Fabrication of cultured oral gingiva by tissue engineering techniques without materials of animal origin

BACKGROUND: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). METHODS: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. RESULTS: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. CONCLUSIONS: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.     

Vascular endothelial growth factor and transforming growth factor-alpha and -beta1 are released from human cultured gingival epithelial sheets

BACKGROUND: It has been demonstrated that human cultured epithelial sheets prepared by tissue engineering techniques provide useful graft material for wound healing and tissue regeneration. However, limited information is available with regard to biological effects such as release of growth factors from human cultured gingival epithelial sheets (HCGES). The purpose of this study was to measure the levels of growth factors released from HCGES into culture medium. METHODS: Twenty patients aged 44 to 71 years with generalized chronic periodontitis were recruited, and their gingival tissues obtained during periodontal flap surgery. The levels of vascular endothelial growth factor (VEGF), transforming growth factor-alpha and -beta1 (TGF-alpha and -beta1), and epidermal growth factor (EGF) released into the culture medium were determined using enzyme-linked immunosorbent assay at the just confluent (T1) and the adequate stratification (T2) culture stages. The medium without cells was collected as a control (T0). Statistical tests were performed by analysis of variance and Sheffé multiple range test among T0, T1, and T2. RESULTS: Significantly higher levels of VEGF and TGF-alpha were observed at T1 and T2 compared to T0 (P<0.001). In addition, there was a significant difference in the TGF-alpha levels between T2 and T1 (P<0.001). TGF-beta1 at T1 was significantly higher in comparison to T0 (P <0.01). EGF had been released only in a small amount at T2. CONCLUSION: This study indicates that meaningful amounts of VEGF and TGF-alpha and -beta1 are released from HCGES, which suggests potential for promoting wound healing and tissue regeneration after grafting.     

A new era in periodontal and periimplant regeneration: use of growth-factor enhanced matrices incorporating rhPDGF

A new, powerful off-the-shelf wound healing and bone regeneration technology termed growth-factor enhanced matrix (GEM) has recently become available for clinical use. This graft material consists of a concentrated solution of pure recombinant human platelet-derived growth factor (rhPDGF-BB), the synthetic form of the body's key natural wound healing stimulator PDGF-BB, and an osteoconductive (bone scaffold) matrix. This is the first available purified, recombinant (synthetic) growth factor product and is the result of over a decade of extensive research. Clinical and animal study results with this graft material demonstrate that it is capable of simultaneously promoting wound healing, regeneration of bone, and acceleration of gingival attach-plent gain in challenging periodontal and periimplant defects.     

Effect of in vitro gingival fibroblast seeding on the in vivo incorporation of acellular dermal matrix allografts in dogs

BACKGROUND: Acellular dermal matrix allograft (ADMA) has been used in various periodontal procedures with successful results. Because ADMA has no blood vessels or cells, slower healing and incorporation are observed compared to a subepithelial connective tissue graft. Fibroblasts accelerate the healing process by regulation of matrix deposition and synthesis of a variety of growth factors. Thus, the objective of this study was to evaluate histologically if gingival fibroblasts affect healing and incorporation of ADMA in dogs when used as a subepithelial allograft. METHODS: Gingival fibroblasts were established from explant culture from the connective tissue of keratinized gingiva collected from the maxilla of seven mongrel dogs. ADMA was seeded with gingival fibroblasts and transferred to dogs. Surgery was performed bilaterally, and the regions were divided into two groups: ADMA+F (ADMA containing fibroblasts) and ADMA (ADMA only). Biopsies were performed after 2, 4, and 8 weeks of healing. RESULTS: The quantity of blood vessels was significantly higher in the ADMA+F group at 2 weeks of healing (Kruskal-Wallis; P <0.05). There was no statistical difference (P >0.05) in the number of cell layers, epithelial area, or inflammatory infiltrate between the two groups at any stage of healing. CONCLUSION: The enhanced vascularization in vivo in early stages supports the important role of fibroblasts in improving graft performance and wound healing of cultured graft substitutes.     

Effect of Emdogain on human periodontal fibroblasts in an in vitro wound-healing model

OBJECTIVE: The aim of this study was to evaluate the influence of Emdogain (EMD) on cultured gingival fibroblasts, periodontal ligament fibroblasts and dermal fibroblasts, using an in vitro model of wound healing. BACKGROUND: Enamel matrix derivative has been demonstrated to promote periodontal regeneration. However, the precise mechanisms by which this agent acts are still unclear. METHODS: The effect of EMD on proliferation of the cells was studied using subconfluent cultures of gingival fibroblasts and periodontal ligament fibroblasts. The cells were made quiescent overnight and then stimulated with various concentrations of EMD (10, 50, 100 and 150 microg/ml) for 24 h. Negative and positive controls were cells cultured in media containing 0.2% and 10% fetal calf serum (FCS). The DNA synthesis was measured by the cellular uptake of [3H]thymidine. For in vitro wounding the cells were cultured, wounded and stimulated with 0.2% FCS, 10% FCS and EMD at a concentration of 20 microg/ml. The percentage of wound fill after treatment was measured after d 1, 4, 6, 12 and 16. The proliferation of cells was also calculated by the extent of incorporation of crystal violet. RESULTS: The results demonstrated that cells in vitro fill an empty space by a combination of proliferation and cell migration. The most rapid closure of a wound area occurred where both proliferation and migration can occur as was seen when wounded cultures were maintained in 10% FCS or at a concentration of 20 microg/ml EMD which promoted proliferation. CONCLUSIONS: Therefore, EMD appears to exert an influence on cells that is compatible with improved wound healing.     

Effect of low-level Er:YAG laser irradiation on cultured human gingival fibroblasts

BACKGROUND: Low-level laser irradiation has been reported to enhance wound healing. Activation of gingival fibroblasts (GF) has a potential for early wound healing in periodontal treatment. The present study aimed to investigate the direct effect of low-level Er:YAG laser irradiation on gingival fibroblasts proliferation in order to clarify the laser effect on healing. METHODS: Cultured human gingival fibroblasts (hGF) were exposed to low-power, pulsed Er:YAG laser irradiation with different energy densities ranging from 1.68 to 5.0 J/cm(2). The cultures were analyzed by means of trypan blue staining and counted under a light microscope. The effect of Er:YAG laser on hGF was also evaluated using a transmission electron microscope (TEM). RESULTS: Cultures irradiated with Er:YAG laser presented faster cell growth when compared with untreated controls. This difference was statistically significant. Transmission electron microscopy revealed rough endoplasmic reticulum, prominent Golgi complexes, and mitochondria after laser irradiation. CONCLUSIONS: Our results showed that the low-level Er:YAG laser irradiation stimulates the proliferation of cultured gingival fibroblasts. The optimal stimulative energy density was found to be 3.37 J/cm(2). This result suggests that Er:YAG laser irradiation may be of therapeutic benefit for wound healing.     

The effect of low level laser irradiation on implant-tissue interaction. In vivo and in vitro studies

Low-level laser therapy (LLLT) is increasingly used in medicine and dentistry. It has been suggested that LLLT may be beneficial in the management of many different medical conditions, including pain, wound healing and nerve injury. The present thesis is based on a series of in vivo and in vitro experimental studies investigating whether LLLT has the potential to enhance titanium-implant interaction. Information about LLLT effect on bone healing is fundamental to understand whether LLLT may improve implant-tissue interaction. Thus in the initial study (I), the effect of LLLT on bone healing and growth in rat calvarial bone defects was investigated. It was found that LLLT may accelerate metabolism and/or mineralization during early bone healing. Based on these findings, study II explored the hypothesis that LLLT can enhance implant integration in the rabbit tibial bone. It was shown that LLLT stimulated the mechanical strength of the interface between the implant and bone after a healing period of 8 weeks. Histomorphometrical and mineral analyses showed that the irradiated implants had greater bone-to-implant contact than the controls. In the in vitro experiments, cellular responses to LLLT were studied in two cell types: primary cultures of human gingival fibroblasts and human osteoblast-like cells, with special reference to attachment, proliferation, differentiation and production of transforming growth factor beta1 (TGF-beta1). The objectives of studies III & IV were to develop a standardized, reproducible in vitro model for testing a GaAlAs diode laser device and to document the influence of single or multiple doses of LLLT, as a guide to defining the optimal laser dose for enhancing cell activity. A further objective was to investigate the effect of LLLT on initial attachment and subsequent behaviour of human gingival fibroblasts cultured on titanium. While both multiple doses (1.5 and 3 J/cm2) and a single dose (3 J/cm2) enhanced cellular attachment, proliferation increased only after multiple doses (1.5 and 3 J/cm2). Study V concerned the response to LLLT of osteoblast-like cells, derived from human alveolar bone cultured on titanium implant material. In this study LLLT significantly enhanced cellular attachment. Greater cell proliferation in the irradiated groups was observed first after 96 h indicating that the cellular response is dose dependent. Osteocalcin synthesis and TGF-beta1 production were significantly stimulated on the samples exposed to 3 J/cm2. The following conclusions are drawn from the results of these five studies: LLLT can promote bone healing and bone mineralization and thus may be clinically beneficial in promoting bone formation in skeletal defects. It may be also used as additional treatment for accelerating implant healing in bone. LLLT can modulate the primary steps in cellular attachment and growth on titanium surfaces. Multiple doses of LLLT can improve LLLT efficacy, accelerate the initial attachment and alter the behaviour of human gingival fibroblasts cultured on titanium surfaces. The use of LLLT at the range of doses between 1.5 and 3 J/cm2 may modulate the activity of cells interacting with an implant, thereby enhancing tissue healing and ultimate implant success.     

Effects of serum on hepatocyte growth factor secretion and activation by periodontal ligament and gingival fibroblasts

BACKGROUND: Hepatocyte growth factor (HGF)/scatter factor is a paracrine growth factor secreted by mesenchymal cells, which exerts an effect on a variety of epithelial cell types. Our recent study revealed that periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) cultured in the presence of serum which contains various stimulants produced HGF or HGF-like factor, a predominant chemoattractant for gingival epithelial cells, and suggested that it could be involved in epithelial down-growth in periodontitis. METHODS: To clarify whether serum in medium stimulates PLF and GF to synthesize or activate HGF, the effect of fetal bovine serum (FBS) on HGF production was determined by enzyme-linked immunoabsorbent assay (ELISA), and its chemotactic activity for gingival epithelial cells was examined by modified Boyden chamber assay. RESULTS: One to 10% FBS in the culture medium stimulated HGF secretion in a dose-dependent manner and the chemotactic activity was decreased by treatment with anti-hHGF neutralizing antibody. Furthermore, fibroblast-conditioned medium incubated with FBS and aprotinin reduced its chemotactic activity. Interestingly, serum-free culture of PLF and GF produced potent chemoattractants for gingival epithelial cells other than HGF. CONCLUSIONS: These results show that FBS stimulates both HGF secretion and activation by PLF and GF.     

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. K.GF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa. from gingival, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal nature of fibroblasts. Cells were first cultured in DMEM with 0.5% fetal calf serum (PCS) and then incubated for 8 h, and 72 h in fresh DMEM with 10% PCS. Total RNA was isolated and used for Northern blotting with a P³²-labeled KGP cDNA. probe. Total RNA from cultured keratinocytes was used as negative controls. KGF mRNA was found in all cultured fibroblasts. Upon addition of 10% PCS to the cell cultures, an increase in KGF mRNA levels was noticed especially after 72 h. Furthermore. RT-PCR analysis of material scraped from the tooth root surface indicated the presence of KGF mRNA even in non cultured periodontal ligament cells.     

Multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair

Fibroblasts are cells of mesenchymal origin. They are responsible for the production of most extracellular matrix in connective tissues and are essential for wound healing and repair. In recent years, it has become clear that fibroblasts from different tissues have various distinct traits. Moreover, wounds in the oral cavity heal under very special environmental conditions compared with skin wounds. Here, we reviewed the current literature on the various interconnected functions of gingival and mucoperiosteal fibroblasts during the repair of oral wounds. The MEDLINE database was searched with the following terms: (gingival OR mucoperiosteal) AND fibroblast AND (wound healing OR repair). The data gathered were used to compare oral fibroblasts with fibroblasts from other tissues in terms of their regulation and function during wound healing. Specifically, we sought answers to the following questions: (i) what is the role of oral fibroblasts in the inflammatory response in acute wounds; (ii) how do growth factors control the function of oral fibroblasts during wound healing; (iii) how do oral fibroblasts produce, remodel and interact with extracellular matrix in healing wounds; (iv) how do oral fibroblasts respond to mechanical stress; and (v) how does aging affect the fetal‐like responses and functions of oral fibroblasts? The current state of research indicates that oral fibroblasts possess unique characteristics and tightly controlled specific functions in wound healing and repair. This information is essential for developing new strategies to control the intraoral wound‐healing processes of the individual patient.     

Light‐Emitting Diode Irradiation Promotes Donor Site Wound Healing of the Free Gingival Graft

Background: This study aims to evaluate the effect of light‐emitting diode (LED) light irradiation on the donor wound site of the free gingival graft. Methods: Rat gingival fibroblasts were chosen to assess the cellular activities and in vitro wound healing with 0 to 20 J/cm² LED light irradiation. Seventy‐two Sprague‐Dawley rats received daily 0, 10 (low‐dose [LD]), or 20 (high‐dose [HD]) J/cm² LED light irradiation on the opened palatal wound and were euthanized after 4 to 28 days; the healing pattern was assessed by histology, histochemistry for collagen deposition, and immunohistochemistry for tumor necrosis factor (TNF)‐α infiltration. The wound mRNA levels of heme oxygenase‐1 (HO‐1), TNF‐α, the receptor for advanced glycation end products, vascular endothelial growth factor, periostin, Type I collagen, and fibronectin were also evaluated. Results: Cellular viability and wound closure were significantly promoted, and cytotoxicity was inhibited significantly using 5 J/cm² LED light irradiation in vitro. The wound closure, reepithelialization, and collagen deposition were accelerated, and sequestrum formation and inflammatory cell and TNF‐α infiltration were significantly reduced in the LD group. HO‐1 and TNF‐α were significantly upregulated in the HD group, and most of the repair‐associated genes were significantly upregulated in both the LD and HD groups at day 7. Persistent RAGE upregulation was noted in both the LD and HD groups until day 14. Conclusion: LED light irradiation at 660 nm accelerated palatal wound healing, potentially via reducing reactive oxygen species production, facilitating angiogenesis, and promoting provisional matrix and wound reorganization.     

Plasma rich in growth factors promote gingival tissue regeneration by stimulating fibroblast proliferation and migration and by blocking transforming growth factor-β1-induced myodifferentiation

Background: Periodontitis involves inflammation and infection of the ligaments and bones that support the teeth. Gingival fibroblasts are the most abundant cells in periodontal tissue, and they play a role in maintaining the structural integrity of the tissue. Plasma rich in growth factors contain a pool of proteins and growth factors that promote wound healing and tissue regeneration. In the present study, we evaluate the potential of different formulations obtained with this approach to stimulate several biologic processes involved in wound healing, including fibroblast proliferation, migration, adhesion, and the autocrine release of some angiogenic factors and extracellular matrix components. Furthermore, the ability of this technology to prevent and inhibit transforming growth factor β1‐induced myodifferentiation was also determined. Methods: Cell proliferation was evaluated through a colorimetric assay, cell migration was performed on culture inserts, and cell adhesion was studied through a fluorescence‐based method. Enzyme‐linked immunosorbent assay was used to determine some of the biomolecules released by gingival fibroblasts. Smooth muscle actin expression was assessed through immunofluorescence microscopy. Results: Results showed that plasma rich in growth factors significantly increased gingival fibroblast proliferation, migration, and cell adhesion on type I collagen matrix. In addition, it stimulated the autocrine expression of vascular endothelial growth factor, hepatocyte growth factor, and hyaluronic acid. The myofibroblast phenotype, which is characterized by expressing α‐smooth muscle actin, was inhibited and reverted by treating with this technology. Conclusion: These findings suggest that plasma rich in growth factors is capable of promoting regeneration of gingival connective tissue by stimulating some of the main processes involved in wound regeneration.     

PHBV补片修补羊皮肤缺损的初步研究

目的：评估纳米级PHBV多聚体支架和PHBV-ECOFLEX复合物支架是否有利于皮肤缺损的愈合。方法：雌性山羊2只,按修复材料不同分为：游离皮片原位移植（对照1组）、无修复材料（对照2组）、纳米级PHBV聚集体（材料1组）、PHBV-ECOFLEX复合物（材料2组）4组。实验动物均切除全层皮肤,造成缺损,同期进行修复。皮肤缺损直径分别为1.0cm、1.5cm和2.0cm,分别于术后1周和3周进行组织学检查。结果：对照1组可见完整上皮细胞及皮肤附件;对照2组见有大量炎性细胞及成纤维细胞;材料1组修复材料无降解,周围炎性细胞浸润,材料内可见吞噬细胞和束状的成纤维细胞;材料2组修复材料基本降解,可见新生的复层鳞状上皮及角化层。结论：PHBV-ECOFLEX复合物支架植入皮肤缺损区后,可诱导上皮细胞增殖、成熟,并且降解速率与上皮细胞生长速率较匹配,有利于皮肤缺损的愈合。     

Effect of biologic mediators on ciprofloxacin accumulation by gingival fibroblasts

BACKGROUND: Human gingival fibroblasts actively accumulate fluoroquinolone antimicrobials. Because fibroblasts are prevalent in gingiva, they may help sustain therapeutic fluoroquinolone levels at that site. The purpose of this study was to determine whether mediators associated with infection or injury can enhance ciprofloxacin accumulation by gingival fibroblasts. METHODS: Quiescent fibroblast monolayers were treated for 1, 6, or 24 hours with several concentrations of tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF)-2, or insulin-like growth factor (IGF)-1. Transport was assayed by measuring cell-associated fluoroquinolone fluorescence. RESULTS: All mediators significantly enhanced ciprofloxacin transport in a dose dependent manner (P < 0.05; ANOVA). Except for TNF, this enhancement was associated with a decrease in the Km of ciprofloxacin transport. Maximal enhancement was observed with 10 ng/ml PDGF or FGF and 30 ng/ml TNF, TGF, or IGF. Brief (1 hour) treatment with TNF or FGF upregulated ciprofloxacin accumulation by a maximum of 13% to 14%, whereas TGF, PDGF, and IGF enhanced this process by 19% to 24%. All of the mediators enhanced ciprofloxacin accumulation by a maximum of 19% to 24% after 6 hours and 30% to 38% after 24 hours. The accumulation of other fluoroquinolones (e.g., gatifloxacin) was also slightly enhanced. CONCLUSIONS: Gingival fibroblasts treated with cytokines or growth factors accumulate significantly more ciprofloxacin than untreated controls. This provides a mechanism by which ciprofloxacin could be preferentially distributed to gingival wound or inflammatory sites, yielding local therapeutic levels that are more sustained than in serum.     

Pluronic polyol effects on human gingival fibroblast attachment and growth

BACKGROUND: Enhanced speed of human gingival fibroblast (HGF) spreading and attachment, as affected by ionic bonding interactions, may facilitate cell orientation and subsequent collagen synthesis to promote early wound healing. The purpose of this study was to determine the in vitro effects of pluronic polyols, a family of widely used surfactants currently used as drug carriers for antibiotic, anti-inflammatory, and anti-neoplastic agents, on the attachment and growth of human gingival fibroblasts (HGF) to dentin and plastic surfaces using established tissue culture techniques. METHODS: Plastic culture wells containing Eagle's minimal essential media (EMEM) with 10% fetal calf serum and Pluronic F-68 or F-127 in concentrations from 1.2 x 10(-2) to 1.2 x 10(-10) M were incubated with HGF and run in replicates of ten. Attached cells were quantified by measuring the optical density of methylene blue-stained cells. Additional experiments were conducted using human dentin sections as a substrate and Pluronic F-68 or F-127 at a concentration of 1.2 x 10(-8) M. In these experiments, HGF were stained with acridine orange and quantified per unit area of dentin by fluorescence microscopy. RESULTS: Attachment and growth of HGF to both plastic and dentin were significantly increased over serum controls by very low concentrations of Pluronic F-68 and F-127 by 30 minutes, with attachment reaching a plateau at 2 hours. CONCLUSIONS: Pluronic polyols, a family of widely used surfactants, in very low dosages may be beneficial in early postsurgical wound healing by facilitating early attachment and enhancing the growth rate of human gingival fibroblasts.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

Tumor Necrosis Factor‐α and Interleukin (IL)‐1β, IL‐6, and IL‐8 Impair In Vitro Migration and Induce Apoptosis of Gingival Fibroblasts and Epithelial Cells,Delaying Wound Healing

Background: Multiple factors affect oral mucosal healing, such as the persistence of an inflammatory reaction. The present study evaluates effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, IL‐6, and IL‐8 on epithelial cells (ECs) and human gingival fibroblasts (GFs) in vitro. Methods: GFs and ECs were seeded in 96‐well plates (1 × 10⁴ cells/well) in plain culture medium (Dulbecco’s modified Eagle’s medium [DMEM]) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum, and incubated for 24 hours. Both cell lines were exposed for 24 hours to the following cytokines: 1) TNF‐α (100 ng/mL); 2) IL‐1β (1 ng/mL); 3) IL‐6 (10 ng/mL); and 4) IL‐8 (10 ng/mL). All cytokines were diluted in serum‐free DMEM. Control cultures were exposed only to serum‐free DMEM. Effects of exposure to inflammatory cytokines were determined by means of: 1) apoptosis (anexin V); 2) cell migration (wound healing assay); 3) inflammatory cytokine synthesis (enzyme‐linked immunosorbent assay). Data were analyzed by Kruskal–Wallis and Mann–Whitney U tests (α = 0.05). Results: Increased apoptosis rates were noted when cells were exposed to inflammatory cytokines, except ECs exposed to IL‐1β. Cell migration was negatively affected by all inflammatory cytokines for both cell lines. ECs and GFs exposed to IL‐6 and IL‐8 significantly increased synthesis of TNF‐α and IL‐1β. Conclusions: Demonstrated results indicate negative effects of tested inflammatory cytokines on ECs and GFs, inducing apoptosis and impairing cell migration. These results can justify delayed oral mucosa healing in the presence of inflammatory reaction.