Abstract
Cyclosporine A (CsA), broadly used in organ transplantation, may contribute to pathogenesis of osteoporosis. The aim of this study was to investigate the effects of CsA on alveolar bone in rats subjected or not to experimental periodontal disease using biochemical, radiographic, and histometric analysis. Forty Wistar rats were divided into 4 equal groups: Group I (Control), Group II (CsA was injected subcutaneously in a daily dose of 10 mg/kg), Group III (Ligature was placed around the mandibular molars), Group IV (Ligature+CsA). After 60 days, rats were decapitated, serum alkaline phosphatase and calcium levels were measured. Radiographic-alveolar bone loss (ABL), histometric-ABL, and percentage of new alveolar bone formation (NABF%) were determined on mandibular molars. Significant increase in serum alkaline phosphatase levels (p < 0.001), no significant difference in calcium levels were observed (p > 0.05) in Group IV compared to Group III. Radiographic and histometric-ABL were significantly less (p < 0.001), NABF% was significantly greater (p < 0.05) in Group IV than in Group III. No significant difference in any of the parameters between Group II and Group I was found. It can be concluded that in the presence of periodontal disease, CsA treatment may bring out an imbalance in the alveolar bone homeostasis by decreasing resorption and stimulating formation of alveolar bone in rats.
Introduction
Cyclosporin A (CsA) is a cyclic endacapeptide that was initally isolated from the fungus Tolypocladium inflatum gams (Keown and Stiller, 1987). CsA has been broadly used in organ transplantation because of its selectively suppressive action on specific T lymphocytes, and blocking synthesis and secretion of certain lymphokines (Calne et al., 1979; Bunjes et al., 1981). CsA has also been used for the treatment of type 2 diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis, malaria, sarcoidosis, and several other immune diseases (Adams and Davies, 1984). The main side effects of CsA include nephrotoxicity, hepatic dysfunction, neurological disturbances, hypertension, and gingival overgrowth (Keown and Stiller, 1987; Seymour et al., 2000). A common and serious side effect of allogenic organ transplantation is osteoporosis, and CsA may also contribute to its pathogenesis (Spolidorio et al., 2004).
Some in vitro studies have indicated that CsA decreases bone resoption by specially inhibiting T lymphocyte-derived messengers such as interleukin-1, -2, -3, and -4, gamma interferon, and tumor necrosis factor-alpha, which are potent bone degrading agents (Canalis et al., 1988). However, some other in vitro studies have reported that CsA can enhance the excretion of prostaglandin E2 and tromboxane, mediators of osteoclastic bone resorption (Whisler et al., 1984; Esa et al., 1988).
There is also some controversy regarding the in vivo effects of CsA on bone metabolism. Some studies have demonstrated that CsA decreased bone resorption and increased bone formation in rats (Orcel et al., 1989), and also the drug normalized the bone remodeling in arthritis-related osteopenia (Dougados et al., 1988). However, some authors have described a severe osteopenia responsible for osteoporotic fractures in transplantation patients receiving CsA therapy (Movsowitz et al., 1988; Muchmore et al., 1991).
Although the effects of CsA on long bones have been carefully examined, the effects of the drug on the maxillofacial skeleton have received limited attention. These limited studies have conflicting results suggesting that alveolar bone formation is decreased (Shen et al., 2001; Nassar et al., 2004), increased (Cox et al., 1987) or unaffected (Fischer and Klinge, 1994; Goncalves et al., 2003) after CsA therapy. Therefore, the purpose of this present study was to investigate the effects of CsA administration at immunosupresive levels on alveolar bone of rats subjected or not to experimental periodontal disease, using biochemical, radiographic, and histometric analysis.
Material and Methods
Animals
Forty male Wistar rats weighing an avarage of 100 g were used in the study. All rats were housed seperately in plastic cages and kept in a temperature-controlled room with a standard 12/12 h light-dark illumination cycle. All animal care and study protocols were in compliance with guidelines approved by the Animal Experiment Commitee with the assignment protocole CAM 10/57-07.16.2001.
The rats were randomly distributed into 4 groups of 10 animals each: Group I (control): 0.9% NaCl was injected daily; Group II (CsA): CsA (Sandimmun IV Solution, Novartis, Sao Paulo, Brazil) was injected subcutaenously for 8 weeks with a daily dose of 10 mg/kg to provide plasma peak and trough levels of 1000 and 750 ng/ml, respectively (Wassef et al., 1985); Group III (ligature): After systemic anesthesia with intraperitoneal injection of 60 mg/kg ketamine-HCl (Warner Lambert, Pfizer Inc., Istanbul, Turkey), 3.0 sterile silk ligatures were tyed on the necks of mandibular first molars which were kept in position to promote microbial dental plaque accumulation and inflammation during the experimental period (Keles et al., 2005); Group IV (ligature and CsA): 3.0 sterile silk ligatures were placed on the necks of mandibular first molars. Also, CsA was injected subcutaenously to the rats in this group for 8 weeks with a daily dose of 10 mg/kg to provide plasma peak and trough levels of 1000 and 750 ng/ml, respectively.
Biochemical Analysis
After the experimental period the animals were anesthetized using ketamine-HCl anesthesia, and blood was obtained by direct cardiac puncture. Blood samples were then centrifuged at 500 × g for 10 min to obtain serum. Serum calcium and serum alkaline phosphatase levels were assessed by the optimized standard method using the Roche-Hitachi MODULAR system (Keles et al., 2005).
Radiographic Analysis
After the cardiac puncture, the rats were decapitated, the mandibles were carefully removed, and soaked in 10% formalin for 48 h. Then in order to measure the amount of bone loss, standardized radiographs were obtained by long cone technique (Kelly et al., 1975) at 70 KvP, 8mA. Radiographic-alveolar bone loss (ABL), expressed by the distance from the alveolar bone crest to the cemento-enamel junction, was measured for each mesial surface of the mandibular first molars (Nassar et al., 2004) using a digimatic caliper (Mitutoyo Digimatic Caliper, Kanagawa, Japan). These measurements were performed three times in different days, by the same examiner on each radiograph.
Histopathologic and Histometric Analysis
Following radiographic examination, the mandibles were decalcified in 10% nitric acid for 48 hours and embedded in parafin. Parafin sections were cut 4 μm-thick in a bucco-lingual direction throughout the mandibular first molars and stained with hematoxylin & eosin (H&E). Histometric-ABL on both buccal and lingual region of mandibular first molars was determined by histometric measuring the distance from the cemento-enamel junction to the alveolar bone crest (Fischer and Klinge, 1994). These measurements were performed under a light microscope (BH2-Research Microscope, Olympus, Tokyo, Japan) transferred to a monitor with a camera apparatus(Objective ×3.3, F10 CCD Camera, Panasonic, Osaka, Japan) at ×200 magnification.
In the H&E-stained sections, the percentage of new alveolar bone formation (NABF%) was determined on both buccal and lingual region of mandibular first molars by comparing volume fractions (Howard and Reed, 1998) of the bone and marrow. The estimation process was performed on a PC monitor by taking advantage of a recently described image acquisition approach (Korkmaz et al., 2003). In brief, glass slides were directly placed into a high-resolution color scanner, scanned at 2400 dpi, and stored as JPEG files. This resulted in × 24 magnified images where our region of interest could be entirely viewed with sufficient resolution for distinguishing the boundaries between bone and marrow. The acquired digital images were then opened in order in a standard graphic program. Sampling of section fields and counting points that fell over the bone and marrow areas were made with the aid of 2 separate grids already prepared in the same program. The field sampling frame, consisted of 1 mm2 areas, and the point grid were superimposed independently on the same image. Point counting was then conducted on every 5th field. Any changes in the amount of the alveolar bone would cause an increase or decrease in the number of points that hit and could certainly be used for comparative purposes.
Statistical Analysis
Normalities of distributions were tested by using the Shapiro-Wilks procedure. In the comparison of the groups, in which the parameters were not in accordance with normal distribution, Kruskal-Wallis and Mann–Whitney U-non-parametric tests were used, whereas in comparison of the groups having normal distribution, One-way ANOVA and Post Hoc Tukey parametric tests were used. These analysis were performed using a statistical software package (SPSS version 12.0, SPSS, Chicago, Illinois, USA).
Results
Biochemical Findings
The serum calcium levels were 11.06 ± 0.04 mg/dl in Group I, 10.86 ± 0.07 mg/dl in Group II, 11.31 ± 0.06 mg/dl in Group III, and 11.16 ± 0.07 mg/dl in Group IV. The serum alkaline phosphatase levels were 903.90 ± 6.76 U/l in Group I, 919.60 ± 6.42 U/l in Group II, 811.0 ± 6.56 U/l in Group III, and 909.70 ± 6.47 U/l in Group IV. Significant decrease in alkaline phosphatase levels (p < 0.001), significant increase in calcium levels (p < 0.05) were observed in ligated Group III compared to Group I, whereas no significant differences in both calcium and alkaline phosphatase levels were found between ligated and CsA-treated Group IV and Group I (p > 0.05). Also, significant increase in serum alkaline phosphatase levels (p < 0.001), no significant difference in calcium levels (p > 0.05) were observed in Group IV compared to Group III (Table 1).
Radiographic Findings
In dental radiographs, there was no sign of alveolar bone resorption in Groups I and II (Figure 1A, B). On the other hand, remarkable alveolar bone resorption was observed in ligated Groups III and IV, more prominent in Group III than those in Group IV (Figure 1C, D). As shown in Table 2, the measurements of radiographic-ABL confirmed the radiographic observations described above that significantly increased ABL was observed in ligated Groups III and IV compared to the control Group I (p < 0.001). Ligature replacement alone (Group III) leads to a significantly higher bone loss (p < 0.001) when compared to ligature replacement combined with CsA treatment (Group IV), whereas CsA treatment alone has no effect on alveolar bone as no significant difference was found between Group I and Group II (p > 0.05).
Histopathologic and Histometric Findings
In the H&E-stained sections a well-organized tooth-gingiva interface was found in unligated Groups I and II (Figure 2A, B), whereas a depressed soft tissue notch was observed in the ligated Groups III and IV (Figure 2C, D). Increased tissue dimensions (including epithelium, connective tissue and total gingiva) and elongated rete pegs were seen especially in the buccal gingiva of CsA-treated Groups II and IV; more prominent in Group IV than in Group II (Figure 2B, D). Remarkable inflammatory cell infiltration was seen in ligated Groups III and IV, while there were no signs of inflammation in unligated Groups I and II. Periodontal breakdown characterized by the connective tissue attachment loss and alveolar bone resorption was observed in ligated Groups III and IV. Root resorption, another sign of periodontal breakdown, was also observed in ligated Group III. Large areas of the bone with numerous osteocytes indicating no alveolar bone resorption were observed in the control Group I and CsA-treated Group II (Figure 3A, B), whereas large areas of the marrow with a few osteocytes indicating alveolar bone resorption were seen in the ligated Group III and ligated and CsA-treated Group IV, more prominent in Group III (Figure 3C, D).
The measurements of histometric-ABL and NABF% are shown in Table 2. Histometric-ABL was significantly greater, NABF% was significantly less in Groups III and IV than those in Groups I and II (p < 0.001). Histometric-ABL was significantly less (p < 0.001), NABF% was significantly greater (p < 0.05) in Group IV compared to those in Group III, while there was no significant difference in these parameters between Groups I and II (p > 0.05).
Discussion
The present study was designed to evaluate the effects of CsA therapy on periodontal breakdown subjected or not to ligature-induced periodontal disease in a well-characterized animal model. This model is characterized by accumulation of dental plaque and increased infiltration of inflammatory cells that lead to a degradation of periodontal connective tissue and alveolar bone (Keles et al., 2005). In recent years, the effects of CsA therapy on soft tissues of the periodontium have been extensively investigated in vitro (Bartold 1989; Schincaglia et al., 1992) and in vivo (Seymour et al., 1987; Somacarrera et al., 1994; Cetinkaya et al., 2006a, 2006b). It has been reported that 10 mg/kg daily doses of the drug provide plasma peak and trough levels of 1000 and 750 ng/ml (Wassef et al., 1985) and blood levels of CsA between 100–400 ng/ml are sufficient to promote immunosupression in animals (rats, dogs, and monkeys) as well as in humans (Fischer and Klinge, 1994). In agreement with our previous studies, this study showed that CsA administration for 60 days, with a daily dose of 10 mg/kg that has been reported to be immunosuppressive in the rat (Dieperink et al., 1986), resulted in evident gingival overgrowth which is characterized by an increase of connective tissue and epithelial tissue in comparison to control. Although the exact mechanisms for the development of CsA-induced gingival overgrowth are still not known, it is becoming clear that many biologic regulators such as cytokines and growth factors are an important part of the overall gingival tissue response (Iacopino et al., 1997; Cetinkaya et al., 2006a).
Besides soft tissues, the effect of CsA on hard tissues has also received some attention. It is believed that in the normal phsiological situation, both bone formation and resorption progress in a balanced regulated manner with osteoclastic bone resorption preceding new alveolar bone formation by osteoblasts (Cueto-Manzano et al., 1999). It has been reported that the presence of CsA brings about an imbalance in this dynamic remodeling cycle (Nassar et al., 2004). However, in our study, no difference was found in radiographic and histometric findings between CsA-treated Group II and control Group I, while increased alveolar bone loss was found in rats with periodontal disease (Groups III and IV) compared to control group. It is important to note that alveolar bone loss was less in CsA-treated rats with periodontal disease (Group IV) compared to the rats with only periodontal disease (Group III). As a result, our radiographic and histometric analysis clearly showed that CsA treatment has no effect on alveolar bone balance of the healthy periodontium whereas in the presence of periodontal disease, CsA leads to a decrease in alveolar bone loss.
There are numerous studies on the effect of CsA on bone tissue, however exact mechanism still remains uncertain. It has been shown that CsA may inhibit bone resorption in vitro (Klaushofer et al., 1987; McCauley et al., 1992). Also, patients treated with CsA in association with renal transplantation or vascularized bone grafts exhibit increased osteoblastic and decreased resorptive parameters (Wilmink et al., 1989). On the other hand, CsA has been shown to inhibit the bone cell activity and to stimulate bone resorption in vitro (Movsowitz et al., 1988; Muchmore et al., 1991). Metabolic heterogeneity among bones has been shown to provide evidence that partly elucidates the controversy (Klein et al., 1990).
Although the effects of CsA on long bones have been carefully examined, the effect of this drug on alveolar bone has received limited attention with contraversial results. The data presented in this study indicate that CsA treatment in rats with periodontal disease (Group IV) leads to a decrease in both radiographic and histometric ABL, and an increase in NABF % compared to the rats with periodontal disease alone (Group III). In contrast with our findings, a distinct pattern of osteopenia, increased osteoclasia, and decreased bone formation around the mandibular incisal region of rats (Fu et al., 1999), decreased bone volume and increased marrow volume around the maxillary secondary molar region of rats have been found after CsA exposure (Shen et al., 2001). Also a tendency of more bone loss in the CsA-medicated animals has been observed as compared to non-medicated ones in an experimental study which has evaluated the effect of CsA on the progression of the periodontal breakdown (Nassar et al., 2004). However, it has been shown that ligature-induced periodontal bone loss is less pronounced in rats treated with CsA, compared to that of control non-medicated rats (Cox et al., 1987). In agreement with this previous study, our results indicated that CsA therapy diminished the alveolar bone loss in rats with periodontal disease. It is well established that the association between the immune system and the pathogenesis of periodontal disease is apparent (Gemmell et al., 1997). For example, approximately 30% of lymphocytes in the sulcular areas in periodontal diease are reported to be T-cells (Okada et al., 1982), and therefore potential targets for CsA. It has been reported that alteration of T-cell mediated activity by CsA may cause a reduction in fibroblast cytotoxicity, a reduction in osteoclast activation with resultant reduction in alveolar bone resorption and in inflammatory infiltrates in humans who have been immunosupressed with convential drugs (Tollefsen et al., 1982; Daley and Wysocki, 1984). Based on these informations, we can suggest that CsA immunosupression might cause a decrease in periodontal breakdown.
In the present study, the biochemical assessment of biomarkers of bone turnover correlated well with the radiographic and histometric measurements as a significant increase in serum alkaline phosphatase level, a marker of osteoblast phenotype (Wong et al., 1990), and a decrease in serum calcium level (although not significant) indicating bone formation was found in ligated and CsA-treated rats (Group IV) compared to only ligated rats (Group III). These biochemical findings could not be compared to any other data from previous studies as in only one study suggesting the diminished periodontal breakdown after CsA therapy, no biochemical assessment has been done (Cox et al., 1987). It has been reported that decreased serum calcium levels could be a non-spesific effect of CsA due to an increased excretion by kidney (Mason, 1990). However, based on the decreased ABL and increased NABF% in Group IV it is relevant to assume that decreased calcium level in this group might be more related to the diminished alveolar bone resorption compared to Group III.
Within the limits of this study, it can be concluded that CsA at immunosupressive levels has no effect on alveolar bone balance of the healthy periodontium. However, in the presence of periodontal disease, CsA treatment may bring out an imbalance in the alveolar bone homeostasis by decreasing resorption and stimulating formation of alveolar bone in rats. Nevertheless, detailed studies are still needed to clarify the possible cellular and molecular mechanisms involved in the effect of immunosupressive drugs on the periodontium.