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.     

Biocompatibility of various collagen membranes in cultures of human PDL fibroblasts and human osteoblast-like cells

The aim of the present study was to evaluate the biocompatibility of differently cross-linked collagen membranes in cultures of human PDL fibroblasts and human osteoblast-like cells. Four collagen membranes [BioGide (BG), BioMend (BM), Ossix (OS) and TutoDent (TD)] were tested. Cells plated on culture dishes (CD) served as positive controls. Six specimens of each membrane were incubated with (1) human PDL fibroblasts [2 x 10(4) cells] (n=24), and (2) human osteoblast-like cells (SaOs-2) [2 x 10(4) cells] (n=24) under standardized conditions. After 7 days, adherent cells were stained with hematoxylin and counted using a reflected light microscope and the cell density per square millimeter was calculated. Additionally, cell morphology was investigated using scanning electron microscopy (SEM). Cell counts were presented as means and standard deviations (cells/mm(2)) and analyzed for statistical difference using the Wilcoxon test: (1) CD (434+/-76)>BG (64+/-19)=OS (61+/-8)>TD (44+/-4)>BM (12+/-5); (2) CD (453+/-92)>BG (94+/-46)=TD (84+/-49)>OS (41+/-23)>BM (0). SEM examination revealed that PDL fibroblasts adherent on BG, TD and OS appeared spindle-shaped and flat, like cells on CD. SaOs-2 osteoblasts adherent on CD were star shaped and flat, but mostly round in shape on BG, OS and TD. BM appeared to be incompatible with the attachment and proliferation of SaOs-2 cells; however, a few PDL fibroblasts were found in a round shape. Within the limits of the present study, it was concluded that (i) BG, TD and OS promoted, and (ii) BM inhibited the attachment and proliferation of human PDL fibroblasts and human SaOs-2 osteoblasts.     

Regulation of HAS expression in human synovial lining cells of TMJ by IL-1beta

Hyaluronan (HA), a major glycosaminoglycan of synovial fluid, is synthesised by a class of membrane-bound HA synthase (HAS) proteins. In the present study, we investigated the regulatory roles of IL-1beta on HAS gene expression and HA production by the fibroblastic synovial lining cells. The synovial lining cells from synovial membrane in human temporomandibular joint (TMJ) were cultured and characterised using immunocytochemistry with CD14, CD44, and vimentin monoclonal antibodies. With or without treatment with IL-1beta, the production of HA was detected with radiometric assay and the expression of HAS mRNAs were analysed with a semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR). HA synthesis was significantly augmented with 1ng/ml of IL-1beta for both 24 and 48h stimulation, however the production of HA declined if stimulated with 10ng/ml of IL-1beta. The expression of HAS2 and 3 mRNA were enhanced about 4.2- and 7.2-fold after 4h stimulation with 1ng/ml of IL-1beta, respectively. From these results, it is concluded that IL-1beta functions on regulating HAS expression and consequently promoting the secretion of HA in synovial lining cells from TMJ.     

尼古丁对人牙周膜成纤维细胞中Fas/Apo-1（CD95）表达的影响

目的：研究尼古丁（nicotine）对体外培养的人牙周膜成纤维细胞（periodontal ligament fibroblasts,PDLFs）中Fas/Apo-1（CD95）表达的影响。方法：采用组织块培养法培养原代人牙周膜成纤维细胞并传代取处于对数生长期的第6代细胞用于实验,采用浓度为2mg/L的尼古丁处理第6代牙周膜成纤维细胞72h,通过免疫组织化学染色法检测尼古丁对人牙周膜成纤维细胞中Fas表达的情况。结果：证实尼古丁作用于人牙周膜成纤维细胞后,胞膜中Fas/Apo-1（CD95）抗原呈阳性表达。结论：提示尼古丁可通过Fas系统这一途径导致人牙周膜成纤维细胞的凋亡,从而加重牙周组织破坏的程度和影响牙周组织的再生。     

The effect of local delivery of PDGF-BB on attachment of human periodontal ligament fibroblasts to periodontitis-affected root surfaces--in vitro

BACKGROUND/AIMS: The purpose of this study was to determine at what concentration does platelet-derived growth factor-BB (PDGF-BB) provide for optimal stimulation of human periodontal ligament fibroblasts (PDL) to adhere to periodontitis affected root surfaces. METHOD: 80 root dentine specimens were prepared from extracted periodontally diseased teeth obtained from patients ranging in age between 35 to 60 years. The root dentine specimens were associated with the subgingival area opposing the periodontal pocket for each extracted tooth. 10 healthy root dentine specimens were obtained from teeth extracted for orthodontic reasons and served as controls. The specimens were distributed into 9 groups (10 specimens in each). In group 1, PDL fibroblasts were cultured on the specimen surface of a diseased treated control. In group 2, PDL fibroblasts were cultured on the specimen surface of a healthy control. In groups 3 to 9, PDL fibroblasts were cultured on a pre-treated specimen surface with concentrations ranging from 5, 10, 20, 50, 100, 200 and 300 ng/ml PDGF-BB, respectively. After 24 h incubation, the media were removed, specimens were fixed, processed for SEM viewing and photographed at 750x. Fibroblast adherence was measured by counting number of cells within a standard test area and cell morphology was scored. RESULTS: Findings suggest dentine specimens pretreated with 5, 10 and 20 ng/ml PDGF-BB were not significantly different in number of adherent cells from the diseased treated control. However, at concentrations of 50, 100, 200 and 300 ng/ml, a highly significant increase in number of adherent fibroblasts was detected when compared to the diseased treated control. At these concentrations, the cell morphology was comparable to that of the healthy control. CONCLUSIONS: PDGF-BB in concentrations equal to or greater than 50 ng/ml demonstrates a significant stimulation of PDL cells adherence to periodontal diseased root surfaces. Since the higher concentrations resulted in similar effects as obtained by 50 ng/ml, it may therefore be considered that this concentration provides for optimal stimulation of human periodontal ligament fibroblasts (PDL) to adhere to periodontitis-affected root surfaces.     

In vitro effect of platelet-derived growth factor-BB on collagen synthesis and proliferation of human periodontal ligament cells

OBJECTIVES: Platelet-derived growth factor (PDGF)-BB is a polypeptide growth factor which has been shown to stimulate periodontal regeneration. In this study, we investigated the time- and dose-dependent effect of PDGF-BB on the proliferation and collagen synthesis of human periodontal ligament (PDL) cells. MATERIALS AND METHODS: For the proliferation assay, PDL cells were cultured in 0.01-10 ng ml(-1) of PDGF-BB for 12 or 24 h, and cell numbers were counted. For the collagen synthesis assay, PDL cells were cultured in 0.1-10 ng ml(-1) of PDGF-BB for 1 to 24 h. The ratio of collagen content in total protein was evaluated, and the gene expression of type I collagen was assessed quantitatively by Northern blotting analysis. RESULT AND CONCLUSIONS: PDGF-BB stimulated the proliferation of PDL cells in a time- and dose-dependent manner with the maximum effect at 10 ng ml(-1). PDGF-BB induced the collagen synthesis of PDL cells with the maximum effect for 24-h treatment, and 1 ng ml(-1) of PDGF-BB. PDGF-BB exhibits an inverse dose-dependent effect on proliferation and collagen synthesis by PDL cells. These findings suggest that PDGF-BB is one of the important regulators of the maintenance of the extracellular matrix in PDL, and may play an important role in the regeneration of PDL.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

Expression of integrins by human periodontal ligament and gingival fibroblasts and their involvement in fibroblast adhesion to enamel matrix-derived proteins

We showed recently that human periodontal ligament (PDL) and gingival fibroblasts adhere and spread on enamel matrix protein (EMP) coatings. In the present study, we investigated whether this interaction can be attributed to integrin expression. Human PDL and gingival fibroblasts were cultured for periods up to 24 h on EMP coatings, in the presence of synthetic RGD-containing peptide or an antibody against the beta1 integrin subunit. The cells were first cultured for 24 h under serum-free conditions and then cultured on EMP coatings for 48 h. Integrin expression levels were assessed by flow cytometry analysis. It was found that attachment and spreading on EMP was inhibited by the synthetic RGD-containing peptide, but not by a synthetic RGE-peptide. Both PDL and gingival fibroblasts showed expression of the integrin subunits, alpha2, alpha5, beta1, and the integrin, alphavbeta3. Incubation with an antibody against the beta1 subunit significantly inhibited the attachment and spreading of PDL and gingival fibroblasts on EMP coatings. We conclude that integrins are involved in the interaction of PDL and gingival fibroblasts with EMP.     

The expression pattern of hyaluronan synthase during human tooth development

In previous studies, hyaluronan (HA) and its major cell surface receptor CD44 have been suggested to play an important role during tooth development. HA synthases (HASs) are the enzymes that polymerize hyaluronan. Data on the expression pattern of HASs during tooth development is lacking and the aim of the present study was to investigate the localisation of HAS by immunohistochemistry in human tooth germs from different developmental stages. The distribution pattern of HAS in the various tissues of the "bell stage" tooth primordia corresponded to that of hyaluronan in most locations: positive HAS immunoreactivity was observed in the dental lamina cells, inner- and outer-enamel epithelium. On the stellate reticulum cells, moderate HAS signal was observed, similar to the layers of the oral epithelium, where faint HAS immunoreactivity was detected. At the early phase of dental hard tissues mineralization, strong HAS immunoreactivity was detected in the odontoblasts and their processes, as well as in the secretory ameloblasts and their apical processes and also, the pulpal mesenchymal cells. The HAS signals observed in odontoblasts and ameloblasts gradually decreased with age. Our results demonstrate that hyaluronan synthesised locally by different dental cells and these results provide additional indirect support to the suggestion that HA may contribute both to the regulation of tooth morphogenesis and dental hard tissue formation.     

三维培养的牙周成纤维细胞在循环张力作用下基质降解的变化

目的：研究培养在胶原胶中的牙周成纤维细胞受循环张力作用后胞外基质降解的变化。方法：植入牙周成纤维细胞的胶原胶受循环张力机械作用24h，应用组化染色、酶凝胶电泳和ALP、DNA分析，观察细胞受力后形态学变化，检测培养基及胶原胶中MMPs的表达变化、细胞的增殖、ALP活性的改变。结果：受力后，细胞沿应力方向排列，DNA含量增加，培养基中MMP-2的表达增加，ALP活性降低。结论：循环张力能改变细胞排列方向，促进了细胞增殖和胞外基质的改建。     

四环素对人牙周膜成纤维细胞的生物学作用

目的探讨四环素对体外培养的人牙周膜成纤维细胞(I1PDLFs)的生物学作用。方法将不同浓度的四环素(1,5,20,100,500,2 500习ml)加人体外培养的HPDLFs中,孵育2d后,在倒置显微镜下观察其对细胞形态的影响,并用M,1法、考马司亮蓝法及“H-TdR掺人法,分别检测四环素对细胞的增殖活性、蛋白合成及DNA合成的影响。结果在1 -. 100 pg/ml的浓度范围内,细胞形态呈正常的梭形或纺锤形。在20- 100留ml的浓度范围内,四环素可促进HPDLFs的增殖及生物合成(P < 0.01)。当四环素的浓度增至2 500 lcg/ml,不仅使细胞的镜下形态发生了明显改变,而且严重抑制了细胞的生物学活性。结论在合适的浓度范围内,四环素能促进HPDLFs的增殖及生物合成,而浓度过高则具有细胞毒性。     

Proliferation and adhesion of periodontal ligament cells on synthetic biominerals

OBJECTIVE: Hydroxiapatite (HA) has been suggested as a useful biomaterial to support the regeneration of tissues. In this study, we investigated the adhesion of periodontal ligament (PDL) cells on octacalcium phosphate (OCP) and its hydrolyzed apatitic product (HL), which are known precursors of HA. METHODS: Rat PDL cells were cultured on OCP or HL-coated dishes. Cell proliferation and adhesion and mRNA expression of collagen I, fibronectin integrin subunits were examined. Cell adhesion inhibition assays were carried out by GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys). RESULTS: In early culture period, the cell number of PDL cells was lower on OCP and HL than that on control without any coating. However, the cell number on OCP or HL caught up with control later period. mRNA expression level of collagen I and fibronectin on OCP and HL were similar among OCP HL and control, although they differed early in the culture period. Integrin subunits were expressed on both OCP and HL as well as on control. Cell adhesion was inhibited by RGD inhibitor peptide. CONCLUSION: Our findings indicated that rat PDL cells produce collagen I and fibronectin on OCP and HL, and then show increased cell numbers depending on adhesion to the matrices through integrins.     

Effects of a fibrin-fibronectin sealing system on proliferation and type I collagen synthesis of human PDL fibroblasts in vitro

Abstract. Fibrin glue (FG) is an agent widely used in many surgical disciplines for achieving hemostasis and tissue adhesion. The aim of this investigation was to determine the effectiveness of a highly concentrated FG (Tissucol®) on the growth and phenotypic expression of human periodontal ligament (PDL)fibroblasts. PDL fibroblast strains were established from cells scraped from PDL, and cultured in the presence and absence of FG for 48 and 72 h. Cell proliferation was studied by counting cells and mitoses, and by immunocytochemical detection of the proliferation-associated Ki-67 nuclear antigen. Type-I collagen production was assessed by radioimmunological assay of the procollagen C-terminal peptide. Results showed that FG treatment was compatible with PDL fibroblast growth and type-I collagen synthesis, although a reduced trend in cell proliferation and collagen production was found in FG-supplemented cultures compared to control cultures. We conclude that FG may represent a suitable substrate for supporting PDL fibroblast growth and function.     

In vitro wound healing responses to enamel matrix derivative

BACKGROUND: Enamel matrix derivative (EMD) contains a variety of hydrophobic enamel matrix proteins and is extracted from developing embryonal enamel of porcine origin. EMD has been associated with the formation of acellular cementum and it has been found to stimulate periodontal regeneration. The present study was established to investigate the influence of EMD on human periodontal ligament (PDL) cells, gingival fibroblasts (GF), and osteosarcoma (MG-63) cells on wound-fill rates using an in vitro wound model. METHODS: Wounds were created by making 3 mm incisions in cell monolayers across the length of tissue culture plates. The wounded PDL, GF, and MG-63 cell monolayers were treated with media containing EMD over a concentration range of 5 to 100 microg/ml, platelet-derived growth factor (PDGF-BB) at 20 ng/ml as a positive control and insulin-like growth factor (IGF-I) at 100 ng/ml as a negative control. PDL cell wounded monolayers also were treated in EMD coated tissue culture plates. After an incubation period (up to 9 days), the cells were fixed and stained and cellular fill was measured across the width of the wound by computer-assisted histomorphometry. RESULTS: When PDL, GF, and MG-63 cells were exposed to EMD in culture medium, an enhanced wound-fill was observed for all cells compared to untreated conditions. At early time points, PDL wound-fill rates in the presence of EMD were statistically greater than the rates of GF and MG-63 treated with EMD (P<0.001). There were no significant differences in wound-fill rates of PDL cells treated with EMD in medium versus EMD coated on culture plates. At days 3 and 6 post-wounding, PDL cells showed a significantly greater response to EMD than to PDGF-BB (P <0.001). EMD also had a greater effect on GF wound-fill rates than PDGF-BB at days 6 and 9. MG-63 cells were less responsive to PDGF-BB and EMD than PDL cells and GF. All 3 cell types treated with IGF-I showed no significant increase of wound-fill rates. CONCLUSION: The present data support the concept that clinical application of enamel matrix derivative may enhance periodontal wound regeneration by specifically modifying periodontal ligament cell proliferation and migration.     

In vitro effect of transforming growth factor-beta on adhesion molecule expression by human gingival fibroblasts cultured in the presence of a titanium abutment.

BACKGROUND: There is little information in the literature on the structural basis mediating gingival cell adhesion to the surface of titanium abutments. We cultured gingival fibroblasts on a titanium abutment creating as closely as possible the in vivo state. We analyzed the constitutive and transforming growth factor (TGF) beta-induced expression of the adhesion molecules CD44, CD49b, CD49c, CD51, CD54, and CD61 and extracellular matrix (ECM) components fibronectin, laminin and collagen IV. METHODS: Three totally edentulous patients underwent implant treatment to anchor the mandibular denture on 2 implants. Gingival mucosa cell specimens were collected from the mandible during the first surgical stage and the gingival fibroblast cultures were prepared. Cells were cultured for 48 hours with or without isoforms TGF-beta1, TGF-beta2, and TGF-beta3. The expression of adhesion molecules and ECM components was analyzed by immunofluorescence staining and flow cytometry. RESULTS: The addition of TGF-beta isoforms to the cell culture over the incubation period had little effect on cell growth rate, but significantly influenced cell orientation, which changed from a sun-burst pattern in control conditions to a more elongated organization and perpendicular to abutment surface. In all fibroblast preparations, a marked expression of CD44 and a moderate positivity for anti-CD49b and CD49c were found. By contrast, CD51, CD54, and CD61 expressions were negligible. When fibroblasts were cultured for 48 hours in the presence of TGF-beta, the expression of most of the receptor molecules increased. The cells expressed constitutively moderate levels of laminin and fibronectin and low amounts of collagen IV. By contrast, treatment with any one of the 3 TGF-beta isoforms greatly enhanced the expression levels of fibronectin, laminin, and, especially, collagen IV. CONCLUSIONS: TGF-beta not only seems to affect the orientation of the cultured gingival fibroblasts, but also to induce a clear-cut modification of their adhesion molecule expression.     

FGF-2 induces proliferation of human periodontal ligament cells and maintains differentiation potentials of STRO-1(+)/CD146(+) periodontal ligament cells

The presence of human STRO-1⁺/CD146⁺ periodontal ligament (PDL) cells has been reported, but obtaining a large amount of these cells is difficult. The purpose of this study was to evaluate the percentages of STRO-1⁺/CD146⁺ cells in PDL cells and determine the effects of FGF-2 on the proliferation and multilineage differentiation potency of these cells. Human PDL (HPDL) cells were individually prepared from 15 extracted teeth. HPDL cells were cultured with or without FGF-2, and the percentages of STRO-1⁺/CD146⁺ cells in each HPDL cell culture was examined using FACSAria?. The STRO-1⁺/CD146⁺ cells were sorted with FACSAria?, and the mRNA expression and differentiation potency of the sorted cells were subsequently examined. The numbers of the STRO-1⁺/CD146⁺ cells in the FGF-2 cultures were significantly higher than those cultured in the absence of FGF-2. The sorted STRO-1⁺/CD146⁺ cells expressed mRNA of PDL markers and differentiated into adipocytes and osteoblast-like cells. The present study shows that FGF-2 augmented the proliferation of the STRO-1⁺/CD146⁺ cells in the HPDL cultures whilst retaining adipogenic and osteogenic differentiation potentials. Thus, it may be useful to culture HPDL cells with FGF-2 for the application of the human STRO-1⁺/CD146⁺ PDL cells in periodontal tissue regeneration.