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JosipJurajStrossmayerUniversityofOsijek
RuđerBoškovićInstitute,Zagreb
UniversityofDubrovnik
UniversityPostgraduateInterdisciplinaryDoctoralStudy
ofMolecularBiosciences
LidijaMilković
Beneficialeffectsoflipidperoxidationinthebonecellgrowth
onbioactiveglass‐Newperspectivesintissueengineering
andregenerativemedicine
PhDTHESIS
Osijek,2014.
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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
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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
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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.)
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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.
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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
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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
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3
6. REFERENCES........................................................................................................................................82
7. SUMMARY.............................................................................................................................................92
8. SAŽETAK...............................................................................................................................................94
9. CURRICULUMVITAE..........................................................................................................................97
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1. INTRODUCTION
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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)
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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
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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
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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/)
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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‐
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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.
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INTRODUCTION
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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].
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INTRODUCTION
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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.
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INTRODUCTION
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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
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INTRODUCTION
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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
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INTRODUCTION
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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.
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INTRODUCTION
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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].
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INTRODUCTION
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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.
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INTRODUCTION
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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.
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INTRODUCTION
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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].
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INTRODUCTION
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Figure5.Molecularmechanismofreactiveoxygenspecies(ROS)formation,involvementof
antioxidativedefenseandimpactofROSoncellularmacromolecules
ETC=electrontransportchain
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INTRODUCTION
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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
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INTRODUCTION
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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].
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INTRODUCTION
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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]
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INTRODUCTION
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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).
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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
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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.
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27
2. MATERIALSANDMETHODS
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MATERIALSANDMETHODS
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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
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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
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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.
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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)
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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.
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MATERIALSANDMETHODS
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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.
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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
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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
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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
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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
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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
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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.
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40
3. RESULTS
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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.
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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
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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
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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.
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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.
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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.
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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
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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).
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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
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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).
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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
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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)
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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.
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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)
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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
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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)
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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.
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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)
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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)
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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.
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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.
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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)
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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
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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).
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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).
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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.
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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
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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.
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69
4. DISCUSSION
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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,
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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
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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‐
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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].
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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].
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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.
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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‐
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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.
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78
5. CONCLUSIONS
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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.
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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.
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81
6. REFERENCES
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[94] Milkovic L, Hoppe A, Detsch R, Boccaccini AR, Zarkovic N. Effects of Cu‐doped 45S5bioactiveglassonthelipidperoxidation‐associatedgrowthofhumanosteoblast‐likecellsinvitro.JBiomedMaterResA2013.
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[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.
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7. SUMMARY
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SUMMARY
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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.
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8. SAŽETAK
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SAŽETAK
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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
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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.
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9. CURRICULUMVITAE
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CURRICULUMVITAE
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9. CURRICULUMVITAE
Personalinformation
Nameandsurname: LidijaMilković
Academictitle: Dipl.ing.ofbiotechnology;Scientificnovice(PhDstudent)
Address: RudjerBoskovicInstitute,LaboratoryforOxidativeStress
Bijenickacesta54,10000Zagreb
Phone: +38514571212
E‐mail: [email protected]
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
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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.
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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
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