Treatment of advanced intrabony defects with an enamel matrix protein derivative

Introduction

The ultimate goal of periodontal therapy is the regeneration of supporting tissues which have been lost following inflammatory periodontal disease ^{(Caton and Greenstein, 1993)}. Various treatment modalities have been used during the last decades in order to accomplish this goal in a predictable way. Up to now the best documented regenerative therapies are the use of various bone grafts and the guided tissue regeneration (GTR) (^{Brunsvold and Mellonig, 1993} ; ^{Karring et al., 1993} ; ^{Tonetti et al., 1998)}. The latter requires the placement of a barrier membrane for covering the periodontal defects, thus allowing periodontal ligament (PDL) cells to selectively repopulate the debrided root surfaces ^{(Nyman et al., 1982} ; ^{Gottlow et al., 1986} ; ^{Karring et al., 1993)}.

Recently, it has been shown that the application of enamel matrix proteins (EMP) onto a debrided and conditioned root surface may also enhance the formation of a new connective tissue attachment with new alveolar bone ^{(Hammarström, 1997)}. Histological findings in monkeys and from a human case report have shown that treatment of acute recession-type defects with EMP may result in the formation of a new attachment apparatus characterized by the presence of acellular cementum with inserting collagen fibers and of new alveolar bone ^{(Hammarström et al., 1997} ; ^{Heijl, 1997)}. Clinically, the treatment of intrabony defects with EMP resulted in a gain of attachment and in radiological hard tissue formation ^{(Heijl et al., 1997} ; ^{Sculean et al., 1999b)}, ^c). However, limited information is available concerning the histological and clinical outcome following treatment of advanced intrabony defects with EMP.

Therefore, the aims of the present study were :

- to evaluate histologically the effect of treating one-wall intrabony defects with EMP ;

- to present the clinical results at one and at two years after treatment with EMP in 20 consecutively treated cases of advanced intrabony defects.

Materials and methods

Pilot study in a monkey

The operative procedures were performed on one adult male monkey (Macaca fascicularis). They were all performed under general anesthesia with Kethalar^® (10 mg/body weight ; Parke Davis Co. Inc., USA). For controlling haemorrhagia in the surgical areas local infiltration anesthesia (Xylocain^®/Adrenalin 2 %, Astra, Copenhagen, Denmark) was additionally delivered.

At the start of the experiment all second premolars were extracted. After a two month healing period intrabony defects with a depth of approximately 6-8 mm measured from the cemento-enamel-junction were produced by means of a slowly rotating cylindric bur at the distal surfaces of all maxillary and mandibular first premolars. In order to enhance dental plaque accumulation and thus mimic chronic periodontal defects, metal strips were placed into the defects and were fixed to the tooth surface in the defect with a composite material. Flap closure was achieved by means of horizontal or vertical mattress sutures. Ten days after surgery the sutures were removed. During the first four weeks following surgery no oral hygiene measures were given. After the fourth week, oral hygiene measures were resumed and consisted of twice per week tooth brushing and topical application of chlorhexidine 0.2 %.

Six weeks after defect creation, full thickness mucoperiosteal flaps were elevated and the metal strips removed. All granulation tissue was removed from the defects and the root surfaces underwent scaling and root planing. Using a small round bur reference notches for histological measurements and indicating the bottom of the defects were prepared on the root surfaces.

The intrabony defects were then randomly treated as follows :

1. enamel matrix proteins derivative (Emdogain^®, Biora AB, Malmö, Sweden) ;

2. or coronally repositioned flaps.

In the defects receiving EMP the root surfaces were conditioned for 2 min with a 24 % EDTA containing gel (Biora AB^®, Malmö, Sweden) according to the instructions given by the manufacturer. Following the removal of the acid rests with copious rinsing with sterile saline, the defects were filled up with EMP. The Emdogain^® gel consists of two components (a vehicle solution and Emdogain^® in a spongy form) which have to be stored in the refrigerator and mixed up fresh before each surgical procedure according to the instructions given by the manufacturer. The Emdogain^® gel was then applied onto the root surfaces and into the defects with a sterile syringe. In order to have a similar situation as in a daily practice in the defects treated with coronally repositioned flaps no conditioning of the root surface was performed. The flaps were repositioned in a vertical position and closed with vertical or horizontal matress sutures.

After the operation the animal received a single dose of intramusculary administred antibiotics (1 ml Clamoxycillin^®, Pfizer, Germany). The sutures were removed after 14 days. Hygiene procedures were performed twice a week including tooth brushing and topical application of 0.2 % chlorhexidine during the first six weeks postsurgery. After this period oral hygiene measures were given once a week as described above. Five months after the last surgical procedure the animal was killed with an overdose of Kethalar^®, and the oral tissues fixed by perfusion with 10 % buffered formalin administered through the carotid arteries. Following this procedure the jaws and surrounding soft tissues were removed and placed in 10 % buffered formalin. The specimens containing the experimental teeth were dissected free, decalcified in EDTA, dehydrated and embedded in paraffin. Mesio-distal serial sections were cut parallel to the long axis of the teeth with the microtome set at 8 µm. Every tenth section was stained with hematoxylin and eosin.

Clinical study

Twenty patients (12 females) aged between 30 and 55 years were included in this study and signed informed consent. The criteria for inclusion in the study were :

a. no systemic diseases ;

b. presence of at least one experimental site showing a clinical attachment loss of at least 10 mm associated with vertical bone loss ;

c. no or moderate mobility at the experimental tooth (mobile teeth were either splinted or included in bridge reconstructions) ;

d. no endodontic problems in the experimental tooth (the treated teeth were either vital or lege artis root canal treated) ;

e. a high level of oral hygiene (Pl I < 1) (^{Silness and Löe, 1964}).

Three months prior to surgery each patient was given thorough oral hygiene instruction, and full mouth supra- and subgingival scaling and root planing under local anaesthesia. The following clinical parameters were assessed by one calibrated investigator (AB) at the experimental site one week prior and twelve months after the surgical procedure using the same manual periodontal probe (PCP 12, Hu-Friedy, USA) : probing pocket depth (PPD), gingival recession (GR) and clinical attachment level (CAL) (^{fig. 5}, ⁹, ¹¹ and ¹⁵). All measurements were estimated at six sites per tooth : mesiovestibular (mb), midvestibular (b), distovestibular (db), mesiolingual (ml), midlingual (l), distolingual (dl). The cemento-enamel junction (CEJ) was used as the reference point. In cases where the CEJ was not visible, a restoration margin was utilized. Only the deepest site per tooth was included in the statistical analysis which was performed with the statistic program SPSS^® for Windows^® using the paired t-test. Hard tissue changes were assessed using conventional radiographs taken with the long cone parallel technique ^{(Sewerin, 1990)} (^{fig. 6}, ¹⁰, ¹², ¹⁶ and ¹⁷).

Surgical procedure

All twenty patients were treated consecutively by the same surgeon (AS). Twelve patients were treated at the Department of Periodontology, University of the Saarland and eight in the private practice of the first author. Under local anaesthesia, intracrevicular incisions were made and full-thickness mucoperiosteal flaps were raised at the vestibular and oral aspect of the experimental tooth. If necessary, vertical releasing incisions were placed mesially or distally in order to obtain optimal access to the defect. After removal of all granulation tissue, the root surfaces underwent a thorough scaling and root planing using hand and ultrasonic instruments (^{fig. 7} and ¹³). No osseous recontouring was performed. The clinical procedures for the conditioning of the root surfaces and application of Emdogain^® were exactly the same for the pilot study is a monkey (^{fig. 8} and ¹⁴). The flaps were repositioned in a vertical position and closed with vertical or horizontal matress sutures. No antibiotics were given post-operatively.

The sutures were removed at 14 days following surgery. The post-operative care included rinses with chlorhexidine digluconate twice per day for the first six weeks post-operatively. During this period no tooth brushing of the operated areas was performed. Tooth brushing was resumed at week seven. No subgingival intrumentation was performed during the first twelve months after surgery. During the first year after therapy recall appointments including oral hygiene reinstruction and professional tooth cleaning were scheduled once per month. After this period the recall appointments were organized once every two months.

Results

Histological results in the monkey

The histological investigation disclosed that in both defects treated with Emdogain^® healing resulted in the formation of a new connective tissue attachment (i.e. new cementum with inserting collagen fibers) and of new alveolar bone coronal to the notch (^{fig. 1}). The newly formed cementum displayed a predominantly acellular character and was only partially attached to the dentin surface (^{fig. 2}). In these both cases the amount of new cementum and of new bone were at the same level and comprised approximately 70 % of the initial defect depth. In the defects treated with flap surgery alone the healing was characterized by the presence of a long junctional epithelium along the instrumented root surfaces and only a minimal amount of periodontal regeneration in the bottom of the defects (^{fig. 3} and ⁴).

Clinical results

Healing was uneventful in all 20 cases. No allergic reactions against Emdogain^® and neither suppuration nor abscesses were observed. However, a slight post-operative swelling at the operated areas was observed in 5 out of the 20 cases. The analysed material consisted of 12 two-wall, 4 one-wall and 4 combined one - and two wall defects. At baseline the mean PPD of the treated defects was 10.0 ± 2.2 mm, the mean GR 1.7 ± 1.6 mm and the mean CAL 11.8 ± 1.9 mm (^{table I}). The clinical parameters twelve months after therapy revealed a highly significant reduction of probing pocket depth and gain of clinical attachment (^{table I} and ^{fig. 10} and ¹⁶). The clinical parameters twelve months after therapy presented a mean PPD of 3.8 ± 1.3 mm, a mean GR of 4.0 ± 1.8 mm and mean CAL of 7.7 ± 1.9 mm. The mean CAL-gain at twelve months after treatment was 4.1 ± 1.7 mm. Two years after therapy the PPD was 4.0 ± 1.3 mm, the GR was 4.1 ± 1.8 mm and the CAL was 8.1 ± 1.8 mm (^{table I}). The CAL-gain at two years was 3.7 ± 1.4 mm. These parameters were highly different compared to the baseline but not statistically significant compared to the one year results (p > 0.05). Radiologically, a new hard tissue formation was observed in all 20 cases. In four cases the radiological hard tissue formation continued over the one year period (^{fig. 16} and ¹⁷).

Discussion

The results of the present study have demonstrated that the application of EMP onto a debrided and conditioned root surface may result histologically in the formation of new cementum with inserting collagen fibers and in alveolar bone regrowth. The fact that in both specimens a periodontal regeneration occured coronal to the notch on the root surface, demonstrates the potential of EMP in enhancing periodontal regeneration. Furthermore, the observation that in these specimens the epithelium downgrowth was very limited, also suggests that EMP may possess some capacity for inhibiting epithelium migration and supports previous in vitro and in vivo observations ^{(Hammarström, 1997} ; ^{Hammarström et al., 1997} ; ^{Gestrelius et al., 1997)}. The fact, however, that in both specimens the newly formed cementum was detached from the root surface does not corroborate previous findings where it was shown that after treatment with EMP the new cementum was always well attached to the root surface and artefacts never occured ^{(Hammarström, 1997} ; ^{Hammarström et al., 1997} ; ^{Heijl, 1997)}. Possible explanations for this discrepancy might be that in the experiments refered to the defects were of an acute-type (i.e. they were surgically created and then immediately treated without a previous dental plaque accumulation period) or that different histological techniques were followed ^{(Caton et al., 1994)}. On the other hand, the observations made in the present study corroborate previous results from a series of animal studies aiming to compare the healing of different types of chronic periodontal defects after treatment with EMP, GTR or combined EMP and GTR ^{(Donos et al., 1998} ; ^{Sculean et al., 1998} ; ^1999a). In these studies the newly formed cementum (although of a predominantly acellular type) was often detached from the root surface and did not appear to be better attached to the root surface than the cementum formed after GTR. Although in all these studies the application of EMP was shown to enhance periodontal regeneration they were not able to prove the superiority of the new attachment formed after treatment with EMP to that formed after treatment with GTR ^{(Donos et al., 1998} ; ^{Sculean et al., 1998} ; ^1999a). On the other hand in a recent study in dogs comparing the treatment of degree III mandibular defects with GTR alone to that of combined EMP and GTR it was shown that although both treatments resulted in comparable amounts of regenerated tissues, certain qualitative differences between the two treatments were present : the cementum formed after the combined treatment was of an acellular type with a higher number of inserting collagen fibers than in the cementum formed after GTR alone ^{(Araújo and Lindhe, 1998)}. Although the clinical relevance of these histological findings is still unclear they indicate that the application of EMP onto a debrided and acid conditioned root surface may selectively enhance acellular cementum formation.

The lack of adverse reactions such as allergies or heavy inflammatory processes as observed in the present study, corroborates previous findings in animals and humans, and demonstrates that EMP is very well tolerated ^{(Hammarström et al., 1997} ; ^{Heijl, 1997} ; ^{Heijl et al., 1997} ; ^{Donos et al., 1998} ; ^{Sculean et al., 1999a)}.^b).^c). Furthermore, recent results from a clinical multicenter study indicate that no adverse reactions to EMP occured in patients which were repeatedly treated with EMP ^{(Zetterström et al., 1997)}. Thus, based on the histological observations it may be assumed that the CAL gain and radiological hard tissue formation does not only represent a clinical improvement but also, to a certain extent, a real periodontal regeneration. The finding that treatment of intrabony defects with EMP results statistically and clinically in an improvement of all clinical parameters has also been reported by others ^{(Heijl et al., 1997} ; ^{Sculean et al., 1999b)}, ^c). In the above mentioned controlled clinical trial comparing the treatment of intrabony defects with EMP to that after conventional periodontal surgery (modified Widman flap), ^Heijl reported a mean CAL gain of 2.1 mm whereas in an other study evaluating the effect of EMP in 32 consecutively treated intrabony defects eight months after treatment a CAL gain of 3.0 mm was reported ^{(Sculean et al., 1999b)}. In a controlled clinical trial comparing the effects of EMP to that of GTR in the treatment of intrabony defects no statistical significant differences between the two treatment modalities were observed ^{(Sculean et al., 1999c)}.

One explanation for the higher CAL gain from the present study compared to the above mentioned studies may be the very deep initial defect depths. Clinical studies have demonstrated that the PPD reduction and the CAL gain obtained after both conventional and regenerative surgery are proportional to the initial defect depth : the deeper the initial defect the greater the PPD reduction and CAL gain ^{(Kaldahl et al., 1996} ; ^{Becker et al., 1988} ; ^{Cortellini et al., 1993)}.

The fact that in all defects treated with EMP a CAL gain as well as a new bone formation occured, suggests that EMP may favor periodontal healing also in defects where a GTR procedure alone would not be indicated due to the collapse of the membrane and emphasizes the clinical relevance of this therapeutical approach.

Other important factors which influences the outcome of regenerative periodontal treatment are the patient selection and post-operative plaque control and the wound stability ^{(Rosling et al., 1976} ; ^{Nyman et al., 1977} ; ^{Wikesjö and Nilvéus, 1990)}. For this reason only patients with a high level of oral hygiene and compliance with the maintenance program were included in the study. In order to ensure post-operative wound stability no mechanical tooth cleaning in the operated areas was performed by the patients in the first six weeks after surgery. During this period both a chemical plaque control and a tight recall program including supragingival tooth cleaning were followed in order to avoid infection due to dental plaque. Thus, it may be assumed that the thorough post-operative maintenance care was responsible not only for the early wound healing events but also in great part for the long-term stability of the achieved results ^{(Rosling et al., 1976} ; ^{Cortellini et al., 1994)}. Furthermore, results from clinical studies indicate that significant clinical improvements in terms of CAL gain and bone regeneration can also be obtained by means of conventional surgery or other regenerative approaches such as GTR alone or in combination with bone grafts, when optimal plaque and infection control are ensured ^{(Rosling et al., 1976} ; ^{Cortellini et al., 1995}, ¹⁹⁹⁶ ; ^{Mora et al., 1996a}, ^b ; ^{Nygaard-Ostby et al., 1996} ; ^{Kim et al., 1996)}. Thus, the post-operative plaque and infection control are at least as important as the surgical procedure itself ^{(Rosling et al., 1976} ; ^{Cortellini et al., 1994} ; ¹⁹⁹⁶⁾.

In conclusion, the present study has histologically shown that EMP has some potential to enhance new cementum and new alveolar bone formation, and to improve the clinical outcome of the surgical treatment of advanced intrabony defects. The achieved clinical results could be maintained stable over a two year period.

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