447
The Histologie Evaluation of New Attachment in Periodontally Diseased Human Roots Treated With Tetracycline-Hydrochloride and Fibronectin* F.A.
Alger,
C. W.
Soit, S. Vuddhakanok, and
. Miles
The purpose of this study was to determine how the treatment of human tooth roots with tetracycline-HQ and fibronectin during periodontal surgery influences the attachment of the gingiva to the root surface. Mucoperiosteal flap surgery was performed on 22 teeth with periodontal disease. Teeth were assigned to three groups. Group one received surgery with degranulation and root planing. Group two received surgery with treatment of roots with tetracycline-HCl. Group three received surgery with treatment of roots with tetracycline-HCl and fibronectin. At 90 days, block sections were taken and teeth, gingiva, alveolar bone, and periodontal ligament were evaluated histologically. Controls healed with a long junctional epithelial attachment. Tetracycline and tetracycline with fibronectin groups demonstrated some reattachment, but only within the notches placed in the root at the original level of the bone. There was a trend for greater connective tissue attachment following tetracycline-HCl treatment of roots. The additional application of fibronectin to tetracycline treated roots appeared to partially negate the enhanced connective tissue attachment observed with tetracycline treatment alone. J Periodontol 1990; 61:447^455.
Key Words: Tooth root; tetracycline/effects; gingival attachment; connective tissue tachment; fibronectin; epithelial attachment.
New attachment has been defined as the reunion of connective tissue with a root surface which has been pathologically exposed.' Conventional periodontal therapy has been shown to arrest the progressive destruction of periodontal tissues, but has demonstrated minimal potential for new
attachment.2"4
Previous researchers have attempted to achieve new attachment. One technique, which has worked more predictably in animals than in humans, has been the topical application of citric acid to roots.5"10 Poison and Proye observed connective tissue attachment to citric acid treated roots in animals. A fibrin linkage preceded connective tissue attachment.11 They speculated that the fibrin linkage resulted from fibronectin binding to collagen fibrils exposed during acid etching. This mechanism of attachment is feasible in that serum transglutaminase covalently links fibronectin with collagen and fibrin.12'13 Demineralized roots bind fibronectin twice as readily as The Ohio State
University, College dontology, Columbus, OH 43210.
of
Dentistry, Department
of Perio-
at-
undemineralized roots. This binding is dependent upon root surface collagen.14-15 Results from in vitro studies have indicated that fibronectin suppressed epithelial cell attachment and growth.16 Fibronectin is also chemoattractant for fibroblasts and periodontal ligament cells.16,17 These findings suggest that procedures which enhance fibronectin binding to roots may have potential for healing with a new attachment. Studies on dogs have demonstrated that the additional application of fibronectin to citric acid treated roots resulted in a 66% greater new attachment.18 Tetracycline-HCl treated roots may bind fibronectin more readily than those treated with citric acid, while exhibiting antimicrobial efficacy for 48 hours.16-19 The purpose of this investigation was to evaluate histologically, the effects of tetracycline-HCl and fibronectin on enhancing new attachment in humans. MATERIALS AND METHODS Prior to treatment, a protocol of the research procedure was approved by The Human Subjects Committee of the Ohio
448
J Periodontol July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT
State University and an Investigational New Drug Exemption was obtained from The Food and Drug Administration for the use of human fibronectin during periodontal surgery. The study sample was comprised of 22 teeth in 5 immediate removable partial denture patients, ages 24 to 51 years of age. Teeth were selected based upon the following criteria: 1. A loss of connective tissue attachment of at least 5 mm. Measurements were determined by probing from the cemento-enamel junction (CEJ) to the base of the periodontal pocket. 2. Radiographic evidence of at least 30% interproximal bone loss. 3. Teeth with a hopeless prognosis, or scheduled to be extracted. Teeth were assigned to one of the following groups: Group 1, a control group, received mucoperiosteal flap surgery with degranulation and root planing. Group 2 received mucoperiosteal flap surgery plus topical root treatment with tetracycline-HCl, pH 2.0, 100 mg/ ml. Group 3 received mucoperiosteal flap surgery, topical root treatment with tetracycline-HCl, and subsequent treatment with human fibronectin, 10 mg/ml. Efforts were made to match experimental and control teeth within the same patient, based upon tooth location and loss of attachment. Patients received oral hygiene instructions and surgery was not scheduled until patients demonstrated the ability to adequately eliminate plaque from their teeth. Ellman mesh splints were bonded to teeth to stabilize them and to assist in coronal suturing of flaps. Inverse bevel incisions were made and mucoperiosteal flaps were elevated well beyond the mucogingival junction to expose the underlying root and alveolar bone. Granulation tissue was removed. Using a diamond bur, reference notches were placed in the tooth. A crown notch was placed at the midfacial of the tooth, extending into dentin. A second notch was placed at the height of the alveolar bone crest or at the depth of an intrabony defect, if present. When calculus was detected, a notch was made on the root surface at the apical extent of the deposit (Fig. 1). Zinc-oxide and eugenol paste was placed as a temporary restoration in the crown notch, after measurements were recovered. Calipers were used to measure the distance from the crown notch to the CEJ, from the crown notch to the root notch at the apical extent of calculus deposits, and from the crown to the alveolar bone notch. The measurements were repeated and recorded three times. Utilizing an electronic digital caliper, the distances were measured and a mean was calculated (Tables 1, 2, and 3). Root surfaces were planed to the alveolar crest or to the base of existing intrabony defects. Surgical sites were irrigated with sterile saline. Teeth in the control group received no additional treatment prior to suturing. Flaps were
Figure 1. Mucoperiosteal flaps were elevated and reference notches were placed into the crown (CN crown notch), and in the root at the level of the alveolar bone crest (BN bone notch). A notch was also placed through the apical extent of calculus detected on the root (RN root =
=
=
notch).
Table 1: Measurements and
Specimen
1
Clinical Clinical CN-RN Means CN-BN Histometric For Attachment
3
Histologie
Means CN-RN CN-BN
RN-Collagen BN-Collagen BN-Collagen: SD
BN-Bone Crest
N/A 4.34
Findings 3 N/A 3.35
in Controls
N/A
3
3 6.02
3 3.70
3 3.30
10.22
8.33
5.16
5.24
N/A
5.78 8.83 0.00 0.20 0.24
3.05 5.71 0.00 0.06 0.08 0.41
0.00 0.00
4
N/A
N/A
3.97
N/A
N/A
N/A
0.00
-.17 0.24 0.75
-.09 0.18 0.77
t 0.48
t
f 0.55
Crown Notch Standard Deviation Root Notch BN Bone Notch Not applicable, no calculus evident on the root. N/A * Crown notch is not observed histologically. Standard deviation is not calculated for mean 0. t Bone crest is not observed histologically. t Number of measurements made to derive a mean.
CN SD RN
=
=
= =
=
= =
=
= =
positioned coronally using resorbable sutures (Fig. 2). A periodontal dressing was placed over the surgical site. The roots of teeth in Group 2 were burnished for 3 minnutes with cotton pellets saturated with a solution of tetracycline-HCl,1' 100 mg/ml. Saliva was excluded from the surgical area by the use of high speed suction and isolation with cotton rolls. Following demineralization the roots were irrigated with sterile saline for 1 minute, and flaps were sutured coronally. tServa Fine Pharmaceuticals, Westbury, NY.
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ALGER, SOLT, VUDDHAKANOK, MILES
Table 2: Measurements and
Specimen Clinical Clinical Means
Findings
in
7
CN-RN CN-BN
-
/
N/A
N/A
4.16
4.48
8.86
Histometric For Attachment
Histologie Means
Tetracycline-Treated
CN-RN CN-BN
CN SD RN BN
Teeth
1
12
13
N/A
2.92 4.25
4.31
4.74
N/A
4
N/A
N/A
N/A
4.35
4.10
8.98
RN-Collagen N/A N/A N/A BN-Collagen 0.88 0.61 0.33 BN-Collagen: SD 0.41 0.05 0.54 BN-Bone Crest
5.84 8.95
449
0.46
0.27
0.72
6.56 9.79 0.00 0.03 0.04 0.70
N/A 4.39 N/A 0.00
N/A 3.93 0.00 0.05 0.09
t 0.57
N/A 0.02 0.02 0.40
Crown notch Standard deviation Root notch Bone notch Not applicable, no calculus evident on the root. N/A * Crown notch is not observed histologically. Standard deviation is not calculated for mean 0. t Bone crest is not observed histologically. Number of measurements made to derive a mean. =
=
=
=
=
=
=
=
Figure
=
2.
Gingival flaps were sutured coronally.
=
Table 3: Measurements and Fibronectin-Treated Teeth
Findings
Specimen
14
Clinical Clinical Means
CN-RN CN-BN
Histometric For Attachment
Histologie Means
CN-RN CN-BN
RN-Collagen BN-Collagen BN-Collagen: SD
BN-Bone Crest
CN SD RN BN
in
Tetracycline-
15
16
17
3
18
3
3
3
N/A 3.36
2.29 4.95
8.98
6
5
4
3
6
N/A
2.32 10.66
N/A
*
N/A
*
3.29
*
8.54
*
N/A
0.00 0.99 0.13 0.51
N/A
N/A 0.01 0.02 0.91
0.00 -.19 0.22 0.70
0.02 0.00 0.90
3
N/A 4.27
*
N/A 0.00
t 0.45
Crown notch Standard deviation Root notch Bone notch Not applicable, no calculus evident on the root. N/A * Crown notch is not observed histologically. Standard deviation is not calculated for mean 0. t Bone crest is not observed histologically. Number of measurements made to derive a mean. =
=
=
=
=
=
=
=
=
=
Group 3 were treated in the same manner as Group 2, plus the application of fibronectin,* 10 mg/ml. The isolated surgical site was air dried and a solution of fibronectin was applied to the root with cotton pellets for 5 minutes. Any solution remaining was applied to the tooth and inside of the flap with a sterile irrigation syringe. Lipidenveloped viruses, including human immunodeficiency vivirus; and non- , non-B hepatitis viruses rus; hepatitis were rendered nonviable by a solvent/detergent virus inactivation system as described by Horowitz.20 At the 1 week post-operative appointment, supragingival Teeth in
New York Blood
Center,
New
York,
NY.
deposits were removed from the teeth. Oral hygiene instrucwere reinforced and supragingival scaling and polishing were performed. These same procedures were repeated at 3, 6, and 10 weeks post-operatively, as needed. At 90 days teeth were removed in block section. A #15 scalpel blade was used to incise the oral tissues, either to periosteum or to tooth structure. These incisions were made along the apical, mesial, and distal portions of the tooth. Facially, the apical extension of the incision was located approximately 2 mm past the alveolar crest, and was determined from caliper measurements made from the crown notch to the bone notch during the initial surgical procedure. Flaps were elevated and a carbide bur was used to extend the incisions through the osseous crest and into tooth structure (Fig. 3). After the block section was outlined in the tooth with the carbide bur, the tooth was carefully extracted along with the soft tissue and crestal bone. The block section, containing gingiva and periodontal ligament attached to alveolar bone and tooth, was carefully removed
tions
and
from the remainder of the tooth with
(Fig. 4).
a
carbide bur
Block sections were fixed in 10% neutral buffered formalin for 48 hours. Décalcification of block sections was accomplished by immersion in a 10% solution of ethylenediaminetetraacetic acid (EDTA) in 0.1 M Tris buffer pH 6.95. The endpoint of specimen décalcification was determined by a chemical test for calcium.21 Decalcified specimens were prepared for embedding after serial dehydrations and perfusion with JB-4 Plus, a glycol-methacrylate plastic. Five sections per specimen, approximately 150 µ apart were sectioned and stained with hematoxylin and eosin. The mounted specimens were evaluated utilizing an Olympus standardized micrometer eyepiece, calibrated to .01 mm. Measurements included the following: from the crown notch to the root notch, from the crown notch to the bone notch, from the bone notch to the alveolar crest, and between both
450
J Periodontol July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT
Figure 3. At 90 days after the first surgical procedure, mucoperiosteal flaps were elevated, leaving a collar of gingiva attached. A carbide bur was used to incise through bone and into tooth. (G Gingiva, Arrow indicates incision through bone) =
Figure 5.
Schematic of Histometric Measurements. Crown notch (CN) to CEJ. b= Crown notch to root notch (RN). c= Crown notch to bone notch (BN). d Root notch to level of collagen attachment. e Bone notch to level of collagen attachment. a
=
=
=
Table 4:
Type
and Number of Teeth Evaluated
Tooth
Type Maxillary 2nd Bicuspid Maxillary 1st Bicuspid Maxillary Canine Maxillary Lateral Incisor Mandibular 1st Bicuspid
Control
Tetracycline Tetracycline/Fibronectin
1
Mandibular Canine Mandibular Lateral Incise
2 2 1
1 2
1 1
RESULTS A total of 22 teeth from 5
imens
The block sections containing gingiva and periodontal ligament attached to alveolar bone and tooth were carefully removed from the remainder of the tooth. Block specimens were obtained at 90 days after the first surgical procedure was performed. (G tooth) gingiva,
Figure 4.
=
=
the root and bone notches and the coronal level of inserting collagen fibers into cementum (Fig. 5). The amount of connective tissue attachment coronal to the bone notch, and the distances from the CEJ to the root notch and bone notch were calculated from the measurements obtained. Statistical findings are not reported here because of the small sample size represented in this study.
were
patients were treated. Four specdamaged during histologie preparation, so that
measurements between reference notches could not be made. The type and number of teeth evaluated can be seen in Table 4.
Clinical Observations At initial presentation, the gingival tissues appeared generally edematous, with marginal redness, blunted papillae, rolled margins, and a loss of resiliency. Plaque and calculus could be detected clinically. Calculus on the root, when present, was used to locate the apical extent of periodontal attachment loss. No calculus was observed on the roots in 11 teeth. At the 1 week post-operative appointment, all patients demonstrated healing without complications. Generally, there was no difference between the rate of healing observed
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ALGER, SOLT, VEDDHAKANOK, MILES
451
clinically between experimental and control sites. At 2 and 3 months, gingival tissues were generally pink, firm, and stippled, without suppuration or bleeding on provocation. The location of the gingiva relative to the cemento-enamel junction indicated that generalized recession of the coronally positioned flaps had occurred. Observations Measurements for connective tissue attachment were made from a mean of 3.9 measurements per specimen. The mean standard deviation between measurements for individual specimens was 0.13 mm. A micrometer eyepiece in an Olympus light microscope was used to measure reattachment and new attachment, relative to reference notches placed into the tooth at the time of surgery. New attachment was considered connective tissue attachment coronal to the root notch placed through calculus at the apical extent of the periodontal pocket. New attachment was not observed in any specimen. In periodontal lesions with horizontal bone loss, there is an area of connective tissue attachment measuring approximately 1 mm between the crest of the supporting alveolar bone and the apical extent of the pocket epithelium.22-23 Therefore, the presence of a connective tissue attachment within 1 mm of the bone crest following periodontal therapy is regarded as reattachment and not new attachment. In this study, reattachment was measured as connective tissue attachment coronal to the notch placed in the root at the level of the alveolar bone crest. Most specimens demonstrated migration of epithelium along the root down to and entering this notch, which represented the level of previous connective tissue attachment. The thickness of the epithelium, at its most apical position on the root, ranged from one cell to over a dozen cells. The oral epithelium was parakeratinized stratified squamous epithelium, with rete pegs. The extent of connective tissue attachment in control specimens ranged from .17 mm apical to the bone notch to .20 mm coronal to the bone notch, with a mean connective tissue reattachment of 0.00 mm. The standard deviation was calculated to be 0.13 mm. New cementum was not observed in any control specimen. Clinical and histologie measurements for control specimens are recorded in Table 1. Figures 6 and 7 show photomicrographs of control block
Histologie
-
specimens. Tetracycline-treated teeth healed with a connective tissue reattachment ranging from 0.00 to 0.88 mm, with a mean
reattachment of 0.27 mm and a standard deviation of 0.35 mm. Connective tissue adjacent to the root, in the area of the bone notch, demonstrated either perpendicular fiber insertion into new cementum or parallel fiber apposition with no new cementum. New cementum was observed in 3 of 7 specimens. Epithelium migrated to the apical extent of the bone notch in 4 of 7 tetracycline treated specimens. Clinical and histologie measurements for tetracycline treated teeth are recorded in Table 2. Figures 8 and 9 show photomicrographs of block specimens.
RN
Figure 6. Control specimen. At 90 days, epithelium has migrated into the root notch (RN) and beyond the bone notch (BN). The arrow indicates the apical extent of epithelium. Original magnification x 40.
Tetracycline and fibronectin-treated teeth healed with a long junctional epithelial attachment and no reattachment in 4 of 5 specimens evaluated. In these specimens, epithelium migrated to the apical extent of the bone notch. A
connective tissue reattachment of 0.99 mm was observed in one specimen. In this specimen, connective tissue adjacent to the root, in the area of the bone notch, demonstrated parallel fiber apposition with no new cementum. When a mean connective tissue reattachment was calculated, it measured 0.17 mm with a standard deviation of 0.47 mm. Clinical and histologie measurements for tetracycline and fibronectin treated teeth are recorded in Table 3. Figures 10 and 11 show photomicrographs of block
specimens.
Differences in
mean
connective tissue attachment be-
control, tetracycline, or tetracycline with fibronectin groups were very small, although there was a trend for tween
greater attachment following tetracycline-HCl
(Tables
5 and
6).
treatment
alone
452
J Periodontol July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT
BN
9
lliii 7. Control specimen. Demonstrates epithelium (E) migrating into the bone notch (BN) and beyond (Arrow). Original magnification x 100.
Figure
DISCUSSION The results of this investigation indicate that the treatment of human roots with tetracycline-HCl or tetracycline-HCl and human fibronectin during periodontal surgery did not result in new attachment. A slight reattachment was observed in both experimental groups, although this gain was very small. Animal studies have indicated that enhanced connective tissue attachment to roots treated with tetracycline-HCl during periodontal surgery is possible.24-25 Our findings indicate that reattachment and new cementum formation can be achieved with root demineralization with tetracyline-HCl in humans. However, reattachment did not reach 1 mm and new attachment was not observed. In vivo studies have reported mixed results when fibronectin was used during periodontal surgery in animals. One study indicated that new attachment could be obtained in beagle dogs, when roots demineralized with citric acid were treated with human fibronectin.18 Other investigators applied canine fibronectin to the roots of dogs which had been demineralized with either a saturated solution of citric acid
A
Figure 8. Tetracycline-treated specimen at 90 days. Epithelium (E) has migrated into the bone notch (BN). Connective tissue fibers can be seen at the apical extent of the bone notch. Periodontal ligament fibers can be seen between the old cementum (O) and alveolar bone (A). Original magnification 40.
tetracycline-HCl, 100 mgs/ml.24 The results of this secstudy are similar to our findings, in that fibronectin application to the demineralized root surface did not enhance the amount of connective tissue repair. A previous study in humans demonstrated that teeth treated with modified Widman flap surgery, citric acid demineralization, and fibronectin application resulted in a mean probing pocket depth reduction that was .22 mm greater than when surgery was performed alone.26 In our study, new attachment was evaluated histologically and histometrically to differentiate between connective tissue attachment and epithelial atttachment. Histologie measurements or
ond
also selected in an attempt to overcome the standardization difficulties inherent in probing attachment
were
measurements.
Patient and tooth selection may be critical to the success of new attachment procedures. Teeth used in this study had been given a hopeless prognosis. These teeth possessed
Volume 61 Number 7
ALGER, SOLT, VUDDHAKANOK, MILES
453
#
^
c Figure 9. Tetracycline-treated specimen. Epithelium (E) is seen within the hone notch. Also within the bone notch, connective tissue fibers (CT) are seen inserting into new cementum (Arrow). Original magnification 100. horizontal bone loss and recession. The most favorable situation for new attachment is found adjacent to 3walled intrabony pockets.27 The premise of this study was that collagen fibrils exposed during tetracycline treatment of roots would bind fibronectin and secure the gingival flap to the root via a fibrin linkage. As fibronectin suppressed the migration of epithelium and attracted fibroblasts and periodontal ligament cells, a new attachment would be facilitated. Instead, there was a trend for less reattachment when fibronectin was applied to the tetracycline-treated tooth. There may have been a flaw in our original premise that fibronectin would inhibit epithelial cell migration. Our findings are consistent with a recent study by Pitaru et al., who'observed that demineralized roots inhibited epithelial migration, but fibronectin partially restored the migration of the epithelium when applied to demineralized roots, in vitro.28 This is one possible explanation why fibronectin did not enhance connective tissue attachment in our study. Other chemotactic factors, such as endothelial growth
Figure 10. Tetracycline-and fibronectin-treated specimen. Epithelium has migrated to the bottom of the bone notch (BN). The apical extent of epithelial migration is indicated (arrow) adjacent to old cementum (C). Original magnification 100.
severe
factor, have been shown in vitro
to
be
more
chemotactic
periodontal ligament fibroblasts than fibronectin.29 Perhaps, in vivo, other chemotactic factors will demonstrate a for
greater potential for connective tissue attachment than fi-
bronectin has. Additional research with fibronectin and other biochemicals is necessary to properly interpret the role that physiological substances have in the healing periodontal lesion. SUMMARY AND CONCLUSIONS Eighteen teeth from four patients were evaluated clinically and histologically for new attachment 90 days after surgical treatment. Teeth selected for this investigation demonstrated severe bone loss and had been given a hopeless
prognosis.
Teeth were assigned to three groups. Group 1 received periodontal surgery, with degranulation, root planing, and coronal flap placement. Group 2 received periodontal surgery, with root demineralization with tetracycline-HCl, 100
454
J Periodontoi July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT Table 5: Mean Connective Tissue Attachment Per Tooth
C;,
'
%
'
\
'
·
MM Reattachment
Specimen Control 1 2 3 4 5 6
0.00 -.17 -.09
0.20 0.06 0.00
Tetracycline 7 8 9 10 11 12 13 Fibronectin 14 15 16 17 18
0.88 0.61 0.33 0.03 0.00 0.05 0.02 0.01 0.99 0.02 -.19 0.00
Table 6: Mean Connective Tissue Attachment Per
Control
Tetracycline Tetracycline
Figure 11. Tetracycline-and fibronectin-treated specimen. Epithelium (E) has migrated to the bottom of the bone notch (BN), but not apical to it. Old cementum (C) and the apical extent of the epithelial migration (arrow) are labelled. Original magnification x 400. for 3 minutes. Group 3 received periodontal surwith gery tetracycline demineralization, followed by human fibronectin application, 10 mgs/ml, for 5 minutes. During surgery, reference notches were placed into the tooth crown, in the root at th apical extent of calculus, and in the root at the level of the alveolar crest. At 90 days teeth were extracted along with a collar of gingival tissue and bone. Teeth were decalcified and embedded in plastic for histologie preparation. Specimens were sectioned at 2 µ and stained with hematoxylin and eosin. A micrometer eyepiece in an Olympus light microscope was used to measure reattachment and new attachment, relative to reference notches placed into the tooth at the time of surgery. New attachment was considered connective tissue attachment coronal to the root notch placed through calculus at the apical extent of the periodontal pocket. New attachment was not observed in any specimen. Reattachment was considered connective tissue attachment that occurred coronal to the bone notch, placed in the
mgs/ml,
and Fibronectin
X X X
=
=
=
0.00 0.27 0.17
Group S.D. S.D. S.D.
= = =
0.13 0.35 0.47
tooth at the alveolar crest, and could extend as far as the root notch. A mean reattachment of 0.00 mm was observed in the control group. The tetracycline-HCl treated group demonstrated a mean reattachment of 0.27 mm. Connective tissue fibers were observed oriented parallel and perpendicular to the root surface. Three of seven tetracycline-HCl treated specimens demonstrated new cementum. The tetracycline-HCl and fibronectin treated group had a mean reattachment of 0.17 mm. No new cementum was observed in the fibronectin group, and connective tissue fibers adjacent to the root demonstrated parallel apposition. A long junctional epithelium, 1 to 12 cells in thickness, extended into the bone notch in 17 of 18 specimens. The addition of fibronectin to tetracycline treated teeth did not enhance connective tissue attachment, and appeared to inhibit it.
REFERENCES 1. American Academy of Periodontology. Current Procedural Terminology for Periodontics. 5th ed. Chicago, IL: The American Academy of Periodontology; 1986:26. 2. Wirthlin MR. The current status of new attachment therapy. J Periodontoi 1981; 52:529. 3. Caton J, Nyman S, Zander H. Histometric evaluation of periodontal surgery. II. Connective tissue attachment levels after four regenerative procedures. J Clin Periodontoi 1980; 7:224. 4. Caton J, Zander HS. Osseous repair of an infrabony pocket without new attachment of connective tissue. J Clin Periodontoi 1976; 3:54. 5. Marks SC, Mehta NR. Lack of effect of citric acid treatment of root surfaces on the formation of new connective tissue attachment. J Clin Periodontoi 1986; 13:109
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8. 9.
10.
ALGER, SOLT, VLJDDHAKANOK, MILES
Register AA. Bone and cementum induction by dentin, demineralized in situ. / Periodontol 1973; 44:49. Register AA, Burdick FA. Accelerated reattachment with cementogénesis to dentin, demineralized in situ. I. Optimum Range. / Periodontol 1975; 46:646. Common J, McFall WT. The effects of citric acid on attachment of laterally positioned flaps. J Periodontol 1983; 54:9. Albair WB, Cobb CM, Killoy WJ. Connective tissue attachment to periodontally diseased roots after citric acid demineralization. J Periodontol 1982; 53:515. Kashani HG, Magner AW, Stahl SS. The effects of root planing and citric acid applications on flap healing in humans A histologie evaluation. / Periodontol 1984; 55:679. Poison AM, Proye MP. Fibrin linkage: A precursor for new attachment. / Periodontol 1983; 54:141. Terranova VP, Martin GR. Molecular factors determine gingival tissue interaction with tooth structure. J Periodont Res 1982; 17:530. Caton JG, Poison AM, Prato GP, Bartolucci EG, Clauser C. Healing after application of tissue-adhesive material to denuded and citric acid—treated root surfaces. J Periodontol 1986; 57:385. Pitaru S, Gray A, Aubin JE, Melcher AH. The influence of the morphological and chemical nature of dental surfaces on the migration, attachment, and orientation of human gingival fibroblasts in vitro. / Periodont Res 1984; 19:408. Karp W, Sodek J, Aubin JE, Melcher AH. A comparison of fibronectin and laminin binding to undemineralized and demineralized tooth root surfaces. / Periodont Res 1986; 21:30. Terranova VP, Franzetti LC, Hic S, et al. A biological approach to periodontal regeneration tetracycline treatment of dentin promotes fibroblast adhesion and growth. J Periodont Res 1986; 21:330. Knox P, Crooks S, Rimmer C. Role of fibronectin in the migration of fibroblasts into plasma clots. J Cell Bio 1986; 102:2318. Caffesse RG, Holden MJ, Kon S, Nasjleti CE. The effect of citric acid and fibronectin application on healing following surgical treatment of naturally occurring periodontal disease in beagle dogs. J Clin Periodontol 1985; 12:578. -
11. 12. 13.
14.
15.
16.
—
17.
18.
19.
455
Wikesjo UME, Baker PJ, Christersson LA, et al. A biochemical approach to periodontal regeneration tetracycline treatment condi-
tions dentin surfaces. J Periodont Res 1986; 21:322. 20. Horowitz B, Weibe M, Lippin A, Stryker MH. Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl) phosphate detergent combinations. Transfusion 1985; 25:516. 21. Seilly DJ. A chemical test to determine the end point of EDTA decalcification. Med Lab Sci 1982; 39:71. 22. Waerhaug J. Healing of the dento-epithelial junction following subgingival plaque control. I. As observed in human biopsy material. J Periodontol 1978; 49:1-8. 23. Garjuilo AW, Wentz FM, Orban B. Dimensions and relations of the dentogingival junction in humans. J Periodontol 1961; 32:261-267. 24. Wikesjo UME, Claffey N, Christersson LA, et al. Repair of periodontal furcation defects in beagle dogs following reconstructive surgery including root surface demineralization with tetracycline-HCl and topical fibronectin application. J Clin Periodontol 1988; 15:73. 25. McLean TN, Smith BA, Caffesse RG, Nasjleti C, Castelli WA. Tetracycline root conditioning in reattachment procedures. / Dent Res 1988; 67:329. 26. Caffesse RG, Kerry GJ, Chaves ES, et al. Clinical evaluation of the use of citric acid and autologous fibronectin in periodontal surgery. J Periodontol 1988; 59:565. 27. Ellegard B, Löe H. New attachment of periodontal tissue after treatment of intrabony lesions. J Periodontol 1971; 42:648. 28. Pitaru S, Hekmati M, Geiger S, Savion . The effects of partial demineralization and fibronectin on migration and growth of gingival epithelial cells on cementum in vitro. J Dent Res, 1988; 67:1386. 29. Terranova V, Hic S, Franzetti L, Lyall R, Wikesjo U. A biochemical approach to periodontal regeneration. J Periodontol 1987; 58:247. —
Send reprint requests to Dr. F. A. Alger, Department of Periodontology, College of Dentistry, Ohio State University, 305 West 12th Avenue, Columbus, OH 43210. Accepted for publication January 23, 1990.
The Histologie Evaluation of New Attachment in Periodontally Diseased Human Roots Treated With Tetracycline-Hydrochloride and Fibronectin* F.A.
Alger,
C. W.
Soit, S. Vuddhakanok, and
. Miles
The purpose of this study was to determine how the treatment of human tooth roots with tetracycline-HQ and fibronectin during periodontal surgery influences the attachment of the gingiva to the root surface. Mucoperiosteal flap surgery was performed on 22 teeth with periodontal disease. Teeth were assigned to three groups. Group one received surgery with degranulation and root planing. Group two received surgery with treatment of roots with tetracycline-HCl. Group three received surgery with treatment of roots with tetracycline-HCl and fibronectin. At 90 days, block sections were taken and teeth, gingiva, alveolar bone, and periodontal ligament were evaluated histologically. Controls healed with a long junctional epithelial attachment. Tetracycline and tetracycline with fibronectin groups demonstrated some reattachment, but only within the notches placed in the root at the original level of the bone. There was a trend for greater connective tissue attachment following tetracycline-HCl treatment of roots. The additional application of fibronectin to tetracycline treated roots appeared to partially negate the enhanced connective tissue attachment observed with tetracycline treatment alone. J Periodontol 1990; 61:447^455.
Key Words: Tooth root; tetracycline/effects; gingival attachment; connective tissue tachment; fibronectin; epithelial attachment.
New attachment has been defined as the reunion of connective tissue with a root surface which has been pathologically exposed.' Conventional periodontal therapy has been shown to arrest the progressive destruction of periodontal tissues, but has demonstrated minimal potential for new
attachment.2"4
Previous researchers have attempted to achieve new attachment. One technique, which has worked more predictably in animals than in humans, has been the topical application of citric acid to roots.5"10 Poison and Proye observed connective tissue attachment to citric acid treated roots in animals. A fibrin linkage preceded connective tissue attachment.11 They speculated that the fibrin linkage resulted from fibronectin binding to collagen fibrils exposed during acid etching. This mechanism of attachment is feasible in that serum transglutaminase covalently links fibronectin with collagen and fibrin.12'13 Demineralized roots bind fibronectin twice as readily as The Ohio State
University, College dontology, Columbus, OH 43210.
of
Dentistry, Department
of Perio-
at-
undemineralized roots. This binding is dependent upon root surface collagen.14-15 Results from in vitro studies have indicated that fibronectin suppressed epithelial cell attachment and growth.16 Fibronectin is also chemoattractant for fibroblasts and periodontal ligament cells.16,17 These findings suggest that procedures which enhance fibronectin binding to roots may have potential for healing with a new attachment. Studies on dogs have demonstrated that the additional application of fibronectin to citric acid treated roots resulted in a 66% greater new attachment.18 Tetracycline-HCl treated roots may bind fibronectin more readily than those treated with citric acid, while exhibiting antimicrobial efficacy for 48 hours.16-19 The purpose of this investigation was to evaluate histologically, the effects of tetracycline-HCl and fibronectin on enhancing new attachment in humans. MATERIALS AND METHODS Prior to treatment, a protocol of the research procedure was approved by The Human Subjects Committee of the Ohio
448
J Periodontol July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT
State University and an Investigational New Drug Exemption was obtained from The Food and Drug Administration for the use of human fibronectin during periodontal surgery. The study sample was comprised of 22 teeth in 5 immediate removable partial denture patients, ages 24 to 51 years of age. Teeth were selected based upon the following criteria: 1. A loss of connective tissue attachment of at least 5 mm. Measurements were determined by probing from the cemento-enamel junction (CEJ) to the base of the periodontal pocket. 2. Radiographic evidence of at least 30% interproximal bone loss. 3. Teeth with a hopeless prognosis, or scheduled to be extracted. Teeth were assigned to one of the following groups: Group 1, a control group, received mucoperiosteal flap surgery with degranulation and root planing. Group 2 received mucoperiosteal flap surgery plus topical root treatment with tetracycline-HCl, pH 2.0, 100 mg/ ml. Group 3 received mucoperiosteal flap surgery, topical root treatment with tetracycline-HCl, and subsequent treatment with human fibronectin, 10 mg/ml. Efforts were made to match experimental and control teeth within the same patient, based upon tooth location and loss of attachment. Patients received oral hygiene instructions and surgery was not scheduled until patients demonstrated the ability to adequately eliminate plaque from their teeth. Ellman mesh splints were bonded to teeth to stabilize them and to assist in coronal suturing of flaps. Inverse bevel incisions were made and mucoperiosteal flaps were elevated well beyond the mucogingival junction to expose the underlying root and alveolar bone. Granulation tissue was removed. Using a diamond bur, reference notches were placed in the tooth. A crown notch was placed at the midfacial of the tooth, extending into dentin. A second notch was placed at the height of the alveolar bone crest or at the depth of an intrabony defect, if present. When calculus was detected, a notch was made on the root surface at the apical extent of the deposit (Fig. 1). Zinc-oxide and eugenol paste was placed as a temporary restoration in the crown notch, after measurements were recovered. Calipers were used to measure the distance from the crown notch to the CEJ, from the crown notch to the root notch at the apical extent of calculus deposits, and from the crown to the alveolar bone notch. The measurements were repeated and recorded three times. Utilizing an electronic digital caliper, the distances were measured and a mean was calculated (Tables 1, 2, and 3). Root surfaces were planed to the alveolar crest or to the base of existing intrabony defects. Surgical sites were irrigated with sterile saline. Teeth in the control group received no additional treatment prior to suturing. Flaps were
Figure 1. Mucoperiosteal flaps were elevated and reference notches were placed into the crown (CN crown notch), and in the root at the level of the alveolar bone crest (BN bone notch). A notch was also placed through the apical extent of calculus detected on the root (RN root =
=
=
notch).
Table 1: Measurements and
Specimen
1
Clinical Clinical CN-RN Means CN-BN Histometric For Attachment
3
Histologie
Means CN-RN CN-BN
RN-Collagen BN-Collagen BN-Collagen: SD
BN-Bone Crest
N/A 4.34
Findings 3 N/A 3.35
in Controls
N/A
3
3 6.02
3 3.70
3 3.30
10.22
8.33
5.16
5.24
N/A
5.78 8.83 0.00 0.20 0.24
3.05 5.71 0.00 0.06 0.08 0.41
0.00 0.00
4
N/A
N/A
3.97
N/A
N/A
N/A
0.00
-.17 0.24 0.75
-.09 0.18 0.77
t 0.48
t
f 0.55
Crown Notch Standard Deviation Root Notch BN Bone Notch Not applicable, no calculus evident on the root. N/A * Crown notch is not observed histologically. Standard deviation is not calculated for mean 0. t Bone crest is not observed histologically. t Number of measurements made to derive a mean.
CN SD RN
=
=
= =
=
= =
=
= =
positioned coronally using resorbable sutures (Fig. 2). A periodontal dressing was placed over the surgical site. The roots of teeth in Group 2 were burnished for 3 minnutes with cotton pellets saturated with a solution of tetracycline-HCl,1' 100 mg/ml. Saliva was excluded from the surgical area by the use of high speed suction and isolation with cotton rolls. Following demineralization the roots were irrigated with sterile saline for 1 minute, and flaps were sutured coronally. tServa Fine Pharmaceuticals, Westbury, NY.
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ALGER, SOLT, VUDDHAKANOK, MILES
Table 2: Measurements and
Specimen Clinical Clinical Means
Findings
in
7
CN-RN CN-BN
-
/
N/A
N/A
4.16
4.48
8.86
Histometric For Attachment
Histologie Means
Tetracycline-Treated
CN-RN CN-BN
CN SD RN BN
Teeth
1
12
13
N/A
2.92 4.25
4.31
4.74
N/A
4
N/A
N/A
N/A
4.35
4.10
8.98
RN-Collagen N/A N/A N/A BN-Collagen 0.88 0.61 0.33 BN-Collagen: SD 0.41 0.05 0.54 BN-Bone Crest
5.84 8.95
449
0.46
0.27
0.72
6.56 9.79 0.00 0.03 0.04 0.70
N/A 4.39 N/A 0.00
N/A 3.93 0.00 0.05 0.09
t 0.57
N/A 0.02 0.02 0.40
Crown notch Standard deviation Root notch Bone notch Not applicable, no calculus evident on the root. N/A * Crown notch is not observed histologically. Standard deviation is not calculated for mean 0. t Bone crest is not observed histologically. Number of measurements made to derive a mean. =
=
=
=
=
=
=
=
Figure
=
2.
Gingival flaps were sutured coronally.
=
Table 3: Measurements and Fibronectin-Treated Teeth
Findings
Specimen
14
Clinical Clinical Means
CN-RN CN-BN
Histometric For Attachment
Histologie Means
CN-RN CN-BN
RN-Collagen BN-Collagen BN-Collagen: SD
BN-Bone Crest
CN SD RN BN
in
Tetracycline-
15
16
17
3
18
3
3
3
N/A 3.36
2.29 4.95
8.98
6
5
4
3
6
N/A
2.32 10.66
N/A
*
N/A
*
3.29
*
8.54
*
N/A
0.00 0.99 0.13 0.51
N/A
N/A 0.01 0.02 0.91
0.00 -.19 0.22 0.70
0.02 0.00 0.90
3
N/A 4.27
*
N/A 0.00
t 0.45
Crown notch Standard deviation Root notch Bone notch Not applicable, no calculus evident on the root. N/A * Crown notch is not observed histologically. Standard deviation is not calculated for mean 0. t Bone crest is not observed histologically. Number of measurements made to derive a mean. =
=
=
=
=
=
=
=
=
=
Group 3 were treated in the same manner as Group 2, plus the application of fibronectin,* 10 mg/ml. The isolated surgical site was air dried and a solution of fibronectin was applied to the root with cotton pellets for 5 minutes. Any solution remaining was applied to the tooth and inside of the flap with a sterile irrigation syringe. Lipidenveloped viruses, including human immunodeficiency vivirus; and non- , non-B hepatitis viruses rus; hepatitis were rendered nonviable by a solvent/detergent virus inactivation system as described by Horowitz.20 At the 1 week post-operative appointment, supragingival Teeth in
New York Blood
Center,
New
York,
NY.
deposits were removed from the teeth. Oral hygiene instrucwere reinforced and supragingival scaling and polishing were performed. These same procedures were repeated at 3, 6, and 10 weeks post-operatively, as needed. At 90 days teeth were removed in block section. A #15 scalpel blade was used to incise the oral tissues, either to periosteum or to tooth structure. These incisions were made along the apical, mesial, and distal portions of the tooth. Facially, the apical extension of the incision was located approximately 2 mm past the alveolar crest, and was determined from caliper measurements made from the crown notch to the bone notch during the initial surgical procedure. Flaps were elevated and a carbide bur was used to extend the incisions through the osseous crest and into tooth structure (Fig. 3). After the block section was outlined in the tooth with the carbide bur, the tooth was carefully extracted along with the soft tissue and crestal bone. The block section, containing gingiva and periodontal ligament attached to alveolar bone and tooth, was carefully removed
tions
and
from the remainder of the tooth with
(Fig. 4).
a
carbide bur
Block sections were fixed in 10% neutral buffered formalin for 48 hours. Décalcification of block sections was accomplished by immersion in a 10% solution of ethylenediaminetetraacetic acid (EDTA) in 0.1 M Tris buffer pH 6.95. The endpoint of specimen décalcification was determined by a chemical test for calcium.21 Decalcified specimens were prepared for embedding after serial dehydrations and perfusion with JB-4 Plus, a glycol-methacrylate plastic. Five sections per specimen, approximately 150 µ apart were sectioned and stained with hematoxylin and eosin. The mounted specimens were evaluated utilizing an Olympus standardized micrometer eyepiece, calibrated to .01 mm. Measurements included the following: from the crown notch to the root notch, from the crown notch to the bone notch, from the bone notch to the alveolar crest, and between both
450
J Periodontol July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT
Figure 3. At 90 days after the first surgical procedure, mucoperiosteal flaps were elevated, leaving a collar of gingiva attached. A carbide bur was used to incise through bone and into tooth. (G Gingiva, Arrow indicates incision through bone) =
Figure 5.
Schematic of Histometric Measurements. Crown notch (CN) to CEJ. b= Crown notch to root notch (RN). c= Crown notch to bone notch (BN). d Root notch to level of collagen attachment. e Bone notch to level of collagen attachment. a
=
=
=
Table 4:
Type
and Number of Teeth Evaluated
Tooth
Type Maxillary 2nd Bicuspid Maxillary 1st Bicuspid Maxillary Canine Maxillary Lateral Incisor Mandibular 1st Bicuspid
Control
Tetracycline Tetracycline/Fibronectin
1
Mandibular Canine Mandibular Lateral Incise
2 2 1
1 2
1 1
RESULTS A total of 22 teeth from 5
imens
The block sections containing gingiva and periodontal ligament attached to alveolar bone and tooth were carefully removed from the remainder of the tooth. Block specimens were obtained at 90 days after the first surgical procedure was performed. (G tooth) gingiva,
Figure 4.
=
=
the root and bone notches and the coronal level of inserting collagen fibers into cementum (Fig. 5). The amount of connective tissue attachment coronal to the bone notch, and the distances from the CEJ to the root notch and bone notch were calculated from the measurements obtained. Statistical findings are not reported here because of the small sample size represented in this study.
were
patients were treated. Four specdamaged during histologie preparation, so that
measurements between reference notches could not be made. The type and number of teeth evaluated can be seen in Table 4.
Clinical Observations At initial presentation, the gingival tissues appeared generally edematous, with marginal redness, blunted papillae, rolled margins, and a loss of resiliency. Plaque and calculus could be detected clinically. Calculus on the root, when present, was used to locate the apical extent of periodontal attachment loss. No calculus was observed on the roots in 11 teeth. At the 1 week post-operative appointment, all patients demonstrated healing without complications. Generally, there was no difference between the rate of healing observed
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ALGER, SOLT, VEDDHAKANOK, MILES
451
clinically between experimental and control sites. At 2 and 3 months, gingival tissues were generally pink, firm, and stippled, without suppuration or bleeding on provocation. The location of the gingiva relative to the cemento-enamel junction indicated that generalized recession of the coronally positioned flaps had occurred. Observations Measurements for connective tissue attachment were made from a mean of 3.9 measurements per specimen. The mean standard deviation between measurements for individual specimens was 0.13 mm. A micrometer eyepiece in an Olympus light microscope was used to measure reattachment and new attachment, relative to reference notches placed into the tooth at the time of surgery. New attachment was considered connective tissue attachment coronal to the root notch placed through calculus at the apical extent of the periodontal pocket. New attachment was not observed in any specimen. In periodontal lesions with horizontal bone loss, there is an area of connective tissue attachment measuring approximately 1 mm between the crest of the supporting alveolar bone and the apical extent of the pocket epithelium.22-23 Therefore, the presence of a connective tissue attachment within 1 mm of the bone crest following periodontal therapy is regarded as reattachment and not new attachment. In this study, reattachment was measured as connective tissue attachment coronal to the notch placed in the root at the level of the alveolar bone crest. Most specimens demonstrated migration of epithelium along the root down to and entering this notch, which represented the level of previous connective tissue attachment. The thickness of the epithelium, at its most apical position on the root, ranged from one cell to over a dozen cells. The oral epithelium was parakeratinized stratified squamous epithelium, with rete pegs. The extent of connective tissue attachment in control specimens ranged from .17 mm apical to the bone notch to .20 mm coronal to the bone notch, with a mean connective tissue reattachment of 0.00 mm. The standard deviation was calculated to be 0.13 mm. New cementum was not observed in any control specimen. Clinical and histologie measurements for control specimens are recorded in Table 1. Figures 6 and 7 show photomicrographs of control block
Histologie
-
specimens. Tetracycline-treated teeth healed with a connective tissue reattachment ranging from 0.00 to 0.88 mm, with a mean
reattachment of 0.27 mm and a standard deviation of 0.35 mm. Connective tissue adjacent to the root, in the area of the bone notch, demonstrated either perpendicular fiber insertion into new cementum or parallel fiber apposition with no new cementum. New cementum was observed in 3 of 7 specimens. Epithelium migrated to the apical extent of the bone notch in 4 of 7 tetracycline treated specimens. Clinical and histologie measurements for tetracycline treated teeth are recorded in Table 2. Figures 8 and 9 show photomicrographs of block specimens.
RN
Figure 6. Control specimen. At 90 days, epithelium has migrated into the root notch (RN) and beyond the bone notch (BN). The arrow indicates the apical extent of epithelium. Original magnification x 40.
Tetracycline and fibronectin-treated teeth healed with a long junctional epithelial attachment and no reattachment in 4 of 5 specimens evaluated. In these specimens, epithelium migrated to the apical extent of the bone notch. A
connective tissue reattachment of 0.99 mm was observed in one specimen. In this specimen, connective tissue adjacent to the root, in the area of the bone notch, demonstrated parallel fiber apposition with no new cementum. When a mean connective tissue reattachment was calculated, it measured 0.17 mm with a standard deviation of 0.47 mm. Clinical and histologie measurements for tetracycline and fibronectin treated teeth are recorded in Table 3. Figures 10 and 11 show photomicrographs of block
specimens.
Differences in
mean
connective tissue attachment be-
control, tetracycline, or tetracycline with fibronectin groups were very small, although there was a trend for tween
greater attachment following tetracycline-HCl
(Tables
5 and
6).
treatment
alone
452
J Periodontol July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT
BN
9
lliii 7. Control specimen. Demonstrates epithelium (E) migrating into the bone notch (BN) and beyond (Arrow). Original magnification x 100.
Figure
DISCUSSION The results of this investigation indicate that the treatment of human roots with tetracycline-HCl or tetracycline-HCl and human fibronectin during periodontal surgery did not result in new attachment. A slight reattachment was observed in both experimental groups, although this gain was very small. Animal studies have indicated that enhanced connective tissue attachment to roots treated with tetracycline-HCl during periodontal surgery is possible.24-25 Our findings indicate that reattachment and new cementum formation can be achieved with root demineralization with tetracyline-HCl in humans. However, reattachment did not reach 1 mm and new attachment was not observed. In vivo studies have reported mixed results when fibronectin was used during periodontal surgery in animals. One study indicated that new attachment could be obtained in beagle dogs, when roots demineralized with citric acid were treated with human fibronectin.18 Other investigators applied canine fibronectin to the roots of dogs which had been demineralized with either a saturated solution of citric acid
A
Figure 8. Tetracycline-treated specimen at 90 days. Epithelium (E) has migrated into the bone notch (BN). Connective tissue fibers can be seen at the apical extent of the bone notch. Periodontal ligament fibers can be seen between the old cementum (O) and alveolar bone (A). Original magnification 40.
tetracycline-HCl, 100 mgs/ml.24 The results of this secstudy are similar to our findings, in that fibronectin application to the demineralized root surface did not enhance the amount of connective tissue repair. A previous study in humans demonstrated that teeth treated with modified Widman flap surgery, citric acid demineralization, and fibronectin application resulted in a mean probing pocket depth reduction that was .22 mm greater than when surgery was performed alone.26 In our study, new attachment was evaluated histologically and histometrically to differentiate between connective tissue attachment and epithelial atttachment. Histologie measurements or
ond
also selected in an attempt to overcome the standardization difficulties inherent in probing attachment
were
measurements.
Patient and tooth selection may be critical to the success of new attachment procedures. Teeth used in this study had been given a hopeless prognosis. These teeth possessed
Volume 61 Number 7
ALGER, SOLT, VUDDHAKANOK, MILES
453
#
^
c Figure 9. Tetracycline-treated specimen. Epithelium (E) is seen within the hone notch. Also within the bone notch, connective tissue fibers (CT) are seen inserting into new cementum (Arrow). Original magnification 100. horizontal bone loss and recession. The most favorable situation for new attachment is found adjacent to 3walled intrabony pockets.27 The premise of this study was that collagen fibrils exposed during tetracycline treatment of roots would bind fibronectin and secure the gingival flap to the root via a fibrin linkage. As fibronectin suppressed the migration of epithelium and attracted fibroblasts and periodontal ligament cells, a new attachment would be facilitated. Instead, there was a trend for less reattachment when fibronectin was applied to the tetracycline-treated tooth. There may have been a flaw in our original premise that fibronectin would inhibit epithelial cell migration. Our findings are consistent with a recent study by Pitaru et al., who'observed that demineralized roots inhibited epithelial migration, but fibronectin partially restored the migration of the epithelium when applied to demineralized roots, in vitro.28 This is one possible explanation why fibronectin did not enhance connective tissue attachment in our study. Other chemotactic factors, such as endothelial growth
Figure 10. Tetracycline-and fibronectin-treated specimen. Epithelium has migrated to the bottom of the bone notch (BN). The apical extent of epithelial migration is indicated (arrow) adjacent to old cementum (C). Original magnification 100.
severe
factor, have been shown in vitro
to
be
more
chemotactic
periodontal ligament fibroblasts than fibronectin.29 Perhaps, in vivo, other chemotactic factors will demonstrate a for
greater potential for connective tissue attachment than fi-
bronectin has. Additional research with fibronectin and other biochemicals is necessary to properly interpret the role that physiological substances have in the healing periodontal lesion. SUMMARY AND CONCLUSIONS Eighteen teeth from four patients were evaluated clinically and histologically for new attachment 90 days after surgical treatment. Teeth selected for this investigation demonstrated severe bone loss and had been given a hopeless
prognosis.
Teeth were assigned to three groups. Group 1 received periodontal surgery, with degranulation, root planing, and coronal flap placement. Group 2 received periodontal surgery, with root demineralization with tetracycline-HCl, 100
454
J Periodontoi July 1990
HISTOLOGIC APPEARANCE OF NEW ATTACHMENT Table 5: Mean Connective Tissue Attachment Per Tooth
C;,
'
%
'
\
'
·
MM Reattachment
Specimen Control 1 2 3 4 5 6
0.00 -.17 -.09
0.20 0.06 0.00
Tetracycline 7 8 9 10 11 12 13 Fibronectin 14 15 16 17 18
0.88 0.61 0.33 0.03 0.00 0.05 0.02 0.01 0.99 0.02 -.19 0.00
Table 6: Mean Connective Tissue Attachment Per
Control
Tetracycline Tetracycline
Figure 11. Tetracycline-and fibronectin-treated specimen. Epithelium (E) has migrated to the bottom of the bone notch (BN), but not apical to it. Old cementum (C) and the apical extent of the epithelial migration (arrow) are labelled. Original magnification x 400. for 3 minutes. Group 3 received periodontal surwith gery tetracycline demineralization, followed by human fibronectin application, 10 mgs/ml, for 5 minutes. During surgery, reference notches were placed into the tooth crown, in the root at th apical extent of calculus, and in the root at the level of the alveolar crest. At 90 days teeth were extracted along with a collar of gingival tissue and bone. Teeth were decalcified and embedded in plastic for histologie preparation. Specimens were sectioned at 2 µ and stained with hematoxylin and eosin. A micrometer eyepiece in an Olympus light microscope was used to measure reattachment and new attachment, relative to reference notches placed into the tooth at the time of surgery. New attachment was considered connective tissue attachment coronal to the root notch placed through calculus at the apical extent of the periodontal pocket. New attachment was not observed in any specimen. Reattachment was considered connective tissue attachment that occurred coronal to the bone notch, placed in the
mgs/ml,
and Fibronectin
X X X
=
=
=
0.00 0.27 0.17
Group S.D. S.D. S.D.
= = =
0.13 0.35 0.47
tooth at the alveolar crest, and could extend as far as the root notch. A mean reattachment of 0.00 mm was observed in the control group. The tetracycline-HCl treated group demonstrated a mean reattachment of 0.27 mm. Connective tissue fibers were observed oriented parallel and perpendicular to the root surface. Three of seven tetracycline-HCl treated specimens demonstrated new cementum. The tetracycline-HCl and fibronectin treated group had a mean reattachment of 0.17 mm. No new cementum was observed in the fibronectin group, and connective tissue fibers adjacent to the root demonstrated parallel apposition. A long junctional epithelium, 1 to 12 cells in thickness, extended into the bone notch in 17 of 18 specimens. The addition of fibronectin to tetracycline treated teeth did not enhance connective tissue attachment, and appeared to inhibit it.
REFERENCES 1. American Academy of Periodontology. Current Procedural Terminology for Periodontics. 5th ed. Chicago, IL: The American Academy of Periodontology; 1986:26. 2. Wirthlin MR. The current status of new attachment therapy. J Periodontoi 1981; 52:529. 3. Caton J, Nyman S, Zander H. Histometric evaluation of periodontal surgery. II. Connective tissue attachment levels after four regenerative procedures. J Clin Periodontoi 1980; 7:224. 4. Caton J, Zander HS. Osseous repair of an infrabony pocket without new attachment of connective tissue. J Clin Periodontoi 1976; 3:54. 5. Marks SC, Mehta NR. Lack of effect of citric acid treatment of root surfaces on the formation of new connective tissue attachment. J Clin Periodontoi 1986; 13:109
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8. 9.
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ALGER, SOLT, VLJDDHAKANOK, MILES
Register AA. Bone and cementum induction by dentin, demineralized in situ. / Periodontol 1973; 44:49. Register AA, Burdick FA. Accelerated reattachment with cementogénesis to dentin, demineralized in situ. I. Optimum Range. / Periodontol 1975; 46:646. Common J, McFall WT. The effects of citric acid on attachment of laterally positioned flaps. J Periodontol 1983; 54:9. Albair WB, Cobb CM, Killoy WJ. Connective tissue attachment to periodontally diseased roots after citric acid demineralization. J Periodontol 1982; 53:515. Kashani HG, Magner AW, Stahl SS. The effects of root planing and citric acid applications on flap healing in humans A histologie evaluation. / Periodontol 1984; 55:679. Poison AM, Proye MP. Fibrin linkage: A precursor for new attachment. / Periodontol 1983; 54:141. Terranova VP, Martin GR. Molecular factors determine gingival tissue interaction with tooth structure. J Periodont Res 1982; 17:530. Caton JG, Poison AM, Prato GP, Bartolucci EG, Clauser C. Healing after application of tissue-adhesive material to denuded and citric acid—treated root surfaces. J Periodontol 1986; 57:385. Pitaru S, Gray A, Aubin JE, Melcher AH. The influence of the morphological and chemical nature of dental surfaces on the migration, attachment, and orientation of human gingival fibroblasts in vitro. / Periodont Res 1984; 19:408. Karp W, Sodek J, Aubin JE, Melcher AH. A comparison of fibronectin and laminin binding to undemineralized and demineralized tooth root surfaces. / Periodont Res 1986; 21:30. Terranova VP, Franzetti LC, Hic S, et al. A biological approach to periodontal regeneration tetracycline treatment of dentin promotes fibroblast adhesion and growth. J Periodont Res 1986; 21:330. Knox P, Crooks S, Rimmer C. Role of fibronectin in the migration of fibroblasts into plasma clots. J Cell Bio 1986; 102:2318. Caffesse RG, Holden MJ, Kon S, Nasjleti CE. The effect of citric acid and fibronectin application on healing following surgical treatment of naturally occurring periodontal disease in beagle dogs. J Clin Periodontol 1985; 12:578. -
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tions dentin surfaces. J Periodont Res 1986; 21:322. 20. Horowitz B, Weibe M, Lippin A, Stryker MH. Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl) phosphate detergent combinations. Transfusion 1985; 25:516. 21. Seilly DJ. A chemical test to determine the end point of EDTA decalcification. Med Lab Sci 1982; 39:71. 22. Waerhaug J. Healing of the dento-epithelial junction following subgingival plaque control. I. As observed in human biopsy material. J Periodontol 1978; 49:1-8. 23. Garjuilo AW, Wentz FM, Orban B. Dimensions and relations of the dentogingival junction in humans. J Periodontol 1961; 32:261-267. 24. Wikesjo UME, Claffey N, Christersson LA, et al. Repair of periodontal furcation defects in beagle dogs following reconstructive surgery including root surface demineralization with tetracycline-HCl and topical fibronectin application. J Clin Periodontol 1988; 15:73. 25. McLean TN, Smith BA, Caffesse RG, Nasjleti C, Castelli WA. Tetracycline root conditioning in reattachment procedures. / Dent Res 1988; 67:329. 26. Caffesse RG, Kerry GJ, Chaves ES, et al. Clinical evaluation of the use of citric acid and autologous fibronectin in periodontal surgery. J Periodontol 1988; 59:565. 27. Ellegard B, Löe H. New attachment of periodontal tissue after treatment of intrabony lesions. J Periodontol 1971; 42:648. 28. Pitaru S, Hekmati M, Geiger S, Savion . The effects of partial demineralization and fibronectin on migration and growth of gingival epithelial cells on cementum in vitro. J Dent Res, 1988; 67:1386. 29. Terranova V, Hic S, Franzetti L, Lyall R, Wikesjo U. A biochemical approach to periodontal regeneration. J Periodontol 1987; 58:247. —
Send reprint requests to Dr. F. A. Alger, Department of Periodontology, College of Dentistry, Ohio State University, 305 West 12th Avenue, Columbus, OH 43210. Accepted for publication January 23, 1990.
