Transcriptome analysis of granulation tissue from periodontal osseous defects

Abstract

Background: Granulation tissue is routinely discarded in periodontal surgical procedures, but few studies have characterized it. The present study aimed to compare global gene expression in granulation tissue derived from different types of periodontal osseous defects.

Methods: Total RNA was isolated from granulation tissue harvested during routine periodontal surgical procedures. Nineteen sites were analyzed-seven infrabony, six suprabony, and six furcation defects. Following quality control checks, samples underwent bulk mRNA sequencing (20-30 million read pairs per sample) before bioinformatic analyses utilizing gene ontology and associated pathway/enrichment analyses.

Results: No statistically significant differentially expressed genes (DEG) were detected between different osseous defect types. An increase in the expression of 11 genes with a false discovery rate of <0.05 was detected when a comparison was made in terms of healing duration post nonsurgical periodontal therapy (NSPT). Notably, the genes involved included those regulating collagen synthesis and osteogenic activity. Analysis based on sex differences revealed 38 DEG. Gene enrichment analysis revealed that 24 DEG without Y-linked attachment are mostly involved in immune regulation.

Conclusions: Routinely discarded periodontal granulation tissue exhibits epithelial characteristics due to a substantial period of maturation post NSPT. Confirmation of ongoing maturation beyond traditional periodontal re-evaluation timepoints warrants further investigation on tissue differentiation potential. More research is needed to elucidate the role of sex as a biological variable affecting periodontal immune response.

Plain language summary: Granulation (wound) tissue is routinely removed during gum (periodontal) surgical procedures, but knowledge on its characteristics is scarce. This study aimed to compare gene expression in granulation tissue derived from different types of periodontal bone defects via high-throughput RNA sequencing. Total RNA was extracted from 95 samples harvested from gum disease patients during surgery. After quality control checks, 19 samples (seven infrabony, six suprabony, and six furcation defects) were eligible for sequencing. Subsequent analyses were done utilizing software with known cell biological pathways and processes. Analysis revealed no differentially expressed genes (DEG) in terms of periodontal defect category. There was statistically significantly increased expression of 11 genes when a comparison was made in terms of healing duration following deep scaling treatment. These genes are involved in collagen synthesis and osteogenic activity. Interestingly, analysis based on sex differences detected 38 DEG. Gene enrichment analysis revealed that 24 DEG without association with Y chromosomes are mostly involved in the regulation of immune system response. Routinely discarded periodontal granulation tissue exhibits lining cell characteristics that change over time following deep scaling treatment. More research is needed to unravel the role of sex as a biological variable affecting periodontal immune response in this type of tissue.

Keywords: RNA sequencing; granulation tissue; periodontitis; regeneration; wound healing.

FIGURE 1

Gene profile of top 20 most highly expressed genes across all defect morphologies ranked by normalized gene count. (A) Bar chart of expressed genes indicated by normalized gene count averaged across all samples. (B) Scatter plot of GO‐annotated biological processes generated using gene ratio and FDR values specific to collective analysis of genes. FDR, false discovery rates; GO, gene ontology.

FIGURE 2

Analysis of expression of genes at FDR < 0.05 when compared over two timepoints of harvesting (<6 vs. ≥6 months after completion of NSPT). Box plots in Figures 2A–C were generated by comparing relative expression of genes with log2 fold difference between two timepoints. Figure 2D lists genes with corresponding functions. FDR, false discovery rates; NSPT, nonsurgical periodontal therapy.

FIGURE 3

Collective analysis of genes expressed at FDR < 0.05 when compared over two timepoints of harvesting (<6 months vs. ≥6 months post NSPT). (A) Heatmap of annotated genes between samples belonging to two timepoints. (B) Scatter plot of GO‐annotated biological processes upregulated at later post‐NSPT healing period. (C) Scatter plot of KEGG‐annotated pathways upregulated at later post‐NSPT healing period. (D) Scatter plot of Reactome‐annotated pathways upregulated at later post‐NSPT healing period. Gene abundance was generated using DESeq2 (v2.11.40.8) and annotated via GRCh38 reference genome. Scatter plots were generated using gene ratio and FDR values specific only to collective analysis of genes. FDR, false discovery rates; GO, gene ontology; GT‐A, granulation tissue harvested at <6 months following completion of NSPT; GT‐B, granulation tissue harvested at ≥6 months following completion of NSPT; KEGG, Kyoto Encyclopedia of Genes and Genomes; NSPT, nonsurgical periodontal therapy.

FIGURE 4

Collective analysis of DEG comparing female and male samples. (A) Heatmap of DEG (female vs. male). (B) Volcano plot of DEG (female vs. male) with red dots indicating upregulated genes in males and blue dots indicating downregulated genes. (C) Scatter plot of GO‐annotated biological processes upregulated in males. (D) Scatter plot of Reactome‐annotated pathways upregulated in males. Scatter plots were generated using gene ratio and p values specific to collective analysis of genes. Y‐linked genes were excluded from enrichment analyses. DEG, differentially expressed genes; F, female; GO, gene ontology; M, male.