Dental Press Journal of Orthodontics

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ORIGINAL ARTICLE • Dental Press J. Orthod. 29 (6) • 2024 • https://doi.org/10.1590/2177-6709.29.6.e242436.oar linkcopy

Changes in salivary biomarkers of pain, anxiety, stress, and inflammation related to tooth movement during orthodontic treatment: a systematic review

Authorship SCIMAGO INSTITUTIONS RANKINGS

Objective: This systematic review aimed to analyze the literature on changes in endogenous salivary biomarkers of pain, anxiety, stress, and inflammation related to tooth movement during orthodontic treatment of children and adolescents.

Material and Methods: An electronic search was performed in nine databases to identify quasi-experimental studies, without restricting publication language and year. Two reviewers extracted the data and assessed the individual risk of bias using the JBI tools, and the certainty of evidence using the GRADE tool.

Results: The electronic search found 7,038 records, of which 12 met the eligibility criteria and were included in the qualitative synthesis. Most studies had a low risk of bias. Biomarkers were grouped into five categories: electrolytes, enzymes, hormones, immunoglobulins, and mediators. Electrolytes showed decreased Ca2+, Pi3+ and K+ levels, and increased Na+ and Cl- levels. All enzymes (ALP, LDH, MMP8, and MMP9) increased over time. Hormones presented a decrease in leptin and some fluctuations in daily cortisol levels. Immunoglobulins (IgA, IgG, IgM, IgD, and IgE) had no significant changes, and salivary IgA showed divergent results among studies. Mediators (sRANKL, OPG, IL‐1β, and PGE2) showed fluctuations at different treatment stages, mainly after orthodontic activation.

Conclusions: Based on a very low certainty level, orthodontic tooth movement had little to no effect on endogenous salivary biomarkers.

Keywords:
Orthodontics; Saliva; Biomarkers

Database	Search strategies (April 2023) and update (September 2023)
Database	Main databases
Embase https://www.embase.com	(‘child’/exp OR ‘child’ OR ‘children’/exp OR ‘children’ OR ‘adolescent’/exp OR ‘adolescent’ OR ‘adolescence’/exp OR ‘adolescence’ OR ‘teen’ OR ‘teenager’/exp OR ‘teenager’ OR ‘youth’/exp OR ‘youth’) AND (‘orthodontics’/exp OR ‘orthodontics’ OR ‘orthodontic treatment’/exp OR ‘orthodontic treatment’ OR ‘orthodontics, preventive’/exp OR ‘orthodontics, preventive’ OR ‘orthodontics, interceptive’/exp OR ‘orthodontics, interceptive’ OR ‘orthodontics, corrective’/exp OR ‘orthodontics, corrective’ OR ‘maxillary expansion’/exp OR ‘maxillary expansion’ OR ‘orthodontic appliances, fixed’/exp OR ‘orthodontic appliances, fixed’ OR ‘orthodontic appliances, functional’/exp OR ‘orthodontic appliances, functional’ OR ‘orthodontic appliances, removable’/exp OR ‘orthodontic appliances, removable’ OR ‘orthodontic appliances’/exp OR ‘orthodontic appliances’) AND (‘saliva’/exp OR ‘saliva’ OR ‘salivary changes’ OR ‘salivary composition’ OR ‘biomarkers’/exp OR ‘biomarkers’ OR ‘salivary biomarkers’ OR ‘pain’/exp OR ‘pain’ OR ‘anxiety’/exp OR ‘anxiety’ OR ‘stress’/exp OR ‘stress’ OR ‘inflammation’/exp OR ‘inflammation’ OR ‘inflammation mediators’/exp OR ‘inflammation mediators’ OR ‘cytokines’/exp OR ‘cytokines’) AND [embase]/lim
LILACS and BBO http://lilacs.bvsalud.org/	((“Child” OR “Children” OR “Adolescent” OR “Adolescence” OR “Teen” OR “Teenager” OR “Youth”) AND (“Orthodontics” OR “Orthodontic Treatment” OR “Orthodontics, Preventive” OR “Orthodontics, Interceptive” OR “Orthodontics, Corrective” OR “Maxillary Expansion” OR “Orthodontic Appliances, Fixed” OR “Orthodontic Appliances, Functional” OR “Orthodontic Appliances, Removable” OR “Orthodontic Appliances”) AND (“Saliva” OR “Salivary Changes” OR “Salivary Composition” OR “Biomarkers” OR “Salivary Biomarkers” OR “Pain” OR “Anxiety” OR “Stress” OR “Inflammation” OR “Inflammation Mediators” OR “Cytokines”)) AND ( db:(“LILACS” OR “BBO”))
MEDLINE (via PubMed) http://www.ncbi.nlm.nih.gov/pubmed	#1 “Child”[Mesh] OR “Children”[tw] OR “Adolescent”[Mesh] OR “Adolescence”[tw] OR “Teen”[tw] OR “Teenager”[tw] OR “Youth”[tw] #2 “Orthodontics”[Mesh] OR “Orthodontic Treatment”[tw] OR “Orthodontics, Preventive”[Mesh] OR “Orthodontics, Interceptive”[Mesh] OR “Orthodontics, Corrective”[Mesh] OR “Maxillary Expansion”[tw] OR “Orthodontic Appliances, Fixed”[Mesh] OR “Orthodontic Appliances, Functional”[Mesh] OR “Orthodontic Appliances, Removable”[Mesh] OR “Orthodontic Appliances”[Mesh] #3 “Saliva”[Mesh] OR “Salivary Changes”[tw] OR “Salivary Composition”[tw] OR “Biomarkers”[Mesh] OR “Salivary Biomarkers”[tw] OR “Pain”[Mesh] OR “Anxiety”[Mesh] OR “Stress”[tw] OR “Inflammation”[Mesh] OR “Inflammation Mediators”[Mesh] OR “Cytokines”[Mesh]
MEDLINE (via PubMed) http://www.ncbi.nlm.nih.gov/pubmed	#1 AND #2 AND #3
SciELO https://scielo.org/	#1 “Child” OR “Children” OR “Adolescent” OR “Adolescence” OR “Teen” OR “Teenager” OR “Youth” #2 “Orthodontics” OR “Orthodontic Treatment” OR “Orthodontics, Preventive” OR “Orthodontics, Interceptive” OR “Orthodontics, Corrective” OR “Maxillary Expansion” OR “Orthodontic Appliances, Fixed” OR “Orthodontic Appliances, Functional” OR “Orthodontic Appliances, Removable” OR “Orthodontic Appliances” #3 “Saliva” OR “Salivary Changes” OR “Salivary Composition” OR “Biomarkers” OR “Salivary Biomarkers” OR “Pain” OR “Anxiety” OR “Stress” OR “Inflammation” OR “Inflammation Mediators” OR “Cytokines”
SciELO https://scielo.org/	#1 AND #2 AND #3
Scopus http://www.scopus.com/	( TITLE-ABS-KEY ( “Child” OR “Children” OR “Adolescent” OR “Adolescence” OR “Teen” OR “Teenager” OR “Youth” ) AND TITLE-ABS-KEY ( “Orthodontics” OR “Orthodontic Treatment” OR “Orthodontics, Preventive” OR “Orthodontics, Interceptive” OR “Orthodontics, Corrective” OR “Maxillary Expansion” OR “Orthodontic Appliances, Fixed” OR “Orthodontic Appliances, Functional” OR “Orthodontic Appliances, Removable” OR “Orthodontic Appliances” ) AND TITLE-ABS-KEY ( “Saliva” OR “Salivary Changes” OR “Salivary Composition” OR “Biomarkers” OR “Salivary Biomarkers” OR “Pain” OR “Anxiety” OR “Stress” OR “Inflammation” OR “Inflammation Mediators” OR “Cytokines” ) )
Web of Science http://apps.webofknowledge.com/	#1 TS=(“Child” OR “Children” OR “Adolescent” OR “Adolescence” OR “Teen” OR “Teenager” OR “Youth”) #2 TS=(“Orthodontics” OR “Orthodontic Treatment” OR “Orthodontics, Preventive” OR “Orthodontics, Interceptive” OR “Orthodontics, Corrective” OR “Maxillary Expansion” OR “Orthodontic Appliances, Fixed” OR “Orthodontic Appliances, Functional” OR “Orthodontic Appliances, Removable” OR “Orthodontic Appliances”) #3 TS=(“Saliva” OR “Salivary Changes” OR “Salivary Composition” OR “Biomarkers” OR “Salivary Biomarkers” OR “Pain” OR “Anxiety” OR “Stress” OR “Inflammation” OR “Inflammation Mediators” OR “Cytokines”)
Web of Science http://apps.webofknowledge.com/	#1 AND #2 AND #3
Gray literature
EASY https://easy.dans.knaw.nl/	(“Orthodontics” OR “Orthodontic Treatment” OR “Orthodontic Appliances”) AND (“Saliva” OR “Salivary Changes” OR “Salivary Composition” OR “Biomarkers” OR “Cytokines”)
ProQuest https://www.proquest.com/	(“Orthodontics” OR “Orthodontic Treatment” OR “Orthodontic Appliances”) AND (“Saliva” OR “Salivary Changes” OR “Salivary Composition” OR “Biomarkers” OR “Cytokines”)

Author, year (Country) Journal of publication (Impact factor) [Analyzed biomarkers]	Study design # Ethical criteria - term $ Funding * Conflicts of interest	Sample size (n) Sex (♀, ♂) Age group (Mean age)	Group (n) \| Group (n) Orthodontic appliance (Usage time)	Method of saliva collection - Preparation - Collection - Storage	Method of saliva analysis
Ganhão et al.²¹, 1984 (South Africa) J Dent Assoc S Afr (N.R.) [IgA; IgG; IgM; IgD; IgE]	Prospective QE # N.R. - N.R.; $ N.R.; * N.R.	(n = 31) (♀ = 22, ♂ = 9) 11-15 yo (N.R.)	Only group (n=31) Fixed orthodontic appliance. (12-32 months)	- Mouth rinses, relax 5min, swallow saliva, lower head, and wait 5min. - UWS collected into a cylinder with 1mg of sodium azide. - Mixed and stored airtight at -10°C until assayed.	- Simplified Radial Immunodiffusion Technique (for IgA, IgG, IgM e IgD); - Phadabas IgE RIA Test Kit.
Li et al.²², 2009 (China) J Oral Rehabil (2.9) [Ca2+; Pi3+; Na+; Cl-; K+]	Prospective QE # Yes - Consent term; $ National Natural Science Foundation of China (No. 30430690); * None.	(n = 24) (♀ = 12, ♂ = 12) 10-16 yo (N.R.)	G1 (n=12) \| Comparator group (n=12) Fixed orthodontic appliance: OPA-K (Tomy Co., Tokyo, Japan) straight archwires for both arches. (6 months)	- Overnight fasting and collection in the morning between 9am and 11am. - UWS (5mL) spit into a cylinder for 10min. - Centrifugation for 5min at 690g at room temperature and the supernatant was analyzed.	- Automated Biochemistry Analyzer (7060 type; Hitachi Co., Tokyo, Japan).
Campos et al.²³, 2010 (Brazil) Med Sci Monitor (3.1) [IgA]	Prospective QE # Yes - Consent term; $ N.R.; * N.R.	(n = 10) (♂ = 10) 11-13 yo (12.2±0.7)	Young group (n=10) Fixed orthodontic appliance: brackets and tubes (Mini Standard Edgewise; American Orthodontics, Sheboygan, WI, USA), 0.014-in straight archwire (Titanium Memory Wire, 857-504; American Orthodontics), and 0.010mm steel wire. (7-14 days)	- Collection on waking, before toothbrushing, drinking, or eating. - Swab under tongue for 2min and transferred to the Salivette Cortisol device (51.1534.500; Sarstedt, Numbrecht, Germany). - Domestic freezer up to 48h, cold boxes with ice for transportation and at -80°C up to analysis.	- Human IgA ELISA Quantitation Kit (Bethyl Laboratories, Montgomery, TX, USA); - Spectramax 190 Microtiter Plate Absorbance Reader (Molecular Device, Sunnyvale, CA, USA).
Gecgelen et al.¹³, 2012 (Turkey) J Oral Rehabil (2.9) [Cortisol]	Prospective QE # Yes - Consent term; $ Süleyman Demirel University Scientific Research Projects Unit (No. 1880- D-09); * N.R.	(n = 40) (♀ = 20, ♂ = 20) 10.9-14.7 yo (N.R.)	Only group (n=40) Peak (n=19) \| Post-peak (n=21) Fixed orthodontic appliance: rapid maxillary expansion appliance with acrylic bonding. (36 days)	- No drinking (only water allowed) or eating for 1h, and sit in normal position. - Saliva collected into a polypropylene tube for 3min. - Storage: N.R.	- Electrochemi-luminescence immunoassay (ECLIA); - Cobas Cortisol Kit (Roche Diagnostics, Indianapolis, IN, USA); - Elecsys 2010 immunoanalyser system (Roche Diagnostics, Mannheim, Germany).
Flórez-Moreno et al.²⁴, 2013 (Colombia) Am J Orthod Dentofac Orthop (3.0) [sRANKL; OPG]	Prospective QE # Yes - Consent term; $ Research Development Committee of the University of Antioquia (No. CODI-Code 8700-1618/2009); * None.	(n = 14) (♀ = 5, ♂ = 9) 11-18 yo (N.R.)	Young group (n=14) Fixed orthodontic appliance: Roth prescription, 0.018x0.025-in brackets and 0.014-in NiTi conventional archwire. (8 weeks)	- Collection before breakfast and any dental hygiene procedure. - UWS (5mL) spit into a 50-mL sterile plastic centrifuge tube (Greiner Bio-one, Frickenhausen, Germany). - Centrifugation for 5min at 800g (IEC Centra CL2 centrifuge; Thermo Electron, Milford, Mass) and supernatants into 500-µL volumes at -75°C until processed.	- Ampli sRANKL Human ELISA and OPG ELISA (Immunodiagnostic Systems, Fountain Hills, AZ, USA); - Microplate Reader (ChroMate 4300; Awareness Technology, Palm City, FL, USA).
Bakar et al.²⁵, 2017 (Malaysia) Braz J Oral Sci (N.R.) [Leptina]	Prospective QE # Yes - Consent term; $ International Islamic University Malaysia (Nos. EDW-B-14-103-0988 e RIGS16-139-0303); * N.R.	(n = 10) (♀ = 7, ♂ = 3) N.R. (16.76±1.1)	Only group (n=10) Fixed orthodontic appliance: fixed upper and lower orthodontic appliances bonded with 0.014-in NiTi wires attached to both arches. (6 weeks)	- Fast from midnight, collection at 8am before toothbrushing. - UWS (5mL) expectorated into disposable tubes every 30sec over 5min. - Centrifugation for 10min at 4000g and samples at -25°C until analyzed.	- Leptin Abnova LEP Human ELISA kit (KA3080).
Al-Khatieeb et al.²⁶, 2018 (Iraq) J Contemp Dent Pract (N.R.) [ALP; LDH]	Prospective QE # N.R. - Consent term; $ None; * None.	(n = 16) (♀ = 6, ♂ = 10) 9-12 yo (N.R.)	Only group (n=16) Removable orthodontic appliance: monoblock. (28 days)	- No drinking or eating for at least 1h and sit in a comfortable position. - UWS (5mL) spit into a 10-mL sterile plastic plane test tube within 10min. - Cooling box, centrifugation for 20min at 3000RPM and supernatants in Eppendorf tubes at -20°C until analysis.	- Semi-Auto Chemistry Analyzer (BA-88A; Mindray, Nanshan, Shenzhen, China).
Aksoy et al.²⁷, 2019 (Turkey) Turk J Orthod (1.1) [Cortisol]	Prospective QE # Yes - Consent term; $ Süleyman Demirel University Scientific Research Projects Unit (No. 2494-M-10); * None.	(n = 20) (♀ = 10, ♂ = 10) 11-14 yo (12.83±0.71)	G1 (n=10) \| G2 (n=10) Fixed orthodontic appliance: 0.016-, 0.016×0.016-, and 0.16×0.22-in (G1) and 0.014-, 0.016-, and 0.016×0.016-in (G2) straight wires (Superelastic NiTi Memory Wire; American Orthodontics, Sheboygan, WI, USA). (8 weeks)	- No drinking (only water allowed) or eating for 1h, and sit in normal position. - Saliva collected into a polypropylene tube for 3min. - Samples at -80°C until analysis.	- Cobas Cortisol Kit (Roche Diagnostics, USA); - Cobas e601 Immunoassay System (Roche Diagnostics, Mannheim, Germany).
Maan & Patil²⁸, 2019 (India) J Orthod Sci (N.R.) [IL-1β; PGE2]	Prospective QE # Yes - Consent term; $ None; * None.	(n = 10) (♀ = 5, ♂ = 5) 12-18 yo (N.R.)	Young group (n=10) Fixed orthodontic appliance: MBT prescription, preadjusted Edgewise brackets (3M Gemini brackets; 3M Unitek Corporation, Monrovia, California) with 0.022-in slots, with 0.014-in (T2), and 0.016-in (T4) NiTi archwires (Ortho Organizers Inc., USA). (1 month)	- Preparation: N.R. - UWS collected into a 45-mL sterile plastic tube. - Samples stored in 2mL Eppendorf tubes at -79°C.	- IL‐1β ELISA Kit (Krishgen Biosystems, India); - PGE2 ELISA Kit (KinesisDx, USA); - Spectrophotometric Microplate Reader (Lisa Plus, India).
Jha et al.²⁹, 2020 (India) J Fam Med Prim Care (1.4) [IgA]	Prospective QE # Yes - Consent term; $ None; * None.	(n = 40) (♀ = N.R. ♂ = N.R.) 8-15 yo (N.R.)	G1 (n=20) \| G2 (n=20) Fixed orthodontic appliance (G1) and removable orthodontic appliance (G2). (6 months)	- No drinking or eating for 1h, collection in the morning between 10am and 11am. - UWS spit into a pre-labeled sterile container. - 1.5mL test tubes, dry ice for transportation, and deep freezer (Samsung RZ90EERS) at-20°C.	- IgA ELISA Kit.
Grgurevic et al.³⁰, 2021 (Croatia) Prog Orthod (4.8) [BMP4]	Prospective QE # Yes - Consent term; $ Scientific Center of Excellence for Reproductive and Regenerative Medicine (project “Reproductive and regenerative medicine - exploration of new platforms and potentials”) and European Union by European Regional Development Fund (No. KK.01.1.1.01.0008); * None.	(n = 18) (♂ = 18) 12-14 yo (N.R.)	G1 (n=12) \| Comparator group (n=6) Fixed orthodontic appliance: MBT prescription, with 0.022-in slot and 0.012-in NiTi straight wire. (30 days)	- No eating or toothbrushing for 1h. - Saliva expectorated into clean Petri dishes. - Samples (1mL) stored in Eppendorf tubes at -80°C, pooled samples centrifugation for 10min at 12000g at 4°C, acetone precipitation, and analysis.	- RC DC Protein Assay Kit II (BioRad); - BMP4 ELISA DuoSet (#DY314, R&D Systems); - Mass Spectrometry (LTQ Orbitrap Discovery Instrument; Thermo Fischer Scientific).
Wazwaz et al.³¹, 2023 (United Kingdom) Sci Rep (4.6) [MMP8; MMP9]	Retrospective QE # Yes - Consent term; $ N.R.; * None.	(n = 16) (♀ = 6, ♂ = 10) 12-18 yo (15.2±1.6)	Only group (n=16) Fixed orthodontic appliance: MBT prescription, 0.022-in brackets (Victory-APC; 3M-Unitek) and specific sequence of archwires (0.014-in NiTi; 0.018-in NiTi; 0.017×0.025-in NiTi; and 0.019×0.025-in stainless steel). (6-18 months)	- Preparation: N.R. - UWS collected. - Centrifugation for 5min at 9200g and samples at -80°C until analysis.	- Human MMP8 e MMP9 ELISA Kit (R&D Systems); - BCA Protein Assay (Termo-Scientific); - LC-MSMS (Q-Exactive-Plus coupled to RSLCnano3000 (Termo-Scientific); - Zymography (ChemiDocTM MP Imaging System; BioRad).

Author, year (Biomarker) [Measurement unit]	Collection time: Reference time	Results (mean ±SD) Group \| Group	Group (n)
Electrolytes
Li et al.²², 2009 (Ca2+) [M]	T1: baseline	1.16 ± 0.36 \| 1.11 ± 0.16	G1* (n=12) \| CG* (n=12)
	T2: 1mo ~	0.95 ± 0.11 \| 1.07 ± 0.15
	T3: 3mo ~	1.02 ± 0.10 \| 1.01 ± 0.11
	T4: 6mo ~	1.01 ± 0.29 \| 1.04 ± 0.15
Li et al.²², 2009 (Pi3+) [M]	T1: baseline	3.02 ± 0.21 \| 2.99 ± 0.34	G1* (n=12) \| CG* (n=12)
	T2: 1mo ~	2.11 ± 0.62 \| 2.95 ± 0.15
	T3: 3mo ~	2.74 ± 0.34 \| 3.00 ± 0.30
	T4: 6mo ~	2.82 ± 0.37 \| 3.01 ± 0.10
Li et al.²², 2009 (Na+) [M]	T1: baseline	9.00 ± 1.71 \| 9.21 ± 1.01	G1* (n=12) \| CG* (n=12)
	T2: 1mo ~	14.93 ± 1.82 \| 8.86 ± 2.01
	T3: 3mo ~	8.71 ± 1.27 \| 9.72 ± 1.16
	T4: 6mo ~	8.57 ± 1.22 \| 9.88 ± 1.58
Li et al.²², 2009 (Cl-) [M]	T1: baseline	21.07 ± 3.61 \| 22.70 ± 3.37	G1* (n=12) \| CG* (n=12)
	T2: 1mo ~	25.86 ± 4.69 \| 21.30 ± 2.75
	T3: 3mo ~	19.64 ± 2.71 \| 21.30 ± 3.33
	T4: 6mo ~	19.86 ± 1.96 \| 20.30 ± 1.70
Li et al.²², 2009 (K+) [M]	T1: baseline	24.51 ± 4.96 \| 24.09 ± 2.93	G1* (n=12) \| CG* (n=12)
	T2: 1mo ~	14.80 ± 1.49 \| 22.54 ± 2.16
	T3: 3mo ~	24.29 ± 5.22 \| 23.41 ± 2.63
	T4: 6mo ~	23.49 ± 4.64 \| 23.98 ± 2.53
Enzymes
Al-Khatieeb et al.²⁶, 2018 (ALP) [IU/L]	T1: baseline	33.94 ± 7.36	OG” (n=16)
	T2: 1h ~	42.38 ± 8.98
	T3: 14d ~	64.00 ± 15.50
	T4: 28d ~	104.06 ± 28.47
Al-Khatieeb et al.²⁶, 2018 (LDH) [IU/L]	T1: baseline	179.63 ± 22.55	OG” (n=16)
	T2: 1h ~	225.81 ± 33.06
	T3: 14d ~	320.19 ± 60.64
	T4: 28d ~	496.31 ± 98.04
Wazwaz et al.³¹, 2023 (MMP8) [pg/mL]	T1: baseline	#9.300 ± N.R.	OG* (n=16)
	T2: 1h ~	#12.400 ± N.R.
	T3: 1w ~	#15.500 ± N.R.
	T4: after alignment conclusion	#12.600 ± N.R.
Wazwaz et al.³¹, 2023 (MMP9) [pg/mL]	T1: baseline	#3.900 ± N.R.	OG* (n=16)
	T2: 1h ~	#5.000 ± N.R.
	T3: 1w ~	#5.950 ± N.R.
	T4: after alignment conclusion	#6.000 ± N.R.
Hormones
Gecgelen et al.¹³, 2012 (Cortisol) [nmol/L]	T1a: baseline (1w before; 09h15)	9.48 ± N.R.	OG* (n=40)
	T1b: baseline (1w before; 09h40)	7.85 ± N.R.
	T1c: baseline (1w before; 10h00)	6.67 ± N.R.
	T1d: baseline (1w before; 10h30)	4.84 ± N.R.
	T2a: 15min before (09h15)	10.55 ± N.R.
	T2b: 10min ~ (09h40)	13.92 ± N.R.
	T2c: 30min ~ (10h00)	11.38 ± N.R.
	T2d: 60min ~ (10h30)	7.26 ± N.R.
	T3a: 1d ~ (09h15)	8.40 ± N.R.
	T3b: 1d ~ (09h40)	8.60 ± N.R.
	T3c: 1d ~ (10h00)	7.29 ± N.R.
	T3d: 1d ~ (10h30)	5.51 ± N.R.
	T4a: 4d ~ (09h15)	9.15 ± N.R.
	T4b: 4d ~ (09h40)	10.02 ± N.R.
	T4c: 4d ~ (10h00)	8.19 ± N.R.
	T4d: 4d ~ (10h30)	6.47 ± N.R.
	T5a: 7d ~ (09h15)	8.84 ± N.R.
	T5b: 7d ~ (09h40)	8.60 ± N.R.
	T5c: 7d ~ (10h00)	7.23 ± N.R.
	T5d: 7d ~ (10h30)	5.65 ± N.R.
	T6a: 15d ~ (09h15)	9.69 ± N.R.
	T6b: 15d ~ (09h40)	9.20 ± N.R.
	T6c: 15d ~ (10h00)	7.88 ± N.R.
	T6d: 15d ~ (10h30)	6.27 ± N.R.
	T7a: 25d ~ (09h15)	9.40 ± N.R.
	T7b: 25d ~ (09h40)	9.27 ± N.R.
	T7c: 25d ~ (10h00)	8.21 ± N.R.
	T7d: 25d ~ (10h30)	6.82 ± N.R.
	T8a: 36d ~ (09h15)	9.83 ± N.R.
	T8b: 36d ~ (09h40)	9.50 ± N.R.
	T8c: 36d ~ (10h00)	8.02 ± N.R.
	T8d: 36d ~ (10h30)	6.07 ± N.R.
	T9a: 3mo + (09h15)	9.28 ± N.R.
	T9b: 3mo + (09h40)	8.04 ± N.R.
	T9c: 3mo + (10h00)	6.41 ± N.R.
	T9d: 3mo + (10h30)	5.19 ± N.R.
Bakar et al.²⁵, 2017 (Leptina) [pg/mL]	T1: baseline (6w before)	7016.457 ± 1344.468	OG* (n=10)
	T2: at bonding	5018.528 ± 901.327
	T3: 6w ~	4901.923 ± 754.657
Aksoy et al.²⁷, 2019 (Cortisol) [μg/dL]	T1: baseline (just before banding)	0.39 ± 0.03 \| 0.41 ± 0.04	G1* (n=10) \| G2* (n=10)
	T2: 1d ~	0.51 ± 0.04 \| 0.52 ± 0.06
	T3a: 2d ~ (before brackets/arch)	0.50 ± 0.07 \| 0.51 ± 0.04
	T3b: 2d ~ (after brackets/arch)	0.57 ± 0.07 \| 0.59 ± 0.03
	T4: 7d ~	0.46 ± 0.03 \| 0.44 ± 0.03
	T5a: 4w ~ (before new arch)	0.44 ± 0.03 \| 0.42 ± 0.05
	T5b: 4w ~ (after new arch)	0.45 ± 0.05 \| 0.42 ± 0.03
	T6a: 8w ~ (before new arch)	0.38 ± 0.03 \| 0.38 ± 0.03
	T6b: 8w ~ (after new arch)	0.41 ± 0.02 \| 0.40 ± 0.02
Immunoglobulins
Ganhão et al.²¹, 1984 (IgA) [IU/mL]			OG-*
	T1: baseline (1mo before banding)	10.3 ± 4.9	T1 (n=31)
	T2: 15min before banding	12.5 ± 3.3	T2 (n=13)
	T3: 1mo ~	9.5 ± 5.3	T3 (n=21)
	T4: 3mo +	6.5 ± 4.9	T4 (n=6)
Ganhão et al.²¹, 1984 (IgG) [IU/mL]			OG-*
	T1: baseline (1mo before banding)	0.31 ± 0.27	T1 (n=31)
	T2: 15min before banding	0.51 ± 0.47	T2 (n=13)
	T3: 1mo ~	0.67 ± 1.52	T3 (n=18)
	T4: 3mo +	0.18 ± 0.23	T4 (n=6)
Ganhão et al.²¹, 1984 (IgM) [IU/mL]			OG-*
	T1: baseline (1mo before banding)	0.43 ± 0.90	T1 (n=30)
	T2: 15min before banding	0.63 ± 1.70	T2 (n=13)
	T3: 1mo ~	0.25 ± 0.92	T3 (n=17)
	T4: 3mo +	0.87 ± 1.50	T4 (n=3)
Ganhão et al.²¹, 1984 (IgD) [IU/mL]			OG-*
	T1: baseline (1mo before banding)	Trace	T1 (n=30)
	T2: 15min before banding	Trace	T2 (n=13)
	T3: 1mo ~	Trace	T3 (n=19)
	T4: 3mo +	Trace	T4 (n=6)
Ganhão et al.²¹, 1984 (IgE) [IU/mL]			OG-*
	T1: baseline (1mo before banding)	0.07 ± 0.19	T1 (n=7)
	T2: 15min before banding	3.40 ± 8.13	T2 (n=6)
	T3: 1mo ~	0.17 ± 0.22	T3 (n=7)
	T4: 3mo +	0.30 ± 0.42	T4 (n=2)
Campos et al.²³, 2010 (IgA) [µg/mL]	T1: baseline (1-7d)	1.211.65 ± 703.24	YG* (n=10)
	T2: at bonding (8-14d)	1.397.66 ± 940.76
	T3: arch initial insertion (15-21d)	1.100.20 ± 703.24
Jha et al.²⁹, 2020 (IgA) [µg/mL]	T1: baseline	137.45 ± 2.5 \| 139.73 ± 2.3	G1* (n=20) \| G2” (n=20)
	T2: 3mo ~	164.0 ± 3.23 \| 144.27 ± 5.32
	T3: 6mo ~	166.4 ± 3.65 \| 145.8 ± 6.02
Mediators
Flórez-Moreno et al.²⁴, 2013 (sRANKL) [pg/mL]	T1: baseline	Md 2.90 (1.65-8.55)	YG* (n=14)
	T2: 24-48h ~	Md 2.70 (1.75-4.15)
	T3: 2w ~	Md 3.10 (1.85-3.10)
	T4: 5w ~	Md 3.10 (1.90-5.55)
	T5: 8w ~	Md 8.40 (3.10-27.4)
Flórez-Moreno et al.²⁴, 2013 (OPG) [pg/mL]	T1: baseline	Md 96.8 (31.0-190.1)	YG* (n=14)
	T2: 24-48h ~	Md 74.3 (46.3-122.6)
	T3: 2 w ~	Md 83.6 (41.1-203.7)
	T4: 5 w ~	Md 77.9 (24.0-124.1)
	T5: 8 w ~	Md 62.0 (2.8-104.4)
Maan & Patil²⁸, 2019 (IL‐1β) [pg/mL]	T1: baseline (before arch)	3.62 ± 1.03 \| 4.06 ± 2.21	YG×* (n=10) ♀=5 \| ♂=5
	T2: 1h ~ (after arch)	12.14 ± 2.86 \| 11.26 ± 3.13
	T3: 1mo ~ (before new arch)	6.82 ± 1.64 \| 5.14 ± 0.88
	T4: 1mo ~ (after new arch)	8.92 ± 1.44 \| 7.10 ± 1.00
Maan & Patil²⁸, 2019 (PGE2) [pg/mL]	T1: baseline (before arch)	41.54 ± 9.85 \| 53.46 ± 15.87	YG×* (n=10) ♀=5 \| ♂=5
	T2: 1h ~ (after arch)	69.90 ± 13.17 \| 134.62 ± 26.06
	T3: 1mo ~ (before new arch)	43.18 ± 5.73 \| 96.22 ± 12.70
	T4: 1mo ~ (after new arch)	51.36 ± 5.16 \| 110.42 ± 10.83

GRADE assessment
Number of studies (participants)	Summary of findings	Certainty
Effects of orthodontic treatment on salivary electrolytes
1 (24 participants)	The evidence showed little effect of the treatment on increasing Ca, Pi and K and reducing Na and Cl, only after the first month. The control group remained stable throughout the study period.	⨁ Very low^a,b,c
Effects of orthodontic treatment on salivary enzymes
2 (32 participants)	The evidence showed moderate effect of the treatment on increasing ALP and LDH during the first month, and increasing MMP8 and MMP9 during all treatment period.	⨁ Very low^a,b,d
Effects of orthodontic treatment on salivary hormones
3 (70 participants)	The evidence showed little effect of rapid maxillary expansion on increasing cortisol during all expansion period. However, the evidence also showed no effect of treatment with fixed orthodontic appliances on cortisol levels. Regarding leptin levels, evidence showed little reduction six weeks after treatment.	⨁ Very low^a,b
Effects of orthodontic treatment on salivary immunoglobulins
3 (81 participants)	The evidence showed little to no effect of orthodontic treatment on salivary immunoglobulin concentrations.	⨁ Very low^a,b,d
Effects of orthodontic treatment on mediators
2 (24 participants)	The evidence showed nonlinear trends in the levels of the biomarkers through time.	⨁ Very low^a,b,d

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