Elucidating salivary biomarkers in post-orthodontic patients using enzyme-linked immunosorbent assay (ELISA)

Tyrosine protein kinase Tec (Tec) is known to activate calcium signalling, which plays a crucial role in bone remodelling. Nuclear envelope spectrin repeat protein 2 (Nesprin-2) is an outer nuclear membrane protein that contributes to cellular mechanosensation, particularly in osteocytes, which influence bone resorption. Zinc finger proteins ZNF729 and ZNF648 are involved in various biological functions and are believed to maintain the dynamic balance of the “pressure tension theory” during orthodontic tooth movement. This study aimed to elucidate the expression patterns of these proteins during the retention phase using the enzyme linked immunosorbent assay (ELISA). Unstimulated whole saliva (5 ml) was collected via passive drooling from 10 post orthodontic patients and 10 healthy controls. Samples from the treated group were collected at debonding (T1), six months post debonding (T2), and one year post debonding (T3). Salivary concentrations of Tec, Nesprin-2, ZNF648, and ZNF729 were analysed using commercially available ELISA kits. Clinical parameters, namely incisor irregularity, intercanine width, and intermolar width, were recorded at each timepoint in the treated group. Independent sample t-tests were used to compare T1 protein levels with controls, while repeated measures ANOVA assessed changes over time. Spearman’s correlation analysis explored associations between protein levels and clinical measurements. All four proteins were active during the post orthodontic retention phase. At T1, Tec, ZNF729, and ZNF648 levels were significantly lower than those in the control group. Nesprin-2 also showed reduced levels at T1, but without statistical significance. Across timepoints, all protein levels increased. Tec exhibited a gradual rise from T1 to T3; Nesprin-2 and ZNF729 showed marked increases between T2 and T3; and ZNF648 rose sharply from T1 to T2. By T3, all protein concentrations had either approached or surpassed control levels. In terms of clinical changes, maxillary parameters including incisor irregularity, intercanine width, and intermolar width remained stable over time. In the mandible, although the irregularity index showed a significant reduction, it was considered clinically acceptable and did not reflect true relapse. Minimal changes were observed in mandibular intercanine and intermolar widths. Since no subject exhibited a relapse greater than 3 mm, a relapse group could not be formed, and comparisons between relapse and non relapse groups were not feasible. Correlations between protein levels and clinical data at T1 were not included in the main analysis, as T1 served as the baseline for both protein expression and clinical parameters. From T2 to T3, some variations in correlations were observed, potentially due to different cellular mechanisms, protein expression dynamics, and local inflammatory responses. The strongest and most significant correlations were observed one year into the retention phase between Tec and ZNF729 with the upper and lower incisors, as well as the intermolar width of the maxilla. This suggests that protein expressions were consistent with changes in tooth movement at T3. The findings of this study indicate that Tec, Nesprin-2, ZNF729, and ZNF648 have potential as biomarkers for monitoring the outcomes and stability of completed orthodontic treatment.