Collection, Storage and Protein Extraction Method of Gingival Crevicular Fluid for Proteomic Analysis

Main Article Content

Harraa S. Mohammed-Salih
Hayder F. Saloom

Abstract

Gingival crevicular fluid (GCF) may reflect the events associated with orthodontic tooth movement. Attempts have been conducted to identify biomarkers reflecting optimum orthodontic force, unwanted sequallea (i.e. root resorption) and accelerated tooth movement. The aim of the present study is to find out a standardized GCF collection, storage and total protein extraction method from apparently healthy gingival sites with orthodontics that is compatible with further high-throughput proteomics. Eighteen patients who required extractions of both maxillary first premolars were recruited in this study. These teeth were randomly assigned to either heavy (225g) or light force (25g), and their site specific GCF was collected at baseline and after 1hr, 1day, 7days, 14days, 21days and 28days post force application. Periostrips were used for GCF collection and subsequent phosphate buffered saline (PBS) was used for immediate protein elution with centrifugal speed of 10000rpm for 5min and stored at -80°C. Protein concentration was estimated using Bradford colorimetric assay. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to resolve the purity of proteins in the collected samples and the method of collection was validated by western immuno-blotting of alpha amylase salivary enzyme. The current collection, storage and protein extraction protocol showed the best protein recovery and purity with validated collection free of salivary contamination. In conclusion, tiny GCF volume from healthy sites and evaporation issues of such promising non-invasive fluid motivate us to investigate a standardized protocol enabling optimal preservation of GCF sample and the currently followed protocol may serve as a reference for future proteomic studies searching for GCF biomarkers in diagnosing and monitoring orthodontic tooth movement.

Downloads

Download data is not yet available.

Article Details

How to Cite
1.
Mohammed-Salih HS, Saloom HF. Collection, Storage and Protein Extraction Method of Gingival Crevicular Fluid for Proteomic Analysis. Baghdad Sci.J [Internet]. [cited 2021Dec.4];19(2):0368. Available from: https://bsj.uobaghdad.edu.iq/index.php/BSJ/article/view/5664
Section
article

References

1. D’Apuzzo F, Cappabianca S, Ciavarella D, Monsurrò A, Silvestrini-Biavati A, Perillo L. Biomarkers of periodontal tissue remodeling during orthodontic tooth movement in mice and men: Overview and clinical relevance. Sci World J. 2013;2013:1–9.
2. Currell SD, Liaw A, Blackmore Grant PD, Esterman A, Nimmo A. Orthodontic mechanotherapies and their influence on external root resorption: A systematic review. Am J Orthod Dentofac Orthop. 2019;155(3):313–29. Available from: https://doi.org/10.1016/j.ajodo.2018.10.015
3. Bostanci N, Belibasakis GN. Gingival crevicular fluid and its immune mediators in the proteomic era. Periodontol 2000. 2018;76(1):68–84.
4. Khurshid Z, Zohaib S, Najeeb S, Zafar MS, Rehman R, Ur Rehman I. Advances of proteomic sciences in dentistry. Int J Mol Sci. 2016;17(5):1–19.
5. Tsuchida S, Satoh M, Takiwaki M, Nomura F. Current status of proteomic technologies for discovering and identifying gingival crevicular fluid biomarkers for periodontal disease. Int J Mol Sci. 2019;20(1):1–11.
6. Preianò M, Falcone D, Maggisano G, Montalcini T, Navarra M, Paduano S, et al. Assessment of pre-analytical and analytical variables affecting peptidome profiling of gingival crevicular fluid by MALDI-TOF mass spectrometry. Clin Chim Acta. 2014;437:120–8. Available from: http://dx.doi.org/10.1016/j.cca.2014.07.022
7. Preianò M, Maggisano G, Murfuni MS, Villella C, Pelaia C, Montalcini T, et al. An Analytical Method for Assessing Optimal Storage Conditions of Gingival Crevicular Fluid and Disclosing a Peptide Biomarker Signature of Gingivitis by MALDI-TOF MS. Proteomics - Clin Appl. 2018;12(5):1–32.
8. Meyer UA, Zanger UM, Schwab M. Omics and Drug Response. Annu Rev Pharmacol Toxicol. 2013;53(1):475–502.
9. Cupp-Sutton KA, Wu S. High-throughput quantitative top-down proteomics. Mol Omi. 2020;16(2):91–9.
10. Vieira GM. Protein biomarkers of external root resorption: A new protein extraction protocol. Are we going in the right direction? Dental Press J Orthod. 2014;19(6):62–9.
11. Saloom H. The influence of obesity on orthodontic tooth movement (A clinical study) [Internet]. King’s College London Dental Institute January; 2017. Available from: https://kclpure.kcl.ac.uk/portal/
12. Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nat Publ Gr. 1970;228:726–34. Available from: http://www.mendeley.com/research/discreteness-conductance-chnge-n-bimolecular-lipid-membrane-presence-certin-antibiotics/
13. Yan JX, Wait R, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption / ionization and electrospray ionization- mass spectrometry. Electrophoresis. 2000;1:3666–72.
14. Helmerhorst EJ. Protective functions of saliva. In: Edgar WM, Dawes C, O’Mullane DM, editors. Saliva and oral health. UK: Stephen Hancocks Limited; 2012.
15. Ghallab NA. Diagnostic potential and future directions of biomarkers in gingival crevicular fluid and saliva of periodontal diseases: Review of the current evidence. Arch Oral Biol. 2018;87:115–24. Available from: https://doi.org/10.1016/j.archoralbio.2017.12.022
16. Kapoor P, Monga N, Kharbanda OP, Kapila S, Miglani R, Moganty R. Effect of orthodontic forces on levels of enzymes in gingival crevicular fluid (GCF): A systematic review. Dental Press J Orthod. 2019;24(2):40.e1-40.e22.
17. Tarallo F, Chimenti C, Paiella G, Cordaro M, Tepedino M. Biomarkers in the gingival crevicular fluid used to detect root resorption in patients undergoing orthodontic treatment: A systematic review. Orthod Craniofacial Res. 2019;22(4):236–47.
18. Goodson J. Gingival crevice fluid flow. Periodontol 2000. 2003;31:43–54.
19. Griffiths GS. Formation, collection and significance of gingival crevice fluid. Periodontol 2000. 2003;31(11):32–42.
20. Messana I, Inzitari R, Fanali C, Cabras T, Castagnola M. Facts and artifacts in proteomics of body fluids . What proteomics of saliva is telling us ? J Sep Sci. 2008;31:1948–63.
21. Griffiths GS, Sterne JAC, Wilton JMA, Eaton KA, Johnson NW. Associations between volume and flow rate of gingival crevicular fluid and clinical assessments of gingival inflammation in a population of British male adolescents. J Clin Periodontol. 1992;19(7):464–70.
22. Buduneli N. Biomarkers in Periodontal Health and Disease/Rationale, Benefits, and Future Directions. 1st ed. Izmir, Turkey: Springer Cham; 2020. 1–90 p.
23. Saloom HF, Papageorgiou SN, Carpenter GH, Cobourne MT. Impact of Obesity on Orthodontic Tooth Movement in Adolescents: A Prospective Clinical Cohort Study. J Dent Res. 2017;96(5):547–54.
24. Saloom HF, Carpenter GH, Cobourne MT. A cross-sectional cohort study of gingival crevicular fluid biomarkers in normal-weight and obese subjects during orthodontic treatment with fixed appliances. Angle Orthod. 2019;89(6):930–5.
25. Jafari M, Mehrneja F, Rahimi F, Asghari SM. The Molecular Basis of the Sodium Dodecyl Sulfate Effect on Human Ubiquitin Structure : A Molecular Dynamics Simulation Study. Sci Rep. 2018;(September 2017):1–15. Available from: http://dx.doi.org/10.1038/s41598-018-20669-7