Periodontal repair in dogs Effect of heparin treatment of the root surface

Ulf M. E. Wikesjo, Noel Claffey and Jan Egelberg ' School of Dentistry, Loma Linda University, Loma Linda, CA, USA

Wikesjo UME, Claffey N and Egelberg J: Periodontal repair in dogs. Effect of heparin treatment of the root surface. J Clin Periodontol 1991; 18: 60-64. Abstract. Studies on periodontal repair to denuded root surfaces have suggested that initial clot adhesion to the root surface may be important for the nature of subsequent heahng. To study this hypothesis, circumferential periodontal defects, approximately 5 mm in vertical dimensions, were surgically created and immediately treated around the mandibular premolars in 4 beagle dogs. Prior to wound closure, the root surfaces were treated with either the anticoagulant heparin or with sahne. Tissue blocks were obtained at sacrifice 4 weeks after surgery. Histometric analysis showed that connective tissue repair to the root surface averaged 50% of the defect height for heparin-treated teeth as compared to 95% for saline-treated teeth. Junctional epithelium amounted to an average of 33% of the defect height in heparin-treated teeth in contrast to 5% following saline treatment. It can be concluded that heparin treatment of the root surface compromises connective tissue repair, confirming clot adhesion as one prerequisite for connective tissue repair of periodontal defects.

The goal of reconstructive periodontal surgery is to achieve connective tissue attachment to denuded root surfaces. It has been assumed that the epithelium hning the surgical flaps needs to be prevented from early access to the root surface during the heahng period to achieve this goal. Thus, surgical techniques have been developed aiming to stop or retard the epithelium from early contact with the root surfaces (Ellegaard et al. 1974, Bogle et al. 1981, Nyman et al. 1982). It appears that a concept of a "race" between epithelium and connective tissue forms the background for these attempts, i.e., maturation of a connective tissue attachment to the root surface should be allowed prior to epithehal interference (Goldman 1948). However, the results of some experimental studies suggest that apical migration of the epithehum may not occur or may be limited under certain conditions (Hiatt et al. 1968, Claffey et al. 1989, Wikesjo & Nilveus 1991). The first requirement for successful regenerative outcome may rest with clot adhesion to the root surface, possibly by virtue of a "fibrin linkage" (Hiatt et al. 1968, Polson & Proye 1983). The rapid adherence of the blood clot to the root surface may thus form a sufficient barrier to epithelial apical migration. The purpose of the present study was to test the hypothesis that clot adhesion to the planed root surface is important

for achieving connective tissue repair following regenerative periodontal surgery. This hypothesis was tested in a beagle dog model by conditioning the planed root surfaces with the anticoagulant heparin in an attempt to compromise the adhesion of the fibrin clot. The amount of connective tissue repair obtained after this procedure was compared to that of root surfaces not given this experimental treatment.

Key words: ani^yiosis; blood coaguiation; new attachment; periodontal regeneration; root resorption. Accepted for publication 10 January 1990

circumferentially. Bone was removed until the teeth became shghtly mobile. The extent of bone removal, measured from the cemento-enamel junction to the reduced bone crest, approximated 4.5 mm for P2 and P3 and 5.5 mm for P4. The surgical procedure included extraction of the 1 st mandibular premolar and amputation of the crown of the 1 st molar. Wound management

Material and Methods Animals

The mandibular right and left 2nd, 3rd and 4th premolars (P2, P3 and P4) in 4 male beagle dogs, 18-24 months old, were utilized for experimentation. Surgical procedures were performed under sodium pentobarbital anesthesia. The dogs received lactated Ringer's solution intravenously during surgery and they were fed a soft dog food diet throughout the study (for detail, see Wikesjo & Nilveus (1990)). Induction of defects

Horizontal periodontal defects were surgically created around the experimental teeth as described by Wikesjo & Nilveus (1991). Briefly, sulcular incisions and elevation of buccal and lingual mucoperiosteal flaps were followed by removal of supporting alveolar bone

Following bone removal, the root surfaces of the experimental teeth were planed with curettes and chisels and the furcation fornices with a flame shaped, water cooled rotating diamond to remove the cementum. The teeth were then isolated with rubber dam and the root surfaces in right and left jaw quadrants were treated with either heparin (HEP-LOCK®, 10 units/ml, Elkins Sinn Inc., Cherry Hill, NJ, diluted to 1 unit/ ml in saline) or sahne. The 2 treatments were alternated between mandibular quadrants in subsequent dogs. Heparin or saline treatment was carried out as a 5-min continuous drop apphcation. Overflow of the heparin solution or the sahne was continuously aspirated. The root surfaces were then allowed to dry for 2 min assisted by a gentle stream of air. After removal of the rubber dam, the periosteum was fenestrated at the base of the buccal and

Heparin treatment of the root surface

NAB ARP Fig. 1. Representative, bucco-hngual section of a heparin (A) and a saline (B) treated root surface. The defect height measured from the cemento-enamel junction (CEJ) to the apical extension of the root planing (ARP) approximates 5 mm. Note the long junctional epithelium (JE) on the heparin treated root surface (open arrow indicates the apical extension of the junctional epithelium) and root resorption (RR) just apical to the junctional epithelium. New alveolar bone (NAB).

lingual mucoperiosteal flaps. The fiaps were replaced to a position 1-2 mm coronal to the cemento-enamel junction and sutured with single interdental sutures. The mesial and distal extensions of the wound were closed with vertical mattress sutures. Sutures were removed after 1 week. An intramuscular broad spectrum antibiotic (Combiotic®, Pfizer Inc., New York, NY) was administered daily during the first 2 weeks after surgery. The next 2 weeks, daily plaque control was maintained by gently wiping the teeth with gauze soaked in a 2% chlorhexidine solution (Hibitane®, ICI Ltd, Macclesfield, Great Britain). Histological procedures

The dogs were sacrificed 4 weeks after surgery by intravenous injection of sodium pentobarbital. Tissue blocks in-

cluding teeth, bone and soft tissues were removed. The blocks were fixed in 10% buffered formalin, decalcified in 10% formic acid, trimmed, dehydrated and embedded in paraffin. Serial sections, 7 //m thick, were cut in a bucco-lingual plane throughout the entire mesio-distal extension of the teeth. Every 14th section, at approximately 100 //m interval, was stained with Masson's trichrome and an adjacent section with hematoxylin and eosin. The most centrally located Masson stained section of the mesial and the distal roots of each tooth was identified by the size of the pulp chamber and the root canal. This section and the 2 step serial sections on either side stained with Masson's trichrome were subjected to histometric analysis. Extent of new cementum formation was additionally confirmed in adjacent hematoxylin and

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eosin stained sections. Thus, 5 subsequent step serial sections, representing approximately 0.4 mm of the mid-portion of the mesial and distal roots from each tooth, were used for analysis. Measurements were performed using a microscope connected to a measuring system for computer aided manual data collection of geometrical structures (Videoplan, Carl Zeiss Inc., Kontron, Eching bei Munchen, West Germany). . . The following measurements were taken for the buccal and lingual surfaces of each root (4 sites per tooth). Defect height: the distance between the apical extension of the root planing and the cemento-enamel junction. Junctional epithelium: the distance from the apical to the coronal extension of the junctional epithelium along the root surface. Connective tissue repair: the distance between the apical extension of the root planing and the apical termination of the junctional epithelium. Cementum formation: the distance between the apical extension of the root planing and the coronal extension of a continuous layer of cementum or a cementum-like deposit on the root surface. Bone formation: the distance between the apical extension of the root planing and the coronal extension of newly formed alveolar bone along the root surface. Root resorption: the combined linear heights of distinct resorption lacunae along the root surface. Ankylosis: the combined linear heights of ankylotic union of newly formed alveolar bone and the root surface. • . Data analysis

Surface, tooth and dog means for each of the measurements were calculated using the 5 selected step serial sections. Differences for treatments were analyzed using the Student ?-test for paired observations (A'^=4). Additionally, the frequency of teeth presenting with root resorption and ankylosis was calculated. Presence of these features in 1 or more of the 10 sections from each tooth resulted in a positive score for the tooth. Results Clinical and histological observations

Postoperatively, healing progressed uneventfully. Additional bleeding was not

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Wikesjo et al. the new cementum layer. Ankylosis was observed in sections from 2 teeth only. Coronally to the cementum, gingival connective tissue interfaced directly with the root surface. Distinct lacunae of root resorption were observed in all teeth at various heights of the connective tissue-root surface interface. Heparin treated teeth further displayed gingival recession and a long junctional epithelium in contrast to the saline treated teeth which healed almost exclusively with connective tissue repair (Figs. 1, 2).

roo%-

75% -

Histometric observations

50% -

25% -

ooo

HEPARIN

SALINE

Fig. 2. Histometric results expressed in percent (%) of the defect height.

observed in quadrants where the root surfaces had been treated with heparin. However, in these quadrants a gradual shght gingival recession was observed during the healing period. Histologically, connective tissue repair at the base of the wound, delineated by the apical extent of the root planing, was similar in nature for the 2 root surface treatments. A thin layer of a cementum-like deposit was observed on the instrumented root surface at the apical portion of most defects. A limited amount of new alveolar bone was usually present, however the bone rarely extended beyond the coronal level of

Buccal and hngual surfaces from all 24 teeth under experimentation were available for the histometric analysis. The heights of the induced periodontal defects and the extent of repair of the various tissue components are shown in Table 1 and Fig. 2. The extent of connective tissue repair to the root surface was significantly smaller for the heparin treated compared to the sahne treated teeth (2.4 ±0.6 versus 4.4 + 0.4 mm). The obvious lack of connective tissue repair in heparin-treated root surfaces was accompanied by a significantly longer junctional epithehum than that observed in saline-treated controls (1.6 + 0.4 versus 0.2 + 0.1 mm). In spite of the difference in total connective tissue repair, cementum and bone formation was limited and similar following both treatments. The extent of root resorption was also similar for both groups, while ankylosis was minimal. Discussion This report concerns the importance of clot adhesion to the root surface to the outcome of periodontal repair following reconstructive surgery. A periodontal wound model was used that heals almost exclusively with connective tissue repair to the surgically denuded root

surface (Wikesjo & Nilveus 1991). To study the importance of clot adhesion, using this model system, a "reversed" approach was used asking the question how an anticoagulant agent may influence the healing between the root surface and the mucoperiosteal fiap. Thus, 2 approaches are readily identifiable, one being systemic alteration of the coagulability of the blood and the other being local alteration of the thromboresistance of the blood-contacting root surface. Clot adhesion following periodontal reconstructive surgery may then be evaluated directly by tensile strength assessments during early wound heahng or indirectly by histology after having subjected the wound to induced (Hiatt et al. 1968) or naturally occurring tensile forces. The tensile forces are assumed to violate the integrity of the root-surfaceadhering blood clot, resulting in wound rupture between the root surface and the mucoperiosteal fiap. The void in the continuity of the wound will then rapidly be lined with epithelium proliferating from the flap margin and consequently result in decreased connective tissue repair to the root surface. The coagulation cascade at the dentin-connective tissue interface will generally follow the extrinsic coagulation pathway and subsequently include activation of various proteolytic enzymes leading to the conversion of prothrombin to thrombin. Briefly, extravascular tissue phospholipids will be released upon tissue damage and initiate the coagulation process. Ca+"^ and tissue factor apoprotein will activate factor VII (proconvertin) and subsequently chain activate the Stuart factor (X) and prothrombin (II) to formation of thrombin. Thrombin then converts fibrinogen (I) to fibrin monomers that cross-hnk to form a fibrin network/clot (for review, see Hemker (1989)). This process may be interfered by heparin, a heterogenous group of straight-chain anionic mucopolysaccarides (glycosaminoglycans)

Table 1. Periodontal repair of circumferential periodontal defects following root surface treatment with heparin or saline; mean + s.d. (mm) N=A

defect height junctional epithelium connective tissue repair cementum formation bone formation : . root resorption .• ankylosis

Heparin

Saline

/>-value heparin versus sahne

4.9 + 0.3

4.6 + 0.4 0.2 + 0.1 4.4 + 0.4 2.0 + 1.7 0.8 + 0.2 0.5 + 0.2 0.02 + 0.03

n.s.

Periodontal repair in dogs. Effect of heparin treatment of the root surface.

Studies on periodontal repair to denuded root surfaces have suggested that initial clot adhesion to the root surface may be important for the nature o...
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