Administration of High Dosesof Caffeine Increases Alveolar Bone Lossin Ligature-Induced Periodontitisin RatsJoyce Pinho Bezerra,* Luiz Ricardo Ferreira da Silva,* Vanessa Adelia de Alvarenga Lemos,†

Poliana Mendes Duarte,* and Marta Ferreira Bastos*

Background: Although some studies have reported a nega-tive influence of caffeine on bone metabolism, there is no in-formation about its effect on the progression of periodontitis.The aim of this study was to evaluate the effect of high dosesof caffeine on ligature-induced periodontitis in rats.

Methods: Twenty-two Wistar rats were assigned to one ofthe following groups: non-caffeine group (n = 12) = animalswithout caffeine ingestion; caffeine group (n = 10) = animalsingesting 10 mg/100 g body weight/day of caffeine via drink-ing water for 56 days. Two weeks after the beginning of caf-feine intake, one of the mandibular molar was randomlyassigned to receive a ligature, whereas the contralateral molarwas left unligated. Forty-two days later, the animals were sac-rificed, and the specimens were processed to obtain decalci-fied sections. The area of periodontal ligament and/or boneloss in the furcation region of the first molars was histometri-cally determined.

Results: Caffeine intake did not have a direct effect on thealveolar bone loss in unligated teeth. But on the ligated tooth,a greater area of bone loss was observed in the animals thatingested caffeine compared to those that did not (P <0.05).

Conclusion: The present study demonstrated that daily in-take of high doses of caffeine may enhance ligature-inducedperiodontitis progression. J Periodontol 2008;79:2356-2360.

KEY WORDS

Alveolar bone; caffeine; periodontitis; rats.

Periodontitis is an infectious diseasecharacterized by the inflammationof the periodontal supporting tis-

sues, resulting in attachment loss andalveolar bone resorption.1 Pathogenicbacteria are well established as the etio-logic agents of the periodontal diseases;2

however previous studies3-6 have demon-strated that systemic and environmentalrisk factors, such as smoking, diabetes,and osteoporosis, may modulate peri-odontal disease progression and severity.

Caffeine, 1,3,7-trimethilxanthine, is oneof the most commonly ingested psy-choactive compounds in the world, beinga major component in beverages, foodsand medications. The hypothesis thatcaffeine may have an influence on bonemetabolism was tested in a series of lab-oratory, animal, and clinical studies.7-12

In general, these studies7-12 have demon-strated some negative effect of caffeine onbone metabolism when evaluations suchas bone cell viability, histomorphometry,and bone mineral index are applied.

Although some systemic factors havebeen recognized as a risk for periodontaldiseases, no study to date has evaluatedthe influence of caffeine consumptionon alveolar bone loss in periodontitis.Because caffeine can have a negativeinfluence on bone tissues, and the lossof alveolar bone is one of the most impor-tant hallmarks of periodontitis, it was

* Department of Periodontics, Dental Research Division, Guarulhos University, Guarulhos,SP, Brazil.

† Biosciences Laboratories, Biological Science Division, Guarulhos University.

doi: 10.1902/jop.2008.080204

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hypothesized that caffeine could bean aggravating factor in periodon-tal bone loss. Therefore, the aimof this study was to evaluate the ef-fect of high doses of caffeine on lig-ature-induced periodontitis in rats.

MATERIALS AND METHODS

AnimalsThe University of Guarulhos Insti-tutional Animal Care and UseCommittee approved the studyprotocol. Twenty-two male Wistarrats obtained from the ButantanInstitute, Sao Paulo, SP, Brazil,were included in this study. Therats were 90 days of age andweighed approximately 330 – 33 gat the beginning of the study. During the acclimatiza-tion (5 days) and experimental period (56 days), eachanimal was housed alone in plastic cages with accessto food‡ and drinking water ad libitum in the Biosci-ence Laboratory of Guarulhos University.

Experimental DesignThe animals were randomly assigned to one of thefollowing two groups: non-caffeine group (n = 12) =animals without caffeine ingestion; caffeine group(n = 10) = animals ingesting 10 mg/100 g bodyweight/day of caffeine§ administered via drinking wa-ter for 56 days. The determined caffeine intake wasmaintained by adjusting its concentration in the wateron days 1, 3, and 5 of each week throughout the ex-perimental period. To ensure complete consumptionof the established dose, the volume of caffeine-sup-plemented water offered was determined by theamount of water consumed on the previous day.

Ligature PlacementTwo weeks after the beginning of caffeine intake, gen-eral anesthesia was obtained by intraperitoneal ad-ministration of xylazin (10 mg/kg) and ketamine(10 mg/kg). One of the mandibular first molars ofeach animal was randomly assigned to receive acotton ligature in a cervical position to induce exper-imental periodontitis (Fig. 1). The contralateral molarof each animal was left unligated to be used as a con-trol.

Histologic and Histometric ProceduresForty-two days after the ligature placement, the ani-mals were euthanized by CO2 inhalation. Subse-quently, the jaws were removed and fixed in 10%buffered formalin for 48 hours. The specimens weredecalcified in a solution containing 10% EDTA, dehy-drated in an ascending series of ethanol solution, and

embedded in paraffin. Serial sections (5 mm) wereobtained in a mesio-distal direction and stained withhematoxylin and eosin solutions. After excluding thefirst and last sections in which the furcation area wasevident, 10 equally distant sections of each molarwere chosen for histometric evaluation. The imagesof the first molar were photographed, and the areaof periodontal ligament and/or bone loss in the furca-tion of both first molars was histometrically deter-mined by the same trained, calibrated, and maskedexaminer using an image system.i

Statistical AnalysesTo test the null hypothesis that caffeine had no influ-ence on interradicular bone loss, an intergroup anal-ysis was performed by the Student t test. In addition,the paired Student t test was used for intragroup com-parisons between ligated and unligated teeth. The sig-nificance level established for all analyses was 1%(P <0.01).

RESULTS

All animals gained weight during the study. The meanbody weight did not show statistically significant dif-ferences between caffeine and non-caffeine groupson day 56 (P = 0.27). The final mean body weightwas 412.8 – 47.6 g and 387.8 – 53.3 g for non-caffeineand caffeine groups, respectively. A significant differ-ence in the bone loss of the furcation area between un-ligated and ligated teeth was observed for the twoexperimental groups (P <0.0001 for the non-caffeinegroup; P = 0.0006 for the caffeine group), showingthat the cotton ligatures around the teeth were ableto induce bone loss. Caffeine intake did not present

Figure 1.A) Cotton ligature in the interproximal region between the first and second molars. B) Cottonligature in a cervical position of the mandibular first molar.

‡ Labina, Purina, Paulinia, SP, Brazil.§ Sigma Aldrich, St. Louis, MO.i Image-Pro, Media Cybernetics, Silver Spring, MD.

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a significant effect on the alveolar bone loss in unli-gated teeth (P = 0.1756). The periodontal ligamentareas observed in unligated teeth were 0.15 – 0.05mm2 and 0.18 – 0.06 mm2 for the non-caffeine andcaffeine groups, respectively. On the other hand, agreater area of bone loss was observed in the ligatedteeth of the animals that ingested caffeine comparedto animals that did not (P = 0.0025). The bone lossareas in the ligated teeth were 0.68 – 0.38 mm2 and1.49 – 0.85 mm2 for the non-caffeine and caffeinegroups, respectively (Fig. 2). Figures 3A through3D illustrate the histologic findings. Figures 3A and3C show the periodontal ligament and absence of in-terradicular bone loss in the furcation area of unli-gated teeth from non-caffeine and caffeine groups,respectively. Figures 3B and 3D illustrate the interra-dicular bone loss induced by ligature in non-caffeineand caffeine groups, respectively, and an increasedbone loss in the caffeine group compared to thenon-caffeine group.

DISCUSSION

It is well recognized that host risk factors such asgenetics, age, gender, lifestyle habits, and certainsystemic diseases can contribute to the pathogenesisof periodontal diseases, increasing soft tissue and al-veolar bone destruction.3-6 Some investigations7-12

have been performed to assess the impact of caffeineand/or coffee consumption on bone metabolism byclinical trials and experimental models. Although

these studies7-12 have already reported a negative in-fluence of caffeine on bone metabolism, the results ofthe present study contribute to the original informa-tion regarding the effect of daily intake of high dosesof caffeine on ligature-induced periodontitis.

Data suggest that caffeine ingestion increases liga-ture-induced periodontitis, even though caffeine wasnot able to induce alveolar bone loss in the absenceof cotton ligature. To the best of our knowledge, therehave been no clinical or animal studies that evaluatethe relationship between caffeine intake and peri-odontitis, hampering a more direct comparison withthe present results. Several studies have demon-strated that bone resorption in periodontitis is medi-ated by increased levels of tumor necrosis factor(TNF)-a, interleukin (IL)-6, IL-1, receptor activatorof nuclear factor-kappa B ligand,13,14 and prosta-glandin (PG) E2,15 which are released from macro-phages and other cells stimulated by pathogens.Sakamoto et al.16 evaluated the effect of coffee inges-tion on TNF-a and IL-6 production after lipopolysac-charide (LPS) stimulation in rats. The authorsdemonstrated that serum levels of these cytokineswere markedly increased at 2 hours after injection ofLPS; however, coffee consumption had no effect onthe production of TNF-a and IL-6 with or withoutLPS injection. It is important to highlight thatSakamoto et al.16 administered a lower dose ofcaffeine compared to our study and performed anacute LPS stimulation, whereas, in the present study,there was a chronic stimulus of the ligature. In supportof our results, Kamagata-Kiyoura et al.17 demon-strated that the association of high or low concentra-tions of caffeine and PGE2 strongly inhibited theproliferation of osteoblast-like cells in vitro. Interest-ingly, high concentrations of both caffeine and PGE2

alone also had inhibitory effects on the proliferationof these cells.

The effects of caffeine on bone metabolism are notfully understood. Similar to the study by Kamagata-Kiyoura et al.,17 other in vitro studies demonstratedthat caffeine inhibits the proliferation of osteoblast-like cells and has deleterious effects on the viabilityof osteoblasts, increasing the rate of apoptosis ofthese cells.11 In addition, because caffeine decreasedthe vitamin D receptor expression in osteoblast-likecell lines, it could be a potential agent to interfere withmolecular mechanisms of bone loss.12 In rats, highdoses of caffeine have been related to increased boneturnover by evaluating the serum osteocalcin levels18

and have been associated with reduction in the bonemineral content.8,10 Epidemiologic studies have dem-onstrated that caffeine consumption has no associa-tion with bone loss in younger women19 but couldexert a negative influence on bone density in elderlywomen.7,9 This phenomenon could be explained by

Figure 2.Mean values and SD (mm2) for the area of periodontal ligament forunligated teeth and area of interradicular bone loss for ligated teeth innon-caffeine and caffeine groups. In the non-caffeine group, * signifiesthat the mean area of bone loss in ligated teeth is significantly higherthan that observed for unligated teeth by the paired Student t test(non-caffeine intragroup analysis). In the caffeine group, † signifies thatthe mean area of bone loss in ligated teeth is significantly higher thanthat observed in unligated teeth by the paired Student t test (caffeineintragroup analysis). In addition, the different symbols also representthat the mean area of bone loss in ligated teeth from the non-caffeinegroup is significantly different than that observed in ligated teeth fromthe caffeine group by the unpaired Student t test (intergroup analysis).

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the fact that chronic ingestion of caffeine increases thelevels of urinary calcium excretion20,21 and intestinalcalcium absorption efficiency decreases with age,which may lead to calcium imbalance in elderly sub-jects.22,23

In the present study, the adjustment of caffeinedosage and its daily administration in drinking waterwere performed to closely simulate chronic caffeineconsumption in humans.21 The high dose of caffeineused in this study is in agreement with other studiesthat have evaluated the effect of caffeine on bonetissue.10,18 According to these previous investiga-tions,16,18 the total amount of caffeine that was admin-istrated daily in our study (10 mg/100 g body weight)wasequivalent to1,360mg/70kg0.75 inhumanswhenthe conversion is based on the metabolic body weight

(kg0.75). Because the averagecaffeine amount in coffee is 85mg/cup, the dosage used wasequivalent to the consumptionof 16 cups of coffee per day.

Clinical studies24,25 evaluat-ing the effects of caffeine on bonemetabolism have faced variousconfounding factors. First, deter-mining a daily caffeine intakelevel isproblematicdue to thevar-iation of caffeine content in com-mercial products (e.g., coffees,teas and sodas) and inaccurateself reporting of caffeine con-sumption by patients. In addition,there is the co-occurrence of con-foundingvariables,suchasosteo-porosis and the consumption ofcigarettes and alcohol with caf-feine intake among the studiedpopulations. It is recognized, forexample, that cigarette smokerstend to consume more caffeinethan non-smokers.22,23 There-fore, in a periodontal clinicalstudy, the relationship betweencaffeine ingestion and periodonti-tis may be difficult to evaluate be-causecoffeeconsumption isoftenassociated with other risk factorsfor periodontal disease, such assmoking habits. Although a vari-ety of statistical analytic tech-niques have been used toidentify and overcome the influ-ence of these factors, in animalstudies, such confounders maybe more precisely controlled.

CONCLUSIONS

The present study demonstrated that daily intake ofhigh doses of caffeine increases ligature-inducedperiodontitis in rats. Therefore, caffeine may be a sig-nificant factor in periodontal tissue breakdown, pos-sibly accelerating the bone loss of patients withperiodontitis.

ACKNOWLEDGMENTS

The authors greatly appreciate the assistance of thelaboratory technicians from the Biological Science Di-vision at Guarulhos University: Paulo Cesar SimoesSilva for helping with the histologic procedures andRogerio Tadeu Barreira for technical support with an-imal care. The authors report no conflicts of interestrelated to this study.

Figure 3.Photomicrographs illustrating periodontal ligament and bone loss in the furcation area of themandibular molars for non-caffeine and caffeine groups: A) unligated tooth (non-caffeine group);B) ligated tooth (non-caffeine group); C) unligated tooth (caffeine group); and D) ligated tooth(caffeine group). Panels A and C demonstrate the presence of the ligament area and absence ofinterradicular bone loss of unligated teeth from non-caffeine and caffeine groups, respectively. PanelsB and D show the interradicular bone loss induced by ligature in non-caffeine and caffeine groups,respectively. In addition, an increased bone loss in the caffeine group (D) compared to thenon-caffeine group (B) can be observed.

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Correspondence: Dr. Marta Ferreira Bastos, Department ofPeriodontics,DentalResearchDivision,GuarulhosUniversity,Rua Dr. Nilo Pecxanha, n. 81, Predio U, 6� Andar, Centro,Guarulhos 07.023-070, SP, Brazil. Fax: 55-11-64641758;e-mail: mfbastos@prof.ung.br.

Submitted April 17, 2008; accepted for publication June16, 2008.

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