case report

CASE REPORT Clinical and Histologic Long-Term Evaluation of a Subepithelial Connective Tissue Graft Used as Treatment for a Mucogingival Defect: A Case Report João Carnio* and Paulo M. Camargo†

Introduction: The objective of this case report is to examine the epithelial surface characteristics of a fully submerged subepithelial connective tissue graft (CTG) that was initially performed to treat a mucogingival problem seven years earlier. Surface keratinization of a fully submerged CTG may be desirable, but its predictability is still subject to debate. Case Presentation: Two adjacent lower teeth were treated for recession and minimal amount of keratinized tissue. The surgical technique included flap advancement and the CTG was completely covered with a flap that had mostly a nonkeratinized surface. At 18 months after surgery, the surface of the healed CTG showed mostly no clinical or histologic signs of epithelial keratinization; at that point, the surface epithelium of the whole grafted area was surgically excised and allowed to heal by secondary intention. The absence of keratinization persisted after the second surgical procedure. Conclusion: When fully submerged, a CTG may not induce keratinization of its overlying epithelial surface. Clin Adv Periodontics 2012;2:224-230. Key Words: Connective tissue; gingivoplasty; periodontics.

Background The attached gingiva plays an important role in protecting the periodontium from mechanical trauma induced by toothbrushing and in facilitating plaque control, therefore contributing to the stabilization of the gingival margin at the level of the cemento-enamel junction (CEJ). The presence of minimal dimensions of attached gingiva or its complete absence constitutes a risk factor for the development of the acquired deformity of gingival recession (GR).1-4 * Department of Periodontics, State University of Londrina Center for the Health Sciences, Londrina, Brazil. †

Section of Periodontics, School of Dentistry, University of California, Los Angeles, Los Angeles, CA.

Submitted June 25, 2011; accepted for publication October 24, 2011 doi: 10.1902/cap.2012.110064

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The subepithelial connective tissue graft (CTG), first described by Langer and Langer in 1985,5 is a widely used and effective mucogingival surgical technique in correcting deficiencies in attached gingiva and covering denuded root surfaces.6 Unlike the free gingival graft, the CTG should be partially or totally covered by the pedicle flap that is elevated in the area receiving treatment. As such, the grafted tissue has an increased chance of surviving because there is a maximal blood supply. There are clinical situations in which the flap covering the CTG is composed mostly or entirely of non-keratinized mucosa. Concerns have been raised with respect to the nature (keratinized versus non-keratinized) of the epithelial surface of the CTG after healing.7,8 This case report presents the long-term clinical and histologic results of a CTG that was performed with flap advancement and, therefore, was completely covered with

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a flap with a mostly non-keratinized surface. By following the case for 7 years, it was possible to study whether the epithelial surface of the grafted area changed its characteristics over time.

Clinical Presentation A 37-year-old female was referred to a private practice (JC) in Londrina, Brazil, for treatment of a mucogingival problem on the facial aspect of teeth #21 and #22 in October, 2003. The treatment objectives included an increase in the apico-coronal dimension of the keratinized tissue, creation of a zone of attached gingiva, and possible coverage of the denuded roots on both teeth (Figs. 1 and 2). The patient provided oral consent before the procedures.

Case Management Preparation of the recipient area for the CTG used an envelope technique9 (Fig. 3). The harvested CTG is shown in Figure 4. Care was exercised to stabilize the CTG at the level of the CEJ. The flap was then advanced to cover the whole CTG and sutured (Fig. 5).

FIGURE 3 Split-thickness flap preparation of the recipient site of the CTG.

FIGURE 4 The subepithelial CTG was harvested from the upper left FIGURE 1 Clinical aspect of teeth #21 and #22 at baseline, which

posterior palate and shaped to the dimensions of the recipient site.

presented with mucogingival problems.

FIGURE 2 Clinical aspect of teeth #21 and #22 at baseline after application

FIGURE 5 The CTG was sutured at the CEJ of teeth #21 and #22. The flap

of Schiller’s solution. Note the presence of minimal keratinized tissue.

was advanced to completely cover the CTG and sutured with 6-0 plain gut.

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Clinical Outcomes At 18 months after surgery, the patient underwent a full periodontal examination of the treated area (Table 1). The improvement in the clinical parameters observed at 18 months was considered of limited clinical significance. Clinical examination of the area, however, clearly revealed an increase in the bucco-lingual dimension (thickness) of the whole mucosal/gingival complex (Fig. 6), which was evaluated by horizontal insertion of a periodontal probe through the tissue in a direction that was perpendicular to the bone under local anesthesia. The apico-coronal increase in the dimensions of the keratinized tissue was minimal (Fig. 7). In an attempt to improve the apico-coronal dimension of the keratinized tissue and since the CTG had already been performed, a superficial layer of the mucosa covering the recipient area was surgically excised by gingivoplasty. According to Edel,10,11 if the CTG is left exposed, keratinization is likely to occur. Before the conclusion of the gingivoplasty, which was eventually extended to the mesial aspect of tooth #20, a full-thickness biopsy was taken from the interproximal area between teeth #21 and #22 (Fig. 8). The biopsied tissue measured z1.5 mm in the mesio-distal direction and

FIGURE 7 Eighteen-month clinical view of the surgically treated area after application of Schiller’s solution. Note the minimal increase in the apicocoronal dimension of the keratinized tissue.

TABLE 1 Periodontal Measurements Taken on the

Straight Facial Aspect Parameter

Baseline

18 Months

7 Years

Tooth #

22

21

22

21

22

21

Probing depth (mm)

1.5

1.0

1.0

1.5

1.0

1.0

Keratinized tissue (mm)

1.5

1.0

1.5

1.5

3.0

2.0

Attached gingiva (mm)

0.0

0.0

0.5

0.0

2.0

1.0

GR (mm)

0.5

2.0

0.5

1.5

0.5

1.5

FIGURE 8 Eighteen-month clinical view of the beginning of the mucosal excision procedure on the facial aspect of teeth #21 and #22. The arrows refer to the region from which a full-thickness biopsy was taken for histologic evaluation. Gingivoplasty was extended to mesial aspect of tooth #20 (not shown in the figure).

9 mm in the apico-coronal direction. Approximately 1 mm of keratinized tissue was included in the coronal end of the specimen.

Management of the Histologic Specimen

FIGURE 6 Healing of the surgical area 18 months after treatment. Note the obvious increase in the thickness of the mucosa in most of the surgically treated area compared to Figure 1.

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Immediately after retrieval, the specimen was fixed in 10% formalin for 48 hours, washed in water, and stored in 70% alcohol. The specimen was then embedded in paraffin and oriented so that bucco-lingual sections were obtained. Sections were 5 mm thick. Staining was performed with hematoxylin and eosin (H&E). The section shown in Figures 9 through 12 was acquired from the center region (mesiodistal direction) of the specimen and is representative of most sections observed throughout the whole biopsy as visualized under a microscope.‡ ‡

Leica DM LB microscope, Leica Microsystems, Wetzlar, Germany.

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After a representative section of the specimen was selected, digital images were obtained using a scanner systemx at 200 magnification. The University California, Los Angeles Translational Pathology Core Laboratory performed the digitization of the histologic sections.

Histologic Findings Figure 9 shows the whole specimen, composed of a core of dense well-organized, connective tissue covered by epithelium. The arrow in Figure 10 denotes the mucogingival junction, which is about 1 mm apical to the gingival margin. Rete pegs are evident above the arrow. On the most coronal aspect (1 mm) of the specimen (Fig. 11), the epithelium demonstrates morphologic characteristics of keratinization. The epithelial cells on the surface are mostly flat and do not present with nuclei. Examination of the specimen farther than 1 mm apical from its coronal end reveals that the connective tissue is covered by epithelium that does not show evidence of keratinization (Fig. 12). The epithelial cells are squamous in shape, as typical of the stratum spinosum in non-keratinized epithelium (Fig. 12). Therefore, the microscopic appearance of this epithelial tissue starting at 1 mm apical from the gingival margin is suggestive of non-

FIGURE 10 Histologic section of the area correspondent to the mucogingival junction in Figure 9. Magnification,3; H&E. Arrow denotes mucogingival junction. Area coronal to the arrow shows flattened epithelial cells, suggesting the formation of a stratum corneum. Area apical to the arrow shows squamous cells, suggesting the absence of keratinization.

FIGURE 11 Histologic section of the coronal area shown in Figure 10. Magnification, 10; H&E. Epithelial cells are flat and lost their nuclei, which is typical of a keratinized surface.

FIGURE 12 Histologic section of the apical area shown in Figure 10.

Magnification, 10; H&E. Squamous cells of the stratum spinosum are present, which is typical of a non-keratinized epithelium.

FIGURE 9 Histologic section of specimen retrieved at 18 months (refer to Fig. 8). Magnification, 1; H&E. Tissue consisted of dense connective tissue covered by epithelium.

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x

ScanScope XT System, Aperio Technologies, Vista, CA.


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keratinized mucosa. The histologic appearance of the epithelium in different areas of the specimen, as described above, corresponded to the clinical description of the area shown in Figure 7.

Final Clinical Outcome At 7 years after treatment, another full clinical examination of the area was conducted (Figs. 13 and 14; Table 1).

Discussion This case report shows that epithelial surface keratinization may not occur when a CTG is performed in a submerged manner. The periodontal flap appears to have remained viable during healing and it conferred a nonkeratinized mucosal surface to the grafted tissue. This suggests that the cells originally present on the flap surface retained their phenotype and were not susceptible to any morphologic change that could be induced by the underlying cells from the CTG.12 The only area in which postsurgical epithelial keratinization was observed was on the most coronal area of the graft, which corresponds to the presurgical narrow band of keratinized epithelium included on the flap.

FIGURE 13 Clinical view of the surgical area 7 years after the CTG.

FIGURE 14 Seven-year postoperative view of teeth #21 and #22 after application of Schiller’s solution. Note that some extra increase in the apico-coronal dimension of the keratinized gingiva was achieved compared to Figures 2 and 7.

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Another possible explanation for the absence of epithelial keratinization in the treated area is the fact that the flap that initially covered the CTG presented with a component, albeit thin, of connective tissue. Therefore, it is possible that the epithelial–mesenchymal interaction remained unchanged during and after healing, despite the fact that palatal connective tissue was placed in contact with the internal aspect of the flap connective tissue. Conserving the original epithelial–mesenchymal interaction may have conferred a non-keratinized surface to the grafted area. This report also presents evidence contrary to the concept that surgical excision of the epithelial tissue present over the healed CTG (epithelial abrasion) may result in the formation of a keratinized surface.13 This further supports the notion that the connective tissue cells from the graft do not play a role in determining the phenotype of the epithelial tissue on its surface. Therefore, it can be assumed that the tissue that first comes in contact with the CTG, which in the case of a submerged CTG is primarily non-keratinized mucosa, confers the surface characteristics of the epithelial surface. This is in agreement with findings reported by Maurer et al.,14 who showed that treatment of a healed CTG with gingivoplasty did not result in keratinization of non-keratinized epithelium. Carnio et al.2 showed that the apico-coronal dimension of keratinized tissue can be augmented by the modified apically repositioned flap (MARF), which is a surgical technique that involves the simple apical positioning of a narrow band of keratinized tissue and leaving the underlying connective tissue exposed, in which healing by the secondary intention occurs. With the MARF, keratinized tissue (and attached gingiva) can be created in an area that previously presented with a non-keratinized mucosal surface. The MARF surgical technique essentially creates an island of exposed connective tissue whose perimeter is completely occupied by keratinized epithelium. Presumably, cell migration starts on the borders of the wound and continues toward the center, resulting in the formation of keratinized epithelium. This suggests that what confers a keratinized nature to the epithelium developing on the connective tissue surface is the type of epithelial cells that are first present in the area. With the MARF, these primary colonizing cells are known to be keratinized based on their origin and location. Based on the MARF wound-healing dynamics, it could be speculated that a CTG needs to be left exposed if keratinization were to be expected on its surface. This thought would be in agreement with the suggestions made by Edel.11 Evidently, this theory needs to be confirmed through in vivo experiments. Although bucco-lingual (tissue thickness) measurements of the treated area were not made, it is obvious that there was an increase in tissue thickness after healing of the CTG (Fig. 9). Therefore, the clinical result observed with surgical treatment was that of a thick, bound-down tissue, with a non-keratinized surface. The effectiveness of such newly formed tissue in the maintenance of attachment levels and gingival health compared to the role exerted by keratinized tissue is not known. It has been suggested that treatment of Subepithelial Connective Tissue Graft for a Mucogingival Defect

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mucogingival defects with acellular dermal matrix results in the formation of tissue that clinically15 and histologically16 resembles bound-down, non-keratinized mucosa, similar to that reported in this case report. The long-term stability and effectiveness in the maintenance of gingival health provided by tissue originated by grafting with acellular dermal matrix has not been determined. Based on the interpretation of the results described in this case report, a fully submerged CTG results in the

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formation of tissue with a non-keratinized epithelial surface. Subsequent surgical epithelial removal from the grafted area and healing of that wound by secondary intention does not induce the formation of keratinized epithelium. Therefore, a fully submerged CTG may not be a reliable technique to increase the apico-coronal dimension of keratinized tissue. These findings need to be confirmed by a case series with a larger number of cases. n

Summary Why is this case new information?

j

This case suggests that a fully submerged CTG may not present with surface keratinization after healing.

What are the keys to successful management of this case?

j

Surgical removal of epithelium via gingivoplasty after treatment with a CTG may not result in formation of keratinized epithelial surface.

What are the primary limitation to success in this case?

j

The long-term clinical relevance of a thick, non-keratinized mucosa in the long-term stability of the position of the gingival margin and overall gingival health in not known.

Acknowledgment The authors report no conflicts of interest related to this case report.

Carnio, Camargo

CORRESPONDENCE: Dr. Paulo M. Camargo, Periodontics, School of Dentistry, University of California, Los Angeles, 10833 Le Conte Ave., CHS 63048, Los Angeles, CA 90095. E-mail: [email protected].


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References

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1. Wennstro¨m J, Lindhe J, Nyman S. Role of keratinized gingiva for gingival health. Clinical and histologic study of normal and regenerated gingival tissue in dogs. J Clin Periodontol 1981;8:311-328.

10. Edel A, Faccini JM. Histologic changes following the grafting of connective tissue into human gingiva. Oral Surg Oral Med Oral Pathol 1977;43:190-195.

2. Carnio J, Camargo PM, Passanezi E. Increasing the apico-coronal dimension of attached gingiva using the modified apically repositioned flap technique: A case series with a 6-month follow-up. J Periodontol 2007;78:1825-1830.

11. Edel A. Clinical evaluation of free connective tissue grafts used to increase the width of keratinised gingiva. J Clin Periodontol 1974;1: 185-196.

3. Wennstro¨m JL. Mucogingival therapy. Ann Periodontol 1996;1:671701. 4. Pini Prato GP. Mucogingival deformities. Ann Periodontol 1999;4:98101. 5. Langer B, Langer L. Subepithelial connective tissue graft technique for root coverage. J Periodontol 1985;56:715-720. 6. Bouchard P, Malet J, Borghetti A. Decision-making in aesthetics: Root coverage revisited. Periodontol 2000 2001;27:97-120. 7. Cordioli G, Mortarino C, Chierico A, Grusovin MG, Majzoub Z. Comparison of 2 techniques of subepithelial connective tissue graft in the treatment of gingival recessions. J Periodontol 2001;72:1470-1476. 8. Han JS, John V, Blanchard SB, Kowolik MJ, Eckert GJ. Changes in gingival dimensions following connective tissue grafts for root coverage: Comparison of two procedures. J Periodontol 2008;79:1346-1354.

12. Ouhayoun JP, Sawaf MH, Gofflaux JC, Etienne D, Forest N. Reepithelialization of a palatal connective tissue graft transplanted in a non-keratinized alveolar mucosa: A histological and biochemical study in humans. J Periodontal Res 1988;23:127-133. 13. Levine RA. Covering denuded maxillary root surfaces with the subepithelial connective tissue graft. Compendium 1991;12:568-578, 570, 572 passim. 14. Maurer S, Hayes C, Leone C. Width of keratinized tissue after gingivoplasty of healed subepithelial connective tissue grafts. J Periodontol 2000;71:1729-1736. 15. Allen EP. AlloDerm: An effective alternative to palatal donor tissue for treatment of gingival recession. Dent Today 2006;25: 48, 50-52; quiz 52. 16. Cummings LC, Kaldahl WB, Allen EP. Histologic evaluation of autogenous connective tissue and acellular dermal matrix grafts in humans. J Periodontol 2005;76:178-186.

indicates key references.

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