Malocclusion is a common dental condition that may be corrected by orthodontic treatment involving fixed or removable appliances.1 Preserving the integrity of the periodontal tissues is a priority during treatment, and has led to the definition of specific hygiene protocols for orthodontic patients.2 The principle of orthodontic treatment is based on corrective tooth movement, which is an occlusal reconstruction process involving alveolar bone remodelling and changes in bone morphology whose objectives are to correct function and establish dental and facial aesthetics.3,4
Fixed appliances may offer conventional-ligation (CLA) or self-ligation (SLA) mechanisms. The main consideration of CLA is to achieve better positional crown control in addition to improving root position following the closure of spaces. To retain an arch wire within a bracket slot, either stainless steel ligatures which can vary in size (0.009 to 0.014 inches) or elastic modules (circular shaped elastomers that can be deformed to engage bracket tiewings and maintain the arch wire), may be used.5 As an alternative to CLA, two types of SLA have been developed: an active closing clip that exerts pressure on the arch wire, which in turn improves rotation control, and a passive bracket with a closing mechanism that transforms an open slot into a tube.6
The biggest challenge of orthodontic therapy is to complete treatment with the least impact on the oral tissues (Figure 1). Periodontal disease is a chronic inflammatory process, initiated by a dysbiotic bacterial biofilm7 that leads to the destruction of investing tissue. Even though periodontal disease has been linked to a defined microbial biofilm composition, the effect of polymicrobial disruption on host homeostasis to periodontitis progression remains poorly understood.8 Recently, metagenomic studies have estimated that the number of bacteria found in the periodontal tissues is up to 600 species.9
Fixed orthodontic appliance materials affect the adhesion of bacteria and the accumulation of biofilm as a result of the roughness of the devices, the free energy of the surfaces, and other physicochemical properties of the biomaterials which influence the retentive capacity of the biofilm.10 In addition to enamel decalcification that leads to incipient caries, periodontal damage can also occur11 with associated local and systemic effects identified as root resorption, psychological disorders, gastrointestinal complications, allergic reactions, infective endocarditis, and chronic fatigue syndrome.12
Knowing which microbial species colonises orthodontic appliances is important to plan strategies and to implement specific preventive control measures during treatment.13 Few studies have investigated periodontal pathogens when different types of fixed appliances have been placed. Previous research has determined that the microorganisms of Socransky’s ‘red’ complex are considered more pathogenic compared to the ‘orange’ complex regarding the aetiology of periodontal disease.14 It is therefore, important to assess the changes in the frequency and load of Socransky’s red and orange complexes that colonise orthodontic appliances and to know the periodontal risk.15 The present study aimed to explore dysbiotic changes in periodontal pathogenic bacteria associated with different types of fixed orthodontic appliances in a Northwestern Mexican population.
Biofilm samples were collected from systemically healthy patients of both genders, who provided signed written informed consent. The study was approved by the ethics committee of the Autonomous University of Sinaloa and complied with the principles of the Declaration of Helsinki. The type of sampling was non-probabilistic. DNA concentrations up to 100 ng or greater were selected using spectrophotometry (nanodrop) and corroborated with electrophoresis tests. In total, 92 biofilm samples from patients were collected which subsequently formed three groups: patients without orthodontic appliances (N:31, group C) patients with conventional-ligating appliances (N:30, group CLA), and patients with self-ligating appliances (N:31, group SLA). The exclusion criteria were patients with an active diagnosis of COVID-19, who had received antibiotic treatment within the previous two weeks, those diagnosed with convulsive diseases, those with injuries or oral pathologies that were not the product of orthodontic treatment (trauma, lacerations, surgeries), those with appliances other than conventional or self-ligating, pregnant patients, or those who, during the previous 2 hours, had carried out oral hygiene, ingested food or administered mouthwash. The clinical characteristics of the patients selected for biofilm sampling were those undergoing orthodontic treatment without extractions, those with mild or moderate crowding whose periodontal status was clinically healthy at the beginning of the treatment and at the time the sample was taken, patients without bleeding at the time of sample collection and who did not require any type of surgical procedure, and patients with a class I molar, class II and III molar relationship of no more than 4 mm.
The patients refrained from eating, drinking, brushing their teeth, and chewing gum for at least 2 hr before the sampling procedure. Sample collection was performed between 8:30 am and 10:30 am before any oral examination or manipulation, thus avoiding disrupting the oral microbiota.16 Biofilm samples were taken from the areas adjacent to the fixed orthodontic appliances on the lower incisors using a CK6 instrument. The biofilm samples were placed in microtubes containing 500 µL of phosphate-buffered saline (PBS) 1X. The samples were kept at 4°C during transportation and stored at -80°C until DNA extraction.
Genomic DNA was obtained from the biofilm samples using the Puregene Gentra Systems kit (DNA Isolation Kit, Minneapolis, MN, USA), according to the manufacturer’s instructions and following their protocol.
The presence and load of periodontal pathogenic bacteria were evaluated by quantitative polymerase chain reaction (qPCR). Sequences of the primers and the conditions applied for real-time PCR are shown in Table I. All species-specific primers targeted the variable regions of the 16S ribosomal RNA gene of the three strains. The qPCR conditions are listed in Table II. All assays were performed using SoFast EvaGreen Supermix (Bio-Rad) in a real-time PCR system (QuantStudio 12K Flex Real-Time PCR System, Thermo Fisher Scientific, Waltham, MA, USA) and the load was determined using the Ct threshold method, assigning 100% of the bacterial load to the C group.14
Primers and qPCR conditions to measure bacterial load.
Specific initiators | Amplified products (pb) | Bacteria |
---|---|---|
FTd 5′-TAATACCGAATGTGCTCATTTACAT-3′ | 316 | |
RTd 5′-TCAAAGAAGCATTCCCTCTTCTTCTTA-3; | ||
FFn 5′-AGAGTTTGATCCTGGCTCAG-3′ | 408 | |
RFn 5′-GTCATCGTGCACACAGAATTGCTG-3 | ||
FPi 5′-TTTGTTGGGGAGTAAAGCGGG-3′ | 576 | |
RPi 5′-TCAACATCTCTGTATCCTGCGT-3′ |
qPCR conditions to measure bacterial load.
Treponema denticola | Fusobacterium nucleatum | Prevotella intermedia | Cycles | Reading melt curve | |
---|---|---|---|---|---|
Desn. initial | 96°C-5 m | 96°C-5 m | 96°C-5 m | 30 | 80°C |
Desn. nat | 95°C-30 s | 95°C-30 s | 95°C-30 s | ||
Align | 55°C-45 s | 56°C-45 s | 56°C-45 s | ||
Ext | 72°C-45 s | 72°C-45 s | 72°C-45 s | ||
Ext final | 72°C-10 m | 72°C-10 m | 72°C-10 m | ||
Conservation | 4°C | 4°C | 4°C |
For a comparison of frequencies between groups, a chi-squared analysis was applied; for bacterial load analysis, a one-way ANOVA was followed by a Dunnet test to compare the conventional and self-ligating groups with the control group.
The presence of periodontal pathogens was observed in all groups, but
The percentages of bacterial load obtained using the Ct threshold method, are summarised in Figure 3. Although the differences in
The CLA and SLA groups were subdivided into those with <12 months and >12 months of treatment. The frequency of
The present results confirm that dysbiotic changes are seen in patients wearing CLA and SLA. A dysbiotic microbiome is present when the diversity and proportions of normal species are disturbed.17 In this regard, dysbiosis generated by fixed orthodontic appliances is responsible for enamel demineralisation but also periodontal disease.18 The current results revealed that all tested bacteria showed a higher load in SLA when compared with the C group.
It is known that after the placement of fixed appliances, an initial biofilm is formed on material surfaces, often resulting in a worsening of the periodontal status. After an initial period of change, the host-microorganism balance can be restored.21 However, the appliance material surface may be altered over time because of contact with food and drink, abrasive oral hygiene measures, or corrosive processes.22 Since the attachment of appliances to the teeth makes it difficult to mechanically remove debris, the environment favours the formation of a biofilm.10,23 Clinically, the bacterial side effects of the appliances become apparent as plaque-associated gingivitis, an increase in pocket probe depth, and bleeding on probing.24 A recent study has explored the presence of bacteria in saliva and while salivary bacterial levels are helpful to indicate risk factors for the development of disease,
The present study focused on metal braces for both CLA and SLA, to minimise the intrinsic variability associated with other materials such as ceramics. Previous studies have reported a greater accumulation of biofilm in CLA metal braces compared to aesthetic material braces.10 A likely reason for a greater accumulation of biofilm on metal braces compared to aesthetic braces might relate to the nature of the ceramic surface, although aesthetic braces are usually larger and therefore have a greater surface area for biofilm adherence. However, ceramic surfaces are smoother, which makes it difficult for the biofilm to adhere and therefore facilitates mechanical removal.26
More recently, Gujar
While
While females displayed a greater positivity for periodontal pathogens than males, except for
The present study is limited by the lack of follow-up of patients after sample collection and by the clinical variables present over time to corroborate that dysbiotic changes lead to periodontal disease. Further studies need to be conducted to support this hypothesis and must consider different types of appliances, their design, orthodontics treatment, environmental, and cultural factors, related to diet, smoking, and oral hygiene.
The periodontal pathogens identified as