Treatment of advanced intrabony defects with enamel matrix proteins (Emdogain®) combined with a bovine-derived xenograft (Bio-oss®)

Introduction

Periodontal regeneration is defined as the healing type following surgical periodontal treatment which results in the formation of a new connective tissue attachment (i.e. new cementum with inserting collagen fibers) and of new alveolar bone (^{Caton and Greenstein, 1993} ; ^{Karring et al., 1997}). Several treatment approaches have been developed during the past decades in order to predictably accomplish this goal (^{Karring et al., 1993} ; ^{Lowenguth and Blieden, 1993} ; ^{Brunsvold and Mellonig, 1993}).

Clinical and histological findings indicate that some types of bone grafts such as intra-or extraoral auto-grafts and demineralized freeze dried bone allograft (DFDBA) may result in periodontal regeneration (^{Schallhorn and Hiatt, 1972} ; ^{Dragoo and Sullivan, 1973} ; ^{Bowers et al., 1989}). On the other hand, treatment of periodontal defects with alloplastic grafts as, for example, various types of synthetic hydroxyapatite results in clinically acceptable responses, but histologically the healing is almost exclusively characterized by connective tissue encapsulation of the graft and a long junctional epithelium without periodontal regeneration (^{Meffert et al., 1985} ; ^{Bowen et al., 1989} ; ^{Yukna, 1993}). Recently, a bovine derived xenograft (BDX) has been successfully used for regenerative periodontal treatment in intrabony defects, ridge augmentation and sinus floor elevation (^{Buser et al., 1990} ; ^{Cohen et al.,1990} ; ^{Clergeau et al., 1996} ; ^{Valentini and Abensur, 1997} ; ^{Camelo et al., 1998} ; ^{Richardson et al., 1999}). The material does not contain any organic components, maintains the natural architecture of bone and resembles most closely to human cancellous bone (^{Valdre et al., 1995} ; ^{Gross, 1997}). BDX shows an excellent osteoconductivity, is very well integrated into bone tissue and is slowly resorbed by osteoclastic activity (^{Chen et al., 1995} ; ^{Skoglund et al., 1997}). Since all organic components are removed, the graft does not elicit any allergic reactions and is clinically very well tolerated (^{Cohen et al., 1990} ; ^{Camelo et al., 1998} ; ^{Richardson et al., 1999}). Furthermore, it does not need a donor site and has an unlimited supply (^{Gross, 1997}). Recent histologic findings in monkeys indicate that BDX is also very well suitable as carrier for BMPs (^{McAllister et al., 1998}). The treatment of intrabony defects with BDX may result in clinically satisfactory results but histologically, it does not have the capacity of preventing epithelial downgrowth (^{Skoglund et al., 1997} ; ^{Camelo et al., 1998}). However, when a GTR membrane was adapted to cover the graft, no epithelium downgrowth occurred and a periodontal regeneration could predictably be achieved (^{Camelo et al., 1998}).

One of the most documented regenerative treatments is at present time GTR which implies the placement of a barrier membrane over the denuded root surfaces and the periodontal defect thus, allowing periodontal ligament and alveolar bone cells to selectively repopulate the isolated space (^{Nyman et al., 1982} ; ^{Gottlow et al., 1986} ; ^{Karring et al., 1993} ; ^{Sculean et al., 1999b} and ^d, and ^2000a and ^b). The clinical and histological outcome of GTR treatment is strongly related to defect configuration, postoperative exposure of the membrane and infection due to subsequent bacterial colonization (^{Selvig et al., 1992} and ¹⁹⁹³ ; ^{Tonetti et al., 1996} ; ^{Karring et al., 1997}, ^{Sculean et al., 2000a} and ^b).

In order to avoid these complications, bioresorbable membranes with similar barrier properties as the non-bioresorbable ePTFE membranes have been developed (^{Cortellini et al., 1996} ; ^{Becker et al., 1996} ; ^{Bouchard et al., 1997} ; ^{Hürzeler et al., 1997}). Controlled histological and clinical studies have further demonstrated that similar results can be obtained with the use of non bioresorbable ePTFE and different bioresorbable membranes (^{Cortellini et al., 1996} ; ^{Becker et al., 1996} ; ^{Bouchard et al., 1997} ; ^{Hürzeler et al., 1997}).

Histological and clinical studies have shown that the use of both non bioresorbable and bioresorbable membranes results predictably in regeneration of 3-and 2-wall intrabony defects as well as of degree II mandibular furcation defects (^{Gottlow et al., 1986} ; ^{Becker et al., 1988} ; ^{Cortellini et al., 1993} and ¹⁹⁹⁶ ; ^{Karring et al., 1993} ; ^{Caffesse et al., 1994} and ¹⁹⁹⁷ ; ^{Hürzeler et al., 1997}).

Recently, enamel matrix protein derivative (EMD) have been shown to promote periodontal regeneration in both animals and humans (^{Hammarström et al., 1997} ; ^{Heijl, 1997} ; ^{Mellonig, 1999} ; ^{Sculean et al., 1999a}, ^b, ^c, ^d and ^2000a, ^b, ^c). Treatment of different types of periodontal defects resulted in the formation of a new periodontal ligament, new cementum with perpendicularly oriented collagen fibers and of new alveolar bone. Furthermore, it was demonstrated that EMD not only stimulates periodontal regeneration but also inhibits the migration of epithelial cells (^{Gestrelius et al., 1997}). The histological and clinical results were comparable to that obtained after GTR treatment, and the obtained clinical results could be maintained over a longer period (^{Pontoriero et al., 1999} ; ^{Sculean et al., 1999b}, ^c, ^d and ^2000a, ^b). Although the regenerative treatment with EMD offers some interesting perspectives to the clinician, some practical problems are still present and need to be solved. One of them is the collapse of the mucoperiosteal flap, which often occurs, especially in deep 1-or 2-wall defects. This in turn may limit the available space for a periodontal regeneration (^{Sculean et al., 1999c}, ^d and ^2000b ). In order to prevent a collapse of the mucoperiosteal flap and to ensure optimal wound stability, it would seem reasonable to combine EMD with a bone graft. In this way, the epithelial downgrowth could be inhibited chemically by EMD and, in the same time, the bone graft would prevent a collapse of the flap, thus, ensuring enough space for the regeneration process.

The aim of the present preliminary study is to present the surgical technique and the one year results following the treatment of intrabony defects with the combination of EMD and BDX.

Material and method

Twelve patients (7 females and 5 males), each of them displaying one advanced intrabony defect were included in this study based on signed informed consent. As disclosed during surgery the intrabony defects displayed a 2-or 3-wall configuration (^{table I}). Criteria for inculsion in the study were :

- no systemic diseases which could influence the outcome of the therapy ;

- good level of oral hygiene (PlI < 1) (Silness and Löe 1964) ;

- compliance with the maintenance program ;

- presence of at least one intrabony defect with a depth of at least 8 mm as detected on the radiographs and measured with a manual periodontal probe (PCP 12, Hu-Friedy, USA) (^{fig. 1}, ⁶, ⁸ and ¹⁰).

Out of the 12 treated patients 2 were smokers (more than 10 cigarettes/day). Three months prior to the surgical procedure all patients went repeatedly through sessions including oral hygiene instructions and professional tooth cleaning. Generally, the Bass' technique ( ^{Bass, 1954}) was recommended, and special efforts were made for achieving a proper interdental cleaning. Additionally to these measures, a full mouth scaling and root planing under local anesthesia was performed. The following clinical parameters were assessed one week prior and one year after the surgical procedure using the same periodontal probe : probing pocket depth (PPD), gingival recession (RG), clinical attachment level (CAL). The surgical procedures were performed by two experienced surgeons (AS and GCC). The measurements were made at 6 sites per tooth - mesiovestibular (mv), midvestibular (v), distovestibular (dv), mesiolingual (ml), midlingual (l), distolingual (dl) - by one, previously calibrated investigator who was not the same as the surgeons. The cemento-enamel junction (CEJ) was used as the reference point. In cases where the CEJ was not visible, a restoration margin was used for these measurements. In the calculations only the deepest site per tooth was included. The statistical analysis was performed with the statistic program SPSS^® for Windows^®. For the statistical analysis the paired t-test was used.

Surgical procedure

All operative procedures were performed under local anesthesia. Following intracrevicular incisions full thickness mucoperiosteal flaps were raised vestibulary and orally. Vertical releasing incisions were performed only if necessary for a better access or to achieve a better closure of the surgical site. All granulation tissue was removed from the defects and the roots were thoroughly scaled and planned using hand and ultrasonic instruments (^{fig. 3} and ⁹). After defect debridement the root surfaces adjacent to the defects were conditioned for 2 minutes with 24 % ETDA (PrefGel^™, Biora-AB, Malmö, Sweden) according to the instructions given by the manufacturer. The defects and the adjacent mucoperiosteal flaps were then thoroughly rinsed with sterile saline in order to remove all EDTA residues. Following root conditioning,, Biora-AB, Malmö, Sweden) was EMD (Emdogain^® applied onto the root surfaces with a sterile syringe. The Emdogain^® gel consisted of two components (a vehicle solution and Emdogain^® in a spongy form) which were stored in the refrigerator and mixed up fresh before each surgical procedure. The remaining Emdogain^® was then mixed with BDX (Bio-Oss^®, Geistlich, Wolhusen, Switzerland). The defects were completely filled with the mixture of Emdogain^® and Bio-Oss^® (^{fig. 4}). Finally, the flaps were repositioned coronally and closed with vertical or horizontal mattress sutures (^{fig. 5}). All patients received antibiotics for 1 week (3 × 500 mg amoxicillin/day). The postoperative care consisted of 0,1 % chlorhexidine (Chlorhexamed^®, Blendamed, Mainz, Germany) rinses twice a day for 6 weeks. The sutures were removed 14 days after the surgery. Recall appointments were scheduled every second week during the first two months after surgery, and once per month following the rest of the observation period. At each recall appointment a reinforcement in oral hygiene measures and professional supragingival tooth cleaning were performed. Neither probing nor subgingival instrumentation were performed during the first year after surgery.

Results

Tooth and defect type, and clinical parameters at baseline and at 1 year after therapy are shown in ^{table I} . At baseline the mean PPD of the treated defects was 10.3 ± 1.6 mm, the mean RG 1.4 ± 1.9 mm and the mean CAL 11.8 ± 3 mm. The postoperative healing was uneventful in all cases. No complications such as allergic reactions, abscesses or infections were observed throughout the study period. At one year after therapy the mean PPD was 4.2 ± 1.9 mm, the mean GR 1.8 ± 1.5 mm and the mean CAL 6.2 ± 3 mm. The mean CAL gain was 5.7 ± 2.1 mm. The PPD reduction and CAL gain were statistically very highly significant (p < 0.0001) whereas the increase in GR was statistically significant (p < 0.01). A hard tissue fill, as observed on the radiographs, occured in all defects (^{fig. 2} and ⁷). Additionally, at one of the sites a reentry procedure was performed and a complete bone fill observed (^{fig. 9} and ¹¹).

Discussion

The results of this preliminary case report study indicate that the treatment of deep intrabony defects with a combination of EMD and BMX may lead to significant PPD reduction and CAL gain. The observation that no adverse reactions such as allergies or abscesses have been observed in any of the patients demonstrates that the combination of these two materials is well tolerated.

This observation is in agreement with the results of other histological and clinical studies which have shown that neither EMD nor BMX elicit allergic or foreign body reactions (^{Cohen et al., 1990} ; ^{Zetterström et al., 1997} ; ^{Heijl et al., 1997} ; ^{Richardson et al., 1999}). The mean PPD reduction and CAL gain observed after the combined treatment was higher than that described after the treatment with EMD alone or BMX alone (^{Heijl et al., 1997} ; ^{Sculean et al., 1999a}, ^b, ^c and ^d ; ^{Richardson et al., 1999}). Results from clinical studies have shown that after treatment with EMD a mean CAL gain ranging from 2.1 mm to 3 mm can be expected (^{Heijl et al., 1997} ; ^{Sculean et al., 1999a} and ^b). In a case report study investigating the treatment with EMD in 32 two-and three-wall intrabony defects a mean CAL of 3 mm was obtained (^{Sculean et al., 1999a}). Similar results were also obtained in a controlled clinical trial comparing the treatment with EMD to that with GTR. At 8 months the CAL gain was 2.9 mm and 2.8 mm respectively, without any statistical significant difference between the two treatments (^{Sculean et al., 1999b}). Histologic findings from monkey and human material indicate that the treatment with EMD does not only result in a clinical improvement, but also to a certain extent in a real periodontal regeneration ( i.e. new periodontal ligament, new cementum with inserting collagen fibers and new alveolar bone) (^{Hammarström et al., 1997} ; ^{Heijl, 1997} ; ^{Mellonig, 1999} ; ^{Sculean et al., 1999c}, ^d and ^2000a, ^b, ^c). The obtained clinical results can be maintained stable, provided that a high level of oral hygiene is ensured (^{Sculean et al., 1999c}). Histological studies have shown that EMD possess the potential for inhibiting or retarding epithelial migration, and also that treatment with EMD results histologically and clinically in comparable results to that of GTR treatment (^{Pontoriero et al., 1999} ; ^{Sculean et al., 1999b}, ^d and ^2000a, ^b). Furthermore, treatment with EMD resulted in less post-operative complications such as membrane exposure and inflammation (^{Sculean et al., 1999b}, ^d and ^2000a, ^b). Evidence from the literature suggests that membrane exposure and subsequent bacterial colonization and infection may jeopardize the outcome of GTR treatment (^{Selvig et al., 1992} ; ^{Simion et al., 1994} ; ^{Sander and Karring, 1995} ; ^{Nowzary et al., 1995}). Thus, the use of EMD instead of a GTR membrane may overcome some of these complications.

In a controlled clinical trial evaluating the outcome of regenerative periodontal treatment in intrabony defects, ^{Richardson et al. (1999)} have treated a total of 30 defects either with DFDBA or with BDX. At 6 months following therapy the soft tissue measurements revealed for the DFDBA group a PPD reduction of 2 ± 1.3 mm and a CAL gain of 2.6 ± 1.6 mm and for the BDX group a PPD reduction of 3 ± 1.7 mm and a CAL gain of 3.6 ± 1.8 mm. These results were statistically and clinically significant compared to baseline. On the other hand, recent results from a human histological study have demonstrated that treatment with BDX alone results only to a limited extent in periodontal regeneration (^{Camelo et al., 1998}). Despite the fact that the graft was very well tolerated and integrated, showing a satisfactory osteo-conductive capacity, it was only to a limited extent able to inhibit epithelial downgrowth (^{Camelo et al., 1998}). In cases where a membrane was additionally adapted in order to cover the graft, no epithelial downgrowth occured and the amount of regenerated tissues was further enhanced. Thus, based on the histological findings it may be assumed that although from a clinical point of view, the use of BDX alone may result in satisfactory results, from a biological point of view, the use of a mechanical or chemical barrier for inhibiting epithelial downgrowth is preferable for obtaining periodontal regeneration with a reasonable predictability. On the other hand, the use of EMD alone may have been limited by an eventual collapse of the mucoperiosteal flap, especially in these deep and large defects. This complication was avoided by combining EMD to BDX and is illustrated by the relatively low increase in recession. The use of BDX may have also accounted for an increase in wound stability which is a crucial factor for obtaining periodontal regeneration (^{Wikesjö and Selvig, 1999}). The clinical relevance of the presented technique is also supported by the high CAL gain measured in this study which was varying from 4 mm to 10 mm (mean 5.7 mm) and is higher than that found in most studies investigating different types of regenerative methods (^{Polson and Heijl, 1978} ; ^{Rosling et al., 1976} ; ^{Bowers et al., 1989} ; ^{Lowenguth and Blieden, 1993} ; ^{Brunsvold and Mellonig, 1993} ; ^{Karring et al., 1997}). It is, however, important to point out that the presented clinical results need to be supported by histological evidence. It is yet not clear whether the CAL gain represents a real periodontal regeneration or only a bone fill without a new connective tissue attachment. Controlled histological and clinical studies are needed to evaluate the quality of the newly formed tissues and to compare this treatment with the single therapies and other regenerative approaches.

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