Proliferation Of Periodontal Ligament Cells Research Articles

Improved Biological Characteristics of Poly(l-Lactic Acid) Electrospun Membrane by Incorporation of Multiwalled Carbon Nanotubes/Hydroxyapatite Nanoparticles

Significant effort has been devoted to fabricating various biomaterials to satisfy specific clinical requirements. In this study, we developed a new type of guided tissue regeneration (GTR) membrane by electrospinning a suspension consisting of poly( l-lactic acid), multiwalled carbon nanotubes, and hydroxyapatite (PLLA/MWNTs/HA). MWNTs/HA nanoparticles were uniformly dispersed in the membranes, and the degradation characteristics were far improved. Cytologic research revealed that the PLLA/MWNTs/HA membrane enhanced the adhesion and proliferation of periodontal ligament cells (PDLCs) by 30% and inhibited the adhesion and proliferation of gingival epithelial cells by 30% also, compared with the control group. After PDLCs were seeded into the PLLA/MWNTs/HA membrane, cell/membrane composites were implanted into the leg muscle pouches of immunodeficient mice. Histologic examinations showed that PDLCs attached on the membranes functioned well in vivo. This new type of membrane shows excellent dual biological functions and satisfied the requirement of the GTR technique successfully in spite of a monolayer structure. Compared with other GTR membranes on sale or in research, the membrane can simplify the manufacturing process, reduce the fabrication cost, and avoid possible mistakes in clinical application. Moreover, it does not need to be taken out after surgery. PLLA/MWNTs/HA membranes have shown great potential for GTR and tissue engineering.

Enamel Matrix Derivative Promotes Human Periodontal Ligament Cell Differentiation and Osteoprotegerin Production in vitro

Enamel matrix derivative (EMD) has been used successfully to aid periodontal repair. We sought to elucidate the mechanism of action of EMD and hypothesized that combined exposure to EMD and parathyroid hormone (PTH), which acts anabolicly when administered intermittently, would enhance periodontal ligament cell proliferation, differentiation, and local factor production. Confluent human periodontal ligament cells were exposed to EMD continuously or to PTH(1-34) intermittently, or a combination of both. Cell number, alkaline phosphatase activity, osteocalcin, and osteoprotegerin production were determined. Continuous challenge with EMD resulted in an increase of the differentiation parameters and osteoprotegerin production, while simultaneously inhibiting proliferation. Intermittent PTH(1-34) administration exerted opposite effects. Combined administration of EMD and PTH(1-34) weakened or even nullified the effects seen for the agents alone. These results suggest that EMD promotes periodontal ligament cell differentiation and osteoprotegerin production, potentially resulting in a microenvironment supporting periodontal repair, whereas combining EMD and PTH(1-34) failed to prove beneficial in this respect.

Effects of enamel matrix derivative and transforming growth factor‐β1 on human periodontal ligament fibroblasts

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. 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. 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. 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.

Comparison of periodontal ligament cells responses to dense and nanophase hydroxyapatite

Hydroxyapatite, a synthetic calcium phosphate ceramic, is used as a biomaterial for the restoration of human hard tissue as well as in techniques which aim to regenerate periodontal tissues. Generally, hydroxyapatite is believed to have osteoconductive effects and to be non-bioresorbable but not to induce to periodontal tissue regeneration. No report has been found on responses of periodontal ligament cells (PDLC), the main contributor to periodontal tissue regeneration, to nanoparticles of hydroxyapatite. The objective of this study was to investigate the possible effects of nanophase powder of hydroxyapatite on proliferation of periodontal ligament cells. Using a sol-gel method, the nanophase hydroxyapatite powders were fabricated. These powders were proved to comprise nanoparticles by transmission electron microscope examination. The primary periodontal ligament cells were cultured on dense particle hydroxyapatite and nanometer particle hydroxyapatite. The effects on proliferation of periodontal ligament cells on dense and nanoparticle hydroxyapatite were examined in vitro using a methyl thiazolil tetracolium (MTT) test. The intercellular effects were studied with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray (EDX). In addition, the influence of the two materials on osteogenic differentiation was determined through measurement of alkaline phosphatase activity and flow cytometry. About 2, 3, and 4 days after treatment with nanoparticles of hydroxyapatite, the proliferation activity of the PDLC increased significantly compared with those proliferating on dense hydroxyapatite and of control PDLC, but no significant difference was found between the PDLC proliferation on dense hydroxyapatite and the control PDLCs. After 3 and 5 days' incubation with nanoparticles of hydroxyapatite, alkaline phosphatase activity was significantly increased as compared to PDLCs incubated with dense hydroxyapatite and control PDLCs. Intracellular engulfment was found in the cultured cells with nanophase hydroxyapatite under electron microscopy. The results suggest that nanophase hydroxyapatite can promote proliferation and osteogenic differentiation of periodontal ligament cells and further that it may be used as a bioresorbable agent in osseous restoration.

A Hepatocyte Growth Factor (HGF)/Receptor Autocrine Loop Regulates Constitutive Self‐Renewal of Human Periodontal Ligament Cells but Reduces Sensitivity to Exogenous HGF

In addition to its prominent role in liver regeneration, hepatocyte growth factor (HGF) is now generally thought to be produced by mesenchymal cells to promote the regeneration of epithelial tissue by a paracrine mechanism. However, it is not known how or if HGF could be involved in the regeneration of periodontal tissues. The purpose of this study was to characterize the ability of normal human periodontal ligament (PDL) cells to produce or respond to HGF. PDL cells derived from healthy young volunteers were used from passages four through 10. HGF receptors were detected both by immunocytochemical staining and Western-blotting analysis. Both DNA synthesis (by bromo-deoxyuridine [BrdU]-incorporation) and secreted HGF were quantified by enzyme-linked immunosorbent assays. Mitogen-activated protein kinase (MAPK) phosphorylation was also analyzed by Western blot. Despite the immunocytochemical demonstration of HGF receptor protein in the cytoplasm and on the plasma membrane of PDL cells, exogenous recombinant human HGF did not exert the mitogenic effects expected. As reported for other mesenchymal cells, PDL cells were found to secrete HGF. Treatments with neutralizing anti-HGF antibody significantly suppressed constitutive PDL cell proliferation and sustained the receptor protein at higher levels than in non-treated cells. Under these conditions, exogenous HGF rapidly phosphorylated extracellular signal-regulated kinase (ERK), an action linked to the cell proliferation and downregulation of cell-surface receptors. Unlike other known mesenchymal or epithelial cells, these findings suggest that normal PDL cells from young donors possess a constitutive HGF/receptor autocrine loop that normally regulates their replacement self-proliferation but reduces sensitivity to exogenously applied HGF by acute receptor downregulation.

Time‐ and dose‐dependent mitogenic effect of basic fibroblast growth factor combined with different bone graft materials: an in vitro study

In periodontal regeneration, the growth factor concentrations and the delivery system used are of great importance. In an attempt to assess the mitogenic effect of basic fibroblast growth factor (bFGF) on periodontal ligament (PDL) cells combined with different bone replacement materials, two allografts of cortical (DFDBA) and cancellous (DFBA) bone and an anorganic bovine material with a synthetic peptide (ABM P-15) were used. The purpose of this study was to evaluate the in vitro mitogenic effect of different doses of bFGF alone or in combination with DFDBA, DFBA and ABM P-15 on human PDL cells in a time-dependent mode. PDL cell cultures were derived from the mid-root of four maxillary premolars. Cells were grown and reached confluence. On day 2 of quiescence, new medium was added along with (1) 1, 5, 10 and 25 ng/ml of bFGF alone, (2) 10 mg of DFDBA, DFBA and ABM P-15 alone and (3) their combination. The mitogenic effect was determined at 24 and 48 h of culture by using a hemocytometer chamber. The cells were counted under a phase contrast microscope. The results revealed that bFGF at the highest concentrations and after 48 h exerted a significant mitogenic effect on PDL cells, and also DFDBA and DFBA supported cell proliferation. Furthermore, DFDBA and DFBA enriched with bFGF had a significant mitogenic effect after 48 h of culture. ABM P-15 with 10 and 25 ng/ml of bFGF up-regulated PDL cell proliferation after 48 h of incubation. The findings of this study demonstrate the beneficial role of bFGF combined with DFDBA and DFBA as carriers in periodontal repair.

Human Periodontal Fibroblast Response to Enamel Matrix Derivative, Amelogenin, and Platelet‐Derived Growth Factor‐BB

The ideal goal of clinical therapy in periodontal defects is regeneration of all lost structures. For regeneration to occur, cell proliferation, migration, and extracellular matrix synthesis are prerequisites. Attempts at regeneration of periodontal defects by guided tissue regeneration using bone grafts and membranes have not always yielded predictable results. Recently, attempts at engineering the defects using various materials have shown promising results. Two such approaches have been used to regenerate periodontal defects, one using extracellular matrix such as enamel matrix proteins and the other using growth factors. However, to our knowledge, no study has looked at combining these two approaches to achieve potentially even greater regeneration. Primary human periodontal ligament (PDL) fibroblasts were explanted, and alkaline phosphatase (ALK PHOS) activity was determined. Phenotypically different cell lines were incubated for 1, 3, 6, and 10 days in 0.2% fetal bovine serum (FBS) media containing different concentrations of either enamel matrix derivative (EMD), amelogenin, platelet-derived growth factor-BB (PDGF-BB), EMD+PDGF-BB, or amelogenin+PDGF-BB. A culture of 0.2% FBS alone served as a negative control, and a culture of 10% FBS served as a positive control. Cell proliferation was measured using a Coulter counter to determine the cell number. The effects on a wound-fill model were evaluated by scraping a 3-mm wide cell-free zone in PDL monolayers across the diameter of the tissue-culture plate and determining PDL cell migration into the cell-free zone using computer assisted histomorphometry. Compared to the control, only EMD+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (-) cell line (P<0.001), and EMD alone, EMD+PDGF-BB, and amelogenin+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (+) cell line (P<0.001) with EMD+PDGF-BB showing a trend for greater proliferation than either PDGF or EMD alone. Individually, EMD and amelogenin had no significant effect on PDL cell proliferation. In the wound-fill experiment, all factors and their combinations except amelogenin significantly enhanced cell migration compared to the control (P<0.05) at the wound edge. In addition, EMD+PDGF-BB had additive effects on the ALK PHOS (-) cell line at the wound edge. At the center of the wound, neither EMD nor amelogenin had a significant wound-fill effect. However, the combination of EMD+PDGF-BB additively increased wound fill for both ALK PHOS (+) and ALK PHOS (-) cells. The combination of EMD and PDGF-BB produces greater proliferative and wound-fill effects on PDL cells than each by themselves. If these combined effects can be translated clinically, one may see greater regeneration in periodontal defects with this combination. However, amelogenin does not have significant effects on PDL cell proliferation or migration by itself. This may suggest that either another enamel matrix component in EMD may be responsible for some of its clinical effects, or that amelogenin alone may not trigger the regenerative potential of periodontal tissues and that it requires a combined interaction with other enamel matrix components of EMD to direct the regenerative process.

Effects of high glucose on cellular activity of periodontal ligament cells in vitro

Periodontal ligament (PDL) cells are the most important cells in the healing of wounds and the regeneration of periodontal tissues. The response of PDL cells regarding cellular activity to high glucose concentration levels could be the key in understanding the events associated with the dental care of brittle diabetes. We studied the effect of high glucose concentration levels on the cellular activity of PDL cells from five non-diabetic patients in vitro. PDL cells were cultured for 14 days in a normal glucose medium (1100 mg/l of glucose) or in a high glucose medium (4500 mg/l of glucose) and a 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for cellular viability was also performed. In order to evaluate the differentiation of PDL cells to osteoblast-like cells, mineralized nodule formation was induced with supplemented media containing 50 μg/ml of ascorbic acid, 10 mM of β-glycerophosphate and 100 nM of dexamethasone for 21 days. High glucose significantly inhibited the proliferation of PDL cells and reduced the optic density of the MTT assay. Concerning the mineralized nodule formation, the percentage of the calcified area to the total culture dish of PDL cells in high glucose level was lower than that in the normal glucose medium. In conclusion, high glucose inhibits the proliferation and differentiation of PDL cells. The data provide an explanation for the delayed periodontal regeneration and healing in diabetic patients.

Amelogenin and ameloblastin show growth‐factor like activity in periodontal ligament cells

Enamel proteins, particularly amelogenin, have been associated with other functions in addition to regulating enamel biomineralization. Extracts of enamel proteins are currently being used to regenerate periodontal tissues, and new studies suggest that enamel proteins might have chondrogenic and osteogenic properties. In this study, we wanted to determine the effect, if any, of purified recombinant amelogenin and ameloblastin on the adhesion, proliferation, and differentiation of periodontal ligament cells in vitro. Immortomouse-derived periodontal ligament (PDL) cells were grown under permissive and differentiation conditions in the presence of different concentrations of mouse recombinant amelogenin, recombinant ameloblastin, or both. Cells were collected after 4 h to determine attachment, after 24 h to determine proliferation, and after 7, 14, 21 and 28 d to determine differentiation using reverse transcription-polymerase chain reaction (RT-PCR). Both amelogenin and ameloblastin had a small, but statistically significant, effect on increasing the cell attachment and proliferation of PDL cells. Both amelogenin and ameloblastin modulated bone morphogenetic protein (BMP) expression, down-regulated the expression of collagen type I, and induced the de novo expression of osteocalcin. Amelogenin also induced the expression of bone sialoprotein. These results suggest that amelogenin, as well as ameloblastin, might have some 'growth factor' activity during periodontium development and regeneration.

Inhibitory Effects of Tumor Necrosis Factor‐Alpha on Migration of Human Periodontal Ligament Cells

Tumor necrosis factor-alpha (TNF-alpha) is associated with chronic gingival inflammation and is suspected to influence periodontal destruction. However, the exact roles of TNF-alpha in wound healing and periodontal tissue regeneration are largely unknown. In the present study, we examined the effects of TNF-alpha on migration and proliferation of human periodontal ligament (PDL) cells. PDL cells were cultured in the presence of TNF-alpha to determine its effects on cellular migration and proliferation. The protein expression profiles of alpha5 and beta1 integrin subunits and their related molecules, paxillin and focal adhesion kinases (FAK), were investigated. Gene expression of fibronectin also was assayed. Further, the activation of Rho-family small guanosine triphosphate (GTP)-binding protein (RhoA) was evaluated using a GTP-loading pull-down assay, and focal adhesion formation by PDL cells after transfection with the expression vector of paxillin-fused green fluorescent protein (GFP) also was observed with confocal microscopy. Cellular migration was impaired by TNF-alpha and recovered following the addition of anti-TNF-alpha antibodies. In contrast, PDL cell proliferation was not affected by TNF-alpha. TNF-alpha upregulated the expression of the alpha5 and beta1 integrin subunits, whereas fibronectin was not overexpressed. Phosphorylation of paxillin and FAK by PDL cells was induced, and RhoA activation also was induced. Confocal microscopic analysis revealed that TNF-alpha induced focal adhesion and stress fiber formation in all parts of the cells. Our results suggested that TNF-alpha impairs cellular migration by enhancing cellular adhesive ability following significant focal adhesion and stress fiber formation.

Effects of enamel matrix protein on the growth of human periodontal ligament cells on root cementum surfaces

To evaluate the effect of enamel matrix protein (EMP) on the attachment and proliferation of periodontal ligament cells (PDLC) on diseased cementum surfaces in vitro. Cementum chips were obtained from diseased roots exposed to periodontal pocket. Thirteen diseased root cementum chips were conditioned with EMP. Meanwhile, 13 diseased and 13 healthy cementum chips were treated with physiological saline as control. The growth and morphology of PDLC on the root surface were observed after 24 hours incubation by scanning electron microscope (SEM). PDLC attachment and proliferation were quantified using MTT assay at 16 or 72 hours. The cells on EMP treated roots under SEM were growing robust like the cells on healthy roots. By contrast, the diseased cementum surface without conditioned with EMP was only partly covered with spindle-shaped cells, with filopodia appearing short and thin. MTT assay indicated that the number of adhered and proliferated cells on diseased cementum chips treated with EMP was significantly greater than that on diseased chips treated with saline (adhesion: 0.45 +/- 0.03 vs. 0.37 +/- 0.05, P < 0.05; proliferation: 0.71 +/- 0.02 vs. 0.55 +/- 0.08, P < 0.01), but less than that on healthy chips (adhesion: 0.45 +/- 0.03 vs. 0.67 +/- 0.08, P < 0.05; proliferation: 0.71 +/- 0.02 vs. 1.05 +/- 0.09, P < 0.05). It was suggested that EMP could promote the growth of PDLC on the diseased root cementum surface.

Apple‐ and Hop‐Polyphenols Protect Periodontal Ligament Cells Stimulated With Enamel Matrix Derivative From Porphyromonas gingivalis

Enamel matrix derivative (EMD) is a tissue regenerative agent used clinically as an adjunct to periodontal surgery. It was previously demonstrated that Porphyromonas gingivalis, a periodontal pathogen, significantly diminished the efficacy of EMD with periodontal ligament (PDL) cells through the proteolytic actions of Arg- and Lys-gingipains (Rgp and Kgp). Thus, antiproteolytic supplements are considered clinically desirable for effective periodontal regenerative therapies. In the present study, we examined apple- (AP) and hop-polyphenols to determine their ability to protect EMD-stimulated PDL cells from P. gingivalis. AP, apple condensed tannin (ACT), hop bract polyphenol (HBP), high and low molecular weight fractions of HBP (HMW-HBP and LMW-HBP), and epigallocatechin gallate (EGCg) were used. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis, and cellular migration and proliferation were evaluated with an in vitro assay of wound healing assay in the presence or absence of the polyphenols. Each polyphenol significantly enhanced the viability of PDL cells infected with P. gingivalis, whereas only EGCg demonstrated cytotoxicity. Further, all polyphenols significantly inhibited Rgp activity, with AP, ACT, and HBP more effective toward Kgp. P. gingivalis markedly diminished the migration and proliferation of EMD-stimulated PDL cells, whereas the addition of AP, ACT, HBP, and HMW-HBP significantly protected the cells from bacterial cytotoxicity. In contrast, EGCg and LMW-HBP did not show protective effects. These results suggest that AP, ACT, AP, HBP, and HMW-HBP protect EMD-stimulated PDL cells from P. gingivalis and may be therapeutically useful supplements for EMD therapy.

Use of Enamel Matrix Protein Derivative Before Dental Reimplantation: A Histometric Analysis

The use of enamel matrix protein in the treatment of periodontal defects has shown a favorable action on the proliferation of periodontal ligament cells, as well as on collagen formation and mineralization. The goal was to evaluate, histologically and histometrically, periodontal tissue regeneration after dental reimplantation using enamel matrix protein derivative (Emdogain, Biora AB, Malmö, Sweden). Male rats (Albinus, Wistar), weighing between 180 and 200 g, were divided in 3 groups. Animals in group I (control) had the upper right incisors extracted, the root canal was sealed with calcium hydroxide, and teeth were reimplanted in their alveoli. Group II underwent the same procedure, but the remaining periodontal ligament was removed from the root surfaces by root planing before reimplantation. In group III,following removal of the periodontal ligament, Emdogain was applied to the root surfaces. Animals were sacrificed 7, 20, and 60 days after reimplantation, and the alveoli were fixed, processed, and stained with hematoxylin and eosin. Formation of periodontal ligament, resorption areas, and ankylosis were analyzed. The results showed that group I (control) was better than groups II and III, with statistically significant differences on days 7 and 20 after reimplantation for formation of periodontal ligament. It may be concluded that with the methodology used, Emdogain was unable to stimulate tissue repair in reimplanted teeth.

Preparation of recombinant human bone morphogenetic protein-2 loaded dextran-based microspheres and their characteristics1

To prepare new pharmaceutical forms with sustained delivery properties of recombinant human bone morphogenetic protein-2 (rhBMP2) for tissue engineering and guided tissue regeneration (GTR) use. rhBMP2-loaded dextran-based hydrogel microspheres (rhBMP2-MPs), which aimed to keep rhBMP2 bioactivity and to achieve long-term sustained release of rhBMP2, were prepared by double-phase emulsified condensation polymerization. The physical, chemical performances and biological characteristics of those microspheres were studied both in vitro and in vivo. The microspheres' average diameter was 30.33+/-4.32 microm with 75.4% ranging from 20 microm to 40 microm and the drug loading and encapsulation efficiency were 7.82% and 82.25%, respectively. The rhBMP2-releasing profiles in vitro showed that rhBMP2 release could be maintained more than 10 d. The rhBMP2-MPs, with good swelling and biodegradation behavior, could be kept for 6 months at below 4 degree without significant characteristic change or bioactivity loss. Cytology studies showed that rhBMP2-MPs could promote the proliferation of periodontal ligament cells (PDLCs) approximately 10 d, while the bioactivity of concentrated rhBMP2 solution could keep no more than 3 d. Scanning electron microscope showed that rhBMP2-MPs could be enchased into the porous structure of calcium phosphate ceremic (CPC) and the eugonic growth of PDLCs in CPC/rhBMP2-MPs scaffolds. Animal experiments indicated that using CPC/rhBMP2-MPs scaffolds could gain more periodontal tissue regeneration than using rhBMP2 compound firsthand with CPC (CPC/rhBMP2). By encapsulating rhBMP2 into dextran-based microspheres, a small quantity of rhBMP2 could achieve equivalent effects to the concentrated rhBMP2 solution and at the same time, could prolong rhBMP2 retention both in vitro and in vivo.

In vitro cell‐type specific biological response of human periodontally related cells to platelet‐rich plasma

Platelet-rich plasma is a blood-derived fraction containing high concentrations of platelets and growth factors. Applied in the form of a gel on surgical wounds, it is able to stimulate hard and soft tissue repair and has been proposed for use in the field of periodontal regeneration. However to date, little is known about the biological interactions between platelet-rich plasma and periodontally related cells. In this study, we investigated the effects between platelet-rich plasma and cell populations involved in periodontal regeneration, namely primary human periodontal ligament cells, gingival fibroblasts and keratinocytes. The proliferation of human periodontal ligament cells, gingival fibroblasts and keratinocytes by [3H]thymidine incorporation was assessed. The alkaline phosphatase activity and type I collagen levels of human periodontal ligament cells were also evaluated by a spectrophotometric assay and western blot analysis, respectively. Incubation of human periodontal ligament cells with platelet-rich plasma resulted in time-dependent growth stimulation (up to fourfold of control at 72 h). Likewise, an increase in the specific activity of alkaline phosphatase (fourfold at 6 days) and collagen (twofold at 7 days) was observed. Platelet-rich plasma also enhanced human gingival fibroblasts proliferation by twofold, whereas it inhibited human keratinocytes growth by 40%, with respect to their own controls at 72 h. Cell populations related to periodontal tissue were differently affected by platelet-rich plasma. In fact, a strong stimulation of human periodontal ligament cells proliferation, a minor increase in the growth rate of human gingival fibroblasts and a marked decrease of human keratinocytes proliferation were evident. In addition, in human periodontal ligament cells increased collagen and alkaline phosphatase activity levels were observed. These findings appear interesting in view of platelet-rich plasma utilization in periodontal regeneration.

Differential effects of estrogen on DNA synthesis in human periodontal ligament and breast cancer cells

It is important to clarify the biological function of the female sex hormones estrogen and progesterone in periodontal ligament cells, as these hormones may affect periodontal health. We have previously shown that human periodontal ligament cells express estrogen receptor beta (ERbeta) but not ERalpha, whereas human breast cancer cells (MCF7) express both ERalpha and ERbeta. Data on progesterone receptor (PgR) expression in human periodontal ligament cells have not been reported. Determine PgR expression in human periodontal ligament and MCF7 cells and to investigate how estrogen affects DNA and collagen synthesis in these two cell types showing different pattern of expression for ERalpha and beta. Periodontal ligament cells were obtained from the periodontal ligament of premolars extracted for orthodontic reasons and MCF7 cells from the American Type Culture Collection (ATCC). PgR expression was determined by immunocytochemistry. DNA and collagen synthesis was determined by [(3)H]thymidine and L-[(3)H]proline incorporation, respectively. PgR immunoreactivity was observed in nuclei of MCF7 but not periodontal ligament cells. Treatment with estrogen (17beta-estradiol, E(2)) at physiological concentrations for 24 h stimulated DNA synthesis by more than two times in MCF7 cells, whereas there was no effect on periodontal ligament cell DNA synthesis. The ER blocker ICI 182780 fully reversed the stimulatory effect of E(2). Not only short-term (24 h) but also long-term (5 days) treatment with E(2) lacked effect on DNA synthesis in periodontal ligament cells. Neither periodontal ligament cell viability nor collagen synthesis was affected by E(2) treatment. Identical results were observed in periodontal ligament cells from male and female subjects. Human MCF7 but not periodontal ligament cells express PgR, suggesting that progesterone via PgR affects MCF7 but not periodontal ligament cells. Further, estrogen stimulates breast cancer MCF7 cell proliferation, whereas it has no effect on proliferation of periodontal ligament cells, probably reflecting cell type specific ER expression pattern in these two cell types.

Simvastatin Promotes Cell Metabolism, Proliferation, and Osteoblastic Differentiation in Human Periodontal Ligament Cells

Simvastatin is one of the cholesterol lowering drugs. Recent studies demonstrated that it has a bone stimulatory effect. Periodontal ligament (PDL) cells are believed to play an important role in periodontal regeneration; that is, they may differentiate into specific cells which make cementum, bone, and attachment apparatus. It would be of interest whether simvastatin has a positive effect on PDL cells. Therefore, effects of simvastatin on cell proliferation and osteoblastic differentiation in PDL cells were analyzed. Human PDL cells were cultured in monolayer with simvastatin for 24 and 72 hours and cell metabolism and proliferation were determined. To analyze osteoblastic differentiation, human PDL cells were cultured in organoid culture for 7, 14, and 21 days and alkaline phosphatase (ALP) activity, osteopontin (OPN), bone morphogenetic protein (BMP) -2, osteocalcin (OCN), and calcium contents were measured. They were co-treated by simvastatin and mevalonate. Simvastatin enhanced cell proliferation and metabolism dose-dependently after 24 hours. Simvastatin also stimulated ALP activity of human PDL cells dose-dependently, and maximum effect was obtained at the concentration of 10(8) M. In time dependent analysis, 10(8) M simvastatin stimulated ALP activity and osteopontin content after 7 days and calcium contents after 21 days. BMP-2 and OCN contents were not detected. Moreover this statin-enhanced ALP activity was abolished by mevalonate. These results suggest that at low concentration, simvastatin exhibits positive effect on proliferation and osteoblastic differentiation of human PDL cells, and these effects may be caused by the inhibition of the mevalonate pathway.

Periodontal regeneration in intrabony defects after application of enamel matrix proteins with guided tissue regeneration: An experimental study in dogs

The aim of this study was to evaluate the effects of enamel matrix proteins (EMP) at the early stage of wound healing in the periodontal tissues by a combination treatment with guided tissue regeneration (GTR). Intrabony defects were produced surgically at the distal aspects of both mandibles in six beagle dogs. At 12 weeks following the surgery, the defects were exposed using a full thickness mucoperiosteal flap procedure. Subsequently, the defects were treated by the following treatments: a control group treated with GTR alone, and an experimental group treated with a combination of GTR and EMP. After one, two, four and eight weeks of the treatment, the animals were sacrificed, and sections of the tissue were stained and evaluated microscopically. After one and two weeks, the proliferating cell nuclear antigen (PCNA)-positive cell ratios of the experimental group were significantly greater than that of the control group. After 2 and 4 weeks, new bone and new cementum formation in the experimental group were significantly greater than those in the control group (P < 0.05). However, after 8 weeks, no statistical difference was found between the two groups in new bone or cementum formation. The study results suggest that a maturation of periodontal ligament cells might contribute, during the early stage of periodontal healing, to stimulate a proliferation of periodontal ligament cells.

Effects of nicotine on the attachment and proliferation of periodontal ligament cells, and reversibility of nicotine-induced cytotoxicity

Cigarette smoking has been shown to alter an individual’s response to various forms of periodontal therapy. In vitro and in vivo studies have indicated that smoking and the use of smokeless tobacco products may significantly contribute to the development and progression of inflammatory periodontal disease. The enhanced periodontal destruction observed in smokers may be caused by an alteration of the host response such as the reduced function of oral and peripheral neutrophils, and the decreased levels of salivary and serum antibodies. Similar effects have been observed at higher concentrations of various tobacco components such as nicotine, acroleine, and cyanide. Another hypothesis regarding the contribution of tobacco, and especially nicotine, to the etiology of periodontitis is that the reparative and regenerative potential of periodontal cells may be affected. It has been documented that nicotine can suppress the growth of osteoblast-like cells in vitro. Additionally, nicotine may be stored in and then released from periodontal fibroblasts. However, most of this cytotoxic substance remains within the fibroblasts, resulting in alterations of the cellular metabolism or various functions. Nicotine is a potent component of tobacco smoke. Nicotinic receptors are widespread and are heterogeneous groups comprised of multiple sub-units. The exact importance of individual receptor sub-units is currently not fully understood. However, this may explain in part the wide variety of physio-

PLATELET-RICH PLASMA PROVIDES NUCLEUS FOR MINERALIZATION IN CULTURES OF PARTIALLY DIFFERENTIATED PERIODONTAL LIGAMENT CELLS

Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-beta1 to modulate proliferation in a cell type- specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.