Healing of recession-type defects following treatment with enamel matrix proteins or guided tissue regeneration

Introduction

The goal of regenerative periodontal treatment is to reestablish the tooths`s supporting apparatus where it has been lost following inflammatory periodontal disease. This goal involves the formation of a new connective tissue attachment (i.e. new cementum with inserting collagen fibers) and new alveolar bone ^{(Caton and Greenstein, 1993)}. Several treatment approaches, such as bone grafting procedures, root surface conditioning, application of growth factors or guided tissue regeneration (GTR) have been used in order to accomplish this goal ^{(Brunsvold and Mellonig, 1993} ; ^{Lowenguth and Blieden, 1993} ; ^{Lynch et al., 1991} ; ^{Karring et al., 1993)}. Until now one of the most predictable treatments is GTR, which implies the placement of a mechanical barrier over the denuded root surfaces and the periodontal defects, thus allowing cells originating from the periodontal ligament and the alveolar bone to selectively repopulate the isolated space ^{(Nyman et al., 1982} ; ^{Gottlow et al., 1986b} ; ^{Karring et al., 1993)}. Histological studies in animals and humans have shown that GTR treatment of recession-type defects results in the formation of new connective tissue attachment and new alveolar bone ^{(Gottlow et al., 1990}, ¹⁹⁹⁴ ; ^{Cortellini et al., 1993} ; ^{Vincenzi et al., 1997} ; ^{Parma-Benfenatti and Tinti, 1998)}. Both non-bioabsorbable and bioabsorbable membranes have been used as barriers with comparable histological and clinical results ^{(Gottlow et al., 1990}, ¹⁹⁹⁴ ; ^{Cortellini et al., 1993} ; ^{Rocuzzo et al., 1996} ; ^{Vincenzi et al., 1997} ; ^{Parma-Benfenatti and Tinti, 1998).}

Recently, it was suggested that the conditioning of the detached root surface with enamel-matrix-proteins (EMD) may also enhance the regeneration of the lost periodontium ^{(Hammarström, 1997)}. Histological findings in monkeys and humans have indicated that treatment of acute-type recession defects and of chronic intrabony defects with EMD may result in the formation of a new attachment apparatus characterized by the presence of new cementum with inserting collagen fibers and of new alveolar bone ^{(Hammarström et al., 1997} ; ^{Heijl, 1997} ; ^{Mellonig, 1999} ; ^{Sculean et al., 1998}, ^1999a, ^1999b)

Thus, the application of EMD in regenerative periodontal treatment seems promising, but no histological studies have yet been published investigating the treatment of chronic recession-type defects with EMD as compared it to the treatment with GTR alone, or the combination of EMD and GTR.

Therefore, the aim of the present pilot study was to present some histological features of the healing of chronic recession-type defects in monkeys following treatment with EMD, GTR and combination of EMD and GTR.

MATERIALS AND METHODS

Three adult male monkeys (Macaca fascicularis) were used in the study. Before the operative procedures the animals were anesthetized with intramuscular injections of Kethalar^® (10 mg/body weight ; Parke Davis Co. Inc., USA). In order to reduce haemorrhagia in the surgical areas local anesthesia was also performed.

Production of experimental defects

Following intracrevicular incisions and elevation of full thickness mucoperiosteal flaps at both the vestibular and oral aspects of the maxillary jaw, recession-type defects were produced at the buccal aspect of the first and second molars in both sides. The vestibular supporting bone was removed to the level of the root length by means of a slowly rotating round bur under irrigation of sterile saline. The exposed root surfaces were then thoroughly curetted and rinsed with sterile saline. In order to prevent spontaneous healing and enhance dental plaque accumulation, cotton floss ligatures were placed into the defects and the flaps were closed with vertical matress sutures. Ten days after surgery the sutures were removed. During the first 4 weeks following surgery, no oral hygiene measures were performed. After the fourth week, the cotton ligatures were removed and oral hygiene measures resumed consisting of tooth-brushing and topical application of chlorhexidine 0.2 % twice per week. Seven weeks following the creation of the defects, full thickness mucoperiosteal flaps were raised, the granulation tissue was removed from the defects and the root surfaces were scaled and planed. Using a small round bur notches indicating the most apical part of each defect were produced in the root surfaces. Thus, any periodontal ligament tissue which later may develop coronally to this notch, will be de novo formed and clearly distinguishable in histological sections. The defects were then randomly assigned to one of the following treatments :

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

2. Bioabsorbable membranes (Resolut^® Regenerative Material, WL, Gore Associates, Inc., Flagstaff, AZ, USA).

3. Combined therapy (Emdogain^® and Resolut^®).

4. Control (coronally repositioned flap).

Prior to the application of Emdogain^® alone or Emdogain^® and Resolut^® the root surfaces were etched for 2 minutes with a 24 % EDTA containing gel according to the instructions given by the manufacturer. The remnants of the gel were removed by copious rinsing with sterile saline. At the sites receiving only GTR or coronally repositioned flaps, no root surface conditioning was performed. The Emdogain^® gel was always freshly prepared and consisted of two components (a vehicle solution and Emdogain^® in a spongy form) which were mixed up just prior to the surgical procedure according to the instructions given by the manufacturer. Immediately after root conditioning and rinsing, Emdogain^® was applied onto the root surfaces by means of a sterile syringe.

At the defects treated with bioabsorbable membranes alone, a Resolut^® membrane of an appropriate configuration was fixed to the tooth at the level of the cemento-enamel-junction (CEJ) with bioabsorbable sutures (Dexon II, Davis & Geck, Inc., Manati, PR).

At the defects receiving the Emdogain^® and Resolut^® treatment the bioabsorbable membranes were first loosely adapted on the root surfaces and not sutured tightly to the tooth until after the application of Emdogain^®.

In all cases the flaps were repositioned coronally and sutured with non-bioabsorbable vertical matress sutures (Gore-Tex^®, Flagstaff, Arizona, USA). At the day of the operation and the following 3 days the animals received intramusculary administrated antibiotics (1 ml Clamoxycillin). 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 after surgery. After this period and for the rest of the experimental period oral hygiene measures were performed once a week as described above. Five months after surgery the animals were killed with an overdose of Kethalar^®, and the oral tissues were fixed by perfusion with 10 % buffered formalin administered through the carotid arteries. Following this procedure the jaws and surrounding soft tissues were removed and immerged in 10 % buffered formalin. Specimens containing the experimental teeth were dissected free, decalcified in EDTA, dehydrated and embedded in paraffin. Vestibulo-oral serial sections were cut parallel to the long axes of the teeth with the microtome set at 8 µm. The sections were stained alternatively with hematoxylin and eosin, or with the oxone-aldehyde-fuchsin-Halmi stain ^{(Sculean et al., 1997)}. Three sections, 100 µm apart, from the central part of the defect were selected for histometric analysis. Linear measurements were performed by one blinded investigator.

Results

Clinical observations

The postoperative healing was uneventful in all three animals. During the second postoperative week, one membrane at a defect treated with EMD plus GTR was exfoliated and exposure of the membrane material occurred in two other defects : one treated with GTR and one with EMD plus GTR. The site where the membrane was exfoliated was excluded from the histometric analysis. The exposed parts of the membranes disintegrated within a few days without any complications. At the sites treated with EMD or coronally repositioned flaps no adverse effects were observed.

Histological observations

EMD treated sites

In the defects treated with EMD new attachment and new bone formation had occurred to a varying extent. The length of the newly formed cementum varied from 1.19 to 3.25 mm (^{table I}) and the amount of new bone varied from 0.65 to 2.64 mm (^{table II}). The newly formed cementum, which was always in continuation with the old cementum was predominantly of an acellular type with inserting collagen fibers oriented perpendicularly to the tooth surface (^{fig. 1}, ², ³ and ⁴). Frequently, a split was seen between the root surface and the newly formed cementum.

GTR treated sites

In the defect where the membrane became exposed during healing a long junctional epithelium was lining the instrumented root surface and only minimal regeneration of cementum (1 mm) and alveolar bone (0.73 mm) was observed in the notch area (^{tables I} and ^II).

In the defects where no exposure of the membrane had occurred, the amount of the newly formed cementum varied from 3.2 to 5.43 mm and the amount of new bone varied from 2.12 to 4.0 mm (^{tables I} and ^II). The new cementum formed following GTR treatment was in continuation with the old cementum apical to the defect and was of a mixed acellular and cellular type with perpendicularly inserting collagen fibers (^{fig. 5} and ⁶). Artifacts (splits between the new cementum and the root surface) were observed in all specimens.

Combined treatment (EMD and GTR)

In the defect where the membrane became exposed, the amount of newly formed cementum measured 1.65 mm whereas it measured 2.9 mm at the site without exposure (^{table I}). The amount of newly formed bone was 1.43 mm at the site exhibiting exposure and 2.5 mm at the site without exposure (^{table II}). The new cementum formed after this treatment was in continuation with the old cementum on the intact root surface and was mostly acellular (^fig.7).

Control treatment

In two out of the three control defects the healing resulted predominantly in the formation of a long junctional epithelium along the instrumented root surfaces. Periodontal regeneration was limited to the apical part of the defects (^{fig. 8}). In these two defects the new cementum varied from 0 to 1.10 mm and the amount of new bone between 0 to 0.33 mm (^{tables I} and ^II). In one of the specimens, however, a considerable amount of new cementum (2.25 mm) and of new bone (1.87 mm) was measured (^{fig. 9} and ¹⁰, ^{tables I} and ^II).

Discussion

The present pilot study has demonstrated that the treatment of chronic recession-type defects with GTR, EMD or EMD plus GTR may lead to new cementum and new alveolar bone formation.

The observation that the treatment of recession-type defects with coronally repositioned flaps may resulted in some, however, unpredictable amount of new attachment and bone corroborates findings from previous studies ^{(Gottlow et al., 1986a)}. Furthermore, it was demonstrated that the treatment of recession-type defects with GTR resulted predictably in higher amounts of newly formed cementum and bone as compared to coronally repositioned flaps alone. This is in agreement with the results of other histological studies with bioabsorbable membranes ^{(Gottlow et al., 1994} ; ^{Vincenzi et al., 1997} ; ^{Parma-Benfenatti and Tinti, 1998)}. However, the amount of regenerated cementum and bone was obviously smaller in cases of membrane exposure. This observation supports the results of previous studies where it was demonstrated that membrane exposure and subsequent bacterial colonization is one of the major complications which affects the outcome of GTR therapy ^{(Selvig et al., 1992} ; ^{Simion et al., 1994} ; ^{Sander and Karring, 1995} ; ^{Nowzary et al., 1995)}. The observation that the outcome was also negatively affected by membrane exposure when GTR was supplemented with EMD indicates that this complication may not be overcome with the application of a biologically active substance such as EMD.

The observation that, in one animal, the treatment with EMD alone resulted in new attachment and new bone formation to a much higher extent than in the controls, suggests that this treatment has a potential for enhancing periodontal regeneration. The regenerated cementum after treatment with EMD alone or a combination of EMD and GTR was predominantly of an acellular type whereas GTR alone resulted in a mixed acellular and cellular type. These findings corroborate previous observations in animals, indicating that EMD might selectively enhance acellular cementum formation ^{(Hammarström, 1997} ; ^{Hammarström et al., 1997} ; ^{Heijl, 1997} ; ^{Araújo and Lindhe, 1998} ; ^{Sculean et al., 1998}, ^1999a).

The fact that artifacts between the root surfaces and the regenerated cementum were always observed, irrespective of the treatment used, suggests that although the new cementum varied regarding the content of cells there are no major qualitative differences between the cementum formed following the various treatment modalities. It is still unknown to what an extent the presence of artifacts might infuence the clinical outcome of the therapy on a long term basis. The observation that artifacts also occurred following root conditioning with EMD is in disagreement with findings from previous studies where it was demonstrated that the cementum formed after this treatment is always firmly attached to the tooth surface ^{(Hammarström et al., 1997} ; ^{Heijl, 1997)}. One explanation for this discrepancy might be that in both these studies, the defects were of an acute-type (i. e. they were immediately treated following their surgical creation) and, therefore, they may not represent the same situation as a chronic, dental plaque infected periodontal defect. This view is supported by histological studies in non-human primates showing that in acute-type defects approximately 50 to 70 % spontaneous regeneration can be expected ^{(Caton et al., 1994)}. On the other hand, the significance of artifacts is still controversially discussed in the literature. Some authors interpret the presence of splits between the newly formed tissues (i. e. new cementum, new periodontal ligament and new bone) to be a result of the decalcification for the histological preparation of the biopsies, and do not reflect a poorer quality of the regenerated tissues ^{(Listgarten, 1972)}. In contrary, other researchers have interpreted similar findings as a healing of an inferior quality which do not necessarily reflect a real regeneration process ^{(Nalbandian and Frank, 1980} ; ^{Hammarström, 1997} ; ^{Hammarström et al., 1997).}

The fact that the application of EMD resulted in varying amounts of new attachment and new alveolar bone formation may suggest that some incontrolled factors are interfering with healing. It cannot be excluded that infection of the wound may play a role, but it is also possible that a lack of space for ingrowth of periodontal ligament cells due to collaps of the mucoperiosteal flap on the root surface in some instances has contributed ^{(Selvig et al., 1993} ; ^{Tonetti et al., 1996)}. In addition, EMD is not able to attach to the root surface for a period longer than two weeks which in some types of defects may be too short a time for optimal healing ^{(Gestrelius et al., 1997)}. Further investigation is needed to reveal the precise acting mechanism of EMD in periodontal and bone regeneration in order to explain the variation in the healing results.

Although on a very limited number of specimens, the present study failed to demonstrate any beneficial effect of the combined EMD plus GTR therapy compared to the EMD or GTR treatments alone. In this context it should be noted that, due to the fact that only a limited number of animals was available for this study, and that the healing pattern may show a great interindividual variability, these results have to be interpreted with caution. Thus, they rather represent findings from individual case reports, than final conclusions based on statistical significance.

However, similar results have been recently presented in a study in monkeys where combined EMD and GTR treatment also failed to improve the predictability of regenerative treatment in intrabony defects ^{(Sculean et al., 1998)}. One the other hand, in recent studies evaluating the healing in mandibular degree III furcation defects following GTR therapy alone and the combined EMD and GTR treatment, showed that the additional application of EMD might enhance periodontal regeneration ^{(Araújo and Lindhe, 1998} ; ^{Donos et al., 1998).}

In conclusion, the observations from the present study indicate that both EMD and GTR may enhance the healing process following regenerative periodontal treatment. However, the mechanisms by which EMD may enhance cementogenesis is still obscure, and remains to be elucidated. Further histological studies using higher numbers of animals and defects are needed in order to clarify the role of enamel matrix proteins on periodontal wound healing and regeneration.

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