Tissue healing under provisional restorations with ovate pontics: A pilothuman histological study

Giovanna Orsini, DDS, MSc, PhD,a Giovanna Murmura, DDS, PhD,b Luciano Artese, MD, DDS,c

Adriano Piattelli, MD, DDS,d Marcello Piccirilli, BSc,e and Sergio Caputi, MD, DDSf

Department of Stomatology and Oral Science, University G. d’Annunzio of Chieti-Pescara, Chieti, Italy

Statement of problem. Ovate pontics mimic the natural tooth contour and provide an esthetic resulthowever, few studies have evaluated the histological changes in underlying tissues.

Purpose. The purpose of this pilot study was to histologically evaluate the healing of gingival tissues in contactwith provisional ovate pontics after 2 weeks.

Material and methods. Three patients requiring fixed partial dentures participated in this study. The provi-sional restorations consisted of a fixed partial denture having 2 ovate pontics: one in acrylic resin (Jet), with anovate shell made of low-fusing ceramic (Duceram LFC); the other made completely of the same acrylic resin(control). After 2 weeks, biopsies of the gingival tissues beneath the pontics were retrieved for histologicalexamination, and immunohistochemistry for evaluation of the vascular endothelial growth factor (VEGF) wasperformed. Data were analyzed using descriptive statistics.

Results. Clinically, all of the ovate pontic–prepared sites showed partial healing. Histologically, the thickness ofthe entire mucosa was similar in both specimens; however, in some regions, the epithelium presented ulcerationsthat were generally deeper and more frequent in control sites than in test sites. Immunohistochemical resultsshowed that tissues beneath LFC pontics seemed to be less inflamed since they demonstrated a lower expressionof VEGF (10.7 6 .8) compared to those beneath acrylic resin ovate pontics (33.9 6 2.5).

Conclusion. This pilot study demonstrated that the placement of provisional LFC ovate pontics may beadvantageous for the reparative processes of the underlying tissues. (J Prosthet Dent 2006;96:252-7.)

CLINICAL IMPLICATIONS

The results of this pilot histological study demonstrated that using low-fusing ceramic to fabricateovate pontics in provisional fixed partial dentures may enhance gingival tissue response. Thismay be clinically advantageous to obtain healthy tissue beneath these pontics in a relatively shorttime (2 weeks).

The ovate pontic design is commonly used to main-tain or enhance the soft tissue contours of fixed partialdentures (FPDs).1 The convex design of this pontichas been recommended to fulfil esthetic, functional,and hygienic demands.1,2 Although ovate pontics havebeen placed in posterior or anterior quadrants withequal success, it is essential to establish exact dimen-sions, and to condition the gingival tissue underlyingovate pontics early by using a provisional restorationwith a convex ovate shape, which acts as a template.1,2

This work was partially supported by the Ministry of Education,University, and Research (M.I.U.R.), Rome, Italy.

aClinical and Research Fellow.bResearcher.cProfessor, Department of Pathology.dProfessor, Department of Oral Pathology; Dean of the Dental School

of Chieti-Pescara.eLab Technician.fProfessor, Department of Prosthodontics; Dean, Department of

Stomatology and Oral Science.

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Moreover, since surface roughness results in a simulta-neous increase in plaque accumulation, thereby height-ening the risk of periodontal inflammation, smoothnessof the pontic materials is advised to maintain healthytissues.3 An early clinical and histological study byTripodakis and Constantinides4 showed that increasedpressure from smooth, polished, and glazed convexpontics in patients with excellent plaque control didnot induce inflammation in the adjacent tissues. Low-fusing ceramic (LFC) represents a highly polished,smooth, and homogenous surface.5 It is a leucite-freerestorative material with hydrogen-bonded hydroxylgroups in a glass matrix that has been demonstrated toshow advantageous mechanical properties.6,7 Whenglazed, LFC exhibits similarities to dental enamel andreduces the wear process of opposing enamel.7

Moreover, LFC has been demonstrated to have a higherflexural strength than feldspathic dental ceramics in theoral environment, and to prevent thermal stresses inmetal substructure.6 Tolboe et al8 demonstrated thatthe mucosa under ovate pontics remained healthy,

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irrespective of the pontic material used, when dentalfloss was used regularly. Zitzmann et al9 performed his-tological evaluation of the alveolar ridge mucosa adja-cent to an ovate pontic after 1 year, showing that thesesites were not associated with overt clinical signs ofinflammation. However, no histological examinationof gingival tissues beneath ovate pontics was performedduring the provisional restoration phase of FPDs.Moreover, no reports were identified in the literaturethat examined the biological properties of LFC andthe histological response at an early phase of healing.Thus, biopsies of the alveolar ridge mucosa underlyingprovisional ovate pontics were performed after 2 weeksand histological and immunohistochemical analyseswere carried out.10 Also, since it is known that the repar-ative mechanism is modulated by a physiological processcalled angiogenesis, which normally occurs during em-bryogenesis, wound healing, inflammation, and tumorprogression,11 the expression of a factor of angiogenesis,vascular endothelial growth factor (VEGF), has been in-vestigated to better characterize the soft tissue responseduring this initial stage of healing.12-16 The aim of thispilot study was to histologically evaluate the use ofLFC during the early phase of healing of soft tissuesbeneath ovate pontics, and to immunohistochemicallycharacterize the tissue response by evaluating VEGF.The purpose of this study was to histologically evaluatethe gingival tissue healing beneath ovate pontics madeof acrylic resin and LFC. The research hypothesis wasthat provisional FPDs with ovate pontics fabricated us-ing LFC may be beneficial for gingival tissue healingafter 2 weeks.

MATERIAL AND METHODS

Three patients requiring maxillary FPDs with 2 adja-cent pontic sites participated in the study. The researchprotocol was approved by the Ethics Committee ofthe Department of Stomatology and Oral Science,University of Chieti-Pescara, Chieti, Italy, and all partic-ipants provided informed consent. Two patients had apreexisting edentulous area in the premolar region,and 1 patient, in the central incisor region. No teethhad been recently extracted, and the alveolar ridgeshad been edentulous for more than 1 year. Under localanesthesia, bone height was gauged with a periodontalprobe, and the soft tissues of the edentulous sites wereprepared for ovate pontic design by the same clinician,using a round diamond rotary cutting instrument(#801-016, medium; Brasseler USA, Savannah, Ga).The dimensions of the sites were created to provideappropriate depth, papillary support, and emergenceprofiles to mimic adjacent teeth. The prepared tissuedepth was no closer than 1 mm from the bone to ensuresuitable healing, and this was assessed by probing witha periodontal probe under local anesthesia.1,17 The

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provisional FPDs had 2 ovate pontics, one made ofacrylic resin (Jet Kit; Lang Dental Mfg Co, Wheeling,Ill), with an ovate shell contacting the tissues made oflow-fusing glass ceramic (LFC-ovate, test, DuceramLFC; Dentsply Ceramco, Burlington, NJ), and theother made completely of acrylic resin (control)(Fig. 1). The ovate pontics had tight but noncompres-sive contact with the underlying mucosa, as it was possi-ble to pass dental floss over the pontics.1 Oral hygieneinstructions were given and patients were advised topass dental floss (Oral-B Laboratories, Redwood City,Calif) beneath the pontics once a day. After 2 weeks,the provisional restorations were removed and soft tis-sues at the pontic site apex were clinically evaluated bya blinded examiner. A modification of indices of Loeand Silness18 and Silness et al19 was used for assessmentof mucosal tissues as the Gingiva Index: 0, absence ofinflammation; 1, mild inflammation; 2, moderate in-flammation; and 3, severe inflammation.9 Then, a soft-tissue biopsy, approximately 2.5 3 2.5 mm in size, wasobtained from the residual ridge gingiva and mucosain contact with test and control sites. All specimenswere immediately fixed in 10% neutral buffered formalin(Bio-Optica, Milan, Italy) and then embedded in paraf-fin (Bio-Optica). Three 100-mm sections were obtainedwith a microtome (Leitz 1512 Profilometer; LeitzGmbH, Wetzlar, Germany) and then stained with he-matoxylin-eosin (Bio-Optica) and observed using a lightmicroscope (Laborlux S; Leitz GmbH).

Immunohistochemistry allowed the localization ofVEGF in tissue sections by the use of labeled antibodiesas specific reagents through antigen-antibody interac-tions visualized by a marker such as a peroxidase. Thestaining of VEGF was performed using the streptavi-din-biotin-peroxidase (SABC) method.20 Paraffin sec-tions were dewaxed using xylene (Farmitalia CarloErba, Milan, Italy), rehydrated, and then washed inphosphate-buffered saline (PBS; Sigma-Aldrich, Milan,Italy; pH: 7.4) for 10 minutes. The following steps

Fig. 1. Provisional restoration with 2 ovate pontics in premo-lar region. Note LFC shell (arrow).

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were optimized by automatic staining (OptiMax;BioGenex, San Ramon, Calif). Sections were incubatedwith primary antibody (Santa Cruz Biotechnology,Santa Cruz, Calif) for 30 minutes at room temperature.Slides were rinsed in buffer (BioGenex), and the immu-noreaction was completed with the SABC method, us-ing a secondary biotinylated antibody (Super sensitiveimmunodetection kit; BioGenex). After incubation atroom temperature, the enzyme formed a colored de-posit at the sites of antibody-antigen binding, revealedusing a substrate chromogen solution (liquid DAB sub-strate pack, OptiMax; BioGenex). Then, the specimenswere counterstained with Mayer’s hematoxylin (Bio-Optica) and coverslips were placed. The quantitativeevaluation of VEGF was performed using a light micro-scope (Laborlux S; Leitz GmbH) connected to a high-resolution video camera (3CCD, JVC KY-F55B; JVC,Yokohama, Japan) and interfaced to a monitor and per-sonal computer (PC) (Intel Pentium III 1200 MMX;

Fig. 2. Ovate pontic-restored sites after 2 weeks demon-strated partially healed tissue and no clinically evident dif-ference between test (left) and control (right) in maxillarypremolar area.

Fig. 4. Control site presenting ulceration and granulation tis-sue characteristic of inflamed zone (arrows). Hematoxylin-eosin staining. Original magnification 310.

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Intel, Santa Clara, Calif). This optical system was associ-ated with a digitizing pad (Matrix Vision GmbH,Oppenweiler, Germany) and a histomorphometry soft-ware package with image-capturing capabilities (Image-Pro Plus 4.5; Media Cybernetics Inc, Silver Spring,Md). The evaluation was done at the level of the endothe-lial cells lining the vessels. For each specimen, 5 high-power fields (HPFs), corresponding to 1.1 mm2, weremeasured. After distinguishing 2 different intensities ofthe expression of VEGF, low and high, the evaluationwas performed in each field. The intensities were recog-nized by the PC software as yellow and red, respectively.20

All patients were clinically reexamined after 8 weeksto assess the level of healing.

For each subject, the mean values and SDs of high-and low-intensity VEGF expression, derived from the5 HPFs, were evaluated.21 For test and control groups,the mean values and SDs for high- and low-intensityVEGF regions were also reported. The analyses wereperformed using statistical software (STATA version8.2; Stata Corp, College Station, Tex).

RESULTS

After the removal of the provisional restorations withthe ovate pontics, the clinical evaluation of the 3 patientsshowed only small amounts of detectable plaque on thepontic surfaces. Both test and control sites in contactwith pontic surfaces, evaluated through a modificationof the Gingiva Index,9,18,19 showed similar clinical fea-tures with signs of partial healing (Fig. 2). Indeed,Gingiva Index scores were almost identical, as all wereequal to 2 (moderate inflammation, moderate redness,edema, and bleeding on probing) except for 1 controlsite, which scored 3 (severe inflammation, marked red-ness and hypertrophy, tendency to spontaneous bleed-ing). At the 8-week recall, inflammation appeared to

Fig. 3. Light micrographs showing test site presenting healthygingival tissue with intact epithelium (between arrows).Hematoxylin-eosin staining. Original magnification 34.

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be reduced and the gingival tissues showed only mildinflammation and a slight change in color.

Histological specimens retrieved after 2 weeks undertest pontics (made of a shell of LFC) were compared tothose obtained from the control pontics (made of con-ventional acrylic resin). The thickness of the mucosaand the epithelium were similar in the specimens

Fig. 5. Immunohistochemical preparations showing VEGFexpression in stromal cells and vessel endothelium (arrows)of test site. Original magnification 340.

Fig. 7. Histomorphometry of immunostaining for VEGF intest site. Original magnification 320. Prevalence of low-intensity (yellow) expression of VEGF is observed.

Table I. Histomorphometry of immunohistochemicalcharacteristics of gingival specimens of patients at2 weeks: high-intensity expression of VEGF

Test Control

Patient Mean (SD) Median (min-max) Mean (SD) Median (min-max)

1 10.0 (1.4) 10.1 (8.2-12.1) 31.4 (3.5) 33.2 (25.8-34.5)

2 11.5 (1.5) 11.4 (9.3-13.4) 36.4 (3.0) 35.3 (33.5-41.2)

3 10.7 (2.2) 11.0 (7.8-13.1) 34.0 (4.8) 35.3 (28.7-39.6)

OCTOBER 2006

harvested from both sites. There were some regions ofthe epithelium that presented ulceration, generallydeeper and more frequent in control than in test sites(Figs. 3 and 4). In the area immediately underlying theepithelium, there were signs of inflammation in all spec-imens, demonstrating abundant newly formed vascularnetworks. The VEFG was positive, in both test and

Fig. 6. Immunohistochemical preparations showing VEGFexpression in stromal cells and vessel endothelium (arrows)of control site. Original magnification 320.

Fig. 8. Histomorphometry of immunostaining for VEGF incontrol site. Original magnification 320. Prevalence ofhigh-intensity (red) expression of VEGF is observed.

Table II. Histomorphometry of immunohistochemicalcharacteristics of gingival specimens of patients at2 weeks: low-intensity expression of VEGF

Test Control

Patient Mean (SD) Median (min-max) Mean (SD) Median (min-max)

1 35.1 (2.6) 34.3 (32.9-39.4) 8.7 (1.1) 9.1 (7.2-10.1)

2 34.7 (3.6) 34.8 (29.2-39.2) 9.9 (2.2) 10.6 (6.4-12.1)

3 29.7 (6.2) 28.7 (21.3-37.2) 11.6 (1.2) 11.1 (10.5-13.2)

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control sites, in endothelial vascular cells, stromal cells,and inflammatory cells (Figs. 5 and 6). For each subject,the VEGF mean values, derived from measuring 5HPFs, are reported in Table I (high-intensity expres-sion) and Table II (low-intensity expression). Themean values and SDs for the high-intensity VEGF re-gions (red cells) were lower for the group of 3 subjects re-ceiving LFC pontics values (10.7 6 0.8) than the grouptreated with acrylic resin ovate pontics (33.9 6 2.5)(Figs. 7 and 8). In contrast, mean values and SDs forthe low-intensity VEGF regions (yellow cells) were higherfor the group of patients receiving LFC pontics (33.2 6

3.0) than controls (10.1 6 1.9).

DISCUSSION

The research hypothesis that fabricating provisionalovate pontics using LFC may benefit early response ofunderlying gingival tissue was partially accepted.Indeed, no evident clinical advantages were seen after2 weeks of ovate pontic provisional restoration in 3 pa-tients, since the sites treated with LFC were clinicallysimilar to those receiving an acrylic resin pontic. In con-trast, histology showed that the residual ridge gingivaltissue under the test LFC pontic appeared less inflamedthan the tissue under the acrylic resin pontic, as mucosalregions presenting ulceration, granulation tissue, andinflammatory cells were more frequently observed incontrol sites than in test sites. Generally, after toothextraction and mechanical preparation of pontic sites,there is a progressive epithelialization of the woundthat results in the formation of a stratified squamousepithelium typical of the gingival tissue.11 The time re-quired for complete healing is variable and depends onvarious factors, one of which is an adequate provisionalmaterial that allows plaque control and ensures healthytissue response.1,8 In postextraction sites conditionedwith ovate pontics, as well as in ovate pontic–preparedsites, a period of at least 3 months is needed for heal-ing.17 The reported histological findings suggest an-other beneficial application of LFC, since this ceramicmay provide a smooth surface that can promote soft tis-sue healing, during the provisional prosthesis, in theearly phase of the reparative process. However, due tothe small sample size, the conclusion that a shell madeof LFC may shorten the waiting period of wound heal-ing required before placing definitive restorations needsfurther investigation.

Another finding suggesting an advantageous re-sponse of the residual ridge gingival tissue induced byLFC may come from the immunohistochemical analysisof angiogenesis. Indeed, angiogenesis is a complex pro-cess that has an important role in developing organs, in-flammation, and wound healing; angiogenesis involvesepithelial cell division, selective degradation of vascularbasement membranes and surrounding extracellular

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matrix, and endothelial cell migration.11-16 Inflamed tis-sues seem to enhance the expression of inflammatorymediators, such as VEGF, which is the most potentand specific agent among the numerous molecules capa-ble of initiating angiogenesis.12 It has been detected invascular endothelial cells, in inflammatory cells, and inthe junctional, sulcular, and gingival epithelium andhas also been implicated in reparative processes.12-16

In this study, both test and control sites expressedVEGF; however, in endothelial cells of control sites, ascompared to those of test sites, there was evidence of ahigher ‘‘high-intensity’’ expression of the VEGF values,as well as lower ‘‘low-intensity’’ expression of the VEGFvalues in the same cells of control sites. Although only3 patients were studied, this pattern may indicate thatangiogenesis is a normal event occurring in wound heal-ing, but that, after 2 weeks, the sites under acrylic resinovate pontics (control) may be more inflamed thanthose underlying LFC ovate shells (test), since they ex-press greater angiogenesis.12,20 Further studies shouldconsider a greater number of patients to assess thepotential clinical advantages of the use of this ceramicin provisional ovate pontics. Moreover, VEGF shouldbe evaluated after a longer period, and future studiesshould be conducted to establish when it would beadvisable to fabricate the definitive FPDs.

CONCLUSIONS

Within the limitations of this pilot study, the resultssuggest that even if no evident clinical advantages canbe definitively determined, a provisional FPD with anovate-shaped pontic made of LFC may be beneficial tothe initial healing process during the interim phase oftreatment.

The authors thank the dental technicians of the Dental Depart-

ments at the University of Chieti-Pescara for their assistance.

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VIA DEI VESTINI 31

66013 CHIETI

ITALY

FAX: 011-39-0871-3554076

E-MAIL: apiattelli@unich.it

0022-3913/$32.00

Copyright � 2006 by The Editorial Council of The Journal of Prosthetic

Dentistry.

doi:10.1016/j.prosdent.2006.08.009

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