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A comparative study of systemic subantimicrobial and topical treatment of minocycline in experimental periodontitis of rats
Archives of Oral Biology (2006) 51, 794—803
www.intl.elsevierhealth.com/journals/arob
A comparative study of systemic subantimicrobial and topical treatment of minocycline in experimental periodontitis of rats Xu Yan, Wei Wei* Key Laboratory of Antiinflammatory-Immunopharmacology in Anhui Province, Institute of Clinical Pharmacology, Anhui Medical University, Hefei 230032, PR China Accepted 27 March 2006
KEYWORDS Periodontitis; Monocycline; Alveolar bone loss; Osteoclasts
Summary Objective: The purpose of this study was to compare the effectiveness of minocycline on treating experimentally induced periodontitis in rats when administered either as a systemic subantimicrobial dose or as a topical ointment. Design: Thirty-two adult male Sprague—Dawley rats in four experimental groups–—(1) model group; (2) systemic subantimicrobial dose of minocycline (5 mg/kg/day) treatment group; (3) topical subgingival dose of minocycline (2 mg/animal/week) treatment group; (4) control group. Experimentally induced periodontitis–—silk ligatures were placed around the crevices of the second molar teeth and the animals fed a 10% sucrose drink. Assessment was carried out at days 28 and 56 using a number of different visual, histological and ultrastructure approaches. (1) Visual assessment–— tooth mobility, gingival index and alveolar bone loss. (2) Histological examination–— monocyte infiltration and resorption lacunae with osteoclasts. (3) Transmission electron microscopy (TEM)–—morphological transformation of fibroblasts and osteoclasts. The collected data were analysed for statistical significance using the analysis of variance statistical test. Results: Minocycline significantly reduced tooth mobility, gingival index and alveolar bone loss when administered either systemically or as a topical ointment compared to the model group (P < 0.01). However, the alveolar bone loss was significantly less (P < 0.01 in the systemic treatment group compared to the local treatment group. Monocyte infiltration and resorption lacunae with osteoclasts were significantly less in the both treatment groups compared to the model group
Abbreviations: HMT, host modulatory therapy; MMPs, matrix metalloproteinases; SRP, scaling and root planing; TEM, Transmission electron microscopy * Corresponding author. Tel.: +86 551 5161208; fax: +86 551 5161208. E-mail address: [email protected] (W. Wei). 0003–9969/$ — see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2006.03.018
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(P < 0.01). The osteoclasts failed to form a ruffled border in the systemic treatment group. Conclusion: Topical treatment significantly reduces gingivitis while systemic treatment is beneficial in terms of inhibiting alveolar bone loss. # 2006 Elsevier Ltd. All rights reserved.
Introduction Traditional periodontal treatment has focused on the reduction of bacterial infection by mechanical removal of infectious agents (scaling and root planing, SRP). Current understanding of the pathogenesis of periodontal diseases has led to a paradigm shift in the way we view the progression of the periodontal disease. It is well recognized that the major component of the soft and hard tissue destruction in periodontitis occurs as a result of the activation of the host’s immune-inflammatory defence mechanisms in response to the presence of bacterial plaque.1 Host-derived inflammatory mediators and enzymes, including matrix metalloproteinases (MMPs), play a significant role in the changes in connective tissue and bone metabolism that lead to the breakdown of the periodontal ligament and alveolar bone resorption. Previous studies have shown that the predominant MMPs found in inflamed human gingiva and gingival crevicular fluid (GCF) are derived from human cells rather than bacteria.2,3 Recent understanding of the pathogenesis of periodontitis has resulted in therapeutic advancements that address both the microbes and the host response in the treatment of periodontitis.4 In 2004, Preshaw et al. proposed a new concept of periodontal therapy which consists of a reduction of the bacterial burden, combined with host modulatory therapy (HMT) and risk factor modification (e.g. smoking cessation therapy), these three aspects constitute a comprehensive treatment strategy for periodontitis.5 Essentially, HMT aims to enhance traditional periodontal therapies by modifying destructive aspects of the immune-inflammatory host response so that periodontal breakdown is reduced and the periodontium is stabilized, slowing the progression of the disease, and allowing for more-predictable management of patients. Currently, host modulatory agents being investigated include tetracyclines, non-steroidal antiinflammatory agents, alendronate (Fosamax) (analogue of pyrophosphate), hormone replacement therapy and anti-arthritic medications. These agents produce their beneficial effects by a variety of mechanisms of action, including inhibition of MMPs, inhibition of prostaglandin production, stimulation of osteoblasts, inhibition of osteoclasts and other antiinflammatory mechanisms.6
Antimicrobial agents are sometimes used as adjuncts for the treatment of aggressive and refractory forms of periodontitis. Tetracyclines have been widely used in periodontal treatment for their antimicrobial and anti-enzymatic effects. Systemic regimen may cause side-effect and adverse host reactions, tetracyclines in subantimicrobial dosage and topical delivery is being studied. Tetracyclines in subantimicrobial dosage attenuate antimicrobial actions but retain effective anti-enzyme and proanabolic. They can inhibit tissue collagenase in the periodontium, as well as directly affect osteoclasts last structure and function.7 These medications can be used as HMT agents to control and suppress the progression of periodontal disease. Clinical trials show that subantimicrobial dose doxycycline combined with mechanical therapy enhances clinical outcomes, such as clinical attachment level (CAL) and probing depths (PDs).5,8—10 Minocycline is more lipid soluble than doxycycline11 and systemic minocycline could be preferentially distributed to gingival wound or inflammatory sites.12 Ramamurthy et al. research13 shows minocycline and chemically modified tetracycline (CMT) can reduce osteoblast collagenase activity. Topical dosage of tetracyclines, due to their ability to bind to the tooth’s surface and slow release, can destroy putative periodontopathic bacteria that penetrate into periodontal connective tissue and have also been demonstrated in clinical studies that local delivery of tetracyclines combined with SRP can improve the effectiveness of mechanical therapy.14,15 The U.S. Food and Drug Administration16 has approved topical minocycline microspheres for the adjunctive treatment of adult periodontitis following scaling and root planing. Also, the pilot study17 shows there is no difference between local delivery of 2% minocycline gel as mono-therapy and traditional subgingival debridement in patients on supportive periodontal therapy. Whilst both regimes have their benefits, the question remains as to which regime is more effective as adjunctive management of conventional mechanical treatments of periodontitis. Recent research18 shows periodontitis is a tertiary vascular infection. On the assumption that treatment of periodontitis by systemic treatment which affects the host’s immune-inflammatory defence mechanisms may be more effective than local antibiotic
796 therapy. Furthermore, previous research parameters focus on MMPs activities in the GCF and clinical indexes, such as CAL and PDs, while the histological changes, in particular, the ultrastructure changes in periodontium have barely been reported. Comparison of clinical indexes with histological changes may give a better understanding of inflammatory progress in periodontitis. The purpose of this study was to evaluate the effectiveness of minocycline hydrochloric in the treatment of experimental periodontitis of rats when administered either as a systemic subantimicrobial dose or as a topical delivery system.
Materials and methods Induction of experimental periodontitis Thirty-two adult male Sprague—Dawley rats (280 26 g) (obtained from Henan experimental animal center, quality series was No. 410112) were used for this study. Experimental periodontitis was induced by placement of a silk ligature around the gingival crevices of the second upper and lower molar teeth utilizing the procedure used by Gyo ¨rfi et al.19 with some modification. Briefly, the rats were anesthetized with chloral hydrate (350 mg/ kg, i.p.) and subjected to surgical silk thread (No. 3) that was doubled around the circumference of the second molars. The rats were fed a 10% sucrose drink. There were four groups each consisting of eight animals. Group 1 was the model group and the rats were subjected to periodontitis and received no pharmacological treatment; group 2 was the systemic subantimicrobial treatment group and the rats were subjected to periodontitis and received systemic subantimicrobial dosage of minocycline hydrochloride (Sigma) orally gavaged with 1 ml boxymethylcellulose (CMC)/100 g body weight containing 0.5 mg minocycline hydrochloride daily; group 3 was the topical treatment group and the rats were subjected to periodontitis and received 0.2 mg minocycline hydrochloride ointment (Periocline, 2% minocycline hydrochloride ointment, Sunstar, Japan) animal 1 week 1 applied to the gingival crevices; group 4 was the blank control group, which were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.), orally gavaged with vehicle alone, 1 ml CMC/100 g weight daily and given tap water to drink. Administration of each treatment commenced on the second day after surgery. The animals were housed in temperature-controlled rooms at 24 8C and the treatments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW
Y. Xu, W. Wei Publication, Bethesda, MD, USA). On day 28/56, all rats were anesthetized with chloral hydrate and sacrificed by neck vertebra dislocation.
Tooth mobility(TM) and gingival index (GI) Before sacrifice, the mobility of the second molar was scored according to the following scale: 0 = no mobility; 1 = slight mobility (vestibular-palatal); 2 = moderate mobility (vestibular-palatal and mesial—distal); or 3 = severe mobility (vertical; the tooth moves in and out of the socket); the gingival index was used to classify inflammation of the gingival and scored on the basis of: 0, normal gingival; 1, mild inflammation, slight change in colour, slight edema, no bleeding on probing; 2, moderate inflammation, redness, edema, and glazing, bleeding on probing; 3, severe inflammation, marked redness and edema, ulceration, tendency to spontaneous bleeding.20
Assessment of alveolar bone loss Immediately after rats had been euthanized, posterior jaws were defleshed, cleaned and soaked in 1 M/L NaOH at room temperature for 60 min to remove the remaining soft tissue debris.21 The jaws were stained with Loeffler’s methylene blue to identify the cemento-enamel junction. An eyepiece micrometer in a dissecting microscope (20 magnification) was used to measure the distance between the cemento-enamel junction and the crest of the alveolar bone. Each second molar had six different measurement sites: mesiobuccal site, buccal site, distobuccal site, mesiolingual site, lingual site and distolingual site.
Histological examination After the rats had been euthanized, block sections of the mandibular dento-alveolar segment containing soft tissue, and the three molars were dissected and immediately fixed in 4% buffered paraformaldehyde (pH 7.5) overnight at 4 8C. The specimens were transferred to a decalcifying solution containing 0.5 M EDTA—Na (pH 7.5—8.0), where they were kept at 4 8C for four weeks. After dehydration the specimens were embedded in paraffin and 5 mm sagittal sections were cut and mounted on slide. The sections were stained with hematoxylin—eosin and analysed by the parameters of monocyte infiltration in gingival tissue and resorption lacunae with osteoclasts on the crest of alveolar bone. The monocytes, which were round, deeply blue stained cells, with condensed nuclei and little cytoplasm were present in the gingival connective tissue (Fig. 1a
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Figure 1 Histological results of the gingival tissue and alveolar bone crests of experimental periodontitis in rats.(1) Monocyte; (2) osteoclast; (3) gingival tissue; (4) alveolar bone; (5) periodontal ligament; (a) day 28, group 2, gingival connective tissue, arrow shows the monocyte infiltration, HE 40. (b) Day 28, group 3, gingival connective tissue, HE 40. (c) Day 28, group 2, resorption lacunae with osteoclasts on the alveolar bone crest, arrow shows osteoclast, HE 40. (d) Day 28, group 3, resorption lacunae with osteoclasts on the alveolar bone crest, arrow shows osteoclast, HE 40.
and b). Osteoclasts are large, motile, multinucleate cells with acidophilic cytoplasm located in areas of bone resorption, were Howship’s lacunae (Fig. 1c and d). From each section, a series of 12 rectangular digital colour images (640 480 pixels) was taken using a CCD video camera (motic cam 1300) connected to the light microscope. Monocytes infiltration and resorption lacunae with active osteoclasts were drawn manually and quantitatively measured using a video image analysis system (motic images advanced Version 3.0).
Transmission electron microscopy (TEM) observation The gingival tissue and alveolar bone segments of the ligatured molars were examined by transmission electron microscopy. Approximately 1 mm3 specimens were fixed in 2.5% glutaraldehyde (4 8C) for 4—6 h and subsequently fixed in osmium tetdroxide. Each section was then subjected to the following process in preparation for examination by TEM. Successive treatment in ethanol 30% and ethanol 50% for 15 min, soaked in 70% uranyl acetate/ethanol for 6—12 h, successive treatment in ethanol 80% and ethanol 95% for 15 min, then treated twice in 100% ethanol for 40 min. Each section was then treated in epoxy dimethyl-
methane for 10 min, a mixture of epoxy dimethylmethane/epoxy resin in a ratio of 1:1 for 2 h, a mixture of epoxy dimethylmethane/epoxy resin in a ratio of 1:2 for 1 h and epoxy resin (Epon812) for 2 h. Each section was then embedded in epoxy resin and heated at 45 8C for 12 h and 65 8C for 48 h. Each section was dissected in 70 nm thick slices using an ultramicrotome (LKB-NOVA, Sweden), followed by staining with a saturated solution of uranyl acetate. After rinsing in distilled water three times for 15 min, the specimens were stained in lead citrate for 15 min. After rinsing in distilled water three times for 10 min each, the sections were examined by TEM apparatus (JEM1230, Japan).
Statistical analysis The statistical significance of the data was evaluated by one-way analysis of variance for randomized design using statistical software. Tukey’s test was used to detect the difference between the individual means using a standard statistical program. Correlation between different dosage and gingival index, tooth mobility, alveolar bone loss, number of monocyte infiltration in gingival tissue and resorption lacunae with osteoclasts on the crest of alveolar bone was worked out with SPSS 10 software.
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Table 1 Visual results from experimental periodontitis in rats treated by systemic or topical minocycline
Group Group Group Group
Gingival index (unit), mean S.D.
Teeth mobility (unit), mean S.D.
Bone resorption (mm), mean S.D.
Day 28
Day 28
Day 28
Day 56
1.46 0.20 0.51 0.08**,$$ 0.92 0.19 ** 0.34 0.08 **
2.17 0.27 0.68 0.09**,$$ 1.18 0.13 ** 0.42 0.10 **
Day 56
Day 56
1 3 0.00 2.125 0.35 2.5 0.53 3.75 0.46 2 1.46 0.36*,$$ 1 000*,$$ 0.5 0.53 ** 0.75 0.00 ** 3 0.125 0.35 ** 0.125 0.35 ** 0.85 0.46 ** 1 0.53 ** ** ** ** 4 0.125 0.35 0.125 0.35 0.25 0.46 0.375 0.52 **
Statistics: One-way ANOVA analysis, all groups compared to group 1, *P < 0.05, **P < 0.01, compared group 2 to group 3, $P < 0.05, $$ P < 0.01.
Results
Histology
Tooth mobility
Histopathological analysis of the region around the second molars in the group 1 stained by hematoxylin and eosin revealed a large number of monocytes infiltration and resorption lacunae with osteoclasts, together with cementum and alveolar bone resorption. At preliminary day 28 results, the gingival papilla had a highly chronic inflammation response, the alveolar bone lacked osteoid and showed resorption lacunae with osteoclasts. In day 56 experiments, the number of monocyte infiltration was lower than that of day 28, but the numbers of osteoclasts were more than those of day 28. Histology of the bone from the control group showed that the periodontal bone surface was covered with a layer of osteoid, few osteoclasts. Both treatment groups can significantly inhibit resorption lacunae with osteoclasts compared to group 1; compare group 2 to group 1, monocyte infiltration was not different in day 28, after day 56 treatment, monocyte infiltration decreased and was significantly different compared to group 1; compare group 2 to group 3, monocyte infiltration was significantly high in day 28, but there was no difference in day 56. The numbers of monocyte infiltration and resorption lacunae with osteoclasts in group 4 (blank control group) were much lower than those of in the other three groups (Fig. 1; Table 2).
(1) TM in groups 2 and 3 was significantly reduced compared to group 1 (P < 0.01, day 28, day 56); (2) TM in groups 2 and 3 was of no significant difference (P > 0.05, day 28, day 56) although TM in the group 2 was lower than the group 3 at day 28; (3) TM in group 4 was significantly less than the other three groups (Table 1).
Gingival index (1) GI in the treatment groups was significantly lower compared to group 1 (P < 0.01, day 28, day 56); (2) GI in group 3 was significantly lower than group 2 (P < 0.01, day 28, day 56); (3) GI in group 4 was significantly less than the other three groups (Table 1).
Alveolar bone loss (ABL) (1) ABL in both treatment groups was significantly lower compared to group 1 (P < 0.01, day 28, day 56); (2) ABL in group 2 was significantly lower than that of group 3 (P < 0.01, day 28, day 56); (3) ABL in group 4 was significantly less than the other three groups (Table 1).
Table 2 Histological results from experimental periodontitis in rats treated by systemic or topical minocycline
Group Group Group Group
1 2 3 4
Monocyte infiltration (number)
Resorption lacunae with osteoclasts (number)
Day 28
Day 56
Day 28
Day 56
35.75 8.06 25.62 2.56$$ 4.50 1.60$$,** 1.87 0.83 **
22.88 3.72 8.88 4.97 ** 3.13 1.46 ** 2.50 1.41 **
16.12 5.06 2.37 0.92 ** 2.25 1.04 ** 0.75 0.71 **
21.13 5.14 3.63 1.3 ** 4.38 1.77 ** 0.75 0.71 **
Statistics: One-way ANOVA analysis, all groups compared to group 1, *P < 0.05, **P < 0.01, compared group 2 to group 3, $P < 0.05, $$ P < 0.01.
A comparative study of systemic subantimicrobial and topical treatment of minocycline
Transmission electron microscopy TEM showed that ultrastructure of fibroblasts in group 4 were rich in collagen fibrils, which consisted of large bundles of collagenous fibers. In contrast, the collagen fibers in group 1 were fragmented, dissolved and arranged out of order. The space between the collagen bundles was filled with mostly amorphous material, instead of being in contact with intact collagen fiber, many of the fibroblasts in the periodontium were surrounded by the debris of the matrix (Fig. 2c). Some of the cells demon-
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strated a rounded rather than elongated morphology and, in some cases, there were aggregates of two or more cells. Fibroblasts in groups 2 and 3 showed that the collagen fibers bundles were less than that of group 4, these intact collagen fibers contacted well and had less amorphous material. The shape of fibroblasts in these two groups showed elongated morphology and contact/interaction with intact collagenous fibers (Fig. 2a and b). Ultrastructural analysis provided evidence of ultrastructural abnormalities to the osteoclasts in alveolar bone. The osteoclasts in groups 1 and 3 had
Figure 2 Transmission electron microscopy results of gingival fibroblasts and alveolar bone osteoclasts of experimental periodontitis in rats. (1) Collagenous fibers; (2) osteoclast’s ruffled border. (a) Day 56, group 2, collagen fibers are intact and arranged orderly, collagen fibers 20,000. (b) Day 56, group 3, collagen fibers are intact and arranged regular. Day 56, collagen fibers, 20,000. (c) Day 56, group 1, collagen fibers are partial or complete degenerate, which were arranged in irregular alignment and some debris around collagen fibers, 20,000. (d) Day 56, group 1, visible and extensive ruffled border present on the margin, osteoclast 8000. (e) Day 56, group 2, small portions of the cell perimeter involved with ruffled border formation and the remainder of the cell surface reveals a smooth profile or no ruffled border formation, osteoclast 8000. (f) Day 56,group 3, ruffled border can been seen, osteoclast 8000.
800 well formed ruffled borders and microfibrils around the ruffled borders. The sealing zone area was rich in vesicles and lysosomes (Fig. 2d and f). The osteoclasts in group 2 had somewhat changed compared to that of group 1, the ruffled borders and associated cytoplasmic vacuoles were generally less extensive, the amount of microfibrils had increased, but the amount of vesicles and lysosome had decreased (Fig. 2e).
Discussion Local delivery minocycline There can be no argument that periodontitis is an infectious disease caused by pathogenic microorganisms.5 Successful prevention and treatment of periodontitis is contingent on effective control of the periodontopathic bacteria, which is accomplished with professional treatment of diseased periodontal sites and patient performed plaque control. Subgingival mechanical debridement, with or without surgery, constitutes the basic means of disrupting the subgingival biofilm and controlling pathogens. Appropriate antimicrobial agents that can be administered systemically or via local delivery may enhance eradication or suppression of subgingival pathogens. But all chemical formulations would only be used as an adjunct to conventional initial phase root debridement in the clinical management of patients. The rationale for the use of local antibiotics in the treatment of periodontal infections is to rapidly suppress periodontal pathogens and favour the establishment of a host compatible microbiota. In order to accomplish this, local application of pharmacological agents must fulfill three criteria: (1) medication must reach the intended site of action; (2) it must remain at adequate concentration and (3) last for a sufficient period of time. Minocycline used in the present study was 2% minocycline hydrochloride ointment as a controlled release device, which is effective for one-week duration.22 The main benefits of a controlled delivery device include improved patient compliance, improved pharmacokinetics, improved drug access to the site of disease and lower total drug dosage.23 The present study showed that local delivery minocycline ointment could significantly inhibit gingival inflammation, but it was less effective in the reduction of alveolar bone loss compared to the systemic subantimicrobial dose minocycline. The results of the current studies imply that local application of minocycline to the periodontal pockets may be limited in superficial area around gingival tissue. However, the
Y. Xu, W. Wei medication concentration in the periodontal ligament and alveolar bone crest was low due to the limited penetration of the medication. Pharmacological agents applied locally for the treatment of periodontitis are limited in effect to controlling bacteria in the periodontal pockets, soft tissue walls of the pocket and the exposed root cementum. Minocycline ointment in topical administration might lack sustenance in being readily squeezed out or diluted with saliva and crevicular fluid. These factors could account for some local delivery discrepancies. Results from the Kim et al. study indicate that the antibiotic effect of doxycycline gel was limited mainly to the subgingival sites.24 The objective of local minocycline delivery is to attempt to eliminate periodontopathogens and their proteinases in the periodontal pockets, however, the proteinases from periodontopathogenic bacteria is only a small part of proteinases which destroy the periodontium during periodontitis, whilst host-derived proteinases are the main cause of destruction of the periodontal tissue. Research data suggest local drug therapy gives only limited benefits.25,26 In addition, considering the potential for drug resistant bacteria and adverse host reactions, high financial costs and non-selfadministration of drugs, topical antibiotic therapy seems to be a less desirable choice.
Systemic subantimicrobial dosage of minocycline Periodontal pathogenesis is mediated by complex interactions between a pathogenic microflora and a susceptible host.1 Host-derived pro-inflammatory mediators and cytokines, together with proteolytic enzymes such as matrix metalloproteinases, play a significant role in the changes in connective tissue and bone metabolism that lead to the breakdown of periodontal ligament (PDL) and alveolar bone resorption. Tetracyclines downregulate MMP activity and reduce collagenase levels in the GCF of patients with periodontitis, in addition to their antimicrobial and antibiotic properties. However, when administered as a subantimicrobial dosage, the antimicrobial or antibiotic functions are lost. Systemic subantimicrobial tetracyclines enter the body via the bloodstream and affect the host’s immune-inflammatory defence mechanisms in response to the presence of bacterial plaque. In the current study, 5 mg/kg/day minocycline hydrochloride was given to rats by orally gavaging as subantimicrobial dosage. Clinical researches5,8—10 show that 20 mg doxycycline twice for humans is subantimicrobial dosage. According to Conversion of Animal Doses to Human Equivalent Doses in FDA,27
A comparative study of systemic subantimicrobial and topical treatment of minocycline the dosage of rat is 4.15 mg/kg/day, so 5 mg/kg/day dosage was chosen as an integer. Similar research28 showed that the subantimicrobial dose of doxycycline range for rats is 2.5 mg—10 mg/kg/day. The results in the present study showed minocycline in 5 mg/kg/day had a good pharmacological dose— effect relationship. The present study shows that minocycline administered either as local delivery or as systemic subantimicrobial dose can significantly inhibit the number of the osteoclasts. However, systemic subantimicrobial treatment of minocycline could significantly reduce alveolar bone loss compared to the local delivery of minocycline. Examination of the ultrastructure of the osteoclasts revealed that osteoclasts in the systemic subantimicrobial minocycline group failed to form ruffled borders which suggest that systemic subantimicrobial minocycline may not only be inhibiting preosteoclasts and multinuclear cells that develop into osteoclasts, but also has a direct inhibitory effect on the bone-resorbing activity of existing osteoclasts. The result is consistent with the other researchers.29 The ruffled border is where decalcification and degradation of bone occurs, the first step in the bone resorption process is acidification of the bone surface microenvironment beneath the osteoclasts ruffled border.30 This acidification is made by vacuolar-type H (+)-ATPase, which is highly expressed from the ruffled border membranes of osteoclasts.31—33 Those osteoclasts lacking a well-organized ruffled border due to systemic subantimicrobial minocycline treatment are thought to have a decreased resorptive function. Where periodontitis is associated with systemic ailments, a systemic subantimicrobial dose of minocycline appears to be a more desirable treatment. Research has revealed that there may be an association between periodontitis and a number of systemic ailments (e.g., diabetes, adverse pregnancy outcomes, preterm low birth weight and cardiovascular disease) are being investigated to determine if there is a cause-and-effect relationship. Sasaki33 study shows that periodontal infections create an oral wound that serves as a portal for the systemic dissemination of oral pathogens. Treatment of periodontitis by systemic treatment affects the host’s immune-inflammatory defence mechanisms, enhances the effectiveness of periodontal outcomes and also may prevent and cure systemic ailments. In addition, a subantimicrobial dose of tetracycline will not cause the development of antibiotic resistance or detrimental shifts in the normal periodontal microflora.8 Relative low financial costs and self-administration makes a systemic subantimicrobial dose of minocycline a preferred option.
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Periodontal disease model in rats In the study of the pathogenesis of periodontal diseases, animal models are useful because human studies are difficult to perform for ethical reasons. Ligature-induced experimental periodontitis in rats has been used to investigate the pathologic processes of periodontal infectious diseases for many years.14 Molars in rats are similar anatomic figuration and structure to humans and ligature-induced periodontitis can imitate the occurrence, progression of periodontal disease in human beings. In the present study, ligature-induced periodontitis in rats of model group showed typical periodontal destruction such as attachment loss and alveolar bone loss. There were a few differences between periodontitis in experimental rats and humans, for example, the molars of rats are smaller than those of humans, so it was difficult to perform any sort periodontal treatment, such as scaling and root planing. However, the principle of conventional clinical periodontal treatment is initial therapy such as plaque control, scaling and root planing. Chemical formulations should be combined with conventional periodontal treatment with the aim being to create the most optimal conditions for stabilization of the periodontium by reducing periodontal putative microbial threshold, inhibiting host destructive processes and increasing wound healing.5 SRP and chemical therapy target different aspects of periodontal pathogenesis. The aims of SRP are to remove subgingival plaque and calculus, and disrupt the biofilm to create a local environment more commensurate with wound healing. Chemical treatment aims to inhibit putative periodontopathic bacteria, to reduce destructive inflammatory host responses. In current study, the most parameters in model group were significantly high compared to the subantimicrobial dose group and topical delivery group. Compared visual results in subantimicrobial group and topical delivery group, GI in topical group was much lower than subantimicrobial dose group while alveolar bone resorption in subantimicrobial dose group was significantly lower than topical delivery group. In histological parameters, monocyte infiltration in subantimicribial dose group was much higher especially in day 28 than topical delivery group. It implied that topical administration reached to periodontal pockets and penetrated to part of periodontal supportive tissue where it eliminated periodontopathogens, therefore, the local external inflammation was reduced. Systemic subantimicrobial dosage of minocycline can be absorbed and be distributed to the whole body and all supportive periodontal tissue, so this low dosage had no antimicrobial effect but modulated
802 host responses such as inhibiting host-derived proteinases, hereby, host destructive response in the whole periodontal tissue was lessened while local gingival tissue represented inflammation situation such as gingiva redness, swelling and bleeding on probing. Studies related to conventional periodontal treatment combined with subantimicrobial dosage doxycycline or topical delivery minocycline show that clinic indexes have been improved, such as probing depth and attachment loss. In the present study, parameters from Tables 1 and 2 showed that all index in group 4 (blank control group) were much lower than the other three operating groups. Minocycline was administrated for days 28 and 56 either in systemic subantimicrobial dose or topical delivery ointment reduced periodontal destruction to a certain degree, but all parameters in groups 2 and 3 were significantly different compared to group 4. It implied that chemical formulations may reduce and retard the progression of periodontal destruction, but they might not cease the progression of periodontal disease. Furthermore, chemical formulations had limited effects or minor effects.
Conclusion Topical dosage of minocycline delivered directly to periodontal pockets can inhibit superficial periodontopathogens, therefore, inflammation of gingiva can be reduced. Systemic subantimicrobial dosage of minocycline can distribute whole periodontium and inhibit destructive aspects of the immuneinflammatory host response. Systemic dosage inhibits the development of osteoclasts and alters the structure of osteoclasts, therefore, the alveolar bone loss is restrained.
Acknowledgements The authors would like to acknowledge the suggestions given by Professor PM Bartold (Dental School, Adelaide University AU) during the editing of this manuscript. The authors are also indebted to Dr. Qiaoer Chen for the help provided analysis software, Dr. Wen Hu excellent technical assistance and Dr. Jiahu Hao helpful statistics assistance. Grant support: This study was supported by a grant from National Natural Science foundation of China (NSFC, 30271606), Department of Education Anhui Province (2002kj146) and Department of Health Anhui Province (2002B009).
Y. Xu, W. Wei
References 1. Offenbacher S. Periodontal diseases: pathogenesis. Ann Periodontol 1996;1:821—78. 2. Sorsa T, Uitto VJ, Suomalainen K, Vauhkonen M, Linda S. Comparison of interstitial collagenases from human gingiva, sulcular fluid and polymorphonuclear leukosteoclastsytes. J Periodontal Res 1988;23:386—93. 3. Golub LM, Sorsa T, Lee HM, Ciancio S, Sorbi D, Ramamurthy NS, et al. Doxycycline inhibits neutrophil (PMN)-type matrix metalloproteinases in human adult periodontitis gingiva. J Clin Periodontol 1995;22:100—9. 4. Ryan ME. Nonsurgical approaches for the treatment of periodontal diseases. Dent Clin North Am 2005;49:611—36. 5. Preshaw PM, Hefti AF, Jepsen S, Etienne D, Walker C, Bradshaw MH. Subantimicrobial dose doxycycline as adjunctive treatment for periodontitis. A review. J Clin Periodontol 2004;31:697—707. 6. Ciancio SG. Systemic medications: clinical significance in periodontics. J Clin Periodontol 2002;29(Suppl. 2):17— 21. 7. Rifkin BR, Vernillo AT, Golub LM. Blocking periodontal disease progression by inhibiting tissue-destructive enzymes: a potential therapeutic role for tetracyclines and their chemicallymodified analogs. J Periodontol 1993;64(8 Suppl.):819—27. [Review]. 8. Caton JG, Ciancio SG, Blieden TM, Bradshaw M, Crout RJ, Hefti AF, et al. Treatment with subantimicrobial dose doxycycline improves the efficacy of scaling and root planing in patients with adult periodontitis. J Periodontol 2000;71: 521—32. 9. Caton JG, Ciancio SG, Blieden TM, Bradshaw M, Crout RJ, Hefti AF, et al. Subantimicrobial dose doxycycline as an adjunct to scaling and root planing: post-treatment effects. J Clin Periodontol 2001;28:782—9. 10. Novak MJ, Johns LP, Miller RC, Bradshaw MH. Adjunctive benefits of subantimicrobial dose doxycycline in the management of severe, generalized, chronic periodontitis. J Periodontol 2002;73:762—9. 11. Burke A. Cunha minocycline versus doxycycline in the treatment of lyme neuroborreliosis. Clin Infect Dis 2000;30: 237—8. 12. Walters JD, Nakkula RJ, Maney P. Modulation of gingival fibroblast minocycline accumulation by biological mediators. J Dent Res 2005;84(4):320—3. 13. Ramamurthy NS, Vernillo AT, Lee HM, Golub LM, Rifikin BR. The effect of tetracyclines on collagenase activity in UM R 106-01 rat osteoblastic osteosarcoma cells. Res Commun Chem Pathol Pharmacol 1990;70(3):323—35. 14. Lessem J, Hanlon A. A post-marketing study of 2805 patients treated for periodontal disease with arestin. J Int Acad Periodontol Osteoclasts 2004;6(4 Suppl.):150—3. 15. Romano F, Tortal I, Debernardi C, Aimetti M. Debridement and local application of tetracycline in the management of persistent periodontitis. Clinical and microbiological results after 12 months. Minerva Stomatol 2005;54:43—51. 16. Preboth M. Minocycline approved for adult periodontitis–— trade name arestin. Am Fam Physician 2001:9. 17. McColl E, Patel K, Dahlen G, Tonetti M, Graziani F, Suvan J, et al. Supportive periodontal therapy using mechanical instrumentation or 2% minocycline gel: a 12 month randomized, controlled, single masked pilot study. J Clin Periodontol 2006;33:141—50. 18. Offenbacher S, Elter JR, Lin D, Beck JD. Evidence for periodontitis as a tertiary vascular infection. J Int Acad Periodontol 2005;7:39—48.
A comparative study of systemic subantimicrobial and topical treatment of minocycline ˆ. Suba ZS, Ender F, Rosivall L. Neurogenic 19. Gyo ¨rfi A, Fazekas AA component in ligature-induced periodontitis in the rat. J Clin Periodontol 1994;21:601—5. 20. Lo ¨e. Silness J. Periodontal disease in pregnancy I. Prevalence and severity. Acta Odontol Scand 1963;21:533—51. 21. Llavaneras A, Ramamurthy NS, Heikkila P, Teronen O, Salo T, Rifkin BR, et al. A combination of a chemically modified doxycycline and a bisphosphonate synergistically inhibits endotoxin-induced periodontal breakdown in rat. J Periodontol 2001;72:1069—77. 22. Yoshinari N. Effect of repeated local minocycline administration on periodontal healing following guided tissue regeneration. J Periodontol 2001;72:284—95. 23. Goodson J. Pharmacokinetic principles controlling efficacy of oral therapy. J Dent Res 1989;68:625—32. 24. Kim TS, Burklin T, Schacher B, Ratka-Kruger P, Schaecken MT, Renggli HH, et al. Pharmacokinetic profile of a locally administered doxycycline gel in crevicular fluid, blood, and saliva. J Periodontol 2002;73:1285—91. 25. Grenier D, Plamondon P, Sorsa T, Lee HM, McNamara T, Ramamurthy T, et al. Inhibition of proteolytic, serpinolytic, and progelatinase-b activation activities of periodontopathogens by doxycycline and the non-antimicrobial chemically modified tetracycline derivatives. J Periodontol 2002;73: 79—85.
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26. Greenstein G, Polson A. The role of local drug delivery in the management of periodontal diseases: a comprehensive review. J Periodontol 1998;69:507—20. 27. FDA. Guidance for industry and reviewers estimating the safe starting dose in clinical trials for therapeutics in adult healthy volunteers. 2002http://www.fda.gov/cber/gdlns/ dose.pdf. p. 12. 28. Bezerra MM, Brito GAC, Ribeiro RA, Rocha FAC. Low-dose doxycycline prevents inflammatory bone resorption in rats. Braz J Med Biol Res 2002;35(5):613—6. 29. Sasaki T, Ohyori N, Debari K, Ramamurthy NS, Golub LM. Effects of chemically modified tetracycline, CMT-8, on bone loss and osteoclast structure and function in osteoporotic states. Ann N Y Acad Sci 1999;878:347—60. 30. Rousselle AV, Heymann D. Osteoclastic acidification pathways during bone resorption. Bone 2002;30:533—40. 31. Sahara T, Sasaki T. Effects of brefeldin-A: potent inhibitor of intracellular protein transport on ultrastructure and resorptive function of cultured osteoclasts. Anat Rec 2001;263:127—38. 32. Sasaki T, Hong MH, Udagawa N, Moriyanma Y. Expression of vacuolar H(+)-ATPase in osteoclasts and its role in resorption. Cell Tissue Res 1994;278:265—71. 33. Sasaki T. Recent advances in the ultrastructural assessment of osteoclastic resorptive functions. Microsc Res Tech 1996; 33:182—91.
www.intl.elsevierhealth.com/journals/arob
A comparative study of systemic subantimicrobial and topical treatment of minocycline in experimental periodontitis of rats Xu Yan, Wei Wei* Key Laboratory of Antiinflammatory-Immunopharmacology in Anhui Province, Institute of Clinical Pharmacology, Anhui Medical University, Hefei 230032, PR China Accepted 27 March 2006
KEYWORDS Periodontitis; Monocycline; Alveolar bone loss; Osteoclasts
Summary Objective: The purpose of this study was to compare the effectiveness of minocycline on treating experimentally induced periodontitis in rats when administered either as a systemic subantimicrobial dose or as a topical ointment. Design: Thirty-two adult male Sprague—Dawley rats in four experimental groups–—(1) model group; (2) systemic subantimicrobial dose of minocycline (5 mg/kg/day) treatment group; (3) topical subgingival dose of minocycline (2 mg/animal/week) treatment group; (4) control group. Experimentally induced periodontitis–—silk ligatures were placed around the crevices of the second molar teeth and the animals fed a 10% sucrose drink. Assessment was carried out at days 28 and 56 using a number of different visual, histological and ultrastructure approaches. (1) Visual assessment–— tooth mobility, gingival index and alveolar bone loss. (2) Histological examination–— monocyte infiltration and resorption lacunae with osteoclasts. (3) Transmission electron microscopy (TEM)–—morphological transformation of fibroblasts and osteoclasts. The collected data were analysed for statistical significance using the analysis of variance statistical test. Results: Minocycline significantly reduced tooth mobility, gingival index and alveolar bone loss when administered either systemically or as a topical ointment compared to the model group (P < 0.01). However, the alveolar bone loss was significantly less (P < 0.01 in the systemic treatment group compared to the local treatment group. Monocyte infiltration and resorption lacunae with osteoclasts were significantly less in the both treatment groups compared to the model group
Abbreviations: HMT, host modulatory therapy; MMPs, matrix metalloproteinases; SRP, scaling and root planing; TEM, Transmission electron microscopy * Corresponding author. Tel.: +86 551 5161208; fax: +86 551 5161208. E-mail address: [email protected] (W. Wei). 0003–9969/$ — see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2006.03.018
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(P < 0.01). The osteoclasts failed to form a ruffled border in the systemic treatment group. Conclusion: Topical treatment significantly reduces gingivitis while systemic treatment is beneficial in terms of inhibiting alveolar bone loss. # 2006 Elsevier Ltd. All rights reserved.
Introduction Traditional periodontal treatment has focused on the reduction of bacterial infection by mechanical removal of infectious agents (scaling and root planing, SRP). Current understanding of the pathogenesis of periodontal diseases has led to a paradigm shift in the way we view the progression of the periodontal disease. It is well recognized that the major component of the soft and hard tissue destruction in periodontitis occurs as a result of the activation of the host’s immune-inflammatory defence mechanisms in response to the presence of bacterial plaque.1 Host-derived inflammatory mediators and enzymes, including matrix metalloproteinases (MMPs), play a significant role in the changes in connective tissue and bone metabolism that lead to the breakdown of the periodontal ligament and alveolar bone resorption. Previous studies have shown that the predominant MMPs found in inflamed human gingiva and gingival crevicular fluid (GCF) are derived from human cells rather than bacteria.2,3 Recent understanding of the pathogenesis of periodontitis has resulted in therapeutic advancements that address both the microbes and the host response in the treatment of periodontitis.4 In 2004, Preshaw et al. proposed a new concept of periodontal therapy which consists of a reduction of the bacterial burden, combined with host modulatory therapy (HMT) and risk factor modification (e.g. smoking cessation therapy), these three aspects constitute a comprehensive treatment strategy for periodontitis.5 Essentially, HMT aims to enhance traditional periodontal therapies by modifying destructive aspects of the immune-inflammatory host response so that periodontal breakdown is reduced and the periodontium is stabilized, slowing the progression of the disease, and allowing for more-predictable management of patients. Currently, host modulatory agents being investigated include tetracyclines, non-steroidal antiinflammatory agents, alendronate (Fosamax) (analogue of pyrophosphate), hormone replacement therapy and anti-arthritic medications. These agents produce their beneficial effects by a variety of mechanisms of action, including inhibition of MMPs, inhibition of prostaglandin production, stimulation of osteoblasts, inhibition of osteoclasts and other antiinflammatory mechanisms.6
Antimicrobial agents are sometimes used as adjuncts for the treatment of aggressive and refractory forms of periodontitis. Tetracyclines have been widely used in periodontal treatment for their antimicrobial and anti-enzymatic effects. Systemic regimen may cause side-effect and adverse host reactions, tetracyclines in subantimicrobial dosage and topical delivery is being studied. Tetracyclines in subantimicrobial dosage attenuate antimicrobial actions but retain effective anti-enzyme and proanabolic. They can inhibit tissue collagenase in the periodontium, as well as directly affect osteoclasts last structure and function.7 These medications can be used as HMT agents to control and suppress the progression of periodontal disease. Clinical trials show that subantimicrobial dose doxycycline combined with mechanical therapy enhances clinical outcomes, such as clinical attachment level (CAL) and probing depths (PDs).5,8—10 Minocycline is more lipid soluble than doxycycline11 and systemic minocycline could be preferentially distributed to gingival wound or inflammatory sites.12 Ramamurthy et al. research13 shows minocycline and chemically modified tetracycline (CMT) can reduce osteoblast collagenase activity. Topical dosage of tetracyclines, due to their ability to bind to the tooth’s surface and slow release, can destroy putative periodontopathic bacteria that penetrate into periodontal connective tissue and have also been demonstrated in clinical studies that local delivery of tetracyclines combined with SRP can improve the effectiveness of mechanical therapy.14,15 The U.S. Food and Drug Administration16 has approved topical minocycline microspheres for the adjunctive treatment of adult periodontitis following scaling and root planing. Also, the pilot study17 shows there is no difference between local delivery of 2% minocycline gel as mono-therapy and traditional subgingival debridement in patients on supportive periodontal therapy. Whilst both regimes have their benefits, the question remains as to which regime is more effective as adjunctive management of conventional mechanical treatments of periodontitis. Recent research18 shows periodontitis is a tertiary vascular infection. On the assumption that treatment of periodontitis by systemic treatment which affects the host’s immune-inflammatory defence mechanisms may be more effective than local antibiotic
796 therapy. Furthermore, previous research parameters focus on MMPs activities in the GCF and clinical indexes, such as CAL and PDs, while the histological changes, in particular, the ultrastructure changes in periodontium have barely been reported. Comparison of clinical indexes with histological changes may give a better understanding of inflammatory progress in periodontitis. The purpose of this study was to evaluate the effectiveness of minocycline hydrochloric in the treatment of experimental periodontitis of rats when administered either as a systemic subantimicrobial dose or as a topical delivery system.
Materials and methods Induction of experimental periodontitis Thirty-two adult male Sprague—Dawley rats (280 26 g) (obtained from Henan experimental animal center, quality series was No. 410112) were used for this study. Experimental periodontitis was induced by placement of a silk ligature around the gingival crevices of the second upper and lower molar teeth utilizing the procedure used by Gyo ¨rfi et al.19 with some modification. Briefly, the rats were anesthetized with chloral hydrate (350 mg/ kg, i.p.) and subjected to surgical silk thread (No. 3) that was doubled around the circumference of the second molars. The rats were fed a 10% sucrose drink. There were four groups each consisting of eight animals. Group 1 was the model group and the rats were subjected to periodontitis and received no pharmacological treatment; group 2 was the systemic subantimicrobial treatment group and the rats were subjected to periodontitis and received systemic subantimicrobial dosage of minocycline hydrochloride (Sigma) orally gavaged with 1 ml boxymethylcellulose (CMC)/100 g body weight containing 0.5 mg minocycline hydrochloride daily; group 3 was the topical treatment group and the rats were subjected to periodontitis and received 0.2 mg minocycline hydrochloride ointment (Periocline, 2% minocycline hydrochloride ointment, Sunstar, Japan) animal 1 week 1 applied to the gingival crevices; group 4 was the blank control group, which were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.), orally gavaged with vehicle alone, 1 ml CMC/100 g weight daily and given tap water to drink. Administration of each treatment commenced on the second day after surgery. The animals were housed in temperature-controlled rooms at 24 8C and the treatments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW
Y. Xu, W. Wei Publication, Bethesda, MD, USA). On day 28/56, all rats were anesthetized with chloral hydrate and sacrificed by neck vertebra dislocation.
Tooth mobility(TM) and gingival index (GI) Before sacrifice, the mobility of the second molar was scored according to the following scale: 0 = no mobility; 1 = slight mobility (vestibular-palatal); 2 = moderate mobility (vestibular-palatal and mesial—distal); or 3 = severe mobility (vertical; the tooth moves in and out of the socket); the gingival index was used to classify inflammation of the gingival and scored on the basis of: 0, normal gingival; 1, mild inflammation, slight change in colour, slight edema, no bleeding on probing; 2, moderate inflammation, redness, edema, and glazing, bleeding on probing; 3, severe inflammation, marked redness and edema, ulceration, tendency to spontaneous bleeding.20
Assessment of alveolar bone loss Immediately after rats had been euthanized, posterior jaws were defleshed, cleaned and soaked in 1 M/L NaOH at room temperature for 60 min to remove the remaining soft tissue debris.21 The jaws were stained with Loeffler’s methylene blue to identify the cemento-enamel junction. An eyepiece micrometer in a dissecting microscope (20 magnification) was used to measure the distance between the cemento-enamel junction and the crest of the alveolar bone. Each second molar had six different measurement sites: mesiobuccal site, buccal site, distobuccal site, mesiolingual site, lingual site and distolingual site.
Histological examination After the rats had been euthanized, block sections of the mandibular dento-alveolar segment containing soft tissue, and the three molars were dissected and immediately fixed in 4% buffered paraformaldehyde (pH 7.5) overnight at 4 8C. The specimens were transferred to a decalcifying solution containing 0.5 M EDTA—Na (pH 7.5—8.0), where they were kept at 4 8C for four weeks. After dehydration the specimens were embedded in paraffin and 5 mm sagittal sections were cut and mounted on slide. The sections were stained with hematoxylin—eosin and analysed by the parameters of monocyte infiltration in gingival tissue and resorption lacunae with osteoclasts on the crest of alveolar bone. The monocytes, which were round, deeply blue stained cells, with condensed nuclei and little cytoplasm were present in the gingival connective tissue (Fig. 1a
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Figure 1 Histological results of the gingival tissue and alveolar bone crests of experimental periodontitis in rats.(1) Monocyte; (2) osteoclast; (3) gingival tissue; (4) alveolar bone; (5) periodontal ligament; (a) day 28, group 2, gingival connective tissue, arrow shows the monocyte infiltration, HE 40. (b) Day 28, group 3, gingival connective tissue, HE 40. (c) Day 28, group 2, resorption lacunae with osteoclasts on the alveolar bone crest, arrow shows osteoclast, HE 40. (d) Day 28, group 3, resorption lacunae with osteoclasts on the alveolar bone crest, arrow shows osteoclast, HE 40.
and b). Osteoclasts are large, motile, multinucleate cells with acidophilic cytoplasm located in areas of bone resorption, were Howship’s lacunae (Fig. 1c and d). From each section, a series of 12 rectangular digital colour images (640 480 pixels) was taken using a CCD video camera (motic cam 1300) connected to the light microscope. Monocytes infiltration and resorption lacunae with active osteoclasts were drawn manually and quantitatively measured using a video image analysis system (motic images advanced Version 3.0).
Transmission electron microscopy (TEM) observation The gingival tissue and alveolar bone segments of the ligatured molars were examined by transmission electron microscopy. Approximately 1 mm3 specimens were fixed in 2.5% glutaraldehyde (4 8C) for 4—6 h and subsequently fixed in osmium tetdroxide. Each section was then subjected to the following process in preparation for examination by TEM. Successive treatment in ethanol 30% and ethanol 50% for 15 min, soaked in 70% uranyl acetate/ethanol for 6—12 h, successive treatment in ethanol 80% and ethanol 95% for 15 min, then treated twice in 100% ethanol for 40 min. Each section was then treated in epoxy dimethyl-
methane for 10 min, a mixture of epoxy dimethylmethane/epoxy resin in a ratio of 1:1 for 2 h, a mixture of epoxy dimethylmethane/epoxy resin in a ratio of 1:2 for 1 h and epoxy resin (Epon812) for 2 h. Each section was then embedded in epoxy resin and heated at 45 8C for 12 h and 65 8C for 48 h. Each section was dissected in 70 nm thick slices using an ultramicrotome (LKB-NOVA, Sweden), followed by staining with a saturated solution of uranyl acetate. After rinsing in distilled water three times for 15 min, the specimens were stained in lead citrate for 15 min. After rinsing in distilled water three times for 10 min each, the sections were examined by TEM apparatus (JEM1230, Japan).
Statistical analysis The statistical significance of the data was evaluated by one-way analysis of variance for randomized design using statistical software. Tukey’s test was used to detect the difference between the individual means using a standard statistical program. Correlation between different dosage and gingival index, tooth mobility, alveolar bone loss, number of monocyte infiltration in gingival tissue and resorption lacunae with osteoclasts on the crest of alveolar bone was worked out with SPSS 10 software.
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Table 1 Visual results from experimental periodontitis in rats treated by systemic or topical minocycline
Group Group Group Group
Gingival index (unit), mean S.D.
Teeth mobility (unit), mean S.D.
Bone resorption (mm), mean S.D.
Day 28
Day 28
Day 28
Day 56
1.46 0.20 0.51 0.08**,$$ 0.92 0.19 ** 0.34 0.08 **
2.17 0.27 0.68 0.09**,$$ 1.18 0.13 ** 0.42 0.10 **
Day 56
Day 56
1 3 0.00 2.125 0.35 2.5 0.53 3.75 0.46 2 1.46 0.36*,$$ 1 000*,$$ 0.5 0.53 ** 0.75 0.00 ** 3 0.125 0.35 ** 0.125 0.35 ** 0.85 0.46 ** 1 0.53 ** ** ** ** 4 0.125 0.35 0.125 0.35 0.25 0.46 0.375 0.52 **
Statistics: One-way ANOVA analysis, all groups compared to group 1, *P < 0.05, **P < 0.01, compared group 2 to group 3, $P < 0.05, $$ P < 0.01.
Results
Histology
Tooth mobility
Histopathological analysis of the region around the second molars in the group 1 stained by hematoxylin and eosin revealed a large number of monocytes infiltration and resorption lacunae with osteoclasts, together with cementum and alveolar bone resorption. At preliminary day 28 results, the gingival papilla had a highly chronic inflammation response, the alveolar bone lacked osteoid and showed resorption lacunae with osteoclasts. In day 56 experiments, the number of monocyte infiltration was lower than that of day 28, but the numbers of osteoclasts were more than those of day 28. Histology of the bone from the control group showed that the periodontal bone surface was covered with a layer of osteoid, few osteoclasts. Both treatment groups can significantly inhibit resorption lacunae with osteoclasts compared to group 1; compare group 2 to group 1, monocyte infiltration was not different in day 28, after day 56 treatment, monocyte infiltration decreased and was significantly different compared to group 1; compare group 2 to group 3, monocyte infiltration was significantly high in day 28, but there was no difference in day 56. The numbers of monocyte infiltration and resorption lacunae with osteoclasts in group 4 (blank control group) were much lower than those of in the other three groups (Fig. 1; Table 2).
(1) TM in groups 2 and 3 was significantly reduced compared to group 1 (P < 0.01, day 28, day 56); (2) TM in groups 2 and 3 was of no significant difference (P > 0.05, day 28, day 56) although TM in the group 2 was lower than the group 3 at day 28; (3) TM in group 4 was significantly less than the other three groups (Table 1).
Gingival index (1) GI in the treatment groups was significantly lower compared to group 1 (P < 0.01, day 28, day 56); (2) GI in group 3 was significantly lower than group 2 (P < 0.01, day 28, day 56); (3) GI in group 4 was significantly less than the other three groups (Table 1).
Alveolar bone loss (ABL) (1) ABL in both treatment groups was significantly lower compared to group 1 (P < 0.01, day 28, day 56); (2) ABL in group 2 was significantly lower than that of group 3 (P < 0.01, day 28, day 56); (3) ABL in group 4 was significantly less than the other three groups (Table 1).
Table 2 Histological results from experimental periodontitis in rats treated by systemic or topical minocycline
Group Group Group Group
1 2 3 4
Monocyte infiltration (number)
Resorption lacunae with osteoclasts (number)
Day 28
Day 56
Day 28
Day 56
35.75 8.06 25.62 2.56$$ 4.50 1.60$$,** 1.87 0.83 **
22.88 3.72 8.88 4.97 ** 3.13 1.46 ** 2.50 1.41 **
16.12 5.06 2.37 0.92 ** 2.25 1.04 ** 0.75 0.71 **
21.13 5.14 3.63 1.3 ** 4.38 1.77 ** 0.75 0.71 **
Statistics: One-way ANOVA analysis, all groups compared to group 1, *P < 0.05, **P < 0.01, compared group 2 to group 3, $P < 0.05, $$ P < 0.01.
A comparative study of systemic subantimicrobial and topical treatment of minocycline
Transmission electron microscopy TEM showed that ultrastructure of fibroblasts in group 4 were rich in collagen fibrils, which consisted of large bundles of collagenous fibers. In contrast, the collagen fibers in group 1 were fragmented, dissolved and arranged out of order. The space between the collagen bundles was filled with mostly amorphous material, instead of being in contact with intact collagen fiber, many of the fibroblasts in the periodontium were surrounded by the debris of the matrix (Fig. 2c). Some of the cells demon-
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strated a rounded rather than elongated morphology and, in some cases, there were aggregates of two or more cells. Fibroblasts in groups 2 and 3 showed that the collagen fibers bundles were less than that of group 4, these intact collagen fibers contacted well and had less amorphous material. The shape of fibroblasts in these two groups showed elongated morphology and contact/interaction with intact collagenous fibers (Fig. 2a and b). Ultrastructural analysis provided evidence of ultrastructural abnormalities to the osteoclasts in alveolar bone. The osteoclasts in groups 1 and 3 had
Figure 2 Transmission electron microscopy results of gingival fibroblasts and alveolar bone osteoclasts of experimental periodontitis in rats. (1) Collagenous fibers; (2) osteoclast’s ruffled border. (a) Day 56, group 2, collagen fibers are intact and arranged orderly, collagen fibers 20,000. (b) Day 56, group 3, collagen fibers are intact and arranged regular. Day 56, collagen fibers, 20,000. (c) Day 56, group 1, collagen fibers are partial or complete degenerate, which were arranged in irregular alignment and some debris around collagen fibers, 20,000. (d) Day 56, group 1, visible and extensive ruffled border present on the margin, osteoclast 8000. (e) Day 56, group 2, small portions of the cell perimeter involved with ruffled border formation and the remainder of the cell surface reveals a smooth profile or no ruffled border formation, osteoclast 8000. (f) Day 56,group 3, ruffled border can been seen, osteoclast 8000.
800 well formed ruffled borders and microfibrils around the ruffled borders. The sealing zone area was rich in vesicles and lysosomes (Fig. 2d and f). The osteoclasts in group 2 had somewhat changed compared to that of group 1, the ruffled borders and associated cytoplasmic vacuoles were generally less extensive, the amount of microfibrils had increased, but the amount of vesicles and lysosome had decreased (Fig. 2e).
Discussion Local delivery minocycline There can be no argument that periodontitis is an infectious disease caused by pathogenic microorganisms.5 Successful prevention and treatment of periodontitis is contingent on effective control of the periodontopathic bacteria, which is accomplished with professional treatment of diseased periodontal sites and patient performed plaque control. Subgingival mechanical debridement, with or without surgery, constitutes the basic means of disrupting the subgingival biofilm and controlling pathogens. Appropriate antimicrobial agents that can be administered systemically or via local delivery may enhance eradication or suppression of subgingival pathogens. But all chemical formulations would only be used as an adjunct to conventional initial phase root debridement in the clinical management of patients. The rationale for the use of local antibiotics in the treatment of periodontal infections is to rapidly suppress periodontal pathogens and favour the establishment of a host compatible microbiota. In order to accomplish this, local application of pharmacological agents must fulfill three criteria: (1) medication must reach the intended site of action; (2) it must remain at adequate concentration and (3) last for a sufficient period of time. Minocycline used in the present study was 2% minocycline hydrochloride ointment as a controlled release device, which is effective for one-week duration.22 The main benefits of a controlled delivery device include improved patient compliance, improved pharmacokinetics, improved drug access to the site of disease and lower total drug dosage.23 The present study showed that local delivery minocycline ointment could significantly inhibit gingival inflammation, but it was less effective in the reduction of alveolar bone loss compared to the systemic subantimicrobial dose minocycline. The results of the current studies imply that local application of minocycline to the periodontal pockets may be limited in superficial area around gingival tissue. However, the
Y. Xu, W. Wei medication concentration in the periodontal ligament and alveolar bone crest was low due to the limited penetration of the medication. Pharmacological agents applied locally for the treatment of periodontitis are limited in effect to controlling bacteria in the periodontal pockets, soft tissue walls of the pocket and the exposed root cementum. Minocycline ointment in topical administration might lack sustenance in being readily squeezed out or diluted with saliva and crevicular fluid. These factors could account for some local delivery discrepancies. Results from the Kim et al. study indicate that the antibiotic effect of doxycycline gel was limited mainly to the subgingival sites.24 The objective of local minocycline delivery is to attempt to eliminate periodontopathogens and their proteinases in the periodontal pockets, however, the proteinases from periodontopathogenic bacteria is only a small part of proteinases which destroy the periodontium during periodontitis, whilst host-derived proteinases are the main cause of destruction of the periodontal tissue. Research data suggest local drug therapy gives only limited benefits.25,26 In addition, considering the potential for drug resistant bacteria and adverse host reactions, high financial costs and non-selfadministration of drugs, topical antibiotic therapy seems to be a less desirable choice.
Systemic subantimicrobial dosage of minocycline Periodontal pathogenesis is mediated by complex interactions between a pathogenic microflora and a susceptible host.1 Host-derived pro-inflammatory mediators and cytokines, together with proteolytic enzymes such as matrix metalloproteinases, play a significant role in the changes in connective tissue and bone metabolism that lead to the breakdown of periodontal ligament (PDL) and alveolar bone resorption. Tetracyclines downregulate MMP activity and reduce collagenase levels in the GCF of patients with periodontitis, in addition to their antimicrobial and antibiotic properties. However, when administered as a subantimicrobial dosage, the antimicrobial or antibiotic functions are lost. Systemic subantimicrobial tetracyclines enter the body via the bloodstream and affect the host’s immune-inflammatory defence mechanisms in response to the presence of bacterial plaque. In the current study, 5 mg/kg/day minocycline hydrochloride was given to rats by orally gavaging as subantimicrobial dosage. Clinical researches5,8—10 show that 20 mg doxycycline twice for humans is subantimicrobial dosage. According to Conversion of Animal Doses to Human Equivalent Doses in FDA,27
A comparative study of systemic subantimicrobial and topical treatment of minocycline the dosage of rat is 4.15 mg/kg/day, so 5 mg/kg/day dosage was chosen as an integer. Similar research28 showed that the subantimicrobial dose of doxycycline range for rats is 2.5 mg—10 mg/kg/day. The results in the present study showed minocycline in 5 mg/kg/day had a good pharmacological dose— effect relationship. The present study shows that minocycline administered either as local delivery or as systemic subantimicrobial dose can significantly inhibit the number of the osteoclasts. However, systemic subantimicrobial treatment of minocycline could significantly reduce alveolar bone loss compared to the local delivery of minocycline. Examination of the ultrastructure of the osteoclasts revealed that osteoclasts in the systemic subantimicrobial minocycline group failed to form ruffled borders which suggest that systemic subantimicrobial minocycline may not only be inhibiting preosteoclasts and multinuclear cells that develop into osteoclasts, but also has a direct inhibitory effect on the bone-resorbing activity of existing osteoclasts. The result is consistent with the other researchers.29 The ruffled border is where decalcification and degradation of bone occurs, the first step in the bone resorption process is acidification of the bone surface microenvironment beneath the osteoclasts ruffled border.30 This acidification is made by vacuolar-type H (+)-ATPase, which is highly expressed from the ruffled border membranes of osteoclasts.31—33 Those osteoclasts lacking a well-organized ruffled border due to systemic subantimicrobial minocycline treatment are thought to have a decreased resorptive function. Where periodontitis is associated with systemic ailments, a systemic subantimicrobial dose of minocycline appears to be a more desirable treatment. Research has revealed that there may be an association between periodontitis and a number of systemic ailments (e.g., diabetes, adverse pregnancy outcomes, preterm low birth weight and cardiovascular disease) are being investigated to determine if there is a cause-and-effect relationship. Sasaki33 study shows that periodontal infections create an oral wound that serves as a portal for the systemic dissemination of oral pathogens. Treatment of periodontitis by systemic treatment affects the host’s immune-inflammatory defence mechanisms, enhances the effectiveness of periodontal outcomes and also may prevent and cure systemic ailments. In addition, a subantimicrobial dose of tetracycline will not cause the development of antibiotic resistance or detrimental shifts in the normal periodontal microflora.8 Relative low financial costs and self-administration makes a systemic subantimicrobial dose of minocycline a preferred option.
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Periodontal disease model in rats In the study of the pathogenesis of periodontal diseases, animal models are useful because human studies are difficult to perform for ethical reasons. Ligature-induced experimental periodontitis in rats has been used to investigate the pathologic processes of periodontal infectious diseases for many years.14 Molars in rats are similar anatomic figuration and structure to humans and ligature-induced periodontitis can imitate the occurrence, progression of periodontal disease in human beings. In the present study, ligature-induced periodontitis in rats of model group showed typical periodontal destruction such as attachment loss and alveolar bone loss. There were a few differences between periodontitis in experimental rats and humans, for example, the molars of rats are smaller than those of humans, so it was difficult to perform any sort periodontal treatment, such as scaling and root planing. However, the principle of conventional clinical periodontal treatment is initial therapy such as plaque control, scaling and root planing. Chemical formulations should be combined with conventional periodontal treatment with the aim being to create the most optimal conditions for stabilization of the periodontium by reducing periodontal putative microbial threshold, inhibiting host destructive processes and increasing wound healing.5 SRP and chemical therapy target different aspects of periodontal pathogenesis. The aims of SRP are to remove subgingival plaque and calculus, and disrupt the biofilm to create a local environment more commensurate with wound healing. Chemical treatment aims to inhibit putative periodontopathic bacteria, to reduce destructive inflammatory host responses. In current study, the most parameters in model group were significantly high compared to the subantimicrobial dose group and topical delivery group. Compared visual results in subantimicrobial group and topical delivery group, GI in topical group was much lower than subantimicrobial dose group while alveolar bone resorption in subantimicrobial dose group was significantly lower than topical delivery group. In histological parameters, monocyte infiltration in subantimicribial dose group was much higher especially in day 28 than topical delivery group. It implied that topical administration reached to periodontal pockets and penetrated to part of periodontal supportive tissue where it eliminated periodontopathogens, therefore, the local external inflammation was reduced. Systemic subantimicrobial dosage of minocycline can be absorbed and be distributed to the whole body and all supportive periodontal tissue, so this low dosage had no antimicrobial effect but modulated
802 host responses such as inhibiting host-derived proteinases, hereby, host destructive response in the whole periodontal tissue was lessened while local gingival tissue represented inflammation situation such as gingiva redness, swelling and bleeding on probing. Studies related to conventional periodontal treatment combined with subantimicrobial dosage doxycycline or topical delivery minocycline show that clinic indexes have been improved, such as probing depth and attachment loss. In the present study, parameters from Tables 1 and 2 showed that all index in group 4 (blank control group) were much lower than the other three operating groups. Minocycline was administrated for days 28 and 56 either in systemic subantimicrobial dose or topical delivery ointment reduced periodontal destruction to a certain degree, but all parameters in groups 2 and 3 were significantly different compared to group 4. It implied that chemical formulations may reduce and retard the progression of periodontal destruction, but they might not cease the progression of periodontal disease. Furthermore, chemical formulations had limited effects or minor effects.
Conclusion Topical dosage of minocycline delivered directly to periodontal pockets can inhibit superficial periodontopathogens, therefore, inflammation of gingiva can be reduced. Systemic subantimicrobial dosage of minocycline can distribute whole periodontium and inhibit destructive aspects of the immuneinflammatory host response. Systemic dosage inhibits the development of osteoclasts and alters the structure of osteoclasts, therefore, the alveolar bone loss is restrained.
Acknowledgements The authors would like to acknowledge the suggestions given by Professor PM Bartold (Dental School, Adelaide University AU) during the editing of this manuscript. The authors are also indebted to Dr. Qiaoer Chen for the help provided analysis software, Dr. Wen Hu excellent technical assistance and Dr. Jiahu Hao helpful statistics assistance. Grant support: This study was supported by a grant from National Natural Science foundation of China (NSFC, 30271606), Department of Education Anhui Province (2002kj146) and Department of Health Anhui Province (2002B009).
Y. Xu, W. Wei
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