J Periodontol July 2006

Case Series
A Histologic Evaluation of Various Stages of Palatal Healing Following Subepithelial Connective Tissue Grafting Procedures: A Comparison of Eight Cases
Kristi M. Soileau* and Robert B. Brannon

Background: It is often necessary to procure tissue from the same area of the palate in cases where connective autogenous grafting procedures are warranted due to limitations caused by anatomical features. The purpose of this study was to determine whether the length of time between a rst procurement and a second would have any bearing on the quality of tissue available for recipient sites. Methods: Eight patients requiring more than one grafting procedure underwent surgery at baseline and again at various intervals ranging from 6 weeks to 11 months. Specimens were taken from the palate and evaluated microscopically, and photographs were taken for purposes of visual comparison. Results: The 6.9- to 7.7-week specimens exhibited complete reepithelialization. The lamina propriae were composed of a cellular proliferation of broblasts with loosely arranged collagen deposition and an occasional thin vascular channel. However, remodeling of the wound appeared complete in the specimens removed at the 9-week interval and beyond. The lamina propria was, in general, composed of thick, dense, interlacing bundles of collagen. Small-caliber blood vessels were interspersed throughout the brous element. Conclusion: Reharvesting of tissue performed earlier than at 9 weeks may result in poorer autogenous graft quality due to indications that remodeling of the connective tissue is still progressing and not as mature as specimens noted at weeks 9 to 47. J Periodontol 2006;77:1267-1273. KEY WORDS Grafts; healing; histology; palate; tissue.

n recent decades, reconstruction of damaged periodontal tissue has taken on an increasingly important role in surgical periodontics. It is because of the similarity of reconstructive periodontal surgery to the medical specialty that many clinicians have referred to these rened approaches as periodontal plastic surgery. Recent emphasis on periodontal reconstruction coincides with restorative dentistrys emphasis on esthetics, and although the emergence of excellent bonding agents has amplied restorative possibilities, bonding alone cannot address mucogingival defects. The restorative dentist should consider reconstructing damaged periodontal structures before recommending cosmetic bonding or altering pontic design.1 Exclusive use of dentin bonding cannot reproduce the functional and esthetic characteristics of soft tissue. This is particularly true in cases of single tooth recession where contour and harmony are essential for optimal esthetics. Once a restoration has been placed on the root, soft tissue grafting becomes difcult without extensive root preparation. Also, in cases of progressive recession, additional loss of attachment can negate the esthetic results of the restorative therapy. Therefore, it is in the patients best interest to include reconstructive periodontal surgery as a principal option in cases of recession. Connective tissue grafting has expanded our options in this area. In cases of gingival recession, periodontal root coverage procedures can create a naturally esthetic result that will blend with the adjacent tissue. Ideal results can be obtained with the replacement of lost periodontal tissues followed by bonding of any residual defects. The two go hand and hand, enhancing our ability to restore form and function in a manner not possible previously. The development of the connective tissue graft has signicantly improved treatment options and predictability. Autogenous subepithelial connective tissue grafting has been used in the eld of periodontics since rst described by Edel2 as a technique for increasing

* Private practice, New Orleans, LA. Department of Oral and Maxillofacial Pathology, Louisiana State University School of Dentistry, New Orleans, LA.

doi: 10.1902/jop.2006.050129

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attached tissue in areas of inadequate gingival width and for root coverage. More recently, with the introduction of the subepithelial connective tissue graft performed by Langer and Calagna3 to build edentulous ridges and by Langer and Langer4 to cover roots, procedures have become more successful, esthetic, and predictable. Several modications in the surgical procedure have been and are currently being made.5-9 The principal applications of the connective tissue graft include the following: 1) root coverage with specic applications in cases of esthetic concern, progressive recession, and recession where marginal tissue inhibits hygiene, causes root sensitivity, and increases risk of root caries; 2) edentulous ridge augmentation or recontouring before restorative therapy; 3) gingival augmentation in conjunction with orthodontics or restorative dentistry; and 4) tissue buildup around implants to enhance esthetics.1 Although the option of donor grafting is a viable alternative for some clinicians, many, and perhaps the majority, still choose to procure tissue directly from the patient undergoing surgical therapy. Regardless of the nal destination, the palate is the most commonly used donor site when considering autogenous soft tissue grafting. The hard palate is composed of the palatal process of the maxillary bone and the horizontal process of the palatine bone. It is covered with masticatory mucosa. The soft tissue extending superiorly from the cemento-enamel junction (CEJ) of the maxillary posterior teeth for ;2 to 4 mm is composed of dense lamina propria. The connective tissue continuing on to the midline of the palate contains loosely organized glandular and adipose tissue. The height, length, and thickness of donor tissue that can be obtained varies with the different anatomic dimensions of the palatal vault. The greatest height (inferior-superior dimension) can be found in the high (U-shaped) palatal vault. The greatest length (anterior-posterior dimension) can be found in a large palate. The thickest tissue can be found in that area from the mesial line angle of the palatal root of the rst molar to the distal line angle of the canine. A thick alveolar process and/or exostosis is often encountered in the molar region, which limits the length and thickness of tissue that may be obtained. The clinician can determine the thickness of the donor tissue by needle soundings following the administration of local anesthesia. The greater and lesser palatine nerves and blood vessels gain entrance into the palate by passing through the greater and lesser palatine foramina. The foramina locations vary, but generally can be identied apical to the third molar at the junction of the vertical and horizontal parts of the palatine bone. These nerves and vessels course anteriorly within a bony groove. The neurovascular bundle may be located 7 to 17 mm from the CEJs of the maxillary pre1268

molars and molars. Prior to making the initial incision to procure the donor tissue from the palate, the surgeon should attempt to palpate the bony groove. This palpation allows the surgeon to determine the maximum apical placement of the incision that is possible before violating the neurovascular bundle. When the palatal vault is shallow, the neurovascular structures will be located more proximally to the CEJ. When the palatal vault is high (U shaped), the structures will be located at a greater distance from the CEJ. The retrieval of donor tissue from the premolar region in the high and average palates offers a greater margin of safety than retrieval from the shallow palatal vault. The mean height of the palatal vault in an adult male is 14.90 mm with a SD of 2.93 mm. The mean height in an adult female is 12.70 mm with a SD of 2.45 mm. Caution must always be exercised not to violate the neurovascular bundle when obtaining the donor tissue, but extreme caution must be exercised when the palate is shallow.10 The area of procurement generally occurs a minimum of 2 mm from the gingival margin in the region located between the distal of the cuspid and mesial to the midpoint of the maxillary rst molar in an effort to stay away from the greater palatine artery and nerve. In the hard palate, mucosal thickness increases with greater distances from the marginal gingiva. The mucosa over the palatal root of the maxillary rst molar is signicantly thinner than at all other positions in the hard palate.11 The desired thickness of the tissue procured depends on the intended use. For root coverage, 1.5 to 2 mm is typically adequate, whereas ridge augmentation procedures require a much thicker graft. Some fatty or glandular tissue may be inadvertently included in the graft, which generally is not considered a problem.1 Predictable root coverage can be anticipated on roots without signicant interproximal loss of attachment, as in those labeled as Class I and II in Millers classication.12 Root coverage becomes increasingly less predictable in areas with greater interproximal bone loss, as in Classes III and IV in Millers system.12 In obtaining autogenous donor tissue, the subepithelial graft technique typically involves the making of two parallel palatal incisions ;2 mm apart, with the incisions carried along to the free gingival margin of the teeth adjacent to the site selected for procurement of donor tissue. Oftentimes, it is necessary to repeatedly use the same area of a patients palate for consecutive grafting procedures due to limitations caused by inadequate tissue or vascular anatomy. Therefore, if there is a shortage of tissue necessary for treating all of the sites, a patient may ultimately need grafting, unless the same palatal donor site is often used on more than one occasion. The purpose of this study was to

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determine whether the length of time between two separate surgical procedures, both using the same palatal donor site, would have any bearing on palatal healing and readiness for a second surgical take. Thus, should there be a minimal healing time prior to reinvading the palate so as to maximize donor tissue value and function? Therefore, the issue is whether or not there should be a 9-week minimal healing time prior to reinvading the palate so as to maximize donor tissue value and function, according to the histologic ndings presented in this study. MATERIALS AND METHODS Patient Population and Study Design Eight patients from a private periodontal practice in New Orleans, Louisiana, were selected to participate in the study (age range: 31 to 62 years; seven females and one male). Subjects were made aware of the case presentation submitted, and the study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000. It was necessary that none of the patients were smokers and that their medical histories were unremarkable. Additionally, all patients required grafting of several teeth such that a one-stage procedure would not have been adequate to achieve an increased gingival width for all of the teeth scheduled for treatment. Two patients underwent surgery at baseline and between 6.9 and 7.7 weeks; one patient had surgery at baseline and at 9.0 weeks; two patients had surgery at baseline and between 12.4 and 13.4 weeks; two patients had surgery at baseline and between 22.9 and 26.3 weeks; and one patient had surgery at baseline and at 47.3 weeks. Therefore, these eight patients were divided into ve groups according to healing intervals (Table 1). All surgeries were completed between 2003 and 2004. Surgical Procedure The presurgical workup included review of allergies, medications being taken, and medical history changes since the patients last visit. All patients were instructed as to the discontinuation of aspirin and aspirin-like products, vitamin E, and alcohol consumption for the 10 days prior to surgery in an effort to prevent bleeding problems at or following surgery. An impression was taken so that a palatal stent could be used postoperatively, and patients were generally given prescriptions for propoxyphene with acetaminophen, alprazolam 0.5 mg, valdecoxib 20 mg, and doxycycline 100 mg, the latter of which was to be taken daily for 2 weeks. At the time of surgery, medical history was again reviewed and monitoring of blood pressure, SaO2, and electrocardiogram (EKG) was performed on all patients throughout the procedure. One hour prior to surgery, patients re-

Table 1.

Time Intervals Between Surgical Procedures

ceived 0.5 mg alprazolam and 20 mg valdecoxib. A general polishing was performed on all teeth, and the patients were given a 0.12% chlorhexidine gluconate solution to swish with for 30 seconds. Marcaine 0.5% with 1:200,000 epinephrine was administered in the recipient surgery site, whereas xylocaine 2% with 1:50,000 epinephrine was administered in the palatal donor site. The donor site received a total of ;0.9 to 1.2 ml per surgery, with some being administered following surgery when deemed necessary for postoperative bleeding control. After preparing the recipient site for accepting the graft, the palatal donor tissue was procured. A doublebladed scalpel was employed, using 1.5 mm of width between the two blades, and an incision was made between the mesial of the rst molar and the distal of the cuspid and ;3 mm from the free gingival margin of the nearby teeth. The incision was made until resistance was felt on the osseous tissue, and the incision was carried horizontally through an amount of tissue necessary to fulll that length of tissue required at the recipient site. The ends of the graft were released by using a 15C blade, and the graft was lifted from the palate. Photographs were taken of each donor graft site, and the last 2 mm of each piece of donor tissue taken from each patient was submitted for histologic evaluation of collagen organization and maturation. The area from which the piece sent for histologic testing came was also measured with a probe and documented so that, with photographs, it would be certain that the same area would be procured at the reharvesting for a comparative histologic evaluation. As stated in Table 1, reharvesting occurred at ;6 weeks and at 2, 3, 6, and 11 months. All of the surgically
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removed specimens were submitted to the Department of Oral and Maxillofacial Pathology, Louisiana State University School of Dentistry, to ensure that an ofcial pathology report was issued. All cases were evaluated microscopically using the standard hematoxylin and eosin (H&E) stain. Following surgery, the palatal site was covered by a surgical acrylic stent, as did Rossmann and Rees13 for bleeding control and minimization of discomfort, which had been fabricated prior to surgery. However, sutures, acrylates, or dressings were not employed on the palatal donor sites in this study. RESULTS Clinical Findings All eight patients healed as expected with no unforeseen adverse occurrences. Discomfort was typically diminished by day 10, at which time wound closure appeared to be clinically complete in all eight cases. An area of slight redness persisted for up to 4 weeks, at which time color began to blend with surrounding palatal tissue, as did tissue contour. Recovery of sensation did not occur for months in some cases. Additionally, a certain shrinkage of all grafts was noted at the recipient sites as maturing occurred. Histologic Findings The histologic ndings of the eight hard palate specimens at the time of the initial surgery (baseline) showed normal palatal mucosa and submucosa. The specimens were composed of orthokeratinized or parakeratinized stratied squamous epithelium with well-formed rete ridges (Figs. 1 and 2). The lamina propria possessed thick dense bundles of collagen with small-caliber blood vessels scattered throughout (Figs. 3 and 4). The group I specimens that were removed at the 6.9-week (48 days) to 7.7-week (54 days) intervals Figure 2.
Normal (baseline) palate from case 8 showing keratinized stratied squamous epithelium overlying dense collagenous tissue. Adipose tissue and small-caliber blood vessels are in the lower one-third of the specimen (H&E; original magnication 40).

Figure 3.
Normal (baseline) palate lamina propria from case 5 composed of dense interlacing collagen bundles (H&E; original magnication 100).

Figure 1.
Normal (baseline) palate from case 5 showing keratinized stratied squamous epithelium overlying dense collagenous tissue (H&E; original magnication 40). 1270

Figure 4.
Normal (baseline) palate lamina propria from case 8 composed of dense interlacing collagen bundles (H&E; original magnication 100).

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showed that reepithelialization of the wound was complete. The lamina propria was composed of a cellular proliferation of broblasts with a loosely arranged deposition of collagen bers and an occasional thin vascular channel (Figs. 5 and 6). These ndings indicated that remodeling of the connective tissues was still in progress. Remodeling of the wound appeared complete in the specimens removed at the 9-week (63 days) interval and beyond. Reepithelialization was complete, and the lamina propria was composed of thick, dense, interlacing bundles of collagen. Small-caliber blood vessels were interspersed throughout the brous element (Figs. 7 through 9). DISCUSSION Regarding the recipient site, Rossmann and Rees13 followed patients an average of 10 months and found

Figure 7.
Reepithelialized wound of case 6 at 160 days. The lamina propria is composed of dense collagenous tissue with several small-caliber blood vessels interspersed throughout (H&E; original magnication 40).

Figure 5.
At 48 days, note the loosely woven collagen bers in the lamina propria of case 1. A thin-walled blood vessel is at the far left of the photograph (H&E; original magnication 100).

Figure 8.
The lamina propria of case 6 at 160 days exhibiting interlacing bundles of dense collagen. There is a capillary in the center and lower left of the photograph (H&E; original magnication 100).

Figure 6.
At 54 days, numerous spindle-shaped brocytic nuclei amid loosely woven collagen bers are noted in the lamina propria of case 2 (H&E; original magnication 100).

Figure 9.
Case 7 at 184 days reveals a densely collagenous lamina propria. A capillary is at the far left of the photograph, and the tip of a rete ridge is at the top left (H&E; original magnication 100). 1271

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a mean shrinkage of recipient grafts of 24% in total surface area. In the palatal donor site, healing is mainly by primary intention, and postoperative discomfort is dramatically decreased compared to the free gingival graft (FGG). In beagle dogs, Squier et al.14 found that actin-containing cells with the ultrastructural characteristics of myobroblasts (contractile broblasts) are present in the granulation tissue of healing skin and oral muscosal wounds and may be responsible for the wound contraction observed clinically in the healing palatal mucoperiosteum. Cornelissen et al.15 found myobroblasts in rat palatal wound tissue between days 4 and 22, with the highest density at 8 days postwounding. The number of collagen type I and type II bers gradually increased until about 8 days postwounding, and thereafter the staining intensity of collagen type III bers decreased. At 60 days postwounding, there were more transversely oriented collagen type I bers and fewer type III bers and elastin present in the submucosa than in normal tissue. This suggests that wound contraction occurs in the mucosa mainly between 4 and 22 days.15 The parallel single incision (PSI) method was used in expectation of a more rapid surface healing/epithelialization due to proposed earlier advanced healing with this technique. In a comparative study of clinical healing in the donor site between patients treated with a trap door (TD) technique or with a PSI, which uses a scalpel with parallel blades, Harris16 recorded a high rate of sloughing of the supercial ap (11 out of 13 cases) for the TD group. Del Pizzo et al.17 showed faster reepithelialization in the SI group than in the TD or FGG groups they studied. However, Del Pizzo et al.17 showed no statistically signicant differences regarding the return of sensibility among the SI, TD, or FGG groups they studied; complete recovery of sensation occurred in at least 8 weeks in a number of patients. Discomfort was absent or minimal in the TD and SI groups, whereas the FGG group reported more problems postoperatively in the rst 2 weeks after surgery. Clinically, eating habits showed a faster return to normal by the second week, whereas 25% of the TD and 34% of the FGG groups still followed a soft food diet.17 Interestingly, Pedlar18 demonstrated that a rugae does not regenerate if it is removed; thus, a return to normality does not occur.18 In the Kahnberg and Thilander study19 on palatal healing in rats, epithelialization was noted to have progressed from the wound borders, and reduction of the wound surface proceeded by contraction of the wound margins and by epithelial cell migration. After 3 weeks, the defects were completely covered with epithelium, although epithelial rete pegs had not yet developed in the central part of the former defect.19 Squier and
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Kremenak20 looked at healing of mucoperiosteal wounds in beagle dogs by use of ultrastructural stereology. When the quantitative data from healing wounds was compared to values from normal, unwounded control tissue, values for the periosteum and submucosa returned close to control values 13 days following wounding, whereas those values for the subepithelial layer and lamina propria were still signicantly different from the controls at 18 days. Thus, Squier and Kremenak20 concluded that there is a gradient of healing between the bone and the surface epithelium, and complete repair and remodeling of the tissue is likely to take longer than 18 days. Donn21 studied three submerged connective tissue autografts that were evaluated at 4, 7, 10, 14, 20, 30, and 90 days, and at 1.5 and 4 years for similarities in a woundhealing study. One wound-healing sequence was actually presented to evaluate the role of the specicity of connective tissue in the creation of a new gingival attachment procedure. In Donns study,21 he admits that difculty often exists in obtaining adequate amounts of donor tissue. He also notes that healing of the palate after 1 month will not leave any depression at the site, and thus the area can usually be entered again for additional connective tissue material.21 It is for this particular reason that this research bears clinical relevance. Should there be a 9-week minimal healing time prior to reinvading the palate so as to maximize donor tissue and function? Because maturation of tissue was notably better at 63 days versus the 48- and 54-day specimens, an additional study following biopsies daily from the 6- to 9-week intervals may further elucidate that particular point at which an acceptable collagen maturation is truly reached. ACKNOWLEDGMENTS The authors thank Dr. James Weir, Department of Oral and Maxillofacial Pathology, Louisiana State University School of Dentistry, for his assistance in photographing the histologic specimens and Dr. David DeGenova (spouse of Dr. Soileau) for contributions toward the layout and design of electronic images. This paper is dedicated to Dr. Roland Meffert, postdoctoral professor and mentor of Dr. Soileau at the Louisiana State University School of Dentistry. REFERENCES
1. Cabrera PO. Connective tissue grafting: An option in reconstructive periodontal surgery. J Am Dent Assoc 1994;125:729-737. 2. Edel A. Clinical evaluation of free connective tissue grafts used to increase the width of keratinized gingiva. J Clin Periodontol 1974;1:185-196. 3. Langer B, Calagna LJ. The subepithelial connective tissue graft. A new approach to the enhancement of anterior cosmetics. Int J Periodontics Restorative Dent 1982;2(2):22-33.

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4. Langer B, Langer L. Subepithelial connective tissue graft technique for root coverage. J Periodontol 1985; 56:715-720. 5. Raetzke PB. Covering localized areas of root coverage employing the envelope technique. J Periodontol 1985;56:397-402. 6. Nelson SW. The subpedicle connective tissue graft. A bilaminar reconstructive procedure for the coverage of denuded root surfaces. J Periodontol 1987;58:95-102. 7. Journee D. Partially buried connective tissue grafts for gingival recession: Clinical results apropos of 22 cases (in French). J Parodontol 1989;8:339-346. 8. Harris RJ. The connective tissue and partial thickness double pedicle graft: A predictable method of obtaining root coverage. J Periodontol 1992;63:477-486. 9. Zabalegui I, Sicilia A, Cambra J, Gil J, Sanz M. Treatment of multiple adjacent gingival recessions with the tunnel subepithelial connective tissue graft: A clinical report. Int J Periodontics Restorative Dent 1999;19: 199-206. 10. Reiser GM, Bruno JF, Mahan PE, Larkin LH. The subepithelial connective tissue palatal donor site: Anatomic considerations for surgeons. Int J Periodontics Restorative Dent 1996;16:130-137. 11. Studer SP, Allen EP, Rees TC, Kouba A. The thickness of the masticatory mucosa in the human hard palate and tuberosity as potential donor sites for ridge augmentation procedures. J Periodontol 1997;68: 145-151. 12. Miller PD. A classication of marginal tissue recession. Int J Periodontics Restorative Dent 1985;5(2):8-13. 13. Rossmann JA, Rees TD. A comparative evaluation of hemostatic agents in the management of soft tissue graft donor site bleeding. J Periodontol 1999;70:13691375.

14. Squier CA, Leranth CS, Ghoneim S, Kremenak CR. Electron microscopic immunochemical localization of action in broblasts of healing skin and palate wounds of beagle dog. Histochemistry 1983;78:513-522. 15. Cornelissen AM, Stoop R, Von den Hoff HW, Maltha JC, Kuijpers-Jagtman AM. Myobroblasts and matrix components in healing palatal wounds in the rat. J Oral Pathol Med 2000;29:1-7. 16. Harris RJ. A comparison of two techniques for obtaining a connective tissue graft from the palate. Int J Periodontics Restorative Dent 1997;17:260-271. 17. Del Pizzo M, Modica F, Bethez N, Priotto P, Romagnoli R. The connective tissue graft: A comparative clinical evaluation of wound healing at the palatal donor site. A preliminary study. J Clin Periodontol 2002;29: 848-854. 18. Pedlar J. Healing following full thickness excision of human palatal mucosa. Br J Plast Surg 1985;38: 347-351. 19. Kahnberg KE, Thilander H. Healing of experimental excisional wounds in the rat palate. (I) Histological study of the interphase in wound healing after sharp dissection. Int J Oral Surg 1982;11:44-51. 20. Squier CA, Kremenak CR. Quantitation of the healing palatal mucoperiosteal wound in the beagle dog. Br J Exp Pathol 1982;63:573-584. 21. Donn BJ. The free connective tissue autograft: A clinical and histologic wound healing study in humans. J Periodontol 1978;49:253-260. Correspondence: Dr. Kristi M. Soileau, 2820 Napoleon Ave., Ste. 470, New Orleans, LA 70115. Fax: 504/8991237; e-mail: [email protected]. Accepted for publication January 6, 2006.

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