development of a novel model for the investigation of implant–soft tissue … · 2011. 1. 9. ·...

Development of a Novel Modelfor the Investigation of Implant–SoftTissue InterfaceWen Lin Chai,*† Keyvan Moharamzadeh,† Ian Michael Brook,‡ Lena Emanuelsson,§

Anders Palmquist,§ and Richard van Noort†

Background: In dental implant treatment, the long-termprognosis is dependent on the biologic seal formed by thesoft tissue around the implant. The in vitro investigation ofthe implant–soft tissue interface is usually carried out usinga monolayer cell-culture model that lacks a polarized-cellphenotype. This study developed a tissue-engineered three-dimensional oral mucosal model (3D OMM) to investigatethe implant–soft tissue interface.

Methods: A 3D OMM was constructed using primary hu-man oral keratinocytes and fibroblasts cultured on a skin-de-rived scaffold at an air–liquid interface. A titanium implant wasinserted into the engineered oral mucosa and further culturedto establish epithelial attachment. The 3D OMM was charac-terized using basic histology and immunostaining for cytoker-atin (CK) 10 and CK13. Histomorphometric analyses of theimplant–soft tissue interface were carried out using a light-mi-croscopy (LM) examination of ground sections and semi-thinsections as well as scanning electron microscopy (SEM).

Results: Immunohistochemistry analyses suggests thatthe engineered oral mucosa closely resembles the normaloral mucosa. The LM and SEM examinations reveal that the3D OMM forms an epithelial attachment on the titanium sur-face.

Conclusion: The 3D OMM provided mimicking peri-implantfeatures as seen in an in vivo model and has the potential tobe used as a relevant alternative model to assess implant–soft tissue interactions. J Periodontol 2010;81:1187-1195.

KEY WORDS

Cell; cultured techniques/methods; dental implants;epithelial attachment; immunohistochemistry; mouthmucosa/anatomy & histology; tissue engineering.

Dental implant systems have beenin clinical use for almost 5 de-cades. The long-term success of

the dental implant treatment was firstreported in the late 1970s by Dr. Per-Ingvar Branemark.1 It is well acceptedthat osseointegration is vital for thesuccess of implant treatment. Althoughthe integrity of the implant–hard tissueinterface is essential, the interactionbetween the gingival/mucosal soft tissueand the implant must also be considered.This biologic soft-tissue seal with theimplant is critical to ensure the long-termprognosis of dental implants.2 The peri-implant mucosa responds to plaque ina fashion similar to the healthy gingivathat surrounds teeth.3,4 When the in-tegrity of the biologic seal around animplant is disturbed by the inflammatoryprocess, the underlying bone is exposedto the oral bacterial environment andleads to peri-implantitis.

Histomorphometric analyses5,6 fromanimal models show that the soft tissueattachment around natural teeth and im-plant surfaces had similar features but dif-fered in terms of the qualityof their biologicseal. In natural teeth, the non-keratinizedjunctional epithelium attaches to theenamel surface via the internal basal lam-ina and hemidesmosomes along the en-tire length of the junctional epithelium.However, the attachment of the non-ker-atinized peri-implant epithelium to theimplant surface is confined at the apical

* Department of General Dental Practice and Oral and Maxillofacial Imaging, University ofMalaya, Malaya, Malaysia.

† Academic Unit of Restorative Dentistry, School of Clinical Dentistry, University ofSheffield, Sheffield, U.K.

‡ Department of Oral & Maxillofacial Surgery and Medicine, School of Clinical Dentistry,University of Sheffield.

§ Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg,Gothenburg Sweden.

doi: 10.1902/jop.2010.090648

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region only.6 A further key difference is the orientationof the connective tissue collagen fibers around im-plants compared to the teeth. In teeth, Sharpey’sfibers attach perpendicularly to the cementum,whereas the peri-implant connective tissue fibersare oriented in a circular arrangement at the innerzone and run in different directions at the outer zone.7

The lack of attachment structures such as hemides-mosomes and connective tissue attachment on theimplant surface could contribute to a weaker biologicseal around implants than that of the natural tooth.

There is increased interest among researchers toinvestigate the factors that influence the soft tissueattachment to the implant. Most of these studies arecarried out in animal models,6-16 whereas limitedstudies are conducted in human subjects.17,18

Alternatively, in studies involving cell-culturemodels, primary or cell lines of keratinocytes and/orfibroblasts are used for the investigation of cell re-sponses on different implant surfaces.19-25 Based onthese in vitro models, information such as histomor-phologic features of cell attachment, cell prolifera-tion,23 activity,22,24 and cell adhesive strength21,24

are reported. Most cell-culture models are based onmonolayer systems that have the disadvantages of:1) a lack of polarized cell phenotype, 2) poor cell dif-ferentiation, and 3) the lack of cell-to-cell contact.26

The importance of cell-to-cell interactions and modu-lation is highlighted.27 Thus, a tissue-engineeredthree-dimensional oral mucosal model (3D OMM),which consists of both keratinocytes and fibroblasts,may provide better evidence than the monolayercell-culture system. A full-thickness, tissue-engi-neered oral mucosal model was previously developedfor thebiologicassessmentofdentalmaterialsandoralhealth products.28-31 To our knowledge, there is nostudy published in the literature that indicates theuse of a tissue-engineered human oral mucosa forevaluation of the implant–soft tissue interface. There-fore, this study further developed the 3D OMM intoa relevant in vitro model for the assessment ofimplant–soft tissue interactions.

MATERIALS AND METHODS

Construction of a 3D OMMThe methods used to develop the new 3D OMM werea modification of a previously reported model.31

Ethical approval was obtained from the North Shef-field Research Ethics Committee for using waste oraltissues of patients. The primary cells, i.e., human oralkeratinocytes (HK) and human oral fibroblasts (HF)were harvested from an oral mucosal biopsy ofa 20-year-old woman who underwent surgical re-moval of her wisdom tooth. Written informed consentwas obtained for this study.

The primary oral keratinocytes were isolated fromthe oral mucosal biopsy and cultured using an explanttechnique. The biopsy was first washed three times infreshly prepared 10 ml Dulbecco’s modified Eagle’smedium (DMEM) that contained 50 U/ml penicillin,50 U/ml streptomycin, and 625 ng/ml fungizone.The biopsy was placed in 2.5 mg/ml dispasei over-night in the fridge. Just before isolation, the biopsywas incubated in a CO2 incubator at 37�C for 30 min-utes. After the enzyme reaction, the epithelium waseasily separated from the connective tissue layer.The separated epithelium was cut into tiny pieces us-ing a scalpel blade #22. Subsequently, the explantswere transferred into a T-75 collagen-coated flask¶

and submerged in a minimal volume of fresh Green’smedium (3 to 5 ml) and left incubated for 3 to 5 hoursin a CO2 incubator to ensure explants were attachedon the flask surface. Green’s medium31 consisted ofDMEM# and Ham’s F-12 medium** in a 3:1 ratioand supplemented with 10% fetal calf serum (FCS),††

10 ng/ml epidermal growth factor,‡‡ 0.4 mg/ml hydro-cortisone,§§ 10-4 mol/l adenine,ii 5 mg/ml insulin,¶¶

5 mg/ml transferrin,## 2 · 10-7 mol/l triiodothyro-nine,*** 2 mM/l glutamine,††† 2.5 mg/ml fungizone,‡‡‡

50 U/ml penicillin,§§§ and 50 U/ml streptomycin.iii

After the attachment, about 7 ml Green’s mediumwas gently added to the flask without disturbing the ex-plants. The Green’s medium was changed every 2 to 3days until the keratinocytes became 80% confluent.

To isolate the HF, the connective tissue of the bi-opsy was incubated in 10 ml 0.05% (weight/volume)collagenase type I¶¶¶ at 37�C overnight. After a 24-hour incubation, the tissue-containing medium wascentrifuged at 1,500 rpm for 4 minutes to form HFpellets in a universal vial. The HF pellets were resus-pended in DMEM that contained 10% FCS and platedin a T75 flask. The medium was changed every 2 to 3days until confluent.

The 3D OMMs were constructed inside 12-mm di-ameter inserts with a 0.4-mm pore size and a 1 · 108

pore density/cm2 in a six-well plate.### An acellularhuman-cadaveric dermis**** (about 0.9-mm thick)

i BD Biosciences, Bedford, U.K.¶ BIOCOAT Cell Environments, Becton Dickinson Labware, Bedford,

U.K.# Sigma-Aldrich, Dorset, U.K.** Sigma-Aldrich.†† Biowest, East Sussex, U.K.‡‡ Sigma-Aldrich.§§ Sigma-Aldrich.ii Sigma-Aldrich.¶¶ Sigma-Aldrich.## Sigma-Aldrich.*** Sigma-Aldrich.††† Sigma-Aldrich.‡‡‡ Sigma-Aldrich.§§§ Sigma-Aldrich.iii Sigma-Aldrich.¶¶¶ Gibco BRL, Life Technologies, Paisley, Scotland.### Costar 12mm Snapwell Insert, Corning Life Sciences, Corning, NY.**** Alloderm, LifeCell, Branchburg, NJ.

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was cut into a 12-mm-diameter disk. It was rehy-drated in 5 ml phosphate buffered saline (PBS) for30 seconds and followed by immersion in 5 ml DMEMfor 15 minutes before placing it onto the insert. A co-culture technique was conducted, in which a mixtureof 5 · 105 of HKs and 5 · 105 HFs were seeded onto thebasement-membrane site of the dermis. The tissuewas submerged in Green’s medium for 3 days. Onday 4, a 4-mm-diameter hole was punched in themiddle of the tissue using a sterile disposabletissue-biopsy punch.†††† Four types of titanium (Ti)surfaces, i.e., polished, machined, sandblasted andTiUnite‡‡‡‡ in disk form, were tested (5-mm diameter· 2.5-mm height; a 5-mm grade IV–diameter Ti rodwas donated by the manufacturer, and polished, ma-chined, sandblasted Ti disks were prepared from thedonated Ti rod using in-house equipment). A Ti diskwas inserted into the prepared hole (Fig. 1). Thismodel was covered with medium and incubatedfor another 5 days. The level of the medium was re-duced to create an air–liquid interface (ALI) and topromote epithelial differentiation over a period of 3to 5 days.

The 4-mm-diameter punched tissue was not dis-carded but was further cultured in a 24-well plate ina parallel condition with the 3D OMM. At the end of

the culture, the punch tissue was processed for waxsectioning, stained with hematoxylin and eosin, andimmunostained with cytokeratin (CK) 10 and CK13.A modified immunostaining protocol was carriedout.11 The punch tissues and normal gingival tissue(positive control) were fixed in 10% formalin beforebeing embedded in wax. The sections were dewaxedin xylene and rehydrated through 100%- and 70%-alcohol series for 5 minutes each. Endogenous perox-idase was quenched with 3% hydrogen peroxide inmethanol for 20 minutes and washed in PBS. The an-tigens were retrieved with 0.1% trypsin§§§§ at 37�C for20 minutes. Unspecific proteins were blocked withhorse normal serumiiii for 40 minutes. The sectionswere then incubated overnight at 4�C with mousemonoclonal primary antibodies¶¶¶¶ at a concentrationof 1:50 and 1:200 for CK10 and CK13, respectively.Subsequently, the sections were treated with biotiny-lated anti-mouse secondary antibodies#### for 1 hourat room temperature and followed by incubation in av-idin-biotinylated peroxidase complex***** for 30minutes at room temperature. The reactions were vi-sualized using 0.05% diaminobenzidine (DAB)†††††

and 0.005% H2O2 in 0.05M Tris buffer, pH 7.6, for10 minutes. The sections were counterstained withhematoxylin for light-microscopy (LM) examination.The negative control was similarly processed exceptwithout the primary-antibody incubation.

Interface ExaminationTo examine the 3D OMM–Ti disk interface under LM,two techniques were used: 1) a ground-sectioningtechnique (the Ti disk was retained in the sections)and 2) an electropolishing technique (the Ti diskwas dissolved electrolytically). In addition, cells at-tached to the Ti disk were examined under a scanningelectron microscope (SEM).Ground sections. At the end of the incubation period,the specimens were fixed with 2.5% glutaraldehyde for2 to 3 days and post-fixed with 1% OSO4 for 2 hours.Dehydration was carried out in serial-ascending etha-nol concentrations at 50%, 70%, 90%, 95%, and100% and with 1,2-propylene oxide for 30 minuteseach with two changes for each solution. The speci-mens were preinfiltrated in 1,2-propylene oxide: ep-oxy resin‡‡‡‡‡ (1:1) for 2 hours followed byinfiltration in pure epoxy resin overnight. After that,the specimens were embedded in new epoxy resin

Figure 1.A) The 3D OMMs were constructed on an acellular dermis, which waspositioned in inserts of a six-well plate. TheTi disks were placed in the center of the oral mucosa after 4 mm ofpunched tissue was removed on each model on day 4 of coculture. B)A schematic diagram of the cross-section of a tissue-engineered oralmucosal model with the Ti disk in place.

†††† Stiefel Laboratories, Bucks, U.K.‡‡‡‡ TiUnite, Nobel Biocare Holding, Zurich-Flughafen, Switzerland.§§§§ Sigma-Aldrich.iiii Vector Labs, Burlingame, CA.¶¶¶¶ Vector Labs.#### Vector Labs.***** Vector Labs.††††† Vector Labs.‡‡‡‡‡ Epon, Agar Scientific, Standsted, U.K.

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and polymerized at 40�C for 15 hours and finally at60�C for 48 hours. The embedded specimens werecut in half using a diamond-band saw.§§§§§ One-halfwas re-embedded in acrylic resiniiiii for the prepara-tion of ground sections.

The embedded specimens were sectioned into thinsections of about 100 mm using a diamond-band saw.The thickness of the sections was further reduced to25 to 30 mm by grinding and polishing with silicon car-bide papers¶¶¶¶¶ of P800, P1200, and P2400 rough-ness using a grinding machine.##### Subsequently,the ground sections were immersed in 10% H2O2 for10 minutes and stained with Richardson’s solution32

(equal parts of 1% azure II and 1% methylene blue in1% borax) for 30 minutes before LM examination.

Electropolishing. The other half of the epoxy resin–embedded specimen was processed with an electro-polishing technique to remove the bulk of Ti.33 Briefly,the surface opposite to the cut Ti surface was exposedby grinding with silicon carbide paper (P80).Thespecimen was immersed in an electrolyte solution,which consisted of a mixture of 5% perchloric acid,35% n-butanol, and 60% methanol, that was cooledto -30�C. The specimen served as the anode, anda platinum ring placed around the specimen servedas the cathode. The electropolishing process wasperformed at 200 mA/cm2 and 24 V for about 4 to 5hours. This process dissolved the bulk of the Ti metaland allowed the preparation of 1- to 1.5-mm-thicksemi-thin sections using glass knives on a micro-tome.****** These sections were stained with tolui-dine blue and examined under LM.††††††

SEM. In a machined Ti surface specimen, after thefixation in 2.5% glutaraldehyde, a pie-shaped sectionof the oral mucosa was removed to expose the inter-face. The specimen was further post-fixed in 1% OSO4

for 2 hours, dehydrated with serial ethanol, and criti-cal-point dried. It was sputter coated with gold beforebeing examined under the SEM.

RESULTS

Characterization of the 3D OMMFigure 2 shows the histologic appearance of a punchtissue stained with hematoxylin and eosin. A well-differentiated stratified squamous oral epithelium,which mimicked that of the normal oral mucosa,was observed. The epithelium consisted of four dis-tinct layers that included the basal, spinous, granular,and superficial keratinized layers. The epithelial cellsshow evidence of terminal differentiation toward thesurface of the epithelium. HFs were present withinthe connective tissue layer.

The immunostained 3D OMM and normal oral mu-cosa are compared in Figure 3. The suprabasal celllayer of the 3D OMM showed a strong expression ofCK13 but a weak expression of CK10, which are the

differentiation markers of the non-keratinized andkeratinized epithelium, respectively. This suggeststhat the 3D OMM reveals features closer to that ofthe non-keratinized stratified epithelium than thekeratinized normal oral mucosa.

The ground sections and semi-thin sections, whichwere obtained from the same specimens are com-pared in Figure 4. The interfacial soft tissue appearedto be intact on the Ti disk. In a semi-thin section, moredetails were revealed; e.g., the apical migration of thecells along the surface of the Ti disk was more obviouscompared to the corresponding ground sections.Some cells had noticeably migrated upward alongthe Ti disk. HFs were sparsely scattered in the connec-tive tissue layer.

Two types of epithelial attachment were identified:pocket and non-pocket types. The pocket type of at-tachment manifests as a gap between the epitheliallayer and the Ti disk. The non-pocket type attachmentrefers to the lack of gap between epithelium and the Tidisk. Clinically, the non-pocket type of attachmentcould indicate a more favorable attachment than thepocket type, as the latter has a higher risk of plaqueretention, which may result in peri-implantitis. In thisstudy, the examination of the ground sections or semi-thin sections showed both types of epithelial attach-ment were present in all types of Ti surfaces (Fig. 4).This suggested that the Ti surface topography did notsignificantly affect the soft tissue attachment.

Figure 2.Hematoxylin and eosin staining of a tissue-engineered oralmucosa, whichhas a stratified squamous epithelial layer with some human fibroblastsscattered in the connective tissue layer (original magnification X40).

§§§§§ Exakt Apparatebau, Norderstedt, Germany.iiiii LR White, London Resin Company, London, U.K.¶¶¶¶¶ Buehler, Dusseldorf, Germany.##### Exakt Apparatebau.****** Ultracut Reichert-Jung, ISS Group Services, Manchester, U.K.†††††† Nikon Eclipse E600, Kawasaki, Kanagawa, Japan.


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SEMIn the SEM specimen, the soft tissue appeared to bedetached from the Ti surface (Fig. 5A) either duringthe sectioning of the soft tissue or during fixationand dehydration procedures. Nevertheless, therewas evidence of cells attached on the machined Tisurface at the exposed area. This indicated that thecells from the 3D OMM formed attachments on theTi surface (Figs. 5B and 5C).

DISCUSSION

Because the biologic seal of the soft tissue around theimplant surface is an important factor in determiningthe long-term success of implant treatment, many re-searchers use different study models to investigate the

implant–soft tissue interface.Animal6-16 and human17,18

models have been the reliablestudy models but are con-strained and governed by strictethical procedures. Although invitro cell-culture models19-25

are simpler, they consist ofmonolayers that limit the abilityto extrapolate information tothe in vivo situation. To over-come these limitations, modelsof tissue-engineered oral mu-cosa, consisting of multiplelayers of epithelial cells, wereused in various in vitro investi-gations such as cytotoxicitytesting28,34,35 and micro-organism infection testing.36

There are some commerciallyavailable oral mucosal mod-els‡‡‡‡‡‡ which are culturedfrom normal oral keratinocytes,and others§§§§§§ which are cul-tured from a squamous cell car-cinoma of the buccal mucosa.However, these commerciallyavailable oral mucosal modelsconsist of an epithelial layeronly and lack the connec-tive tissue component. Lockeet al.27 reported that fibroblastsplay an important role in modu-lating epithelial cell differen-tiation. In the present study,a full-thickness model that con-tained both types of cells (i.e.,keratinocytes and fibroblasts)was used to construct the 3DOMM. To our knowledge, thisis the first study that used a tis-

sue-engineered 3D OMM for the investigation of theimplant–soft tissue interface.

The present 3D OMM was modified from an earlierin-house model. The previous model was used forthe investigation of the biocompatibility for dentalmaterials.28,29,31 However, the basement membranematrixiiiiii attached to the Ti surface in the absenceof any cells. Thus, a modification of this model byreplacing the basement membrane matrixiiiiii withan acellular dermis had to be made for the investi-gation of the implant–soft tissue interface.

Figure 3.Immunostaining of the tissue-engineered and normal oral mucosa with CK10 and CK13. A and B =negative control; C and D = CK10; E and F = CK13. The tissue-engineered oral mucosa was positivelystained with CK13 but weakly stained with CK10 (original magnifications x20).

‡‡‡‡‡‡ EpiOral, EpiGingival, MaTek Corporation, Ashland, MA.§§§§§§ SkinEthic HGE, SkinEthic HOE, SkinEthic Laboratories, Nice,

France.iiiiii Matrigel, BD Biosciences, San Jose, CA.


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Using acellular dermis asa scaffold and cocultured withHK and HF, the tissue-engi-neered oral mucosa had fea-tures that mimicked thoseseen in normal oral mucosa(Fig. 3). The primary cellmodels were cultured for 2weeks with 3 to 5 days ofALI toward the end of culture.An optimal duration of ALI fa-cilitates the differentiation ofthe epithelial layer, as was ev-ident in the immunostainingof the tissue in this study.The maindisadvantageofpri-mary cells is that there canbe batch-to-batch variations.Thus, it is suggested that thesame pool of primary cellsshould be used in the sameexperiment for different testsurface comparisons.

In the present model,Green’s medium enrichedwith FCS was used. However,the influence of different cul-ture media on the structuresand adhesion molecules in-volved in the interface wasnot obvious. Because theoral mucosal models pro-duced in this study were nottransplanted to patients, theuse of a serum-containingculture medium was of lessconcern. One study37 sug-gested the addition of 0.5mM calcium into the mediumto enhance the differentiationof the epithelial cells.

Several techniques havebeen explored to examinethe implant–soft tissue inter-face. Ground sectioning, whichinvolves cutting through andgrinding with the Ti in situ, isthe most commonly usedtechnique.6-16 This techniqueis a highly labor-intensive pro-cedure. The advantage ofground sections is that theyprovide information about theposition of the peri-implant tis-sue in relation to the implantsurface at the interface.

Figure 4.A comparison of the soft tissue attachment on four different types of Ti surfaces using ground section and semi-thin section techniques. Two types of soft tissue attachment were identified, i.e., ‘pocket’ and ‘non-pocket’(original magnification x20).


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In contrast, semi-thin sections do not provide therelationship between the soft tissue and Ti surfaceas do ground sections. However, semi-thin sectionsshowed more detail of the structure (Fig. 4). Apicalepithelial migration was more noticeable in thesemi-thin section compared to the correspondingground section because of their lower thickness.

Two types of epithelial attachment were observed(i.e., pocket and non-pocket) on all Ti surfaces, butthe pattern of attachment could not be correlated tothe type of surface topography. In sandblasted andTiUnite specimens, the ground sections showeda pocket type of attachment but appeared as a non-pocket type of attachment in semi-thin sections. Itseems that the 3D OMM formed different types of ep-ithelial attachment on the Ti surface.

SEM is another method that allows in situ exami-nation of the specimens without cutting through theimplant. The advantage of SEM is that it providesa three-dimensional architecture of the implant–tissue interface compared to the LM and TEM, whichonly show two-dimensional features of the inter-face.17 We found that there were residual cells stillattached to the Ti disks after the soft tissue wasremoved (Fig. 5). This observation was in accor-dance with another study14 and suggested that thebond strength of the epithelial cells to the implantsurface was greater than the cell–cell bonding withinthe epithelial layer. However, a major limitation of anSEM examination is that the detailed information onthe nature of the cell organelles involved in the inter-face between epithelial cells and the implant materialis not available.38

Based on several techniques used in the presentstudy to examine the implant–soft tissue interface, itwas found that the 3D OMM formed an epithelialattachment on Ti surfaces. Thus, the model can beused to test different implant surfaces. The histomor-phometric analysis of the interface provides informa-tion on the nature of the soft tissue response to

different surfaces. For example, if there is a gap be-tween the epithelial layer and test surface, it indicatesthat the biologic seal is not as favorable compared tosurfaces that have a close epithelial attachment. Thisnew 3D OMM provides more detailed informationof the soft tissue response to implant surfaces thancan be obtained using a monolayer cell-culturemodel. Therefore, this model is more clinically rele-vant than monolayer cultures and could reduce theneed for animal testing. Further studies on quantita-tive approaches to assess soft tissue attachment toTi surfaces using this 3D OMM are in progress.

CONCLUSION

The 3D OMM provides peri-implant features thatmimick those seen in vivo and therefore, has the po-tential to be used as a relevant alternative model toassess implant–soft tissue interactions.

ACKNOWLEDGMENTS

Dr. Chai was offered a scholarship by The Govern-ment of Malaysia to pursue her PhD study at the Uni-versity of Sheffield. The authors thank the followingsurgeons in the School of Clinical Dentistry, Univer-sity of Sheffield, who helped obtained the oral muco-sal biopsies: Prof. Peter Robinson; Julian Yates; Mr.Raj Patel; and Drs. Simon Atkin, Tariq Khan, GhadaEl Hassey, and Issan Bakri. We appreciate the invalu-able advice from Prof. Peter Thomsen and the techni-cal support from Birgitta Norlindh, who are both fromBIOMATCELL VINN Excellence Center of Biomate-rials and Cell Therapy, University of Gothenburg.The Ti disks and the commercially pure Ti were kindlyprovided by Nobel Biocare Holding, Zurich-Flughafen,Switzerland (Gen 77943 rev00). No financial supportwas received for this study from any commercial firm.The authors report no conflicts of interest related tothis study.

Figure 5.A scanning electron micrograph of an exposed soft tissue interface after the removal of a section of the 3D OMM. A) A segment of 3D OMM was detachedfrom the machined Ti surface. B and C) The residual cells attached on the Ti surface at low and high magnifications, respectively. (Original magnification: A·49; B ·47; C ·376.)


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Correspondence: Dr. Wen Lin Chai, Department of GeneralDental Practice and Oral and Maxillofacial Imaging,University of Malaya, Kuala Lumpur 50603, Malaysia.E-mail: [email protected]; [email protected].

Submitted November 18, 2009; accepted for publicationMarch 19, 2010.


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