284
Longitudinal Observation of Cementum Regeneration Through Multiple Fluorescent Labeling* Nobuya Ogura, Toyotune Mera,
Fernando Sato, and Isao Ishikawa
THE ASSESSMENT OF NEW ATTACHMENT AFTER PERIODONTAL TREATMENT has been the focus of continuous research. An approach to longitudinally examine the deposition of cementum was devised by using fluorescence microscopy (FL), contact microradiography (CMR), and toluidine blue staining (TBS) after the injection of three labeling agents known to be incorporated within newly mineralized tissues with different tones: tetracycline, calcein, and alizarin complexion. Three adult Japanese monkeys (male, 6.0 to 8.3 Kg weight) were used for this experiment. Bone defects were surgically created in 24 mandibular sites and a copper plate was inserted for a period of 4 weeks to promote microbial colonization to form periodontal pockets. Scaling and root planing (baseline) were then performed, and the fluorescent agents were administered twice weekly leaving a 1 week interval between the different agents. The mandibular specimens were fixed in neutralized formalin and embedded in polyester resin. Undecalcified sections were prepared 3, 6, and 9 weeks after baseline. Cementum regeneration was confirmed in 18 out of 24 sites; in 6 samples only epithelial proliferation was observed. Regeneration could be seen as early as 2 weeks after debridement. Cementum was identified by observation under FL of a labeled structure, discrimination in the degree of mineralization of dentin by CMR, and by the presence of functional collagen fibers and location of the epithelial border by TBS. In this study the use of three different labeling agents using the three observation techniques was shown to be effective for the longitudinal assessment of cementum regeneration. J Periodontol 1991; 62:284-291.
Key Words: Cementum regeneration, fluorescent labeling; cein; alizarin complexion; tetracycline. The
contact
microradiology; cal-
regeneration of pathologically involved tooth-supporting tissues is one of the final goals of periodontal therapy. Although there have been many attempts to regenerate con-
Mineralized tissue labeling observation using fluorescent agents is a method which has been used to study the regeneration of osseous and dental tissues. Nalbardian3 employed tetracycline fluorescent labeling to observe human tooth cementum. More recently, Blömlof and co-workers4'5 reported that when using a tetracycline labeling method they were unable to observe regeneration following root planing alone; nevertheless, they found new attachment after cleansing the root surface with detergents even in the absence of root planing. Three different fluorescent labeling agents known to be incorporated within newly mineralized tissues5 7 were used to longitudinally record the development of new cementum and to evaluate the significance of fluorescent labeling in the observation of cementum regeneration.
actually regenerated.
MATERIALS AND METHODS Three adult Japanese monkeys (male, 6.0 to 8.3 Kg weight) were used for the experiment. Hemisepta-type one wall osseous defects (24 sites) were surgically prepared with a
nective tissue attachment to the root surface, the methods of assessing a newly formed attachment system (such as clinical attachment level measuring procedures, radiographic analysis, and re-entry surgery) fail to provide information on the various processes which occur during periodontal tissue regeneration.1,2 Currently, the only method recognized for recording new attachment is through histological measurement from a conformed notch on the surface of the exposed root.1 This method has revealed important information for evaluating the effects of periodontal treatment. However, it is impossible to obtain accurate longitudinal data, since the samples can only be observed at one stage during the healing process. Furthermore, this method is unable to clearly determine whether the measured tissues
'Department of Periodontology, School of Dentistry, Tokyo Medical and University, Tokyo, Japan.
Dental
Volume 62 Number 4 •
•
OGURA, MERA, SATO, ISHIKAWA Bone defect Metal plate
•
•
Fluorescent
plate labelings
-
Monkey
Ligature 0(
baseline
I
3,6,or9
)
Time
(weeks)
3W
5W
cd——
cd—-
OW
2W
—.
Development
of
Plaque
control
OW
Monkey
experimental Periodontitis
chisel and steel bur on the mesial aspect of the lower premolare and molars. A copper plate was inserted and retained with orthodontic wire in each defect to delay osseous repair and allow bacterial plaque colonization. The gingival flap was sutured using 4-0 silk suture material, such that the plate was covered. Plaque control was ceased for 4 weeks. After this period, the copper plates were removed and scaling and root planing procedures were performed eliminating plaque-exposed cementum (baseline). Plaque control was instituted following debridement by toothbrushing 3 times a week. The pocket depth was probed to record the clinical gingival variations at baseline and 1, 3, 6, and 9 weeks after scaling and root planing had been performed. Three fluorescent agents were prepared to longitudinally record the regeneration process of cementum (Fig. 1): 1) Teramycin (TC)f dose: 30mg/kg; 2) Calcein (CAL)
(3,3'-Bis N,N-di(carboxymethyl) aminomethyl-fluorescein (C30 H26 N2 013)*, 2% solution in normal saline; dose: 8mg/kg.6; and 3) Dotite alizarin complexion (AC)* (1, 2 di- hydroxyanthraquinon-3-yl-methylamine-N, N-diacetic acid, 2% solution in normal saline; dose: 20 mg/kg.7 The agents were successively administered by intramuscular injection at 2, 3, 4, 5, 6, and 9 weeks after scaling and root planing as shown in Figure 2. The labeling injections were given twice a week while the two mandibular quadrants of each animal were assigned a different baseline period. The labeling agents were set in order, first tetracycline, then calcein, and finally alizarin complexion. The interval between each injection was extended to 1 week whenever a different labeling agent was administered. The animals were sacrificed 4 days after the series of injections had been completed. The mandibular excised and fixed in neutralized formalin, then dehydrated using different alcohol concentrations and finally embedded in polyester resin.§ The undecalcified specimens were sectioned bucco-lingually at 550 µ intervals with a Crystal Cutter.11 Each serial section was then thinned to 50 µ sections with the HT Lapping System.11
9W
6W
cd——
4W
cd—
OW —
OW
II
3w
µ.--—
R
Monkey
9W
6W
.
CD—
L
Figure 1: Experimental outline. Periodontal defects were prepared and a baseline was established after scaling and root planing were performed. Fluorescent labeling agents were injected and plaque control instituted until the 3rd, 6th, or 9th weeks in which each of the animals was assigned for sacrifice.
segments
1 Sacrifice
Cal
Sacrifice
Removal of metal
285
3W
5W
cd*—*
cd—
OW
2W
6W
3W
Figure 2: Scheme of labeling agent administration. Each site received an individual frame of labeling period. The continuous arrows represent tetracycline, segemented arrows calcein, and dot-segmented arrows alizarin complexion. The injections were repeated twice a week, leaving a 1 week interval between the different agents. The three animals were assigned a different baseline period (indicated by the week number beneath the continuous line) for each mandibular quadrant and the injections ceased 4 days before sacrifice. Table 1:
Probing Depth
Reduction
Observation
Baseline
Final
Monkey*_Period_Data_Data_
tMean
±
Baseline vs. 3 weeks
3.0±0.5
2.0±0.2
P
Longitudinal Observation of Cementum Regeneration Through Multiple Fluorescent Labeling* Nobuya Ogura, Toyotune Mera,
Fernando Sato, and Isao Ishikawa
THE ASSESSMENT OF NEW ATTACHMENT AFTER PERIODONTAL TREATMENT has been the focus of continuous research. An approach to longitudinally examine the deposition of cementum was devised by using fluorescence microscopy (FL), contact microradiography (CMR), and toluidine blue staining (TBS) after the injection of three labeling agents known to be incorporated within newly mineralized tissues with different tones: tetracycline, calcein, and alizarin complexion. Three adult Japanese monkeys (male, 6.0 to 8.3 Kg weight) were used for this experiment. Bone defects were surgically created in 24 mandibular sites and a copper plate was inserted for a period of 4 weeks to promote microbial colonization to form periodontal pockets. Scaling and root planing (baseline) were then performed, and the fluorescent agents were administered twice weekly leaving a 1 week interval between the different agents. The mandibular specimens were fixed in neutralized formalin and embedded in polyester resin. Undecalcified sections were prepared 3, 6, and 9 weeks after baseline. Cementum regeneration was confirmed in 18 out of 24 sites; in 6 samples only epithelial proliferation was observed. Regeneration could be seen as early as 2 weeks after debridement. Cementum was identified by observation under FL of a labeled structure, discrimination in the degree of mineralization of dentin by CMR, and by the presence of functional collagen fibers and location of the epithelial border by TBS. In this study the use of three different labeling agents using the three observation techniques was shown to be effective for the longitudinal assessment of cementum regeneration. J Periodontol 1991; 62:284-291.
Key Words: Cementum regeneration, fluorescent labeling; cein; alizarin complexion; tetracycline. The
contact
microradiology; cal-
regeneration of pathologically involved tooth-supporting tissues is one of the final goals of periodontal therapy. Although there have been many attempts to regenerate con-
Mineralized tissue labeling observation using fluorescent agents is a method which has been used to study the regeneration of osseous and dental tissues. Nalbardian3 employed tetracycline fluorescent labeling to observe human tooth cementum. More recently, Blömlof and co-workers4'5 reported that when using a tetracycline labeling method they were unable to observe regeneration following root planing alone; nevertheless, they found new attachment after cleansing the root surface with detergents even in the absence of root planing. Three different fluorescent labeling agents known to be incorporated within newly mineralized tissues5 7 were used to longitudinally record the development of new cementum and to evaluate the significance of fluorescent labeling in the observation of cementum regeneration.
actually regenerated.
MATERIALS AND METHODS Three adult Japanese monkeys (male, 6.0 to 8.3 Kg weight) were used for the experiment. Hemisepta-type one wall osseous defects (24 sites) were surgically prepared with a
nective tissue attachment to the root surface, the methods of assessing a newly formed attachment system (such as clinical attachment level measuring procedures, radiographic analysis, and re-entry surgery) fail to provide information on the various processes which occur during periodontal tissue regeneration.1,2 Currently, the only method recognized for recording new attachment is through histological measurement from a conformed notch on the surface of the exposed root.1 This method has revealed important information for evaluating the effects of periodontal treatment. However, it is impossible to obtain accurate longitudinal data, since the samples can only be observed at one stage during the healing process. Furthermore, this method is unable to clearly determine whether the measured tissues
'Department of Periodontology, School of Dentistry, Tokyo Medical and University, Tokyo, Japan.
Dental
Volume 62 Number 4 •
•
OGURA, MERA, SATO, ISHIKAWA Bone defect Metal plate
•
•
Fluorescent
plate labelings
-
Monkey
Ligature 0(
baseline
I
3,6,or9
)
Time
(weeks)
3W
5W
cd——
cd—-
OW
2W
—.
Development
of
Plaque
control
OW
Monkey
experimental Periodontitis
chisel and steel bur on the mesial aspect of the lower premolare and molars. A copper plate was inserted and retained with orthodontic wire in each defect to delay osseous repair and allow bacterial plaque colonization. The gingival flap was sutured using 4-0 silk suture material, such that the plate was covered. Plaque control was ceased for 4 weeks. After this period, the copper plates were removed and scaling and root planing procedures were performed eliminating plaque-exposed cementum (baseline). Plaque control was instituted following debridement by toothbrushing 3 times a week. The pocket depth was probed to record the clinical gingival variations at baseline and 1, 3, 6, and 9 weeks after scaling and root planing had been performed. Three fluorescent agents were prepared to longitudinally record the regeneration process of cementum (Fig. 1): 1) Teramycin (TC)f dose: 30mg/kg; 2) Calcein (CAL)
(3,3'-Bis N,N-di(carboxymethyl) aminomethyl-fluorescein (C30 H26 N2 013)*, 2% solution in normal saline; dose: 8mg/kg.6; and 3) Dotite alizarin complexion (AC)* (1, 2 di- hydroxyanthraquinon-3-yl-methylamine-N, N-diacetic acid, 2% solution in normal saline; dose: 20 mg/kg.7 The agents were successively administered by intramuscular injection at 2, 3, 4, 5, 6, and 9 weeks after scaling and root planing as shown in Figure 2. The labeling injections were given twice a week while the two mandibular quadrants of each animal were assigned a different baseline period. The labeling agents were set in order, first tetracycline, then calcein, and finally alizarin complexion. The interval between each injection was extended to 1 week whenever a different labeling agent was administered. The animals were sacrificed 4 days after the series of injections had been completed. The mandibular excised and fixed in neutralized formalin, then dehydrated using different alcohol concentrations and finally embedded in polyester resin.§ The undecalcified specimens were sectioned bucco-lingually at 550 µ intervals with a Crystal Cutter.11 Each serial section was then thinned to 50 µ sections with the HT Lapping System.11
9W
6W
cd——
4W
cd—
OW —
OW
II
3w
µ.--—
R
Monkey
9W
6W
.
CD—
L
Figure 1: Experimental outline. Periodontal defects were prepared and a baseline was established after scaling and root planing were performed. Fluorescent labeling agents were injected and plaque control instituted until the 3rd, 6th, or 9th weeks in which each of the animals was assigned for sacrifice.
segments
1 Sacrifice
Cal
Sacrifice
Removal of metal
285
3W
5W
cd*—*
cd—
OW
2W
6W
3W
Figure 2: Scheme of labeling agent administration. Each site received an individual frame of labeling period. The continuous arrows represent tetracycline, segemented arrows calcein, and dot-segmented arrows alizarin complexion. The injections were repeated twice a week, leaving a 1 week interval between the different agents. The three animals were assigned a different baseline period (indicated by the week number beneath the continuous line) for each mandibular quadrant and the injections ceased 4 days before sacrifice. Table 1:
Probing Depth
Reduction
Observation
Baseline
Final
Monkey*_Period_Data_Data_
tMean
±
Baseline vs. 3 weeks
3.0±0.5
2.0±0.2
P
