Anti-inflammatory effects of hesperidin on human gingival fibroblasts stimulated by lipopolysaccharide of Porphyromonas gingivalis in vitro.

Periodontal disease is an inflammatory disease caused by periodontopathogenic bacteria, the inflammatory response generated against them, and host factors. Furthermore, environmental factors can lead to disease progression. Using lipopolysaccharide (LPS)-stimulated human gingival fibroblast (HGF), this study investigated the bioactivity of HGF after exposure to hesperidin (Hesp) and the anti-inflammatory activity of Hesp against early periodontitis. HGF were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum. They were exposed to LPS for 6 h, followed by Hesp (1, 10, 30, and 50 µM) exposure for 4 h. Cell proliferation was evaluated using reduction staining with alamerBlue™. Inflammatory cytokines [interleukin (IL)-6 and IL-8] and Toll-like receptor 4 (TLR4) levels were assessed using reverse transcription quantitative polymerase chain reaction. Hesp 50 µM + LPS inhibited cell proliferation. The Hesp exposure group inhibited the expression of IL-8 and IL-6. No significant difference in TLR4 expression was observed. Hesp significantly suppressed IL-6 and IL-8 expression by inhibiting downstream signaling without inhibiting TLR4 activation.

Human gingival fibroblast response on zirconia and titanium implant abutment: A systematic review.

PURPOSE: The peri-implant region, where restoration interfaces with mucosal tissue, plays an essential role in overall implant success and is just as important as osseointegration. The implant abutment materials are in intimate contact with human gingival fibroblasts (HGFs). This study compares the proliferation of HGFs between zirconia and titanium abutments used in dental implants. METHODS: An electronic search was performed using PubMed, EMBASE, and Web of Science databases. English articles based on in vitro studies testing HGFs proliferation on zirconia and titanium implant abutment materials were included. A quality assessment of the selected study was performed using the web-based Science in Risk Assessment and Policy (SciRAP) tool. The HGFs proliferation and cellular morphology tests on zirconia and titanium materials from the included studies were summarized, exploring the role of material surface characteristics. RESULTS: The electronic search yielded 401 studies, of which 17 were selected for inclusion. Zirconia exhibited comparable or superior efficacy in promoting the proliferation of HGFs compared to titanium. Observations on cellular morphology showed similar outcomes for both materials. Establishing a definitive relationship between contact angle, surface roughness, and their influence on cellular response remains challenging due to the varied methodological approaches in the reviewed studies. CONCLUSION: Based on the findings of this systematic review, zirconia shows comparable reliability to titanium as an abutment material for HGFs proliferation, with comparable or superior HGFs proliferative outcomes.

Magnolia kobus Extract Suppresses Porphyromonas gingivalis LPS-Induced Proinflammatory Cytokine and MMP Expression in HGF-1 Cells and Regulates Osteoclastogenesis in RANKL-Stimulated RAW264.7 Cells.

Clinical prevention is of utmost importance for the management of periodontal diseases. Periodontal disease starts with an inflammatory response in the gingival tissue, and results in alveolar bone destruction and subsequent tooth loss. This study aimed to confirm the anti-periodontitis effects of MKE. To confirm this, we studied its mechanism of action using qPCR and WB in LPS-treated HGF-1 cells and RANKL-induced osteoclasts. We found that MKE suppressed proinflammatory cytokine protein expression by inhibiting the TLR4/NF-κB pathway in LPS-PG-induced HGF-1 cells and blocking ECM degradation by regulating the expression of TIMPs and MMPs. We also confirmed that TRAP activity and multinucleated cell formation were reduced in RANKL-stimulated osteoclasts after exposure to MKE. These results were confirmed by inhibiting TRAF6/MAPK expression, which led to the suppression of NFATc1, CTSK, TRAP, and MMP expression at the gene and protein levels. Our results confirmed that MKE is a promising candidate for the management of periodontal disease based on its anti-inflammatory effects and inhibition of ECM degradation and osteoclastogenesis.

Proliferative effect of cannabidiol in human gingival fibroblasts via the mitogen-activated extracellular signal-regulated kinase (MEK) 1/2.

BACKGROUND AND OBJECTIVES: Cannabidiol exerts its anti-inflammatory and anti-oxidant activities in various human cells. However, its proliferative effect has not been extrapolated to human gingival fibroblasts (HGFs). This study aimed to determine the proliferative and promigratory effects of cannabidiol in HGFs and to elucidate the signaling mechanism(s). MATERIALS AND METHODS: HGFs, characterized by their CD73, CD90, and CD105 expressions by flow cytometry, were treated with cannabidiol at 0.01-30 µM. The cytotoxicity was determined by the MTT assay, while the proliferative effect was examined by the BrdU assay, immunoblot and immunofluorescence for cyclin D1 and Ki-67 expressions, respectively, and cell cycle analysis. The promigratory effect of cannabidiol was investigated by a wound healing assay. Phosphorylation of the p38 MAPK, JNK, and ERK upon treatment with cannabidiol was explored, and their involvement in cell proliferation and cyclin D1 and Ki-67 expressions was studied using pharmacological inhibitors. RESULTS: No toxicity was found in HGFs treated with any doses of cannabidiol up to 30 µM. The mean percentage of cell proliferation was significantly enhanced by treatment with cannabidiol at 3 or 10 µM (p < .001), consistent with upregulated expressions of cyclin D1 and Ki-67 and increased percentages of HGFs in the S and G2/M phases. Moreover, treatment with cannabidiol significantly induced cell migration (p < .05). The p38 MAPK and ERK1/2 were significantly activated by cannabidiol (p < .05), but only pretreatment with UO126, a MEK1/2 inhibitor, significantly inhibited cell proliferation and cyclin D1 and Ki-67 expressions (p < .05). CONCLUSION: Treatment with cannabidiol at non-toxic doses promotes HGFs' proliferation and migration.

Regulation of osteogenesis in bone marrow-derived mesenchymal stem cells via histone deacetylase 1 and 2 co-cultured with human gingival fibroblasts and periodontal ligament cells.

OBJECTIVE: This study aimed to determine the regulatory mechanism of bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation mediated by humoral factors derived from human periodontal ligament (HPL) cells and human gingival fibroblasts (HGFs). We analyzed histone deacetylase (HDAC) expression and activity involved in BM-MSC differentiation and determined their regulatory effects in co-cultures of BM-MSCs with HPL cells or HGFs. BACKGROUND: BM-MSCs can differentiate into various cell types and can, thus, be used in periodontal regenerative therapy. However, the mechanism underlying their differentiation remains unclear. Transplanted BM-MSCs are affected by periodontal cells via direct contact or secretion of humoral factors. Therefore, their activity is regulated by humoral factors derived from HPL cells or HGFs. METHODS: BM-MSCs were indirectly co-cultured with HPL cells or HGFs under osteogenic or growth conditions and then analyzed for osteogenesis, HDAC1 and HDAC2 expression and activity, and histone H3 acetylation. BM-MSCs were treated with trichostatin A, or their HDAC1 or HDAC2 expression was silenced or overexpressed during osteogenesis. Subsequently, they were evaluated for osteogenesis or the effects of HDAC activity. RESULTS: BM-MSCs co-cultured with HPL cells or HGFs showed suppressed osteogenesis, HDAC1 and HDAC2 expression, and HDAC phosphorylation; however, histone H3 acetylation was enhanced. Trichostatin A treatment remarkably suppressed osteogenesis, decreasing HDAC expression and enhancing histone H3 acetylation. HDAC1 and HDAC2 silencing negatively regulated osteogenesis in BM-MSCs to the same extent as that achieved by indirect co-culture with HPL cells or HGFs. Conversely, their overexpression positively regulated osteogenesis in BM-MSCs. CONCLUSION: The suppressive effects of HPL cells and HGFs on BM-MSC osteogenesis were regulated by HDAC expression and histone H3 acetylation to a greater extent than that mediated by HDAC activity. Therefore, regulation of HDAC expression has prospects in clinical applications for effective periodontal regeneration, mainly, bone regeneration.

Inhibition of gingival fibroblast necroptosis mediated by RIPK3/MLKL attenuates periodontitis.

AIM: Necroptosis participates in the pathogenesis of many inflammatory diseases, including periodontitis. Here, we aimed to investigate the role and mechanism of necroptosis inhibitors in attenuating periodontitis. MATERIALS AND METHODS: The Gene Expression Omnibus (GEO) dataset GSE164241 was re-analysed to identify the role of necroptosis in periodontitis. Gingival specimens from healthy subjects or periodontitis patients were collected to evaluate the expression level of necroptosis-associated proteins. The therapeutic effect of necroptosis inhibitors on periodontitis was assessed in vivo and in vitro. Moreover, Transwell assays and Western blotting and siRNA transfection were used to identify the effects of necroptotic human gingival fibroblasts (hGFs) on THP-1 macrophages. RESULTS: Re-analysis revealed that gingival fibroblasts (GFs) in periodontitis gingiva showed the highest area under the curve score of necroptosis. Elevated levels of necroptosis-associated proteins were identified in GFs in periodontitis gingiva collected from patients and mice. In ligature-induced periodontitis mice, local administration of receptor interacting protein kinase 3(RIPK3) inhibitor GSK'872 or sh-mixed-lineage kinase domain-like pseudokinase (Mlkl) markedly abrogated necroptosis and rescued periodontitis. Analogously, necroptosis inhibitors alleviated the inflammatory response and release of damage-associated molecular patterns in lipopolysaccharide- or LAZ (LPS + AZD'5582 + z-VAD-fmk, necroptosis inducer)-induced GFs and then reduced THP-1 cell migration and M1 polarization. CONCLUSIONS: Necroptosis in GFs aggravated gingival inflammation and alveolar bone loss. Necroptosis inhibitors attenuate this process by modulating THP-1 macrophage migration and polarization. This study offers novel insights into the pathogenesis and potential therapeutic targets of periodontitis.

A Sequential Micro-Immunotherapy Medicine Increases Collagen Deposition in Human Gingival Fibroblasts and in an Engineered 3D Gingival Model under Inflammatory Conditions.

Periodontal therapies use immune mediators, but their side effects can increase with dosage. Micro-immunotherapy (MI) is a promising alternative that employs immune regulators at low and ultralow doses to minimize adverse effects. In this study, the effects of 5 capsules and the entire 10-capsule sequence of the sequential MI medicine (MIM-seq) were tested in two in vitro models of periodontitis. Firstly, human gingival fibroblasts (hGFs) exposed to interleukin (IL)-1ß to induce inflammation were treated with five different capsules of MIM-seq for 3 days or with MIM-seq for 24 days. Subsequently, MIM-seq was analyzed in a 3D model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus. Simultaneously, a non-IL-1ß-treated control and a vehicle were included. The effects of the treatments on cytotoxicity, collagen deposition, and the secreted levels of IL-1α, IL-6, prostaglandin E2 (PGE2), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were evaluated. None of the tested items were cytotoxic. The complete sequence of MIM-seq decreased PGE2 release and restored collagen deposition levels induced by IL-1ß treatment in hGFs exposed to IL-1ß. MIM-seq treatment restored collagen production levels in both models. These promising preclinical findings suggest that MIM-seq should be further investigated for periodontitis treatment.

Effect of Bacterial Infection on Ghrelin Receptor Regulation in Periodontal Cells and Tissues.

The effect of bacterial infection on the expression of growth hormone secretagogue receptor (GHS-R) was investigated in periodontal cells and tissues, and the actions of ghrelin were evaluated. GHS-R was assessed in periodontal tissues of rats with and without periodontitis. Human gingival fibroblasts (HGFs) were exposed to Fusobacterium nucleatum in the presence and absence of ghrelin. GHS-R expression was determined by real-time PCR and immunocytochemistry. Furthermore, wound healing, cell viability, proliferation, and migration were evaluated. GHS-R expression was significantly higher at periodontitis sites as compared to healthy sites in rat tissues. F. nucleatum significantly increased the GHS-R expression and protein level in HGFs. Moreover, ghrelin significantly abrogated the stimulatory effects of F. nucleatum on CCL2 and IL-6 expressions in HGFs and did not affect cell viability and proliferation significantly. Ghrelin stimulated while F. nucleatum decreased wound closure, probably due to reduced cell migration. Our results show original evidence that bacterial infection upregulates GHS-R in rat periodontal tissues and HGFs. Moreover, our study shows that ghrelin inhibited the proinflammatory actions of F. nucleatum on HGFs without interfering with cell viability and proliferation, suggesting that ghrelin and its receptor may act as a protective molecule during bacterial infection on periodontal cells.

Poly(ethylene-Co-vinyl Alcohol)/Titanium Dioxide Nanocomposite: Preparation and Characterization of Properties for Potential Use in Bone Tissue Engineering.

A series of poly(ethylene-co-vinyl alcohol)/titanium dioxide (PEVAL/TiO2) nanocomposites containing 1, 2, 3, 4 and 5 wt% TiO2 were prepared by the solvent casting method. These prepared hybrid materials were characterized by Fourier-transform infrared (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The pores and their interconnections inside these nanocomposites were created using naphthalene microparticles used as a porogen after having been extracted by sublimation under a high vacuum at temperatures slightly below the glass transition temperature. A cellular activity test of these hybrid materials was performed on human gingival fibroblast cells (HGFs) in accordance with ISO 10993-5 and ISO 10993-12 standards. The bioviability (cell viability) of HGFs was evaluated after 1, 4 and 7 days using Alamar Blue®. The results were increased cell activity throughout the different culture times and a significant increase in cell activity in all samples from Day 1 to Day 7, and all systems tested showed significantly higher cell viability than the control group on Day 7 (p < 0.002). The adhesion of HGFs to the scaffolds studied by SEM showed that HGFs were successfully cultured on all types of scaffolds.

Injectable Human Hair Keratin-Fibrinogen Hydrogels for Engineering 3D Microenvironments to Accelerate Oral Tissue Regeneration.

Traumatic injury of the oral cavity is atypical and often accompanied by uncontrolled bleeding and inflammation. Injectable hydrogels have been considered to be promising candidates for the treatment of oral injuries because of their simple formulation, minimally invasive application technique, and site-specific delivery. Fibrinogen-based hydrogels have been widely explored as effective materials for wound healing in tissue engineering due to their uniqueness. Recently, an injectable foam has taken the spotlight. However, the fibrin component of this biomaterial is relatively stiff. To address these challenges, we created keratin-conjugated fibrinogen (KRT-FIB). This study aimed to develop a novel keratin biomaterial and assess cell-biomaterial interactions. Consequently, a novel injectable KRT-FIB hydrogel was optimized through rheological measurements, and its injection performance, swelling behavior, and surface morphology were investigated. We observed an excellent cell viability, proliferation, and migration/cell-cell interaction, indicating that the novel KRT-FIB-injectable hydrogel is a promising platform for oral tissue regeneration with a high clinical applicability.

RNAseq of TGF-ß receptor type I kinase-dependent genes in oral fibroblast exposed to milk.

BACKGROUND: Milk is a rich source of natural growth factors that may support oral tissue homeostasis and wound healing. We had shown earlier that blocking TGF-ß receptor type I kinase with the inhibitor SB431542 abolished the expression of IL11 and other genes in human gingival fibroblasts exposed to the aqueous fraction of milk. Our aim was to identify the entire signature of TGF-ß receptor type I kinase-dependent genes regulated by the aqueous fraction of human milk. RESULT: RNAseq revealed 99 genes being strongly regulated by milk requiring activation of the SB431542-dependent TGF-ß receptor type I kinase. Among the SB431542-dependent genes is IL11 but also cadherins, claudins, collagens, potassium channels, keratins, solute carrier family proteins, transcription factors, transmembrane proteins, tumor necrosis factor ligand superfamily members, and tetraspanin family members. When focusing on our candidate gene, we could identify D609 to suppress IL11 expression, independent of phospholipase C, sphinosine-1 phosphate synthesis, and Smad-3 phosphorylation and its nuclear translocation. In contrast, genistein and blocking phosphoinositide 3-kinases by wortmannin and LY294002 increased the milk-induced IL11 expression in gingival fibroblasts. CONCLUSION: Taken together, our data revealed TGF-ß receptor type I kinase signaling to cause major changes of the genetic signature of gingival fibroblasts exposed to aqueous fraction of human milk.

Regulation of osteogenesis via miR-101-3p in mesenchymal stem cells by human gingival fibroblasts.

INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into various types of cells and can thus be used for periodontal regenerative therapy. However, the mechanism of differentiation is still unclear. Transplanted MSCs are, via their transcription factors or microRNAs (miRNAs), affected by periodontal cells with direct contact or secretion of humoral factors. Therefore, transplanted MSCs are regulated by humoral factors from human gingival fibroblasts (HGF). Moreover, insulin-like growth factor (IGF)-1 is secreted from HGF and regulates periodontal regeneration. To clarify the regulatory mechanism for MSC differentiation by humoral factors from HGF, we identified key genes, specifically miRNAs, involved in this process, and determined their function in MSC differentiation. MATERIALS AND METHODS: Mesenchymal stem cells were indirectly co-cultured with HGF in osteogenic or growth conditions and then evaluated for osteogenesis, undifferentiated MSC markers, and characteristic miRNAs. MSCs had their miRNA expression levels adjusted or were challenged with IGF-1 during osteogenesis, or both of which were performed, and then, MSCs were evaluated for osteogenesis or undifferentiated MSC markers. RESULTS: Mesenchymal stem cells co-cultured with HGF showed suppression of osteogenesis and characteristic expression of ETV1, an undifferentiated MSC marker, as well as miR-101-3p. Over-expression of miR-101-3p regulated osteogenesis and ETV1 expression as well as indirect co-culture with HGF. IGF-1 induced miR-101-3p and ETV1 expression. However, IGF-1 did not suppress osteogenesis. CONCLUSIONS: Humoral factors from HGF suppressed osteogenesis in MSCs. The effect was regulated by miRNAs and undifferentiated MSC markers. miR-101-3p and ETV1 were the key factors and were regulated by IGF-1.

Phenytoin sodium-ameliorated gingival fibroblast aging is associated with autophagy.

BACKGROUND AND OBJECTIVE: Human gingival fibrolasts aging is an important cause of periodontal disease. Phenytoin sodium (phenytoin) has a side effect of gingival hyperplasia and an effect on the autophagy progress. This study investigated whether the effect of phenytoin on aging gingival fibroblast is related to the autophagy pathway. MATERIAL AND METHODS: The aging model of gingival fibroblast cell line HGF-1 was induced by hydrogen peroxide (H2 O2 ), and the treatment of phenytoin and 3-methyladenine (3-MA) was performed simultaneously. Cell viability, cell cycle, and intracellular calcium ion were measured by flow cytometry. Changes in expression of basic fibroblast growth factor (bFGF), P16INK4A , P21cip1 , and bFGF, P16INK4A , P21cip1 , LC3II, p62, and Beclin were tested by using reverse transcription polymerase chain reaction, western blot, and immunofluorescence staining. RESULTS: The results showed that aging HGF-1 proliferation was inhibited by H2 O2 , gene, protein expression of bFGF, P16INK4A , and P21cip1 were decreased, autophagy-related proteins LC3II, p62, and Becline were decreased, and the proportion of G0/G1 phase and intracellular calcium ion of cell cycle was increased. Phenytoin treatment could recovery above changes, but the effect of phenytoin could be blocked by 3-MA. CONCLUSION: We propose that phenytoin alleviates the aging of gingival fibroblasts induced by H2 O2 . This condition is related to the enhancement of autophagy pathway.

Regulation of matrix metalloproteinase-1 by Filifactor alocis in human gingival and monocytic cells.

OBJECTIVES: Periodontitis is a highly prevalent chronic inflammatory disease caused by periodontopathogens, such as Filifactor alocis. This study sought to examine the matrix metalloproteinase (MMP)-1 synthesis by monocytic and fibroblastic cells in response to F. alocis and to unravel the underlying cellular mechanisms. MATERIAL AND METHODS: Gingival biopsies from periodontally healthy and periodontitis individuals were analyzed for the presence of F. alocis and MMP-1 by RT-PCR. Human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells were stimulated with F. alocis in the presence and absence of a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. MMP-1 expression and protein levels were studied by RT-PCR and ELISA, respectively. RESULTS: F. alocis was highly prevalent in biopsies from periodontitis patients but barely present in the healthy gingiva. Significantly higher MMP-1 expression levels were found in the inflamed gingiva as compared with healthy biopsies. F. alocis caused a significant and dose-dependent MMP-1 upregulation in both cells. The stimulatory effect of F. alocis on MMP-1 was TLR2- and MAPK-dependent and more pronounced on THP-1 cells as compared with HGF-1 cells. CONCLUSIONS: Our results demonstrate that F. alocis and MMP-1 are more prevalent at periodontitis sites. Additionally, our study provides original evidence that F. alocis can stimulate MMP-1 production by fibroblastic and monocytic cells, suggesting that F. alocis may contribute to periodontal breakdown through MMP-1. CLINICAL RELEVANCE: F. alocis and MMP-1 are linked to each other and key players in periodontitis, which may have significant implications for future diagnostic and treatment strategies.

Glutathione catabolism by Treponema denticola impacts its pathogenic potential.

Treponema denticola is a spirochete that is etiologic for periodontal diseases. This bacterium is one of two periodontal pathogens that have been shown to have a complete three step enzymatic pathway (GTSP) that catabolizes glutathione to H2S. This pathway may contribute to the tissue pathology seen in periodontitis since diseased periodontal pockets have lower glutathione levels than healthy sites with a concomitant increase in H2S concentration. In order to be able to demonstrate that glutathione catabolism by the GTSP is critical for the pathogenic potential of T. denticola, allelic replacement mutagenesis was used to make a deletion mutant (Δggt) in the gene encoding the first enzyme in the GTSP. The mutant cannot produce H2S from glutathione since it lacks gamma-glutamyltransferase (GGT) activity. The hemolytic and hemoxidation activities of wild type T. denticola plus glutathione are reduced to background levels with the Δggt mutant and the mutant has lost the ability to grow aerobically when incubated with glutathione. The Δggt bacteria with glutathione cause less cell death in human gingival fibroblasts (hGFs) in vitro than do wild type T. denticola and the levels of hGF death correlate with the amounts of H2S produced. Importantly, the mutant spirochetes plus glutathione make significantly smaller lesions than wild type bacteria plus glutathione in a mouse back lesion model that assesses soft tissue destruction, a major symptom of periodontal diseases. Our results are the first to prove that T. denticola thiol-compound catabolism by its gamma-glutamyltransferase can play a significant role in the in the types of host tissue damage seen in periodontitis.

Effect of interleukin-1ß on ghrelin receptor in periodontal cells.

OBJECTIVES: Periodontopathogens induce immunoinflammatory responses characterized by the release of inflammatory mediators, e.g., interleukin (IL)-1ß, IL-6, and IL-8. Ghrelin (GHRL) is an appetite hormone which mediates its effect via the functional receptor GHS-R1a. This study was to examine the effect of an inflammatory insult on GHS-R1a in human periodontal cells. MATERIALS AND METHODS: Periodontal ligament (PDL) cells and gingival fibroblasts (HGFs) were exposed to IL-1ß in the presence and absence of GHRL. Cells were also pre-incubated with specific inhibitors of NF-κB or MEK1/MEK2 signaling. Gene expression of GHS-R1a and proinflammatory mediators was assessed by real-time PCR, GHS-R1 protein level by immunocytochemistry, and NF-κB nuclear translocation by immunofluorescence. RESULTS: IL-1ß increased significantly the GHS-R1a expression in both cell types in a dose-dependent manner. The stimulatory effect of IL-1ß involved the NF-κB and MAPK pathways. Exposure of cells to IL-1ß also resulted in an increased production of GHS-R1 protein in both cell types. Furthermore, GHRL counteracted significantly the stimulatory actions of IL-1ß on IL-6 and IL-8 in PDL cells. CONCLUSIONS: This study demonstrates for the first time that IL-1ß upregulates the functional ghrelin receptor in periodontal fibroblastic cells. Moreover, these results further support the assumption that the GHRL/GHS-R system exerts anti-inflammatory effects. Therefore, the upregulation of ghrelin receptor in periodontal cells in response to an inflammatory stimulus may represent a negative feedback mechanism to attenuate the initial inflammatory process in periodontal diseases. CLINICAL RELEVANCE: The anti-inflammatory GHRL/GHS-R system may serve as a promising target for the prevention and therapy of periodontal diseases.

HGF-1 proliferation on titanium dental implants treated with laser melting technology.

BACKGROUND: Titanium and its alloys are currently the most common dental implant materials. For the best bone-implant contact, machined titanium is subjected to various surface treatments. In the present study, proliferation of human gingivial fibroblast (HGF-1) cells on Grade 5 titanium disks covered with Grade 23 titanium by selective laser melting technology was evaluated. AIM: The main aim was to provide a novel surface procedure providing more biocompatible external structure with a biomechanically intact inner structure and increasing cell proliferation. MATERIALS AND METHODS: Forty-eight titanium Grade 5 machined disks with 5 mm of diameter was divided into four groups. Group 1, also known as the control group, was not subjected to any surface treatment. Group 2 was treated with sand-blasted, large-grid, acid-etching (SLA) technique and Group 3 was treated with selective laser melting (SLM) method. Group 4 was treated with both SLM and SLA. The surface topography was analyzed using scanning electron microscope and the roughness of the samples was evaluated via optic profilometer. Additionally, optical tensiometer was used to measure the surface wettability. To obtain further insights on biocompability of the samples, HGF-1 cell viability at 48 h was assessed with MTT assay. These results were also confirmed by fluorescent staining. Results: Although Ra value and wettability of Group 4 were the highest amongst the samples handled, based on 48 h MTT results and fluorescent staining, highest cell proliferation was observed in Group 3. CONCLUSIONS: It was verifed that the surface topography, roughness, and wettability are all crucial factors on healthy cell populations. Therefore, it was concluded that disks treated with SLM were shown to express the most suitable condition for biocompability.

Human gingival fibroblast feeder cells promote maturation of induced pluripotent stem cells into cardiomyocytes.

The use of human induced pluripotent stem (iPS) cells has been investigated in multiple regenerative medicine studies. However, although methods for efficient differentiation of iPS cells into heart tissues have been devised, it remains difficult to obtain cardiac tissue with high contractility. Herein, we established a method for differentiating iPS cells into highly contractile cardiomyocytes (CMs), and demonstrate that the use of human gingival fibroblasts (HGFs) as a feeder cells promotes maturation of iPS-derived CMs (iPS-CMs) in vitro. After CM differentiation of iPS cells, iPS-CMs showed increased mRNA expression of the CM specific maker cardiac troponin T (cTnT) in the absence and presence (on-feeder condition) of cocultured HGFs, and decreased expression of pluripotent markers was observed under both conditions. Protein expression of cTnT was also observed in immunocytochemical analyses, although on-feeder CMs showed comparatively robust sarcomere structure and significantly stronger contractility than feederless cardiomyocytes, suggesting that HGF feeder cells facilitate CM differentiation of iPS cells.

Biotherapeutic Effect of Gingival Stem Cells Conditioned Medium in Bone Tissue Restoration.

Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal stem cells (hGMSCs) may present advantages compared to other MSCs, including the easier isolation. However, MSCs' secretome has attracted much attention for its potential use in tissue regeneration, such as conditioned medium (CM) that contains different soluble factors proved to be useful for the regenerative purposes. In this study, we evaluated the osteogenic capacity of a poly-(lactide) (3D-PLA) scaffold enriched with hGMSCs and hGMSCs derived CM and its ability to regenerate bone defects in rat calvarias. 3D-PLA alone, 3D-PLA + CM or 3D-PLA + hGMSCs with/without CM were implanted in Wistar male rats subjected to calvarial defects. We observed that 3D-PLA scaffold enriched with hGMSCs and CM showed a better osteogenic capacity, being able to repair the calvarial defect as revealed in vivo by morphological evaluation. Moreover, transcriptomic analysis in vitro revealed the upregulation of genes involved in ossification and regulation of ossification in the 3D-PLA + CM + hGMSCs group. All of these results indicate the great osteogenic ability of 3D-PLA + CM + hGMSCs supporting its use in bone regenerative medicine, in particular in the repair of cranial bone defects. Especially, hGMSCs derived CM played a key role in the induction of the osteogenic process and in bone regeneration.

Comparison of Cytomorphometry and Early Cell Response of Human Gingival Fibroblast (HGFs) between Zirconium and New Zirconia-Reinforced Lithium Silicate Ceramics (ZLS).

New zirconia-reinforced lithium silicate ceramics (ZLS) could be a viable alternative to zirconium (Y-TZP) in the manufacture of implantological abutments-especially in aesthetic cases-due to its good mechanical, optical, and biocompatibility properties. Although there are several studies on the ZLS mechanical properties, there are no studies regarding proliferation, spreading, or cytomorphometry. We designed the present study which compares the surface, cellular proliferation, and cellular morphology between Y-TZP (Vita YZ® T [Vita Zahnfabrik (Postfach, Germany)]) and ZLS (Celtra® Duo [Degudent (Hanau-Wolfgang, Germany)]). The surface characterization was performed with energy dispersive spectroscopy (EDS), scanning electron microscopy (SEM), and optical profilometry. Human gingival fibroblasts (HGFs) were subsequently cultured on both materials and early cellular response and cell morphology were compared through nuclear and cytoskeletal measurement parameters using confocal microscopy. The results showed greater proliferation and spreading on the surface of Y-TZP. This could indicate that Y-TZP continues to be a gold standard in terms of transgingival implant material: Nevertheless, more in vitro and in vivo research is necessary to confirm the results obtained in this study.