Year : 2017 | Volume
: 9 | Issue : 2 | Page : 80--84
Detection of molecular biomarkers as a diagnostic tool in the planning and progression of orthodontic treatment
Aditi Gaur, Sandhya Maheshwari, Sanjeev K Verma
Department of Orthodontics and Dental Anatomy, Dr. Z. A. Dental College, Aligarh Muslim University, Aligarh, India
Dr. Aditi Gaur
Department of Orthodontics and Dental Anatomy, Dr. Z. A. Dental College, Aligarh Muslim University, Aligarh
Orthodontic treatment focuses on providing patient care at the appropriate timing to utilize the growth potential for best results. It involves growth modification of the craniofacial region along with alveolar bone remodeling during tooth movement. The dynamic process of bone metabolism involves the release of biochemical mediators in the circulation. These molecules are indicative of the bone remodeling activity of osteoblastic deposition and osteoclastic resorption. Such biomarkers when detectable in the systemic circulation highlight the skeletal maturity of orthodontic patients and when detected locally as, in gingival crevicular fluid (GCF) and saliva, indicate the progression of orthodontically induced alveolar bone remodeling. Assessment of molecular biomarkers of bone remodeling in the body fluids would aid the clinicians in planning orthodontic treatment at the ideal timing and evaluating the advent of the treatment.
|How to cite this article:|
Gaur A, Maheshwari S, Verma SK. Detection of molecular biomarkers as a diagnostic tool in the planning and progression of orthodontic treatment.J Orofac Sci 2017;9:80-84
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Gaur A, Maheshwari S, Verma SK. Detection of molecular biomarkers as a diagnostic tool in the planning and progression of orthodontic treatment. J Orofac Sci [serial online] 2017 [cited 2023 Jan 30 ];9:80-84
Available from: https://www.jofs.in/text.asp?2017/9/2/80/222393
Orthodontic treatment is a comprehensive treatment involving growth modification of the craniofacial region and alveolar bone remodeling effecting tooth movement. The underlying mechanism for bone remodeling involves an interaction between biochemical mediators which are released in the circulation. Detection of such molecules would aid the clinicians in assessing the growth status of the orthodontic patient and the efficacy of orthodontic care being rendered. Assaying these biomarkers would also help in evaluating the speed of treatment and local tissue reactions to the forces being applied. The present article aims at highlighting the molecular biomarkers for bone metabolism and their significance in orthodontic treatment.
Molecules detectable in serum indicate the general growth status of an individual and, thus, highlight the skeletal maturity level. Researchers have performed a number of studies providing an array of molecules indicating skeletal growth turnover.
Serum levels for osteocalcin, bone alkaline phosphatase (ALP), and serum carboxy-terminal telopeptide were recorded and correlated to bone mineral density by Silva et al., showing higher values in pubertal age group. Biomarker values were shown to be decreased with advancing bone age and sexual maturation and showed parallelism with peak height velocity.
Insulin-like growth factor-1 (IGF-1) working in parallel with the growth hormone has been shown to be a biomarker in the evaluation of skeletal maturity.,, Serum IGF-1 concentrations have been found to increase slowly in pre-pubertal children with a further steep increase during puberty. After puberty, a subsequent continuous fall in circulating IGF-1 levels suggests that there is an increase in IGF-1 activity in period of increased skeletal growth.
Gupta et al. evaluated serum IGF-1 levels in females of the age group 8–23 years. They showed that IGF-1 can be correlated with the status of the cervical vertebrae maturation and MP3 radiographs, thus, suggesting its use as a reliable marker for assessing the skeletal age.
In a recent study by Jain et al., it was suggested that IGF-1 and insulin like growth factor binding protein (IGFBP-3) serum levels can be used as biochemical markers for skeletal maturation. It was also shown that mean serum IGF-1 and IGFBP-3 levels were increased at cervical vertebrae maturation index (CVMI) stage 3 and 4, respectively.
Bone metabolism is associated with ALP and acid phosphatase, expressed, respectively, by osteoblasts and osteoclasts. ALP is a ubiquitous tetrameric enzyme, localized outside cell membrane. Himes et al. showed that serum ALP levels are associated with skeletal activity in adolescents.
Hussain et al. evaluated the levels of parathyroid hormone-related protein (PTHrP) in serum at varying cervical vertebrae maturation stages. The results showed that serum PTHrP levels had a positive correlation with cervical vertebrae maturation stages from the pre-pubertal to the late pubertal stages.
The disadvantage of using serum as an analytic fluid is that the collection procedure is invasive. Also, it provides the status of systemic activity but cannot highlight the local bone activity.
Gingival crevicular fluid is a mixture of substances derived from serum, host inflammatory cells, structural cells of the periodontium, and oral bacteria.
Gingival crevicular fluid (GCF) composition reflects the metabolic state of the deeper-seated tissues of the periodontium, for example, alveolar bone turnover.,
The collection and analysis of GCF have provided a noninvasive and site-specific means to assess the biochemical status of the marginal periodontium. Collection by filter-paper strips is currently most frequently used in GCF studies in periodontal and orthodontic research. To monitor orthodontic tooth movement noninvasively, changes have been examined in the profile and levels of various molecules in GCF.
Cytokines are the local biochemical signal molecules involved in cell-to-cell signaling and also act as mediators of mechanically induced bone remodeling.
These include the interleukin 1 (IL-1), interleukin 2 (IL-2) interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor α (TNF-α), and interferon γ (IFNγ).,,,
Cytokine profile alteration on application of orthodontic forces have been shown by many researchers, in the GCF of orthodontic patients.
Uematsu et al. showed elevated levels of the concentrations of interleukin (IL)-1β, IL-6, TNF-α, and β2-microglobulin in the experimental group of 12 subjects undergoing orthodontic treatment.
Ren et al. found levels of proinflammatory cytokines in the early stage of tooth movement but at different time points. IL-1β and IL-6 and TNF-α reached significant levels at 24 h; IL-8 reached a significant elevation at 1 month.
Iwasaki et al. showed in their study that, the ratio of IL-1 to IL-1RA plays an important role in the speed and amount of tooth movement.
A study was conducted by to show the levels of biomarkers in GCF of patients undergoing orthodontic treatment using aligners. It was shown that there was an increase in the concentration of bone modeling and remodeling mediators at the pressure sites [IL-1β, receptor activator of nuclear factor-kappa ligand (RANKL)] and tension sites [transforming growth factor (TGF)-β1, osteoprotegerin (OPN)], thus, indicating the role of these biomarkers in the orthodontic tooth movement.
Grant et al. found significant increases in levels of IL-1β, IL-8, TNF-α, matrix metalloproteinase (MMP-9), and tissue inhibitors of matrix metalloproteinase (TIMPs) 1 and 2 in tension regions, while compression sites exhibited increases in IL-1β and IL-8 after 4 h, MMP-9.
The TNF-related ligand RANKL and its two receptors, receptor activator of nuclear factor Kappa-B ligand (RANK) and osteoprotegrin (OPG), have been suggested to be involved in the remodeling process. RANKL is a downstream regulator of osteoclast formation and OPG is a decoy receptor which competes with RANK for RANKL binding, thus suppressing osteoclastic activity.
In a study by Wellington et al. in adolescent and adult patients, it was shown that the ratio of RANKL to OPG peaked in adolescents 6 weeks, after the first rectangular archwire was tied in. The significant elevation of the ratio of RANKL to OPG at this stage in adolescents only is reflective of a higher rate of alveolar bone turnover in the periodontium of adolescents compared with adults.
Similar results were shown by Nishijima et al. in periodontal ligament cells obtained from the GCF of experimental subjects after an application of retraction force on canines.
Collagenases, MMP-1 and MMP-8, degrade collagen fibers, whereas gelatinases such as MMP-2 and MMP-9 degrade denatured collagen, complementing the collagenases.
Increased levels of MMP-1 and MMP-2 levels have been quantified by Western immunoblot assay at the pressure and tension sides of retracted canines 1, 2, 3, 4, and 8 h after activation of an orthodontic appliance.
Apajalahti et al. showed consistently enhanced levels of MMP-8, in GCF from orthodontically treated teeth at 4–8 h after force application relative to baseline values and control teeth.
Ingman et al. demonstrated that GCF levels of MMP-8 measured over 28 days of orthodontic movement were significantly elevated around orthodontically moved teeth in comparison with control teeth. High levels of MMP-9 were found throughout the observation period.
Capelli et al. conducted a study demonstrating an increase in the levels of MMP-3, MMP-9, and MMP-13 on compression site during orthodontic tooth movement.
Bone contains abundant amounts of TGF-β1, insulin-like growth factors, which regulate bone remodeling. Uematsu et al. in their study, determined the levels of TGF-β1 in GCF of 12 patients undergoing a distal movement of canine. TGF-β1 concentrations calculated by enzyme-linked immunosorbent assay were found significantly higher in the experimental group indicating its role in bone remodeling. Toia et al. observed increased IGF-1 levels 4 h after an application of orthodontic force, but they were significantly reduced 10 days after the start of treatment.
Prostaglandins increase the level of cellular cyclic adenosine mono-phosphate (cAMP) and activation of osteoclasts, thus, promoting bone remodeling., Grieve et al. observed significant elevations from baseline in GCF for prostaglandin E (PGE) with radioimmunoassay analysis of GCF collected from 10 subjects undergoing orthodontic treatment showing the inflammatory nature of the process.
Orthodontic tooth movement affects the number, functional properties, and distribution of both mechanosensitive and nociceptive periodontal nerve fibers.
Substance P has been shown to cause vasodilatation and increased vascular permeability, contributing to increased local blood flow that occurs during inflammation. Yamaguchi et al. demonstrated significantly higher GCF levels of serratiopeptidase (SP) and IL-1β for the treated teeth than for the corresponding control teeth from 8 to 72 h indicating the inflammatory response of tissues to mechanical stress.
A biomarker of primary granule release from polymorphonuclear leukocytes is the lysosomal enzyme β-glucuronidase. Increased levels of this enzyme have been found in the GCF of adolescents treated with rapid maxillary expander.
The GCF-aspartate aminotransferase (AST) activity was found to be significantly elevated in both tension and compression sites at days 7 and 14. This rise has been explained as a consequence of a controlled trauma which produces cell death as a consequence of mechanical force exerted on alveolar bone and periodontal ligament.
GCF lactate dehydrogenase levels reflect the biological activity that takes place in the periodontium during orthodontic movement as shown by Perinetti et al. in orthodontic patients.
Batra et al. found variation in levels of ALP in GCF depending upon the amount of tooth movement.
Significantly elevated levels of cathepsin B, a lysosomal cysteine proteinase, have been detected in GCF from teeth exposed to orthodontic force.
Pentraxin (PTX-3) is a “long” pentraxin produced especially by fibroblasts, neutrophils and macrophages, cells abundantly found in periodontal tissues during orthodontic movements. Surlin et al. showed an increase of GCF levels of PTX-3 from 1 h before the orthodontic appliance to a maximum at 24 h, suggesting its involvement, the aseptic inflammation induced by the orthodontic forces.
Apart from biomarkers for tooth movement, molecules for root resorption during treatment have also been detected in the GCF. Dentin sialoprotein was found to be raised in GCF of tooth sites undergoing physiological root resorption and those under orthodontic forces, portraying the importance of this molecule as a biomarker for root resorption during orthodontic treatment. Balducci et al. reported finding elevated levels of dentin sialoprotein (DSP) and dentin phosphophoryn in the GCF of patients undergoing orthodontic treatment, in whom there were radiographic signs of root resorption. Lombardo et al. have suggested a newer method using a micro-bead approach for the detection of dentin sialoprotein as an early biomarker for root resorption.
The identification of salivary biomarkers and its use as a diagnostic tool has many advantages. It is much easier to collect, and sufficient quantities can easily be obtained for analysis. The collection of saliva is also far less invasive compared to other bodily fluids such as GCF, serum, and urine.
Biochemical mediators of orthodontic tooth movement have been evaluated in salivary samples of orthodontic patients.
Flórez-Moreno et al. evaluated levels of RANKL and OPG in unstimulated saliva samples of 20 orthodontic patients. They found that values of RANKL showed significant increases, OPG salivary values showed a significant downward trend, and the sRANKL/OPG ratio tended to increase significantly over time after the activation visit. Their results indicate that variations in salivary concentrations of RANKL and OPG, and their ratios might be linked to the different phases of orthodontic tooth movement.
Batool et al. showed significant elevation in median salivary levels of IL-8 and granulocyte macrophage-colony stimulating factor after 2 weeks and after 4 weeks from placement of orthodontic appliance compared to their levels at baseline time.
Abdul Wahab et al. evaluated the salivary enzyme levels during orthodontic tooth movement with self-ligating brackets. They reported that AST, tartrate-resistant acid phosphatase, and ALP are potential biomarkers for orthodontic tooth movement.
Saadi et al. calculated the value of both salivary IL-1β and TNF-α. The values were highest at 1 h after the placement of round nitinol wire during orthodontic therapy, which decreased following 2 weeks.
A study looking into the effects of orthodontic treatment on salivary proteins has been performed by Zhang et al. using a surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF) approach. They found mass peak in intensities of peptide profiles in saliva obtained from orthodontic patients.
Burke et al. evaluated changes in the total protein concentration in whole saliva and GCF on an application of force, and demonstrated a significant difference in cyclic adenosine monophosphate-dependent protein kinase regulatory subunit II (RII) after orthodontic separator placement, indicating that the cAMP pathway is activated after tooth movement is initiated.
Flórez-Moreno et al. studied the levels of deoxypyridinoline and bone-specific ALP in saliva during orthodontic tooth movement, which are the biomarkers of osteoclastic and osteoblastic activity respectively. They found that deoxypyridinoline (DPD) dominated the earlier phases of orthodontic tooth movement (OTM), whereas bone-specific alkaline phosphatase (BAP) might serve as indicator of bone formation as soon as the tooth movement stopped.
Ellias et al. conducted a proteomic analysis of saliva to identify potential biomarkers for orthodontic tooth movement. Protein S100-A9 (S100-A9), a calcium-binding protein, immunoglobulin J chain, and Ig α-1 chain C region were found associated with orthodontic treatment.
Very few studies have been conducted evaluating the biomarkers in saliva because of the low sensitivity of saliva as a diagnostic fluid. Further research is required to expand the scope for the use of saliva as a diagnostic fluid in orthodontic patients.
A vast range of biomarkers for bone turnover have been detected in body fluids. These biomarkers help the clinicians in assessing treatment timing and efficacy in orthodontic patients. The methods for detecting these biomarkers still lack accuracy and, thus, are not routinely used in orthodontic practice. Further research is required to detect orthodontic biomarkers with accuracy and to design chair-side diagnostic kits to assess the progression of orthodontic treatment. This will provide the clinicians with additional tools to consistently diagnose and manage orthodontic patients in a better way.
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Conflicts of interest
There are no conflicts of interest.
|1||Silva CC, Goldberg TB, Nga HS, Kurokawa CS, Capela RC, Teixeira AS, Dalmas JC. Impact of skeletal maturation on bone metabolism biomarkers and bone mineral density in healthy Brazilian male adolescents. J Pediatr 2011;87:450-60.|
|2||Ishaq RA, Soliman SA, Foda MY, Fayed MM. Insulin-like growth factor I: A biologic maturation indicator. Am J Orthod Dentofacial Orthop 2012;142:654-61.|
|3||Gupta S, Deoskar A, Gupta P, Jain S. Serum insulin-like growth factor-1 levels in females and males in different cervical vertebral maturation stages. Dental Press J Orthod 2015;20:68-75.|
|4||Sinha M, Tripathi T, Rai P, Gupta SK. Serum and urine insulin-like growth factor-1 as biochemical growth maturity indicators. Am J Orthod Dentofacial Orthop 2016;150:1020-7.|
|5||Juul A, Bang P, Hertel NT, Main K, Dalgaard P, Jorgensen K et al. Serum insulin-like growth factor-I in 1030 healthy children, adolescents, and adults: Relation to age, sex, stage of puberty, testicular size, and body mass index. J Clin Endocrinol Metab 1994;78:744-52.|
|6||Gupta S, Jain S, Gupta P, Deoskar A. Determining skeletal maturation using insulin-like growth factor I (IGF-I) test. Prog Orthod 2012;13:288-95.|
|7||Jain N, Tripathi T, Gupta SK, Rai P, Kanase A, Kalra S. Serum IGF-1, IGFBP-3 and their ratio: Potential biochemical growth maturity indicators. Prog Orthod 2017;18:11.|
|8||Himes JH, Huang Z, Haas JD, Rivera R, Pineda O. Serum alkaline phosphatase activity and skeletal maturation in Guatemalan adolescents. Ann Hum Biol 1993;20:39-46.|
|9||Hussain MZ, Talapaneni AK, Prasad M, Krishnan R. Serum PTHrP level as a biomarker in assessing skeletal maturation during circumpubertal development. Am J Orthod Dentofacial Orthop 2013;143:515-21.|
|10||Lamster IB, Ahlo JK. Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases. Ann N Y Acad Sci 2007;1098:216-29.|
|11||Embery G, Waddington R. Gingival crevicular fluid: Biomarkers of periodontal tissue activity. Adv Dent Res 1994;8:329-36.|
|12||Guentsch A, Kramesberger M, Sroka A, Pfister W, Potempa J. Comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis. J Periodontol 2011;82:1051-60.|
|13||Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop 2006;129:469.e1-32.|
|14||Suda T, Nakamura I, Jimi E, Takahashi N. Regulation of osteoclast function. J Bone Mineral Res 1997;12:869-79.|
|15||Baggiolini M, Clark-Lewis I. Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett 1992;307:97-101.|
|16||Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S et al. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 2000;191:275-86.|
|17||Kohara H, Kitaura H, Yoshimatsu M, Fujimura Y, Morita Y, Eguchi T et al. Inhibitory effect of interferon-γ on experimental tooth movement in mice. J Interferon Cytokine Res 2012;32:426-31.|
|18||Uematsu S, Mogi M, Deguchi T. Interleukin (IL)-1 beta, IL-6, tumornecrosis factor-alpha, epidermal growth factor, and beta 2-microglobulin levels are elevated in gingival crevicular fluid during human orthodontic tooth movement. J Dent Res 1996;75:562–7.|
|19||Ren Y, Vissink A. Cytokines in crevicular fluid and orthodontic tooth movement. Eur J Oral Sci 2008;116:89-97.|
|20||Iwasaki LR, Chandler JR, Marx DB, Pandey JP, Nickel JC. IL-1 gene polymorphisms, secretion in gingival crevicular fluid, and speed of human orthodontic tooth movement. Orthodont Craniofacial Res 2009;12:129-40.|
|21||Castroflorio T, Gamerro EF, Caviglia GP, Deregibus A. Biochemical markers of bone metabolism during early orthodontic tooth movement with aligners. Angle Orthod 2017;87:74-81.|
|22||Grant M, Wilson J, Rock P, Chapple I. Induction of cytokines, MMP9, TIMPs, RANKL and OPG during orthodontic tooth movement. Eur J Orthod 2013;35:644-51.|
|23||Yamaguchi M. RANK/RANKL/OPG during orthodontic tooth movement. Orthod Craniofacial Res 2009;12:113-9.|
|24||Rody WJ Jr., Wijegunasinghe M, Wiltshire WA, Dufault B. Differences in the gingival crevicular fluid composition between adults and adolescents undergoing orthodontic treatment. The Angle Orthod 2014;84:120-6.|
|25||Nishijima Y, Yamaguchi M, Kojima T, Aihara N, Nakajima R, Kasai K. Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro. Orthod Craniofacial Res 2006;9:63-70.|
|26||Canavarro C, Teles RP, Capelli J Jr. Matrix metalloproteinases-1, −2, −3, −7, −8, −12, and −13 in gingival crevicular fluid during orthodontic tooth movement: A longitudinal randomized split-mouth study. Eur J Orthod 2013;35:652-8.|
|27||Cantarella G, Cantarella R, Caltabiano M, Risuglia N, Bernardini R, Leonardi R. Levels of matrix metalloproteinases 1 and 2 in human gingival crevicular fluid during initial tooth movement. Am J Orthod Dentofacial Orthop 2006;130:568.e11-16.|
|28||Apajalahti S, Sorsa T, Railavo S, Ingman T. The in vivo levels of matrix metalloproteinase-1 and −8 in gingival crevicular fluid during initial orthodontic tooth movement. J Dent Res 2003;82:1018-22.|
|29||Ingman T, Apajalahti S, Mantyla P, Savolainen P, Sorsa T. Matrix metalloproteinase-1 and −8 in gingival crevicular fluid during orthodontic tooth movement: A pilot study during 1 month of followup after fixed appliance activation. Eur J Orthod 2005;27:202-7.|
|30||Capelli J Jr, Kantarci A, Haffajee A, Teles RP, Fidel R Jr, Figueredo CM. Matrix metalloproteinases and chemokines in the gingival crevicular fluid during orthodontic tooth movement. Eur J Orthod 2011;33:705-11.|
|31||Uematsu S, Mogi M, Deguchi T. Increase of transforming growth factor-beta 1 in gingival crevicular fluid during human orthodontic tooth movement. Arch Oral Biol 1996;41:1091-5.|
|32||Toia M, Galazzo R, Maioli C, Granata R, Scarlatti F. The IGF-I/IGFBP-3 system in gingival crevicular fluid and dependence on application of fixed force. J Endocrinol Investig 2005;28:1009-14.|
|33||Kaji H, Sugimoto T, Kanatani M, Fukase M, Kumegawa M, Chihara K. Prostaglandin E2 stimulates osteoclast-like cell formation and bone-resorbing activity via osteoblasts: Role of cAMP-dependent protein kinase. J Bone Mineral Res 1996;11:62-71.|
|34||Yamasaki K, Shibata Y, Imai S, Tani Y, Shibasaki Y, Fukuhara T. Clinical application of prostaglandin E1 (PGE1) upon orthodontic tooth movement. Am J Orthod 1984;85:508-18.|
|35||Grieve W 3rd, Johnson G, Moore R, Reinhardt R, DuBois L. Prostaglandin e (PGe) and interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid during human orthodontic tooth movement. Am J Orthod Dentofacial Orthop 1994;105:369-74.|
|36||Norevall LI, Forsgren S, Matsson L. Expression of neuropeptides (CGRP, substance) during and after orthodontic tooth movement in the rat. Eur J Orthod 1995;17:311-25.|
|37||Yamaguchi M, Yoshii M, Kasai K. Relationship between substance P and interleukin-1β in gingival crevicular fluid during orthodontic tooth movement in adults. Eur J Orthod 2006;28:241-6.|
|38||Tzannetou S, Efstratiadis S, Nicolay O, Grbic J, Lamster I. Comparison of levels of inflammatory mediators IL-1β and βG in gingival crevicular fluid from molars, premolars, and incisors during rapid palatal expansion. Am J Orthod Dentofacial Orthop 2008;133:699-707.|
|39||Perinetti G, Paolantonio M, D’Attilio M. Aspartate aminotransferase activity in gingival crevicular fluid during orthodontic treatment. A controlled short-term longitudinal study. J Periodontol 2003;74:145-52.|
|40||Perinetti G, Serra E, Paolantonio M. Lactate dehydrogenase activity in human gingival crevicular fluid during orthodontic treatment: A controlled, short-term longitudinal study. J Periodontol 2005;76:411-7.|
|41||Batra P, Kharbanda OP, Duggal R, Singh N, Parkash N. Alkaline phosphatase activity in gingival crevicular fluid during canine retraction. Orthod Craniofacial Res 2006;9:44-51. doi: 10.1111/j.1601-6343.2006.00358.x|
|42||Sugiyama Y, Yamaguchi M, Kanekawa M, Yoshii M, Nozoe T, Nogimura A et al. The level of cathepsin B in gingival crevicular fluid during human orthodontic tooth movement. Eur J Orthod 2003;25:71-6.|
|43||Surlin P, Rauten AM, Silosi I, Foia L. Pentraxin-3 levels in gingival crevicular fluid during orthodontic tooth movement in young and adult patients. Angle Orthod 2012;82:833-8.|
|44||Kereshanan S, Stephenson P, Waddington R. Identification of dentine sialoprotein in gingival crevicular fluid during physiological root resorption and orthodontic tooth movement. Eur J Orthod 2008;30:307-14. doi: 10.1093/ejo/cjn024|
|45||Balducci L, Ramachandran A, Hao J, Narayanan K, Evans C, George A. Biological markers for evaluation of root resorption. Arch Oral Biol 2007;52:203-8. doi: 10.1016/j.archoralbio.2006.08.018|
|46||Lombardo L, Carinci F, Martini M, Gemmati D, Nardone M, Siciliani G. Quantitive evaluation of dentin sialoprotein (DSP) using microbeads − A potential early marker of root resorption. Oral Implantol (Rome) 2016;9:132-42.|
|47||Kaufman E, Lamster IB. The diagnostic applications of saliva – A review. CROBM 2002;13:2197-212.|
|48||Flórez-Moreno GA, Isaza-Guzmán DM, Arroyave S. Time-related changes in salivary levels of the osteotropic factors sRANKL and OPG through orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2013;143:92-100.|
|49||Batool H, Al-Ghurabi, Harraa S, Mohammed-Salih, Ghazi A, Saloom H. Evaluation of salivary levels of proinflammatory cytokines (IL-1α, IL-8 and GM-CSF) in adult orthodontic patients. IOSR J Dent Med Sci 2014;13:75-8.|
|50||Abdul Wahab RM, Abu Kasim N, Senafi S, Jemain AA, Zainol Abidin IZ, Shahidan MA et al. Enzyme activity profiles and ELISA analysis of biomarkers from human saliva and gingival crevicular fluid during orthodontic tooth movement using self-ligating brackets. Oral Health Dent Manag 2014;13:194-9.|
|51||Saadi N, Ghaib NH. Effect of orthodontic tooth movement on salivary levels of interleukin-1beta, tumor necrosis factor-alpha, and C-reactive protein. J Bagh College Dent 2013;25.|
|52||Zhang J, Zhou S, Zheng H, Zhou Y, Chen F, Lin J. Magnetic bead-based salivary peptidome profiling analysis during orthodontic treatment durations. Biochem Biophys Res Commun 2012;421:844-9.|
|53||Burke JC, Evans CA, Crosby TR, Mednieks MI. Expression of secretory proteins in oral fluid after orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2002;121:310-5.|
|54||Flórez-Moreno GA, Marín-Restrepo LM, Isaza-Guzmán DM, Tobón-Arroyave SI. Screening for salivary levels of deoxypyridinoline and bone-specific alkaline phosphatase during orthodontic tooth movement: A pilot study. Eur J Orthod 2013;35:361-8. doi: 10.1093/ejo/cjr138|
|55||Ellias MF, Ariffin S, Karsani SA, Rahman MA, Senafi S, Wahab R. Proteomic analysis of saliva identifies potential biomarkers for orthodontic tooth movement. Sci World J 2012;2012:647240.|
|56||Nunes LAS, Mussavira S, Bindhu OS. Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid:a systematic review. Biochem Med 2015;25:177-92.|