Reichert Et Al 2009_Modulation of Osteogenic Cell Morphology

8/12/2019 Reichert Et Al 2009_Modulation of Osteogenic Cell Morphology

1/14

Acta Stomatol Croat. 2009;43(3):188-201.

IZVORNI ZNANSTVENI RADORIGINAL SCIENTIFIC PAPER

Christoph Reichert 1, Marcus Klein 2, Adrian Kasaj 3, Sebastian Kuhn 4, Hermann Gtz 5, Gerhard Hommel 6,Heinz Duschner 5, Bilal Al-Nawas 2

Modulacija morfologije osteogenih stanicapomou ECM liganada i derivata caklinskogmatriksa

Modulation of Osteogenic Cell Morphology byECM Ligands and Enamel Matrix Derivative

Zaprimljen: 22. lipanj 2009.Prihvaen: 24. srpanj 2009.

Adresa za dopisivanjeOA. priv. doc. dr.med.dent. Adrian KasajDepartment of Operative Dentistry andPeriodontology Johannes Gutenberg-UniversityAugustusplatz 2, 55131 MainzTel.: 0049/6131/173064Faks: 0049/6131/[email protected]

Saetak Preduvjet za uspjenu regeneraciju parodontnog tkiva jest odgovarajua aktivacijapopulacije za to odgovornih stanica, poput osteoblasta. U tom su sluaju staninaadhezija i sazrijevanje usko povezani s morfologijom stanica i f-aktinskom organi-zacijom citoskeleta. Mogunost pojaavanja parodontalnog cijeljenja pomou po -jedinih komponenti izvanstaninog matriksa (ECM) i derivata caklinskog matriksadobro je dokumentirana u literaturi. Svrha rada : Svrha ovog istraivanja bila je te -stirati uinak ECM-bjelanevina, kolagena tipa I, laminina-1 te komercijalnog proi -zvoda EMD-a na morfoloku i citoskeletnu organizaciju osteogenih stanica. Ispita -nici i postupci: Tijekom promatranja analizirano je ukupno 2450 osteogenih stanicaiz pet razliitih staninih linija (etiri primarnih i jedne komercijalne) kultiviranih napojedinim supstratima, a analizirali su ih tri neovisna promatraa. Nakon bojenja zaf-aktin staninog citoskeleta i automatizirane CLSM-vizualizacije, stanice su podije -ljene u tri skupine ovisno o njihovim morfolokim svojstvima (nezrele, prijelazne izrele). Osim deskriptivne analize obavljena je bila i multivarijantna logika regresi -ja radi identi ciranja odgovarajuih parametara koji utjeu na staninu morfologijui organizaciju citoskeleta. Rabljeni pojedinani ligandi kolagena i laminina te pose -bice EMD poticali su stvaranje zrelog osteogenog fenotipa, premda su bile uoenei odreene razlike meu koritenim staninim linijama. Rezultati: Analiza morfolo-gije i citoskeleta pouzdan je nain skupljanja prvih podataka o biokompatibilnosti ibioaktivaciji stanica na razliitim supstratima. Nai rezultati upuuju na mogui po -tencijal istraivanih liganada u pojaavanju osteogenoga staninog privrstka i sa -

zrijevanju te time i pomaganju cijeljenja parodontnog tkiva.

Kljune rijeiosteoblasti; citoskelet; parodontnebolesti; izvanstanini matriks; caklinski

matriks, proteini

1 Ortodontska klinika Sveuilita Friedrich-Wilhelm, NjemakaRheinische Friedrich-Wilhelms-University, Department of Orthodontics

2 Oralna i maksilofacijalna klinika Sveuilita Johannes Gutenberg, Mainz, Njemaka Johannes Gutenberg-University Mainz, Department of Oral and Maxillofacial Surgery

3 Kirurka i parodontoloka klinika Sveuilita Johannes Gutenberg, Mainz, Njemaka Johannes Gutenberg-University Mainz, Department of Operative Dentistry and Periodontology

4 Traumatoloka klinika Sveuilita Johannes Gutenberg, Mainz, Njemaka Johannes Gutenberg University, Department of Trauma Surgery5 Institut za mikroanalizu Sveuilita Johannes Gutenberg, Mainz, Njemaka Johannes Gutenberg-University Mainz, Institute of Applied Structure- and Microanalysis

6 Institut za medicinsku biostatiku, epidemiologiju i informatiku (IMBEI) Sveuilita Johannes Gutenberg, Mainz, Njemaka Johannes Gutenberg-University Mainz, Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI)

ACTASTOMATOLOGICACROATICAwww.ascro.hr


2/14

Reichert et al. Modulation of osteogenic cell morphology 189

Uvod

Uspjena regeneracija intraoralnih kotanih i pa-rodontnih defekata zahtijeva reakciju tkiva domai-na s regrutiranjem i aktivacijom (i zatitom) odgo-varajuih susjednih populacija stanica. Za stvaranjenovoga mineraliziranog kotanog tkiva i reorganiza-ciju potpornih tkiva zuba nune su osteogene stani-ce (1). U tom su kontekstu postavljeni temelji tera-pijskih strategija voene regeneracije tkiva (GTR-a)(2-5). Ako se koristimo aloplastinim biomaterija-lima kao membranama ili zamjenama za kost, onemoraju zadovoljavati suene kriterije, u prvom redubiokompatibilnost. Osim toga, potrebna je nekakvavrsta bio-aktivacije kako bi se potaknulo sazrijeva-nje. Kao mogui rizini imbenici za GTR identi-cirana je nedovoljna integracija tkiva i/ili bakterij-

ska kolonizacija aloplastinih materijala (6).Preduvjet za regeneraciju tkiva dobro su urav-

noteene interakcije stanica i izvanstaninog ma-triksa (ECM-a). Kod kotanog tkiva sastav ECM-aima vanu regulacijsku zadau za osteogene stani-ce poput adhezije, pokretljivosti, umnaanja i sazrijevanja (7-9). Osim neizostavnog kolagena, lamini-ni kao sastavni dijelovi bazalne membrane, imajudodatnu vanu zadau u kotanom cijeljenju (1, 8,10, 11).

Premda proiene kotano morfogenetske bje-lanevine nisu prihvaene u svakodnevnoj rutinskojparodontnoj terapiji, koritenje derivata caklinskogamatriksa (EMD-a) je kao terapijska mogunost u li-teraturi dobro dokumentirana (4, 12 18). Biolokiaktivni sastojci identi cirani u caklinskom matrik-su sastoje se od niza bjelanevina poput ameloge-nina, tufelina, ameloblastina i enamelina te takoerproteaze koje su prije toga imale ulogu u razvojuzuba i potpornih tkiva (19-23). Jedini ECM koji jedostupan na tritu jest svinjski Emdogain (Stra-umann, vicarska). Emdogain je kiseli ekstrakt

svinjskoga caklinskog matriksa i sadrava uglav-nom amelogeninsku frakciju. Uporabom te biomi-metiki aktivne bjelanevine ustanovljena su, i kli-niki i radioloki, dugorona poboljanja klinikogstanja, poput smanjenja kotanih defekata, dubinesondiranja i poboljanja razine privrtska (4, 14, 18,24). Na ivotinjskim modelima uoeno je pobolj-anje parodontne regeneracije, osteogeneza i angi-ogeneza (25, 26). Nedavno tiskan revijalni lanakna staninoj je i molekularnoj razini saeo uinkeEMD-a na: (1) stanini pripoj, irenje i kemotaksi-

ju; (2) proliferaciju stanica i preivljavanje; (3) izra-aj transkripcijskih imbenika; (4) izraaj imbeni-ka rasta, citokine, sastavne dijelove izvanstaninog

Introduction

Successful regeneration of intra-oral osseousand periodontal defects requires adequate host tis-sue reactions with recruitment and subsequent ac-tivation (and protection) of relevant adjacent cellpopulations. Indispensable for the formation of newmineralised bony tissue and re-organisation of toothsupporting structures are osteogenic cells (1). Inthis context, fundamental therapeutic strategies inthe eld of guided tissue regeneration (GTR) havebeen introduced (2-5). However, in case of utilisa-tion of alloplastic biomaterials like occlusive mem-branes or bone substitute materials, the applied ma-terials have to meet stringent requirements, rst ofall biocompatibility. Furthermore, some kind of bio-activation with promotion of cell maturation is de-sirable. Insuf cient tissue integration and/or bacteri-al colonisation of the alloplastic materials have beenidenti ed as potential risk factors for GTR (6).

Prerequisite for tissue regeneration are well-bal-anced cell to extracellular matrix (ECM) interac-tions. In bony tissue, the composition of the ECMplays a regulatory role for osteogenic cell functionslike adhesion, motility, proliferation and differenti-ation (7-9). Besides the ubiquitous protein collagen,laminins as components of the basement membraneplay an additional important role in bone healing (1,

8, 10, 11).While the use of puri ed growth factors like bonemorphogenetic proteins has not been established indaily routine periodontal therapy, the applicationof enamel matrix derivative (EMD) is an evidence-based therapeutic option (4, 12-18). The biologicalactive ingredients identi ed in enamel matrix con-sist of a variety of speci c matrix proteins like ame-logenin, tuftelin, ameloblastin and enamelin as wellas proteases, contributing a pivotal role in the de-velopment of teeth and supporting structures (19-

23). The only EMD currently commercially avail-able is the porcine derived Emdogain (Straumann,Switzerland). Emdogain represents an acidic ex-tract of porcine enamel matrix and contains mainlythe amelogenin fraction. A positive long term effecton bony defects, probing depth and attachment levelcould be documented both clinically or radiograph-ically after application of these biomimetric activeproteins (4, 14, 18, 24). In animal models, an im-provement of periodontal regeneration, osteogene-sis and angiogenesis has been observed (25, 26). On

a cellular and molecular level, a recent systematicreview summarises effects of EMD on: (1) cell at-tachment, spreading, and chemotaxis; (2) cell prolif -


3/14

Modulacija morfologije osteogenih stanica190 Reichert i sur.

eration and survival; (3) expression of transcriptionfactors; (4) expression of growth factors, cytokines,extracellular matrix constituents, and other mac-romolecules; and (5) expression of molecules in-volved in the regulation of bone remodelling for

different cell types including epithelial cells, gingi-val broblasts, periodontal ligament cells, cement-oblasts and osteogenic cells (20). It was concludedthat the analysed data provided strong evidence forEMD to support wound healing and new periodon-tal tissue formation. For osteogenic cells in detail, ithas been suggested that EMD promotes cell viabili-ty and cell proliferation in a dose-dependent manner(27, 28), furthermore cell attachment (29) and cellmotility (7) were increased. Schwartz et al. showedthat EMD affects early states of cell maturation by

stimulating cell proliferation and alkaline phospha-tase activity as well as late states by stimulating os-teocalcin expression (30). Other authors con rmedthe principal promotion of osteogenic cell differen-tiation by EMD (29, 31). A summarization of thecurrent literature, to include divergent results, leadsto the assumption that the effects of EMD dependsto a degree on the investigated cell type. Neverthe-less, the overall effect of EMD on osteoblastic cellsis stimulatory rather than inhibitory.

The actin-cytoskeleton imparts as connective link

between extracellular matrix and intercellular reac-tions (32). It is of vital importance to maintain cellu-lar homeostasis and indispensable for cell adhesionand migration (33-35). Furthermore, over the actin-supported signalling pathway between extracellularmatrix and intracellular compartments, the outside-in signalling for proliferation and cellular differenti-ation is regulated (36). Various studies show that thementioned osteoblastic cell attributes adhesion anddifferentiation are closely associated with charac-teristic cell morphologies (37, 38). Osteogenic cells

cultivated on biological active biocompatible sur-faces represented a stretched, spindle-shaped phe-notype with development of lammelipodias and -lopodias, whereas less compatible surfaces resultedin rather roundish cell forms (37-40).

Various studies reveal a close correlation be-tween f-actin cytoskeletal organisation, cell adhe-sion and osteogenic cell maturation (differentiation)with associated matrix production (39, 41, 42). Inthese studies, cell phenotypes and cytoskeletal or-ganisation were expediently categorised (round cell

shape with low cytoskeletal organisation vs. polari-sized cell shape with intermediate cytoskeletal or-ganisation vs. spreaded cell shape with highly so-

matriksa i ostale makromolekule; i (5) izraaj mo-lekula ukljuenih u remodeliranje kostiju za razli-ite vrste stanica, ukljuujui epitelne, gingivalne

broblaste, stanice parodontnog ligamenta, cemen-toblaste i osteogene (20). Zakljueno je da svi ti po-

daci daju vrstu potporu tezi da EMD poboljava ci- jeljenje rana i stvaranje novoga parodontnog tkiva.Podaci o osteogenim stanicama potanko dokazujuda se poveava preivljavanje i proliferacija stani-ca u dozom ovisnom obliku (27, 28), zatim pove-ani su stanini pripoj (29) i pokretljivost stanica(7). Schwartz i suradnici pokazali su da EMD utje-e na rane faze sazrijevanja stanica zahvaljujui sti-mulaciji stanine proliferacije preko aktivnosti al-kalne fosfataze, a u kasnijim fazama stimulacijomizraaja osteokalcina (30). Drugi autori potvrdili su

primarnu ulogu EMD-a u diferencijaciji osteogenihstanica (29, 31). Ako prouimo raspoloivu litera-turu, doi emo do pretpostavke da utjecaj EMD-adjelomice ovisi o promatranim vrstama stanica. Nakraju, mora se rei da je uinak EMD-a na osteobla-stne stanice vie stimulatoran negoli inhibitoran.

Aktinski citoskelet je spoj izmeu izvanstani-nog matriksa i unutarstanine reakcije (32). Vital-no je vaan za odravanje stanine homeostaze i ne-zamjenjiv za staninu adheziju i migraciju (33-35).Osim toga, regulacija prenoenja signala za prolife-

raciju i staninu diferencijaciju u unutranjost sta-nice obavlja se preko aktinskoga signalnog puta odizvanstaninog matriksa do odgovarajuih unutar-staninih odjeljaka (36). Mnogobrojna istraivanjapokazala su da su spomenuta svojstva osteoblastnihstanica, poput adhezije i diferencijacije, usko pove-zana s njihovom karakteristinom staninom mor-fologijom (37, 38). Osteogene stanice kultivirane nabioloki aktivnim biokompatibilnim povrinamapredstavljaju izduene stanice vretenastog fenoti-pa s razvojem lamelipodiza i lopodiza, a kultivaci-

ja na manje kompatibilnih povrina rezultira stvara-njem okruglastih stanica (37-40).Mnoga istraivanja otkrila su usku povezanost

izmeu f-aktinske organizacije citoskeleta, stani-ne adhezije i osteogenog sazrijevanja (diferencijacije) sa stvaranjem matriksa (39, 41, 42). U tim istra-ivanjima stanini fenotip i organizacija citoskeletapotanko su opisani i kategorizirani (okrugli staninioblik s niskom organizacijom citoskeleta nasuprotpolariziranim stanicama sa srednjim stupnjem orga-nizacije citoskeleta te proirenim stanicama s viso-

koorganiziranom strukturom citoskeleta). U nedav-nim istraivanjima Titushkin i Cho (2007.) tvrde dadiferencijaciju osteogenih stanica prati poveanje


4/14


phisticated cytoskeletal organisation). In a recentinvestigation, Titushkin and Cho (2007) stated thatosteogenic cell differentiation goes along with an in-crease in membrane-cytoskeleton interaction whichis typical for mature osteoblasts. These modulations

are related to remodelling of the actin cytoskeletonfrom thick (solitary) stress bers in osteogenic pre-cursor cells into a thinner lamentous network inmature osteoblasts (43).

As an implication, capturing of cell morphologyand cytoskeletal organisation can be identi ed as ahelpful approach to get an idea on the in uence offunctionalised substrates on the promotion of osteo-genic cell maturation.

The aim of the present study was to determinethe in uence of classical solitaire functional ECM-ligands (collagen type I, laminin-1) as well as com-mercially available EMD (Emdogain) on osteo-genic cell attachment and maturation by assessingcell morphology and cytoskeletal architecture. Inconcordance with the relevant literature, we test-ed the assumption that EMD promotes the develop-ment of a differentiated osteogenic phenotype.

Materials and Methods

Cell lines and cell cultureA commercial hipbone-derived osteoblastic cell

line (line P) HHOB-c (Fa. Promocell, Heidel-berg, Germany) and four primary human cell lines(lines K, N, S, T) were examined. The lat-ter were obtained from excess material of routinesurgical (orthognatic) procedures at the Departmentof Oral and Maxillofacial Surgery, Johannes Guten-berg-University of Mainz. In accordance to the reg-ulations of the local ethics committee, no patient-related data were recorded and the samples wereanonymised. The patients were free of systemic orlocal alterations of bone homeostasis (osteoporosis

or other bone diseases, neoplasies, systemic infec-tions, steroidal medication, radiation therapy etc.).The patients were three female and one male pa-tient, age ranging from 25 to 40 years. The bonespecimens were thoroughly washed in phosphate-buffered saline solution (PBS, PAA Laboratories,Pasching, Austria) and seeded onto culture dish-es in standard osteoblast cultivation medium, con-sisting of 10% fetal calf serum (FCS, Gibco Invit-rogen, Karlsruhe, Germany), Dulbeccos modi edEagles medium (DMEM, Gibco Invitrogen), dexa-

methasone (100 nmol/l, Serva Bioproducts, Heidel-berg, Germany), L-glutamin (Gibco Invitrogen) andstreptomycin (100 mg/ml, Gibco Invitrogen). Culti-

interakcije citoskeletnih membrana, to je tipinaznaajka zrelih osteoblasta. Te modulacije su u ve-zi s remodelacijom aktinskih citoskeletnih debelih(pojedinanih) niti kod osteogenih prekursora u ta-nje vlaknaste mree u zrelim osteoblastima (43).

Zbog toga se odreivanje morfologije stanica iorganizacije citoskeleta moe iskoristiti kao poka-zatelj za dobivanje prvih naznaka utjecaja funkci-onalnih supstrata na pojaano sazrijevanja osteoge-nih stanica.

Svrha ovog istraivanja bila je odrediti utjecajklasinih pojedinanih funkcionalnih ECM-liga-nada (kolagen tip I, laminin-1) te komercijalnogEMD-a (Emdogain) na osteogeni stanini pripoj isazrijevanje pomou procjene morfologije stanica iarhitekture citoskeleta. U skladu s relevantnom lite-raturom testirali smo pretpostavku da pospjeuju ra-zvoj i diferencijaciju osteoblastnog fenotipa.

Materijali i metode

Stanine linije i kultura stanicaIstraivane su komercijalna osteoblastna stani-

na linija dobivena iz kosti kuka (line P) HHOB-c(Fa. Promocell, Heidelberg, Njemaka) i etiri pri-marne humane stanine linije (linije K, N, S,T). Posljednje navedene stanine linije dobivenesu iz vika kotanog materijala tijekom rutinskih ki-rurkih operacija (ortognatske) na Odjelu za oral-nu i maksilofacijalnu kirurgiju Sveuilita JohannesGutenberg u Mainzu. U skladu s odredbama lokal-noga Etikog povjerenstva, uzorci su bili anonimnii nisu bili zabiljeeni nikakvi osobni podaci o pa-cijentima. Pacijenti nisu imali nikakve poremeajekotane homeostaze (osteoporozu ili ostale bolestikostiju poput: neoplazmi, sistemskih infekcija, ste-roidnih lijekova, terapija radijacijom i sl.). U skupi-ni su bile tri ene i jedan mukarac u dobi od 25 do40 godina. Uzorci kostiju temeljito su oprani u fos-fatima puferiranoj otopini (PBS, PAA Laboratories,Pasching, Austrija) i zasaeni u petrijeve posudicesa standardnim medijem za kultivaciju osteoblastauz 10% fetalnoga teleeg seruma (FCS, Gibco Invi-trogen, Karlsruhe, Njemaka), Dulbeccos modi edEagles medium (DMEM, Gibco Invitrogen), dek-

sametazon (100 nmol/l, Serva Bioproducts, Heidel-berg, Njemaka ), L-glutamin (Gibco Invitrogen) istreptomicin (100 mg/ml, Gibco Invitrogen). Kul-


5/14


vation was carried out at 37C in a constant humidi-ed atmosphere of 95% air and 5% CO2.

Before our investigations, all ve cell lines werequalitatively characterized by immunohistochemi-cal expression of alkaline phosphatase (AP) and os-

teocalcin (labelled streptavidinbiotin/horseradishperoxidase). Cells were passaged at regular inter-vals depending on their growth characteristics us-ing 0.25% trypsin (Seromed Biochrom KG, Berlin,Germany).

All trials were taken out between the 3rd and 5 th passage. Glass slides coated with the respective li-gands (see below) were cultivated with the differ-ent osteogenic cell lines (seeding density of 2 x 104/ml). After 48h of cultivation under constant cultureconditions xation and cytoskeleton-speci c stain-

ing was performed (see below).

Test substrates and coatingAs representatives of solitaire ECM-proteins,

collagen type I (human skin; Sigma, St.Louis,USA) and laminin-1 (human placenta; Sigma,St.Louis, USA) were applied. As commerciallyavailable EMD, Emdogain (Straumann, Basel,Switzerland) was utilised. All components weredissolved in PBS supplemented with 1mmol Ca-Cl 2 and adjusted to a concentration of 100 mg/ml.Bovine serum albumin (BSA, Sigma, St.Louis,USA) at a concentration of 10mg/ml served as thecontrol. As previously described, sterilized glassslides, diameter 2cm, (Hecht-Assistant, Sond-heim, Germany) were incubated with 200l of therespective stock solutions for 1h at 37C, thus al-lowing the proteins to precipitate on the glass sur-face (7). Soluble remnants were removed by rins-ing gently with PBS. Potential remaining freeadhesion sites were blocked with a solution of1% BSA for 30 minutes at room temperature. Un-treated glass slides served as negative control. Af -ter placing the differently prepared glass slides in90mm tissue culture petri dishes (Greiner HoldingAG, Kremsmuenster, Austria) further cultivationwas performed.Visualisation of the cell cytoskeleton andcategorisation of cell morphology

After a cultivation period of 48 hours the cellswere xed on the glass slides with paraformalde-hyd (4%) at room temperature for 10 minutes and

permeabilized with 0,1% Triton-X for three min-utes. Speci c staining for the f-actin cytoskeletonwas done with BODIPY FL Phallacidin (Molecu-

tura stanica bila je obavljena na 37C u konstantnovlanoj atmosferi uz 95% zraka i 5% CO2.

Prije poetka naeg istraivanja svih pet stani-nih linija kvalitativno je karakterizirano koritenjemimunohistokemijskog izraaja alkalne fosfataze

(AP-a) i osteokalcina (obiljeen streptavidinbi-otin/horseradish peroksidazom). Presaivanja re-dovito ovise o njihovim karakteristikama rasta uzpomo 0,25% trypsina (Seromed Biochrom KG,Berlin, Njemaka).

Svi pokusi bili su obavljeni izmeu treeg i pe-tog presaivanja. Predmetna stakalca presvuenaodgovarajuim ligandima (pogledati ispod) kultivi-rana su s razliitim osteogenim staninim linijama(gustoa saenja 2 x 104/ml). Nakon 48 sati kultiva-cije u standardnim uvjetima, bila je obavljena ksa-

cija i speci no bojenje za citoskelet (pogledati is-pod).Testirani supstrati i premazi

Kao predstavnici pojedinanih ECM-bjelane-vina koriteni su: kolagen tip I (humana koa; Si-gma, St.Louis, SAD) i laminin-1 (humana placenta;Sigma, St.Louis, SAD). Od komercijalnih EMD-auporabljen je bio Emdogain (Straumann, Basel,vicarska). Sve komponente otopljene su u PBS-ukojemu je dodan 1 mmol CaCl 2 te prilagoena kon-centracija na 100 mg/ml. Kao kontrola uporabljen je kravlji serumski albumin (BSA, Sigma, St.Louis,SAD) s koncentracijom od 10 mg/ml. Kao to je veistaknuto, sterilizirana predmetna stakalca promje-ra 2 cm (Hecht-Assistant, Sondheim, Njemaka) in-kubirana su s 200 l odgovarajue otopine jedan satna temperaturi od 37 stupnjeva kako bi se omogu-ila precipitacija bjelanevina na staklenu povrinu(7). Topivi ostatci blago su isprani PBS-om. Poten-cijalno slobodna vezna mjesta blokirana su pomou1-postotne otopine BSA 30 minuta na sobnoj tem-peraturi. Kao negativna kontrola rabila su se nepre-parirana predmetna stakalca. Kultura stanica na takopripremljenim razliitim predmetnim stakalacimanastavljena je njihovim postavljanjem u 90 mm pe-trijeve posudice (Greiner Holding AG, Kremsmu-enster, Austrija).

Vizualizacija staninog citoskeleta ikategorizacija stanine morfologije

Nakon kultivacije od 48 sati stanice su parafor-maldehidom (4%) ksirane na predmetnim stakal-cima na sobnoj temperaturi 10 minuta te su uinje-

na propusnima uz pomo 0,1% Triton-X-a tijekom3 minute. Obavljeno je bilo i speci no bojenje zacitoskeletni f-aktin pomou BODIPY FL Phalla-


6/14


lar Probes, Carlsbad, USA) according to manufac-tures guidelines. Visualisation was performed byconfocal laser scanning microscopy (CLSM: Lei-ca TCS SP2 X1, Wetzlar, Deutschland). The excita-tion of the green uorescenting dye was done in the

uorescence mode of the microscope with a blueargon laser at a wave length =488nm. For detec-tion, an Acusto Optic Beam Splitter (AOBS) in a de-tection channel at Langpass >500nm was utilised.With a Fluotar 10x04 oil-objective (Leica, Wetzlar,Deutschland) it was possible to scan a detail of 1,5x 1,5 mm (Fig. 1). The resolution of the scanned im-ages was 1024 x 1024 pixels. After automated de-tection of 25 adjacent scanning areas (5 x 5), it waspossible to merge them to a complete image of 7.5x 7.5 mm (Fig. 2).

According to the method of Sinha et al. (42) andEl-Amin et al. (39), we performed a descriptive cat-

cidina (Molecular Probes, Carlsbad, SAD) pre-ma uputama proizvoaa. Vizualizacija je obavlje-na laserskim konfokalnim skening-mikroskopom(CLSM: Leica TCS SP2 X1, Wetzlar, Njemaka).Poticanje zelene uorescentne boje uinjeno je u

uorescentnom modu rada mikroskopa pomouplavog argonskog lasera valne duljine =488nm. Zadetekciju se rabio Acusto Optic Beam Splitter (AO-BS) u detekcijskom kanalu pri Langpassu >500 nm.Koritenjem Fluotar 10x04 uljnog objektiva (Leica,Wetzlar, Njemaka) bilo je mogue snimiti podrujaveliine 1,5 x 1,5 mm (Slika 1.). Razluivost skeni-ranih slika jest 1024x1024 piksela. Nakon automati-zirane detekcije 25 dodirnih skeniranih podruja (5x 5), bilo ih je mogue spojiti u krajnje slike velii-ne 7,5 x 7,5 mm (Slika 2.).

Prema opisanoj metodi Sinha i suradnika (42) teEl-Amina i njegovih kolega (39), obavili smo deskrip-

Slika 3. Primjer osteogenih stanica podtipa I, II i III (phallacidinsko bojenje, originalno poveanje 40 x). Obratiti pozornost narazliita poveanja koja odgovaraju mjernoj oznaki omjera.

Figure 3 Exemplary representation of osteogenic cells of subtype I, II and III (phallacidin staining, original magni cation 40x).Note the different digital enlargements with corresponding scale bars.

Slika 1. Osteogene stanice S stanine linije nakon 48-satne kultivacije na kolagenu tipa I. Phallacidinskobojenje aktinskog citoskeleta (originalnopoveanje 10x; duina ruba: 1,5mm).

Figure 1 Osteogenic cells of line S after 48h cultivationtime on collagen type I. Phallacidin staining of theactin cytoskeleton (original magni cation 10x;

edge length: 1.5mm).

Slika 2. Osteogene stanice S linije nakon 48-satnekultivacije na kolagenu tipa I. Sloena slika od25 pojedinanih slika (originalno poveanje 10x;duina ruba: 7,5mm).

Figure 2 Osteogenic cells of line S after 48h incubationon collagen type I. Merged image of 25 singlepictures (original magni cation 10x; edge length:

7.5mm).


7/14


egorisation of cell morphology and cytoskeleton in-to three maturation subtypes (Fig. 3): type I (immature): round, small cells ( 50m),

the actin cytoskeleton is poorly differentiatedwith stress bers at the margin of the cell,

type II (intermediate): long, spindle shaped cells(>50m) with formation of peripheral actinstress bers,

type III (mature): large cells (> 100m) withmany differentiated lopodias and lamellipodiasand a highly organized cytoskeleton with a ne,sophisticated actin network.

Evaluation of cell morphology and statisticalanalysis

The stained and CLSM-recorded cells were sub- jected to further statistical evaluation if they met thefollowing inclusion criteria: the whole cell is displayed in a sharp manner (in

focus, no overlay caused by artefacts etc.), isolated position of the cell (low cell-cell-con-

tacts, no cellular aggregation).Beginning from the upper left side of a merged

image, the rst 100 cells (for each cell line and eachsubstrate) which ful lled the listed inclusion crite-ria where numbered consecutively (1 - 100). Theassignment of the cells into the above de ned cat-egories (type I - III) was conducted in a semi-quan-titative manner by three independent observers. Inpreliminary tests, the observers were trained aboutthe relevant features of the respective cell categoriesin order to achieve a calibration.

The statistical analysis was performed by the In-stitute of Medical Biostatistics, Epidemiology andInformatics (IMBEI), Johannes Gutenberg-Uni-versity Mainz. The software program SPSS (SPSSInc., Chicago, USA) was utilised. Only cells with100% agreement between the observers (inter-ob-server concordance) were further processed. For theanalysis of potential in uence factors (substratum,cell line) a multivariate logistic regression on cellmorphology and cytoskeleton organisation was per-formed. As references we de ned the neutral sub-strate BSA and the cell line K, which showed thelowest overall maturation. All p-values 50m) te stvaranje perifernih aktinskih stre-snih niti;

tip III (zreo): velike stanice (> 100m) uz mnogediferencirane lopode (stanine izdanke) i lame-lopode i visokoorganizirani citoskelet s tankom,so sticiranom (sloenom) mreom aktina.

Ocjenjivanje stanine morfologije i statistikaanaliza

Obojene i CLSM-zabiljeene stanice bile su pod-vrgnute daljnjoj statistikoj procjeni ako su zadovo-ljavale sljedee uvjete: cijela stanica otro je ocrtana i struktura dobro

vidljiva (u fokusu nema artefaktnog preklapanjaslojeva);

izolirani poloaj stanice (malo stanica-stanicakontakata, bez staninog nakupljanja).Poevi od gornjega lijevog kuta sloene slike, pr-

vih 100 stanica (za svaku staninu liniju i svaki sup-strat) koje su zadovoljavale navedene kriterije uklju-ivanja, pobrojeno je redom (1 - 100). Uvrtavanjestanica u de nirane skupine (tip I - III) obavila su po-lukvantitativno tri neovisna promatraa. Radi kali-bracije, oni su na preliminarnim testovima uvjeba-vali prepoznavanje kljunih znaajki za svrstavanjestanica u navedene skupine.

Statistika analiza bila je obavljena u Institutu zamedicinsku biostatistiku, epidemiologiju i informa-tiku (IMBEI) Sveuilita Johannes Gutenberg u Ma-inzu. Koriten je programski paket SPSS-a (SPSSInc., Chicago, SAD). Samo stanice sa 100% podu-darnom klasi kacijom meu promatraima (meu-promatrako preklapanje) uporabljene su za daljnjuobradu. Za analizu utjecaja pojedinih vanih imbe-nika (supstrata, staninih linija) na staninu morfo-logiju i organizaciju citoskeleta, rabila se multivarijantna logistika regresija. Kao referentnu polaznutoku odredili smo neutralni supstrat BSA i stani-nu liniju K koja je imala prosjeno najmanji stu-panj sazrijevanja. Sve dobivene vrijednosti na razinip


8/14


line T. For all 5 cell lines, a total of 2450 cellswere analysed by independent observers. Out ofthis, 1507 cells (60.3%) met the criterion of inter-observer concordance. Between the different celllines, the ratio ranged from 58.0% (line S) to

66.2% (line K).Figures 4a-4e show the ratio of each maturationsubtype (types I-III) for the analysed lines cultivatedon the respective substrates. Figure 4f shows the re-sults for the whole population (n=1507). For all celllines, the majority of the type-I cell phenotype wasfound on the negative control (untreated glass). Itranged from 31.4% (line P) to 50.8% (line K).

ljanih stanica. U pet staninih linija svaki neovisnipromatra analizirao je ukupno 2450 stanica. Odtoga broja je 1507 stanica (60,3%) zadovoljavalouvjet meupromatrakog podudaranja. Izmeu ra-zliitih staninih linija omjer se kretao od 58,0% (li-

nija S) do 66,2% (linija K).Slike od 4a do 4e pokazuju omjere svakog ma-turacijskog podtipa (tipovi I-III) za analiziranu sta-ninu liniju i odgovarajui supstrat. Na Slici 4f surezultati za cijelu populaciju (n=1507). Meu svimstaninim linijama najvei udjel tip-I staninog fe-notipa pronaen je u negativnoj kontroli (netretira-na predmetna stakalca). Kretao se od 31,4% (linijaP) do 50,8% (linija K).

Slike od 4a do 4f. Omjeri razliitih podtipova zrelosti stanica (I: nezrela, II: prijelazna, III: zrela) za testirane supstrate. Slike od4a do 4e prikazuju razliite istraivane stanine linije, a na Slici 4f je ukupna analizirana stanina populacija (n=1507).

Figures 4a - 4f Ratios of the different maturation subtypes (I: immature, II: intermediate, III: mature) for the investigatedsubstrates. Figures 4a - 4e display the different investigated cell lines, gure 4f displays the cumulative cellpopulation (n=1507).

a

c

e

b

d

f


9/14


Najvie je stanica, bez obzira na supstrat i stani-nu liniju, imalo obiljeja podtipa II. Jedine iznimkebile su stanice linije K na staklu (50,8% podtip I)i linija T na EMD (54,5% podtip III).

U negativnoj kontroli stakla opazili smo ukupno

manje staninog fenotipa tipa III (raspon od 1,5%za liniju K do 10,0% za liniju P). U uspored-bi s BSA-kontrolnom skupinom supstrata, za pojedinane ECM-bjelanevine kolagen i laminin teEMD, pronaene su vie vrijednosti tipa III stani-nog fenotipa. Jedina iznimka identi cirana je u liniji K s niim vrijednostima za kolagen. Za kolagen je omjer tipa III staninog fenotipa bio od 3,1% zaliniju K do 34,9% za liniju N, za laminin omjer je bio od 11,.8% za linju K do 47,2% za liniju S,te za EMD omjer je iznosio od 15,4% za liniju Kdo 54,5% za liniju T.

Na Slici 5. su kumulativni rezultati za sve ispi-tane supstrate na pet istraenih staninih linija. Op-enito je bio visok udjel staninog fenotipa tipaII (raspon od 56,% za liniju S do 69,% za linijuK). Ukupan omjer za tip II staninog fenotipa usvim staninim linijama iznosio je 64,6%. Omjer zatip III bio je u rasponu od 8,% za liniju K do 26,%za liniju N, uz ukupni omjer od 19,9% za sve sta-nine linije.

Primjenjujui logistiku regresiju, izraunat jerizik za odgovarajue supstrate u odnosu pre-ma BSA-u, bez obzira na to je li stanica pokazivalasvojstva tipa I, II ili III (Tablica 1.). Prema tome jeizraunata vjerojatnost omjera (Exp(B)) kao mjere.Negativna kontrola stakla je statistiki znatno po-dupirala izraaj podtipa I, a razvoj podtipa III bio je jako inhibiran. Pojedinane ECM-bjelanevinekolagen i laminin te EMD pojaale su izraaj pod-tipa III (Eksp(B) kolagen: 1,745; Eksp(B) laminin:2,181; Eksp(B) EMD: 2,491) i znatno smanjile pod-tip I.

Regardless of substratum and cell line the major-ity of the cells expressed subtype II. The only excep-tions were assessed for line K on glass (50.8% sub-type I) and line T on EMD (54.5% subtype III).

For the negative control glass we observed a

collective lower expression of type III cell pheno-types (ranging from 1.5% for line K to 10.0%for line P). Compared to the substratum controlgroup BSA, for the solitaire ECM-proteins collagenand laminin as well as for EMD, higher levels oftype III cell phenotypes were found. The only ex-ception was identi ed for line K with lower val-ues for collagen. For collagen, the ratio of type IIIcell phenotypes ranged from 3.1% for line K upto 34.9% for line N, for laminin, the ratio rangedfrom 11.8% for line K up to 47.2% for line Sand for EMD, the ratio ranged from 15.4% for lineK up to 54.5% for line T.

Figure 5 shows the cumulative results of all test-ed substrates for the investigated 5 cell lines. In gen-eral, a high proportion of type II cell phenotypes(ranging from 56.9% for line S up to 69.9% forline K) was found. The ratio of type II cell phe-notypes for all lines amounted 64.6%. The ratio oftype III cell phenotypes ranged from 8.5% for lineK up to 26.6% for line N with a total ratio of19.9% for all cell lines.

Employing logistic regression, a risk-calcula-tion for the respective substrates - referred to BSA -was performed, whether a cell was likely to expresscell phenotypes I, II or III (Table 1). Therefore, oddsratio (Exp(B)) was calculated as a measure. Thenegative control glass supported the expression ofsubtype I in a signi cant manner, whereas the devel-opment of subtype III was signi cantly inhibited.The solitaire ECM-proteins laminin and collagen aswell as EMD led to a signi cant higher expressionof subtype III (Exp(B) collagen: 1.745; Exp(B) lam-inin: 2.181; Exp(B) EMD: 2.491) with a respectivesigni cant down regulation of subtype I.

Slika 5. Omjeri razliith podtipova tipovastanine zrelosti (I: nezrela, II: prijelazna,III: zrela) za razliite stanine linije nasvim testiranim supstratima.

Figure 5 Ratios of the different maturationsubtypes (I: immature, II: intermediate,III: mature) for the different investigatedcell lines on all tested substrates.


10/14


Compared to the reference cell line K, celllines N, S and T showed a signi cant low-er ratio of type I cell phenotypes, for cell line Sa signi cant lower ratio of type II cell phenotypeswas found additionally. Compared to line K, forall cell lines an increased expression of cell type IIIwas recorded (from Exp(B) line T: 2.626 up toExp(B) line P: 4.066).

Discussion

Precondition for successful and timely periodon-tal tissue regeneration is a well balanced interactionof relevant cell populations at the defect site. For theosteogenic cell lineage in special a distinct sequenceof (1)osteoconduction (the growth of bony tissueinto the structure of an implant or graft) and (2)denovo bone formation (osteogenic cell proliferationand differentiation) has been described (1). In vitro ,assignment of speci c osteogenic cell attributes like

cell motility, cell proliferation and cell differentia-tion serve as an indicator of the state of cell matu-ration: in consecutive osteogenic maturation stagescell motility and proliferation decreases while celldifferentiation increases, nally resulting in post-mitotic mature osteoblasts (44-46). Furthermore, afast and plane cell adhesion is mandatory precondi-tion for further cell differentiation. In this context,various studies clearly assigned cell morphologyand cytoskeletal organisation to distinct osteogen-ic cell maturation stages and substrate biocompati-

bility (37-42). According to the method of Sinha etal. (42) and El-Amin et al. (39) in this observation-al study, we categorized osteogenic cell morpholo-

U usporedbi s referentnom staninom linijomK, stanine linije N, S i T imale su mnogonii omjer tipa I staninog fenotipa, a kod staninelinije S naen je dodatno i dosta nii omjer tipa IIstaninog fenotipa. U usporedbi sa staninom lini- jom K, kod svih drugih linija bio je zabiljeen po- jaan izraaj tipa III (iz Eksp(B) linija T: 2,626 doEksp(B) linija P: 4,066).

Rasprava

Preduvjet za uspjenu i pravodobnu parodontnuregeneraciju jest dobro uravnoteena interakcija od-govarajuih stanica na mjestu oteenja. Za stani-ce osteogene linije opisani su speci ni procesi kojise dogaaju u odreenom redoslijedu (1):osteokon -dukcija (uratanje kotane strukture u strukturu im-plantata ili grafta) i (2)novo stvaranje kostiju (pro-liferacija i diferencijacija osteogenih stanica) (1). Invitro pridruivanje odreenih staninih svojstava,

poput stanine pokretljivosti, proliferacije i diferen-cijacije, slui kao pokazatelj staninog sazrijevanja:u slijedu sazrijevanja osteogenih stanica smanjujuse stanina pokretljivost i proliferacija, a poveavadiferencijacija, to rezultira stvaranjem zrelih post-mitotikih osteoblasta (44-46). Osim toga, brza iuinkovita stanina adhezija obvezatna je za daljnjudiferencijaciju stanica. U tom kontekstu mnoga suistraivanja jasno dodijelila odreenu staninu mor-fologiju i organizaciju citoskeleta odreenim stup-njevima sazrijevanja osteogenih stanica i biokom-

patibilnosti supstrata (37-42). Prema metodi Sinha isuradnika (42) te El-Amina i njegovih kolega (39),mi smo u ovom istraivanju klasi cirali morfolo-

Tablica 1. Rezultati logistike regresije koritene za procjenu stanine morfologije i organizacije citoskeleta za odgovarajueskupine (tipovi I III) ovisno o supstratu BSA i staninoj liniji K. (df = stupnjevi slobode; Exp(B) = omjervjerojatnosti)

Table 1 Results of the logistic regression aimed on cell morphology and cytoskeleton organisation for the referring categories(types I III) in dependence of substrate BSA and cell line K. (df = degrees of freedom; Exp(B) = odds ratio)

Varijable

Variables

A n a

l i z a r i z i

k a

t i p

I

R i s k a n a

l y s i s

T y p e

I

df Znaajnost

Signi canceExp(B)

A n a

l i z a r i z i

k a

t i p

I I

R i s k a n a

l y s i s

T y p e

I I

df Znaajnost

Signi canceExp(B)

A n a

l i z a r i z i

k a

t i p

I I I

R i s k a n a

l y s i s

T y p e

I I I

df Znaajnost

Signi canceExp(B)

Svi supstrati All substrates 4 < 0.001 4 < 0.001 4 < 0.001

Staklo Glass 1 0.000 3.175 1 0.002 0.606 1 0.000 0.337Kolagen Collagen 1 0.000 0.315 1 0.420 1.153 1 0.009 1.745Laminin 1 0.000 0.205 1 0.667 1.079 1 0.000 2.181EMD 1 0.000 0.077 1 0.688 1.074 1 0.000 2.491

Sve stanine linije All cell lines 4 0.000 4 0.012 4 0.000

Line N 1 0.000 0.389 1 0.066 0.73 1 0.000 4.037Line P 1 0.123 0.264 1 0.251 0.821 1 0.000 4.066Line S 1 0.000 0.706 1 0.002 0.589 1 0.000 3.685Line T 1 0.000 0.406 1 0.893 0.997 1 0.000 2.626


11/14


giju i organizaciju osteogenih stanica u tri podtipa,ovisno o stupnju sazrijevanja stanica.

Loa strana te metode jest ovisnost o promatra-evoj subjektivnosti. Osim toga, preporueno 100-postotno meupromatrako preklapanje rezultiralo

je iskljuivanjem mnotva stanica (943 stanice ili39,7%) iz daljnje statistike analize. Automatiziranikompjutorski algoritmi za procjenu i analizu stani-ne morfologije i organizacije citoskeleta ve posto- je (47, 48) i moda bi mogli biti korisni u obavlja-nju tih pokusa. Naalost, ti automatizirani algoritmizahtijevaju visoke standarde pripreme uzoraka i ja-ko su osjetljivi na povrinske, ksacijske i procesneartefakte. Poluautomatizirana metoda opisana u na-oj studiji uspjela je kompenzirati takve artefakte zamiljenje promatraa o obliku i boji.

Nai rezultati upuuju na to da na istraivanimstaninim linijama N, S i T pojedinani ECMligandi: kolagen i laminin te derivati caklinskog ma-triksa (EMD-a) pomau dobro priljubljeno diferen-ciranje u osteogeni fenotip ve nakon 48 sati, to jepremailo staklo i BSA. To pokazuje da ti supstra-ti pozitivno djeluju na staninu adheziju i sazrije-vanje.

Multivarijantna logistika regresija je kod svihispitanih stanica dodatno potvrdila te nalaze (bezobzira na staninu liniju) s najveim stupnjem sa-

zrijevanja za EMD (Eksp(B) EMD: 2,491, Eksp(B)laminin: 2,181, Eksp(B) kolagen: 1,745). Nai serezultati slau s onima iz literature (8, 9, 20, 27-29,31). Osim toga, u naem radu su ispitivani ligandipokazali da potiu i staninu pokretljivost, premdakod EMD-a u manjoj mjeri nego kod kolagena i la-minina (7). injenica da je stanina migracija obi-no prije diferencijacije (44), podupire pretpostavkuda osteogene stanice na EMD-u predstavljaju viediferencirane stanice, a time i manje pokretnu sub-populaciju.

Bez obzira na ispitivane ligande, multivarijan-tna logistika regresija otkrila je da linija K op-enito ima najmanje zreo osteogeni fenotip, a li-nije N i P pokazuju naveu tendenciju premazrelim osteoblastima. Ti nalazi podupiru hipotezuda se diferencirani uinci EMD-a mogu povezatisa stanjem sazrijevanja istraivane stanine linije(49) i istiu potrebu za poznavanjem intrinzinihsvojstava stanica za interpretaciju rezultata kultu-ra stanica (50).

Paljivo prenoenje naihin vitro podataka u in

vivo klinike situacije pokazuje potencijal istraiva-nih liganada u podupiranju parodontne regeneracije,te istie osteogeni stanini privrstak i sazrijevanje

gy and cytoskeletal architecture into three subtypes,re ecting the stage of cell maturation.

A drawback of the utilised methodology is thesubjective observer-dependence. Furthermore, thepresupposed 100% inter-observer concordance re-

sulted in an exclusion of a larger cell population(943 cells or 39.7%) from further statistical anal-ysis. Computer-based, automated algorithms to as-sess and analyse cell morphology and cytoskele-tal organisation have already been established (47,48) and might be an useful completion of this trial.However, such automated algorithms demand highprocessing standards have been proven to be vulner-able to surface-, xation- and processing artefacts.The semi-automated analysis routine described inthe present study was able to compensate such ar-

tefacts by the observers abstract opinion of shapeand colour.Our results show for the investigated cell lines

N, S and T that the solitaire ECM ligands col-lagen and laminin as well as enamel matrix deriva-tive (EMD) promote a well-attached, differentiatedosteogenic phenotype after 48 hours, which exceed-ed glass and BSA. This indicates a promotion of celladhesion and cell maturation for these substrates.

Multivariate logistic regression of all investi-gated cells (regardless cell line) additionally under-

lined these ndings with highest cell maturation forEMD (Exp(B) EMD: 2.491, Exp(B) laminin: 2.181,Exp(B) collagen: 1.745). Our results are concordantwith current literature (8, 9, 20, 27-29, 31). Further-more, in a recent observation by our group, the in-vestigated ligands proved to promote osteogeniccell motility as well, however for EMD to a lowerextend than for collagen and laminin (7). The factthat cell migration generally precedes cell differen-tiation (44) underlines the assumption that the os-teogenic cells on EMD represent a more differenti-

ated and therefore less mobile subpopulation.Regardless the investigated ligands, multivari-ate logistic regression revealed for line K gener-ally the least mature osteogenic phenotype, where-as lines N and P showed the highest tendenciestowards mature osteoblasts. These ndings supportthe hypothesis that differential effects of EMD canbe well related to the maturation state of the inves-tigated cell line (49) and emphasizes the need ofknowledge of intrinsic cell properties for the inter-pretation of cell culture results (50).

A careful translation of ourin vitro data into thein vivo clinical situation highlights the potential ofthe investigated ligands to support periodontal re-


12/14


na mjestu oteenja. To se odnosi kako na proie-ni EMD tako i na primjenu kolagenih membrana tepotvruje dosadanje klinike spoznaje (5, 18).

Zakljuak

Prve naznake biokompatibilnosti i bioaktivacijerazliitih supstrata mogu se zapaziti tijekom analizestanine morfologije i organizacije citoskeleta do-bivene iz jednostavnih operativnih postupaka. Akoprihvatimo djelomino heterogene rezultate za ra-zliite istraivane stanine linije, naa zapaanja ja-sno upuuju na potencijal kako pojedinanih ECMliganada (kolagen tipa I, laminin-1), tako i EMD-a upodupiranju osteogene stanine adhezije i sazrijeva-nja. Rezultatima se moemo koristiti kao osnovomza daljnja opsena istraivanja in vitro i in vivo .

generation by enhancing osteogenic cell attachmentand maturation at the defect site. This applies to boththe use of puri ed EMD as well as the employmentof occlusive collagen membranes and supports re-spective clinical observations (5, 18).

Conclusion

First indications of biocompatibility and bio-ac-tivation of different substrates can be observed usinganalysis of cell morphology and cytoskeletal organ-isation derived from low operative intense proce-dures. Accepting the partly heterogeneous resultsfor the different investigated cell lines, our ndingsclearly indicate the potential of both solitaire ECMligands (collagen type 1, laminin-1) as well as EMDto promote osteogenic cell adhesion and maturation.

The obtained results might serve as basis for furtherdetailed in vitro and in vivo studies.

Received: June 22, 2009 Accepted: July 24, 2009

Address for correspondenceOA. Priv.-Doz. Dr.med.dent. Adrian Kasaj Johannes Gutenberg-UniversityDepartment of Operative Dentistry andPeriodontologyAugustusplatz 2, 55131 MainzTel.: 0049/6131/173064Fax: 0049/6131/[email protected]

AbstractThe precondition for successful periodontal regeneration is adequate activation ofrelevant cell populations like osteogenic cells. Here, cell adhesion and maturationare closely associated with cell morphology and f-actin cytoskeletal organisation.The potential of solitaire extracellular matrix (ECM) components as well as enamelmatrix derivative (EMD) to enhance periodontal healing is well documented. Objec -tive: The aim of the study was to test the impact of the ECM proteins collagen type 1and laminin-1 as well as commercially available EMD on osteogenic cell morphologyand cytoskeletal organisation. Material and methods : In an observational study, atotal of 2450 osteogenic cells of 5 different cell lines (4 primary ones and 1 commer-cial one) cultivated on the respective substrates were analysed by 3 independentobservers. After staining for the f-actin cytoskeleton and automated CLSM visuali-sation, cells were assigned to 3 different categories depending on morphologicalcell attributes (immature vs. intermediate vs. mature). Besides descriptive analy-sis, a multivariate logistic regression was performed to identify relevant in uenceparameters on cell morphology and cytoskeletal organisation. Results: The appliedsolitaire ligands collagen and laminin and especially EMD promoted a mature osteo-genic phenotype. Nevertheless, considerable differences between the investigatedcell lines could be identi ed as well. Analysis of cell morphology and cytoskeletalorganisation offers a reliable method of acquiring the rst hints of biocompatibilityand bio-activation on different substrates. Conclusion: Our results highlight the po-tential of the investigated ligands to support periodontal regeneration by enhanc-ing osteogenic cell attachment and maturation.

Key wordsOsteoblasts; Extracellular Matrix;Enamel Matrix Proteins; Cytoskeleton;Periodontal Diseases

References1. Davies JE, Hosseini MM. Histodynamics of endosseous

wound healing. In: Davies JE, editor. Bone engineering.Toronto: em squarred inc.; 1999. p. 1-14.

2. Cortellini P, Pini Prato G, Tonetti MS. Periodontal regen-eration of human infrabony defects. I. Clinical measures. J Periodontol. 1993;64(4):254-60.

3. Sculean A, Chiantella GC, Windisch P, Arweiler NB, BrecxM, Gera I. Healing of intra-bony defects following treat-ment with a composite bovine-derived xenograft (Bio-Oss Collagen) in combination with a collagen membrane(Bio-Gide PERIO). J Clin Periodontol. 2005;32(7):720-4.

4. Sculean A, Schwarz F, Miliauskaite A, Kiss A, Arweiler N,Becker J, et al. Treatment of intrabony defects with an

enamel matrix protein derivative or bioabsorbable mem-brane: an 8-year follow-up split-mouth study. J Periodon-tol. 2006;77(11):1879-86.

5. Sculean A, Nikolidakis D, Schwarz F. Regeneration ofperiodontal tissues: combinations of barrier membranesand grafting materials - biological foundation and pre-clinical evidence: a systematic review. J Clin Periodontol.2008;35(8 Suppl):106-16.

6. Tonetti MS, Prato GP, Cortellini P. Factors affecting thehealing response of intrabony defects following guidedtissue regeneration and access ap surgery. J Clin Peri -odontol. 1996;23(6):548-56.


13/14


7. Klein MO, Reichert C, Koch D, Horn S, Al-Nawas B. In vitroassessment of motility and proliferation of human osteo-genic cells on different isolated extracellular matrix com-ponents compared with enamel matrix derivative by con-tinuous single-cell observation. Clin Oral Implants Res.2007;18(1):40-5.

8. Roche P, Goldberg HA, Delmas PD, Malaval L. Selec-tive attachment of osteoprogenitors to laminin. Bone.1999;24(4):329-36.

9. Liu G, Hu YY, Zhao JN, Wu SJ, Xiong Z, Lu R. Effect of type Icollagen on the adhesion, proliferation, and osteoblasticgene expression of bone marrow-derived mesenchymalstem cells. Chin J Traumatol. 2004;7(6):358-62.

10. Timpl R, Brown JC. The laminins. Matrix Biol.1994;14(4):275-81.

11. Boskey AL, Paschalis E. Matrix proteins and biomineral-ization. In: Davies JE, editor. Bone engineering. Toronto:em squarred incorporated; 1999. p. 44-63.

12. Cortellini P, Tonetti MS. A minimally invasive surgicaltechnique with an enamel matrix derivative in the regen-erative treatment of intra-bony defects: a novel approach

to limit morbidity. J Clin Periodontol. 2007;34(1):87-93.13. Heijl L, Heden G, Svrdstrm G, Ostgren A. Enamel ma-trix derivative (EMDOGAIN) in the treatment of intrabonyperiodontal defects. J Clin Periodontol. 1997;24(9 Pt2):705-14.

14. Pietruska MD. A comparative study on the use of Bio-Oss and enamel matrix derivative (Emdogain) in thetreatment of periodontal bone defects. Eur J Oral Sci.2001;109(3):178-81.

15. Sculean A, Schwarz F, Becker J, Brecx M. The applica-tion of an enamel matrix protein derivative (Emdogain)in regenerative periodontal therapy: a review. Med PrincPract. 2007;16(3):167-80.

16. Sculean A, Schwarz F, Chiantella GC, Arweiler NB, Beck-er J. Nine-year results following treatment of intrabonyperiodontal defects with an enamel matrix derivative:report of 26 cases. Int J Periodontics Restorative Dent.2007;27(3):221-9.

17. Tonetti MS, Lang NP, Cortellini P, Suvan JE, Adriaens P,Dubravec D, et al. Enamel matrix proteins in the regener-ative therapy of deep intrabony defects. J Clin Periodon-tol. 2002;29(4):317-25.

18. Trombelli L, Farina R. Clinical outcomes with bioactiveagents alone or in combination with grafting or guidedtissue regeneration. J Clin Periodontol. 2008;35(8 Sup-pl):117-35.

19. Bgue-Kirn C, Krebsbach PH, Bartlett JD, Butler WT. Den-tin sialoprotein, dentin phosphoprotein, enamelysin andameloblastin: tooth-speci c molecules that are distinc -

tively expressed during murine dental differentiation. Eur J Oral Sci. 1998;106(5):963-70.20. Bosshardt DD. Biological mediators and periodontal re -

generation: a review of enamel matrix proteins at the cel-lular and molecular levels. J Clin Periodontol. 2008;35(8Suppl):87-105.

21. Bosshardt DD, Nanci A. Hertwigs epithelial root sheath,enamel matrix proteins, and initiation of cementogene-sis in porcine teeth. J Clin Periodontol. 2004;31(3):184-92. 22. Fincham AG, Moradian-Oldak J, Simmer JP. The structural biology of the developing dental enam-el matrix. J Struct Biol. 1999;126(3):270-99.

23. Veis A. Amelogenin gene splice products: potential sig -naling molecules. Cell Mol Life Sci. 2003;60(1):38-55.

24. Sculean A, Kiss A, Miliauskaite A, Schwarz F, Arweiler NB,Hannig M. Ten-year results following treatment of intra-bony defects with enamel matrix proteins and guided tis-sue regeneration. J Clin Periodontol. 2008;35(9):817-24.

25. Shimizu-Ishiura M, Tanaka S, Lee WS, Debari K, Sasaki T.Effects of enamel matrix derivative to titanium implanta-tion in rat femurs. J Biomed Mater Res. 2002;60(2):269-76.

26. Donos N, Sculean A, Glavind L, Reich E, Karring T. Woundhealing of degree III furcation involvements followingguided tissue regeneration and/or Emdogain. A histolo-gic study. J Clin Periodontol. 2003;30(12):1061-8.

27. Guida L, Annunziata M, Carinci F, Di Feo A, Passaro I, Oli-va A. In vitro biologic response of human bone marrowstromal cells to enamel matrix derivative. J Periodontol.2007;78(11):2190-6.

28. Schwarz F, Rothamel D, Herten M, Sculean A, ScherbaumW, Becker J. Effect of enamel matrix protein derivativeon the attachment, proliferation, and viability of humanSaOs(2) osteoblasts on titanium implants. Clin Oral In-vestig. 2004;8(3):165-71.

29. Rincon JC, Xiao Y, Young WG, Bartold PM. Enhanced pro-liferation, attachment and osteopontin expression byporcine periodontal cells exposed to Emdogain. ArchOral Biol. 2005;50(12):1047-54.

30. Schwartz Z, Carnes DL Jr, Pulliam R, Lohmann CH, Syl-via VL, Liu Y, et al. Porcine fetal enamel matrix derivativestimulates proliferation but not differentiation of pre-os-teoblastic 2T9 cells, inhibits proliferation and stimulatesdifferentiation of osteoblast-like MG63 cells, and increas-es proliferation and differentiation of normal human os-teoblast NHOst cells. J Periodontol. 2000;71(8):1287-96.

31. He J, Jiang J, Safavi KE, Spngberg LS, Zhu Q. Emdogainpromotes osteoblast proliferation and differentiation andstimulates osteoprotegerin expression. Oral Surg OralMed Oral Pathol Oral Radiol Endod. 2004;97(2):239-45.

32. Carpenter CL. Actin cytoskeleton and cell signaling. CritCare Med. 2000;28(4 Suppl):N94-9.

33. Yamada KM. Integrin signaling. Matrix Biol.1997;16(4):137-41.

34. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, GinsbergMH, Borisy G, et al. Cell migration: integrating signalsfrom front to back. Science. 2003;302(5651):1704-9.

35. Zamir E, Geiger B. Components of cell-matrix adhesions. J Cell Sci. 2001;114(Pt 20):3577-9.

36. Pienta KJ, Coffey DS. Nuclear-cytoskeletal interactions:evidence for physical connections between the nucleusand cell periphery and their alteration by transformation. J Cell Biochem. 1992;49(4):357-65.

37. Kim HJ, Kim SH, Kim MS, Lee EJ, Oh HG, Oh WM, et al. Vary-ing Ti-6Al-4V surface roughness induces different ear-ly morphologic and molecular responses in MG63 osteo-blast-like cells. J Biomed Mater Res A. 2005;74(3):366-73.

38. Park BS, Heo SJ, Kim CS, Oh JE, Kim JM, Lee G, et al. Effectsof adhesion molecules on the behavior of osteoblast-likecells and normal human broblasts on different titaniumsurfaces. J Biomed Mater Res A. 2005;74(4):640-51.

39. El-Amin SF, Lu HH, Khan Y, Burems J, Mitchell J, Tuan RS,et al. Extracellular matrix production by human osteo-blasts cultured on biodegradable polymers applicablefor tissue engineering. Biomaterials. 2003;24(7):1213-21.

40. Klein MO, Tapety F, Sandhfer F, Gtz H, Schfer B, Wag-ner W, et al. Visualization of vital osteogenic cells on dif-ferently treated titanium surfaces using Confocal LaserScanning Microscopy (CLSM) a pilot study. Z ZahnrztlImpl. 2006(2):145-53.

41. Shah AK, Lazatin J, Sinha RK, Lennox T, Hickok NJ, TuanRS. Mechanism of BMP-2 stimulated adhesion of osteo-blastic cells to titanium alloy. Biol Cell. 1999;91(2):131-42.


14/14


42. Sinha RK, Morris F, Shah SA, Tuan RS. Surface composi-tion of orthopaedic implant metals regulates cell attach-ment, spreading, and cytoskeletal organization of pri-mary human osteoblasts in vitro. Clin Orthop Relat Res.1994;(305):258-72.

43. Titushkin I, Cho M. Modulation of cellular mechanics dur-ing osteogenic differentiation of human mesenchymalstem cells. Biophys J. 2007;93(10):3693-702.

44. Aubin JE. Advances in the osteoblast lineage. BiochemCell Biol. 1998;76(6):899-910.

45. Aubin JE. Bone stem cells. J Cell Biochem Suppl. 1998;30-31:73-82.

46. Malaval L, Liu F, Roche P, Aubin JE. Kinetics of osteopro-genitor proliferation and osteoblast differentiation in vi-tro. J Cell Biochem. 1999;74(4):616-27.

47. Krtolica A, Ortiz de Solorzano C, Lockett S, Campisi J.Quanti cation of epithelial cells in coculture with -broblasts by uorescence image analysis. Cytometry.2002;49(2):73-82.

48. Lichtenstein N, Geiger B, Kam Z. Quantitative analysis ofcytoskeletal organization by digital uorescent micros -copy. Cytometry A. 2003;54(1):8-18.

49. Dean DD, Lohmann CH, Sylvia VL, Cochran DL, Liu Y, Boy-an BD, et al. Effect of porcine fetal enamel matrix deriv-ative on chondrocyte proliferation, differentiation, andlocal factor production is dependent on cell maturationstate. Cells Tissues Organs. 2002;171(2-3):117-27.

50. Balls M, Coecke S, Bowe G, Davis J, Gstraunthaler G, Har-tung T, et al. The Importance of Good Cell Culture Prac-tice (GCCP). ALTEX. 2006;23 Suppl:270-3.

Reichert Et Al 2009_Modulation of Osteogenic Cell Morphology

Documents

Transcript of Reichert Et Al 2009_Modulation of Osteogenic Cell Morphology