Identification of Salivary Exosome-Derived miRNAs as Potential Biomarkers of Bone Remodeling During Orthodontic Tooth Movement

Abstract

Orthodontic tooth movement (OTM) is a complex process involving bone remodeling, and is regulated by various molecular factors, including microRNAs (miRNAs). These small, non-coding RNAs are critical in post-transcriptional gene regulation and have been implicated in the modulation of osteoclast and osteoblast activity during OTM. This study aimed to explore the expression profiles of salivary exosome-derived miRNAs during OTM to identify potential biomarkers that could provide insights into the biological processes involved in orthodontic tooth movement. Saliva samples were collected from 15 patients at three time points: before treatment (Day 0), 7 days after the treatment's onset (Day 7), and 40 days after the treatment's onset (Day 40). The exosomes were isolated, and the miRNAs were extracted and sequenced. A differential expression analysis and gene ontology (GO) enrichment were performed to identify the miRNAs involved in osteoblast and osteoclast differentiation. Out of the 1405 detected miRNAs, 185 were analyzed. Several miRNAs were associated with bone-remodeling processes. The statistically significant finding was the downregulation of hsa-miR-4634 after 40 days of treatment. These findings contribute to the understanding of miRNA regulation in orthodontics and may have broader implications for skeletal disorders, such as osteoporosis.

Keywords: extracellular vesicles; gene expression regulation; mechanical stress; osteogenesis; saliva.

Figure 1

TEM image showing EVs isolated from saliva fluid pooled from multiple patient samples. The scale bar represents 500 nm. Arrows point to two representative structures.

Figure 2

Nanoparticle tracking analysis (NTA) of EVs isolated from pooled saliva samples collected from multiple patients, presenting a size distribution plot and the mean size of each peak.

Figure 3

Heatmap of the 25 most variably expressed miRNAs ranked by p-value < 0,05. The expression levels (log2CPM) are shown across saliva samples with the miRNA. The color scale ranges from blue (low expression) to red (high expression).

Figure 4

MDS plot showing the separation of samples across three time points (Day 0, Day 7, and Day 40) based on the log fold change (logFC). Each point represents a sample, with the colors indicating the corresponding time points: orange for Day 0, green for Day 7, and purple for Day 40. The leading dimensions 1 and 2 explain 29% and 12% of the variance, respectively.

Figure 5

Boxplot of hsa-miR-4634 expression levels (log2 CPM) at three time points (Day 0, Day 7, and Day 40). The red square represents the mean expression level, and the black dots represent individual samples. The black line in each box indicates the median value, and the density plot on each side shows the distribution of expression values.

Figure 6

Bar plots showing the log fold changes (logFC) of miRNAs ranked by p-value < 0.05 across different time point comparisons: Day 0 to Day 7, Day 7 to Day 40, and Day 0 to Day 40. Each bar represents an miRNA, with positive and negative values indicating upregulation and downregulation, respectively, between the compared time points. The data presented represent the combined analysis of all the included samples rather than individual patient data.