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Association of Interleukin 4 Gene Polymorphisms With Dental Implant Loss Suzane C. Pigossi, DDS, MSc,* Fabiano Alvim-Pereira, PhD,† Claudia C. K. Alvim-Pereira, PhD,‡ Paula C. Trevilatto, PhD,§ and Raquel M. Scarel-Caminaga, PhDk

ental implants have become the therapeutic modality of choice for oral rehabilitation. The term osseointegration, referring to a direct contact between live bone and titanium surface, was coined by Brånemark in the 60s. Successful osseointegration achievement is the result of the majority of implant procedures but, in some cases, environmental and host factors may interfere and lead to an osseointegration failure, even though a gold standard technique and an apparent ideal indication occurred.1 Those failure outcomes have a huge impact in absolute numbers because millions of implant surgeries are conducted worldwide each year. Failure in the osseointegration process is the main cause of implant loss.2 Failures can be classified as early, when osseointegration fails to occur before occlusal function, or late, when the achieved osseointegration is lost after a period in function.3 No matter the time of failure, implant loss consists in a complex situation to be managed by many implant surgeons.

D

*PhD Student, Department of Morphology, School of Dentistry at Araraquara, UNESP-São Paulo State University, Araraquara, Brazil. †Adjunct Professor, Department of Dentistry, University Federal of Sergipe, Lagarto, Brazil. ‡Adjunct Professor, University Federal of Sergipe, Department of Medicine, Lagarto, Sergipe, Brazil. §Full Professor, Center for Health and Biological Sciences, Pontifícia University Católica of Paraná, Curitiba, Brazil. kAdjunct Professor, Department of Morphology, School of Dentistry at Araraquara, UNESP-São Paulo State University, Araraquara, Brazil.

Reprint requests and correspondence to: Raquel M. Scarel-Caminaga, PhD, Department of Morphology, School of Dentistry in Araraquara, UNESP-Univ Estadual Paulista, P. 331, Araraquara CEP 14801-903, Brazil, Phone: (+55)16.3301.6504, Fax: (+55)16.33016433, E-mail: [email protected] ISSN 1056-6163/14/02306-723 Implant Dentistry Volume 23  Number 6 Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0000000000000157

Purpose: The purpose of this study was to investigate the association between interleukin 4 (IL4) polymorphisms/haplotypes and dental implant loss. Materials and Methods: Two hundred and seventy eight (n ¼ 278) unrelated patients were divided into 2 groups: (1) control group (C) composed of 186 individuals presenting at least 1 osseointegrated implant and (2) study group (S) composed of 94 individuals presenting at least 1 implant loss. After DNA collection, IL4 polymorphisms were investigated by polymerase chain reaction (PCR)restriction fragment length polymorphism and for the variable number of tandem repeat (VNTR) only by PCR. Results: No association between alleles/genotypes of −590 (C/T) (P ¼

0.9704/P ¼ 0.5992) and VNTR (P ¼ 0.7155/P ¼ 0.8789) polymorphisms and implant loss were found between the groups. Regarding +33 (C/T) polymorphism, no difference was found in genotype frequency (P ¼ 0.1288), but the C allele was associated with implant loss (P ¼ 0.0236, odds ratio ¼ 1.61, 95% confidence interval ¼ 1.1–2.4). Haplotype analysis showed no statistical differences between the groups. Conclusion: The C allele of the +33 (C/T) polymorphism in the IL4 gene was associated with susceptibility to dental implant loss in Brazilians in the studied population. (Implant Dent 2014;23:723–731) Key Words: dental implant loss, interleukin-4, polymorphisms, haplotypes

A surgery procedure is mandatory to implant placement and a host inflammatory response is expected to occur. Also, the covering material of the implants, considered inert, can stimulate immunogenic cells to produce inflammatory mediators, such as interleukins (ILs).4 These cytokines have been considered to have a key role in the regulation of the inflammatory and immunologic host response.5 An intense inflammatory process could impair osseointegration.6 To clarify the failure mechanisms, several studies have investigated tissues surrounding unsuccessful dental implants.7 An abnormal immune-

inflammatory response involving different cell types, such as macrophages, polymorphonuclear neutrophils, T and B lymphocytes, endothelial cells, fibroblasts, keratinocytes, osteoclasts, and osteoblasts, can destroy the peri-implant and periodontal tissues.8 If activated, these cells can synthesize and release cytokines,9 which mediate both the inflammatory and the osteolytic processes. IL-4 is a potent downregulator of macrophage function that inhibits the secretion of pro-inflammatory cytokines, such as IL-1, IL-6, and tumor necrosis factor (TNF). After stimulation with lipopolysaccharides of oral bacteria, IL-4

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Table 1. PCR Primers, Assay Conditions, and Restriction Enzymes for Genotyping the Polymorphisms in the IL4 Gene SNP

Primers

−590

(F) 50 TAA ACT TGG GAG AAC ATG GT 30 (R) 50 TGG GGA AAG ATA GAG TAA TA 30 (F) 50 CTC ATT TTC CCT CGG TTT CAG C 30

+33

VNTR

PCR Annealing Temperature

Restriction Enzyme

51°C

AvaII

60°C

Mnl

C T C

55°C

d

T 3R

(R) 50 GAA GCA GTT GGG AGG TGA GA 30 (F) 50 TAG GCT GAA AGG GGG AAA GC 30 (R) 50CTG TTC ACC TCA ACT GCT CC 30

acts as a mitogen of B cells and enhances the secretion of immunoglobulin G (IgG) and immunoglobulin E (IgE).10 Lack of IL-4 in periodontal tissues may cause the accumulation of macrophages, increased CD14 expression, and high production of IL-1b, TNF-a, and prostaglandin E2 in human monocytes, which leads to bone resorption.11 Auxiliary T lymphocyte (Th2) cells secrete IL-4, IL-10, and IL-13, which are associated with humoral immune responses and induce antibody production.12 In an attempt to establish diagnostic markers for monitoring implant health status, levels of ILs have been measured in diseased implant sites.13 IL4 gene, which encodes the antiinflammatory cytokine IL-4, is present on chromosome 5q31.114 and comprises 4 exons in approximately 10 kb (GenBank accession no. M23442). Polymorphisms in the IL4 gene have been largely investigated in case-control studies on inflammatory and autoimmune diseases.15 Three polymorphisms in the IL4 gene presenting physical proximity on the chromosome are in linkage disequilibrium (LD), forming haplotypes.16 The variation rs2243250 is a single-nucleotide polymorphism (SNP) in the promoter region of the IL4 gene at the position −590 (C/T). The second SNP, rs2070874, is located in the 50 untranslated region of the IL4 gene at position +33 (C/T).17 The third one, located in the third intron, is a variable number of tandem repeat (VNTR) polymorphism of 70 bp, with one allele presenting a 70-bp deletion (183 bp) and another with a 70-bp insertion (253 bp), which were also referred to as B1 (70 bp Deletion) and B2 (70 bp Insertion).18 Haplotypes formed by those polymorphisms have been recently associated with susceptibility to chronic periodontitis.19 Because IL-4 downregulates pro-inflammatory

Fragment Sizes (bp)

Reference

177, 18, 1995

24

83, 43, 20, 4, 126, 20, 4

Shibata et al9a

253, 186

Michel, et al9b

2R

cytokine secretion and enhances IgG and IgE, interfering with the immuneinflammatory response, the aim of this study was to investigate if polymorphisms in the IL4 gene are associated with dental implant loss in a Brazilian population.

MATERIALS

Allele

AND

METHODS

Selection of Subjects

A total of 3578 patient records from the Latin-American Dental Research Institute (ILAPEO) of Curitiba, Brazil, were analyzed in this study. All patients were implant treated (NEODENT Implante Osteointegrável) between 1996 and 2006, and of the 3578 subjects treated, 126 patients (3.5%) presented implant loss (early failure represented the majority of cases, 88.2%; 187 of 212 implants failed). From those 126 individuals presenting that implant loss, 94 were evaluated (32 were not evaluated because of death or address change). Thus, the study group (S) composed of 94 individuals presenting at least 1 implant loss. The control group (C) was then composed of 186 patients treated with osseointegrated implants, which were in function for at least 6 months and without any loss, matched with (S) by gender, age, and smoking habits (Table 2). Therefore, the total sample was composed of 280 unrelated individuals of both genders with a mean age of 56.10 6 11.32 years (range, 29.5–89.3 years). The study sample was from the south region of Brazil, in Paraná State. According to the Brazilian Institute of Geography and Statistics (IBGE), in 2005, 73% of the population from Paraná State was Caucasians, 23.3% were of mixed ancestry, 2.5% were Afro-Americans, and 1.2% was Asiatic descents. All patients were previously advised about the nature of the study

and signed a consent form within a protocol approved by an Institutional Review Board (Ethical Committee in Research at Pontifícia University Católica of Paraná, protocol 323). Subjects answered a personal, medical, and dental history anamnesis and also had their socioeconomic profile assessed according to the Brazilian Economical Classification Criteria.20 The subjects in good general health could not have any of the following exclusion criteria: HIV infection, current pregnancy or lactation, orthodontic appliances, present necrotizing ulcerative gingivitis and periodontitis, and history of aggressive periodontitis (AgP). Besides, it also excluded patients that submitted a precocious prosthesis load or regenerative surgery, such as bone grafting, and postsurgical complications like infection. These strict criteria in obtaining the samples aim to reduce the influence of systemic factors in the loss of the implant to analyze the influence of the polymorphism alone.21 This study evaluated clinical and socioeconomic variables, such as gender, age, smoking status, ethnic group, socioeconomic profile, presence of systemic diseases, medical treatment, current medication, drugs within 3 months, dental appointments, brushing daily frequency, use of dental floss, and number of teeth and periodontal parameters (tooth mobility, clinical attachment level, gingival plaque, and calculus index). Measurements of probing pocket depth (PPD) and clinical attachment loss (CAL) were recorded at 4 points around each tooth. The periodontal parameters were recorded from each site using a conventional periodontal probe (Hu-Friedy, Chicago, IL). All clinical data of the selected patients were collected by one examiner (F.A.P.).22

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Table 2. Results of Social and Clinical Variables Analyzed by Univariated and Multivariated Model Between Control and Study Groups Control Group Study Group General data Gender, n (%) Male Female Age

n ¼ 186

n ¼ 94

64 (34.4) 122 (65.6) 54.94 6 11.55 44 (23.7)

36 (38.3) 58 (61.7) 56.97 6 10.76 18 (19.1)

Smoke, n (%) Ethnic group, n (%) Caucasian 176 (95.1) 92 (98.9) Non-Caucasian 9 (4.9) 1 (1.1) Socioeconomic profile, n (%) A1/A2/B1 96 (51.6) 48 (51.1) B2/C/D 90 (48.4) 46 (48.9) Systemic data, n (%) n ¼ 186 n ¼ 94 Presence of systemic 127 (68.3) 69 (73.4) disease Medical treatment 75 (40.3) 44 (46.8) Drugs of continuous 78 (41.9) 43 (45.7) use Drugs within 3 months 64 (34.4) 33 (35.1) Odontologic data, n (%) n ¼ 186 n ¼ 94 Dental appointments 140 (75.3) 75 (79.8) over 3 times per year Brushing daily over 3 135 (72.6) 72 (76.6) times Dental floss use 149 (80.1) 70 (74.5) Mouthwash 85 (45.7) 46 (48.9) Edentulism 34 (18.3) 8 (8.5) Number of installed 4.44 6 3.12 5.85 6 3.62 implants Number of remaining 20.21 6 6.33 18.35 6 7.00 teeth Periodontal index n ¼ 151 n ¼ 86 Probing pocket depth 2.72 6 0.46 2.55 6 0.47 Clinical attachment 3.61 6 0.85 3.67 6 1.07 level Gengival index 0.63 6 0.38 0.64 6 0.28 Plaque index 0.12 6 0.23 0.23 6 0.41 Calculus index 0.07 6 0.12 0.13 6 0.24 Denta mobility 19 (12.5) 16 (18.6)

Univariated (P)

Univariated OR

Multivariated (P)

Multivariated OR

2.405 (1.065–5.429) -

0.006‡ ,0.001‡

3.372 (1.429–7.959) 1.159 (1.072–1.254)

0.521* 0.156† 0.391* 0.172

0.931*

0.377* 0.300* 0.543* 0.908* 0.398* 0.470* 0.280* 0.608* 0.031*

0.735§

0.002§ 0.766§ 0.500§ 0.689§ 0.2369§ 0.201*

*Person chi-square test. †Student t test. ‡Binary logistic regression model, the variables with P , 0.200 are inputted in the multivariate model. §Mann-Whitney U test. Bold values indicate statistically significant results (P , 0.05).

In relation to the implant clinical parameters, a total of 1367 implants were installed in patients of this study by experienced professionals from the ILAPEO. The placed implants were classified as control (C; n ¼ 1232) or study (S; n ¼ 135). The following clinical characteristics were accessed and compared between C and S, such as

implant position, primary stability, type of platform, surgical technique, loading aspects, presence of graft, and both bone quantity and quality.23 Analysis of Genetic Polymorphisms

IL4 gene polymorphisms (C−590T) and (C+33T) were investigated by polymerase chain reaction (PCR)-

restriction fragment length polymorphism techniques and the polymorphism VNTR 70 bp (del/ins) was investigated by PCR. The assay conditions are summarized in Table 1. Polymorphism analyses were performed after separation of DNA fragments by electrophoresis on a 10% polyacrylamide gel stained with silver

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Table 3. Clinical Findings (n ¼ 1367) for Control and Study Implants Control Implants (n ¼ 1232), n (%) Position Maxilla Mandible Anterior region Posterior region Primary stability Good (.40) Poor (,40) Platform Internal Hex External Hex Surgical technique One-step surgery Two-step surgery Loading time Immediate load Yes No Time to load (wk) Maxilla† Mandible† Load Load implants Unload implants Graft procedure Yes No Bone quantity Good Poor Bone quality Good Poor

Study Implants (n ¼ 135), n (%)

P

(37.5) (62.5) (35.3) (64.7)

0.003*

652 (68.8) 295 (31.2)

49 (52.7) 44 (47.3)

0.001*

353 (28.7) 832 (67.7)

37 (27.4) 95 (70.4)

0.536*

456 (46.2) 532 (53.8)

44 (40.4) 65 (59.6)

0.250*

150 (12.2) 1075 (87.8)

9 (6.7) 126 (93.3)

0.056*

627 605 550 682

(50.9) (49.1) (44.6) (55.4)

43.39 6 37.08 (608) 40.50 6 46.88 (570)

50 85 47 88

43.93 6 52.79 (10) 29.44 6 56.07 (15)

0.037*

0.964‡ 0.370‡

1178 (95.6) 54 (4.4)

25 (18.7) 109 (81.3)

0.001*

235 (19.1) 997 (80.9)

22 (16.3) 113 (83.7)

0.433*

1096 (89.0) 136 (11.0)

123 (91.1) 12 (8.9)

0.445*

1092 (88.6) 140 (11.4)

118 (87.4) 17 (12.6)

0.671*

*Chi-square test. †Mean 6 SD. ‡Mann-Whitney U test. Bold values indicate statistically significant results (P , 0.05).

nitrate. The gels were photographed with a GDS 8000 System. Statistical Analysis

Differences between clinical categorical variables were analyzed by Pearson chi-square test or Student t test when indicated; odds ratio (OR) with 95% confidence intervals (CIs) were calculated when possible. Continuous variables were previously normality tested by Kolmogorov-Smirnov test. Differences between the groups were assessed by Student t test or MannWhitney U test as indicated. Binary logistic regression model was applied. All clinical variables with P , 0.200 in univariate tests were inputted in the

model. Differences in the allelic and genotypic frequencies of polymorphisms in IL4 gene between the control and implant loss groups were analyzed using the Pearson chi-square test. The ARLEQUIN version 3.1 program25 was used to calculate Hardy-Weinberg (H-E) equilibrium, reconstruct haplotypes by the expectation-maximization algorithm because of the unknown gametic phase, and evaluate a likelihood ratio test of LD. Differences in the haplotype distributions between the studied groups were assessed by the CLUMP program, which uses the Monte Carlo simulations.26 The risk associated with individual alleles and genotypes of each polymorphism, and with the computationally inferred

haplotypes, was calculated as the OR with 95% CI, accompanied by the Fisher exact test. Statistical analyses were performed using the BioEstat statistical package version 5. Differences were considered significant when P , 0.05.

RESULTS Regarding clinical and socioeconomic variables, such as gender, age, smoking status, ethnic group, socioeconomic profile, presence of systemic diseases, medical treatment, current medication, drugs within 3 months, dental appointments, brushing daily frequency, use of dental floss, and number of teeth and periodontal parameters (tooth mobility, clinical attachment level, gingival plaque, and calculus index), there were no statistical significant differences between the groups. Statistical significant differences between the groups were found in edentulism (P ¼ 0.031) and PPD (P ¼ 0.002). After binary multivariate model, the variables edentulism (P ¼ 0.006) and OR ¼ 3.372 (1.429–7.959) remain presenting statistical significant differences between the groups (Table 2). Table 3 presented data regarding implant clinical findings.23 Comparing control and study implants, some clinical parameters seem to contribute to dental implant loss. About position of implant placement, in mandible, there was higher prevalence of study (85/135) than control (605/1232) implants. Moreover, posterior region showed more study (88/ 135) than control (682/1232) implants. A good primary stability was shown to be greater in control (652/947) than in study (49/93) implants. Finally, implants that received load were more frequent in control (1178/1232) than in study (25/134) implants. Genotype distributions of the −590 and VNTR polymorphisms were consistent with H-E equilibrium in the control and failed implant groups but not +33 SNP. Genotype and allelic distributions of IL4 −590 SNP, +33 SNP, and VNTR polymorphisms in the control and study (at least 1 failed dental implant) groups are presented in Table 4. There was no significant difference in the genotype distribution between patients with successful and failed implants for −590

IMPLANT DENTISTRY / VOLUME 23, NUMBER 6 2014

Table 4. Distribution of Alleles and Genotypes of the −590 (C/T), +33 (C/T) and VNTR 70 bp (Del/Ins) Polymorphisms in the IL4 in the Studied Groups SNP −590 (C/T) Allele T C Genotype TT TC CC H-W equilibrium +33 (C/T) Allele T C Genotype TT TC CC H-W equilibrium VNTR Allele I D Genotype II ID DD H-W equilibrium

Control

Study

P

n ¼ 372 91 (24.45) 281 (75.54) n ¼ 186 13 (6.98) 65 (34.95) 108 (58.07) P ¼ 0.458

n ¼ 184 44 (23.92) 140 (76.08) n ¼ 92 4 (4.34) 36 (39.13) 52 (56.52) P ¼ 0.470

0.9704

n ¼ 372 n ¼ 184 132 (35.48) 47 (25.55) 240 (64.52) 137 (74.45) OR ¼ 1.61; 95% CI, 1.1–2.4 n ¼ 186 n ¼ 92 39 (20.96) 12 (13.05) 54 (29.04) 23 (25) 93 (50.00) 57 (61.95) P , 0.0001 P ¼ 0.001

0.0236

n ¼ 372 268 (72.05) 104 (27.95) n ¼ 186 92 (49.46) 84 (45.16) 10 (5.38) P ¼ 0.098

0.7155

0.5992

0.1288

n ¼ 184 136 (73.92) 48 (26.08) n ¼ 92 48 (52.18) 40 (43.47) 4 (4.35) P ¼ 0.221

0.8789

Values are represented as n (%). Bold values indicate statistically significant results (P , 0.05); H-W equilibrium, Hardy-Weinberg Equilibrium. P values were calculated by the chi-square test. Alleles or genotypes in the parenthesis represent frequencies percentage values (%).

(P ¼ 0.5992), +33 (P ¼ 0.1288), and VNTR (P ¼ 0.8789) polymorphisms. However, the allelic distribution of the +33 SNP was significantly different between the control and failed implants groups; individuals carrying the C allele

were susceptible to lose dental implants (OR ¼ 1.61, 95% CI ¼ 1.1–2.4, P ¼ 0.0236). Significant LD was observed for all the investigated polymorphisms. The distribution of haplotypes showed

Table 5. Distribution of the IL4 Haplotypes in the Studied Groups Haplotypes −590, +33, VNTR

Control, n ¼ 372 n (%)

TCI 11 CCI 208 TTD 58 TCD 6 CTI 37 TTI 13 CTD 16 CCD 23 P* ¼ 0.4555 CLUMP test

(2.95) (55.91) (15.6) (1.62) (9.94) (3.5) (4.3) (6.18)

Failed Implants, n ¼ 184 n (%) 11 110 27 4 14 1 5 12

(5.97) (59.78) (14.7) (2.17) (7.6) (0.54) (2.72) (6.52)

P

OR

95% CI

0.13 0.43 0.87 0.89 0.45 0.07 0.49 0.97

2.08 1.17 0.93 1.35 0.74 0.15 0.62 1.05

0.88–4.90 0.81–1.67 0.56–1.52 0.37–4.86 0.39–1.41 0.01–1.16 0.22–1.72 0.51–2.17

Differences between the groups were analyzed using the Fisher exact test. OR (95% CI): odds ratio values with the respective 95% of confidence intervals. *P value obtained from the T4 value of the CLUMP program.

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no significant differences between the control and failed implant groups (P ¼ 0.4555, Table 5; P ¼ 0.3886, Table 6).

DISCUSSION In the present study, the analyzed clinical and socioeconomic data seem not to influence dental implant loss, with exception of edentulism. Higher percentage of edentulism was found in the control group. Implant success may be compromised by site-specific persistence of bacterial biofilm co-aggregations contaminating healed alveolar bone.27 Complete edentulation results in a significant reduction of bacteria related to periodontitis and peri-implantitis.28 In fact, a major percentage of edentulism was found in patients who did not present implant failure. Regarding periodontal parameters of patients, although higher plaque (PI), gingival (GI), and calculus index (CI), increased CAL, and tooth mobility may indicate implant morbidity,29 they did not seem to contribute to implant loss in this study. However, statistical significant difference between the groups was found for the PPD. In a bivariate logistic regression model, the edentulism variable maintained statistical significant difference between groups; it may reinforce the influence on implant loss in this study. However, other studies enrolling higher number of patients belonging to different ethnicities are necessary to confirm the obtained results. Considering clinical aspects of individual implants it could be noted by results of the present study that the implantation site influences implant failure rates. The majority of lost implants were found in posterior regions. This may be attributable to a combination of unfavorable factors present in the posterior region. First, an insufficient quantity of available bone because of the anatomical limitations is present in this area (the maxillary sinus and the inferior alveolar nerve).30 The cortical layer of both jaws tends to become thinner and more porous posteriorly. Furthermore, from a biomechanical standpoint, implants placed in the posterior region have to support the higher functional load increasing the risk of failure.31 Indeed, in this study, implants placed in the

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Table 6. Distribution of IL4 Haplotypes (Arranged as Genotypes) in Studied Groups Haplotypes −590, +33, VNTR CCI/CCI CTI/CCI CTI/CTI CCI/CCD CCI/CTD CCD/CCD CTI/CTD CTD/TTD CTD/CTD CCI/TCD TTD/CTI TTD/CCI TCD/TCD TCD/CCI TCI/TCD TCI/CCI TCI/TCI TTD/TCI TTI/CCI TTD/TTD TTD/TTI TTI/CTI TTD/CCD TTI/TTI P† ¼ 0.3886

Control, n ¼ 186 n (%) 66 9 3 17 8 2 4 2 1 1 17 26 0 5 0 6 1 3 4 3 2 1 2 3

(35.48) (4.83) (1.62) (9.13) (4.3) (1.08) (2.15) (1.08) (0.58) (0.58) (9.13) (13.97) (0.0) (2.68) (0.0) (3.22) (0.58) (1.62) (2.15) (1.61) (1.08) (0.58) (1.08) (1.62)

Failed Implants, n ¼ 92 n (%)

P

OR

95% CI

35 (38.04) 2 (2.17) 2 (2.17) 10 (10.9) 3 (3.26) 1 (1.08) 0 (0.0) 2 (2.17) 0 (0.0) 0 (0.0) 8 (8.7) 17 (18.5) 1 (1.08) 1 (1.08) 1 (1.08) 6 (6.52) 2 (2.17) 0 (0.0) 1 (1.08) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

0.77 0.45 0.88 0.80 0.92 0.54 0.37 0.85 0.71 0.71 0.91 0.42 0.71 0.67 0.71 0.33 0.53 0.54 0.88 0.54 0.80 0.71 0.80 0.54

1.11 0.43 1.35 1.21 0.75 1.01 * 2.04 * * 0.94 1.39 * 0.39 * 2.09 4.11 * 0.5 * * * * *

0.66–1.87 0.09–2.06 0.22–8.25 0.53–2.76 0.19–2.89 0.09–1.29 0.28–14.75

0.39–2.28 0.71–2.72 0.04–3.45 0.65–6.67 0.36–45.94 0.05–4.53

Differences between the groups were analyzed using the Fisher exact test. OR (95% CI): odds ratio values with the respective 95% of confidence intervals. *OR not calculated because of the presence of zero. †P value obtained from the T4 value of the CLUMP program.

mandible tended to fail more, in agreement with other previous studies.32–35 Primary stability is associated with the mechanical engagement of an implant with the surrounding bone. This allows the mechanical adaptation of the implant to the bone site until secondary stability is acquired.36 Therefore, in agreement with what was observed in the present study, it is essential to assess the primary implant stability to ensure a successful osseointegration.37 Another significant result presented here was that most failures occurred before loading (81.3%), suggesting that a host response role on the individual healing process influenced osseointegration.22,38,39 Polymorphisms have been shown to modulate host response and susceptibility to numerous complex diseases and traits,40–42 including periodontal disease43 and dental implant failure.44 The present study showed, for the first time, that the +33 C allele of the IL4 gene was

associated with susceptibility to dental implant loss. Individuals from the south region of Brazil carrying at least 1 copy of allele C were significantly susceptible to lose dental implants. Allele C and CC genotype of the +33 (C/T) polymorphism was associated with susceptibility to chronic periodontitis in individuals from the southeastern region of Brazil.19 In addition, the present study showed no association of the −590 (C/T) polymorphism in the IL4 gene with implant failure. The −590T allele was previously associated with asthma/atopy in the American45 and Japanese24 population and with chronic periodontitis in Brazilians.19 Regarding the VNTR polymorphism, no association with early dental implant loss was identified in the present study. The lack of association was also found with chronic periodontitis in a Czech population. Nevertheless, IL4 haplotype [T(−590)/T(−33)/(insertion)VNTR] was associated with an increased risk for chronic periodontitis.46 It was reported

that the 70-bp insertion was associated with chronic periodontitis.19 Significant LD was observed between all investigated polymorphisms, similarly to what was observed in the literature,46–48 but the analysis of haplotypes indicated no association with susceptibility to dental implant loss. In contrast with this result, for another Brazilian population,19 it was noted that the TCI haplotype was associated with susceptibility to chronic periodontitis. Considering that the second allele of the haplotype referred to the +33 (C/T) SNP, it can be noted that this haplotype related with chronic periodontitis contain the C allele, similar to the findings of the same C allele associated with dental implant loss in the present study. In other words, only the +33C allele of the IL4 gene was associated with susceptibility to dental implant loss, but when this SNP is included in the analysis of IL4 haplotype (the second position of the haplotype), the significance with the loss of dental implants was not maintained. This could be explained because only the +33 (C/T) polymorphism was not consistent with H-E equilibrium in the control group and in the implant loss group. There are several causes for deviation from H-E equilibrium, including nonrandom mating, differential survival of marker carriers, genetic drift, population stratification, or a combination of these factors.48,49 A previous study of a European Caucasian population focusing on patients with AgP and the same polymorphisms in the IL4 gene that was investigated here demonstrated a similar lack of H-E equilibrium in the AgP group.49 Those researchers hypothesized that the result of H-E equilibrium might have been obtained because of the selection criteria of patients with AgP. Another explanation for the association of +33 (C/T) SNP with dental implant losses differently to the haplotype finding is related to the investigated polymorphisms in the IL4 gene to form the haplotype. Actually, despite the majority of reports that study the same polymorphisms investigated here, there are other SNPs in the IL4 gene beyond −590 and VNTR that could be investigated as haplotypes, and there is the possibility that another haplotype show significant results regarding dental implant loss.

IMPLANT DENTISTRY / VOLUME 23, NUMBER 6 2014 Previous studies have investigated functional polymorphisms (especially those influencing protein levels) in genes encoding inflammatory molecules, as well as extracellular matrix and bone metabolism mediators. For example, studies regarding IL1,50–52 IL2, IL6,53 IL10,54 TNFA,55 and vitamin D receptor22 showed no association with early dental implant failure. However, significantly higher frequency of the allele 2 for IL1RN gene (coding for IL-1 receptor antagonist) was observed in subjects with multiple implant losses than in the control group.23 This allele has been associated with diminished production of IL-1RA protein (anti-inflammatory cytokine) and IL-1RA/IL-1 ratio with a gene dosage effect.56 Multiple losses were associated with the homozygous genotype 2/2 in IL1RN gene.23 Furthermore, a recent study demonstrated association of risk alleles (minor allele) in the IL1A, IL1B, IL1RN, and TNFA genes with titanium implant loss, considering in the analyses of all these genes.57 This finding supports the hypothesis that genetic variations of inflammatory pathways contribute to the clustering of implant failure in subsets of individuals23 and strengthens the validity of IL1A, IL1B, IL1RN, and TNFA polymorphisms as predictive markers.57 Moreover, bone morphogenetic protein-4 (BMP4) gene polymorphisms were associated with marginal bone loss before second-stage surgery (implant loading).58 In another study, a positive correlation was found between a calcitonin receptor gene polymorphism and marginal bone loss at second-stage surgery.59 Polymorphisms in the promoter region of human matrix metalloproteinase 1 gene (MMP1) was also associated with early implant failure in nonsmokers; patients presenting allele 2G were 3 times more likely to lose dental implants.60 Another study also associated haplotypes formed by polymorphisms in MMP1 gene with dental implant loss.61 This allele was also shown to augment transcriptional activity and can potentially increase the level of protein expression.62 However, a sequence variation in the gelatinase B (MMP9) gene showed no association with early dental implant failure.60 Recently, a study found that the C−799T polymorphism in the promoter

region of MMP8 gene was associated with early implant failure in nonsmokers. Patients bearing C/T genotype or T allele seem to be more likely to have implant loss. This allele can provide the molecular basis for a more intense degradation of extracellular matrix, which might indicate an increased susceptibility to osseointegration failure.21 Multifactorial traits are conceptually defined as resultant from the interaction between several genes and environmental factors.63 A wide variety of multifactorial traits have demonstrated that inflammatory response represents the “common soil” in the etiopathogenic process.64 In this context, together with other immuneinflammatory genes that underlie the host response, IL-4 polymorphisms may have an impact in the modulation of osseointegration.

CONCLUSION For the first time, to our knowledge, the C allele of the +33 (C/T) (rs2070874) polymorphism in the IL4 gene was significantly associated with susceptibility to dental implant loss in the Brazilian population studied.

DISCLOSURE The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.

ACKNOWLEDGMENTS This work was supported in part by the State of São Paulo Foundation Research (FAPESP) Grant 2009/ 09932-8. The studied patients were provided by ILAPEO, Curitiba-PR. The authors report no conflicts of interest related to this study.

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