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Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study

Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by... original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study 1 2 3 Vitória de Oliveira Chami , Livia Nunes , Jonas Capelli Júnior DOI: https://doi.org/10.1590/2177-6709.23.5.041-046.oar Introduction: The search for more aesthetic and comfortable orthodontic devices has led to an increase in the use of clear aligners. Objective: To increase knowledge on biological mechanisms of orthodontic tooth movement using Invisalign align- ers. Methods: This study included 11 patients with a mean age of 23.6 ± 4.8 years. Cases planning included alignment and leveling of lower incisors using Invisalign aligners. Gingival crevicular fluid samples were collected from the lower incisors on the day of delivery of aligner number 1 (T0) and after 1 (T24h), 7 (T7d), and 21 (T21d) days. During the observation period of the study, the patients used only the aligner number 1. Levels of nine cytokines were quantified using Luminex’s multi-analysis technology. Non-parametric tests were used for comparisons between cytokine expression levels over time. Results: Cytokine expression levels remained constant after 21 days of orthodontic activation, except those of MIP-1β, which presented a statistical difference between T24h and T21d with a decrease in the concentration levels. IL-8, GM-CSF, IL-1β, MIP-1β, and TNF-α showed the highest concentrations over time. Conclusions: The different behavior in the levels of the investigated cytokines indicates a role of these biomarkers in the tissue remodeling induced by Invisalign. Keywords: Invisalign. Gingival crevicular fluid. Cytokines. Introdução: a busca por dispositivos ortodônticos mais estéticos e confortáveis gerou um aumento no uso de alinhadores transparentes. Objetivo: ampliar o conhecimento sobre os mecanismos biológicos associados ao movimento dentário orto- dôntico promovido por alinhadores Invisalign . Métodos: a amostra foi constituída por 11 pacientes, com idade média de 23,6 ± 4,8 anos. O planejamento dos casos incluiu alinhamento e nivelamento de incisivos inferiores usando os alinhadores. O fluido gengival crevicular foi coletado na superfície vestibular de incisivos inferiores no dia da entrega do alinhador número 1 (T0) e após 1 (T24h), 7 (T7d) e 21 (T21d) dias. Durante o período de observação do estudo, os pacientes utilizaram apenas o alinhador número 1. Os níveis de nove citocinas foram quantificados por meio do sistema Luminex de multianálise. Testes não paramétricos foram realizados para comparações entre os níveis de expressão de citocinas ao longo do tempo. Resultados: a concentração das citocinas manteve-se constante após 21 dias de ativação ortodôntica, exceto a MIP-1β, que apresentou uma redução estatisticamente significativa entre os tempos T24h e T21d. As IL-8, GM-CSF, IL-1β, MIP-1β e TNF-α apresenta- ram as maiores concentrações ao longo do tempo. Conclusão: a constância na expressão dos níveis das citocinas parece estar compatível com o estímulo mecânico induzido por alinhadores. Palavras-chave: Invisalign. Fluido crevicular gengival. Citocinas. Universidade Federal de Santa Maria, Programa de Pós-Graduação em Ciências How to cite: Chami VO, Nunes L, Capelli Júnior J. Expression of cyto- Odontológicas (Santa Maria/RS, Brazil). kines in gingival crevicular fluid associated with tooth movement induced Universidade do Estado do Rio de Janeiro, Programa de Pós-Graduação em by aligners: a  pilot study. Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6. Odontologia (Rio de Janeiro/RJ, Brazil). DOI: https://doi.org/10.1590/2177-6709.23.5.041-046.oar Universidade do Estado do Rio de Janeiro, Departamento de Odontologia Preventiva e Comunitária (Rio de Janeiro/RJ, Brazil). » Patients displayed in this article previously approved the use of their facial and in- traoral photographs. Submitted: July 19, 2017 - Revised and accepted: February 17, 2018 Contact address: Livia Nunes » The authors report no commercial, proprietary or financial interest in the products Av. 28 de Setembro, 157, sl. 230, Vila Isabel – Rio de Janeiro/RJ or companies described in this article. CEP: 20.551-030 – Email: liviakfn@gmail.com © 2018 Dental Press Journal of Orthodontics 41 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study INTRODUCTION G-(CSF), GM-(CSF), monocyte chemoattractant protein(MCP)-1, macrophage inflammatory protein The mechanical stimulus associated with in- duced tooth movement must be performed (MIP)-1β, and tumor necrosis factor (TNF)-α, in orthodontically moved teeth using aligners. smoothly and continuously, promoting a series of tissue and cell changes in the periodontium, ac- MATERIAL AND METHODS companied by an increased release of inflammatory mediators, such as cytokines. Sample selection These biochemical mediators are low molecu This study sample consisted of 11 patients lar weight proteins that are involved in all phases of (6  women [54.5%] and five men [45.5%]) with a mean age of 23.63 ± 4.88 years, undergoing treat inflammation. Proinflammatory cytokines induce - the classic characteristics of inflammation, through ment at the Or thodontic Clinic of the Dental School vasodilation and invasion of tissues by leukocytes, of Universidade do Estado do Rio de Janeiro(Brazil). while anti-inflammatory cytokines are involved in The inclusion criteria were: full permanent den- the resolution of inflammatory process. tition; Little’s Irregularity Index (Little, 1975) be- The cytokines are characterized by overlapping tween 3 and 5 mm in the anterior segment of the and interlocking functions, and provide informa - lower arch; absence of caries or restorations in the tion on local cell metabolism, reflecting periodontal anterior teeth; and full-mouth plaque score ≤ 30% 2,3 and full-mouth bleeding score ≤ 10%. Patients with health status and bone remodeling. Thereby, the evaluation of this mediators is important to eluci- autoimmune diseases and those who were pregnant or lactating were not eligible. Additionally, partici date the molecular mechanisms that occur during - orthodontic movement. pants exhibiting a prolonged use of medication (e.g., To monitor the biological responses during orth - antibiotics, antihistamines, cortisone, hormones, odontic therapy, gingival crevicular fluid (GCF) anal- and any other medication that may interfere with the inflammatory process or may adversely affect ysis is the method of choice because it is noninvasive and feasible to collect the fluid at different time inter- the periodontium) three months before and during the study period were excluded. This study protocol vals without producing sequelae. In addition, cyto- kine expression in GCF can provide an indirect mea- was approved by the Research Ethics Committee of 4,5 Hospital Universitário Pedro Ernesto/UERJ. A signed in surement of changes in the periodontal ligament. - The Invisalign system induces tooth movement formed consent was obtained from patients’ parents using plastic trays called aligners. In selected cases, prior to the study. these aligners represent an alternative to the tradi- Periodontal control tional metal or ceramic brackets. They produce in- termittent forces on the teeth because they are re- One week before the start of orthodontic treat- movable and their strength levels fluctuate over the ment and during collection times, all patients re- course of treatment. Kuncio et al suggested that ceived oral hygiene instructions to demonstrate the teeth moved with aligners are not subjected to the correct use of brush and floss to control possible t ypical stages of orthodontic movement as described gingival inflammation. by Krishnan and Davidovitch. Dur ing each day of GCF collection, a per iodontal There are only few studies in the orthodon- evaluation was performed. Specifically, the visible plaque index (VPI) and bleeding on probing (BOP) tic literature that have clarified the biological phe- nomena of orthodontic movement using aligners. were recorded of the buccal, lingual, mesial, and distal aspects of the lower incisors, using a manual Thus, the aim of the present study was to detect and quantify nine cytokines: interleukin (IL)-β, periodontal probe, type Goldman-Fox/Williams (IL)-7, (IL)-8, (IL)-17, colony stimulating factors (Hu-Friedy, Chicago, IL, USA). © 2018 Dental Press Journal of Orthodontics 42 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 Chami VO, Nunes L, Capelli Júnior J original article Orthodontic devices the biotinylated antibodies was added before the fi- The Invisalign aligner (Align Technology, San nal washing step. The Luminex analyzer was used Jose, California) was the orthodontic device used to determine the magnitude of the phycoerythrin- in the study. Using the ClinCheck soft ware, patient derived signal in a microparticle-specific manner. planning, including alignment and leveling of the anterior teeth, was performed by a single operator. Statistical analysis The inferior arch was selected to be evaluated con- The Statistical Package for Social Sciences v. 13.0 (SPSS Inc., Chicago, IL, USA) was used for data sidering that the Irregularity Index of lower inci- sors was one of the inclusion criteria. analysis. The normality of the sample was verified using the Shapiro-Wilk test; therefore, non-para During the 21-day observation period of the - study, the patients used only the number 1 align- metr ic tests were selected for analysis. Differences in er. Before insertion of the aligner, patients were the expression levels of each mediator over time were instructed on the use and hygiene of the device. evaluated using the Friedman’s test. When signifi- It should be noted that there was no bonding or in - cant interactions were observed, a Bonferroni-cor- terproximal reduction in the teeth dur ing this st a ge rected Wilcoxon paired signed-rank test was used to of treatment. evaluate the significance of the difference between baseline and the other time points. A p-value of less Immunological monitoring than 0.05 was considered as statistically significant. The GCF samples were collected f rom the vestib- ular face of lower central incisors and lateral incisor (teeth #31, #41, and #42) at different times: on the day of delivery of the aligner (T0) and at 1  (T24h), 7 (T7d), and 21 (T21d) days after orthodontic acti- vation (Fig 1). Prior to GCF collection, the supragingival plaque was carefully removed. The collection sites were isolated using cotton rolls and dried with light air jets. GCF was collected using absorbent paper strips (Periopaper , Interstate Drug Exchange, Ami- tyville, NY, USA), which were inserted 1–2 mm into the gingival sulcus and maintained for 30s. Samples contaminated with blood were discarded, and a new sample was collected a few minutes later from the same site. Paper tips from the same patient were pooled in a sealed Eppendorf plastic tube and stored at -80 C until analysis. Thereafter, the GCF samples were thawed, and biomarkers were detected using Luminex multi-an TM alyte technology (Bio-Plex Pro Human Cytokine Grp I Panel 17-Plex, Catalog no. M50-00031YV, BIO-RAD, Hercules, CA, USA) according to the manufacturer’s instruction. Samples were incubated with antibodies immobilized on color-coded mic- roparticles, washed to remove unbound material, and then incubated with biotinylated antibodies to the molecules of interest. After further washing, the Figure 1 - Samples were collected from the vestibular face of lower central streptavidin-phycoerythrin conjugate that binds to incisors and a lateral incisor (teeth #31, #41, #42). © 2018 Dental Press Journal of Orthodontics 43 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study RESULTS Inflammatory mediators The levels of the different GCF biomarkers are Periodontal During the study period and GCF collection, all summarized in Table 1. patients exhibited a clinically satisfactory oral hygiene. All cytokines, IL-1β, IL-7, IL-8, IL-17, G-CSF, Data collected for the BOP showed values below 30% GM-CSF, MCP-1, MI P-1β, and TNF-α were iden- of the evaluated sites and VPI below 10% (Fig 2). In tified and quantified at all times. The levels of all addition, the evaluated indices decreased over time. mediators, except MIP-1β, were similar over time with no significant change. In particular, decreases over time were seen for all these GCF biomarkers. At the pairwise comparisons, the level of MIP-1β was significantly lower at 21 days as compared to the 24 hours (p <0.05). IL-17, MCP-1, MIP-1β and TNF-α showed greater levels at 24 hours as com- pared to the corresponding baseline scores. DISCUSSION Invisalign aligners are an alternative treatment that provides greater aesthetics and more comfort for orthodontic patients. Thus, the demand for this type of device has significantly increased in the re- cent years; however, studies related to the biology of VPI BOP movement using aligners are scarce. When assessing cytokine concentration levels dur- ing the study period, all mediators, except MIP-1β, showed fluctuations over time, with no statistically significant differences. MIP-1β, which is considered a chemoattractant Figure 2 - Descriptive analysis of the percentage of sites with visible plaque cytokine, is related to the activation and recr uitment (VPI) and gingival bleeding (BOP). of monocyte/macrophage cell lines. Furthermore, Table 1 - Total amount of cytokines throughout the collection times. T0 T24h T7d T21d Cytokines P interquartile interquartile interquartile interquartile Median Median Median Median range range range range IL-1β 15.23 27.03 11.91 32.46 14.15 23.56 8.55 11.13 0.654 IL-7 0.18 0.63 0.0 0.89 0.34 0.34 0.34 0.34 0.992 IL-8 151.44 249.52 137.16 302.48 85.46 115.37 104.64 48.03 0.138 IL-17 1.29 2.66 2.59 2.61 2.59 2.84 1.91 2.16 0.405 G-CSF 2.05 4.28 2.05 3.99 2.36 4.07 3.28 3.37 0.504 GM-CSF 60.84 15.79 58.69 22.52 56.53 11.98 59.64 30.74 0.921 MCP-1 0.0 1.42 1.04 1.42 0.84 1.04 0.61 1.04 0.981 MIP-1β 10.03 17.77 12.95 13.79 15.45 10.29 10.03 5.44 0.020* TNF-α 0.78 3.47 1.44 2.22 1.44 3.47 0.78 3.00 0.625 Data are presented as median and interquartile deviations (pg). p: differences over time, p ≤ 0.05 represents a statistical difference. * significant difference between T24h and T21d. © 2018 Dental Press Journal of Orthodontics 44 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 Chami VO, Nunes L, Capelli Júnior J original article this chemokine may play an important role in host levels of IL-17 over the observation period, similar to a previous study that applied intrusive forces on response by recruiting inflammatory cells into ac- tive inflammation and inducing the release of other the premolar teeth wherein no statistically signifi- 10 20 cellular mediators. Proinflammatory cytokines, cant changes in IL-17 levels were obser ved. such as IL-1, IL-6, and TNF-α, and resident cells, Despite its importance in bone remodeling as a pro- such as fibroblasts and osteoblasts, appear to induce inflammatory cytokine, the production of GM-CSF dur - chemokines such as MIP-1β. Thus, the role of ing orthodontic movement remains unclear. In this study, chemokines in orthodontic tooth movement may fluctuations in results were observed with no statistically be associated with the fact that these mediators are significant differences, which is consistent with the re - involved in the migration of monocytes to the peri - sults of a study conducted on adult patients using forces odontal ligament, where they will differentiate into for the distalization of canines. However, in the ado- osteoclasts and macrophages, essential cells for orth- lescent patients of this same study, concentration levels odontic movement. were higher in 24h; which was also identified after 4 h In this study, the expression levels of MIP-1β in a study using forces for canine distalization. In  an- were statistically different at T24h and T21d. This other study, GM-CSF could not be evaluated because its 12 22 finding is contrary to that of Capelli et al, who expression was below detection levels. showed a consistency in the concentration of this MCP-1 has been described as chemokine that stimu- cytokine, without statistically significant differ - lates osteoclast formation and bone resorption. The pres- ences, during the distalization of canines with re- ent study observed no changes in its levels over time, and 12,16 traction loops. Thus, the greater level of MIP-1β this was in agreement with previous studies . at T24h would be consistent with the release of in- IL-7 mediator was identified at low expression levels at all times and there are no human studies in flammatory markers in early hours after a exposure to orthodontic forces. literature that focus on the response of IL-7 to orth- The proinflammatory cytokines, such as TNF-α, odontic treatment. IL-1β and IL-8, are related to the acute phase of in- Despite the possible influence of periodontal in- flammatory response associated with tooth move- flammation on the results of cytokine analysis in the ment; therefore, their expression levels are increased GCF, since there is a possibility of increased secre- during the initial hours after orthodontic force ap- tion due to periodontal plaque related inflamma- plication. This increase in TNF-α and IL-8 ex- tion; the periodontal indices in the present study pression levels was observed in the first 24 h after remained low and presented a decrease over the the aligner was installed. These results are consistent studied time. with those of a previous study that used conventional A possible explanation for the present findings orthodontic appliances, with the insertion of NiTi relates to the type of device used in this study: it is wires and mechanical distal movement of canine be possible to remove the device for oral hygiene. In ad - - 1,14-17 fore or after dental alignment and leveling. dition, the patients were submitted to oral hygiene In contrast, IL-1β expression levels showed fluc- control by providing them with brushing instruc- tuations over time, which is consistent with the tions throughout the study per iod. Thus, the chang- findings by Iwasaki et al, where a peak concentra- es obser ved are strongly associated with stimulation tion of this cytokine was not characterized within of tooth movement. In this study, concentration levels of inflamma the first 24 h. Castrofolio et al demonstrated that - a peak concentration of IL-1β occurred 21 d after tory mediators were analyzed and longitudinally orthodontic activation with Invisalign. identified in a single sample using immunoenzymat- IL-17, considered a pro-inflammatory cyto- ic multi-analysis assay with microspheres. This  as- kine, acts synergistically with other cytokines, such say has the abilit y to simultaneously measure a large as IL-1, IL-6, TNF-α. In this study, the results number of targets with a small sample, thereby re- showed no significant changes in the concentration sulting in higher yields and lower costs. © 2018 Dental Press Journal of Orthodontics 45 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study Considering the wide range of biological re- CONCLUSIONS sponses and factors interfering with GCF, the re- When using aligners (Invisalign), cytokine ex- duced number of participants may be considered a pression levels involved in cell recruitment indicates limitation of the present study. However, this study a role of these biomarkers in the tissue remodeling analyzed a total of 180 sites and a large number of induced by light forces. Thus, further studies are 1,12-17,19-20,22 previous studies used similar sample sizes. needed to increase the knowledge regarding inter Another important limitation was that GCF is a ferences and biological influences during orthodon- transudate that is used to remotely study phenomena tic treatment with Invisalign. 1,21,24 that occur in the periodontal ligament. Thus, these results should be interpreted with caution. Finally, the present results warrant further inves- tigations to clarify the biological mechanisms be- hind orthodontic aligners. REFERENCES 1. Başaran G, Ozer T, Kaya FA, Hamamci O. Interleukins 2, 6, and 8 levels 14. Başaran G, Ozer T, Kaya FA, Kaplan A, Hamamci O. Interleukine-1beta and in human gingival sulcus during orthodontic treatment. Am J Orthod tumor necrosis factor-alpha levels in the human gingival sulcus during Dentofacial Orthop. 2006 July;130(1):7.e1-6. orthodontic treatment. Angle Orthod. 2006 Sept;76(5):830-6. 2. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue level reactions to 15. Karacay S, Saygun I, Bengi AO, Serdar M. Tumor necrosis factor-alpha levels orthodontic force. Am J Orthod Dentofacial Orthop. 2006 Apr;129(4):469. during two different canine distalization techniques. Angle Orthod. 2007 e1-32. Jan;77(1):142-47. 3. Krishnan V, Nair A, Ranjit A, Davidovitch Z. Research in tooth movement 16. Alikhani M, Raptis M, Zoldan B, Sangsuwon C, Lee YB, Alyami B, et al. Effect biology: the current status. Semin Orthod. 2012 Dec;18(4):308-16. of micro-osteoperforations on the rate of tooth movement. Am J Orthod 4. Perinetti G, Primozic J, Castaldo A, Di Lenarda R, Contardo L. Is gingival Dentofacial Orthop. 2013 Nov;144(5):639-48. crevicular fluid volume sensitive to orthodontic tooth movement? 17. Grant M, Wilson J, Rock P, Chapple I. Induction of cytokines, MMP9, TIMPs, A systematic review of split-mouth longitudinal studies. Orthod Craniofac RANKL and OPG during orthodontic tooth movement. Eur J Orthod. 2013 Res. 2013 Feb;16(1):1-19. Oct;35(5):644-51. 5. Capelli J Jr, Fidel Jr R, Figueredo CM, Teles RP. Change in the gingival fluid 18. Iwasaki LR, Chandler JR, Marx DB, Pandey JP, Nickel JC. IL-1 gene volume during maxillary canine retraction. Dental Press J Orthod. 2010 Mar- polymorphisms, secretion in gingival crevicular fluid, and speed of Abr;15(2):52-7. human orthodontic tooth movement. Orthod Craniofac Res. 2009 6. Site Invisalign Brasil. 2016 [Acesso em: 1 Mar 2016]. Disponível em: http:// May;12(2):129-40. doutor.invisalign.com.br/sobre. 19. Castroflorio T, Gamerro EF, Caviglia GP, Deregibus A. Biochemical markers 7. Kuncio D, Maganzini A, Shelton C, Freeman K. Invisalign and traditional of bone metabolism during early orthodontic tooth movement with aligners. orthodontic treatment postretention outcomes compared using the Angle Orthod. 2017 Jan;87(1):74-81. American Board of Orthodontics Objective Grading System. Angle Orthod. 20. Madureira DF, Silva JM, Teixeira AL, Abreu MH, Pretti H, Lages EM, et 2007 Sept;77(5):864-9. al. Cytokine measurements in gingival crevicular fluid and periodontal 8. Little RM. The irregularity index: a quantitative score of mandibular anterior ligament: are they correlated? Am J Orthod Dentofacial Orthop. 2015 alignment. Am J Orthod. 1975 Nov;68(5):554-63. Aug;148(2):293-301. 9. Rossini G, Parrini S, Castroflorio T, Deregibus A, Debernardi CL. Efficacy 21. Ren Y, Maltha JC, Van’t Hof MA, Von Den Hoff JW, Kuijpers-Jagtman AM, of clear aligners in controlling orthodontic tooth movement: a systematic Zhang D. Cytokine levels in crevicular fluid are less responsive to orthodontic review. Angle Orthod. 2015 Sept;85(5):881-9. force in adults than in juveniles. J Clin Periodontol. 2002 Aug;29(8):757-62. 10. Emingil G, Atilla G, Başkesen A, Berdeli A. Gingival crevicular fluid EMAP-II, 22. Van Gastel J, Teughels W, Quirynen M, Struyf S, Van Damme J, Coucke W, MIP-1alpha and MIP-1beta levels of patients with periodontal disease. J Clin et al. Longitudinal changes in gingival crevicular fluid after placement Periodontol. 2005 Aug;32(8):880-5. of fixed orthodontic appliances. Am J Orthod Dentofacial Orthop. 2011 11. Alhasimi N, Frithiof L, Brudvik P, Bakhiet M. Chemokines are upregulated June;139(6):735-44. during orthodontic tooth movement. J Interferon Cytokine Res. 1999 23. Khalifian S, Raimondi G, Brandacher G. The use of Luminex assays to Sept;19(9):1047-52. measure cytokines. J Invest Dermatol. 2015 Apr;135(4):1-5. 12. Capelli J Jr, Kantarci A, Haffajee A, Teles RP, Fidel R Jr, Figueredo CM. Matrix 24. Perinetti G, Primožič J, Castaldo A, Di Lenarda R, Contardo L. Is gingival metalloproteinases and chemokines in the gingival crevicular fluid during crevicular fluid volume sensitive to orthodontic tooth movement? orthodontic tooth movement. Eur J Orthod. 2011 Dec;33(6):705-11. A systematic review of split-mouth longitudinal studies. Orthod Craniofac 13. Nunes L, Quintanilha L, Perinetti G, Capelli J Jr. Effect of orthodontic force Res. 2013 Feb;16(1):1-19. on expression levels of ten cytokines in gingival crevicular fluid. Arch Oral Biol. 2017 Apr;76:70-5. © 2018 Dental Press Journal of Orthodontics 46 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Dental Press Journal of Orthodontics Unpaywall

Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study

Dental Press Journal of OrthodonticsOct 1, 2018

Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study

Abstract

original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study 1 2 3 Vitória de Oliveira Chami , Livia Nunes , Jonas Capelli Júnior DOI: https://doi.org/10.1590/2177-6709.23.5.041-046.oar Introduction: The search for more aesthetic and comfortable orthodontic devices has led to an increase in the use of clear aligners. Objective: To increase knowledge on biological mechanisms of orthodontic tooth movement using Invisalign align- ers. Methods: This study included 11 patients with a mean age of 23.6 ± 4.8 years. Cases planning included alignment and leveling of lower incisors using Invisalign aligners. Gingival crevicular fluid samples were collected from the lower incisors on the day of delivery of aligner number 1 (T0) and after 1 (T24h), 7 (T7d), and 21 (T21d) days. During the observation period of the study, the patients used only the aligner number 1. Levels of nine cytokines were quantified using Luminex’s multi-analysis technology. Non-parametric tests were used for comparisons between cytokine expression levels over time. Results: Cytokine expression levels remained constant after 21 days of orthodontic activation, except those of MIP-1β, which presented a statistical difference between T24h and T21d with a decrease in the concentration levels. IL-8, GM-CSF, IL-1β,

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original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study 1 2 3 Vitória de Oliveira Chami , Livia Nunes , Jonas Capelli Júnior DOI: https://doi.org/10.1590/2177-6709.23.5.041-046.oar Introduction: The search for more aesthetic and comfortable orthodontic devices has led to an increase in the use of clear aligners. Objective: To increase knowledge on biological mechanisms of orthodontic tooth movement using Invisalign align- ers. Methods: This study included 11 patients with a mean age of 23.6 ± 4.8 years. Cases planning included alignment and leveling of lower incisors using Invisalign aligners. Gingival crevicular fluid samples were collected from the lower incisors on the day of delivery of aligner number 1 (T0) and after 1 (T24h), 7 (T7d), and 21 (T21d) days. During the observation period of the study, the patients used only the aligner number 1. Levels of nine cytokines were quantified using Luminex’s multi-analysis technology. Non-parametric tests were used for comparisons between cytokine expression levels over time. Results: Cytokine expression levels remained constant after 21 days of orthodontic activation, except those of MIP-1β, which presented a statistical difference between T24h and T21d with a decrease in the concentration levels. IL-8, GM-CSF, IL-1β, MIP-1β, and TNF-α showed the highest concentrations over time. Conclusions: The different behavior in the levels of the investigated cytokines indicates a role of these biomarkers in the tissue remodeling induced by Invisalign. Keywords: Invisalign. Gingival crevicular fluid. Cytokines. Introdução: a busca por dispositivos ortodônticos mais estéticos e confortáveis gerou um aumento no uso de alinhadores transparentes. Objetivo: ampliar o conhecimento sobre os mecanismos biológicos associados ao movimento dentário orto- dôntico promovido por alinhadores Invisalign . Métodos: a amostra foi constituída por 11 pacientes, com idade média de 23,6 ± 4,8 anos. O planejamento dos casos incluiu alinhamento e nivelamento de incisivos inferiores usando os alinhadores. O fluido gengival crevicular foi coletado na superfície vestibular de incisivos inferiores no dia da entrega do alinhador número 1 (T0) e após 1 (T24h), 7 (T7d) e 21 (T21d) dias. Durante o período de observação do estudo, os pacientes utilizaram apenas o alinhador número 1. Os níveis de nove citocinas foram quantificados por meio do sistema Luminex de multianálise. Testes não paramétricos foram realizados para comparações entre os níveis de expressão de citocinas ao longo do tempo. Resultados: a concentração das citocinas manteve-se constante após 21 dias de ativação ortodôntica, exceto a MIP-1β, que apresentou uma redução estatisticamente significativa entre os tempos T24h e T21d. As IL-8, GM-CSF, IL-1β, MIP-1β e TNF-α apresenta- ram as maiores concentrações ao longo do tempo. Conclusão: a constância na expressão dos níveis das citocinas parece estar compatível com o estímulo mecânico induzido por alinhadores. Palavras-chave: Invisalign. Fluido crevicular gengival. Citocinas. Universidade Federal de Santa Maria, Programa de Pós-Graduação em Ciências How to cite: Chami VO, Nunes L, Capelli Júnior J. Expression of cyto- Odontológicas (Santa Maria/RS, Brazil). kines in gingival crevicular fluid associated with tooth movement induced Universidade do Estado do Rio de Janeiro, Programa de Pós-Graduação em by aligners: a  pilot study. Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6. Odontologia (Rio de Janeiro/RJ, Brazil). DOI: https://doi.org/10.1590/2177-6709.23.5.041-046.oar Universidade do Estado do Rio de Janeiro, Departamento de Odontologia Preventiva e Comunitária (Rio de Janeiro/RJ, Brazil). » Patients displayed in this article previously approved the use of their facial and in- traoral photographs. Submitted: July 19, 2017 - Revised and accepted: February 17, 2018 Contact address: Livia Nunes » The authors report no commercial, proprietary or financial interest in the products Av. 28 de Setembro, 157, sl. 230, Vila Isabel – Rio de Janeiro/RJ or companies described in this article. CEP: 20.551-030 – Email: liviakfn@gmail.com © 2018 Dental Press Journal of Orthodontics 41 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study INTRODUCTION G-(CSF), GM-(CSF), monocyte chemoattractant protein(MCP)-1, macrophage inflammatory protein The mechanical stimulus associated with in- duced tooth movement must be performed (MIP)-1β, and tumor necrosis factor (TNF)-α, in orthodontically moved teeth using aligners. smoothly and continuously, promoting a series of tissue and cell changes in the periodontium, ac- MATERIAL AND METHODS companied by an increased release of inflammatory mediators, such as cytokines. Sample selection These biochemical mediators are low molecu This study sample consisted of 11 patients lar weight proteins that are involved in all phases of (6  women [54.5%] and five men [45.5%]) with a mean age of 23.63 ± 4.88 years, undergoing treat inflammation. Proinflammatory cytokines induce - the classic characteristics of inflammation, through ment at the Or thodontic Clinic of the Dental School vasodilation and invasion of tissues by leukocytes, of Universidade do Estado do Rio de Janeiro(Brazil). while anti-inflammatory cytokines are involved in The inclusion criteria were: full permanent den- the resolution of inflammatory process. tition; Little’s Irregularity Index (Little, 1975) be- The cytokines are characterized by overlapping tween 3 and 5 mm in the anterior segment of the and interlocking functions, and provide informa - lower arch; absence of caries or restorations in the tion on local cell metabolism, reflecting periodontal anterior teeth; and full-mouth plaque score ≤ 30% 2,3 and full-mouth bleeding score ≤ 10%. Patients with health status and bone remodeling. Thereby, the evaluation of this mediators is important to eluci- autoimmune diseases and those who were pregnant or lactating were not eligible. Additionally, partici date the molecular mechanisms that occur during - orthodontic movement. pants exhibiting a prolonged use of medication (e.g., To monitor the biological responses during orth - antibiotics, antihistamines, cortisone, hormones, odontic therapy, gingival crevicular fluid (GCF) anal- and any other medication that may interfere with the inflammatory process or may adversely affect ysis is the method of choice because it is noninvasive and feasible to collect the fluid at different time inter- the periodontium) three months before and during the study period were excluded. This study protocol vals without producing sequelae. In addition, cyto- kine expression in GCF can provide an indirect mea- was approved by the Research Ethics Committee of 4,5 Hospital Universitário Pedro Ernesto/UERJ. A signed in surement of changes in the periodontal ligament. - The Invisalign system induces tooth movement formed consent was obtained from patients’ parents using plastic trays called aligners. In selected cases, prior to the study. these aligners represent an alternative to the tradi- Periodontal control tional metal or ceramic brackets. They produce in- termittent forces on the teeth because they are re- One week before the start of orthodontic treat- movable and their strength levels fluctuate over the ment and during collection times, all patients re- course of treatment. Kuncio et al suggested that ceived oral hygiene instructions to demonstrate the teeth moved with aligners are not subjected to the correct use of brush and floss to control possible t ypical stages of orthodontic movement as described gingival inflammation. by Krishnan and Davidovitch. Dur ing each day of GCF collection, a per iodontal There are only few studies in the orthodon- evaluation was performed. Specifically, the visible plaque index (VPI) and bleeding on probing (BOP) tic literature that have clarified the biological phe- nomena of orthodontic movement using aligners. were recorded of the buccal, lingual, mesial, and distal aspects of the lower incisors, using a manual Thus, the aim of the present study was to detect and quantify nine cytokines: interleukin (IL)-β, periodontal probe, type Goldman-Fox/Williams (IL)-7, (IL)-8, (IL)-17, colony stimulating factors (Hu-Friedy, Chicago, IL, USA). © 2018 Dental Press Journal of Orthodontics 42 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 Chami VO, Nunes L, Capelli Júnior J original article Orthodontic devices the biotinylated antibodies was added before the fi- The Invisalign aligner (Align Technology, San nal washing step. The Luminex analyzer was used Jose, California) was the orthodontic device used to determine the magnitude of the phycoerythrin- in the study. Using the ClinCheck soft ware, patient derived signal in a microparticle-specific manner. planning, including alignment and leveling of the anterior teeth, was performed by a single operator. Statistical analysis The inferior arch was selected to be evaluated con- The Statistical Package for Social Sciences v. 13.0 (SPSS Inc., Chicago, IL, USA) was used for data sidering that the Irregularity Index of lower inci- sors was one of the inclusion criteria. analysis. The normality of the sample was verified using the Shapiro-Wilk test; therefore, non-para During the 21-day observation period of the - study, the patients used only the number 1 align- metr ic tests were selected for analysis. Differences in er. Before insertion of the aligner, patients were the expression levels of each mediator over time were instructed on the use and hygiene of the device. evaluated using the Friedman’s test. When signifi- It should be noted that there was no bonding or in - cant interactions were observed, a Bonferroni-cor- terproximal reduction in the teeth dur ing this st a ge rected Wilcoxon paired signed-rank test was used to of treatment. evaluate the significance of the difference between baseline and the other time points. A p-value of less Immunological monitoring than 0.05 was considered as statistically significant. The GCF samples were collected f rom the vestib- ular face of lower central incisors and lateral incisor (teeth #31, #41, and #42) at different times: on the day of delivery of the aligner (T0) and at 1  (T24h), 7 (T7d), and 21 (T21d) days after orthodontic acti- vation (Fig 1). Prior to GCF collection, the supragingival plaque was carefully removed. The collection sites were isolated using cotton rolls and dried with light air jets. GCF was collected using absorbent paper strips (Periopaper , Interstate Drug Exchange, Ami- tyville, NY, USA), which were inserted 1–2 mm into the gingival sulcus and maintained for 30s. Samples contaminated with blood were discarded, and a new sample was collected a few minutes later from the same site. Paper tips from the same patient were pooled in a sealed Eppendorf plastic tube and stored at -80 C until analysis. Thereafter, the GCF samples were thawed, and biomarkers were detected using Luminex multi-an TM alyte technology (Bio-Plex Pro Human Cytokine Grp I Panel 17-Plex, Catalog no. M50-00031YV, BIO-RAD, Hercules, CA, USA) according to the manufacturer’s instruction. Samples were incubated with antibodies immobilized on color-coded mic- roparticles, washed to remove unbound material, and then incubated with biotinylated antibodies to the molecules of interest. After further washing, the Figure 1 - Samples were collected from the vestibular face of lower central streptavidin-phycoerythrin conjugate that binds to incisors and a lateral incisor (teeth #31, #41, #42). © 2018 Dental Press Journal of Orthodontics 43 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study RESULTS Inflammatory mediators The levels of the different GCF biomarkers are Periodontal During the study period and GCF collection, all summarized in Table 1. patients exhibited a clinically satisfactory oral hygiene. All cytokines, IL-1β, IL-7, IL-8, IL-17, G-CSF, Data collected for the BOP showed values below 30% GM-CSF, MCP-1, MI P-1β, and TNF-α were iden- of the evaluated sites and VPI below 10% (Fig 2). In tified and quantified at all times. The levels of all addition, the evaluated indices decreased over time. mediators, except MIP-1β, were similar over time with no significant change. In particular, decreases over time were seen for all these GCF biomarkers. At the pairwise comparisons, the level of MIP-1β was significantly lower at 21 days as compared to the 24 hours (p <0.05). IL-17, MCP-1, MIP-1β and TNF-α showed greater levels at 24 hours as com- pared to the corresponding baseline scores. DISCUSSION Invisalign aligners are an alternative treatment that provides greater aesthetics and more comfort for orthodontic patients. Thus, the demand for this type of device has significantly increased in the re- cent years; however, studies related to the biology of VPI BOP movement using aligners are scarce. When assessing cytokine concentration levels dur- ing the study period, all mediators, except MIP-1β, showed fluctuations over time, with no statistically significant differences. MIP-1β, which is considered a chemoattractant Figure 2 - Descriptive analysis of the percentage of sites with visible plaque cytokine, is related to the activation and recr uitment (VPI) and gingival bleeding (BOP). of monocyte/macrophage cell lines. Furthermore, Table 1 - Total amount of cytokines throughout the collection times. T0 T24h T7d T21d Cytokines P interquartile interquartile interquartile interquartile Median Median Median Median range range range range IL-1β 15.23 27.03 11.91 32.46 14.15 23.56 8.55 11.13 0.654 IL-7 0.18 0.63 0.0 0.89 0.34 0.34 0.34 0.34 0.992 IL-8 151.44 249.52 137.16 302.48 85.46 115.37 104.64 48.03 0.138 IL-17 1.29 2.66 2.59 2.61 2.59 2.84 1.91 2.16 0.405 G-CSF 2.05 4.28 2.05 3.99 2.36 4.07 3.28 3.37 0.504 GM-CSF 60.84 15.79 58.69 22.52 56.53 11.98 59.64 30.74 0.921 MCP-1 0.0 1.42 1.04 1.42 0.84 1.04 0.61 1.04 0.981 MIP-1β 10.03 17.77 12.95 13.79 15.45 10.29 10.03 5.44 0.020* TNF-α 0.78 3.47 1.44 2.22 1.44 3.47 0.78 3.00 0.625 Data are presented as median and interquartile deviations (pg). p: differences over time, p ≤ 0.05 represents a statistical difference. * significant difference between T24h and T21d. © 2018 Dental Press Journal of Orthodontics 44 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 Chami VO, Nunes L, Capelli Júnior J original article this chemokine may play an important role in host levels of IL-17 over the observation period, similar to a previous study that applied intrusive forces on response by recruiting inflammatory cells into ac- tive inflammation and inducing the release of other the premolar teeth wherein no statistically signifi- 10 20 cellular mediators. Proinflammatory cytokines, cant changes in IL-17 levels were obser ved. such as IL-1, IL-6, and TNF-α, and resident cells, Despite its importance in bone remodeling as a pro- such as fibroblasts and osteoblasts, appear to induce inflammatory cytokine, the production of GM-CSF dur - chemokines such as MIP-1β. Thus, the role of ing orthodontic movement remains unclear. In this study, chemokines in orthodontic tooth movement may fluctuations in results were observed with no statistically be associated with the fact that these mediators are significant differences, which is consistent with the re - involved in the migration of monocytes to the peri - sults of a study conducted on adult patients using forces odontal ligament, where they will differentiate into for the distalization of canines. However, in the ado- osteoclasts and macrophages, essential cells for orth- lescent patients of this same study, concentration levels odontic movement. were higher in 24h; which was also identified after 4 h In this study, the expression levels of MIP-1β in a study using forces for canine distalization. In  an- were statistically different at T24h and T21d. This other study, GM-CSF could not be evaluated because its 12 22 finding is contrary to that of Capelli et al, who expression was below detection levels. showed a consistency in the concentration of this MCP-1 has been described as chemokine that stimu- cytokine, without statistically significant differ - lates osteoclast formation and bone resorption. The pres- ences, during the distalization of canines with re- ent study observed no changes in its levels over time, and 12,16 traction loops. Thus, the greater level of MIP-1β this was in agreement with previous studies . at T24h would be consistent with the release of in- IL-7 mediator was identified at low expression levels at all times and there are no human studies in flammatory markers in early hours after a exposure to orthodontic forces. literature that focus on the response of IL-7 to orth- The proinflammatory cytokines, such as TNF-α, odontic treatment. IL-1β and IL-8, are related to the acute phase of in- Despite the possible influence of periodontal in- flammatory response associated with tooth move- flammation on the results of cytokine analysis in the ment; therefore, their expression levels are increased GCF, since there is a possibility of increased secre- during the initial hours after orthodontic force ap- tion due to periodontal plaque related inflamma- plication. This increase in TNF-α and IL-8 ex- tion; the periodontal indices in the present study pression levels was observed in the first 24 h after remained low and presented a decrease over the the aligner was installed. These results are consistent studied time. with those of a previous study that used conventional A possible explanation for the present findings orthodontic appliances, with the insertion of NiTi relates to the type of device used in this study: it is wires and mechanical distal movement of canine be possible to remove the device for oral hygiene. In ad - - 1,14-17 fore or after dental alignment and leveling. dition, the patients were submitted to oral hygiene In contrast, IL-1β expression levels showed fluc- control by providing them with brushing instruc- tuations over time, which is consistent with the tions throughout the study per iod. Thus, the chang- findings by Iwasaki et al, where a peak concentra- es obser ved are strongly associated with stimulation tion of this cytokine was not characterized within of tooth movement. In this study, concentration levels of inflamma the first 24 h. Castrofolio et al demonstrated that - a peak concentration of IL-1β occurred 21 d after tory mediators were analyzed and longitudinally orthodontic activation with Invisalign. identified in a single sample using immunoenzymat- IL-17, considered a pro-inflammatory cyto- ic multi-analysis assay with microspheres. This  as- kine, acts synergistically with other cytokines, such say has the abilit y to simultaneously measure a large as IL-1, IL-6, TNF-α. In this study, the results number of targets with a small sample, thereby re- showed no significant changes in the concentration sulting in higher yields and lower costs. © 2018 Dental Press Journal of Orthodontics 45 Dental Press J Orthod. 2018 Sept-Oct;23(5):41-6 original article Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study Considering the wide range of biological re- CONCLUSIONS sponses and factors interfering with GCF, the re- When using aligners (Invisalign), cytokine ex- duced number of participants may be considered a pression levels involved in cell recruitment indicates limitation of the present study. However, this study a role of these biomarkers in the tissue remodeling analyzed a total of 180 sites and a large number of induced by light forces. 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