Volume 74 • Number 10

A Comparative Study on the Effectof Nicotine Administration and CigaretteSmoke Inhalation on Bone HealingAround Titanium ImplantsJoão B. César-Neto,* Poliana M. Duarte,* Enilson A. Sallum,* Deise Barbieri,† Heitor Moreno Jr.,†and Francisco H. Nociti Jr.*

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Background: A series of isolated studies has focused on theinfluence of smoking on bone around titanium implants. Thisstudy proposes to investigate the impact of two conditions, i.e.,nicotine administration and cigarette smoke inhalation, on thehealing around implants.

Methods: Forty-five Wistar rats were used. After anesthesia,the tibiae surface was exposed and a screw-shaped titaniumimplant was placed bilaterally. The animals were randomly assignedto one of the following groups: Group 1: control, n = 19; Group 2:intermittent cigarette smoke inhalation, n = 15; and Group 3: sub-cutaneous administration of nicotine (3 mg/kg) twice daily, n = 11.After 60 days, the animals were sacrificed. The degree of bone-to-implant contact (BIC) and the bone area (BA) within the lim-its of the threads of the implant were measured in the cortical(zone A) and cancellous bone (zone B) areas.

Results: In zone A, cigarette smoke presented a significantnegative influence on BIC and BA (Kruskal-Wallis test, P <0.05).In contrast, the administration of nicotine did not influence eitherparameter (P >0.05). In zone B, cigarette smoke inhalation alsoresulted in a decreased percentage of BIC compared to the con-trol group (P <0.05). In addition, the BA was significantly decreasedin groups 2 and 3 when compared to controls (P >0.05).

Conclusion: The negative impact of smoking on implant out-comes may be related to more than one molecule present in thecigarette smoke and nicotine seems to partially contribute, espe-cially in the cancellous bone. J Periodontol 2003;74:1454-1459.

KEY WORDSDental implants; nicotine/adverse effects; osseointegration;smoking/adverse effects; tobacco/adverse effects.

* Department of Prosthodontics and Periodontics, Division of Periodontics, School ofDentistry at Piracicaba, UNICAMP, Piracicaba, São Paulo, Brazil.

† Department of Pharmacology, Faculty of Medical Sciences, UNICAMP, CidadeUniversitária Zeferino Vaz, Campinas, São Paulo, Brazil.

The long-term success of implanttherapy has been reported by sev-eral authors,1-4 however, some sys-

temic conditions have been correlatedwith higher rates of failure.5 Smoking isone of the factors often discussed in rela-tion to implant failure. It is well recognizedthat cigarette smoking is associated withimpaired wound healing following surgeryin the oral cavity,6 resulting in reducedbone height,7 increased bone loss rate,8

increased resorption of the alveolar ridge,8

higher incidence of periodontitis,9 andtype IV bone.10 A greater incidence ofimplant failures before loading in the max-illae of smokers than in non-smokers (9%and 1%, respectively)11 and higher ratesof later failures (11.28% and 4.76% forsmokers and non-smokers, respectively)12

have been reported. Gorman et al.13 eval-uated the relationship between smokingand the failure rates of dental implants atsecond-stage surgery. They suggestedthat smoking is detrimental to implantsuccess. Haas et al.14 suggested thatsmokers suffer detrimental effects aroundsuccessfully integrated maxillary implantsand Lindquist et al.15 reported that smok-ing was the most important factor affect-ing the rate of peri-implant bone loss inthe mandible. Esposito et al.5 reviewedthe literature regarding factors associatedwith the loss of oral implants and con-cluded that smoking was one of the fac-tors associated with biological failures of

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the implants. Lambert et al.16 reported a longitudinalclinical study on the outcome of osseointegrated den-tal implants in smokers and non-smokers and concludedthat smoking resulted in increased implant failure rate.

In an attempt to understand and illustrate suchimpaired clinical outcomes, some animal model stud-ies were performed to provide histological data. Stefaniet al.17 and Nociti et al.18 investigated the influence ofnicotine administration on the osseointegration andbone density around dental implants. In both studies,the authors were not able to demonstrate a significantinfluence of nicotine on bone healing around titaniumimplants. It was then hypothesized that nicotine, byitself, was not able to interfere with the bone healingaround titanium implants. Therefore, a cigarette smokeexposure chamber was designed in order to investigatein rats the influence of the cigarette smoke as a wholeon the bone around titanium implants.19,20 It wasshown that intermittent cigarette smoke inhalationaltered the proportion of mineralized tissue around tita-nium implants in the cancellous area.19 In addition, itwas observed that the animals exposed to cigarettesmoke presented a minor bone filling of the threads inboth regions.20 These studies were the only histolog-ical studies we found on the influence of smoking andtitanium implants. Two relevant aspects have not beenexplored. First, the serum levels of nicotine and coti-nine were not assessed, and a parallel study withhuman serum levels of both substances is necessaryto allow comparisons. Second, nicotine administrationand cigarette smoke inhalation were evaluated sepa-rately in the studies and did not allow comparisonsbetween the two.

In the present study we hypothesize that nicotinealone may not reproduce the negative impact of cig-arette smoke inhalation around titanium implantsinserted in rats with serum levels of nicotine and coti-nine similar to those reported for smokers.

MATERIALS AND METHODSAnimalsForty-five male Wistar rats (300 to 400 g) were includedin the study. The animals were kept in plastic cageswith access to food and water ad libitum. Prior to thesurgical procedures all animals were allowed to accli-matize to the laboratory environment for 5 days. Theprotocol was approved by the University of CampinasInstitutional Animal Care and Use Committee.

Implant SurgeryGeneral anesthesia was obtained by intramuscularadministration of ketamine (0.5 ml/kg). Skin wascleaned with iodine surgical soap. An incision approx-imately 1 cm long was made and the bone surface ofthe tibiae surgically exposed by blunt dissection. Underprofuse saline irrigation, bicortical implant beds were

drilled at a rotary speed not exceeding 1500 rpm. Onescrew-shaped commercially available pure titaniumimplant, 4.0 mm long and 2.2 mm in diameter, wasplaced bilaterally until the screw thread had been com-pletely introduced into the bone cortex. Finally, softtissues were replaced and sutured. Postoperatively, theanimals received an antibiotic‡ through a single intra-muscular injection.

Experimental DesignAfter the implant surgery, the animals were randomlyassigned to one of the following treatment groups:Group 1: control (n = 19); Group 2: intermittent ciga-rette smoke inhalation (n = 15); and Group 3: subcu-taneous injections of nicotine (3 mg/kg) twice daily(n = 11). The group 2 animals were intermittentlyhoused in an animal cigarette smoke exposure cham-ber as previously described.19,20 Briefly, the device con-sisted of a 45 × 25 × 20 cm3 clear acrylic chamber, anair-pump, and two inflow/outflow tubes. Five animals(group 2) were housed in the chamber at the sametime and the smoke from 10 cigarettes, containing1.3 mg of nicotine each, was pumped into the cham-ber. The animals were forced to breath the cigarettesmoke-contaminated air for 8 minutes, three times dailyuntil they were sacrificed. Group 3 animals receivedsubcutaneous injections of 3 mg/kg twice daily untilsacrifice. Group 1 animals were neither exposed to thecigarette smoke nor received subcutaneous injectionsat any time. All animals were sacrificed at 60 days.

Nicotine and Cotinine Serum Levels: AnalyticalMethodsBlood samples were taken before the implant surgeryand at 30 and 60 days. The procedure was systemati-cally performed 15 minutes after cigarette smoke inhala-tion or nicotine adminstration. Serum samples wereassayed for concentrations of nicotine and cotinine byhigh-pressure liquid chromatography (HPLC), composedof two pumps,§ programmed by a system controller,� aUV-Vis detector¶ set at 260 nm, and a reversed-phasecolumn# (150 mm × 4.6 mm I.D. × 5 µm). The mobilephase consisted of 20 mM dibasic potassium phosphate,20 mM monobasic potassium phosphate containing 0.1%of triethylamine. The pH of the solution was adjustedto 6.3 with phosphoric acid and 10% of acetonitrile wasadded to the final solution. The flow rate was 1.0 ml/minute. Two-phenylimidazole was used as internal stan-dards. All the reagents used were HPLC grade.

The extraction of the samples followed the method-ology previously described by Nakajima et al.,21 but

‡ Pentabiótico, Wyeth-Whitehall Ltda, São Paulo, SP, Brazil.§ LC-10ADvp, Shimadzu Corporation, Tokyo, Japan.� SCL-10ADvp, Shimadzu Corporation.¶ SPD-10ADvp, Shimadzu Corporation.# Column Luna, Phenomenex, Torrance, CA.

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we evaporated to dryness under nitrogen atambient temperature. The injection volumewas 20 µl and the limit of quantification was10 ng/ml.

Histometric ProcedureAfter sacrifice, the tibiae were removed andfixed in 4% neutral formalin for 48 hours. Unde-calcified sections were prepared as previouslydescribed;22 i.e., the blocks were dehydratedusing an ascending series of ethanol (60% to100%) and embedded in glycolmethacrylate.**Subsequently, 20 to 30 µm sections wereobtained and stained using 1% toluidine blue.The percentage of bone-to-implant contact(BIC) and bone area (BA) within the threadsof the implants was obtained bilaterally†† and the datafrom both sides were averaged to give a mean scorefor each animal. The data were arranged in cortical(zone A) and cancellous bone (zone B) areas as previ-ously described.19,20

Statistical AnalysisThe null hypothesis was tested by an intergroup analy-sis using the non-parametric Kruskal-Wallis test (alpha =0.05), for zones A and B separately (group 1 versusgroup 2 versus group 3). Pairwise multiple comparisonswere carried out by Tukey test (alpha = 0.05) if theKruskal-Wallis test showed significant differences.

RESULTSClinical ObservationsFifty-five animals were initially involved in this exper-iment; however, one animal from group 1 died, as didfive from group 2 (three during the first 2 days of expo-sure as a consequence of smoke inhalation and two dur-ing blood collection); and four from group 3, all duringblood collection. All surviving group 2 animals demon-strated breathing difficulties. A non-significant weightloss was observed for group 2 and group 3 animals.

Serum Levels of Nicotine and CotinineThe serum levels of nicotine and cotinine were lowerthan the detectable limit for all groups before the implantplacement. Group 1 (control) had no detectable values atthe second and third evaluations (30 and 60 days afterimplant insertion). Groups 2 and 3 presented detectablevalues of nicotine and cotinine in both evaluations. Themean values of nicotine were 346.1 ng/ml ± 114.3 and376.03 ng/ml ± 53.85 at day 30 for groups 2 and 3,respectively; and 174.9 ng/ml ± 32.2 and 401.0 ng/ml ±64.0 at day 60 for groups 2 and 3, respectively. Themean values of cotinine were 265.4 ng/ml ± 109.8 and294.38 ng/ml ± 41.24 at day 30 for groups 2 and 3,respectively; and 149.9 ng/ml ± 27.4 and 181.2 ng/ml ±17.8 at day 60 for groups 2 and 3, respectively.

Histometric AnalysisStatistical analysis showed a significant difference in BICbetween groups 1 and 2 in cortical or cancellous bone;no differences were observed between groups 1 and3, and groups 2 and 3 in either zone (Table 1). Similarresults were observed concerning BA in zone A; e.g.,a significant influence of smoke inhalation comparedto control group and no differences between nicotineadministration and the other groups. Moreover, in thecancellous bone cigarette smoke inhalation and nico-tine administration decreased the percentage of miner-alized bone within the implant threads (Table 1). Figure 1illustrates typical histological findings in each group.

DISCUSSIONThe present investigation evaluated the impact of sub-cutaneous injections of nicotine compared to intermit-tent cigarette smoke inhalation on bone healing aroundtitanium implants inserted in the tibiae of rats using his-tological methods; e.g., degree of BIC and BA within theimplant threads. The data for the cortical (zone A) andcancellous (zone B) regions were evaluated separatelybecause of the anatomic and metabolic differencesbetween these areas and because previous studiesreported that the bone in the medullar compartmentmay be more susceptible to systemic conditions.23,24

The results of the present study demonstrated that cig-arette smoke inhalation negatively influenced bone heal-ing for both histometric parameters in cortical andcancellous bone. It would appear that part of the effectsproduced by cigarette smoke is caused by nicotine andthis is of great importance in cancellous bone.

A previous report from our laboratory,19 using a sim-ilar protocol of cigarette smoke inhalation, observed anegative influence of this treatment on the propor-tion of mineralized bone in cancellous zone lateral tothe implant. We also demonstrated that intermittent

** Technovit 7200, Heraeus Kulzer GmbH, Wehrheim, Germany.†† Image-Pro, Media Cybernetics, Silver Spring, MD.

Table 1.

Histometric Parameters (mean ±± SD)

Zone 1 Zone 2

Groups BIC BA BIC BA

1 55.60 ± 11.00a 86.47 ± 4.80a 36.67 ± 7.11a 32.01 ± 6.62a

2 41.39 ± 15.64b 79.85 ± 6.17b 25.55 ± 13.34b 20.71 ± 8.57b

3 45.54 ± 3.06ab 81.54 ± 5.06ab 27.95 ± 11.93ab 21.91 ± 6.48b

P value 0.003 0.003 0.012 <0.001

Letters should be considered by columns (Kruskal-Wallis test). If two different groups havethe same letters, then they are not statistically significant, and vice versa.

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Figure 1.Photomicrograph illustrating the histological aspect observed around the implants placed (toluidine blue; original magnification ×12.5; bar = 0.32 mm).A) Group 1; B) group 2; C) group 3.

cigarette smoke inhalation produces a minor bone fill-ing of titanium implant threads inserted in the tibiae ofrats.20 In addition, the present study demonstrated thatintermittent cigarette smoke inhalation might affect BIC.

Using a mechanism by which rabbits were exposedto cigarette smoke, Ueng et al.25,26 reported that inter-mittent cigarette smoke exposure delayed the mineral-ization during the bone healing process of distractionosteogenesis. In the present investigation, the deviceused to expose the animals to the cigarette smoke wasmodified from the Ueng et al. reports25,26 to allow theinclusion of 5 animals at a time in a chamber measur-ing 45 × 25 × 20 cm3. The use per exposure of 10 cig-arettes was determined by our former studies19,20 whichdemonstrated that this was the volume of smoke theanimals could support for 8 minutes, three times a dayfor 60 days. The assessment of serum level of cotininedemonstrated that this protocol of intermittent cigarettesmoke inhalation, and the nicotine administration pro-duced serum levels correlated with human smokers whoconsume between 10 to 20 cigarettes/day.27 The inter-val between the exposures/injections and the blood col-lections was based on the half-lives of nicotine andcotinine,28 the time necessary to metabolize nicotineinto cotinine (half-life formation of the metabolite: 20 to28 minutes)29 and the time necessary for the animal torecover its normal functions of breathing and movement.Special care was taken with the animals, mainly in the

intermediate collection, because pilot studies revealed ahigh incidence of death during this procedure.

In the present study, regarding nicotine and cotinineserum levels, a degree of variability, and changes at 30and 60 days were observed. The variability might beexplained by two events. First, because special carewas taken during the blood collection, it presented dif-ferent duration according to the chance of death for eachanimal. Second, since it was not possible to standard-ize the animals’ blood flow, different time intervals wererequired to collect the amount of blood necessary foranalysis. Moreover, changes in nicotine absorption, asa consequence of emphysema as previously reported,30

may have caused different serum concentrations.In contrast to a previous report,17 the present study

showed that nicotine administration produced a nega-tive influence on BA in the cancellous zone. Since thedosage (3 mg/kg) and frequency of administration(twice daily) used in the present study are higher thanthose in the earlier study17 (0.93 mg/kg, once daily,eight animals), these data seem to confirm a previousreport showing that nicotine may present a dose-depen-dent response in vivo.31

In vitro studies may lead us to, mechanistically,approach the data presented by the present investi-gation. Several authors have reported that nicotine isa cytotoxic agent that produces negative effects onfibroblasts32-34 and osteoblasts cell cultures.35,36 It has

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also been shown that acrolein and acetaldehyde, volatilecomponents of cigarette smoke, have negative effectson fibroblasts cultures.37-39 Furthermore carbon monox-ide, another compound of cigarette smoke, has a greataffinity for hemoglobin.40 This link is 200 times strongerthan the link to oxygen, consequently decreasing theoxygen-carrying capacity of hemoglobin.40 This maybe of great importance mainly for healing areas, wherethe cells have a high metabolism and require a greatamount of oxygen. Another investigation reported thathydrogen cyanide, a volatile component of cigarettesmoke, inhibits enzyme systems necessary for oxida-tive metabolism and oxygen transport at the cellularlevel.41 Taken together, the effects of these events mayhave the potential to jeopardize bone healing.

In contrast to most of clinical studies11,14-16 thatreported early implant failure in smokers, in the presentstudy, the observation of a lower bone-to-implant con-tact and poor bone quality around the implants guidesus to correlate cigarette consumption with late implantfailure. A similar finding can also be inferred from aretrospective study by Ekfeldt et al.42 in which theyobserved that the number of lost implants in smokerswas significantly higher after loading (22) than before (9).

Smokers have likely been smoking for many yearsbefore implant placement; i.e., the bone tissue has beenexposed to cigarette smoke components for a long time.It remains to be investigated whether different resultswould be observed in animals exposed for a longer periodof time before implant insertion. In addition, despitethe findings of the present investigation, the effect ofnicotine-based products as an adjunct to smoking ces-sation protocols on implant outcomes remains unclear.Finally, a relevant aspect currently under investigationconcerns the benefits that a smoking cessation protocolcould provide to the observations of the present study.

In conclusion, within the limits of the present study,intermittent cigarette smoke inhalation may result inlower bone-to-implant contact and less bone areawithin the threads and part of these effects seems tobe caused by nicotine, particularly in cancellous bone.

ACKNOWLEDGMENTSThe authors greatly appreciate the assistance of ASTechnology, São José des Campos, S.P., Brazil, forsupplying the implants. This study was supported inpart by Fundação de Amparo à Pesquisa do Estadode São Paulo (98/5298-6, FAPESP, Brazil) and by agrant from Conselho Nacional de Pesquisa(301119/97, CNPq, Brazil) to Dr. Moreno Jr.

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Correspondence: Dr. Francisco H. Nociti Jr., Av. Limeira901, Caixa Postal: 052, CEP: 13414-018, Piracicaba, SP, Brazil.Fax: 55-19-3412-5218; e-mail: nociti@fop.unicamp.br.

Accepted for publication March 28, 2003.

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