Effects of HB-EGF and epiregulin on wound healing of gingival cells in vitro

Oral Diseases (2011) 17 , 785–793 Objective: Gingival wound healing is important to periodontal disease and surgery. This in vitro study was conducted to assess the manner in which heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and epiregulin cooperatively participate in the wound‐healing process in the gingival epithelial and fibroblast cells of the oral mucosa. Material and Methods: Gingival epithelium and fibroblast were separated from gingival tissue biopsies and prepared to primary cultures. The changes in the mRNA expression were evaluated via real‐time PCR. The effects on cell proliferation, migration, and repopulation were evaluated in vitro. Results: The different regulation of expressions of HB‐EGF, epiregulin, and epidermal growth factor receptors was observed over time and with different gingival cell types. HB‐EGF exerted a cell migration‐inducing effect on both epithelial and fibroblast cells, whereas epiregulin did not. Both growth factors functioned as mitogens for epithelial cell proliferation, but not for fibroblast proliferation. HB‐EGF strongly promoted epithelial cell repopulation and mildly promoted fibroblast repopulation, whereas epiregulin promoted only fibroblast repopulation. Conclusion: These results indicated that both growth factors might function importantly in the wound‐healing process of human gingival tissue via the different regulation of the expression, cell migration, proliferation, and repopulation.     

富血小板血浆和贫血小板血浆处理的生物降解膜对牙周韧带成纤维细胞影响的扫描电镜观察

目的 :探索富血小板血浆 (platelet -richplasma ,PRP)和贫血小板血浆 (platelet -poorplasma,PPP)处理后的生物降解膜对附着的人牙周韧带成纤维细胞形态的影响。方法 :体外培养人牙周韧带成纤维细胞 ,应用梯度密度离心 ,从人新鲜全血中获取PRP和PPP ,并分别预处理两种生物降解膜。体外细胞培养 2 4h ,扫描电镜观察PRP和PPP对两种生物降解膜的附着效果 ,以及附着于膜上人牙周韧带成纤维细胞的超微结构。结果 :PRP和PPP能有效地贴附于生物降解膜 ,在未处理和处理后的生物降解膜上均可见附着的呈梭形或长扁形的人牙周韧带成纤维细胞 ,但在处理膜上人牙周韧带成纤维细胞明显地被包裹于PRP和PPP中 ,仅见增大的胞体、胞浆突起的轮廓 ,细胞彼此重叠或融合成片状。结论 :有效地贴附于生物降解膜的PRP和PPP能将人牙周韧带成纤维细胞网织在一起 ,并促进细胞的增殖。提示经PRP和PPP处理的生物降解膜可能更利于引导牙周组织再生。     

新型牙周再生细胞传递载体的体外建立

目的 体外建立新型细胞传递载体,观察富血小板血浆（PRP）和贫血小板血浆（PPP）对鼠牙龈成纤维细胞、牙周膜成纤维细胞和牙槽骨成骨细胞在生物降解膜上附着及增殖的影响。方法 体外培养鼠牙龈成纤维细胞、牙周膜成纤维细胞和牙槽骨成骨细胞。应用梯度密度离心从鼠全血中获取PRP和PPP,并分别预处理生物降解膜。体外细胞培养48 h,检测鼠3种细胞在处理和未处理膜上的附着、增殖和形态。结果 鼠3种细胞在PRP和PPP处理膜上的附着数均较未处理组有显著增加（P<0.05）;鼠3种细胞在PRP处理膜上的附着数虽较PPP处理组略有增加,但统计学检验无意义（P＞0.05）;在同一处理或未处理组中,鼠3种细胞间附着数比较无统计学意义（P＞0.05）。体外培养1 h和48 h,鼠3种细胞在PRP和PPP处理膜上的增殖均显著强于未处理组（P<0.05）;体外培养48 h,鼠3种细胞在PRP和PPP处理膜上的增殖显著强于体外培养1 h（P<0.05）;在同一处理或未处理组中,鼠3种细胞间的增殖则无统计学意义（P＞0.05）。附着于PRP和PPP处理膜上的鼠3种细胞均不同程度显示胞体增大,胞浆扩展,有的形成丝状伪足。结论 PRP和PPP处理的生物降解膜能明显加强鼠各型细胞的附着和增殖,这为建立细胞传递载体和促进牙周再生奠定实验基础。     

Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factor-beta and modulates the proliferation of periodontally related cells in vitro

BACKGROUND: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined. METHODS: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5-bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated. RESULTS: In the PRP preparations, platelets were concentrated up to 70.9 x 10(4) cells/microl (283.4% of the unconcentrated plasma). The levels of PDGF-AB and TGF-beta1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells. CONCLUSIONS: These data demonstrated that both PDGF-AB and TGF-beta1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-beta1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy.     

Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferation in vitro

Bertrand‐Duchesne M‐P, Grenier D, Gagnon G. Epidermal growth factor released from platelet‐rich plasma promotes endothelial cell proliferation in vitro. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01205.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: The therapeutic benefits of platelet‐rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation. Material and Methods: The levels of vascular endothelial growth factor (VEGF), platelet‐derived growth factor BB (PDGF‐BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium‐ and thrombin‐activated PRP samples from five donors were quantified by enzyme‐linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody‐coated beads on HUVEC proliferation was also tested. Results: Average concentrations of VEGF, PDGF‐BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody‐coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose‐dependent manner. Conclusion: This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds.     

Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts

Wada N, Wang B, Lin N‐H, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival and periodontal ligament fibroblasts. J Periodont Res 2011; 46: 438–447. © 2011 John Wiley & Sons A/S Background and Objective: Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. Material and Methods: iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c‐MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM‐2, TG30 and TRA‐1‐60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real‐time RT‐PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. Results: Human gingival fibroblast‐ and periodontal ligament fibroblast‐derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell‐associated cell‐surface antigens, SSEA3, SSEA4, GCTM‐2, TG30 (CD9) and Tra‐1‐60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. Conclusion: These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.     

Molecular events associated with ciclosporin A-induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts

Sobral LM, Aseredo F, Agostini M, Bufalino A, Pereira MCC, Graner E, Coletta RD. Molecular events associated with ciclosporin A‐induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts. J Periodont Res 2012; 47: 149–158. © 2011 John Wiley & Sons A/S Background and Objective: Ciclosporin A (CsA)‐induced gingival overgrowth is attributed to an exaggerated accumulation of extracellular matrix, which is mainly due to an increased expression of transforming growth factor‐β1 (TGF‐β1). Herein, the in vitro investigation of effects of overexpression of Smad7, a TGF‐β1 signaling inhibitor, in the events associated with CsA‐induced extracellular matrix accumulation was performed. Material and Methods: The effects of Smad7 were assessed by stable overexpression of Smad7 in fibroblasts from normal gingiva. Smad7‐overexpressing cells and control cells were incubated with CsA, and synthesis of type I collagen, production and activity of MMP‐2 and cellular proliferation were evaluated by ELISA, zymography, growth curve, bromodeoxyuridine incorporation assay and cell cycle analysis. The effects of CsA on cell viability and apoptosis of fibroblasts from normal gingiva were also evaluated. Western blot and immunofluorescence for phospho‐Smad2 were performed to measure the activation of TGF‐β1 signaling. Results: Although the treatment with CsA stimulated TGF‐β1 production in both control and Smad7‐overexpressing fibroblasts, its signaling was markedly inhibited in Smad7‐overexpressing cells, as revealed by low levels of phospho‐Smad2. In Smad7‐overexpressing cells, the effects of CsA on proliferation, synthesis of type I collagen and the production and activity of MMP‐2 were significantly blocked. Smad7 overexpression blocked CsA‐induced fibroblast proliferation via p27 regulation. Neither CsA nor Smad7 overexpression induced cell death. Conclusion: The data presented here confirm that TGF‐β1 expression is related to the molecular events associated with CsA‐induced gingival overgrowth and suggest that Smad7 overexpression is effective in blocking these events, including proliferation, type I collagen synthesis and MMP‐2 activity.     

Periodontal tissue engineering after tooth replantation

Background: Blood‐derived products, platelet‐poor plasma (PPP) and platelet‐rich plasma (PRP), constitute an approach in the enhancement of tissue healing. PRP has also been used as a scaffold for bone marrow stem cells in tissue engineering. This study evaluates the effect of PPP, calcium chloride–activated PRP (PRP/Ca), calcium chloride– and thrombin‐activated PRP (PRP/Thr/Ca), and bone marrow mononuclear cells and PRP/Ca (BMMCs/PRP/Ca) on the healing of replanted dog teeth. Methods: After 30 minutes of extraction, teeth were replanted with 1) no material (control); 2) PPP; 3) PRP/Ca; 4) PRP/Thr/Ca; or 5) BMMCs/PRP/Ca. Histologic, histomorphometric, and immunohistochemical analysis was assessed 120 days after replantation. Data from histomorphometric analysis were analyzed statistically (analysis of variance, Tukey; P <0.05). Quantitative immunohistochemical analysis was analyzed by Kruskal‐Wallis and Dunn post hoc test (P <0.05). Results: Flow cytometry analysis showed 55.98% of CD34⁺ and 32.67% of CD90/Thy‐1 for BMMCs sample. BMMCs/PRP/Ca presented the largest areas of replacement resorption characterized by osseous ingrowth into cementum (P <0.05), with intense immunomarcation for tartrate‐resistant acid phosphatase. The PRP/Ca group also showed areas of replacement resorption with significant immunomarcation for osteopontin. PRP/Thr/Ca presented no replacement resorption. PPP showed areas of inflammatory resorption, with immunomarcation for tartrate‐resistant acid phosphatase. Conclusions: The results suggest that platelets activated with thrombin play an important role in the healing of tissues after tooth replantation. Additional studies are necessary to test other materials, because PRP/Ca did not present an appropriate scaffold for undifferentiated cells in the treatment of avulsed teeth.     

Bioactive antioxidant mixtures promote proliferation and migration on human oral fibroblasts

Objective

Antioxidants (AOs) are the first line of defence against free radical damage and are critical for maintaining optimum health and well being. The need for AOs becomes even more critical with increased exposure to free radicals generated by pollution, cigarette smoke, drugs, illness, stress and exercise. Antioxidant supplementation is an excellent way of improving free radical protection. The aim of this study was to provide cytotoxicity, proliferation and migration data on the in vitro effects of bioactive AO mixtures on human oral fibroblasts.

Methods

Human oral fibroblasts were obtained from human gingival (HGF) and periodontal (HPDL) tissues. Each of these oral fibroblasts was cultured separately in three concentrations of the bioactive pure polyphenol and turmeric derivative mixtures; resveratrol (R), ferulic acid (F), phloretin (P) and tetrahydrocurcuminoids (T); [(RFT), (PFR), and (PFT)]. Cell viability, proliferation, morphology and migratory behaviour were analysed in vitro using high throughput in vitro 96 well plate wound assay.

Results

RFT decreased (10⁻³ M) and increased (10⁻⁵ M) cell number in HGF cells. Three concentrations (10⁻³, 10⁻⁴, and 10⁻⁵ M) of PFR and PFT increased DNA synthesis in HGF cells. PFT promoted cell migration but PFR and RFT had no significant change in HGF wound healing rates in a 96 well plate assay monolayer wound. In the HPDL cells, the 10⁻⁴ M concentration of both RFT and PFT increased cell number at 72 h and 96 h whereas the lower concentration 10⁻⁵ M of RFT significantly stimulated cell number at 96 h. PFR (10⁻³ M and 10⁻⁵ M) and PFT (10⁻³ M) increased DNA synthesis after 48 h treatment in HPDL cells.

Conclusions

High and low concentrations (10⁻³–10⁻⁵ M) of these AOs (RFT, PFR) may have beneficial effects on functional mechanisms regulating fibroblast migration and proliferation during gingival healing or periodontal repair.     

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.     

Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts

Background and Objective: Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen‐degrading ability of human gingival fibroblasts and its mechanism. Material and Methods: Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water‐soluble tetrazolium‐1 and lactate dehydrogenase, respectively. The collagen‐degrading ability of human gingival fibroblasts was evaluated in collagen‐coated six‐well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Results: Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 μg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 μg/mL. Cigarette smoke condensate increased the collagen‐degrading ability of human gingival fibroblasts, especially at a concentration of 100 μg/mL (1.5‐fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP‐1, proMMP‐2, MMP‐14 and TIMP‐1, and decreased the production of TIMP‐2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP‐2, MMP‐14 and TIMP‐2 in membrane extracts, especially at concentrations of 50–100 μg/mL. Conclusion: Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of ≥ 400 μg/mL. Cigarette smoke condensate can increase the collagen‐degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.     

Platelet‐Poor and Platelet‐Rich Plasma Stimulate Bone Lineage Differentiation in Periodontal Ligament Stem Cells

Background: Plasma‐derived fractions have been used as an autologous source of growth factors; however, limited knowledge concerning their biologic effects has hampered their clinical application. In this study, the authors analyze the content and specific effect of both platelet‐rich plasma (PRP) and platelet‐poor plasma (PPP) on osteoblastic differentiation using primary cultures of human periodontal ligament stem cells (HPLSCs). Methods: The authors evaluated the growth factor content of PRP and PPP using a proteome profiler array and enzyme‐linked immunosorbent assay. HPLSCs were characterized by flow cytometry and differentiation assays. The effect of PRP and PPP on HPLSC bone differentiation was analyzed by quantifying calcium deposition after 14 and 21 days of treatment. Results: Albeit at different concentrations, the two fractions had similar profiles of growth factors, the most representative being platelet‐derived growth factor (PDGF) isoforms (PDGF‐AA, ‐BB, and ‐AB), insulin‐like growth factor binding protein (IGFBP)‐2, and IGFBP‐6. Both formulations exerted a comparable stimulus on osteoblastic differentiation even at low doses (2.5%), increasing calcium deposits in HPLSCs. Conclusions: PRP and PPP showed a similar protein profile and exerted comparable effects on bone differentiation. Further studies are needed to characterize and compare the effects of PPP and PRP on bone healing in vivo.     

The growth and osteoclastogenic effects of fibroblasts isolated from keratocystic odontogenic tumor

Oral Diseases (2012) Objectives: To investigate the growth characteristics and effects on osteoclastogenesis in fibroblasts isolated from keratocystic odontogenic tumor (KCOT) fibrous capsule. Materials and Methods: Fibroblasts isolated from KCOT fibrous capsule and normal gingival mucosa were cultured in vitro. Their colony‐forming units and proliferative activity were investigated, and the osteoclastogenic effects were also observed by a co‐culture system with osteoclast precursor cell line Raw264.7. The mRNA of several genes related to bone resorption (IL‐6, VEGF, COX‐2, and M‐CSF) was analyzed by real‐time PCR. Results: Keratocystic odontogenic tumor fibroblasts developed fewer CFU and had longer population doubling time than gingival fibroblasts (P < 0.05). In contrast to gingival fibroblasts, KCOT fibroblasts expressed less IL‐6, COX‐2, and M‐CSF (P < 0.05); however, the Raw264.7 co‐cultured with KCOT fibroblasts developed more osteoclast‐like cells and expressed higher level of nfatc1 than that co‐cultured with gingival fibroblasts. Increased COX‐2 expression and VEGF expression were detected in KCOT fibroblasts and Raw264.7 co‐culture system (P < 0.05). Conclusion: Although KCOT fibroblasts showed lower level of cell proliferation than gingival fibroblasts, higher osteoclastogenic ability was detected when co‐cultured with Raw264.7. These results suggest that the cell–cell interaction in the co‐culture system, possibly by increasing COX‐2 and VEGF expression, may be responsible for the increased osteoclastogenic effects of KCOT fibroblasts.     

Nicotine-induced damage affects gingival fibroblasts in the gingival tissue of rats

Background: Very limited information is available from in vivo studies about whether smoking and/or nicotine affect gingival tissues in the absence of plaque. The purpose of this study is to evaluate the effect of the systemic administration of nicotine in the proliferation and counting of fibroblast‐like cells in the gingival tissue of rats. Methods: Thirty adult male Wistar rats were randomly assigned into two groups to receive subcutaneous injections of a saline solution (control group = group C) or nicotine solution (group N; 3 mg/kg) twice a day. The animals were euthanized 37, 44, or 51 days after the first subcutaneous injection. Specimens were routinely processed for serial histologic sections. Five fields of view in the connective tissue adjacent to the gingival epithelium and above the alveolar bone crest of the maxillary first molar were selected for the counting of fibroblast‐like cells. Data were statistically analyzed (P <0.05). Results: The intergroup analysis detected a lower number of fibroblast‐like cells in group N compared to group C on days 37 (2.65 ± 1.41 and 6.67 ± 3.25, respectively), 44 (2.70 ± 1.84 and 8.57 ± 2.37, respectively), and 51 (2.09 ± 1.41 and 7.49 ± 2.60, respectively) (P <0.05). The quantification of fibroblast‐like cells showed no significant difference (P >0.05) in the intragroup analysis of control and nicotine throughout experimental periods. In the intergroup analysis, group N had reduced proliferating cell nuclear antigen–positive fibroblasts compared to group C in all periods (P <0.05). Conclusion: The daily systemic administration of nicotine negatively affected, in vivo, the number and proliferation of fibroblast‐like cells in the gingival tissue of rats.     

Optimum force and duration of toothbrushing to enhance gingival fibroblast proliferation and procollagen type I synthesis in dogs

BACKGROUND: Toothbrushing enhances gingival fibroblast proliferation, which promotes wound healing. Optimum force and duration of toothbrushing for stimulation of fibroblast proliferation are key factors in maximizing effects of toothbrushing on periodontal wound healing. We therefore evaluated the effects of different durations and forces of toothbrushing on proliferative activity and procollagen synthesis of gingival fibroblasts. METHODS: Twelve dogs were used. In each dog, buccal gingivae of 12 teeth were examined for 3 weeks. Nine of these 12 teeth were each assigned to 1 of 9 different combinations of brushing force (0.98, 1.96, or 2.45 N) and duration (10, 20, or 40 seconds). The remaining 3 teeth received plaque removal without brushing, via a scaler. RESULTS: Force and duration of toothbrushing affected both proliferating cell nuclear antigen (PCNA)-positive and procollagen Type I C-peptide (PIP)-positive fibroblast ratios (P < 0.05). The highest ratio of PCNA-positive fibroblasts was produced by brushing at 1.96 N for 20 seconds. The highest ratio of PIP-positive fibroblasts was produced by brushing at 1.96 N for 10 seconds. CONCLUSIONS: Toothbrushing at certain forces and durations enhanced the proliferative activity and procollagen synthesis of gingival fibroblasts. The toothbrushing duration that increased procollagen synthesis (10 seconds) was shorter than that which increased fibroblast proliferative activity (20 seconds).     

Antimicrobial Activity of Platelet‐Rich Plasma and Other Plasma Preparations Against Periodontal Pathogens

Background: In addition to releasing a pool of growth factors during activation, platelets have many features that indicate their role in the anti‐infective host defense. The antimicrobial activities of platelet‐rich plasma (PRP) and related plasma preparations against periodontal disease–associated bacteria were evaluated. Methods: Four distinct plasma fractions were extracted in the formulation used commonly in dentistry and were tested for their antibacterial properties against three periodontal bacteria: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. The minimum inhibitory concentration of each plasma preparation was determined, and in vitro time‐kill assays were used to detect their abilities to inhibit bacterial growth. Bacterial adhesion interference and the susceptibility of bacterial adherence by these plasma preparations were also conducted. Results: All plasma preparations can inhibit bacterial growth, with PRP showing the superior activity. Bacterial growth inhibition by PRP occurred in the first 24 hours after application in the time‐kill assay. PRP interfered with P. gingivalis and A. actinomycetemcomitans attachment and enhanced exfoliation of attached P. gingivalis but had no influences on F. nucleatum bacterial adherence. Conclusions: PRP expressed antibacterial properties, which may be attributed to platelets possessing additional antimicrobial molecules. The application of PRP on periodontal surgical sites is advisable because of its regenerative potential and its antibacterial effects.     

Differential effects of prostaglandin E2 and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes

Weinberg E, Topaz M, Dard M, Lyngstadaas P, Nemcovsky C, Weinreb M. Differential effects of prostaglandin E ₂ and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes. J Periodont Res 2010; 45: 731–740. © 2010 John Wiley & Sons A/S Background and Objective: Elevated levels of prostaglandins contribute to periodontal destruction but can impair gingival healing by affecting local fibroblasts. Enamel matrix derivative (EMD) has beneficial effects on supporting and gingival tissues. We showed that prostaglandin E₂ (PGE₂) inhibits the proliferation of human gingival fibroblasts (hGFs) and that EMD stimulates it. Prostaglandins and EMD may also affect skin healing by targeting dermal fibroblasts (DFs). Thus, we compared the effects of these two agents on the proliferation of hGFs, human gingival keratinocytes (hGKs) and hDFs. Material and Methods: Cells from healthy human gingiva or skin were treated with PGE₂ and/or EMD, and proliferation was assessed by measuring cell number and DNA synthesis. Results: In hGFs, PGE₂ (1 μm ) inhibited proliferation while EMD stimulated it. When present together, EMD abolished the PGE₂‐induced inhibition. Serum increased (by a factor of 10) the amount of phosphorylated extracellular signal‐regulated kinase (p‐ERK), PGE₂ reduced it (by 70–80%) and EMD restored it when present with PGE₂. Prostaglandin E₂ stimulated cAMP production in hGFs while serum or EMD did not. Enamel matrix derivative stimulated hDF proliferation, but the inhibitory effect of PGE₂ was milder than with hGFs. When present together, EMD abolished the PGE₂‐induced inhibition. Enamel matrix derivative inhibited the proliferation of primary hGKs, but PGE₂ had no effect. Finally, we found that hDFs contained about five times less prostaglandin EP₂ receptor mRNA than hGFs, while hGKs contained none. Conclusion: Prostaglandin E₂ inhibits and EMD stimulates hGF proliferation via distinct pathways. The different sensitivities of hDFs and hGKs to PGE₂ can be explained by the levels of EP₂ expression.     

Cytopathologic effects of arecoline on human gingival fibroblasts in vitro

Arecoline, a major betel nut alkaloid, has been detected in saliva obtained during betel nut chewing in concentrations up to 140 μg/ml, corresponding to 0.9 mM. Arecoline in the millimolar concentration range might participate in the initiation and/or progression of periodontal disease during the long-term effects of betel nut chewing. In this study, cell growth, cell proliferation, assessment of cytoplasmic enzyme lactate dehydrogenase (LDH) and collagen synthesis were used to investigate the effects of human gingival fibroblasts exposed to arecoline levels of 0–200 μg/ml. Control culture exhibited a normal monolayer of long spindle-shaped fibroblast morphology. Arecoline-treated human gingival fibroblasts showed a more rounded appearance and detached at the higher concentrations. At concentrations higher than 75 μg/ml, many cells had detached from the surface of the petri dish and numerous floating cells could be seen under the inverted microscope. At a concentrations higher than 25 μg/ml, arecoline inhibited cell growth, proliferation and collagen synthesis and increased LDH leakage in a dose-dependent manner (P<0.05). These results indicate that arecoline is a cytotoxic agent to human gingival fibroblasts. Repeated and long-term exposure to arecoline could impair gingival fibroblast function. Betel quid chewers might be more susceptible to destruction of the periodontium and less responsive to a regeneration procedures during periodontal therapy. Received: 31 August 1998 / Accepted: 3 November 1998     

The effect of platelet-rich plasma on the cellular response of rat bone marrow cells in vitro

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation and the differentiation of rat bone marrow cells (RBMCs). PRP, platelet-poor plasma (PPP), and bone marrow cells were derived from the rats (hearts and tibia) and the cells were cultured with or without PRP or PPP (0 [control]), 0.2 approximately 10 microL/mL). The proliferation of RBMCs was measured on days 2 and 4, and alkaline phosphatase (ALP) staining and activity measurement were evaluated to determine the effect of PRP on the differentiation on days 4 and 8. PRP enhanced the proliferation significantly compared to the control group (P < .05). These enhancements were greater than ones induced by the addition of PPP. ALP staining appeared to show that PRP decreased the number of ALP positive cells and ALP activity significantly (P < .05). Our results demonstrate that PRP stimulates the proliferation but suppresses the differentiation of RBMCs.     

Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction

Background and Objective: Platelet‐derived growth factor‐BB is a potent mediator of tooth‐supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet‐derived growth factor‐BB application is its short half‐life in vivo. Gene therapy has shown strong promise for the long‐term delivery of platelet‐derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet‐derived growth factor‐B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad‐PDGF‐B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell‐surface proteins associated with cellular signaling. Material and Methods: HGFs from human subjects were treated by adenoviral PDGF‐B, PDGF‐1308 (a dominant negative mutant of PDGF) and recombinant human platelet‐derived growth factor‐BB, and then incubated in serum‐free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF‐B was measured by RT‐PCR and Western blot. Cell proliferation was evaluated by [methyl‐³H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF‐B gene transfer. The expression of α and β PDGFR and Akt were measured by Western blot analysis. Results: Sustained in vitro expression of PDGF‐B in HGFs by Ad‐PDGF‐B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF‐BB and Ad‐PDGF‐1308, Ad‐PDGF‐B maintained cell growth in serum‐free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF‐B also prolonged downregulation of the growth arrest specific gene (gas) PDGFαR. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long‐term cell signaling effect as a result of the over‐expression of Ad‐PDGF‐B, compared with pulse recombinant human platelet‐derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad‐PDGF‐B, for at least up to 96 h. Conclusion: These findings further demonstrate that gene delivery of PDGF‐B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet‐derived growth factor‐BB protein. These data on platelet‐derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.