RNAseq of gingival fibroblasts exposed to cross-linked hyaluronic acid

Hyaluronic acid was proposed to support soft tissue recession surgery and guided tissue regeneration. Nevertheless, the molecular mechanisms through which hyaluronic acid modulates the response of the connective tissue cells remain elusive. To elucidate the impact of hyaluronic acid on the connective tissue cells, we employed bulk RNA sequencing to unravel the changes in the genetic signature gingival fibroblasts exposed to 1.6% cross-linked hyaluronic acid and 0.2% natural hyaluronic acid hyaluronic acid. Transcriptome-wide changes were modest, even when implementing a minimum of 1.5 log2 fold-change and a significance threshold of 1.0 -log10; only a dozenth gene was differentially expressed and ranked according to the Manhattan distance. Upregulated were PLK3, SLC16A6, IL6, HBEGF, DGKE, DUSP4, PTGS2, FOXC2, ATAD2B, NFATC2, and downregulated were MMP24 and PLXNA2. RT-PCR analysis supported the impact of hyaluronic acid on increasing expression of the selected gene panel. The findings from bulk RNA sequencing suggest that gingival fibroblasts only show a weak response when exposed to hyaluronic acid. Overall design: Explant cultures of gingiva from three healthy individuals who provided informed consent were used to isolate gingival fibroblasts. Ethical approval from the Ethical Committee of the Medical University of Vienna (EK #631/2007) is available. These cells were isolated and expanded in DMEM supplemented with 10% fetal calf serum (FCS) and 1% antibiotics (Invitrogen Corporation, Carlsbad, CA). A pool of the three donors of gingival fibroblasts, all at low passage, were seeded at 30.000 cells/cm2 into 24-well plates and exposed to hyaDENT BG (Regedent AG; Zurich, Switzerland) a medical device consistent with 16.0?mg/mL of hyaluronic acid cross-linked and 2.0?mg/mL of hyaluronic acid with sodium chloride. The gingival fibroblasts were exposed to a 4.5-fold diluted hyaDENT BG, resulting in a final concentration of a total of 4 mg/mL cross-linked and native HA for 6 hours in serum-free DMEM followed by isolation of total RNA.