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Purpose: To investigate the biocompatibility of silver nanoparticle (AgNP)-doped Ti-6Al-4V surfaces by evaluating the viability and proliferation rate of human gingival fibroblasts (HGFs)--as the dominant cells of peri-implant soft tissues-- seeded on the modified surfaces. Materials and Methods: AgNPs (sizes 8 nm and 30 nm) were incorporated onto Ti-6Al-4V specimen surfaces via electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations of 100 ppm, 200 ppm, and 300 ppm. One control and six experimental groups were included in the study: (1) control (Ti-6Al- 4V), (2) 8 nm/100 ppm, (3) 8 nm/200 ppm, (4) 8 nm/300 ppm, (5) 30 nm/100 ppm, (6) 30 nm/200 ppm, and (7) 30 nm/300 ppm. HGF cell primary cultures were isolated from periodontally healthy donor patients and cultured in direct contact with the group specimens for 24 and 72 hours. The cytotoxicity of AgNP-doped Ti-6Al-4V specimens toward HGF was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and BrdU (5-bromo-2'-deoxyuridine) assay tests. Calcein AM and ethidium homodimer (EthD-1) fluorescent stains were used to determine the live and dead cells. The morphology and attachment properties of the HGFs were determined via scanning electron microscopy (SEM). Results: Energy dispersive x-ray (EDX) analysis confirmed the presence of AgNPs on the specimens. The MTT test revealed that AgNPs of both sizes and all concentrations presented a decreased cellular metabolic activity compared to the control discs. All concentrations of both sizes of AgNPs affected the cell proliferation rate compared to the control group, as revealed by the BrdU assay. Overall, cytotoxicity of the modified Ti-6Al-4V surfaces depended on cell exposure time. Observation via confocal microscopy confirmed the results of the MTT and BrdU assay tests. Specifically, most cells remained alive throughout the 72-hour culture period. SEM images revealed that adjacent cells form bonds with each other, creating confluent layers of conjugated cells. Conclusions: The findings of the present study indicate that Ti-6Al-4V surfaces modified with 8 nm and 30 nm AgNPs at concentrations of 100 ppm, 200 ppm, and 300 ppm do not produce any serious cytotoxicity toward HGFs. The initial arrest of the HGF proliferation rate recovered at 72 hours. These results on the antibacterial activity against common periodontal pathogens, in combination with the results found in a previous study by the same research group, suggest that AgNP-doped Ti-6Al-4V surfaces are potential candidates for use in implant abutments for preventing peri-implant diseases. [ABSTRACT FROM AUTHOR]

Background: Decellularized extracellular matrix (dECM) has been proposed as a useful source of biomimetic materials for regenerative medicine due to its biological properties that regulate cell behaviors. The present study aimed to investigate the influence of decellularized ECM derived from dental pulp stem cells (DPSCs) on gingival fibroblast (GF) cell behaviors. Cells were isolated from dental pulp and gingival tissues. ECM was derived from culturing dental pulp stem cells in growth medium supplemented with ascorbic acid. A bioinformatic database of the extracellular matrix was constructed using Metascape. GFs were reseeded onto dECM, and their adhesion, spreading, and organization were subsequently observed. The migration ability of the cells was determined using a scratch assay. Protein expression was evaluated using immunofluorescence staining. Results: Type 1 collagen and fibronectin were detected on the ECM and dECM derived from DPSCs. Negative phalloidin and nuclei were noted in the dECM. The proteomic database revealed enrichment of several proteins involved in ECM organization, ECM–receptor interaction, and focal adhesion. Compared with those on the controls, the GFs on the dECM exhibited more organized stress fibers. Furthermore, cultured GFs on dECM exhibited significantly enhanced migration and proliferation abilities. Interestingly, GFs seeded on dECM showed upregulation of FN1, ITGB3, and CTNNB1 mRNA levels. Conclusions: ECM derived from DSPCs generates a crucial microenvironment for regulating GF adhesion, migration and proliferation. Therefore, decellularized ECM from DPSCs could serve as a matrix for oral tissue repair. [ABSTRACT FROM AUTHOR]

Periodontal disease presents a significant challenge in oral health due to its chronic inflammatory nature and subsequent degradation of tooth-supporting structures. Natural compounds have attracted attention for their potential therapeutic effects in alleviating symptoms of periodontitis (PD). In this study, we investigated the impact of Ganoderma lucidum spore oil (GLSO), a lipid component extracted from broken-walled GLS using the supercritical CO2 extraction method, on PD pathogenesis in vitro and in vivo. Treatment of human gingival fibroblasts with GLSO resulted in a significant reduction in the expression of inflammatory factors, including matrix metalloproteinase (MMP)-1 and interleukin (IL)-8, upregulated by lipopolysaccharide or IL-1β. Molecular mechanism studies revealed that the observed decrease in inflammatory factor expression may be attributed to the inhibition of phosphorylated c-Jun N-terminal kinase activity by GLSO. Furthermore, intraperitoneal injection of GLSO in a ligature-induced PD mouse model led to a notable reduction in periodontal inflammation and alveolar bone loss, accompanied by decreased levels of MMP-1 and IL-8. These in vivo results support the potential therapeutic efficacy of GLSO in alleviating PD symptoms. Overall, our study provides novel insights into the beneficial effects of GLSO in PD management. Further research is warranted to elucidate the underlying molecular mechanisms and explore the clinical applicability of GLSO as a promising therapeutic agent for PD treatment. [ABSTRACT FROM AUTHOR]

The efficacy of pregabalin in pain treatment has led to the search for new formulations for its use through different routes of administration. This study aimed to prepare, characterize, and evaluate the cytotoxicity of pregabalin (PG) gels for topical application in the oral cavity. Solutions with three different concentrations of PG were prepared and added to a 1.0% carbopol gel base. Thermal analyses (TG and DSC) and FTIR were performed on the gel and pure pregabalin. Stability (preliminary and accelerated) and rheology studies were also conducted on the gels. Cytotoxicity was evaluated in human gingival fibroblasts in the following groups: WG (1.0% carbopol gel base), PG2G (2.0% pregabalin gel), PG5G (5.0% pregabalin gel), and PG10G (10% pregabalin gel). A transparent and homogeneous gel with a pH of 6 was obtained. The formulations showed stability, and the different drug concentrations did not influence the product's characteristics. None of the tested groups showed cytotoxicity for the analyzed cells. The pregabalin gels exhibited favorable and non-toxic characteristics for human gingival fibroblasts in vitro. Therefore, this product may be a promising therapeutic alternative for topical application in the oral mucosa. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer's. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-κB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Periodontitis is a leading chronic oral disorder and poses a serious burden on public health. O-GlcNAc glycosylation (O-GlcNAcylation) is regulated only by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and participates in the regulation of human gingival fibroblasts (HGFs) function. Hence, the purpose of this study is to investigate whether HGFs cell function and periodontitis pathogenesis are regulated by O-GlcNAcylation. Herein, we first established cell model of periodontitis induced by lipopolysaccharide (LPS). The cell viability was measured with CCK-8 assay. Pyroptosis was measured by flow cytometry and western blot. The inflammatory factors levels were detected with ELISA kits. Afterward, our findings indicated that LPS elevated the O-GlcNAcylation level of HGFs and inhibition of O-GlcNAcylation improved LPS-induced pyroptosis of HGFs. Mechanistically, LPS heightened the expression of OGT to induce the O-GlcNAcylation of NLRP3. Subsequently, we certified that Thr542 was the O-GlcNAcylation site of NLRP3. More importantly, upregulation of NLRP3 reversed the effects of OGT knockdown on LPS-induced pyroptosis. In general, the current research demonstrated that LPS contributed to the pyroptosis of HGFs by enhancing the OGT expression to promote O-GlcNAcylation of NLRP3, which suggested that O-GlcNAcylation of NLRP3 was a driving factor for periodontitis and offered a novel insight into the treatment of this disease. [ABSTRACT FROM AUTHOR]

The development of healthy peri-implant soft tissues is critical to achieving the esthetic and biological success of implant restorations throughout all stages of healing and tissue maturation, starting with provisionalization. The purpose of this study was to investigate the effects of eight different implant provisional materials on human gingival fibroblasts at various stages of cell settlement by examining initial cell attachment, growth, and function. Eight different specimens-bis-acrylic 1 and 2, flowable and bulk-fill composites, self-curing acrylic 1 and 2, milled acrylic, and titanium (Ti) alloy as a control-were fabricated in rectangular plates (n = 3). The condition of human gingival fibroblasts was divided into two groups: those in direct contact with test materials (contact experiment) and those in close proximity to test materials (proximity experiment). The proximity experiment was further divided into three phases: pre-settlement, early settlement, and late settlement. A cell culture insert containing each test plate was placed into a well where the cells were pre-cultured. The number of attached cells, cell proliferation, resistance to detachment, and collagen production were evaluated. In the contact experiment, bis-acrylics and composites showed detrimental effects on cells. The number of cells attached to milled acrylic and self-curing acrylic was relatively high, being approximately 70% and 20-30%, respectively, of that on Ti alloy. There was a significant difference between self-curing acrylic 1 and 2, even with the same curing modality. The cell retention ability also varied considerably among the materials. Although the detrimental effects were mitigated in the proximity experiment compared to the contact experiment, adverse effects on cell growth and collagen production remained significant during all phases of cell settlement for bis-acrylics and flowable composite. Specifically, the early settlement phase was not sufficient to significantly mitigate the material cytotoxicity. The flowable composite was consistently more cytotoxic than the bulk-fill composite. The harmful effects of the provisional materials on gingival fibroblasts vary considerably depending on the curing modality and compositions. Pre-settlement of cells mitigated the harmful effects, implying the susceptibility to material toxicity varies depending on the progress of wound healing and tissue condition. However, cell pre-settlement was not sufficient to fully restore the fibroblastic function to the normal level. Particularly, the adverse effects of bis-acrylics and flowable composite remained significant. Milled and self-curing acrylic exhibited excellent and acceptable biocompatibility, respectively, compared to other materials.

Abstract Background Decellularized extracellular matrix (dECM) has been proposed as a useful source of biomimetic materials for regenerative medicine due to its biological properties that regulate cell behaviors. The present study aimed to investigate the influence of decellularized ECM derived from dental pulp stem cells (DPSCs) on gingival fibroblast (GF) cell behaviors. Cells were isolated from dental pulp and gingival tissues. ECM was derived from culturing dental pulp stem cells in growth medium supplemented with ascorbic acid. A bioinformatic database of the extracellular matrix was constructed using Metascape. GFs were reseeded onto dECM, and their adhesion, spreading, and organization were subsequently observed. The migration ability of the cells was determined using a scratch assay. Protein expression was evaluated using immunofluorescence staining. Results Type 1 collagen and fibronectin were detected on the ECM and dECM derived from DPSCs. Negative phalloidin and nuclei were noted in the dECM. The proteomic database revealed enrichment of several proteins involved in ECM organization, ECM–receptor interaction, and focal adhesion. Compared with those on the controls, the GFs on the dECM exhibited more organized stress fibers. Furthermore, cultured GFs on dECM exhibited significantly enhanced migration and proliferation abilities. Interestingly, GFs seeded on dECM showed upregulation of FN1, ITGB3, and CTNNB1 mRNA levels. Conclusions ECM derived from DSPCs generates a crucial microenvironment for regulating GF adhesion, migration and proliferation. Therefore, decellularized ECM from DPSCs could serve as a matrix for oral tissue repair.

Abstract Periodontal disease presents a significant challenge in oral health due to its chronic inflammatory nature and subsequent degradation of tooth-supporting structures. Natural compounds have attracted attention for their potential therapeutic effects in alleviating symptoms of periodontitis (PD). In this study, we investigated the impact of Ganoderma lucidum spore oil (GLSO), a lipid component extracted from broken-walled GLS using the supercritical CO2 extraction method, on PD pathogenesis in vitro and in vivo. Treatment of human gingival fibroblasts with GLSO resulted in a significant reduction in the expression of inflammatory factors, including matrix metalloproteinase (MMP)-1 and interleukin (IL)-8, upregulated by lipopolysaccharide or IL-1β. Molecular mechanism studies revealed that the observed decrease in inflammatory factor expression may be attributed to the inhibition of phosphorylated c-Jun N-terminal kinase activity by GLSO. Furthermore, intraperitoneal injection of GLSO in a ligature-induced PD mouse model led to a notable reduction in periodontal inflammation and alveolar bone loss, accompanied by decreased levels of MMP-1 and IL-8. These in vivo results support the potential therapeutic efficacy of GLSO in alleviating PD symptoms. Overall, our study provides novel insights into the beneficial effects of GLSO in PD management. Further research is warranted to elucidate the underlying molecular mechanisms and explore the clinical applicability of GLSO as a promising therapeutic agent for PD treatment.

Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments. [ABSTRACT FROM AUTHOR]

Selenium nanoparticles (SeNPs) have specific properties that result from their biosynthesis particularities. Chitosan can prevent pathogenic biofilm development. A wide palette of bacterial nanocellulose (BNC) biological and physical-chemical properties are known. The aim of this study was to develop a hydrogel formulation (SeBNCSFa) based on ferulic acid-grafted chitosan and bacterial nanocellulose (BNC) enriched with SeNPs from Kombucha fermentation (SeNPsK), which could be used as an adjuvant for oral implant integration and other applications. The grafted chitosan and SeBNCSFa were characterized by biochemical and physical-chemical methods. The cell viability and proliferation of HGF-1 gingival fibroblasts were investigated, as well as their in vitro antioxidant activity. The inflammatory response was determined by enzyme-linked immunosorbent assay (ELISA) of the proinflammatory mediators (IL-6, TNF-α, and IL-1β) in cell culture medium. Likewise, the amount of nitric oxide released was measured by the Griess reaction. The antimicrobial activity was also investigated. The grafting degree with ferulic acid was approximately 1.780 ± 0.07% of the total chitosan monomeric units, assuming single-site grafting per monomer. Fourier-transform infrared spectroscopy evidenced a convolution of BNC and grafted chitosan spectra, and X-ray diffraction analysis highlighted an amorphous rearrangement of the diffraction patterns, suggesting multiple interactions. The hydrogel showed a high degree of cytocompatibility, and enhanced antioxidant, anti-inflammatory, and antimicrobial potentials. [ABSTRACT FROM AUTHOR]

Chlorhexidine (CHX) represents one of the most commonly used antiseptics in dentistry and other medical–pharmaceutical fields due to its broad-spectrum antimicrobial activity. However, the potential toxic events arising from its common use in practice has become a subject of increasing concern. Thus, the present study was designed to investigate the potential toxicity of CHX digluconate at concentrations covering its antibacterial properties (0.0002–0.2%) in HGF primary gingival fibroblasts, HaCaT immortalized human keratinocytes, and JB6 Cl 41-5a epidermal cells, as well as its irritant action in ovo. Our results indicate that CHX exerted a concentration- and time-dependent cytotoxicity in all cell lines, which was evidenced by the reduction in cell viability, number, and confluence, damaged cell membrane integrity, impaired cell morphology, and specific apoptotic nuclear shape. The highest cytotoxicity was caused by CHX digluconate 0.02% and 0.2%, concentrations, at which an irritant effect on the chorioallantoic membrane was also observed. The novel findings revealed in this research contribute to the overall safety profile of CHX and stand as a basis for further investigations in this regard. [ABSTRACT FROM AUTHOR]

To address the challenges posed by biofilm presence and achieve a substantial reduction in bacterial load within root canals during endodontic treatment, various irrigants, including nanoparticle suspensions, have been recommended. Berberine (BBR), a natural alkaloid derived from various plants, has demonstrated potential applications in dentistry treatments due to its prominent antimicrobial, anti-inflammatory, and antioxidant properties. This study aimed to produce and characterize a novel polymeric nanoparticle of poly (lactic-co-glycolic acid) (PLGA) loaded with berberine and evaluate its antimicrobial activity against relevant endodontic pathogens, Enterococcus faecalis, and Candida albicans. Additionally, its cytocompatibility using gingival fibroblasts was assessed. The polymeric nanoparticle was prepared by the nanoprecipitation method. Physicochemical characterization revealed spheric nanoparticles around 140 nm with ca, −6 mV of surface charge, which was unaffected by the presence of BBR. The alkaloid was successfully incorporated at an encapsulation efficiency of 77% and the designed nanoparticles were stable upon 20 weeks of storage at 4 °C and 25 °C. Free BBR reduced planktonic growth at ≥125 μg/mL. Upon incorporation into PLGA nanoparticles, 20 μg/mL of [BBR]-loaded nanoparticles lead to a significant reduction, after 1 h of contact, of both planktonic bacteria and yeast. Sessile cells within biofilms were also considered. At 30 and 40 μg/mL, [BBR]-loaded PLGA nanoparticles reduced the viability of the sessile endodontic bacteria, upon 24 h of exposure. The cytotoxicity of BBR-loaded nanoparticles to oral fibroblasts was negligible. The novel berberine-loaded polymeric nanoparticles hold potential as a promising supplementary approach in the treatment of endodontic infections. [ABSTRACT FROM AUTHOR]

The periodontium comprising periodontal ligament (PDL), gingiva, and epithelium play crucial roles in maintaining tooth integrity and function. Understanding tissue cellular composition and gene expression is crucial for illuminating periodontal pathophysiology. This study aimed to identify tissue-specific markers via scRNA-Seq. Primary human PDL, gingiva, and epithelium tissues (n = 7) were subjected to cell hashing and sorting. scRNA-Seq library preparation using 10× Genomics protocol and Illumina sequencing was conducted. The analysis was performed using Cellranger (v3.1.0), with downstream analysis via R packages Seurat (v5.0.1) and SCORPIUS (v1.0.9). Investigations identified eight distinct cellular clusters, revealing the ubiquitous presence of epithelial and gingival cells. PDL cells evolved in two clusters with numerical superiority. The other clusters showed varied predominance regarding gingival and epithelial cells or an equitable distribution of both. The cluster harboring most cells mainly consisted of PDL cells and was present in all donors. Some of the other clusters were also tissue-inherent, while the presence of others was environmentally influenced, revealing variability across donors. Two clusters exhibited genetic profiles associated with tissue development and cellular integrity, respectively, while all other clusters were distinguished by genes characteristic of immune responses. Developmental trajectory analysis uncovered that PDL cells may develop after epithelial and gingival cells, suggesting the inherent PDL cell-dominated cluster as a final developmental stage. This single-cell RNA sequencing study delineates the hierarchical organization of periodontal tissue development, identifies tissue-specific markers, and reveals the influence of environmental factors on cellular composition, advancing our understanding of periodontal biology and offering potential insights for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the in vitro biocompatibility of human gingival fibroblasts with preformed paediatric crowns and resistance to acid exposure at levels that simulate the oral environment. Methods: This laboratory study investigated primary HGFs viability, metabolic activity, cytotoxicity, and apoptotic events on preformed metal crown discs, composite resin-coated wells, and monolithic zirconia fragments at 24, 48, and 72 h using the ApoTox-Glo Triplex assay. The PPCs were also immersed in 0.1% lactic acid, 0.2% phosphoric acid, or 10% citric acid for 7 days at 37 °C to reproduce conditions associated with dietary intake or gastric reflux. Samples were then subject to inductively coupled plasma optical emission spectrometry to quantitate the release of ions. Results: The viability of HGFs on stainless steel and CR significantly declined at 48 and 72 h, representing potential cytotoxicity (p < 0.05). Cytotoxicity of HGFs was also higher for stainless steel and ZR compared to control (p < 0.05). PMCs and ZR crowns gave minimal ion release. Meanwhile, significant quantities of metallic ions, including copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn), were present in eluates from veneered-preformed metal crowns. Conclusion: As PPCs can be exposed to highly acidic environments for many years, thus the release of metallic ions from V-PMCs should form the further investigation in future studies. [ABSTRACT FROM AUTHOR]

This research investigates the suitability of printed polyamide 12 (PA12) and its dyed version to support cells in bioengineering applications. For this purpose, human gingival fibroblasts (hGF06) were cultured on PA-12 scaffolds that were 3D-printed by Multi Jet Fusion (MJF). The study examined the direct cultivation of cells on MJF-printed cell culture scaffolds and the effect of leachate of PA-12 printed by MJF on the cultured cells. The article presents research on the surface treatment of PA12 material used in 3D printing and the effect of automatic staining on cell vitality and proliferation in vitro. The study presents a unique device designed exclusively for staining prints made of the biocompatible material PA12 and demonstrates the compatibility of 3D-printed polyamide 12 parts stained in the novel device with a nutrient culture medium and cells. This novel PA12 surface treatment for biomedical purposes does not affect the compatibility with the culture medium, which is essential for cell viability and proliferation. Fluorescence microscopy revealed that mitochondrial fitness and cell survival were not affected by prolonged incubation with clear or dyed PA12 3D-printed parts. [ABSTRACT FROM AUTHOR]

Background. The market for wound healing biomaterials is a rapidly growing industry globally. Moringa oleifera Lam. (MO) is a plant that has gained attention for its benefit in buccal mouthwashes and antiseptic products. This study aimed to assess the cytotoxic potential of MO hydroalcoholic extract on human primary gingival fibroblasts. Methods. The gingival tissue was collected from a healthy adult male at Umm Al-Qura University Dental Teaching Hospital. Gingival fibroblasts were isolated and cultured in Dulbecco’s Modified Eagle Medium (DMEM). MO leaves were dried, powdered, and extracted with 80% ethanol. Various concentrations of MO alcohol extract were used to assess the cell viability of gingival fibroblasts using the MTT assay. Results. The gingival fibroblasts treated with higher concentrations of MO hydroalcoholic extract (10, 5, and 2.5 mg/ml) showed significant morphological changes, including cytoplasm swelling, ruptured cells, nuclear changes, and apoptotic bodies. At these concentrations, cell viability decreased sharply. However, at a lower concentration of 2.5 mg/ml, the cells showed better viability. MO alcohol extract at concentrations of 1.25 mg/ml or lower did not have cytotoxic effects on fibroblasts, with cell viability comparable to that of the control. Conclusions. MO alcohol extract at concentrations of 1.25 mg/ml or lower is not cytotoxic and does not induce dystrophic changes in morphology. Further research is needed to understand MO’s impact on oral health and to assess its potential use in oral care products in vivo.

In periodontitis, gingival fibroblasts (GF) appear to produce a multitude of paracrine factors. However, the influence of GF-derived soluble factors on osteoclastogenesis remains unclear. In this case study, production of paracrine factors by GF was assessed under inflammatory and non-inflammatory conditions, as well as their effect on osteoclastogenesis. Human primary GF were cultured in a transwell system and primed with a cocktail of IL-1β, IL-6 and TNF-α to mimic inflammation. GF were co-cultured directly and indirectly with human peripheral blood mononuclear cells (PBMC). Cytokines and chemokines in supernatants (flow cytometry based multiplex assay), osteoclastogenesis (TRAcP staining) and gene expression (qPCR) were quantified on days 7 and 21.Results from this case study showed that GF communicated via soluble factors with PBMC resulting in a two-fold induction of osteoclasts. Reversely, PBMC induced gene expression of IL-6, OPG and MCP-1 by GF. Remarkably, after priming of GF with cytokines, this communication was impaired and resulted in fewer osteoclasts. This could be partly explained by an increase in IL-10 expression and a decrease in MCP-1 expression. Intriguingly, the short priming of GF resulted in significantly higher expression of inflammatory cytokines that was sustained at both 7 and 21 days.GF appear to produce paracrine factors capable of stimulating osteoclastogenesis in the absence of physical cell-cell interactions. GF cultured in the presence of PBMC or osteoclasts had a remarkably inflammatory phenotype. Given profound expression of both pro- and anti-inflammatory cytokines after the inflammatory stimulus, it is probably the effector hierarchy that leads to fewer osteoclasts.

ABSTRACTBackground Methionine adenosyl transferase II alpha (MAT2A) is the key enzyme to transform methionine into S-adenosylmethionine (SAM), the main methylgroup donor involved in the methylation. The purpose of our study wasto explore whether MAT2A-mediated methionine metabolism affected theexpression of inflammatory cytokines in human gingival fibroblasts(hGFs).Methods Both healthy and inflamed human gingiva were collected. HGFs werecultured and treated with P. gingivalis, with or without MAT2Ainhibitor (PF9366), small interference RNA (siRNA), or extrinsic SAMpretreatment. The levels of inflammatory cytokines were detected byreal-time PCR, western blotting, and ELISA. SAM levels were detectedby ELISA. The nuclear factor-kappa B (NF-κB) and mitogen-activatedprotein kinase (MAPK) pathway was explored by western blotting.Results The expression of MAT2A was increased in the inflamed tissues. P.gingivalis infection promoted the expression of MAT2A and SAM inhGFs. Meanwhile, PF9366 and MAT2A-knockdown significantly decreasedexpression of inflammatory cytokines and SAM production. PF9366inhibited activation of NF-κB/MAPK pathway in P. gingivalis-treatedhGFs.Conclusions MAT2A-mediated methionine metabolism promoted P. gingivalis-inducedinflammation in hGFs. Targeting MAT2A may provide a novel therapeuticmethod for modulating periodontitis.

The aim of this study was to assess the influence of eucalyptol and menthol on the cell viability, migration, and reactive oxygen species production of human gingival fibroblasts (GFs) in vitro. Three different concentrations of eucalyptol and menthol were prepared following ISO 10993-5 guidelines (1, 5, and 10 mM). GFs were isolated from extracted teeth from healthy donors. The following parameters were assessed: cell viability via MTT, Annexin-V-FITC and 7-AAD staining, and IC50 assays; cell migration via horizontal scratch wound assay; and cell oxidative stress via reactive oxygen species assay. Data were analyzed using one-way ANOVA and Tukey's post hoc test. Statistical significance was established at p < 0.05. Eucalyptol and Menthol exhibited high cytotoxicity on gingival fibroblasts, as evidenced by cytotoxicity assays. Eucalyptol showed lower levels of cytotoxicity than menthol, compared to the control group. The cytotoxicity of the tested substances increased in a concentration-dependent manner. The same occurred in a time-dependent manner, although even 10 min of exposure to the tested substances showed a high cytotoxicity to the GFs. Commercially available products for oral application with these substances in their composition should be tested for cytotoxicity before their use. [ABSTRACT FROM AUTHOR]

Objective: To investigate how delayed blood centrifugation affects the composition of the resultant platelet rich fibrin membrane (PRF, a concentrated growth factor preparation) and its biological effects towards gingival fibroblasts. Materials and methods: Blood samples were collected from 18 healthy individuals and centrifuged immediately (T-0), or after a 1–6-minute delay (T-1–6, respectively), to generate PRF. Each PRF membrane was weighed. T-0 and T-6 membranes were incubated for 48 h in cell culture medium at 37 °C to create PRF “releasates” (soluble factors released from the PRF). Human gingival fibroblasts were incubated for 48 h with or without the releasates, followed by RNA isolation and real-time polymerase chain reaction to measure expression of select genes associated with granulation tissue formation, angiogenesis and wound contraction. Additional T-0 and T-6 membranes were used for visualization of leucocyte nuclei and platelets by immunostaining. Results: Immediate centrifugation (T-0) resulted in the largest membranes, T-6 membranes being on average 29% smaller. Leucocytes and platelets were significantly more abundant in T-0 than in T-6 samples. Majority of the fibroblast genes studied were consistently either upregulated or downregulated by the T-0 PRF releasates. However, centrifugation after a 6-minute delay significantly weakened the fibroblast responses. Conclusions: Delayed centrifugation resulted in smaller PRF membranes with fewer leucocytes and platelets and also significantly reduced on the expression of a set of healing-related gingival fibroblast genes. Clinical relevance: The higher expression of wound healing-related genes in gingival fibroblasts by the immediately-centrifuged PRF membranes may increase their biological properties in clinical use. [ABSTRACT FROM AUTHOR]

Objective: To describe the existing knowledge on the efficacy of the different gingival retraction systems (GRSs) in gingival displacement, to know their effects on biological functions of human gingival fibroblasts (HGFs), and on the expression of inflammatory mediators (TNF-α and MCP-1) in gingival crevicular fluid (GCF), and saliva. Methods: The protocol used for this systematic review was registered in INPLASY: 202410005. A digital search was performed in the databases PubMed/MEDLINE, Scopus, Science Direct, Web of Science, and Google Scholar of the literature published in the English language in the last 17 years (from December 10th, 2006, to May 15th, 2023), and included retrospective randomized clinical studies, prospective, and in vitro experimental studies. In addition, PRISMA criteria were followed. The methodological validity of the selected articles was assessed using Joanna Briggs Institute (JBI) critical appraisal tool, and the modified Consolidated Standards of Reporting Trials checklist (CONSORT). Results: 27 articles published between 2006 and 2023 were evaluated. Six hundred 32 subjects, aged between 18 and 65, participated in the clinical studies. 93.7% of the studies assessed periodontally healthy patients, and only 6.3% evaluated patients with mild gingivitis. Also, 882 teeth were samples, of which the majority were posterior teeth (54%). The most commonly used GRSs was aluminum chloride gingival retraction paste (74%). The GCF samples were taken in 67% of the studies, and ELISA was used in all studies (100%) to determine inflammatory mediators. The most frequently analyzed marker was TNF-α (67%). Conclusion: The system Merocel Strips (Mystic, conn, USA) achieved the highest level of gingival displacement (1.66 ± 3.7 mm). In addition, the braided cords produced the lowest TNF-α levels (0.43 ± 0.08pg/mL). Astringent systems such as ferric sulfate had higher toxicity in HGFs. [ABSTRACT FROM AUTHOR]

Objective Methods Results Conclusion This study aimed to clarify the difference in Fusobacterium nucleatum (F. nucleatum) induced inflammatory cytokines and nod‐like receptor protein 3 (NLRP3) inflammasomes dysregulation among three periodontal cells.Oral epithelial cells (HIOECs), THP‐1 macrophages, and human gingival fibroblasts (HGFs) were exposed to F. nucleatum with/without adenosine triphosphate (ATP) and nigericin (Nig). Cell morphology was assessed by scanning electron microscopy. qRT‐PCR, protein microarrays, and bioinformatic methods were used to evaluate the cytokines and their complex interplay. NLRP3 inflammasomes activation was detected by western blotting and ELISA.F. nucleatum adhered to and invaded cells. In HIOECs, F. nucleatum enhanced interleukin (IL)‐1α/1β/6/10/13, TNF‐α, and interferon (IFN)‐γ expression. In THP‐1 macrophages, F. nucleatum up‐regulated IL‐1α/1β/6/10 and TNF‐α levels. In HGFs, F. nucleatum increased IL‐6 levels. F. nucleatum and ATP synergistically boosted IFN‐γ level in THP‐1 macrophages and IL‐13 level in HGFs. IL‐1α/1β/6, and TNF‐α served as epicenters of the inflammatory response. Additionally, F. nucleatum activated NLRP3 inflammasomes in HIOECs, and ATP/Nig boosted the activation. F. nucleatum also triggered NLRP3 inflammasomes in THP‐1 macrophages, but in HGFs, only NLRP3 and caspase‐1 levels were elevated.F. nucleatum infiltrated periodontal supporting cells and dysregulated inflammatory cytokines and NLRP3 inflammasomes. [ABSTRACT FROM AUTHOR]

Reversine is a purine derivative that has been investigated with regard to its biological effects, such as its anticancer properties and, mostly, its ability to induce the dedifferentiation of adult cells, increasing their plasticity. The obtained dedifferentiated cells have a high potential for use in regenerative procedures, such as regenerative dentistry (RD). Instead of replacing the lost or damaged oral tissues with synthetic materials, RD uses stem cells combined with matrices and an appropriate microenvironment to achieve tissue regeneration. However, the currently available stem cell sources present limitations, thus restricting the potential of RD. Based on this problem, new sources of stem cells are fundamental. This work aims to characterize mouse gingival fibroblasts (GFs) after dedifferentiation with reversine. Different administration protocols were tested, and the cells obtained were evaluated regarding their cell metabolism, protein and DNA contents, cell cycle changes, morphology, cell death, genotoxicity, and acquisition of stem cell characteristics. Additionally, their teratoma potential was evaluated after in vivo transplantation. Reversine caused toxicity at higher concentrations, with decreased cell metabolic activity and protein content. The cells obtained displayed polyploidy, a cycle arrest in the G2/M phase, and showed an enlarged size. Additionally, apoptosis and genotoxicity were found at higher reversine concentrations. A subpopulation of the GFs possessed stem properties, as supported by the increased expression of CD90, CD105, and TERT, the existence of a CD106+ population, and their trilineage differentiation capacity. The dedifferentiated cells did not induce teratoma formation. The extensive characterization performed shows that significant functional, morphological, and genetic changes occur during the dedifferentiation process. The dedifferentiated cells have some stem-like characteristics, which are of interest for RD. [ABSTRACT FROM AUTHOR]

We investigated the effects of low‐level Er:YAG laser irradiation on proliferation and alternations in early gene expression of gingival fibroblasts. Mice primary gingival fibroblasts were irradiated with an Er:YAG laser (1.8, 3.9, and 5.8 J/cm2). Irradiation at 3.9 J/cm2 promoted cell proliferation without significant changes in lactate dehydrogenase or Hspa1a expression. Three hours after irradiation at 3.9 J/cm2, the Fn1 expression level was significantly increased. RNA‐seq identified 15 differentially expressed genes between irradiated and non‐irradiated cells, some of which belonged to immediate early genes (IEGs). Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated MAPK pathway enhancement, and gene set enrichment analysis showed enrichment in the TGF‐β signaling gene set. Enhanced proliferation via laser irradiation disappeared upon inhibition of Dusp4, Dusp5, and Tgfr1 expression. Low‐level Er:YAG laser irradiation, especially at 3.9 J/cm2 without a major temperature elevation, enhanced fibroblast proliferation, via TGF‐β and the MAPK signaling pathway following IEG expression. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the ability of photodynamic therapy, based on the use of a gel containing 5% delta aminolaevulinic acid (ALAD) for 45′ followed by irradiation with 630 nm LED (PDT) for 7′, to eradicate Candida albicans strains without damaging the gingiva. C. albicans oral strains and gingival fibroblasts (hGFs) were used to achieve these goals. The potential antifungal effects on a clinical resistant C. albicans S5 strain were evaluated in terms of biofilm biomass, colony forming units (CFU/mL) count, cell viability by live/dead analysis, and fluidity membrane changes. Concerning the hGFs, viability assays, morphological analysis (optical, scanning electronic (SEM), and confocal laser scanning (CLSM) microscopes), and assays for reactive oxygen species (ROS) and collagen production were performed. ALAD-mediated aPDT (ALAD-aPDT) treatment showed significant anti-biofilm activity against C. albicans S5, as confirmed by a reduction in both the biofilm biomass and CFUs/mL. The cell viability was strongly affected by the treatment, while on the contrary, the fluidity of the membrane remained unchanged. The results for the hGFs showed an absence of cytotoxicity and no morphological differences in cells subjected to ALAD-aPDT expected for CLSM results that exhibited an increase in the thickening of actin filaments. ROS production was augmented only at 0 h and 3 h, while the collagen appeared enhanced 7 days after the treatment. [ABSTRACT FROM AUTHOR]

Background: Periodontal disease is a major cause of tooth loss in adults and is a representative oral disease commonly suffered by most people around the world. Mainly the proliferation of Gram-negative bacteria and secreted virulence factors cause an inflammatory response and destroy periodontal tissue. Gossypetin, isolated from Hibiscus sabdariffa L, is known to have various pharmacological effects, including antibacterial and anticancer activities. We aimed to confirm the anti-inflammatory effect of gossypetin through interleukin-6 (IL-6) regulation in human gingival fibroblasts (HGFs) stimulated with lipopolysaccharide (LPS) of Porphyromonas gingivalis, a major cause of adult periodontitis.Methods: CCK-8 assay was performed to confirm the concentration-dependent cytotoxicity of gossypetin against HGFs. The secretion level and mRNA expression of IL-6, an inflammation-related cytokine, and the effect of gossypetin on these in HGFs stimulated with P. gingivalis LPS were confirmed by ELISA and qRT-PCR analysis, respectively. Results: Up to a concentration of 100 μM gossypetin with or without P. gingivalis LPS, the survival rate for HGFs was maintained at over 95% and showed no toxicity. ELISA and qRT-PCR analysis results showed that P. gingivalis LPS increased IL-6 secretion and mRNA levels in HGFs compared to the control group. However, this increase in IL-6 was significantly down-regulated by gossypetin treatment in a dose-dependent manner. In particular, 80 μM gossypetin inhibited IL-6 production to the level of the control group. Conclusion: These results indicated that gossypetin attenuated IL-6 production in HGFs stimulated by P. gingivalis LPS, which may ultimately suppress the inflammatory response in periodontal tissue. Therefore, gossypetin may have potential as a natural ingredient for the prevention and treatment of periodontal disease.

The efficacy of pregabalin in pain treatment has led to the search for new formulations for its use through different routes of administration. This study aimed to prepare, characterize, and evaluate the cytotoxicity of pregabalin (PG) gels for topical application in the oral cavity. Solutions with three different concentrations of PG were prepared and added to a 1.0% carbopol gel base. Thermal analyses (TG and DSC) and FTIR were performed on the gel and pure pregabalin. Stability (preliminary and accelerated) and rheology studies were also conducted on the gels. Cytotoxicity was evaluated in human gingival fibroblasts in the following groups: WG (1.0% carbopol gel base), PG2G (2.0% pregabalin gel), PG5G (5.0% pregabalin gel), and PG10G (10% pregabalin gel). A transparent and homogeneous gel with a pH of 6 was obtained. The formulations showed stability, and the different drug concentrations did not influence the product’s characteristics. None of the tested groups showed cytotoxicity for the analyzed cells. The pregabalin gels exhibited favorable and non-toxic characteristics for human gingival fibroblasts in vitro. Therefore, this product may be a promising therapeutic alternative for topical application in the oral mucosa.

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.

The development of healthy peri-implant soft tissues is critical to achieving the esthetic and biological success of implant restorations throughout all stages of healing and tissue maturation, starting with provisionalization. The purpose of this study was to investigate the effects of eight different implant provisional materials on human gingival fibroblasts at various stages of cell settlement by examining initial cell attachment, growth, and function. Eight different specimens—bis-acrylic 1 and 2, flowable and bulk–fill composites, self-curing acrylic 1 and 2, milled acrylic, and titanium (Ti) alloy as a control—were fabricated in rectangular plates (n = 3). The condition of human gingival fibroblasts was divided into two groups: those in direct contact with test materials (contact experiment) and those in close proximity to test materials (proximity experiment). The proximity experiment was further divided into three phases: pre-settlement, early settlement, and late settlement. A cell culture insert containing each test plate was placed into a well where the cells were pre-cultured. The number of attached cells, cell proliferation, resistance to detachment, and collagen production were evaluated. In the contact experiment, bis-acrylics and composites showed detrimental effects on cells. The number of cells attached to milled acrylic and self-curing acrylic was relatively high, being approximately 70% and 20–30%, respectively, of that on Ti alloy. There was a significant difference between self-curing acrylic 1 and 2, even with the same curing modality. The cell retention ability also varied considerably among the materials. Although the detrimental effects were mitigated in the proximity experiment compared to the contact experiment, adverse effects on cell growth and collagen production remained significant during all phases of cell settlement for bis-acrylics and flowable composite. Specifically, the early settlement phase was not sufficient to significantly mitigate the material cytotoxicity. The flowable composite was consistently more cytotoxic than the bulk–fill composite. The harmful effects of the provisional materials on gingival fibroblasts vary considerably depending on the curing modality and compositions. Pre-settlement of cells mitigated the harmful effects, implying the susceptibility to material toxicity varies depending on the progress of wound healing and tissue condition. However, cell pre-settlement was not sufficient to fully restore the fibroblastic function to the normal level. Particularly, the adverse effects of bis-acrylics and flowable composite remained significant. Milled and self-curing acrylic exhibited excellent and acceptable biocompatibility, respectively, compared to other materials. [ABSTRACT FROM AUTHOR]

To investigate biological processes of the periodontium, in vitro primary cell models have been established. To study the biology of the gingiva, primary gingival fibroblast cell models are widely used. For such experiments, cells need to be expanded and passaged. A key assumption is that primary cells maintain most of their original characteristics they have in situ. The aim of this research is to explore the impact of early passaging on selected gene expression of human gingival fibroblast cells. For this purpose, gene expression from the outgrowth of the resected tissues until the fourth passage was followed for nine tissue samples, from both healthy and diseased sites. Micrographs were taken from the cultures, RNA was extracted from the samples of each passage and quantitative PCR was performed for selected genes representing various biological processes. Epithelial cells were present during the first outgrowth, but were no longer present in the second passage. Our results indicate that the morphology of the gingival fibroblast cells does not change with passaging and that passages 2–4 contain only gingival fibroblasts. Gene expression of M-CSF, TNF-α, TLR4, POSTN and FAPα was unchanged by passaging, the expression of IL-6, IL-1β and TLR2 decreased due to passaging and the expression of in particular the selected osteogenesis genes (ALP, RUNX2, Osteonectin, COL1A), OPG and MKI67 increased with passaging. Worldwide, use of the same passage in laboratory experiments using primary cell cultures is the standard. Our results support this, since for certain genes, in particular osteogenesis genes, expression may alter solely due to passaging. [ABSTRACT FROM AUTHOR]

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK—as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-β (TGF-β) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-β immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-β nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-β receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-β are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts. [ABSTRACT FROM AUTHOR]

Non-invasive physical plasma (NIPP), an electrically conductive gas, is playing an increasingly important role in medicine due to its antimicrobial and regenerative properties. However, NIPP is not yet well established in dentistry, although it has promising potential, especially for periodontological applications. The aim of the present study was to investigate the effect of NIPP on a commercially available human gingival fibroblast (HGF) cell line and primary HGFs in the presence of periodontitis-associated bacteria. First, primary HGFs from eight patients were characterised by immunofluorescence, and cell numbers were examined by an automatic cell counter over 5 days. Then, HGFs that were preincubated with Fusobacterium nucleatum (F.n.) were treated with NIPP. Afterwards, the IL-6 and IL-8 levels in the cell supernatants were determined by ELISA. In HGFs, F.n. caused a significant increase in IL-6 and IL-8, and this F.n.-induced upregulation of both cytokines was counteracted by NIPP, suggesting a beneficial effect of physical plasma on periodontal cells in a microbial environment. The application of NIPP in periodontal therapy could therefore represent a novel and promising strategy and deserves further investigation. [ABSTRACT FROM AUTHOR]

Chlorhexidine (CHX) represents one of the most commonly used antiseptics in dentistry and other medical–pharmaceutical fields due to its broad-spectrum antimicrobial activity. However, the potential toxic events arising from its common use in practice has become a subject of increasing concern. Thus, the present study was designed to investigate the potential toxicity of CHX digluconate at concentrations covering its antibacterial properties (0.0002–0.2%) in HGF primary gingival fibroblasts, HaCaT immortalized human keratinocytes, and JB6 Cl 41-5a epidermal cells, as well as its irritant action in ovo. Our results indicate that CHX exerted a concentration- and time-dependent cytotoxicity in all cell lines, which was evidenced by the reduction in cell viability, number, and confluence, damaged cell membrane integrity, impaired cell morphology, and specific apoptotic nuclear shape. The highest cytotoxicity was caused by CHX digluconate 0.02% and 0.2%, concentrations, at which an irritant effect on the chorioallantoic membrane was also observed. The novel findings revealed in this research contribute to the overall safety profile of CHX and stand as a basis for further investigations in this regard.

Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments.

To address the challenges posed by biofilm presence and achieve a substantial reduction in bacterial load within root canals during endodontic treatment, various irrigants, including nanoparticle suspensions, have been recommended. Berberine (BBR), a natural alkaloid derived from various plants, has demonstrated potential applications in dentistry treatments due to its prominent antimicrobial, anti-inflammatory, and antioxidant properties. This study aimed to produce and characterize a novel polymeric nanoparticle of poly (lactic-co-glycolic acid) (PLGA) loaded with berberine and evaluate its antimicrobial activity against relevant endodontic pathogens, Enterococcus faecalis, and Candida albicans. Additionally, its cytocompatibility using gingival fibroblasts was assessed. The polymeric nanoparticle was prepared by the nanoprecipitation method. Physicochemical characterization revealed spheric nanoparticles around 140 nm with ca, −6 mV of surface charge, which was unaffected by the presence of BBR. The alkaloid was successfully incorporated at an encapsulation efficiency of 77% and the designed nanoparticles were stable upon 20 weeks of storage at 4 °C and 25 °C. Free BBR reduced planktonic growth at ≥125 μg/mL. Upon incorporation into PLGA nanoparticles, 20 μg/mL of [BBR]-loaded nanoparticles lead to a significant reduction, after 1 h of contact, of both planktonic bacteria and yeast. Sessile cells within biofilms were also considered. At 30 and 40 μg/mL, [BBR]-loaded PLGA nanoparticles reduced the viability of the sessile endodontic bacteria, upon 24 h of exposure. The cytotoxicity of BBR-loaded nanoparticles to oral fibroblasts was negligible. The novel berberine-loaded polymeric nanoparticles hold potential as a promising supplementary approach in the treatment of endodontic infections.

The periodontium comprising periodontal ligament (PDL), gingiva, and epithelium play crucial roles in maintaining tooth integrity and function. Understanding tissue cellular composition and gene expression is crucial for illuminating periodontal pathophysiology. This study aimed to identify tissue-specific markers via scRNA-Seq. Primary human PDL, gingiva, and epithelium tissues (n = 7) were subjected to cell hashing and sorting. scRNA-Seq library preparation using 10× Genomics protocol and Illumina sequencing was conducted. The analysis was performed using Cellranger (v3.1.0), with downstream analysis via R packages Seurat (v5.0.1) and SCORPIUS (v1.0.9). Investigations identified eight distinct cellular clusters, revealing the ubiquitous presence of epithelial and gingival cells. PDL cells evolved in two clusters with numerical superiority. The other clusters showed varied predominance regarding gingival and epithelial cells or an equitable distribution of both. The cluster harboring most cells mainly consisted of PDL cells and was present in all donors. Some of the other clusters were also tissue-inherent, while the presence of others was environmentally influenced, revealing variability across donors. Two clusters exhibited genetic profiles associated with tissue development and cellular integrity, respectively, while all other clusters were distinguished by genes characteristic of immune responses. Developmental trajectory analysis uncovered that PDL cells may develop after epithelial and gingival cells, suggesting the inherent PDL cell-dominated cluster as a final developmental stage. This single-cell RNA sequencing study delineates the hierarchical organization of periodontal tissue development, identifies tissue-specific markers, and reveals the influence of environmental factors on cellular composition, advancing our understanding of periodontal biology and offering potential insights for therapeutic interventions.

Background: Although microbial infection is direct cause of periodontal disease, various environmental factors influence the disease severity. Aging is considered a risk factor for oral diseases, with the prevalence of periodontal diseases increasing with age. Moreover, senescence-associated secretory phenotype (SASP) expressed in age-related diseases is a key marker of chronic inflammation and aging phenotypes. Therefore, this study aimed to understand the relevance of senescent cells to periodontal health and disease, investigate the possibility of regulating the expression of aging- and osteolysis-related factors in gingival fibroblasts, and investigate the effect of senescence induction in gingival fibroblasts on osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). Methods: After stimulation with 400 nM hydrogen peroxidase, human gingival fibroblasts (HGFs) were examined for senescenceassociated ‐galactosidase. Western blot and enzyme-linked immunosorbent assays were performed to assess the expression of SASP. Osteoclast formation was assessed in BMMs using a conditioned medium (CM) from hydrogen peroxide-stimulated HGFs. Osteoclastic differentiation was investigated using tartrate-resistant acid phosphatase (TRAP) staining and activity. Data analysis was performed using SPSS version 25.0. Results: The expression of senescence-related molecules, including p53, p16, and p21, and the expression of osteolytic factors, including IL-6, IL-8, and IL-17, were found to be significantly higher in the hydrogen peroxide-stimulated HGF than in the control group. Regarding the indirect effects of senescent gingival cells, the number of osteoclasts and TRAP activity increased according to the differentiation of BMM cultured in CM. Conclusion: Our results on the of between osteolytic factors and cellular senescence in gingival fibroblast cells helped to reveal evidence of pathological aging mechanisms. Furthermore, our results suggest that the development of novel therapies that target specific SASP factors could be an effective treatment strategy for periodontal disease.

Abstract Background Long-term use of anti-resorptive or anti-angiogenic drugs in cancer patients with odontogenic infections may lead to medication-related osteonecrosis of the jaw (MRONJ). This study investigated whether anti-angiogenic agents aggravate MRONJ occurrence in anti-resorptive-treated patients. Methods The clinical stage and jawbone exposure of MRONJ patients caused by different drug regimens were analyzed to ascertain the aggravation effect of anti-angiogenic drugs on anti-resorptive drug-based MRONJ. Next, a periodontitis mice model was established, and tooth extraction was performed after administering anti-resorptive and/or anti-angiogenic drugs; the imaging and histological change of the extraction socket were observed. Moreover, the cell function of gingival fibroblasts was analyzed after the treatment with anti-resorptive and/or anti-angiogenic drugs in order to evaluate their effect on the gingival tissue healing of the extraction socket. Results Patients treated with anti-angiogenic and anti-resorptive drugs had an advanced clinical stage and a bigger proportion of necrotic jawbone exposure compared to patients treated with anti-resorptive drugs alone. In vivo study further indicated a greater loss of mucosa tissue coverage above the tooth extraction in mice treated with sunitinib (Suti) + zoledronate (Zole) group (7/10) vs. Zole group (3/10) and Suti group (1/10). Micro-computed tomography (CT) and histological data showed that the new bone formation in the extraction socket was lower in Suti + Zole and Zole groups vs. Suti and control groups. In vitro data showed that the anti-angiogenic drugs had a stronger inhibitory ability on the proliferation and migration function of gingival fibroblasts than anti-resorptive drugs, and the inhibitory effect was obviously enhanced after combining zoledronate and sunitinib. Conclusion Our findings provided support for a synergistic contribution of anti-angiogenic drugs to anti-resorptive drugs-based MRONJ. Importantly, the present study revealed that anti-angiogenic drugs alone do not induce severe MRONJ but aggravate the degree of MRONJ via the enhanced inhibitory function of gingival fibroblasts based on anti-resorptive drugs.

Periodontal disease is a chronic inflammatory disease that leads to the gradual destruction of the supporting structures of the teeth including gums, periodontal ligaments, alveolar bone, and root cementum. Recently, interests in alleviating symptoms of periodontitis (PD) using natural compounds is increasing. Avenanthramide-C (Avn-C) is a polyphenol found only in oats. It is known to exhibit various biological properties. To date, the effect of Avn-C on PD pathogenesis has not been confirmed. Therefore, this study aimed to verify the protective effects of Avn-C on periodontal inflammation and subsequent alveolar bone erosion in vitro and in vivo. Upregulated expression of catabolic factors, such as matrix metalloproteinase 1 (MMP1), MMP3, interleukin (IL)-6, IL-8, and COX2 induced by lipopolysaccharide and proinflammatory cytokines, IL-1β, and tumor necrosis factor α (TNF-α), was dramatically decreased by Avn-C treatment in human gingival fibroblasts and periodontal ligament cells. Moreover, alveolar bone erosion in the ligature-induced PD mouse model was ameliorated by intra-gingival injection of Avn-C. Molecular mechanism studies revealed that the inhibitory effects of Avn-C on the upregulation of catabolic factors were mediated via ERK (extracellular signal-regulated kinase) and NF-κB pathway that was activated by IL-1β or p38 MAPK and JNK signaling that was activated by TNF-α, respectively. Based on this study, we recommend that Avn-C may be a new natural compound that can be applied to PD treatment. [ABSTRACT FROM AUTHOR]

Objective: Exploring the correlation between human β‐defensins (HBDs) and immune infiltration in periodontitis, and whether it is regulated by vitamin D3. Background: The human body produces essential antimicrobial peptides called HBDs, which are associated with periodontitis. There is a strong link between periodontal tissue destruction and the immune cell infiltration. Moreover, vitamin D3 has been reported to regulate the expression of immune cell chemokines. However, the relationship between vitamin D3, HBDs, and immune infiltration in periodontitis remains to be investigated. Methods: The Gene Expression Omnibus database was accessed to obtain transcriptomic information of gingival samples taken from periodontitis patients. The expression value of HBD‐2 and HBD‐3 was calculated. Additionally, using the online program ImmuCellAl, 10 immune cells were scored for immune infiltration in the high‐HBDs‐expression group and the low‐HBDs‐expression group, separately. After that, transcriptome sequencing was done based on human gingival fibroblasts that had received vitamin D3 treatment. Furthermore, hGFs were treated by vitamin D3, tumor necrosis factor‐α (TNF‐α), and Porphyromonas gingivalis lipopolysaccharide (Pg‐LPS). The expressions of HBD‐2, HBD‐3, interleukin‐8 (IL‐8), and monocyte chemoattractant protein‐1 (MCP‐1) were detected. To seek the potential mechanism, CYP27A1 siRNA was employed to reduce the expression of CYP27A1, and nuclear factor‐gene binding protein 65 (NF‐κB p65) was examined. Results: In GSE10334, the expressions of HBD‐2 and HBD‐3 were down‐regulated in periodontitis group. Meanwhile, monocyte, macrophage, and CD4_T cell were less infiltrated in low‐HBD‐2‐expression group, while less Gamma‐delta T‐cell infiltration was found in low‐HBD‐3‐expression group. Transcriptome sequencing found that 21 genes were significantly expressed, of which the function was enriched in response to bacterial origin and TNF signal pathway. Vitamin D3 could significantly up‐regulate the expression of HBD‐2 and HBD‐3, which could be controlled by knocking down CYP27A1 mRNA expression. With prolonged vitamin D3 stimulation, the expression of HBD‐2 and HBD‐3 increased. TNF‐α/Pg‐LPS could significantly increase the expression of HBD‐2, HBD‐3, IL‐8, MCP‐1, and p65, all of which were reduced by vitamin D3. Conclusion: HBDs are correlated with immune infiltration in periodontitis. Vitamin D3 inhibits the expression of HBDs and chemokines induced by TNF‐α/Pg‐LPS, possibly through NF‐κB pathway, in human gingival fibroblasts. [ABSTRACT FROM AUTHOR]

Objectives: A beneficial effect of cross-linked hyaluronic acid (cHA) on periodontal wound healing and regeneration has recently been demonstrated. The present in vitro study was designed to obtain deeper knowledge on the effect of cHA when applied in the gingival sulcus (serum-rich environment) during non-surgical periodontal therapy. Materials and methods: The influence of cHA, human serum (HS), and cHA/HS on (i) a 12-species biofilm formation, (ii) the adhesion of periodontal ligament fibroblasts (PDLF) to dentine surface, (iii) the expression and secretion of interleukin-8, and (iv) the expression of receptors of HA in PDLF and gingival fibroblasts (GF) were evaluated. Results: At 4 h of biofilm formation, cHA and HS in combination (cHA/HS) slightly decreased the colony-forming unit counts in biofilm whereas the metabolic activity of biofilm was reduced in all test groups (cHA, HS, cHA/HS) vs. control. At 24 h, the quantity of biofilm was reduced in all test groups vs. untreated control. The test substances did not affect adhesion of PDLF to dentin. HS increased the expression of IL-8 by PDLF and GF which was partially downregulated by cHA. HS and/or cHA promoted the expression of the HA receptor RHAMM in GF but not in PDLF. Conclusions: In summary, the present data indicate that serum neither negatively affect the activity of cHA against periodontal biofilm nor had any unwanted influence on the activity of PDLF. Clinical relevance: These findings lend additional support for the positive effects of cHA on cells involved in periodontal wound healing, thus pointing to its potential use in non-surgical periodontal therapy. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is an oral pathogen that promotes dysbiosis by quenching the bactericidal activity of the host immune system while maintaining chronic inflammation, leading to periodontitis. This involves the secretion of virulence factors such as P. gingivalis peptidyl arginine deiminase (PPAD), which converts the C‐terminal Arg residues of bacterial and host‐derived proteins and peptides into citrulline. We have previously shown that PPAD activity and major fimbriae (containing FimA) are necessary for P. gingivalis to activate Toll‐like receptor 2 (TLR2). TLR2 is an important component of the innate immune system and plays a predominant role in the recognition of P. gingivalis by host cells. Here, we extend those findings to show that P. gingivalis strains deficient for PPAD and fimbriae induced almost identical transcriptional profiles in infected primary human gingival fibroblasts (PHGFs), but these differed substantially from the transcriptome elicited by the wild‐type ATCC 33277 strain. Apparently, PPAD‐modified fimbriae trigger the host cell response to P. gingivalis, as confirmed by showing that the proinflammatory host cell response mediated by TLR2 is dependent on PPAD activity and the presence of fimbriae, with type I fimbriae as the most potent TLR2 activators. We also found that PPAD‐modified accessory fimbrial subunits (FimC, FimD, and FimE) alone or in combination are TLR2 ligands in a reporter cell line. Although FimA polymerization to form the fimbrial shaft was not required for TLR2 activation, the secretion and proteolytic maturation of FimA were necessary for signaling by accessory Fim proteins. This was supported by showing that the proinflammatory activation of PHGFs is dependent on PPAD and accessory fimbrial subunits. We conclude that accessory fimbrial subunits are modified by PPAD and stimulate the response to P. gingivalis infection in a TLR2‐dependent manner. [ABSTRACT FROM AUTHOR]

Nonsteroidal anti-inflammatory drugs are suggested to have anti-cancer effects in many studies, but elucidation of mode of action remains a challenging task. The present study was aimed to evaluate the effect of an Aryl hydrazone derivative of Naproxen (AHD) on HepG2 liver cancer cells and HGF gingival fibroblasts. The mentioned cell lines were randomly divided into control groups and test groups exposed to 1, 10, 20, 50, and 100 μg/mL of Naproxen and (AHD). The cytotoxic effects of the compounds were measured using the MTT assay. The rate of apoptosis and cell cycle were also evaluated using flow cytometry at 24 and 48 h. The data were compared between groups using one-way analysis of variance. The results showed a significantly reduced viability of liver cancer cells by 20, 50, and 100 μg/mL of Naproxen, and AHD after 24 h (IC50 = 51.35 μg/ mL and IC50 = 87.05 μg/ mL) and 48 h (IC50 = 24.92 μg/ mL and IC50 = 20.82 μg/ mL) (p < 0.0001). Exposure to Naproxen and AHD increased the late apoptosis rate 9.10 and 6.42 times respectively in HepG2 cancer cells compared to the control group at 48 h. (**** p < 0.0001). In the group treated with AHD, a significant decrease in G1 and a significant increase in S and G2 phases occurred in the percentage of arrested cells in HepG2 cancer at 24 h (****p < 0.0001). A significant decrease in G1 and a significant increase in S phases of Naproxen and AHD occurred in the percentage of arrested cells on HepG2 cancer at 48 h (***p < 0.001, **p < 0.01, ****p < 0.0001 ***p < 0.001 respectively). The results of this study demonstrate that Naproxen and the evaluated AHD induce cytotoxic effects in the HepG2 cancer cell line through the apoptosis pathway by cell arrest in the G2/M phase. The less cytotoxicity was observed in gingival fibroblasts than in cancer cells. [ABSTRACT FROM AUTHOR]

Background: /purpose: Periodontal disease is a chronic inflammatory disease that occurs in the tissues that support and attach teeth. There is considerable evidence of a relationship between diabetes and periodontal disease. Emerging studies have reported that myeloid-derived growth factor (MYDGF) can inhibit apoptosis and inflammation. The purpose of this study was to investigate whether MYDGF mediates the role of hyperglycemia in fibroblasts in periodontitis tissues. Materials and methods: Fibroblasts were isolated and cultured from normal gums. Gene expression levels were detected by RT-PCR. The protein level was detected by western blotting. Cell viability was determined by MTT assay. To investigate the role of MYDGF, the plasmid was transfected into fibroblasts. The expression levels of cytokines were determined by ELISA. Results: High glucose can down-regulate the expression of MYDGF in human gingival fibroblasts in a time-dependent manner, and decrease the fibroblast activity. SOD level was decreased and MDA level was increased in gingival fibroblasts by high glucose. High glucose up-regulates pro-apoptotic indicator Bax, down-regulates anti-apototic indicator Bcl-2, and increased endoplasmic reticulum stress related indicators Nox 2, GRP78, ATF6, and PERK. In addition, high glucose increased TNF-α, IL-1β, IL-8 and CXCL1 protein levels in fibroblasts. Our study also found that high glucose inhibits the AKT signaling pathway and activates the nuclear factor κB (NF-κB) pathway. Interestingly, overexpression of MYDGF reversed these effects. Conclusion: MYDGF is down-regulated in gingival fibroblasts induced by high glucose. Overexpression of MYDGF inhibits apoptosis induced by high glucose, inhibits oxidative stress and cytokine secretion of gingival fibroblasts induced by high glucose, and induces AKT pathway activation and NF-κB pathway inhibition.

Abstract Background When infected with Porphyromonas gingivalis, gingival fibroblasts undergo metabolic reprogramming, and rely on aerobic glycolysis rather than oxidative phosphorylation for rapid energy replenishment. Hexokinases (HKs) are catalysts for glucose metabolism, and HK2 constitutes the major HK inducible isoform. The objective of this study is to determine whether HK2-mediated glycolysis promotes inflammatory responses in inflamed gingiva. Methods Levels of glycolysis-related genes were assessed in normal and inflamed gingiva. Human gingival fibroblasts were harvested and infected with Porphyromonas gingivalis in order to mimic periodontal inflammation. 2-deoxy-d-glucose, an analogue of glucose, was used to block HK2-mediated glycolysis, while small interfering RNA was used to knock down HK2 expression. The mRNA and protein levels of genes were analyzed by real-time quantitative PCR and western blotting, respectively. HK2 activity and lactate production were assessed by ELISA. Cell proliferation was assessed by confocal microscopy. The generation of reactive oxygen species was assessed by flow cytometry. Results Elevated expression of HK2 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 was observed in the inflamed gingiva. P. gingivalis infection was shown to promote glycolysis in human gingival fibroblasts, as evidenced by increased gene transcription of HK2 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, cell glucose consumption, and HK2 activity. Inhibition and knockdown of HK2 resulted in reduced cytokine production, cell proliferation, and reactive oxygen species generation. Furthermore, P. gingivalis infection activated the hypoxia-inducible factor-1α signaling pathway, thus promoting HK2-mediated glycolysis and proinflammatory responses. Conclusions HK2-mediated glycolysis promotes inflammatory responses in gingival tissues, and therefore glycolysis can be targeted in order to inhibit the progression of periodontal inflammation.

The aim of this study was to assess the influence of eucalyptol and menthol on the cell viability, migration, and reactive oxygen species production of human gingival fibroblasts (GFs) in vitro. Three different concentrations of eucalyptol and menthol were prepared following ISO 10993-5 guidelines (1, 5, and 10 mM). GFs were isolated from extracted teeth from healthy donors. The following parameters were assessed: cell viability via MTT, Annexin-V-FITC and 7-AAD staining, and IC50 assays; cell migration via horizontal scratch wound assay; and cell oxidative stress via reactive oxygen species assay. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. Statistical significance was established at p < 0.05. Eucalyptol and Menthol exhibited high cytotoxicity on gingival fibroblasts, as evidenced by cytotoxicity assays. Eucalyptol showed lower levels of cytotoxicity than menthol, compared to the control group. The cytotoxicity of the tested substances increased in a concentration-dependent manner. The same occurred in a time-dependent manner, although even 10 min of exposure to the tested substances showed a high cytotoxicity to the GFs. Commercially available products for oral application with these substances in their composition should be tested for cytotoxicity before their use.

Due to the desirable aesthetic performance, zirconia abutments attract tremendous attention in implant related restoration of the anterior zone. However, the inadequate soft tissue integration originated from the inertia surface of zirconia severely limits its application. Fabricating bionic-patterned zirconia abutments is believed to be a promising strategy to solve this puzzle. The main obstacle to fabricating bionic-pattern lies in that conventional zirconia manufacturing technology cannot precisely engineer complex micro-pattern. Notably, modern 3D printing presents a feasible way to obtain various sophisticated topological architecture. In the present study, series of bionic-patterned zirconia were fabricated by 3D printing, simulating lotus leaf, butterfly wings, shark skin, and gecko foot. The features of micro-patterns and quality of 3D printing were systematically characterized. In addition, the effect of various bionic-patterned zirconia on the behaviors of gingival fibroblasts was investigated. Results showed that zirconia with various micro-scale bionic patterns was successfully and precisely fabricated by 3D printing. Among the four types of bionic patterns, the ones that resembled the geckos' feet or lotus leaves were the most recommended topography for cell growth and spreading, while the pattern mimicked shark skin upgraded the expression of fibroblast functional genes including Col-I, Fn, and Itgb- 1 most obviously. The bionic-patterned zirconia can efficiently promote adhesion, proliferation, and related gene expression of gingival fibroblasts, indicating that bionic-patterned zirconia has great potential to promote soft tissue integration. [Display omitted] • Constructing micro-bionic patterns via 3D printing from zirconia slurry was proved to be possible. • The printed bionic-patterned zirconia rendered great potential to enhance soft tissue integration used as dental abutments. • The micro-patterns facilitated proliferation, spreading, and increased the related gene expression of gingival fibroblasts. [ABSTRACT FROM AUTHOR]