Platelet-rich fibrin (PRF) is prepared by spontaneous coagulation of fractionated blood. When squeezed between two plates, PRF is separated into solid PRF membranes and a liquid exudate, the PRF serum. The question arises regarding how much the overall activity remains in the PRF membranes and what is discarded into the PRF serum. To this end, we have exposed gingival fibroblasts to lysates prepared from PRF membranes and PRF serum, followed by bulk RNA sequencing. A total of 268 up- and 136 down-regulated genes in gingival fibroblasts exposed to PRF membrane lysates were significantly regulated under the premise of a minimum log2 with 2.5-fold change and a minus log10 significance level of two, respectively. PRF serum only caused 62 up- and 32 down-regulated genes under these conditions. Among the 46 commonly up-regulated genes were CXCL1, CXCL5, CXCL6, CXCL8, IL33, IL6, and PTGS2/COX2, stanniocalcin-1—all linked to an inflammatory response. PRF membrane lysates further increased chemokines CCL2, CCL7, CXCL2, CXCL3, and IL1R1, IL1RL1, and IL1RN, as well as the paracrine factors IL11, LIF, IGF1, BMP2, BMP6, FGF2, and CCN2/CTGF, and all hyaluronan synthases. On the other hand, PRF serum increased DKK1. The genes commonly down-regulated by PRF membrane lysates and PRF serum included interferon-induced protein with tetratricopeptide repeats (IFIT1, IFIT2, IFIT3) and odd-skipped-related transcription factors (OSR1 and OSR2), as well as FGF18 and GDF15, respectively. Taken together, PRF membrane lysates, compared to PRF serum, cause a more complex response in gingival fibroblasts, but each increased chemokine expression in gingival fibroblasts.

Tannic acid (TA), a natural polyphenol with antiphlogistic and crosslinking properties, is a versatile component of hydrogel that can be delivered to inflammatory sites in oral applications. However, the impact of TA dampening an inflammatory response in oral cells remains to be shown. We, therefore, established a bioassay where chemokine expression is induced by exposing gingival fibroblasts and HSC2 oral squamous carcinoma cells to IL1β and TNFα. Additionally, gingival fibroblasts were stimulated with saliva and poly I:C HMW to trigger chemokine production. Our findings demonstrate that TA effectively reduced the expression of CXCL1, CXCL2, CXCL8, and CXCL10—in gingival fibroblasts and HSC2 cells—without affecting cell viability. This effect was further confirmed by immunoassays for CXCL8. Moreover, we observed that TA decreased ERK, JNK, and p65 phosphorylation in gingival fibroblasts and partially inhibited NF-κβ/p65 nuclear translocation. Notably, TA released from a gellan gum hydrogel retained its ability to suppress chemokine expression in gingival fibroblasts. These in vitro findings provide insights into the anti-inflammatory properties of TA in oral cells and introduce gellan gum hydrogel as a delivery vehicle paving the way for future preclinical research.

Platelet-rich fibrin (PRF), the coagulated plasma of fractionated blood, is widely used to support tissue regeneration in dentistry, and the underlying cellular and molecular mechanisms are increasingly being understood. Periodontal connective tissues steadily express CXCL8, a chemokine that attracts granulocytes and lymphocytes, supporting homeostatic immunity. Even though PRF is considered to dampen inflammation, it should not be ruled out that PRF increases the expression of CXCL8 in gingival fibroblasts. To test this hypothesis, we conducted a bioassay where gingival fibroblasts were exposed to PRF lysates and the respective serum. We show here that PRF lysates and, to a lesser extent, PRF serum increased the expression of CXCL8 by the gingival fibroblasts, as confirmed by immunoassay. SB203580, the inhibitor of p38 mitogen-activated protein kinase, reduced CXCL8 expression. Consistently, PRF lysates and, to a weaker range, the PRF serum also caused phosphorylation of p38 in gingival fibroblasts. Assuming that PRF is a rich source of growth factors, the TGF-β receptor type I kinase inhibitor SB431542 decreased the PRF-induced expression and translation of CXCL8. The findings suggest that PRF lysates and the respective serum drive CXCL8 expression by activating TGF-β and p38 signaling in gingival fibroblasts.

Dimethyl fumarate (DMF), originally proposed to treat multiple sclerosis, is considered to have a spectrum of anti-inflammatory effects that effectively control periodontitis, mainly when applied with a hydrogel delivery system. Chemokine expression by gingival fibroblasts is a significant driver of periodontitis; thus, hydrogel-based strategies to deliver DMF, which in turn dampen chemokine expression, are of potential clinical relevance. To test this approach, we have established a bioassay where chemokine expression is induced by exposing gingival fibroblast to IL1β and TNFα, or with saliva. We show herein that DMF effectively reduced the expression of CXCL8, CXCL1, CXCL2, and CCL2—and lowered the phosphorylation of ERK and JNK—without affecting cell viability. This observation was confirmed by immunoassays with CXCL8. Consistently, the forced chemokine expression in HSC2 oral squamous epithelial cells was greatly diminished by DMF. To implement our hydrogel-based delivery system, gingival fibroblasts were cocultured with gellan gum hydrogels enriched for DMF. In support of our strategy, DMF-enriched gellan gum hydrogels significantly reduced the forced chemokine expression in gingival fibroblasts. Our data suggest that DMF exerts its anti-inflammatory activity in periodontal cells when released from gellan gum hydrogels, suggesting a potential clinical relevance to control overshooting chemokine expression under chronic inflammatory conditions.

Hyaluronic acid was proposed to support soft tissue recession surgery and guided tissue regeneration. The molecular mechanisms through which hyaluronic acid modulates the response of connective tissue cells remain elusive. To elucidate the impact of hyaluronic acid on the connective tissue cells, we used bulk RNA sequencing to determine the changes in the genetic signature of gingival fibroblasts exposed to 1.6% cross-linked hyaluronic acid and 0.2% natural 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 of genes were differentially expressed. Upregulated genes were PLK3, SLC16A6, IL6, HBEGF, DGKE, DUSP4, PTGS2, FOXC2, ATAD2B, NFATC2, and downregulated genes were MMP24 and PLXNA2. RT-PCR analysis supported the impact of hyaluronic acid on increasing the expression of a selected gene panel. The findings from bulk RNA sequencing suggest that gingival fibroblasts experience weak changes in their transcriptome when exposed to hyaluronic acid.

Objective: Titanium surface modifications improve osseointegration in dental and orthopedic implants. However, soft tissue cells can also reach the implant surface in immediate loading protocols. While previous research focused on osteogenic cells, the early response of soft tissue cells still needs to be better understood. Material and methods: We have established a bioassay to this aim where human gingival fibroblasts, HSC2 oral squamous carcinoma cells, and murine bone marrow cells were cultured onto titanium discs or exposed to the respective supernatants for overnight. Modifications were double acid-etching (SLA), and coating with simulated body fluid (SBF) with or without odanacatib (ODN), a selective cathepsin K inhibitor reducing bone resorption. Results: Our findings indicate that direct contact with titanium discs, with all surface modifications, slightly reduces cell viability. Growing gingival fibroblasts on discs consistently showed a trend toward increased IL8 expression. In HSC2 cells, this setting significantly increased IL1 and IL8 expression, confirmed by the immunoassay. Murine bone marrow macrophages also showed an increase in IL1 and IL6 expressions. Supernatants of the respective discs failed to cause these changes. Although ODN coating inhibited cathepsin K, osteoclastogenesis remained unchanged. Conclusions: These findings suggest that titanium discs do not provide a favorable in vitro surface for oral soft tissue cells as they lose viability and respond with a moderately increased expression of inflammatory cytokines. Clinical relevance: The soft tissue surrounding a dental implant can impact rehabilitation success. Understanding how soft tissue cells respond to titanium surface is potentially relevant to understand clinical outcomes.