Human Oral Fibroblasts Research Articles

Corrosion Resistance of Graphene oxide/Silver Coatings on Ni\u2013Ti alloy and Expression of IL-6 and IL-8 in Human Oral Fibroblasts

Graphene based materials (GBMs) have potentials for dental and medical applications. GBMs may cause changes in the levels of cytokine released in the body. This study aimed to study the corrosion resistance of graphene oxide (GO) and GO/silver (GO/Ag) nanocomposite coated nickel-titanium (NiTi) alloy by electrophoretic deposition and to access the viability of human pulp fibroblasts, and the interleukin (IL)-6 and IL-8 expression level. The bare and coated NiTi samples were characterized by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), Raman spectroscopy, surface profilometry, and X-ray diffraction (XRD). The corrosion resistance of the bare NiTi and coated NiTi samples were investigated by potentiodynamic polarization and electrochemical impedance spectroscopy in 3.5% NaCl solution. The cell viability of human pulp fibroblasts was accessed by the treated culture medium of the bare NiTi and coated NiTi alloys containing 1% fetal bovine serum. IL-6 and IL-8 expression levels were studied by human enzyme-linked immunosorbent assay (ELISA). Data were analyzed using One-way ANOVA (α = 0.05). Both the GO-coated NiTi and GO/Ag-coated NiTi alloys showed better corrosion resistance, a lower rate of corrosion, and higher protection efficiency than the bare NiTi alloy. The coated NiTi alloys were biocompatible to human pulp fibroblasts and showed upregulation of IL-6 and IL-8 levels.

Inhibition of HIF1A-AS1 impedes the arecoline-induced migration activity of human oral mucosal fibroblasts

Long non-coding RNA hypoxia-inducible factor 1α-antisense RNA 1 (HIF1A-AS1) has been known to participate in various types of malignancies, but its role in the development of precancerous oral submucous fibrosis (OSF) has not been investigated. In the current study, we first observed the aberrant upregulation of HIF1A-AS1 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) isolated from OSF specimens. Next, we demonstrated that administration of arecoline, a natural alkaloid that is found in areca nut, induced the elevation of HIF1A-AS1 in BMFs. This finding showed that the habit of areca nut chewing may lead to an increase of HIF1A-AS1 in oral mucosa. Moreover, we found that knockdown of HIF1A-AS1 hindered the arecoline-stimulated migration capacity in BMFs, suggesting HIF1A-AS1 was critical to the transdifferentiation of BMFs into myofibroblasts. Altogether, our results demonstrated that overexpression of HIF1A-AS1 in OSF tissues may result from the use of areca nut and lead to activation of BMFs, which contribute to the progression of OSF.

Detection of hydrogen sulfide using BODIPY based colorimetric and fluorescent on-off chemosensor

A colorimetric and fluorescence turn-off probe 10-(4-azido phenyl)-5,5-difluoro-5h-dipyrrolo[1,2-c:1’,2’-f][1,3,2] diazaborinin-4-ium-5-uide, 1, for selective detection of H2S is reported. The probe displayed a robust decrease in fluorescence intensity with high sensitivity, specificity and least toxicity to detect exogenous H2S and also in live normal human oral fibroblast cells loaded with probe 1. The detection limit of probe 1 being 0.17 µm for H2S. A colorimetric and fluorescence turn-off BODIPY probe 1, for selective detection of exogenous H2S in live normal human oral fibroblast cells loaded with probe 1 and poisonous H2S gas formed under biodegradation in sewage waters and water tanks and corroded pipe lines. The detection limit of probe 1 being 0.17 μM

Effects of electronic cigarette liquid on monolayer and 3D tissue-engineered models of human gingival mucosa.

Background There is limited data available on potential biological effects of E-cigarettes on human oral tissues. The aim of this study was to evaluate the effects of E-cigarette liquid on the proliferation of normal and cancerous monolayer and 3D models of human oral mucosa and oral wound healing after short-term and medium-term exposure. Methods Normal human oral fibroblasts (NOF), immortalized OKF6-TERET-2 human oral keratinocytes, and cancerous TR146 keratinocyte monolayer cultures and 3D tissue engineered oral mucosal models were exposed to different concentrations (0.1%, 1%, 5% and 10%) of E-cigarette liquid (12 mg/ml nicotine) for 1 hour daily for three days and for 7 days. Tissue viability was monitored using the PrestoBlue assay. Wounds were also produced in the middle surface of the monolayer systems vertically using a disposable cell scraper. The alterations in the cell morphology and wound healing were visualized using light microscopy and histological examination.Results Statistical analysis showed medium-term exposure of TR146 keratinocytes to 5% and 10% E-liquid concentrations significantly increased the viability of the cancer cells compared to the negative control. Short-term exposure of NOFs to 10% E-liquid significantly reduced the cell viability, whereas medium-term exposure to all E-liquid concentrations significantly reduced the NOF cells’ viability. OKF6 cells exhibited significantly lower viability following short-term and mediumterm exposure to all E-cigarette concentrations compared to the negative control. 3D oral mucosal model containing normal oral fibroblasts and keratinocytes showed significant reduction in tissue viability after exposure to 10% E-liquid, whereas medium-term exposure resulted in significantly lower viability in 5% and 10% concentration groups compared to the negative control. There was a statistically significant difference in wound healing times of both NOF and OKF6 cells after exposure to 1%, 5% and 10% E-cigarette liquid.Conclusion Medium-term exposure to high concentrations of the E-cigarette liquid had cytotoxic effects on normal human oral fibroblasts and OKF6 keratinocytes, but a stimulatory cumulative effect on the growth of cancerous TR146 keratinocyte cells as assessed by the PrestoBlue assay and histological evaluation of 3D oral mucosal models. In addition, E-liquid exposure prolonged the wound healing of NOF and OKF6 oral mucosa cells.

Abstract 30: The development of a 3D cell-culture model of drug-induced oral mucositis

Abstract Purpose: Mucositis is a debilitating disease affecting cancer patients. Mucositis is an adverse effect of radiation or chemotherapy. In the past, our lab has developed a model of radiation-caused mucositis using a validated human tissue substitute of oral mucosa and studied the post radiation cellular and molecular stresses and their prevention. In this project, we wished to develop an in-vitro model of chemical-, or drug-induced oral mucositis. For this purpose, we used a three dimensional (3-D) cell culture model of oral mucosa and as the drug to induce mucositis we used everolimus. Everolimus belongs to rapalogs which represent important therapies against cancer but one of its adverse-effects is mucositis. Mucositis caused by everolimus develops early in therapy and progresses quickly to grade 3 making patients to postpone life-saving therapies. Thus, it is very important to understand and prevent the development of everolimus-caused mucositis. Methods: The 3-D cell cultures of primary human oral keratinocytes grown on top of primary human oral fibroblasts were purchased from MatTek Corporation. These 3-D cell cultures (tissues) are similar to human oral mucosal tissue and have been validated as its substitutes. Different 3-D tissues were treated for 24, 40 and 60 hours with 8, 16, 32, and 64 ng/ml of everolimus. Tissues which received no treatment were used as controls. We then evaluated the tissue histology using H and E staining, and gene expression using RNA-sequencing. Results: Tissues which were treated with everolimus show signs of distress based on the concentration of everolimus and the duration of the treatment. Low and medium concentration of everolimus (8ng/ml) and (16 or 32 ng/ml), respectively, show no, or minimal tissue distress after 24 hours of treatment duration. Tissues which received a high concentration of everolimus (64 ng/ml) even at a short duration of treatment, show signs of distress, as evidenced by atypical proliferation at the keratinous epithelium, vacuoles, or shredded keratin layers, as well as, higher apoptosis, and expression of pro-inflammatory cytokine genes. In general, the higher the concentration of everolimus and the longer the duration of the treatment, the more the signs of the stress in tissues. Conclusion: The cell and molecular stress that the 3-D tissue cultures of human oral mucosa experience depends on the drug concentration and the duration of treatment. Acknowledgment We thank Novartis for their support. Citation Format: Maria P. Lambros, QinQin Fei, Jonathan Moreno. The development of a 3D cell-culture model of drug-induced oral mucositis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 30.

Titanium dioxide-based scanning powder can modulate cell activity of oral soft tissue - Insights from in vitro studies with L929 cells and periodontal fibroblasts

PurposeTo reveal the impact of titanium dioxide-based scanning powder for intraoral digital impression on the biological activity of oral fibroblasts. MethodsMurine L929 cells and human periodontal ligament (PDLF) and gingival fibroblasts (GF) were treated with ten-fold serial dilutions of scanning powder and the corresponding conditioned medium (filtrate of overnight incubation of powder in medium) starting with 30mg/ml. Bicinchoninic acid protein assay, formazan- and resazurin-based toxicity assays, live/dead and annexin V/propidium iodide (PI) staining and immunoassays for interleukin (IL)-6 and IL-8 were performed. Powder composition was analyzed using energy dispersive X-ray spectroscopy (EDS). ResultsFormazan and resazurin conversion was lesser in L929 cells than PDLF and GF in the presence of scanning powder. Induction of cell death was caused by 30mg/ml of powder in L929 cells but not in PDLF and GF. No pronounced impact of the conditioned medium was seen in cytotoxicity assays or live/dead-, and annexin V/PI staining. In PDLF and GF IL-6 expression was increased by the powder, while there was a decrease in IL-8. Powder particles did not deplete protein from medium. EDS showed a heterogeneous mixture consisting predominantly of titanium dioxide. ConclusionsScanning powder decreased cell activity and induced cell death in L929 cells at high concentrations. Human oral fibroblasts showed an increase in IL-6 levels but more resistance to the cytotoxicity of the powder. Within the limitations of an in vitro study our results suggest that proper cleaning after scanning is of clinical relevance to avoid potential unwanted effects of the powder.

Construction of Vascularized Oral Mucosa Equivalents Using a Layer-by-Layer Cell Coating Technology.

There have been many advances in tissue engineering with respect to in vitro and in vivo models of oral mucosa equivalents (OMEs). To apply in vitro reconstructed oral mucosa models to regenerative medicine and alternatives to animal testing, it is necessary to develop the technology of reconstructing different types of oral tissues, such as control of epithelial differentiation and introduction of appendages. We previously reported that functional three-dimensional (3D) tissue models could be quickly constructed by using a layer-by-layer (LbL) cell coating technique that assembles extracellular matrix (ECM) nanofilms to a cell surface. In this study, 3D human OMEs composed of lamina-propria, keratinized or non-keratinized epithelium, and blood capillaries were constructed by using the LbL cell coating technology. Human oral mucosal fibroblasts (HOMFs) were coated with ECM nanofilms and accumulated for the construction of oral mucosal lamina-propria. To construct OMEs with keratinized or non-keratinized epithelium, human oral keratinocytes isolated from gingiva (human oral gingival keratinocytes: HOGKs) or human oral keratinocytes isolated from oral mucosa (human oral mucosal keratinocytes: HOMKs) were used in this study. We further studied the construction of epithelialized OMEs with density- and size-controlled blood capillary networks by using human umbilical vein endothelial cells (HUVECs). It was revealed that these constructions had barrier functions in accordance with their histological characterization. The OMEs with keratinization (K-OMEs) showed higher transepithelial electrical resistance (TEER) values compared with OMEs with non-keratinization (N-OMEs). The constructed epithelialized OMEs with blood capillaries are useful for in vitro/ex vivo research models and regenerative medicine as in oral tissue regeneration. The results suggest that OMEs with oral tissue appendages are more promising alternatives to animal testing and can be applied to the design of in vitro oral models that mimic human tissue organs.

Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

Extracellular lysyl oxidases (LOX and LOXL1–LOXL4) are critical for collagen biosynthesis. LOXL2 is a marker of poor survival in oral squamous cell cancer. We investigated mechanisms by which tumor cell secreted LOXL2 targets proximal mesenchymal cells to enhance tumor growth and metastasis. This study identified the first molecular mechanism for LOXL2 in the promotion of cancer via its enzymatic modification of a non-collagenous substrate in the context of paracrine signaling between tumor cells and resident fibroblasts. The role and mechanism of active LOXL2 in promoting oral cancer was evaluated and employed a novel LOXL2 small molecule inhibitor, PSX-S1C, administered to immunodeficient, and syngeneic immunocompetent orthotopic oral cancer mouse models. Tumor growth, histopathology, and metastases were monitored. In vitro mechanistic studies with conditioned tumor cell medium treatment of normal human oral fibroblasts were carried out in the presence and absence of the LOXL2 inhibitor to identify signaling mechanisms promoted by LOXL2 activity. Inhibition of LOXL2 attenuated cancer growth and lymph node metastases in the orthotopic tongue mouse models. Immunohistochemistry data indicated that LOXL2 expression in and around tumors was decreased in mice treated with the inhibitor. Inhibition of LOXL2 activity by administration of PXS-S1C to mice reduced tumor cell proliferation, accompanied by changes in morphology and in the expression of epithelial to mesenchymal transition markers. In vitro studies identified PDGFRβ as a direct substrate for LOXL2, and indicated that LOXL2 and PDGF-AB together secreted by tumor cells optimally activated PDGFRβ in fibroblasts to promote proliferation and the tendency toward fibrosis via ERK activation, but not AKT. Optimal fibroblast proliferation in vitro required LOXL2 activity, while tumor cell proliferation did not. Thus, tumor cell-derived LOXL2 in the microenvironment directly targets neighboring resident cells to promote a permissive local niche, in addition to its known role in collagen maturation.

Extracellular vesicles derived from cancer-associated fibroblasts induce the migration and invasion of oral squamous cell carcinoma

ABSTRACTAs one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.

Relevance of Topographic Parameters on the Adhesion and Proliferation of Human Gingival Fibroblasts and Oral Bacterial Strains.

Dental implantology allows replacement of failing teeth providing the patient with a general improvement of health. Unfortunately not all reconstructions succeed, as a consequence of the development of infections of bacterial origin on the implant surface. Surface topography is known to modulate a differential response to bacterial and mammalian cells but topographical measurements are often limited to vertical parameters. In this work we have extended the topographical measurements also to lateral and hybrid parameters of the five most representative implant and prosthetic component surfaces and correlated the results with bacterial and mammalian cell adhesion and proliferation outcomes. Primary human oral gingival fibroblast (gum cells) and the bacterial strains: Streptococcus mutans, Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans, implicated in infectious processes in the oral/implant environment were employed in the presence or absence of human saliva. The results confirm that even though not all the measured surface is available for bacteria to adhere, the overall race for the surface between cells and bacteria is more favourable to the smoother surfaces (nitrided, as machined or lightly acid etched) than to the rougher ones (strong acid etched or sandblasted/acid etched).

Comparison of the in vitro and in vivo toxic effects of thymoquinone using oral cancer HSC-3 and HSC-4 cell lines, oral fibroblasts, HACAT cell line, and Zebrafish embryos

Abstract The discovery and development of anticancer agents were made through long processes from screening new compounds in various cell lines, animal experimental studies, until clinical trials Majority of cancer chemotherapeutic agents have been initially screened in an in vitro cell-based system and then subsequently studied in an in vivo model using small animals destined for pharmacokinetic properties. Zebrafish (Danio rerio) has become one of the most popular animal model systems used for chemical toxicity tests and drug discovery. Thymoquinone (TQ) is the main constituent of black seed (Nigella sativa, spp) essential oil which shows anti-neoplastic activities in various tumor cells. However, to date, studies on the toxicity of TQ on normal cells or animal model are still insufficient. Here we report that TQ is selective toxic to different human cell lines and show negligible effect on the Zebrafish. The MTT assay demonstrated that TQ induced cytotoxicity on oral cancer cells (HSC-3 and HSC-4) but not on the normal cells (human oral fibroblast and HACAT cells). Moreover, no significant morphological change was observed in the Zebrafish model following TQ exposure at the concentration (0.003 mg/mL), similar to those in the cancer cells. The study suggests that TQ as a cancer chemotherapeutic candidate without apparent adverse effects.

Cytotoxic and genotoxic potential of the type I photoinitiators BAPO and TPO on human oral keratinocytes and V79 fibroblasts

ObjectivesPhenylbis(acyl) phosphine oxide (BAPO) and diphenyl(acyl) phosphine oxide (TPO) are alternative photoinitiators to camphorquinone (CQ) in dental resinous materials. Aim of this study was to investigate their cytotoxic/genotoxic potential in human oral keratinocytes (OKF6/Tert2) and Chinese hamster lung fibroblasts (V79) in comparison to CQ. MethodsCells were exposed to different concentrations of BAPO and TPO (1–50μM). Cytotoxicity was evaluated using H33342 and MTT assay, cell proliferation by BrdU proliferation assay and microscopy. Effects on cellular redox homeostasis were assessed by detecting intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) using the DCFH2 assay and by quantification of mRNA expression of oxidatively regulated, cyto-protective enzymes. Genotoxic potential was determined by use of micronucleus (MN) assay. ResultsBAPO and TPO induced a concentration-dependent decrease of cell number. BAPO and TPO showed 50- to 250-fold higher cytotoxicity than CQ. In contrast to CQ, both photoinitiators revealed no increase of intracellular ROS/RNS. However, BAPO (10μM) at least significantly induced mRNA-expression of redox-regulated proteins after 24h similar to 2.5mM CQ. Additionally, BAPO significantly raised the number of micronuclei, but only in V79 cells (10μM: 12±1, 2.5mM CQ: 15±1, medium control: 6±3). However, it also significantly decreased proliferation of these cells (10μM BAPO: 19.8%±7.3% compared to controls). SignificanceBAPO and TPO revealed concentration-dependent cytotoxic effects in human oral keratinocytes and V79 cells. However, in contrast to CQ, no generation of intracellular ROS/RNS was found. Only BAPO induced genotoxicity in V79 cells.

Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model

Porphyromonas gingivalis-induced inflammatory effects are mostly investigated in monolayer cultured cells. The aim of this study was to develop a 3D spheroid model of gingiva to take into account epithelio-fibroblastic interactions. Human gingival epithelial cells (ECs) and human oral fibroblasts (FBs) were cultured by hanging drop method to generate 3D microtissue (MT) whose structure was analyzed on histological sections and the cell-to-cell interactions were observed by scanning and transmission electron microscopy (SEM and TEM). MTs were infected by P. gingivalis and the impact on cell death (Apaf-1, caspase-3), inflammatory markers (TNF-α, IL-6, IL-8) and extracellular matrix components (Col-IV, E-cadherin, integrin β1) was evaluated by immunohistochemistry and RT-qPCR. Results were compared to those observed in situ in experimental periodontitis and in human gingival biopsies. MTs exhibited a well-defined spatial organization where ECs were organized in an external cellular multilayer, while, FBs constituted the core. The infection of MT demonstrated the ability of P. gingivalis to bypass the epithelial barrier in order to reach the fibroblastic core and induce disorganization of the spheroid structure. An increased cell death was observed in fibroblastic core. The development of such 3D model may be useful to define the role of EC–FB interactions on periodontal host-immune response and to assess the efficacy of new therapeutics.

Resins for stereolithographic 3D printing – the impact on L929 cells and human oral fibroblasts

Resins for stereolithographic 3D printing – the impact on L929 cells and human oral fibroblasts

Activity of two hyaluronan preparations on primary human oral fibroblasts.

Background and ObjectiveThe potential benefit of using hyaluronan (HA) in reconstructive periodontal surgery is still a matter of debate. The aim of the present study was to evaluate the effects of two HA formulations on human oral fibroblasts involved in soft tissue wound healing/regeneration.Material and MethodsMetabolic, proliferative and migratory abilities of primary human palatal and gingival fibroblasts were examined upon HA treatment. To uncover the mechanisms whereby HA influences cellular behavior, wound healing‐related gene expression and activation of signaling kinases were analyzed by qRT‐PCR and immunoblotting, respectively.ResultsThe investigated HA formulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding type III collagen and transforming growth factor‐β3, characteristic of scarless wound healing. The HAs upregulated the expression of genes encoding pro‐proliferative, pro‐migratory, and pro‐inflammatory factors, with only a moderate effect on the latter in gingival fibroblasts. In palatal but not gingival fibroblasts, an indirect effect of HA on the expression of matrix metalloproteinases 2 and 3 was detected, potentially exerted through induction of pro‐inflammatory cytokines. Finally, our data pointed on Akt, Erk1/2 and p38 as the signaling molecules whereby the HAs exert their effects on oral fibroblasts.ConclusionBoth investigated HA formulations are biocompatible and enhance the proliferative, migratory and wound healing properties of cell types involved in soft tissue wound healing following regenerative periodontal surgery. Our data further suggest that in gingival tissues, the HAs are not likely to impair the healing process by prolonging inflammation or causing excessive MMP expression at the repair site.

Smokeless tobacco extract inhibits proliferation and promotes apoptosis in oral mucous fibroblasts.

The consumption of smokeless tobacco extract (STE) is growing rapidly, and it has been implicated in several human diseases including diabetes, inflammation and a number of types of cancer. The toxicity of STE requires evaluation, as it is known to induce numerous public health issues. To investigate whether STE serves a role in cultured human oral mucosa fibroblasts (hOMFs), the present study examined HOMF morphology with inverted microscopy and immunofluorescence staining. The cell viability was measured with MTT assays, which detected the cell apoptosis rate via flow cytometry. The activities of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured via flow cytometry and commercial kits, subsequent to exposing the cells to various concentrations of STE. Reverse transcription quantitative polymerase chain reaction and western blot analyses were used to demonstrate that the mRNA and the protein expression levels of cell cycle-associated genes (cyclin-dependent kinase inhibitor 1 and cyclin D1), apoptosis-associated genes [B cell lymphoma 2 (Bcl-2) and Bcl-2-associatied X protein], tumor protein (p53), nuclear factor kappa light chain enhancer of activated B cells (NF-κB)-transcription factor (p65) signaling pathways, NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H: quinoneoxidoreductase1 (NQO1). The results indicated that the hOMF cells were positive for cytokeratin staining. STE induced G1-S cell cycle progression and cell apoptosis by regulating the cell cycle or apoptosis-associated proteins. STE treatment increased the concentrations of ROS and MDA, and decreased the concentrations of SOD and CAT. STE unregulated phosphorylated-p53, NF-κB p65, Nrf2, HO-1, and NQO1 expression levels in the hOMF cells. The present study demonstrated that STE appears to promote oral disease.

Abstract 458: Relationship between autophagy, activation and senescence in normal and cancer-associated human oral fibroblasts

Abstract Cancer-associated fibroblasts (CAFs) isolated from oral squamous cell carcinoma (OSCC) display an activated phenotype in that they express α-smooth muscle actin (αSMA) and are frequently prematurely senescent. OSCC cell lines can induce senescence in normal fibroblasts in vitro by a mechanism that involves TGF-β and reactive oxygen species. Another phenotype closely associated with senescence is autophagy, but the relationship between autophagy, activation and senescence in CAFs has not been examined in detail. We first examined autophagosome accumulation in normal human oral fibroblasts (NHOFs; n=3) and in a panel of CAFs (n=8) that exhibited different degrees of senescence. Autophagosome accumulation was observed in senescent CAFs, as shown by the increased expression of the LC3B-II protein and by the number of LC3B cytoplasmic puncta, which were quantified using immunofluorescence. The number of LC3B puncta was significantly greater (p<0.01) in senescent CAFs compared to NHOFs, and senescent CAFs contained a higher fraction of autophagic fibroblasts than NHOFs, as indicated by increased number of LC3B puncta per cell. Further, the number of LC3B puncta correlated positively with the degree of senescence, as demonstrated by senescence-associated β-galactosidase (SA β-Gal) activity. To investigate whether autophagy, activation and senescence develop simultaneously when fibroblasts are stimulated to undergo senescence, we examined the temporal induction of these phenotypes in NHOFs in response to TGF-β1. NHOFs were treated with TGF-β1 (4 ng/ml) for up to 10 days to induce autophagy, αSMA expression and SA β-Gal activity. The results showed that autophagic flux preceded fibroblast activation, which in turn was followed by senescence. In summary, our results demonstrate that CAFs from OSCC concomitantly display autophagic, activated and senescent phenotypes and that the induction of autophagy occurs before fibroblast activation and sensecence. Taken together, these data suggest that autophagy, activation and sensecence in fibroblasts may be part of a unified programme leading to the development of CAFs. Citation Format: May Leng Tan, Eric K. Parkinson, Lee Fah Yap, Ian C. Paterson. Relationship between autophagy, activation and senescence in normal and cancer-associated human oral fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 458.

Bone-Conditioned Medium Obtained From Calvaria, Mandible, and Tibia Cause an Equivalent TGF-β1 Response In Vitro.

Bones with different embryological origin and mode of ossification are supposed to vary in their capacity for supporting graft consolidation. The aim of the current pilot study was to assess the TGF-β1 activity of bone chips obtained from distinct anatomic locations. Conditioned medium was prepared from bone chips harvested from pig calvaria, mandible, and tibia. Human oral fibroblasts were exposed to bone-conditioned medium (BCM) followed by reverse transcriptase polymerase chain reaction of the TGF-β1 target genes. Also an immunoassay for interleukin 11 (IL-11) and TGF-β1 was performed. The impact of BCM on alkaline phosphatase activity was determined with murine MC3T3-E1 osteogenic cells. The authors report here that BCM contains TGF-β1 in the ng/mL range. Bone chips prepared from pig calvaria, mandible, and tibia femur had a similar capacity for increasing the expression of the TGF-β1 target genes IL-11, NOX4, and PRG4. Correspondingly, immunoassays revealed similar production of IL-11 by human oral fibroblasts. Furthermore, conditioned medium obtained from the 3 bones decreased alkaline phosphatase activity in MC3T3-E1 osteogenic cells. These preliminary data demonstrate that particulated bone grafts, regardless of embryological origin, mode of ossification and morphology, release a similar TGF-β1 activity.

Tanshinone Suppresses Arecoline-Induced Epithelial-Mesenchymal Transition in Oral Submucous Fibrosis by Epigenetically Reactivating the p53 Pathway.

Oral submucous fibrosis (OSF) induced by chewing of the areca nut has been considered to be a precancerous lesion with a high probability of developing oral squamous cell carcinoma. Tanshinone (TSN) is the main component extracted from Salvia miltiorrhiza, a traditional Chinese medicine, which was found to have diverse pharmacological effects, such as anti-inflammatory and antitumor. In the current study, we aimed to identify the inhibitory effects and the underlying mechanism of TSN on OSF progress. We found that treatment with TSN inhibited the arecoline-mediated proliferation of primary human oral mucosal fibroblasts and reversed the promotive effects of arecoline on the EMT process. By RNA deep sequencing, we screened two possible targets for TSN: LSD1 and p53. We confirmed that p53 is much lower in OSF than in normal mucous tissues. In addition, p53 and its downstream molecules were decreased by arecoline treatment in oral mucosal fibroblasts, which was reversed by treatment with TSN in a dose-dependent manner. Our results also revealed that arecoline stimulation resulted in hypermethylation of the promoter of TP53 and subsequent downregulation of p53 levels, which was reversed by TSN. Furthermore, we identified that LSD1 could epigenetically activate TP53 by recruiting H3K27me1 and H3K4m2 to its promoter. Our findings provide new insights into the mechanism by which TSN influences arecoline-induced OSF and rationale for the development of clinical intervention strategies for OSF and even oral squamous cell carcinoma.

Lysophosphatidic Acid Regulates the Vitamin D Receptor and its Metabolism‐Related Genes in Human Gingival Fibroblasts

Our laboratory has established that lysophosphatidic acid (LPA) regulates the biology of human oral fibroblasts, and that gingival fibroblasts (GF) express the LPA1–5 receptor subtypes. We have also shown that LPA 18:1 controls immediate‐early GF gene transcription of inflammatory cytokines, and of their receptors and regulators. Vitamin D3 is a key hormone in decreasing inflammation, and has been shown by Nastri et al. (2018) to decrease pro‐inflammatory and increase anti‐inflammatory cytokine production by P. gingivalis‐ and S. pyogenes‐infected human GF. Therefore, we hypothesized that LPA would also modulate transcription of its receptor, VDR, since chronic periodontal disease (PD) is an inflammatory condition in which we have reported that LPA is elevated in human gingival crevicular fluid and saliva (Bathena et al., 2011). After GF treatment with 1 × 10−5 M LPA 18:1 for 2h or 8h, mRNA was extracted. Agilent Whole Human Genome Oligonucleotide Microarray analyses [n=3; each n= pooled mRNA derived from 3 healthy young donors; (9 total donors; the study conformed to the Declaration of Helsinki guidelines, was approved by the Creighton University IRB, and informed written consent was obtained from all donors)] revealed that LPA consistently and significantly down‐regulated the VDR vs. control (≤ −2.0 ± 0.5‐fold at 2h, and −3.1 ± 1.1 fold at 8h, respectively). Of great interest, the receptor cubilin (CUBN), which has been shown in other systems to bind vitamin D binding protein (DBP), was also down‐regulated in groups 1 (−2.0) and 2 (−4.1) at 8h; it may be involved in the secretion of vitamin D by GF, as postulated by Liu et al., (2012). LPA treatment also significantly affected transcription of the cytochromes (CYP) CYP27A1 [−3.0 ± 0.6 (2h); −3.2 ± 1.4 (8h)] and CYP27B1 (induction or repression varied by group) which is involved in the production of metabolically active metabolites and which catalyzes the hydroxylation at the C‐1α position of 25(OH)D3 and 24R, 25(OH)2D3, respectively. These results for the VDR (minimum level of significance, p < 1×10−7) provide preliminary evidence that LPA likely exerts complex actions during periodontal inflammation by regulating it, along with other key receptors and enzymes in the vitamin D pathway.Support or Funding InformationHealth Future Foundation (D.R.C).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.