Biomarker changes in gingival crevicular fluid during

orthodontic treatment with clear aligners - a systematic review

Aušrinė Kuzmauskaitė

1

, Alvyda Žarovienė

2

1

Independent researcher

2

crevicular fluid associated with orthodontic treatment using clear aligners.

Material and methods. This systematic review was conducted in accordance with the Preffered Reporting Items

for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive search was conducted on

PubMed, Science Direct, ProQuest, Web of Science and Cohrane Library electronic databases from July 1st until

July 31 2025. In accordance with the predefined inclusion and exclusion criteria, studies assessing changes in

inflammatory cytokines and bone metabolism biomarkers during orthodontic treatment with clear aligners were

included in this review.

Results. A total of 403 records were identified through electronic database searches, and after duplicate removal

and screening, 9 studies were included in the review. All examined inflammatory cytokines (interleukin-1 beta

(IL-1β), interleukin-1 alpha (IL-1α), tumor necrosis factor- alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-

biological activity of aligner therapy and highlight the need for further studies to clarify the clinical significance

of these molecular changes.

Keywords: gingival crevicular fluid, clear aligners, orthodontic treatment, cytokines, inflammatory biomarkers,

bone metabolism biomarkers.

1. Introduction

Orthodontic tooth movement (OTM) is a

mediators, which together orchestrate alveolar bone

resorption by osteoclasts and bone formation by

osteoblasts, ultimately allowing tooth movement

[1]. As these molecular changes are reflected in the

periodontal environment, gingival crevicular fluid

(GCF) serves as a valuable medium for detecting

cytokines and other biomarkers related to

orthodontic tooth movement [2].

Gingival crevicular fluid (GCF) is an exudate of

inflammatory origin that derives from the gingival

plexus and adjacent periodontal tissues. It contains

proteins, tissue breakdown products, growth factors,

electrolytes, cytokines, as well as other low-

underlying OTM [4]. Cytokines are low-molecular-

weight proteins (<25 kDa) secreted predominantly

in an autocrine or paracrine manner, and many of

them participate in the recruitment and activation of

immune cells at sites of localized force application

or stress within the physiological environment [4,5].

Moreover, bone metabolism biomarkers represent

valuable tools for monitoring the complex dynamics

of bone remodeling. By mirroring the interplay

between resorption and formation, they provide

involved in OTM and treatment-induced bone

responses [4,6].

better oral hygiene maintenance, and increased

patient comfort [9]. Unlike fixed appliances,

aligners produce intermittent forces on teeth due to

their removability, and these forces tend to fluctuate

over time [10]. According to Kuncio et al. [11], tooth

movement achieved with aligners may not

correspond to the typical stages of orthodontic

movement outlined by Krishnan and Davidovitch

[12]. However, a major limitation of clear aligners is

that they are not suitable for treating all types of

malocclusions, particularly those requiring complex

tooth movements [13,14].

It is well established that cytokine activity and bone

of the molecular mechanisms underlying OTM with

aligners. Therefore, the aim of this systematic

review was to synthesize the available evidence on

cytokines and BMBs changes in GCF during

orthodontic treatment with clear aligners.

2. Materials and Methods

This systematic review was conducted in accordance

with the Preferred Reporting Items for Systematic

Reviews and Meta-Analyses (PRISMA) guidelines

Reviews (PROSPERO) under the identifier

CRD420251120919.

orthodontic treatment (P) with clear aligners (I),

where changes in gingival crevicular fluid were

assessed across multiple treatment time points (C).

The outcomes (O) were defined as quantitative

alterations in the composition of GCF, particularly

in biomarkers related to inflammation and bone

metabolism. The review included prospective

clinical studies, comprising pilot, comparative and

cohort designs (S).

2.2 Eligibility Criteria

The inclusion criteria included:

2.4 Search strategy and study selection

aligners OR aligners OR invisalign) AND

(inflammatory OR cytokine OR bone metabolism

OR biomarkers). The search strategy was

appropriately modified and applied to the other

electronic databases included in this review.

The process of study selection was independently

conducted by two reviewers. The procedure

involved an initial screening of titles and abstracts,

followed by full-text assessment of potentially

eligible articles. Duplicate records were identified

and removed using Zotero reference management

software version 7.0.24 (Corporation for Digital

Scholarship, USA). Any disagreements that arose

during this process were resolved through discussion

population details (number of participants, sex

distribution, and mean age). Information was also

collected on aligner type, GCF sampling method,

and the GCF collection site. In addition, data

regarding the biomarkers analyzed, their temporal

changes during aligner therapy, and the main

conclusions were extracted.

2.6 Risk of bias assessment

Risk of bias in the included non-randomized studies

3.1. Results

3.1 Study selection

were included in this systematic review.

3.2 Study charasteristics

The main characteristics of the included studies are

summarized in Table 1. All included articles were

designed as prospective studies, comprising pilot

studies [18-20], comparative studies [4,21,22],

observational studies [25,26], and one cohort study

[25]. Collectively, the studies assessed 152 patients

undergoing orthodontic treatment with clear

and in several cases, age was presented for overall

study population rather than specifically for the clear

aligner group. Two studies did not specify the sex

distribution of participants [20, 25].

Most studies specified the use of Invisalign® clear

aligners, while one study investigated aligners

manufactured by K Line [20]. A few studies did not

report the manufacturer or brand [21,22,24].

Gingival crevicular fluid (GCF) was collected using

either PerioPaper® strips or microcapillary pipettes,

with sample isolation achieved using cotton rolls,

sterile gauze, or gentle air-drying. Sampling sites

varied widely, including maxillary and mandibular

Table 1. Characteristics of included studies.

3.3 Results of individual studies

The main outcomes of the nine included studies are

The following sections provide a detailed synthesis

of the findings, grouped by biomarker category.

observed a sharp elevation as early as day 1, while

Other inflammatory cytokines were evaluated less

frequently across the included studies. IL-1α and IL-

Two of the nine included studies did not identify any

statistically significant changes in the analyzed

biomarkers. Chami et al. [19] reported no significant

alterations in IL-1β, IL-8, TNF-α, IL-17, or GM-

CSF levels during the observation period, while

Chen et al. [23] also reported no significant

differences in IL-1β or TNF-α concentrations.

OPG and elevated OPN levels during the same

3.4 Quality assessment

The results of the risk of bias assessment for the

included non-randomized studies are summarized in

Table 3. According to the ROBINS-I tool, two

studies were identified as having a serious overall

risk of bias due to inadequate control of confounding

factors [22,27], while the remaining studies were

consistently rated as having moderate overall risk of

Table 2. Outcomes.

GM-CSF, granulocyte-macrophage colony-stimulating factor; IL 1α, interleukin-1 alpha; IL 1β, interleukin-1

beta; IL-2, interleukin-2; Il-6, interleukin-6; Il-8, interleukin-8; IL-17, interleukin-17; OPG, osteoprotegerin;

Risk of bias domains

Study

+

+