559

Periodontal Repair in Dogs: Effect of Saliva Contamination of the Root Surface* UlfM.E. Wikesjö,

Karin

Hagen,

and David D. Nielsen

This study evaluated whether periodontal repair following reconstructive surgery may be compromised by saliva contamination of the root surfaces during the surgical procedure. Circumferential periodontal defects, 5 to 6 mm in vertical dimension, were surgically created in the mandibular premolars in 9 beagle dogs and immediately treated. The denuded root surfaces in left and right jaw quadrants in 4 dogs were treated with either filter sterilized saliva or saline, while in the remaining 5 dogs the root surfaces were treated with unfiltered saliva or saline. The wounds were closed and allowed to heal for 4 weeks. The dogs were sacrificed and tissue blocks prepared for histometric analysis. Results showed no difference between teeth treated with filtered or unfiltered saliva and the saline treated controls. Connective tissue repair to the root surfaces exceeded 70% of the defect height in all experimental groups. Regeneration of cementum and alveolar bone was limited and did not exceed 30% of the defect height. The results indicate that contamination of the root surface by saliva during the surgical procedure does not necessarily compromise connective tissue repair to the root surface. / Periodontal 1990; 61:559-563.

root/surgery; saliva/adverse effects; periodontal diseases/surgery Key

Words: Tooth

connective tissue attachment;

Clinical procedures aiming at reconstruction of periodontal defects have generally met limited success in terms of meaningful gain of periodontal support.1 The reasons for these limitations are not clear, but healing events may have been compromised by factors like wound and root preparation, extent of primary wound closure, wound stabilization, and postoperative infection control. Another factor that possibly may have an impact on periodontal healing may be the contamination of the root surface with saliva. Recent in vitro experimentation may be interpreted to support this notion. Attachment and locomotion of gingival fibroblasts have been found to be inhibited on tissue culture plastic and on cementum following coating of these surfaces with saliva.2-3 Furthermore, dentin blocks, which first have been superficially demineralized and then coated with fibronectin, exhibit a decreased attachment of gingival mesenchymal cells after treatment with saliva.4 Recent in vivo experimentation has suggested that factors preceding the attachment of fibroblastic cells under certain circumstances may determine whether a long junctional epithelium or a new connective tissue attachment will interface the root surface following periodontal reconstructive *School of

Dentistry,

Loma Linda

University,

Loma

Linda, CA.

surgery.5 In this experiment the root surfaces of experimental periodontal defects in dogs were "contaminated" with heparin prior to wound closure. Significantly less connective tissue repair was observed following this treatment compared to the saline control, presumably due to interference with blood clot adhesion during the initial healing phase. It seems possible that contamination of the root surface by saliva may also compromise periodontal repair by leaving a proteinaceous coating which may affect clot adhesion. Thus, the present study was designed to histologically evaluate healing of circumferential periodontal defects in dogs following flap surgery which included root surface contamination with saliva. MATERIAL AND METHODS

Animals and Surgical Procedures Nine male beagle dogs (18 to 24 months old) were divided into 2 experimental groups; Group 1 (4 dogs) to be treated with filtered saliva and Group 2 (5 dogs) with unfiltered saliva. Filtered saliva was chosen since preceding in vitro experiments indicated decreased attachment and locomotion of fibroblastic cells to such saliva treated surfaces.2-4 Unfiltered saliva was chosen since it represented a possible clinical scenario.

560

J Periodontol 1990

EFFECTS OF ROOT SALIVARY CONTAMINATION ON HEALING

Circumferential periodontal defects were created in right and left mandibular premolare (P2, P3, and P4) as earlier described.6 Briefly, following flap elevation, alveolar bone was removed around the experimental teeth to a level approximately 5.0, 5.5, and 6.0 mm from the cemento-enamel junction for P2, P3, and P4 respectively. In 1 dog in Group 2, left and right P3 were removed during surgery due to severe crowding. An intravenous administration of sodium pentobarbital anesthesia and a lactated Ringer's solution was used for the surgeries. The animals were fed a soft dog food diet throughout the study. This experimentation was part of an institutionally approved protocol in periodontal wound healing. Saliva Samples A saliva sample was obtained from each dog immediately prior to anesthesia for surgery. A crystal of citric acid was positioned on the back of the tongue to activate salivation. Saliva, dripping from the mouth, was collected in a sterile glass beaker till a volume of approximately 5 ml was obtained. The individual saliva samples from the 4 dogs of Group 1 were separately aspirated through polysulfone 0.45 µ pore size filters.* Saliva samples from the 5 dogs of Group 2 remained unprocessed. Until use during surgery, saliva was stored at room temperature in a sterile plastic

syringe.

Bacterial load of filtered and unfiltered saliva samples analyzed in separate experiments. Following serial 10fold dilutions, 0.1 ml from each dilution was spread onto enriched blood-agar plates.* The plates were incubated anaerobically at 37°C for 7 days.§ Plates with 30 to 300 colonies were selected for counting and the results expressed as number of colony forming units (CFU) per ml of saliva.

was

Wound

Management

elevation and removal of alveolar bone as described above, the exposed root surfaces of the experimental teeth were instrumented with curettes, chisels, and water cooled rotating diamonds to remove the cementum and plane the roots. The teeth were next isolated with rubber dam and the root surfaces in 1 jaw quadrant were treated with either filtered (Group 1) or unfiltered saliva (Group 2). The remaining jaw quadrant in each dog was treated with saline. The saliva or saline was applied as a 5-minute continuous drop application. Excess saliva and saline was aspirated. After air drying the root surfaces for 2 minutes and removal of the rubber dam, the wound was closed by replacing the flap margins slightly coronal to the cementoenamel junction as earlier described.6 Sutures were removed after 1 week. A broad spectrum antibiotic11 was ad-

Following flap

Acrodisc, #4184, Gelman Sciences Inc., Ann Arbor, MI. Trypticase Soy Agar, BBL Microbiological Systems, Becton Dickinson & Co., Cockeysville, MD. SBBL GasPak Anaerobic Systems, Becton Dickinson & Co., Cockeysville, MD. "Combiotic, Pfizer Inc.,

New

York,

NY.

September

ministered intramuscularly daily for 2 weeks following surgery. Plaque control was maintained by daily application of a 2% Chlorhexidine solution.1'

Histological Procedures The dogs were sacrificed 4 weeks after surgery. Tissue blocks were removed, fixed in 10% buffered formalin, decalcified in 10% formic acid, trimmed, dehydrated, and embedded in paraffin. Serial sections, 7 µ thick, were cut in a bucco-lingual plane throughout the entire mesiodistal extension of the teeth. Every 14th section was stained with Masson's trichrome and the adjacent section with hematoxylin and eosin. The most centrally located Masson stained section of the mesial and the distal root 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 eosin stained sections. Thus, 5 subsequent step serial sections, representing approximately 0.4 mm of the mid-portion of both the mesial and distal root from each tooth, were analyzed. Measurements were performed using a microscope connected to a measuring system for computer aided manual data collection.* The following measurements were taken for the buccal and lingual surfaces of each root (4 sites per tooth; Fig. 1): 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 résorption 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 measurecalculated using the 5 selected step serial sections. Differences for treatments were analyzed using 4 Student's i-test for paired observations (Group 1: 2: the 5 dogs). Additionally, frequency dogs; Group

ments were

=

=

'Hibitane, ICI Ltd., Macclesfield, Great Britain. "Videoplan, Carl Zeiss Inc., Kontron, Eching bei München, West Germany.

Volume 61 Number 9

WIKESJÖ, HAGEN, NIELSEN

561

Table 1. Group 1. Periodontal Repair of Circumferential Periodontal Defects Following Root Surface Treatment With = 4 Filtered Saliva or Saline. Mean ± s.d. in mm, dogs

Saline

5.0±0.6 0.4 ±0.4 4.7 ±0.5 1.2 ±1.9 0.8 ±0.4 1.3 ±1.1 0.1 ±0.1

5.2±0.2 0.4 ±0.5 4.7 ±0.5 1.5±1.5 1.2 + 0.8 1.4±0.6 0.1 ±0.1

Defect Height Junctional Epithelium Connective Tissue Repair Cementum Formation Bone Formation Root Resorption

Ankylosis n.s.

Figure

1. Histometric parameters and landmarks in

buceo-lingual

P-value Saliva vs. Saline

Filtered Saliva

n.s. n.s.

n.s. n.s. n.s.

n.s. n.s.

P>0.05

sec-

tions.

(CEJ) cemento-enamel junction, (JE) junctional epithelium, (RR) root résorption, (NAB) new alveolar bone, (NC) new cementum Icementum-like deposit, (ARP) apical extension of root planing.

of teeth presenting with root résorption 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. FILTERED

SALIVA

RESULTS Bacterial Load of Filtered and Unfiltered Saliva Single colonies were found in the culture of 1 filtered saliva sample. The 3 other samples exhibited no growth. Mean number of cultivable bacteria (±s.d.) for the unfiltered saliva samples amounted to 3.4 ± 4.0 x10s (range 0.8 to 9.1 105) CFU/ml.

Figure 2. Group defect height.

Healing generally progressed uneventfully following surgery and included a slight gingival recession. One premolar (P2) treated with filtered saliva became progressively looser and exfoliated during the healing period leaving 11 teeth

treated with filtered saliva and 12 contralateral teeth treated with saline available for histometric analysis in Group 1. In Group 2, a total of 14 teeth treated with unfiltered saliva and 14 contralateral teeth treated with saline were analyzed.

Histological and Histometric Observations Group 1 (filtered saliva): The heights of the induced periodontal defects were 5.0 ± 0.6 and 5.2 ± 0.2 mm for the filtered saliva and saline treated teeth respectively (Table

expressed

in

percent (%) of the

Table 2.

Groups 1 and 2. Proportions of Teeth with Root Resorption and Ankylosis Following Root Surface Treatment With Filtered

or

Unfiltered Saliva

or

Group Root

Clinical Observations

1. Histometric results

Resorption Ankylosis

Saline

Group

1

2

Filtered Saliva

Saline

Unfiltered Saliva

Saline

10/11 2/11

12/12 3/12

14/14 4/14

14/14 6/14

1). Mean connective tissue repair to the root surface comprised more than 90% of the defect height (Fig. 2). The connective tissue repair was limited with respect to cementum formation and regeneration of alveolar bone (Table 1 and Fig. 2). All but 1 tooth presented with root résorption (Table 2). Mean linear root résorption for the treatments amounted to approximately 30% of the connective tissue repair (Table 1). Ankylosis was observed in less than 25% of the teeth (Table 2). Mean linear ankylosis amounted to only a small fraction (2%) of the connective tissue repair (Table 1).

562

EFFECTS OF ROOT SALIVARY CONTAMINATION ON HEALING

Table 3. Group 2. Periodontal Repair of Circumferential Periodontal Defects Following Root Surface Treatment with Unfiltered Saliva or Saline. Mean ± s.d. in mm, = 5 dogs

Defect Height Junctional Epithelium Connective Tissue Repair Cementum Formation Bone Formation Root Resorption

Ankylosis n.s.

Unfiltered Saliva 5.6±0.2 1.2±1.2 4.0±1.7 0.8±1.0 1.0 ±0.4 1.0±0.8 0.2 ±0.5

Saline

n.s.

A periodontal wound model earlier defined was used.6 This model allows for connective tissue repair to the denuded root surface rather than a long junctional epithelium and may be utilized to study the nature of the healing dentin connective tissue interface as well as how healing may be altered by implant materials and various root surface

n.s.

treatments.

n.s.

In a preceding study we used this model to elucidate the influence of heparin on periodontal repair.5 Root surfaces treated with the anticoagulant exhibited a long junctional epithelium rather than an extensive connective tissue repair observed in saline treated controls. The reduced connective tissue repair following the use of heparin was suggested to be related to a compromised blood clot adhesion to the root surface. Tensile forces on the wound, that did not affect the saline treated controls, may have caused wound rupture between the flap and the root surface in the heparin treated teeth which subsequently epithelialized. The compromised clot adhesion may be related to the anticoagulant effect of heparin at the interface, but may also relate to an altered immediate adsorption of plasma proteins to the root surface. Any of these effects, alone or combined, may have been decisive for the results reported. By analogy, it seemed possible that "contamination" of the root surfaces with saliva prior to wound closure may similarly compromise the adsorption of plasma proteins and thus possibly affect the outcome of periodontal repair. This study, however, in a limited number of dogs, did not provide any evidence that saliva contamination of the root surface during periodontal reconstructive surgery will influence periodontal healing. Almost complete connective tissue repair was observed following root surface treatments with filtered saliva and saline. Apparently the adsorbed saliva did not adversely affect the clot adhesion to the root surface and the subsequent wound maturation. The additional microbial insult from the unfiltered saliva did not seem to interfere with the healing process. Contaminating bacteria may have been neutralized by host response factors and/or the systemic antimicrobial regimen during the first 2 weeks of healing. However, the lower mean connective tissue repair and larger standard deviation following this treatment may indicate a trend towards compromised healing. One may speculate that the wound stability in this experimental model may, in part, explain the results, although it did not prevent a reduced attachment level following the heparin treatment. Any induced compromise on the fragile attachment factors of the early healing events by saliva may have been overshadowed by the stability of the wound, allowing connective tissue to mature to the root surface rather than resulting in wound rupture and subsequent

P-value Saliva vs. Saline

5.6±0.5 0.6 + 0.5 4.7±0.7 0.9±0.6 0.9 ±0.5 1.2±0.5 0.2 ±0.3

n.s.

n.s. n.s.

n.s.

P>0.05

UNFILTERED SALINE

SALIVA

Figure 3. Group defect height.

2. Histometric results

expressed

in percent

J Periodontol 1990

September

(%) of the

Group 2 (unfiltered saliva): The periodontal defect heights for teeth treated with unfiltered saliva and the contralateral saline controls were 5.6 ± 0.2 and 5.6 ± 0.5 mm respectively (Table 3). On the average, connective tissue repair relative to the defect height comprised 72% and 85% respectively for saliva and saline treated teeth and mean junctional epithelium amounted to 21% and 11% respectively (Fig. 3). Formation of cementum and regeneration of alveolar bone was limited and did not extend coronal to the apical 1/5 of the defect height (Table 3 and Fig. 3). All teeth exhibited root résorption (Table 2). Mean linear root résorption amounted to approximately 25% of the connective tissue repair for both treatments (Table 3). Less than 40% of the teeth exhibited ankylosis (Table 2). Mean linear ankylosis approximated 5% of the connective tissue repair

(Table 3).

DISCUSSION This study evaluated how saliva contamination of the root surface during reconstructive surgery may alter periodontal

healing.

epithelialization.

In conclusion, it should be emphasized that, although no evidence was found for saliva contamination of the root surface being detrimental to connective tissue repair to the root surface, the results do not exclude that this may be of more critical importance in other periodontal wounds.

Volume 61 Number 9

WIKESJÖ, HAGEN, NIELSEN

periodontal surgery may still be commendable.

Acknowledgments

REFERENCES 1.

Egelberg J. Regeneration and repair of periodontal tissues. J Periodont

Res 2.

1987; 22:233-242. Heaney TG, Embery G,

3. 4.

The authors express their gratitude to Julie Cranfill for secretarial assistance; Norman Medina, for data management; Ellis Jones, Cynthia Jensvold, and Richard Tinker for graphical presentation; and Douglas Chancellor for histotechnical preparation.

inhibit adhesion and locomotion of human gingival fibroblast-like cells in vitro. J Periodont Res 1986; 21:266-278. Heaney TG. Inhibition of fibroblast attachment. / Clin Periodontal 1986; 13:987-994. Terranova VP, Franzetti LC, Hie S, et al. A biochemical approach to periodontal regeneration: Tetracycline treatment of dentin promotes fibroblast adhesion and growth. J Periodont Res 1986; 21:330-337. Wikesjö UME, Claffey N, Egelberg J. Periodontal repair in dogs: Effect of heparin treatment of the root surface. J Clin Periodontal 1991; 18: in press. Wikesjö UME, Nilveus R. Periodontal repair in dogs: Healing patterns in large circumferential periodontal defects. / Clin Periodontal 1991; 18: in press.

coprotein

Therefore, minimizing such wound contamination during reconstructive

5.

6.

reprint requests to: Dr. Ulf .E. Wikesjö, University, Loma Linda, CA 92350. Accepted for publication February 27, 1990. Send

Loma Linda

Green D. Saliva and

salivary sulphated gly-

563

School of

Dentistry,

Periodontal repair in dogs: effect of saliva contamination of the root surface.

This study evaluated whether periodontal repair following reconstructive surgery may be compromised by saliva contamination of the root surfaces durin...
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