405
Effect of Exogenously Applied Prostaglandin E2 on Alveolar Bone Loss—Histometric Analysis Mutsumi Miyauchi, * Naokuni Ito, * and Ikuko Ogawaf
Ijuhin, * Hiromasa Nikai, * Takashi Takata,f Hiroshi
The effect of Prostaglandin E2 (PGE2) on alveolar bone résorption was examined in 8-week old Wistar rats by histometric analysis. One mg/ml PGE2 topically applied to gingival sulcus induced a marked increase in osteoclasts. The number of osteoclasts increased progressively and reached a maximum at 12 hours. Ultrastructurally, these osteoclasts were in active form with well developed ruffled borders and clear zones. The changes in numbers of osteoclasts after application of various concentrations of PGE2 were dose-dependent (0.001 to 1.0 mg/ml), but higher concentrations of PGE2 (2 mg/ ml) were less effective. In addition, the number of osteoclasts in groups treated with both PGE2 and endotoxin was higher than those that received PGE2 only. These results indicate that bone résorption caused by PGE2 depends on activation and increase of osteoclasts, and suggests that endogenous PGE2 production by host cells stimulated by plaque-associated bacterial endotoxin may be an important pathogenetic factor in periodontal disease. / Periodontol 1992; 63:405-411.
Key Words: Prostaglandin E2; bone résorption; osteoclasts; alveolar process; periodontal
disease/etiology.
One of the principal clinical and biologic features of Periodontitis is alveolar bone loss. It is generally considered that alveolar bone résorption in Periodontitis is primarily caused by osteoclasts stimulated by various chemical mediators.1 Recently, details of biological aspects of osteoclasts have been elucidated. But, the mechanisms of bone destruction by osteoclasts in chronic inflammatory lesions, such as rheumatoid arthritis and chronic periodontal disease, are not still well understood.2 It has been reported that a variety of products from activated inflammatory cells, such as Prostaglandins (PGs),1"8 osteoclast activating factor (OAF),1'2 interleukin1 (IL-1),1'2'9'10 and heparin1'11 stimulate osteoclastic bone résorption in vitro. In particular, PGs, which are biologically active derivatives of long-chain polyunsaturated fatty acid and known to be a potent soluble mediator of several chronic inflammatory diseases, have been the focus of intense research. They have initially been described as a stimulator of bone résorption in vitro by Klein and Raisz.3 On the other hand, it has been shown that PGE2 levels are high in inflamed human gingiva12'13 and the gingival fluid associated with Periodontitis.14 Furthermore indomethacin,
'Department of Oral Pathology, Hiroshima University School of tistry, Hiroshima, Japan. ,Clinical Laboratory, Hiroshima University Dental Hospital.
Den-
inhibitor of Prostaglandin synthesis, has been reported reduce alveolar bone loss in experimental Periodontitis.15'16 In contrast to the numerous biological, pharmacological, and physiological investigations on PGs in vitro, little information is available regarding their effects on in vivo periodontal tissues. In this study, we attempt to examine the effects of exogenously applied PGE2 on osteoclastic résorption of alveolar bone by morphometric methods. After topical application of PGE2 on the gingiva, we counted the number of osteoclasts along with the margin of alveolar bone in the rat molar periodontium and statistically analyzed their changes with time and dose. Furthermore, we attempted to examine the interaction of PGEj and bacterial endotoxin on the change in number of osteoclasts by means of serial application of PGE2 and endotoxin. an
to
MATERIALS AND METHODS A total of 69 8-week old (about 210 gm), male Wistar strain rats were used in these studies. They were fed a conventional feed through each experimental period. The animals were divided into 3 groups and treated with PGE2 under each experimental condition described below. The PGE2 treatment was done as follows. The animal was sedated with an intraperitoneal injection of 20% ethyl car-
406
EFFECTS OF
PGE2
barriate*
(0.5ml/100 gr body weight) and fixed on his back stand. PGE2§ was dissolved in 0.05% ethanol (AL) in sterile physiological salt solution (PS).11 A sterile cotton applicator (2 mm in diameter and 1 cm in length) was saturated with the solution and placed on the on an
J Periodontol May 1992
ON ALVEOLAR BONE
experimental
occlusal surfaces of upper molar teeth for 1 hour. The cotton applicator was changed every 20 minutes.
Experiment 1 The aim of this experiment was to study the temporal changes in the number of osteoclasts observed after topical application of PGE2. Thirty-six rats were divided into 12 subgroups of 3 each. One mg/ml PGE2 was administered to the rats in 7 subgroups as described above. Each subgroup was sacrificed at 1, 3, or 12 hours, or 1, 2, 3, or 7 days after the PGE2 treatment. As a control, 4 other subgroups were treated with 0.05% v/v ethanol in sterile physiological salt solution (AL/PS) and sacrificed at 12 hours and 1, 2, or 7 days after treatment. The remaining subgroup was an untreated control group.
ethanols, and embedded in Epon 812. Semithin sections (2µ ) parallel to the long axis of the tooth, including the
tooth apex, were made from the mesial and distal roots and stained with toluidine blue. Some thin sections stained with uranyl acetate and lead citrate were examined in JEOL100S electron microscope at 80kV. The toluidine blue stained semithin sections were used for histometric evaluation of osteoclasts. The number of osteoclasts seen along the 1 mm of inner alveolar wall contour (periodontal ligament side) were counted under a light microscope equipped with an ocular-micrometer. The mononucleated or multinucleated giant cells with pale foamy cytoplasm contacting the bone surface were judged as osteoclasts. On the average, we counted the number of osteoclasts at 15 different sites in each experimental and control group (5 in each animal). The student t test was used to determine if there was a statistically significant difference between experimental and control groups (AL/PS treated animals), and between the experimental and untreated control group. The results were given as mean ± standard error
(M
±
SE).
Experiment 2 In this experiment, the different number of osteoclasts seen after topical application of various concentrations of PGE2 were examined. Twelve rats were divided into 4 subgroups of 3 each. PGE2 was dissolved in concentrations of 0.001, 0.01, 0.1, and 2.0 mg/ml in 0.05% v/v AL/PS and each concentration of PGE2 was applied for 1 hour in the same method as experiment 1. The rats were sacrificed 12 hours after application.
Experiment 3 The interaction of PGE2 and bacterial endotoxin on osteoclastic activity and alveolar bone résorption was studied in the third experiment. Twenty-one rats, divided into 7 subgroups of 3 each, were treated with 1 mg/ml PGE2 solution in 0.05% v/v AL/PS for 1 hour as described above, followed by topical application of 5 mg/ml endotoxin (Edx)1 from Escherichia coli in PS for 1 hour. Materials from each subgroup were obtained at 1, 3, and 12 hours and 1, 2, 3, and 7 days after Edx application. Materials were taken from the right and left upper molar regions and fixed for 3 hours in full strength Karnovsky fixative (0.1 M cacodylate buffered 4% w/v paraformal-
dehyde-5% w/v glutaraldehyde solution, pH 7.4, 4°C), and decalcified for 2 weeks in 10% EDTA solution (pH 7.4). The decalcified materials
into slices at the buceach copalatal plane parallel palatal root under a dissecting microscope. These slices were postfixed for 1 hour in cacodylate buffered with 1% w/v osmium tetroxide, stained in block with 1% uranyl acetate, dehydrated in graded were cut
to
*Katayama Co., Osaka, Japan. §Funakoshi Co., Tokyo, Japan. "Otsuka Co., Tokyo, Japan. 'Sigma Chemical Co., St. Louis,
MO.
RESULTS
Histologie Findings
Control animals. The histologie findings of rat molar palatal periodontal tissues after topical application of 0.05% v/v AL/PS were identical to those of untreated controls. There were few osteoclasts in the periodontal ligament facing the alveolar bone. Rather smaller and round preosteoclast-like mononuclear cells, exhibiting the similar cytoplasmic appearance to an osteoclast, were often seen around blood vessels or in the region near the alveolar bone
margin (Fig. 1). Experimental animals. PGE2 applied topically to the gingival tissue caused various changes in the periodontal tissues. The cells of JE were more loosely connected. Large numbers of polymorphonuclear leukocytes (PMN) migrated into both the wide intercellular spaces of the JE and subepithelial connective tissue. In the subepithelial connective tissue, edematous changes with dilation of blood vessels was prominent. Osteoclastic bone résorption was seen along the margin of alveolar bone facing the periodontal ligament. Ultrastructurally, most of observed osteoclasts possessed well-developed brush borders on the cell surface facing the resorbed cavity of bone. Preosteoclast-like mononuclear cells also increased. Figure 2 is representative of the light microscopic features of the rat molar palatal periodontal ligament tissue after PGE2 application. In addition to these findings, the animals receiving higher concentrations of PGE2 (2 mg/ml) showed degenerative changes of various cells, such as JE, gingival fibroblasts,
and osteoblasts located at the alveolar crest (Fig. 3). Although many osteoclasts were still observed, most of them were present apart from the irregularly resorbed bone margin.
Volume 63 Number 5
MIYAUCHI, UUHIN, NIKAI, TAKATA, ITO,
Figure 1. Preosteoclast-like cells (POC) 12 hours after topical application of 0.05% ethanol in sterile physiological salt solution were small and round and located apart from bone margin. Epoxy section stained with toluidine blue; original magnification x 200.
an
More detailed histologie observations will be another article.
Histometric Findings A representative palatal
reported in
periodontal tissue area used for histometrical measurement is shown in Figure 4. Experiment 1. The temporal changes in the number of osteoclasts in untreated, AL/PS-treated, and PGE2-treated groups are illustrated in Figure 5. The mean number of osteoclasts of the untreated group was 3.47 ± 0.68. At 12 hours, 1, 2, and 7 days after 0.05% AL/PS application, the mean number of osteoclasts was 4.40 ± 0.81, 2.50 ± 0.97, 3.00 ± 1.04, and 3.91 ± 1.18, respectively. Statistically there was no significant difference between these values and those of the untreated control group. After topical application of 1 mg/ml PGE2, the mean number of osteoclasts increased distinctly when compared with that of untreated control animals (Fig. 5), reaching the maximum (9.20 ± 1.23) at 12 hours and then decreasing gradually. A significant increase of osteoclasts (P
Effect of Exogenously Applied Prostaglandin E2 on Alveolar Bone Loss—Histometric Analysis Mutsumi Miyauchi, * Naokuni Ito, * and Ikuko Ogawaf
Ijuhin, * Hiromasa Nikai, * Takashi Takata,f Hiroshi
The effect of Prostaglandin E2 (PGE2) on alveolar bone résorption was examined in 8-week old Wistar rats by histometric analysis. One mg/ml PGE2 topically applied to gingival sulcus induced a marked increase in osteoclasts. The number of osteoclasts increased progressively and reached a maximum at 12 hours. Ultrastructurally, these osteoclasts were in active form with well developed ruffled borders and clear zones. The changes in numbers of osteoclasts after application of various concentrations of PGE2 were dose-dependent (0.001 to 1.0 mg/ml), but higher concentrations of PGE2 (2 mg/ ml) were less effective. In addition, the number of osteoclasts in groups treated with both PGE2 and endotoxin was higher than those that received PGE2 only. These results indicate that bone résorption caused by PGE2 depends on activation and increase of osteoclasts, and suggests that endogenous PGE2 production by host cells stimulated by plaque-associated bacterial endotoxin may be an important pathogenetic factor in periodontal disease. / Periodontol 1992; 63:405-411.
Key Words: Prostaglandin E2; bone résorption; osteoclasts; alveolar process; periodontal
disease/etiology.
One of the principal clinical and biologic features of Periodontitis is alveolar bone loss. It is generally considered that alveolar bone résorption in Periodontitis is primarily caused by osteoclasts stimulated by various chemical mediators.1 Recently, details of biological aspects of osteoclasts have been elucidated. But, the mechanisms of bone destruction by osteoclasts in chronic inflammatory lesions, such as rheumatoid arthritis and chronic periodontal disease, are not still well understood.2 It has been reported that a variety of products from activated inflammatory cells, such as Prostaglandins (PGs),1"8 osteoclast activating factor (OAF),1'2 interleukin1 (IL-1),1'2'9'10 and heparin1'11 stimulate osteoclastic bone résorption in vitro. In particular, PGs, which are biologically active derivatives of long-chain polyunsaturated fatty acid and known to be a potent soluble mediator of several chronic inflammatory diseases, have been the focus of intense research. They have initially been described as a stimulator of bone résorption in vitro by Klein and Raisz.3 On the other hand, it has been shown that PGE2 levels are high in inflamed human gingiva12'13 and the gingival fluid associated with Periodontitis.14 Furthermore indomethacin,
'Department of Oral Pathology, Hiroshima University School of tistry, Hiroshima, Japan. ,Clinical Laboratory, Hiroshima University Dental Hospital.
Den-
inhibitor of Prostaglandin synthesis, has been reported reduce alveolar bone loss in experimental Periodontitis.15'16 In contrast to the numerous biological, pharmacological, and physiological investigations on PGs in vitro, little information is available regarding their effects on in vivo periodontal tissues. In this study, we attempt to examine the effects of exogenously applied PGE2 on osteoclastic résorption of alveolar bone by morphometric methods. After topical application of PGE2 on the gingiva, we counted the number of osteoclasts along with the margin of alveolar bone in the rat molar periodontium and statistically analyzed their changes with time and dose. Furthermore, we attempted to examine the interaction of PGEj and bacterial endotoxin on the change in number of osteoclasts by means of serial application of PGE2 and endotoxin. an
to
MATERIALS AND METHODS A total of 69 8-week old (about 210 gm), male Wistar strain rats were used in these studies. They were fed a conventional feed through each experimental period. The animals were divided into 3 groups and treated with PGE2 under each experimental condition described below. The PGE2 treatment was done as follows. The animal was sedated with an intraperitoneal injection of 20% ethyl car-
406
EFFECTS OF
PGE2
barriate*
(0.5ml/100 gr body weight) and fixed on his back stand. PGE2§ was dissolved in 0.05% ethanol (AL) in sterile physiological salt solution (PS).11 A sterile cotton applicator (2 mm in diameter and 1 cm in length) was saturated with the solution and placed on the on an
J Periodontol May 1992
ON ALVEOLAR BONE
experimental
occlusal surfaces of upper molar teeth for 1 hour. The cotton applicator was changed every 20 minutes.
Experiment 1 The aim of this experiment was to study the temporal changes in the number of osteoclasts observed after topical application of PGE2. Thirty-six rats were divided into 12 subgroups of 3 each. One mg/ml PGE2 was administered to the rats in 7 subgroups as described above. Each subgroup was sacrificed at 1, 3, or 12 hours, or 1, 2, 3, or 7 days after the PGE2 treatment. As a control, 4 other subgroups were treated with 0.05% v/v ethanol in sterile physiological salt solution (AL/PS) and sacrificed at 12 hours and 1, 2, or 7 days after treatment. The remaining subgroup was an untreated control group.
ethanols, and embedded in Epon 812. Semithin sections (2µ ) parallel to the long axis of the tooth, including the
tooth apex, were made from the mesial and distal roots and stained with toluidine blue. Some thin sections stained with uranyl acetate and lead citrate were examined in JEOL100S electron microscope at 80kV. The toluidine blue stained semithin sections were used for histometric evaluation of osteoclasts. The number of osteoclasts seen along the 1 mm of inner alveolar wall contour (periodontal ligament side) were counted under a light microscope equipped with an ocular-micrometer. The mononucleated or multinucleated giant cells with pale foamy cytoplasm contacting the bone surface were judged as osteoclasts. On the average, we counted the number of osteoclasts at 15 different sites in each experimental and control group (5 in each animal). The student t test was used to determine if there was a statistically significant difference between experimental and control groups (AL/PS treated animals), and between the experimental and untreated control group. The results were given as mean ± standard error
(M
±
SE).
Experiment 2 In this experiment, the different number of osteoclasts seen after topical application of various concentrations of PGE2 were examined. Twelve rats were divided into 4 subgroups of 3 each. PGE2 was dissolved in concentrations of 0.001, 0.01, 0.1, and 2.0 mg/ml in 0.05% v/v AL/PS and each concentration of PGE2 was applied for 1 hour in the same method as experiment 1. The rats were sacrificed 12 hours after application.
Experiment 3 The interaction of PGE2 and bacterial endotoxin on osteoclastic activity and alveolar bone résorption was studied in the third experiment. Twenty-one rats, divided into 7 subgroups of 3 each, were treated with 1 mg/ml PGE2 solution in 0.05% v/v AL/PS for 1 hour as described above, followed by topical application of 5 mg/ml endotoxin (Edx)1 from Escherichia coli in PS for 1 hour. Materials from each subgroup were obtained at 1, 3, and 12 hours and 1, 2, 3, and 7 days after Edx application. Materials were taken from the right and left upper molar regions and fixed for 3 hours in full strength Karnovsky fixative (0.1 M cacodylate buffered 4% w/v paraformal-
dehyde-5% w/v glutaraldehyde solution, pH 7.4, 4°C), and decalcified for 2 weeks in 10% EDTA solution (pH 7.4). The decalcified materials
into slices at the buceach copalatal plane parallel palatal root under a dissecting microscope. These slices were postfixed for 1 hour in cacodylate buffered with 1% w/v osmium tetroxide, stained in block with 1% uranyl acetate, dehydrated in graded were cut
to
*Katayama Co., Osaka, Japan. §Funakoshi Co., Tokyo, Japan. "Otsuka Co., Tokyo, Japan. 'Sigma Chemical Co., St. Louis,
MO.
RESULTS
Histologie Findings
Control animals. The histologie findings of rat molar palatal periodontal tissues after topical application of 0.05% v/v AL/PS were identical to those of untreated controls. There were few osteoclasts in the periodontal ligament facing the alveolar bone. Rather smaller and round preosteoclast-like mononuclear cells, exhibiting the similar cytoplasmic appearance to an osteoclast, were often seen around blood vessels or in the region near the alveolar bone
margin (Fig. 1). Experimental animals. PGE2 applied topically to the gingival tissue caused various changes in the periodontal tissues. The cells of JE were more loosely connected. Large numbers of polymorphonuclear leukocytes (PMN) migrated into both the wide intercellular spaces of the JE and subepithelial connective tissue. In the subepithelial connective tissue, edematous changes with dilation of blood vessels was prominent. Osteoclastic bone résorption was seen along the margin of alveolar bone facing the periodontal ligament. Ultrastructurally, most of observed osteoclasts possessed well-developed brush borders on the cell surface facing the resorbed cavity of bone. Preosteoclast-like mononuclear cells also increased. Figure 2 is representative of the light microscopic features of the rat molar palatal periodontal ligament tissue after PGE2 application. In addition to these findings, the animals receiving higher concentrations of PGE2 (2 mg/ml) showed degenerative changes of various cells, such as JE, gingival fibroblasts,
and osteoblasts located at the alveolar crest (Fig. 3). Although many osteoclasts were still observed, most of them were present apart from the irregularly resorbed bone margin.
Volume 63 Number 5
MIYAUCHI, UUHIN, NIKAI, TAKATA, ITO,
Figure 1. Preosteoclast-like cells (POC) 12 hours after topical application of 0.05% ethanol in sterile physiological salt solution were small and round and located apart from bone margin. Epoxy section stained with toluidine blue; original magnification x 200.
an
More detailed histologie observations will be another article.
Histometric Findings A representative palatal
reported in
periodontal tissue area used for histometrical measurement is shown in Figure 4. Experiment 1. The temporal changes in the number of osteoclasts in untreated, AL/PS-treated, and PGE2-treated groups are illustrated in Figure 5. The mean number of osteoclasts of the untreated group was 3.47 ± 0.68. At 12 hours, 1, 2, and 7 days after 0.05% AL/PS application, the mean number of osteoclasts was 4.40 ± 0.81, 2.50 ± 0.97, 3.00 ± 1.04, and 3.91 ± 1.18, respectively. Statistically there was no significant difference between these values and those of the untreated control group. After topical application of 1 mg/ml PGE2, the mean number of osteoclasts increased distinctly when compared with that of untreated control animals (Fig. 5), reaching the maximum (9.20 ± 1.23) at 12 hours and then decreasing gradually. A significant increase of osteoclasts (P
