Influence of Novel Nano-titanium

7/30/2019 Influence of Novel Nano-titanium

1/12

Influence of Novel Nano-Titanium Implant

Surface on Human Osteoblast Behaviorand Growth

Stefano Tete, MD, DDS,* Filiberto Mastrangelo, MD, DDS, PhD, Raimondo Quaresima, Eng, Raffaele Vinci, MD,

Gilberto Sammartino, MD, DDS, Liborio Stuppia, MD, and Enrico Gherlone, MD#

L

ong-term integration of endosse-ous implants in bone tissue isguaranteed by an intimate contact

between the titanium surface and thehost structure.1,2 Recent in vitro and invivo studies36 have shown that physicaland chemical characteristics, derivedfrom surface coating treatment, have acentral role during initial phases of boneto implant integration, conditioning thequality and quantity of cellular responseand the time of healing processes.7,8 Infact, during initial phases of machinedand macroroughness coating endosse-ous integration, new bone formation

was observed around the fixture only onhost bone (distance growth osteogene-sis), whereas during microroughnessimplant integration, it is possible to notea significant enhancement of new boneapposition on the host bone and on thetitanium surface (contact growth osteo-genesis), increasing stability and reduc-

ing the healing period.9 An essential roleof osteogenesis processes is played by os-teoblast progenitor stem cells duringrecruitment, adhesion, proliferation, differ-

entiation, and mineralized matrix deposi-tion during bone regeneration phases.1012

Adult human dental pulp and young hu-man dental papilla cultured in osteogenicmedium show cellular phenotypes andmineralizing capabilities similar to thoseof osteoblasts.13,14 Numerous bone regen-erative studies1518 have founded that hu-man dental papilla stem cells (hD-PaSCs), similar to adult stem cells,can differentiate into specialized celltypes both in vivo and in vitro situa-tions,19 confirming the feasibility of these

cell line types for experimental regener-

ation and clinical application.12,20 In den-tal cell therapy, the technique for manip-ulation of the isolated dental progenitor/stem cell growth and induction of 3D

tissue formation in vitro21

and in vivoneeds to be developed.22,23 The aim of ourstudy is to analyze the physicochemicalmodification of new implant surfaces, thetreatment modification of coatingroughness, and their influence on os-teoblast progenitor stem cell mor-phology, adhesion, and proliferation.

MATERIALS AND METHODShDPaSCs Expansion and Mesenchymal

Stem Cell Sorting

Six healthy patients (3 males and

3 females), in orthodontic treatment

*Associate Professor of Oral Surgery, Department of OralScience, University G. dAnnunzio, Chieti, Italy.Post Doctoral Fellow, Young Researcher, Department of OralScience, University G. Dannunzio, Chieti, Italy.Associated Professor, Department of Biomaterials, University

of lAquila, Italy.Head of Advanced Oral Surgery Unit, Department of OralScience, University S. Raffaele, Ateneo Vita e Salute, Milan, Italy.Professor of Oral and Maxillofacial Surgery, Head ofDepartment of Oral Surgery, Faculty of Medicine, University ofNaples Federico II, Naples, Italy.Professor, Genetic Department of UniversityG. dAnnunzio,Chieti, Italy.#Chief Professor, Oral Science Department, University S.Raffaele, Ateneo Vita e Salute, Milan, Italy.

Reprint requests and correspondence to: Stefano Tete,MD, DDS, Dott. Filiberto Mastrangelo, Department ofOral Science, University G. dAnnunzio, Via deiVestini, Chieti 66013, Italy, Tel: 39 08713554095,

E-mail: [email protected]/[email protected]

ISSN 1056-6163/10/01906-520Implant Dentistry

Volume 19 Number 6Copyright 2010 by Lippincott Williams & Wilkins

DOI: 10.1097/ID.0b013e3182002eac

Purpose: The aim of the study is toinvestigate human osteoblast-like cell

behavior and growth in the presence of

3 different titanium implant surfaces.

Materials: Human stem cells werefirst obtained and then sorted by

fluorescence-activated cell sorter from

mesenchymal stem cell clusters of hu-

man dental papilla. Theobtained human

dental papilla stem cells were induced to

differentiate into osteoblast-like cells

and were then analyzed by reverse tran-

scriptase polymerase chain reaction and

Western blot analyses. The cells pro-

liferated and were cultured onto 3 dif-ferent titanium discs (sandblasted,

sandblasted and large-grit acid-

etched, and full contact coverage

[FCC]) and analyzed by scanning

electron microscope.

Results: In all analyses samples,a high cell activity was observed,

with typical osteoblast mature mor-

phostructural response on rough

surface. The high number of osteoblast-

like cells was found on titanium FCC

discs. At the same time, scanning

electron microscope analysis con-

firmed the high biocompatibility of

this surface.

Conclusion: The rapid maturationof the osteoblast-like cells on FCC tita-

nium surface suggests that this structure

could play a central role during initial

phases of bone healing processes.(Implant Dent 2010;19:520531)

Key Words: human dental papilla stemcells, osteogenic differentiation, tita-nium surface, full contact coveragesurface

520 INFLUENCE OF NANO-TITANIUM SURFACE TET ET AL


2/12

for Classes II and III malocclusion,hyperdivergence, and overcrowding,average age 16 years, were recruited atthe Oral Science Department of G.dAnnunzio University, Chieti, forthird molar germ extraction. In accor-

dance with the Ethics Committee ofthe University of Chieti, all patients(or their parents in case of minors)expressed their informed written con-sent before surgery. Six impacted thirdmolar buds were surgically removed,and the dental papillae were delicatelyremoved and immediately were im-mersed in a digestive solution (4mg/mL Dispase I [Roche, Basel, Swit-zerland]; 3 mg/mL collagenase[Sigma, St. Louis, MO]; 1% penicil-lin/streptomycin [Invitrogen]; 1% cla-

rithromycin in 1.25 mL of minimumessential medium Eagle, alpha modi-fication [-MEM; Sigma-Aldrich]) at37C, for 1 hour. After enzymatic di-gestion, cells were passed through a70-m BD Falcon strainer (BectonDickinson, Franklin Lakes, NJ), andthe single-cell suspensions wereseeded onto plates in standard growthmedium (-MEM, 100 M 2-phospho-L-ascorbic acid, 2 mM L-glutamine[Sigma], 100 g/mL streptomycin, and100 U/mL penicillin supplemented with20% fetal bovine serum [Invitrogen], at37C in a 5% CO2). The medium wasrenewed every 3 days to remove cellsdebris. The adherent cells reached con-fluence after 20 days. For mesenchy-mal cell sorting, an immunophenotypicanalysis was performed on first-passagecells. Approximately 1.5 106 cellswere harvested, washed 3 times withphosphate-buffered saline (PBS), sus-pended in 0.7 mL PBS, and then in-cubated with 10 L fluorescein

isothiocyanate- or phycoerythrin-labeled monoclonal antibodies againstCD5, CD22, CD29, CD90, CD146,CD166, and STRO-1 and 2 mL fixativesolution for 20 minutes at room temper-ature. Cells were centrifuged at 300g for10 minutes, dissolved the pellet into 1mL PBS, and then analyzed by using afluorescence-activated cell sorter Van-tage flow cytometer and CellQuest Soft-ware (Becton Dickinson). Cells positivefor mesenchymal markers CD29, CD90,CD146, CDE166, and STRO-1 were

sorted and collected for the osteogenic

differentiation. Nonsorted cells wereused as controls.

In Vitro Osteogenic Differentiation and

Microscopic Observation

For 40 days, sorted cells were col-

lected and seeded under a controlledatmosphere (5% CO2, at 37C) onto10 mm dishes in Owen-modified os-teogenic medium (-MEM, 15% fetalbovine serum [Invitrogen], 1%L-glutamine, 50 M 2-phospho-L-ascorbic acid trisodium salt [Sigma], 3mM glycerol-2-phosphate disodiumsalt [Sigma], and 10 nM dexametha-sone) at a density of no less than 2 107/mL.24 Microscopic analyses (lightmicroscope and electron microscopeanalyses) of differentiating cells were

performed at day 7 (before the cellconfluence) and at day 40 (after thecell confluence). For light microscopeanalysis, cells were stained with tolu-idine blue and observed under an Axi-olab microscope (Zeiss, Oberkochen,Germany) connected to a digital cam-era, Fuji FinePixS2Pro (Fujifilm Cor-poration, Tokyo, Japan). The imageswere stored in RAF format with3032 2035 grid of pixels. The cellswere then observed by electron micro-scope analysis with a scanning elec-tron microscope (SEM) LEO 435 VPafter being subjected to fixing proce-dure: the cells were treated with 3%glutaraldehyde in 0.15 M PBS (pH 7.4) for 30 minutes and then washed in0.15 M PBS for 15 minutes and post-fixed in 2% phosphate-buffered os-mium with the addition of 0.15 Msucrose, at room temperature for 5hours. The samples were gradually de-hydrated in increasing concentrationsof propylene oxide (from 50% to

100%, 10% steps), saturated withamyl acetate, carried through criticalpoint drying according to standardprocedure using liquid carbon dioxide,and sputtered with gold-palladiumcoating.

Reverse Transcriptase Polymerase Chain

Reaction and Western Blot Analyses

To confirm that cells were differ-entiating toward an osteoblast-likepathway, the expression of typicalmarkers was investigated. Reverse

transcriptase polymerase chain reac-

tion (RT-PCR) and Western blot anal-yses were performed on days 3, 5, 10,and 40 from the beginning of culturein osteogenic conditions to evaluatealkaline phosphatase (ALP), osteocal-cin (OCN), and matrix extracellular

phosphoglycoprotein (MEPE) expres-sion variations. For RT-PCR analysis,total RNA was extracted from differ-entiated cells on the established days,using 1 mL TRIzol (Life Technolo-gies) and then amplified following themanufacturers instructions. In brief,cDNA synthesis was performed withRNA by using RETROscript kit (Am-bion, Inc.): 10 g purified RNA wereincubated at 4C for 1 hour with 10mM Tris-HCl pH 8.3, 50 mM KCl, 1.5nM MgCl2, 0.8 mM oligo-dT (Applied

Biosystem), 5 M random primers,0.5 U RNAsi inhibitor, and 5U reversetranscriptase. Then, 5 M of each spe-cific oligonucleotide, 0.8 mM oligo-dT, 0.2 mM of each primer, and 2U/50 L Taq polymerase (AppliedBiosystem) were added to 2 L of thecDNA previously obtained. Samplesunderwent a preliminary 5-minute de-naturation step at 94C, followed by35 cycles of 94C for 1 minute, an-nealing at 55C (ALP and MEPE) or58C (OCN and GAPDH) for 1minute, and elongation at 72C for 1minute, with a final elongation step at72C for 10 minutes. The amplifica-tion products were subjected to elec-trophoresis on agarose gel, at roomtemperature, applying a potential dif-ference of 1 to 3 V/cm. The identity ofthe products was confirmed by cyclesequencing the amplified cDNA.

For Western blot analysis, pro-teins were harvested at the indicatedtimes, using 25 mM Tris buffer pH 7.4

(containing 150 mM NaCl, 100 Msodium orthovanadate, 1.5 mMMgCl2, 1.0 mM ethylenediamine tet-raacetic acid, 1% NP40, 10% glycerol,1 mM phenylmethylsulfonyl fluoride,5 g/mL leupeptin, and 5 g/mLaprotinin) at 4C. A part was takenfrom each sample to evaluate the totalprotein contents, according to theBradford method.25 Approximately 30g/mL proteins were taken from thesupernatant, loaded onto 12.5% to15% polyacrylamide gel, and sepa-

rated by electrophoresis. Proteins were

IMPLANT DENTISTRY / VOLUME 19, NUMBER 6 2010 521


3/12

then moved to a polyvinylidene fluo-ride membrane, blocked with PBS/0.1% Tween20/5% nonfat milk (Bio-Rad Laboratories) for 2 hours at 4C,incubated overnight at 4C with spe-cific primary antibodies (polyclonalrabbit anti-human ALP, dilution1:10,000; anti-human OCN, dilution1:200 [Abcam, Cambridge, UnitedKingdom]; and anti-human MEPE [1g/mL; R&D, Minneapolis, MN]),and then repeatedly washed and ex-posed to donkey anti-rabbit horserad-ish peroxidase-conjugated secondaryantibody for 1 hour at room tempera-ture (final dilution 1:5,000; GEHealthcare Life Sciences). To deter-mine the equal loading of samples perlane, the blots were stripped and re-

probed with an anti--actin antibody(dilution 1:100, incubation for 1 hourat room temperature; Santa Cruz Bio-technologies). Immunocomplexeswere visualized using the enhancingchemiluminescence detection system(GE Healthcare Life Sciences) andquantified by densitometric analysis(Molecular Analyst System; Bio-RadLaboratories).

Alizarin Red Staining

To assess the presence of miner-alized depositions in the extracellularmatrix (ECM), culture of differenti-ated cells were stained at days 5, 10,and 40 with Alizarin red S solution(Sigma-Aldrich, Milano, Italy), ac-cording to the method described byBuser et al.26

Description and Evaluation of

Physicochemical Characteristics of

Titanium Surfaces

The titanium test discs to be used

as scaffold for cell proliferation wereprovided by WinSix (BioSAF srl,London, United Kingdom). Sand-blasted surface samples (SAB) weretreated only with applied TiO2 parti-cles on the disc surface. The sand-blasted and large-grit acid-etched(SLA) disc samples were obtained by2 acid etching processes with fluorid-ric acid and sulfuric-hydrochloric acid(H2S-HCl) after one sandblasting TiO2process. The full contact coverage(FCC) surface samples were obtained

by a galvanotactic anodizing process

in a phosphate-sulfate bath. All samplesurface profilometry and morphologywere analyzed with SEM LEO 420(LEO Electron Microscopy, Ltd., Cam-bridge, United Kingdom) and commercialprofilometer (Hommel T 20; HommelGmbH, Villingen-Schwermingen,Germany), equipped with a 5-m ra-dius diamond tip and sensitive to ver-tical movements to an accuracy of10.01 m. All points of absolute val-ues of average profile (Ra), all pointsof values of root mean square (Rq) andthe average value of the absoluteheight of the 5 highest peaks, and theabsolute depth of the 5 deepest valleys(Rz) were taken. All surface sampleswere scanned at 5 different profilo-metric positions, and the data were

automatically determined using soft-ware Statistica 8 (Stat Soft, srl, Italy),

and the variance was analyzed withpost hoc turkey comparative test withthe Ra roughness sample parameter.The chemical surface composition wasevaluated by x-ray photoelectron spec-troscopy (EDAX; Perkin Elmer PHI5400 ESCA System) to analyze 5nm of the most external layer and ex-pressed in atomic percentage.

Proliferation of Osteoblast-Like Cells

Onto Titanium Surface

After the differentiation phase,osteoblast-like cells were replatedonto the titanium test discs and Thecells were selected into 3 groups, ap-proximately 3.7 104 cells wereseeded onto each of the 3 different testdiscs and cultured in standard growth

medium for a total of 20 days andwere observed by SEM.

Fig. 1. Flow cytometric analysis of primary cultured hDPaSCs performed at day 20.

Closed histograms, in red, represent staining for each marker expression. The hDPaSCsresulted negative for hematopoietic markers (CD5 and CD22) but positive for cell surfaceantigens exhibited by mesenchymal stem cells (CD29, CD90, CD146, CD166, andSTRO-1).



4/12

RESULTSCell Sorting and Cell Behavior During In

Vitro Osteogenic Differentiation

The hDPaSCs were isolated by en-zymatic digestion. By day 1 and allthrough the first week in culture, cells

were aggregated in groups with somecellular debris. At day 15, the cells were80% confluent. Immunophenotypicanalysis by fluorescence-activated cellsorter allowed to discriminate a sub-population of cells negative to hemato-poietic markers CD5 and CD22(hematopoietic stem/progenitor cells/endothelium) but positive to cell surfaceantigens exhibited by mesenchymalstem cells (CD29, CD90, CD146,CD166, and STRO-1; Fig. 1). The pat-tern of marker expression of the dental

papillae obtained from different donorsdid not vary significantly.

The cells were then induced toosteogenic differentiation by addingspecific supplements to culture medi-um.19 Cells were observed by light andelectron microscopes at day 7 (beforethe confluence) and at day 40 (afterthe confluence) to asses changes incell morphology after cell differentia-tion. Moreover, to evaluate changes intypical osteoblastic marker expres-sions during the differentiation, cellswere investigated at days 3, 5, 10, and40 by RT-PCR and Western blot.

At light microscope observation af-ter 7 days in vitro, cells from the initialaggregates started to expand, exhibitinga high proliferation rate and assuming astarry and a fibroblast-like morphology(Fig. 2, A). After 40 days, the sampleswere stained with toluidine blue, and atlow light microscope magnification, thecells showed a diversified morphologyand a thick net multilayer organization

(Fig. 2, B). At SEM analysis, the cellsshowed a mesenchymal stem cell-likemorphostructure with rounded nuclei.Long filopodia and lamellipodia sur-rounded the cell surfaces and formed athick extracellular net by connecting thecells to each other. Fiber depositionscould be observed on culture plates aftercell confluence (Fig. 2, C and D).

Both RT-PCR and Western blotanalyses on differentiating cells showedthat the mRNA expression and the pro-tein content of the investigated markers

changes according to their specific be-

Fig. 2. Light (upper panel) and SEM (lower panel) observation of hDPaSCs after 7 (A and C)

and 40 days (B and D) in vitro, under osteogenic conditions.

Fig. 3. Detection of ALP, OCN, and MEPE in hDPaSCs cultured under osteogenic differenti-ation condition by RT-PCR (left panel) and Western blot (right panel) at 3, 5, 10, and 40 daysin vitro. Graphics represent the levels of ALP, OCN, and MEPE analyzed by scanning densi-tometry and normalized to GAPDH and to-actin levels, respectively. In each panel, the valuesare expressed as mean SEM (n 3) versus values detected in cells at 3 days after seeding(*P 0.05, **P 0.01, ***P 0.001).



5/12

havior. ALP increased to its maximumat day 5 and decreased during the fol-lowing days, and its expression at day40 was quite comparable with that dur-ing the first week. On the contrary, theanalysis of OCN expression showed asignificant, continuous increase during

the experimental time. Finally, RT-PCR

and Western blot indicated that MEPEexpression was downregulated, begin-ning from the fifth day after the additionof the osteogenic supplements (Fig. 3).Quantitative Alizarin red staining, ondifferentiating hDPaSCs at 5, 10, and 40days confirmed cell differentiation into

an osteoblast-like cell lineage (Fig. 4)

and revealed the increasing amount ofmineralized nodule with high levels ofcalcium.

Titanium Surface Observation

Macroscopic and microscopic im-

plant surfaces analysis showed typicalspecimens topography differences. Atlow (300) SEM analysis magnifica-tion, SAB implant surface showed amacroroughness morphology with ir-regularly distributed porosity. Athigher (5000) magnification, irregu-lar areas could be observed with ele-vated sharp crests alternated to deepvalleys. SEM analysis of SLA implantsurface at low (1000) magnificationprovided a microrough topographywith regularly distributed cavities. At

higher magnification, it was possibleto observe more homogeneous surfaceporosity with bevelled peaks alternat-ing with microvalleys. With FCC elec-trochemical treatment implant surfaceat low (1000) magnification SEManalysis, it was possible to noticemore regular nanorough topography.At higher (5000) magnification,FCC surface showed specific mor-phology with regularly distributedcrests similar to volcanoes, nanopo-

rosities, and low valleys. Specimenprofilometric evaluation confirmeddifferent surface roughness with statis-tical parameters. SLA and FCC rough-ness surfaces exhibited statisticallyhigher values (P 0.0001) than theSAB samples. The average roughnesssamples (Ra) of the sandblasted tita-nium sample (1.35 0.16 m) wasobserved similar to the SLA specimen(1.41 0.20 m). FCC surfaceshowed a lower Ra value (0.73 0.09m; Fig. 5). In Rz analysis, it could be

observed that the SAB specimens(14.11 1.09 m) showed very sim-ilar values to SLA (12.87 1.40 m).Moreover, the FCC titanium samplesprovided a significant lower Rz value(5.03 1.07 m), indicating a moreregular surface (Fig. 5; Table 1) Inaccordance with the surface treatmentprocess, the root mean square (Rq) val-ues showed the same range of rough-ness values for SLA (21.79 1.30m) and FCC (25.37 0.95 m).Once again, in SAB implants, the Rqvalue (51.66 1.89 m) showed a

Fig. 4.Alizarin red staining of hDPASCs during osteogenic differentiation:A, day 5; B, day 10;and C, day 40.

Fig. 5. Graphic representation of the profilometric data on each surface of Ra, Rz, and Rq.



6/12

higher difference (Table 1; Fig. 5),indicating an irregular surface. Thesandblasting treatment left typicalmacroroughness on the surfaces, andSEM back scattered analysis of SABsurface specimens showed typicaltreatment morphology with differentcomposition areas (gray colour), in-clusions (particles of 1020 m) forhigher sandblasted pressure, and bro-ken border areas. In SLA specimens, itwas possible to notice more homoge-

neous microroughness according toSLA treatment with more micro- andnanopores (2 m150 nm), roughnesscrest (100200 nm), and deep macro-pores (25 m). SEM back scatteredanalysis of FCC specimens showedregular and plane surfaces with circu-lar pores (10 m700 nm), crests sim-ilar to volcanoes, and macrocircularpores (10 m; Figs. 6A, 6B).

At EDAX analysis in all SABsamples, it was possible to observe

higher values of titanium (Ti), alumi-nium (Al), calcium (Ca), and oxygen(O2) and lower values of silicon (Si),sulphur (S), and chrome (Cr) residuesof sandblasted treatment. The SLAimplant group showed higher values

only of Ti and lower value of O2.EDAX analysis of all FCC specimensdetected higher values of Ti and phos-phorous (P), and lower values of O2and Ca on the surfaces (Fig. 5).

Osteoblast-Like Cells Onto

Titanium Surfaces

SEM analysis was used to evalu-ate cell morphology, cell distribution,and cell adhesion rate on different ti-

tanium coating. After culturing for 7days onto SAB titanium coating, it ispossible to notice a low number ofosteoblast cells (200 m), strongly ad-hering to the surface. At higher mag-nification (5020 m), the osteoblastcells appeared extremely flattened,with a typical shuttle morphology andlong filopodia in close contact witheach other and the material surface.After 7 days, SLA titanium specimensshowed an increased number of cells

on the coating (200 m) with conflu-ent monolayer cells in different smallareas. At higher magnification in thoseareas, osteoblast-like cells showed aninitial confluence phase and adherenceonto the sample surfaces with an in-creased number of elongated filopodiaand lamellipodia (5010 m). After 7days, culturing onto FCC titaniumcoating was possible to evaluate ahigher number of cells growing on thetitanium surface, distributed aroundmore samples areas, and the typicalnet morphology tended to form a con-fluent layer on the surface. At highermagnification (5020 m), the osteo-blast cells network showed a greatcoating adhesion. A great number offilopodia and lamellipodia indicated aclose contact to each other and is pos-sible to observe an initial ECM deposi-tion. At 20 m magnification, it waspossible to evaluate the flattened cellmorphology and strongly adhered osteo-blast cell net on the nanostructure of the

volcanoes FCC surface (Fig. 7).

Fig. 6A. Upper panel: SEMand EDAX analysis of SAB implant surface. Middle panel: SEM andEDAX analysis of SLA implant surface. Lower panel: SEM and EDAX analysis of FCC implantsurface.

Table 1. Mean Values of the Profilometric Analysis Lead on the Three Different

Implant Samples

Specimens Ra

(m) Rz (m) Rq (m)

SAB 1.35 0.15 14.11 1.08 51.66 1.89

SLA 1.41 0.19 12.87 1.39 21.91 1.30

FCC 0.75

0.09 6.23

1.06 25,37

0.94



7/12

DISCUSSIONThe osseointegration process is a

complex mechanism for predictableclinical results of implant dentistry.

Recent studies show that surface treat-

ment and macro- and microroughnessmorphology may direct the process ofosseointegration by influencing im-portant initial phases of differentiation

of osteoblasts, cell adhesion onto the

surface, full cell proliferation, andECM mineralized deposition to sur-faces both in vitro and in vivo.3,8,27

Some studies have shown that dentalimplants with a high nanoroughnessvalue may promote bone formation

around the implant fixture, maximizebone healing, and improve bone bond-ing for predictable clinical results.5,6

The SEM and the profilometric Tisamples scans found a high roughnessvalue in all the specimens examined.The Rq value of SAB implants con-firmed the irregular macroroughnessof the samples, with highest peaks anddeepest valleys according to the spe-cific surface treatment. The profilo-metric FCC and SLA average showed

some Rq range values, with homoge-neous microtopography with regularcavity distribution. In FCC samples,bevelled peaks appear alternated withmicrovalleys and nanoporosities. Inthis study, FCC testing demonstratedvalues of roughness (Ra, Rz, and Rq) ofmostly regular surfaces comparablewith values of sandblasted large-gritsurfaces and sandblasted and acid-etched surfaces. Moreover, EDAXanalysis showed the presence of alarge number of residual molecules(Al, Ca, O2, Si, S, and Cr) of SABtreatment on the sample surfaces,whereas SLA and FCC surfaces werevery clean and pure with the presenceof residual products (Ca and P) morebiocompatible with bone tissue. Totest the ability of osteoblast-like cellsobtained from hDPaSCs to proliferateonto surfaces commonly used forcraniofacial implantology and to eval-uate their usefulness for tissue engi-neering, we tested these cells on discswith SAB, SLA, and FCC titaniumsurfaces. SEM observation showed agood osteoblast-like cells growth andadherence on this latter surface. Os-teoblast culture on titanium discs after20 days found few cell colonies aggre-gated in a restricted areas of SABcoating and a lot of cell colonies ontoSLA and FCC samples. With highermagnification, it was possible to notethe presence of few cells nearer to theSAB specimens. Typical osteoblastic

morphotype with poor lamellipodia

Fig. 6B. Upper Panel: SEM and EDAX analysis of SAB implant surface. Middle panel: SEMand EDAX analysis of SLA implant surface. Lower panel: SEM and EDAX analysis of FCCimplant surface.



8/12

and long filopodia intracellular con-nections could be observed. After 20days, the specimens, with high-magnification SEM analysis, showedareas with cell aggregates arranged al-most uniformly and forming a cellculture layer on the disc with moreintracellular connections. At the sametime, high-magnification SEM analy-sis of FCC coating showed a greatamount of cell proliferation adherentto the surface and many filopodia andlamellipodia connected to each otherto form a thick net. At higher magni-fication SEM analysis, it is possible toobserve the presence of typical exocy-tosis bubbles, evidence of large cel-lular activities, and, moreover, thepresence of initial (ECM) matrix gran-ule deposition onto the osteoblastic

stem cell surfaces.

CONCLUSIONOur research demonstrates that

nanostructured surfaces can influence invitro the early stage of osteogenesis withintense cellular reactivity. The FCCtreatment eliminates surface contami-nants and results in a consistent and

reproducible Ti oxide surface layer withtotal biocompatible residues (Ca and P),which is characterized by unique nano-topography. The interacting osteoblast-like cell coatings exhibit the typicalmorphology of migrating cells and ad-hesion with a big cell network, broadlamellar leading, and long filamentoustraining edges well supported by the cy-toskeleton. However, biologicalosteoblast-like cell activity is clear forthe comparison of the proliferationnumber, the closed adhesion behavior

onto the surface, the cellular secretion

with numerous typical bubbles ofexocytosis, and the presence of initialextracellular nodule of matrix deposi-tion. The enhanced early cellular be-havior and the surface nanostructureinfluence the kinetics of the initialphases of bone healing. Therefore, itwill be interesting to follow-up thetitanium nanostructure effects duringformation and the later stage of invitro osteogenesis and in vivo bonerepair for a new generation of dentalimplants in which nanocoating, aloneor with cells, can lead to faster and morepredictable results of bone healing.

Disclosure

The authors claim to have no finan-cial interest in any company or in any of

the products mentioned in this article.

REFERENCES1. Branemark PI, Adell R, Breine U, et

al. Intra-osseous anchorage of dentalprostheses. I. Experimental studies. Scand

J Plast Reconstr Surg. 1969;3:81-100.2. Branemark PI, Zarb GA, Albrektsson

T, eds. Tissue Integrated Prostheses: Os-seointegration in Clinical Dentistry. Chicago,IL: Quintessence Publishers; 1985:87-102.

3. Martin J, Schwartz Z, Hemmert T, etal. Effect of titanium surface roughness onproliferation, differentiation, and protein syn-thesis of human osteoblast-like cells (MG63).

J Biomed Mater Res. 1995;29:389-401.4. Wennerberg A, Albrektsson T, Jo-

hansson C, et al. Experimental study ofturned and grit-blasted screw-shaped im-plants with special emphasis on effects ofblasting material and surface topography.Biomaterials. 1996;17:15-22.

5. Cochran D, Schenk R, Lussi A, et al.Bone response to unloaded and loaded tita-nium implants with sandblasted and acid-etched surface: A histometric study in thecanine mandible. J Biomed Mater Res.1998;40:1-11.

6. Cooper LF. A role for surface topog-raphy in creating and maintaining bone attitanium endosseous implants. J ProsthetDent. 2000;84:522-534.

7. Wen X, Wang X, Zhang N. Micro-rough surface of metallic biomaterials: Aliterature review. Biomed Mater Eng. 1996;6:173-189.

8. Buser D. Titanium for dental applica-tions (II): Implants with roughened sur-faces. In: Brunette DM, Tengvall P, TextorM, et al, eds. Titanium in Medicine. Berlin,Germany: Springer; 2001:876-888.

9. Davies JE. Understanding peri-implant endosseous healing. J Dent Educ.

2003;67:932-949.

Fig. 7. SEM observation of SAB (Aand B), SLA (C and D), and FCC (E and F) implant surface

after osteoblast-like cells culturing.



9/12

10. Smith DC. Surface characteriza-tion of implants: Biological implications. In:Davies JE, ed. Bone Biomaterial Interface.

Tor onto, Canada: Tor onto UniversityPress; 1991:9-17.

11. Bowers K, Keller J, Randolph B, etal. Optimization of surface micromorphol-ogy for enhanced osteoblast responses invitro. Int J Oral Maxillofac Implants. 1992;7:302-310.

12. Gronthos S, Brahim J, Li W, et al.Stem cells properties of human dental pulpstem cells. J Dent Res. 2002;81:531-535.

13. Antonucci I, Iezzi I, Morizio E, et al.Isolation of osteogenic progenitors from hu-man amniotic fluid using a single step cultureprotocol. BMC Biotechnol. 2009;9:9.

14. Miura M, Gronthos S, Zhao M, etal. SHED: stem cells from human exfoli-ated deciduous teeth. Proc Natl Acad SciU S A. 2003;100:5807-5812.

15. Kasugai S, Shibata S, Suzuki S, et

al. Character izat ion of a system of mineralized-tissue formation by rat dentalpulp cells in culture. Arch Oral Biol. 1993;38:769-777.

16. Shi S, Robey PG, Gronthos S.Comparison of human dental pulp andbone marrow stromal stem cells by cDNAmicroarray analysis. Bone. 2001;29:532-539.

17. Tete S, Nargi E, Mastrangelo F, etal. Changes in matrix extracellular phos-phoglycoprotein expression before andduring in vitro osteogenic differentiation ofhuman dental papilla mesenchymal cells.Int J Immunopathol Pharmacol. 2008;21:309-318.

18. Mastrangelo F, Nargi E, Carone E,et al. Tri-dimensional response of humandental follicular stem cells onto a synthetichydroxyapatite scaffold. J Health Sci.2007;20:819-826.

19. De Coppi P, Bartsch G Jr, SiddiquiMM, et al. Isolation of amniotic stem celllines with potential for therapy. Nat Bio-technol. 2007;25:100-106.

20. Laino G, DAquino R, Graziano A,et al. A new population of human adultdental pulp stem cells: An useful source ofliving autologous fibrous bone tissue (LAB).

J Bone Miner Res. 2005;20:230-239.21. Nakamura Y, Hammarstrom L,

Matsumoto K, et al. The induction of repar-ative dentine by enamel proteins. IntEndod J. 2002;35:407-417.

22. Iohara K, Nakashima M, Ito M, et al.Dentin regeneration by dental pulp stemcell therapy with recombinant human bonemorphogenetic protein 2. J Dent Res.2004;83:590-595.

23. Owen TA, Aronow M, Shalhoub V,

et al. Progressive development of the ratosteoblast phenotype in vitro: reciprocalrelationships in expression of genes asso-ciated with osteoblast proliferation and dif-ferentiation during formation of the boneextracellular matrix. J Cell Physiol. 1990;143:420-430.

24. Bradford MM. A rapid and sensitivemethod for the quantitation of microgramquantities of protein utilizing the principle ofprotein-dye binding. Anal Biochem. 1976;72:248-254.

25. Gregory CA, Gunn WG, Peister A,et al. An Alizarin red-based assay of min-eralization by adherent cells in culture:Comparison with cetylpyridinium chlo-ride extraction.Anal Bioch em. 2004;329:77-84.

26. Buser D, Schenk R, Steinemann S,et al. Influence of surface characteristicson bone integration of titanium implants.

A histomorph ometri c study in miniaturepigs. J Biomed Mater Res. 1991;25:889-902.

27. Wennerberg A, Albrektsson T,Andersson B, et al. A histomorphometricand removal torque study of screw-shapedtitanium implants with three different surfacetopographies. Clin Oral Implants Res. 1995;6:24-30.

Abstract TranslationsGERMAN / DEUTSCH

AUTOR(EN): Stefano Tete, MD, DDS, Filiberto Mas-trangelo, MD, DDS, PhD, Raimondo Quaresima, RaffaeleVinci, MD, Gilberto Sammartino, MD, DDS, Liborio Stup-pia, MD, and Enrico Gherlone, MDEinfl uss einer neuartigen Nano-Titan-Implant at-

Oberflache auf das Verhalten und das Wachstum men-

schlicher Osteoblasten

ZUSAMMENFASSUNG: Zielsetzung: Die vorliegendeStudie zielt darauf ab, das Verhalten sowie das Wachstum men-schlicher Osteoblastartiger Zellen bei Vorliegen dreier unter-schiedlicher Titan-Implantat-Oberflachen zu untersuchen.Materialien und Methoden: Zuerst wurden menschlicheStammzellen entnommen, dann gema FACS aus mesenchy-malen Stammzell-Clustern menschlicher Zahnpapillenaussortiert. Die so gewonnenen menschlichen Zahnpapillen-Stammzellen (hDPaSCs) wurden zur Ausdifferenzierung in Os-teoblastartige Zellen gebracht und dann mittels RT-PCR undWestern-Blot-Test analysiert. Die Zellen teilten sich und wurdenauf drei unterschiedliche Titanscheiben (Sab, SLA,FCC) aufge-

tragen und mittels Rasterelektronenmikroskopie analysiert.

Ergebnisse: Bei allen Analyseproben konnte eine groe Zellak-tivitat festgestellt werden. Dabei war eine fur Osteoblastentypische reife morphostrukturelle Antwort auf rauen Oberfla-chen zu beobachten. Auf den Titan-FCC-Scheiben fand sicheine hohe Anzahl an Osteoblastartigen Zellen. Gleichzeitig er-wies die Rasterelektronenanalyse eine groe Biokompatibilitatdieser Oberflache. Schlussfolgerung: Die schnelle Reifung derOsteoblastartigen Zellen auf FCC-Titan-Oberflachen lassen denSchluss zu, dass diese Struktur eine zentrale Rolle bei den erstenStufen des Knochenheilungsprozesses spielen konnte.

SCHLUSSELWORTER: Menschliche Zahnpapillen-Stammzellen (hDPaSCs), Osteogenetische Differen-zierung, Titanoberflache, FCC-Oberflache (Full ContactCoverage)

SPANISH / ESPAOLAUTOR(ES): Stefano Tete, MD, DDS, Filiberto Mas-trangelo, MD, DDS, PhD, Raimondo Quaresima, RaffaeleVinci, MD, Gilberto Sammartino, MD, DDS, Liborio Stup-

pia, MD, y Enrico Gherlone, MD



10/12

Influencia de la nano superficie de un novedoso implante

de titanio en el comportamiento y crecimiento de osteoblas-

tos humanos

ABSTRACTO: Proposito: El objetivo de este estudio esinvestigar el comportamiento y crecimiento de celulas huma-nas parecidas a osteoblastos ante la presencia de tres super-ficies diferentes de implantes de titanio. Materiales yMetodos: Celulas humanas madre se obtuvieron primero yluego se organizaron por FACS grupos de celulas madremesenquimales de la papila dental humana. Las celulas de lapapila dental humana obtenidas (hDPaSCs) fueron inducidasa que se diferencien entre celulas parecidas a osteoblastos yluego analizadas por RT-PCR y Western blot. Las celulasproliferaron y fueron cultivadas en tres discos diferentes detitanio (Sab, SLA, FCC) y analizadas por SEM. Resultados:En todos los analisis, se observaron muestras de alta actividadcelular, con la respuesta morfoestructural tpica de osteoblas-tos maduros sobre una superficie aspera. El numero alto de

celulas parecidas a osteoblastos se encontro en discos detitanio FCC. Al mismo tiempo, el analisis SEM confirmo laalta biocompatibilidad de esta superficie. Conclusion: Larapida maduracion de las celulas parecidas a osteoblastos enla superficie de titanio FCC sugiere que esta estructura podrajugar un papel central durante las fases iniciales de los pro-cesos de curacion del hueso.

PALABRAS CLAVES: Celulas madres de la papila dentalhumana (hDPaSCs), diferenciacion osteogenica, superficie detitanio, superficie con cobertura completa de contacto (FCC)

PORTUGUESE / PORTUGUSAUTOR(ES): Stefano Tete Medico, Cirurgiao-Dentista,Filiberto Mastrangelo, Medico, Cirurgiao-Dentista, PhD, Rai-mondo Quaresima, Raffaele Vinci, Medico, GilbertoSammartino, Medico, Cirurgiao-Dentista, Liborio Stuppia,Medico e Enrico Gherlone, MedicoInfluencia de Superfcie Recente de Implante de Nano

Titanio sobre o Comportamento e o Crescimento de Osteo-

blasto Humano

RESUMO: Objetivo: O objetivo do estudo e investigar o

comportamento e o crescimento de celulas tipo osteoblastohumano na presenca de tres diferentes superfcies de implantede titanio. Material e Metodos: Celulas-tronco humanas fo-ram primeiro obtidas, entao separadas por FACS a partir deagrupamentos de celulas-tronco mesenquimais de papilasdentarias humanas. As Celulas-Tronco de Papila Dentariahumana obtidas (hDPaSCs) foram induzidas a diferenciar-seem celulas tipo osteoblasto e foram entao analisadas porRT-PCR e Western blot. As celulas proliferaram e foramcultivadas em tres diferentes discos de titanio (Sab, SLA,FCC) e analisadas por microscopia por varredura de eletrons.Resultados: Em todas as amostras de analise uma alta ativ-

idade celular foi observada, com tpica resposta morfoestru-

tural madura de osteoblasto em superfcie aspera. O altonumero de celulas tipo osteoblasto foi encontrado em discosde Titanio FCC. Ao mesmo tempo, a analise da microscopiapor varredura de eletrons confirmou a alta biocompatibilidadedesta superfcie. Conclusao: A rapida maturacao das celulastipo osteoblasto em superfcie de titanio FCC sugere que essa

estrutura poderia desempenhar um papel central durante asfases iniciais de processos de cura do osso.

PALAVRAS-CHAVE: Celulas-Tronco de Papila DentariaHumana (hDPaSCs), diferenciacao osteogenica, superfcie detitanio, superfcie de Cobertura de Contato Total (FCC)

RUSSIAN /: Stefano Tet, , , Filiberto Mastrangelo, , -

, , Raimondo Quaresima, RaffaeleVinci, , Gilberto Sammartino, , , Li-borio Stuppia, , Enrico Gherlone, -

-

. .

- . . ,

(Fluorescence Activated CellSorter, FACS) . (human Dental PapillaStem Cells, hDPaSC) , , - -. , a

(Sab, SLA, FCC),

. .

. FCC. ,

. . -

FCC



11/12

,

.

: (hDPaSC), , - , (Full Contact Coverage, FCC)

TURKISH / TU RKCEYAZARLAR: Stefano Tete, MD, DDS, Filiberto Mas-trangelo, MD, DDS, PhD, Raimondo Quaresima, RaffaeleVinci, MD, Gilberto Sammartino, MD, DDS, Liborio Stup-pia, MD, and Enrico Gherlone, MDYeni Nano Titanyum Implant Yuzeyinin Insan Osteoblast-

larnn Davrans ve Buyumesi Uzerine Etkisi

OZET: Amac: Bu calsmann amac, uc adet degisik tita-

nyum implant yuzeyinin varlgnda insan osteoblastlarnabenzer hucrelerin davranslarn ve buyumesini arastrmakt.

Gerec ve Yontem: Once insan kok hucreleri alnd ve ardn-dan FACS yontemi ile insan dental papillasndaki mezenki-mal kok hucre kumelerinden sraland. Elde edilen insanDental Papilla Kok Hucrelerinin (iDPKH) osteoblast-benzerihucrelere farkllasmas saglandktan sonra bunlar RT-PCR veWestern blot yontemleri ile analiz edildi. Hucreler cogald ve

uc degisik titanyum diskte (Sab, SLA, FCC) kultur yaplpSEM ile analiz edildi. Bulgular: Tum analiz orneklerindepuruzlu yuzeyde tipik olgun osteoblast morfo-yapsal yantile birlikte yuksek hucre aktivitesi goruldu. En yuksek saydaosteoblast-benzeri hucreler, titanyum FCC disklerinde bu-lundu. Ayrca, SEM analizi bu yuzeyin buyuk olcude biyo-uyumlu oldugunu teyit etti. Sonuc: FCC titanyum yuzeyindeosteoblast-benzeri hucrelerin hzla olgunlasmas, bu yapnnkemik iyilesme surecinin baslangc evrelerinde onemli bir roloynayabilecegini dusundurmektedir.

ANAHTAR KELIMELER: Insan Dental Papilla Kok Hucre-

leri (iDPKH), osteojenik ayrmlasma, titanyum yuzey, FullContact Coverage (FCC) yuzey

JAPANESE /



12/12

CHINESE /

KOREAN /


Date post:	04-Apr-2018
Category:	Documents
Upload:	sergio-di-vitantonio
View:	220 times
Download:	0 times

Influence of Novel Nano-titanium

Documents