,etal.TheeffectsofangiotensinIIandtheoxidativestressmediator4‐hydroxynonenalonhumanosteoblast‐likecellgrowth:possiblerelevancetootosclerosis.FreeRadicBiolMed2012;57C:22–8.

[65] UchidaK. 4‐Hydroxy‐2‐nonenal: a product andmediator of oxidative stress. Prog LipidRes2003;42:318–43.

[66] Zarkovic N, Ilic Z, Jurin M, Schaur RJ, Puhl H, Esterbauer H. Stimulation of HeLa cellgrowth by physiological concentrations of 4‐hydroxynonenal. Cell Biochem Funct1993;11:279–86.

[67] ZarkovicN.4‐hydroxynonenalasabioactivemarkerofpathophysiologicalprocesses.MolAspectsMed2003;24:281–91.

[68] Siems W, Grune T. Intracellular metabolism of 4‐hydroxynonenal. Mol Aspects Med2003;24:167–75.

[69] ZarkovicN, CipakA, JaganjacM,Borovic S, ZarkovicK. Pathophysiological relevance ofaldehydicproteinmodifications.JProteomics2013.

[70] Kakishita H, Hattori Y. Vascular smooth muscle cell activation and growth by 4‐hydroxynonenal.LifeSci2001;69:689–97.

[71] BorovicS, Sunjic I,WildburgerR,MittelbachM,WaegG,ZarkovicN.Lipidperoxidationend product as a modulator of cell growth. In: Hayashi T, editor. Prog. Cell GrowthProcessRes.,NewYork:NovaSciencePublishers,Inc.F.Columbus,NewYork,Inc.;2008,p.237.

[72] Allevi P, Anastasia M, Cajone F, Ciuffreda P, Sanvito AM. Structural requirements ofaldehydesproducedinLPOfortheactivationoftheheat‐shockgenesinHeLacells.FreeRadicBiolMed1995;18:107–16.

[73] BarreraG,PizzimentiS,LauroraS,BriatoreF,ToaldoC,DianzaniMU.4‐hydroxynonenalandcellcycle.Biofactors2005;24:151–7.

[74] Kreuzer T, Grube R, Wutte A, Zarkovic N, Schaur RJ. 4‐Hydroxynonenal modifies theeffects of serum growth factors on the expression of the c‐fos proto‐oncogene and theproliferationofHeLacarcinomacells.FreeRadicBiolMed1998;25:42–9.

[75] RuefJ,RaoGN,LiF,BodeC,PattersonC,BhatnagarA,etal.Inductionofrataorticsmoothmuscle cell growth by the lipid peroxidation product 4‐hydroxy‐2‐nonenal. Circulation1998;97:1071–8.

[76] BarreraG,Pizzimenti S, SerraA, FerrettiC, FazioVM, SaglioG, et al. 4‐hydroxynonenalspecifically inhibitsc‐mybbutdoesnotaffectc‐fosexpressions inHL‐60cells.BiochemBiophysResCommun1996;227:589–93.

REFERENCES

87  

[77] Laurora S, Tamagno E, Briatore F, Bardini P, Pizzimenti S, Toaldo C, et al. 4‐Hydroxynonenal modulation of p53 family gene expression in the SK‐N‐BEneuroblastomacellline.FreeRadicBiolMed2005;38:215–25.

[78] ZamaraE,NovoE,MarraF,GentiliniA,RomanelliRG,CaligiuriA,etal.4‐Hydroxynonenalasaselectivepro‐fibrogenicstimulusforactivatedhumanhepaticstellatecells.JHepatol2004;40:60–8.

[79] Zanetti D, Poli G, Vizio B, Zingaro B, Chiarpotto E, Biasi F. 4‐Hydroxynonenal andtransforming growth factor‐β1 expression in colon cancer. Mol Aspects Med2003;24:273–80.

[80] Zarkovic N, Cipak A, Cindric M, Waeg G, Borovic Sunjic S, Mrakovcic L, et al. 4‐Hydroxynonenal‐protein adducts as biomarkers of oxidative stress, lipid peroxidationandoxidativehomeostasis.FreeRadicals,HealLifestyle2009:37–45.

[81] Kreuzer T, Grube R, Wutte A, Zarkovic N, Schaur RJ. 4‐Hydroxynonenal modifies theeffects of serum growth factors on the expression of the c‐fos proto‐oncogene and theproliferationofHeLacarcinomacells.FreeRadicBiolMed1998;25:42–9.

[82] Lushchak VI. Glutathione homeostasis and functions: potential targets for medicalinterventions.JAminoAcids2012;2012:736837.

[83] AwasthiS,SinghalSS,AwasthiYC,MartinB,WooJ‐H,CunninghamCC,etal.RLIP76andCancer.ClinCancerRes2008;14:4372–7.

[84] RamanaKV,BhatnagarA,SrivastavaS,YadavUC,AwasthiS,AwasthiYC,etal.Mitogenicresponses of vascular smooth muscle cells to lipid peroxidation‐derived aldehyde 4‐hydroxy‐trans‐2‐nonenal(HNE):roleofaldosereductase‐catalyzedreductionoftheHNE‐glutathioneconjugatesinregulatingcellgrowth.JBiolChem2006;281:17652–60.

[85] Leonarduzzi G, Robbesyn F, Poli G. Signaling kinasesmodulated by 4‐hydroxynonenal.FreeRadicBiolMed2004;37:1694–702.

[86] WildburgerR,ZarkovićN,EggerG,PetekW,Meinitzera,BorovićS,etal.Comparisonofthevaluesofbasicfibroblastgrowthfactordeterminedbyanimmunoassayintheseraofpatients with traumatic brain injury and enhanced osteogenesis and the effects of thesameseraonthefibroblastgrowthinvitro.EurJClinChemClinBiochem1995;33:693–8.

[87] WildburgerR,ZarkovicN,LebG,BorovicS,ZarkovicK,TatzberF.Post‐traumaticchangesininsulin‐likegrowthfactortype1andgrowthhormoneinpatientswithbonefracturesandtraumaticbraininjury.WienKlinWochenschr2001;113:119–26.

[88] WildburgerR,Borović S, ZarkovićN,Tatzber F. Post‐traumaticdynamic changes in theantibody titer against oxidized low density lipoproteins. Wien Klin Wochenschr2000;112:798–803.

[89] Sunjic SB, CipakA,RabuzinF,WildburgerR, ZarkovicN.The influenceof4‐hydroxy‐2‐nonenal on proliferation, differentiation and apoptosis of human osteosarcoma cells.Biofactors2005;24:141–8.

REFERENCES

88  

[90] GerhardtL‐C,BoccacciniAR.BioactiveGlassandGlass‐CeramicScaffoldsforBoneTissueEngineering.Materials(Basel)2010;3:3867–910.

[91] Hayashi T, Shishido N, Nakayama K, Nunomura A, Smith M a, Perry G, et al. Lipidperoxidation and 4‐hydroxy‐2‐nonenal formationby copper ionbound to amyloid‐betapeptide.FreeRadicBiolMed2007;43:1552–9.

[92] Waeg G, Dimsity G, Esterbauer H. Monoclonal antibodies for detection of 4‐hydroxynonenalmodifiedproteins.FreeRadicRes1996;25:149–59.

[93] Zarkovic K, Zarkovic N, Schlag G, Redl H. Histological Aspects of Sepsis Induced BrainChangesinaBaboonModel. In:SchlagG,RedlH,TraberD,editors.Shock.SepsisOrganFail.BrainDamageSecond. toHemorrhagic‐TraumaticShock.SepsisTrauma.Brain Inj.,Heilderberg:Springer‐Verlag;1997,p.146–68.

[94] Milkovic L, Hoppe A, Detsch R, Boccaccini AR, Zarkovic N. Effects of Cu‐doped 45S5bioactiveglassonthelipidperoxidation‐associatedgrowthofhumanosteoblast‐likecellsinvitro.JBiomedMaterResA2013.

[95] BradfordMM.Arapidandsensitivemethodforthequantitationofmicrogramquantitiesofproteinutilizingtheprincipleofprotein‐dyebinding.AnalBiochem1976;72:248–54.

[96] Jørgensen P,Milkovic L, Zarkovic N,Waeg G, Rattan SIS. Lipid peroxidation‐derived 4‐hydroxynonenal‐modified proteins accumulate in human facial skin fibroblasts duringageinginvitro.Biogerontology2014;15:105–10.

[97] GartlandA,BuckleyKA,DillonJP,CurranJM,HuntJA,GallagherJA.IsolationandCultureofHumanOsteoblasts.In:PicotJ,editor.Hum.CellCult.Protoc.(fromMethodsMol.Med.Ser.,HumanaPress;2005,p.29–54.

[98] CicioneC,Muiños‐LópezE,Hermida‐GómezT,Fuentes‐BoqueteI,Díaz‐PradoS,BlancoFJ.Effects of severe hypoxia on bone marrow mesenchymal stem cells differentiationpotential.StemCellsInt2013;2013:232896.

[99] SchneiderS,YochimJ,BrabenderJ,UchidaK,DanenbergKD,MetzgerR,etal.Osteopontinbut not osteonectin messenger RNA expression is a prognostic marker in curativelyresectednon‐smallcelllungcancer.ClinCancerRes2004;10:1588–96.

[100] ZhangY,BaY,LiuC,SunG,DingL,GaoS,etal.PGC‐1alphainducesapoptosisinhumanepithelial ovarian cancer cells through a PPARgamma‐dependent pathway. Cell Res2007;17:363–73.

[101] PillonNJ, Soulage CO. Lipid Peroxidation by‐Products and theMetabolic Syndrome. In:CatalaA,editor.Biochem.Genet.Mol.Biol.»“LipidPeroxidation,”InTech;2012,p.409–36.

[102] BedardK, KrauseK‐H. TheNOX family of ROS‐generatingNADPH oxidases: physiologyandpathophysiology.PhysiolRev2007;87:245–313.

[103] KamataH,HirataH.Redoxregulationofcellularsignalling.CellSignal1999;11:1–14.

REFERENCES

89  

[104] ChenKC‐W,ZhouY,ZhangW,LouMF.ControlofPDGF‐inducedreactiveoxygenspecies(ROS) generation and signal transduction in human lens epithelial cells. Mol Vis2007;13:374–87.

[105] Henrotin Y. The role of reactive oxygen species in homeostasis and degradation ofcartilage.OsteoarthrCartil2003;11:747–55.

[106] HenchLL.ThestoryofBioglass.JMaterSciMaterMed2006;17:967–78.

[107] Ralay Ranaivo H, Hodge JN, Choi N, Wainwright MS. Albumin induces upregulation ofmatrix metalloproteinase‐9 in astrocytes via MAPK and reactive oxygen species‐dependentpathways.JNeuroinflammation2012;9:68.

[108] Allen R., Tresini M. Oxidative stress and gene regulation. Free Radic Biol Med2000;28:463–99.

[109] YanJ,HalesBF.DepletionofGlutathioneInduces4‐HydroxynonenalProteinAdductsandHydroxyureaTeratogenicity in theOrganogenesis StageMouseEmbryo 2006;319:613–21.

[110] AwasthiYC,AnsariGAS,Awasthi S.Regulationof4‐hydroxynonenalmediated signalingbyglutathioneS‐transferases.MethodsEnzymol2005;401:379–407.

[111] UemuraT,NemotoA,LiuY,KojimaH,DongJ,YabeT,etal.Osteopontininvolvementinboneremodelinganditseffectsoninvivoosteogenicpotentialofbonemarrow‐derivedosteoblasts/poroushydroxyapatiteconstructs.MaterSciEngC2001;17:33–6.

[112] Deschaseaux F, Sensébé L, Heymann D. Mechanisms of bone repair and regeneration.TrendsMolMed2009;15:417–29.

[113] Hoppe A, Güldal NS, Boccaccini AR. A review of the biological response to ionicdissolution products from bioactive glasses and glass‐ceramics. Biomaterials2011;32:2757–74.

[114] Mattson MP, Meffert MK. Roles for NF‐kappaB in nerve cell survival, plasticity, anddisease.CellDeathDiffer2006;13:852–60.

[115] LynchSM,FreiB.Mechanismsof copper‐ and iron‐dependentoxidativemodificationofhumanlowdensitylipoprotein.JLipidRes1993;34:1745–53.

[116] Gaetke L. Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology2003;189:147–63.

[117] Awasthi YC, Yang Y, Tiwari NK, Patrick B, Sharma A, Li J, et al. Regulation of 4‐hydroxynonenal‐mediated signaling by glutathione S‐transferases. Free Radic BiolMed2004;37:607–19.

[118] MattsonMP.Hormesisdefined.AgeingResRev2008;7:1–7.

REFERENCES

90  

[119] Siow RCM, Ishii T, Mann GE. Modulation of antioxidant gene expression by 4‐hydroxynonenal: atheroprotective role of the Nrf2/ARE transcription pathway. RedoxRep2007;12:11–5.

[120] ParolaM,BellomoG,RobinoG,BarreraG,DianzaniMU.4‐Hydroxynonenalasabiologicalsignal: molecular basis and pathophysiological implications. Antioxid Redox Signal1999;1:255–84.

[121] CalabreseV,CorneliusC,Dinkova‐KostovaAT,CalabreseEJ,MattsonMP.Cellularstressresponses, the hormesis paradigm, and vitagenes: novel targets for therapeuticinterventioninneurodegenerativedisorders.AntioxidRedoxSignal2010;13:1763–811.

 

91  

7. SUMMARY

SUMMARY

92  

7. SUMMARY

Complexbonefracturesrequiresurgicalinterventionanduseofbonegraftsinorderto

aidproperhealing.Thebioactiveglasses(BGs)aresyntheticbonegraftswithosteoinductiveand

osteoconductiveproperties,which stimulate thebonehealingprocess.While theprinciplesof

BGactionsareyetnotcompletelyunderstood,norarethecomplexprocessesoffracturerepair,

continuous research is attempting to improve the BG properties with addition of bioactive

molecules,orbyincorporationofspecifictherapeuticions.

Our previous research has shown that lipid peroxidation (LPO), and especially its

product and mediator 4‐hydroxynonenal (HNE), known as signalling molecule in the

proliferation,differentiationandapoptosisofcells,cancontrolthegrowthofbonecells.Theaim

ofthisstudywastoinvestigatetheroleofLPOinthegrowthofbonecellsontheBGs.

The obtained findings of an involvement of lipid peroxidation in the process of enhanced

osteogenesis assisted with the BGs will lead to improved understanding of the principles

governing the bone repair, which may result in creation of new, improved biomaterials and

applicationsfortheenhancedregenerationofthebonetissue.

 

93  

8. SAŽETAK

SAŽETAK

94  

8. SAŽETAK

Frakture kostiju, osobito one dugih kostiju, velikih zglobova te višestruke frakture,

predstavljaju značajan medicinski, ali i socijalni problem zbog dugotrajnog oporavka i

mogućnosti invaliditeta, koje je posebice naglašeno kod starijih ljudi. Veliki dio tih fraktura

zahtjevakirurškuintervencijutekorištenjenekihodkoštanihusadakakojiomogućujupotporu,

ispunjavajunastaluprazninuipotičubiološkocijeljenjenastalogdefekta.Zbogtogajedanašnja

regenerativnamedicinausmjerenakrazvojuinženjerstvatkivakosti,pričemujevelikinaglasak

pridan biomaterijalima. Kao jedni od atraktivnih predstavnika biomaterijala, pojavila su se

bioaktivna stakla koja imajupogodnupovršinukojapotiče rast stanica i stvaranjenove kosti,

odnosno posjeduju osteokonduktivna i osteoinduktivna svojstva. Obzirom da tijekom procesa

cijeljenja frakturebitnuulogu igraju imolekularnimehanizmipotaknutiodređenimsignalnim

molekulama, citokinima i faktorima rasta, današnja istraživanja u tkivnom inženjerstvu

usmjerena su k poboljšanju svojstava postojećih bioaktivnih stakala tako da ih kombiniraju s

bioaktivnim molekulama ili im se dodaju metalni ioni, kao na primjer bakar (Cu), koji će se

postepenootpuštatiuokolinidefektaitakododatnostvaratiterapeutskiučinak.

Prijašnjaistraživanjaotkrilasuvezuizmeđusistemskogodgovorapacijenatastraumatskom

ozljedom glave i fenomena ubrzane osteogeneze fraktura kostiju gdje je kao bitan parametar

definirana lipidna peroksidacija (LPO). Jedan od produkata LPO, 4‐hidroksinonenal (HNE)

prvotnosmatrankaotoksičnainepoželjnamolekulavezanauzrazličitapatološkastanja,danas

je poznat i po svojoj fiziološkoj ulozi djelujući kao signalna molekula. HNE, ovisno o

koncentraciji,usmjeravastanicekproliferaciji,diferencijaciji iliusmrtapoptozom.Stogajecilj

ovogradabiorazjasnitikojuuloguimajuLPOilipidnaperoksidacijakodpoboljšaneosteogeneze

prirastustanicanaBS.

Kakobisetoispitalonapravljenajeimunocitokemijskaanalizakojajeukazalanajednujako

zanimljivučinjenicu, a to jeda stanice stvarajuendogeniHNEkad rastunaBS.Uzevšiuobzir

novo saznanje, a i činjenicu da je danas veliki dio istraživanja usmjeren ka pronalasku novih

načinaimolekulakojećepoboljšatipostojećasvojstvaBS,uradusekrenulokafunkcionalizaciji

BS,45S5i13‐93salbuminom(o,5mg/ml),fiziološkomkoncentracijomHNEa(2,5μM)odnosno

njihovomkombinacijom.

Dobivenirezultatisuukazalidakorištenitretmaninisutoksičnitedajelipidnaperoksidacija

uključena u rast stanica na BS. Nadalje pokazano je poticajno djelovanje HNEa na rast i

SAŽETAK

95  

diferencijacijustanicaterazjašnjenodajep44/p42(ERK1/2)MAPKsignalniputaktiviran,aput

p38nije.

Drugidioispitivanjajenapravljennabiostaklu45S5sdodatkombakra(Cu;0.1,1i2.5%))te

jepotvrđenaulogalipidneperoksidacijeuprocesurastastanicanabioaktivnomstaklustimeda

jekodstakalas1i2.5%Cuonabilanegativnatj.tekoncentracijesusepokazaletoksičnimadok

jekodBSs0.1%Cuonastimuliralastanicenarast.

StogamožemozaključitidalipidnaperoksidacijaigrabitnuuloguurastustanicanaBStebi

dobiveni rezultatimogli poslužiti u stvaranju novih, poboljšanih biomaterijala i postupaka za

poticanjeregeneracijetkivakosti.

 

96  

9. CURRICULUMVITAE

CURRICULUMVITAE

97  

9. CURRICULUMVITAE

 

Personalinformation

Nameandsurname: LidijaMilković

Academictitle: Dipl.ing.ofbiotechnology;Scientificnovice(PhDstudent)

Address: RudjerBoskovicInstitute,LaboratoryforOxidativeStress

Bijenickacesta54,10000Zagreb

Phone: +38514571212

E‐mail: lidija.milkovic@irb.hr

Citizenship: Croatian

Dateandplaceofbirth: 27.11.1977.Zagreb,Croatia

Workexperience

Since1stofJune,2008.‐ Scientificnovice,PhDstudentintheLaboratoryforoxidative

stressattheRudjerBoskovicInstitute

2004.–2008. R&DassociateinKvasacLtd.

1996.‐1997. Laboratorytechnicianinthelaboratoryformicrobiologyatthe

ClinicalHospital"Dr.FranMihaljevic"

Education

2008.‐2014. Specialist graduate professional study Project management,

joinedstudyofRudjerBoskovicInstituteandGraduateschoolfor

businessandmanagement''BaltazarAdamKrcelic''

2007.‐ UniversityPostgraduateInterdisciplinaryDoctoralStudyof

MolecularBiosciences;JosipJurajStrossmayerUniversityof

Osijek,UniversityofDubrovnikandRudjerBoskovicInstitute

inZagreb

1997.–2003. UniversityofZagreb,FacultyofFoodTechnologyand

Biotechnology;Study:Biotechnology‐Biochemicalengineering

CURRICULUMVITAE

98  

Researchandotherprojects

2013.–2014. ResearcherontheCroatian‐Germanbilateralproject:“Theroleof

lipidperoxidationproductsingrowthregulationofthebonecells

stimulatedbythenoveltypesofCu‐doped45S5bioactiveglass”

2012.–2013. Researcheron theCroatian‐Austrianbilateral project: “Effects of

oxidativelymodified collagen on breast cancer stem cell growth

anddifferentiation”

2012. ResearcherontheCroatian‐Germanbilateralproject:“Effectof

novel Cu‐bioglass and lipid peroxidation products on bone

regeneration”

2010.–2011. ResearcherontheCroatian‐Slovenianbilateralproject:

“Improvementof adhesivepropertiesofbiomedicalmaterialsby

plasmatreatment”

2007.‐2008. Researcher on the Croatian‐French bilateral project: “The use of

novel, genuine assays for lipid peroxidation evaluation in

experimentalcarcinogenesis”

2006.‐2008. Researcheronaproject„Novelmethodtosupportbonegrowthby

bioactive glass coatedwith protein/lipid peroxidation products“

supported by Austrian bank Jubiläumsfonds der

OesterreichischenNationalbank.

Membershipinscienceorganizationsandbodies

CroatianSocietyforCancerResearch(HDIR),

TheEuropeanAssociationforCancerResearch(EACR)

TheInternationalHNE‐Club

COSTCM1106StemchemAction

Publications

1. Milkovic,Lidija;Hoppe,Alexander;Detsch,Rainer;Boccaccini,AldoR.;Zarkovic,Neven.

EffectsofCu‐doped45S5bioactiveglassonthelipidperoxidation‐associatedgrowthof

human osteoblast‐like cells in vitro. Journal of biomedical materials research. Part A.

(2013)inpress.

CURRICULUMVITAE

99  

2. Jaganjac,M;Vesel,A;Milkovic,L;Recek,N;Kolar,M;Zarkovic,N;Latiff,A;Kleinschek,

K.‐S; Mozetic, M. Oxygen‐rich coating promotes binding of proteins and

endothelialization of polyethylene terephthalate polymers. Journal of biomedical

materialsresearch.PartA.(2013)inpress.

3. Jørgensen, Peter;Milkovic, Lidija; Zarkovic, Neven; Waeg, Georg; Rattan, Suresh I.S.

Lipid peroxidation‐derived 4‐hydroxynonenal‐modified proteins accumulate in human

facial skin fibroblasts during ageing in vitro. Biogerontology (Dordrecht). (2013) in

press.

4. Weber,D*;Milkovic,L*;Bennett,S.J;Griffiths,H.R;Zarkovic,N;Grune,T.Measurement

of HNE‐protein adducts in human plasma and serum by ELISA‐Comparison of two

primaryantibodies.RedoxBiology.1(2013);226‐233.

5. Cindrić,Marina;Čipak,Ana;Zapletal,Emilija;Jaganjac,Morana;Milković,Lidija;Waeg,

G.;Stolc,S.;Žarković,Neven;BorovićŠunjić,Suzana.Stobadineattenuatesimpairmentof

anintestinalbarriermodelcausedby4‐hydroxynonenal.Toxicologyinvitro.27(2013),

1;426‐432.

6. Recek, Nina; Jaganjac, Morana; Kolar, Metod;Milkovic,Lidija; Mozetič, Miran; Stana‐

Kleinschek, Karin; Vesel, Alenka. Protein Adsorption on Various Plasma‐Treated

PolyethyleneTerephthalateSubstrates.Molecules.18(2013),10;12441‐12463.

7. Recek, N; Mozetič, M; Jaganjac, M; Milković, L; Žarković, N; Vesel, A. Improved

proliferation of human osteosarcoma cells on oxygen plasma treated polystyrene.

Vacuum.98(2013);116‐121.

8. Rudić,M;Milković,L; Žarković, K; Borović‐Šunjić, S; Sterkers, O;Waeg, G; Ferrary, E;

Bozorg Grayeli, A; Žarković, N. The effects of angiotensin II and the oxidative stress

mediator4‐hydroxynonenalonhumanosteoblast‐likecellgrowth:Possiblerelevanceto

otosclerosis.Freeradicalbiology&medicine.57(2013);22‐28.

9. Jaganjac, Morana; Prah, Iva Ozana; Čipak, Ana; Cindrić, Marina; Mrakovčić, Lidija;

Tatzber, Franz; Ilinčić, Petar;Rukavina, Vinko; Spehar, Branka; ParlovVuković, Jelena;

Telen,Sanda;Uchida,Koji;Lulić,Zoran;Žarković,Neven.EffectsofBioreactiveAcrolein

fromAutomotiveExhaustGasesonHumanCells InVitro.Environmentaltoxicology.27

(2012),11;644‐652.

10. Rudić, Milan; Nguyen, C.; Nguyen, Y.;Milković, Lidija; Žarković, Neven; Sterkers, O.;

Ferrary, E.; Grayeli, A.B. Effect of Angiotensin II on Inflammation Pathways in Human

PrimaryBoneCellCulturesinOtosclerosis.Audiologyandneuro‐otology.17(2012),3;

169‐178.

CURRICULUMVITAE

100  

11. Jaganjac,Morana;Milković,Lidija;ČipakGašparović,Ana;Mozetič,Miran;Recek,Nina;

Žarković, Neven; Vesel, Alenka. Cell adhesion on hydrophobic polymer surfaces.

MaterialiinTehnologije.46(2012);53‐56.

12. Lovaković,Tomislava;Poljak‐Blaži,Marija;Duburs,Gunars;Čipak,Ana;Cindrić,Marina;

Vigante, Brigita; Bisenieks, Egils; Jaganjac, Morana; Mrakovčić, Lidija; Dedić, Azra;

Žarković,Neven.Growthmodulationofhumancellsinvitrobymildoxidativestressand

1, 4‐dihydropyridine derivative antioxidants. Collegium antropologicum. 35 (2011), 1;

137‐141.

13. Vesel,Alenka;Mozetič,Miran;Jaganjac,Morana;Milković,Lidija;ČipakGašparović,Ana;

Žarković, Neven. Biocompatibility of oxygen‐plasma‐treated polystyrene substrates.

Europeanphysicaljournal‐appliedphysics.56(2011),2;24024‐1‐24024‐4.

14. Breusing, Nicolle; Grune, Tilman; Andrišić, Luka; Atalay, Mustafa; Bartosz, Grzegorz;

Biasi, Fiorella; Borović, Suzana; Bravo, Laura; Casals, Isidre; Casillas, Rosario;

Dinischiotu, Anca; Drzewinska, Joanna; Faber, Heidemarie; Mohd Fauzi, Norsyahida;

Gajewska,Agnieszka;Gambini,Juan;Gradinaru,Daniela;Kokkola,Tarja;Lojek,Antonin;

Luczaj,Wojciech;Margina,Denisa;Mascia, Cinzia;Mateos, Raquel;Meinitzer, Andreas;

Mitjavila, María Teresa; Mrakovčić, Lidija; Munteanu, Maria Cristina; Podborska,

Martina;Poli,Giuseppe;Sicinska,Paulina;Skrzydlewska,Elzbieta;Vina,Jose;Wiswedel,

Ingrid; Žarković, Neven; Zelzer, Sieglinde; Spickett, Corinne M. An inter‐laboratory

validationofmethodsoflipidperoxidationmeasurementinUVA‐treatedhumanplasma

samples.Freeradicalresearch.44(2010),10;1203‐1215

15. Cindrić, Marina; Čipak, Ana; Serly, Julianna; Plotniece, Aiva; Jaganjac, Morana;

Mrakovčić, Lidija; Lovaković, Tomislava; Dedić, Azra; Soldo, Ivo; Duburs, Gunars;

Žarković,Neven;Molnár,József.ReversalofMultidrugResistanceinMurineLymphoma

Cells by Amphiphilic Dihydropyridine Antioxidant Derivative. Anticancer research. 30

(2010),10;4063‐4070.

16. Mrakovčić,Lidija;Wildburger, Renate; Jaganjac,Morana; Cindrić,Marina; Čipak, Ana;

BorovićŠunjić,Suzana;Waeg,Georg;MogusMilanković,Andrea;Žarković,Neven.Lipid

peroxidation product 4‐hydroxynonenal as factor of oxidative homeostasis supporting

bone regenerationwithbioactiveglasses.ActabiochimicaPolonica.57 (2010),2; 173‐

178.

17. Čipak,Ana;Mrakovčić,Lidija;Ciz,Milan;Lojek,Antonin;Mihaylova,Boryana;Goshev,

Ivan; Jaganjac,Morana;Cindrić,Marina;Sitić, Sanda;Margaritoni,Marko;Waeg,Georg;

Balić, Marija; Žarković, Neven. Growth suppression of human breast carcinoma stem

CURRICULUMVITAE

101  

cellsby lipidperoxidationproduct4‐hydroxy‐2‐nonenalandhydroxyl radical‐modified

collagen.ActabiochimicaPolonica.57(2010),2;165‐171.

18. Jaganjac, Morana; Matijević, Tanja; Cindrić, Marina; Čipak, Ana; Mrakovčić, Lidija;

Gubisch, Wolfgang; Žarković, Neven. Induction of CMV‐1 promoter by 4‐Hydroxy‐2‐

nonenalinhumanembryonickidneycellsinvitro.ActabiochimicaPolonica.57(2010),

2;179‐183.

19. Mrakovčić, Lidija; Cindrić, Marina; Žarković, Neven; Borović Šunjic, Suzana; Moguš

Milanković, Andrea; Wildburger, Renate. In vitro model of bone regeneration with

bioactive glass and lipid peroxidation. Clinical and Experimental Medical Journal. 4

(2010);73‐78.

20. Čipak, Ana; Borović, Suzana; Jaganjac, Morana; Bresgen, Nikolaus; Kirac, Iva; Grbeša,

Ivana;Mrakovčić,Lidija;Cindrić,Marina;Šćukanec‐Špoljar,Mira;Gall‐Trošelj,Koraljka;

Ćorić,Marijana;Eckl,Peter;Žarković,Neven.Influenceof4‐hydroxynonenalandspleen

cells on primary hepatocyte culture and a novel liver‐derived cell line resembling

hepatocytestemcells.ActabiochimicaPolonica.57(2010),2;185‐191.

21. Cindrić, Marina; Čipak, Ana;Mrakovčić, Lidija; Žarković, Kamelija; Borović, Suzana;

Wilburger,Renate;Žarković,Neven.Homeostasis, Stress&Aging. TürkiyeKlinikleri=

JournalofMedicalSciences.29(2009),5‐S1;S3‐S5.

22. Wildburger,Renate;Mrakovčić,Lidija;Štroser,Marina;Andrišić,Luka;BorovićŠunjić,

Suzana;Žarković,Kamelija;Žarković,Neven.PeroxidationandAge‐AssociatedDiseases‐

CauseorConsequence?.TürkiyeKlinikleri=JournalofMedicalSciences.29(2009)189‐

193.