Mechanical forces alter extracellular matrix synthesis by human periodontal ligament fibroblasts

Periodontal ligament fibroblasts (PDLFs) are a heterogeneous population of cells that are involved in the normal maintenance, repair and regeneration of both the ligament and adjacent hard tissues. Additionally, the ability of these cells to respond to mechanical stimulation suggests that they have a central role in mediating the osseous remodeling that underlies physiological and orthodontic tooth movement. To characterize their role further in this process, the current study evaluated the effect of tensional stress on the biosynthesis of extracellular matrix (ECM) proteins by human PDLFs. Cell strains were established from extracted human premolars and third molars. Cells exposed to 5% biaxial deformation (strain) at a frequency of 30 times/min for 24 hr exhibited statistically significant increases in type I collagen and fibronectin synthesis, and a statistically significant decrease in tropoelastin production relative to unstretched controls. Cells exposed to 10% strain exhibited similar responses for fibronectin and tropoelastin while the amount of type I collagen synthesized by stretched cells did not differ from control levels. These results indicate that mechanical stimulation of PDLFs alters type I collagen, tropoelastin and fibronectin production and that these cells are differentially responsive to varying levels of mechanical stress. The ability of these cells to alter ECM protein synthesis in response to specific magnitudes of tensional stress may in part explain how PDLFs regulate ligament and hard tissue remodeling.     

Adhesiveness of human ligament fibroblasts to laminin

The adhesiveness of fibroblasts from the human anterior cruciate and medial collateral ligaments to the laminin molecule was studied, with particular emphasis on the intrinsic differences between fibroblasts from the two ligaments. Cellular adhesion strength, adhesion area, laminin concentration, and seeding time were examined. Cell adhesion to laminin anchored with poly-D-lysine to a cleaned cover glass was measured with a micropipette micromanipulation system after seeding. The adhesion strength of fibroblasts from the anterior cruciate ligament to laminin was greater than and significantly different from that of fibroblasts from the medial collateral ligament, depending on the laminin concentration. Fibroblasts from the anterior cruciate ligament also exhibited an increase in adhesion strength, dependent on laminin concentration of as much as 30 micrograms/ml, at which the laminin receptors were thought to be saturated. Fibroblasts from the medial collateral ligament did not show such an increase except at laminin concentrations of 5-10 micrograms/ml. There was no significant difference in adhesion area between fibroblasts from the two ligaments except after 45 minutes at a laminin concentration of 40 micrograms/ml. For both, the adhesion to laminin showed little correlation to seeding time during periods of as long as 60 minutes. Measurements of adhesion area also failed to show a significant correlation to seeding time for fibroblasts from either ligament at laminin concentrations of 20 and 40 micrograms/ml. Adhesion strength normalized by adhesion area had no correlation to seeding time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

The dietary effect of 1,3-biseicosapentaenoyl-2-gamma-linolenoyl glycerol (STG) on the fatty acid composition of guinea pigs was examined and compared with that of an eicosapentaenoic acid ethyl ester (EPA-E) and of a soybean oil (SBO) diet. In terms of content of plasma lipid, EPA-E had a greater hypolipidemic effect than STG. On the other hand, in terms of EPA incorporation, contents of EPA in liver lipid were almost the same in the STG and EPA-E groups. Considering that the amount of EPA administered in the EPA-E group was almost 1.5 times that of the STG group, EPA may be absorbed more effectively as the glycerol ester than as the ethyl ester in guinea pigs. In all the tissue lipids, the STG group had a higher unsaturation index (UI) than the EPA-E group even though there is a lower UI in the STG diet than the EPA-E diet. These results suggest that greater amounts of desaturase products as a whole were synthesized in the STG group than in the other two groups. The dihomo-gamma-linolenic acid/arachidonic acid (DGLA/AA) ratio in plasma total lipids in the STG group was 3.5 times that of SBO group, and the DGLA/AA ratio in the EPA-E group was half that of the SBO group. In liver lipid, the ratios of DGLA/AA and EPA/AA in the STG group were 0.687 and 0.488 (phosphatidylcholine fraction) and 0.237 and 0.752 (phosphatidylethanolamine fraction), respectively. The ratio of DGLA/AA as well as the high EPA/AA ratio obtained in the present study with the STG diet may lead to physiological alterations, including enhanced synthesis of 1- and 3-series eicosanoids.     

CD40 mediated activation of gingival and periodontal ligament fibroblasts

Lipids and lipoproteins have been associated with breast cancer risk; however, published results have been inconsistent. To clarify these associations, we measured fasting lipids in women undergoing breast biopsies. A case-control study examined the association of fasting levels of lipids with histologically defined breast cancer risk. Four groups of premenopausal women were assembled on the basis of histological appearance of breast tissue: 1) no epithelial proliferation (n = 102), 2) proliferation without atypia (n = 53), 3) atypical hyperplasia or carcinoma in situ (n = 53), and 4) node-negative invasive cancer (n = 102). A postoperative fasting blood specimen was analyzed for cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Demographics, risk factors, diet, physical activity, fasting weight, and skin-fold thickness were measured. Triglyceride levels were significantly higher in women with node-negative invasive cancer (0.94 +/- 1.04 mg/ml) than in those with no epithelial proliferation (0.83 +/- 1.04 mg/ml, p = 0.03). This association persisted after adjustment for age, body size, lipids, reproductive and familial risk factors, and previous benign breast problems (p < 0.01), in keeping with an independent association of elevated triglycerides with breast cancer risk.     

In vitro attachment of human gingival fibroblasts to endosseous implant materials

In recent years, the focus of dental implant research has been the nature of the bone-implant interface associated with osseointegration, yet the transgingival portion of endosseous dental implants has received little attention. The purpose of this study was to determine the attachment of human gingival fibroblasts to three different implant materials: commercially pure titanium, non-porous hydroxyapatite, and porous hydroxyapatite. Cell attachment was quantified by radiolabeling gingival fibroblasts with tritiated thymidine and counting attached cells by liquid scintillation following incubation for periods of 20, 40, and 60 minutes. Additional studies coating implant surfaces with fibronectin were also performed. The nature of the implant material itself appeared to affect the number of attached cells. Determined on a surface area basis, fibroblast attachment was greatest to titanium followed by non-porous hydroxyapatite. Porous hydroxyapatite demonstrated the least amount of fibroblast attachment. When incubated with fibronectin at a concentration of 50 micrograms/ml, no increase in the number of cells attached to the various implant materials was observed. A small but statistically significant increase in the number of fibroblasts attached to porous hydroxyapatite at 40 minutes was observed when implant materials were pre-treated with fibronectin.     

Human periodontal ligament fibroblast response to PDGF-BB and IGF-1 application on tetracycline HCI conditioned root surfaces

The purpose of this study was to investigate the effect of 2 growth factors, platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor-1 (IGF-1), alone or in combination, on the adherence of human periodontal ligament fibroblast (PDL) to tetracycline HCl (TTC) conditioned and nonconditioned periodontally involved root surfaces. There were 80 root dentine chips from 80 patients, ranging from 35 to 70 years of age, each with one periodontally involved tooth requiring extraction. A root dentine chip was obtained from the subgingival surface opposite to the periodontal pocket of each extracted tooth. The dentine chips were randomly distributed into one of 8 groups. In group 1, PDL fibroblasts were cultured and allowed to attach on the dentine surface. In group 2, PDL fibroblasts were cultured on a PDGF-BB pre-treated dentine surface and in group 3, they were cultured on a IGF-1 pre-treated dentine surface. In group 4, PDL fibroblasts were cultured on a dentine surface pretreated with a combination of PDGF-BB and IGF-1. In group 5, PDL fibroblasts were cultured and allowed to attach on the TTC conditioned dentine surfaces. In groups 6 and 7, surface of dentine chips were conditioned with TTC and then were treated with PDGF-BB or IGF-1 respectively, followed by placement of PDL fibroblast and cultured. In group 8, dentine surfaces were conditioned with TTC and then pre-treated with a combination of PDGF-BB and IGF-1 before the fibroblasts were cultured. After 24 h of incubation, the media was removed and samples were fixed and processed for SEM at magnifications of x34, x750, x2000. Photographing and evaluation of samples was performed at x750 in which fibroblast adherence was measured by counting cells within a standard test area. The results of the non-TTC conditioned root surfaces demonstrated a significant increase in fibroblasts adherence in the PDGF-BB and combination PDGF-BB/IGF-I treatment groups (groups 2, 4) when compared to the control (group 1) as well as the TTC control (group 5). The combination of PDGF-BB/IGF-1 (group 4) did not significantly improve the adhesion of cells compared to PDGF-BB alone (group 2), but did significantly improve adhesion when compared to IGF-1 alone (group 3). There were no significant differences in cell morphology between the growth factor groups (groups 2, 3, 4) and control (group 1). In general, the cells demonstrated a flat, stellate-shaped morphology. The results of the TTC conditioned root surfaces, showed a statistically significant increase of cellular adherence in the PDGF-BB group (group 6) when compared to the TTC control (group 5), similar to the non-TTC group (group 2). However, the morphology of the cells in groups 5, 6, 7, and 8 demonstrated generally a rounded or oval shape with only an occasional cell exhibiting a flat form. In the experimental system of this study, the inclusion of PDGF-BB on the surface of dentine chips increased the number of adhering PDL cells, and the addition of TTC conditioning had little effect except to change the morphology of adhering cells.     

The effects of oestrogen on osteocalcin production by human periodontal ligament cells

BACKGROUND: Free radical production has been reported to be increased in patients with diabetes mellitus, and it has been suggested that hyperglycaemia may directly contribute to the generation of oxidative stress. The aim of the present study was to evaluate the effects of an acute increase in glycaemia on plasma antioxidant defences. RESULTS: During the oral glucose tolerance test (OGTT), plasma concentration of protein-bound sulphydryl (SH) groups, vitamin C, vitamin E and uric acid significantly decreased in normal as well as non-insulin-dependent diabetes mellitus (NIDDM) subjects. Total plasma radical-trapping activity, which evaluates plasma antioxidant capacity due to known and unknown antioxidants present in the plasma as well as their mutual co-operation, was also significantly reduced. CONCLUSION: This finding supports the hypothesis that hyperglycaemia may, even acutely, induce an oxidative stress.     

The response of periodontal ligament cells to fibronectin

Fibronectin (fn) is an extracellular matrix (ECM) molecule important in cell adhesion and migration and in wound healing. It is also likely important in periodontal ligament (PDL) cell-ECM interactions, and thus in regenerating periodontal tissues. In this study we characterized PDL cells and their interactions with FN, testing different PDL cell isolates taken from healthy and diseased conditions. PDL cells were characterized by their morphology, integrin profile, motility, and bone nodule formation. Cells were then assayed for adhesion, proliferation, and chemotaxis in response to FN or FN fragments. Cell isolates were morphologically heterogeneous and fibroblastic, had a normal-appearing actin cytoskeleton and a wide range of migration potentials, and formed bone-like nodules in vitro. They expressed alpha5, beta1, alpha v, and alpha4 integrin subunits, known receptors for FN, and in fact they bound FN preferentially at 5 and 10 microg/ml. Intact FN induced greater PDL cell proliferation and chemotaxis than did FN fragments (120-kDa cell-binding, 60-kDa heparin-binding, and 45-kDa collagen-binding). PDL cells harvested from diseased and healthy conditions were no different on the basis of these assays. These data demonstrate that PDL cells are a mixed population of fibroblastic cells, capable of forming a mineralized matrix. They also suggest that maximal proliferation and chemotaxis require specific FN domains that are present on the intact molecule but not its fragments.     

Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells

Basic fibroblast growth factor (FGF-2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF-2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [125I]-labeled FGF-2 to human PDL cells was studied by radioreceptor assay. The binding of [125I]-FGF-2 to PDL cells reached a plateau after 2.5 h incubation at 4 degrees C and was inhibited by the addition of unlabeled FGF-2 and acidic FGF (FGF-1; aFGF), but not insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor-beta 1. Scatchard analysis revealed the presence of approximately 1.0 x 10(5) FGF-2 binding sites per cell with an apparent Kd of 1.2 x 10(-10) M. Interestingly, the binding of [125I]-FGF-2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF-2 and its receptor was not. The responsiveness of PDL cells to FGF-2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF-2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodifferentiation of PDL cells into mineralized tissue forming cells.     

Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblasts

Human placental trophoblast expresses as unusual repertoire of major histocompatibility complex (MHC) class I products that appears to reflect the unique role of this epithelium in mediating feto-maternal relations during pregnancy. Trophoblast is devoid of human leucocyte antigen (HLA)-A,-B antigens but can express one or more non-HLA-A,-B class I proteins. The human choriocarcinoma cell lines JEG-3, BeWo and JAR are widely used as models to study trophoblast. During attempts to isolate non-HLA-A,-B class I from JEG-3 and BeWo by immunoaffinity chromatography using a monoclonal antibody to beta 2-microglobulin we observed a 55,000 MW protein co-purifying with class I. N-terminal amino acid sequencing and immunoblotting using a specific antiserum identified this product as calreticulin, a molecule recently shown to be involved in the assembly of classical class I in human B-lymphoblastoid cells. In our hands JEG-3 and BeWo were found to express 45,000 MW non-HLA-A,-B class I proteins while the 40,000 MW HLA-G product was identified only in JEG-3. Our data suggest that calreticulin associates with non-HLA-A,-B class I heterodimers and with free 45,000 MW non-HLA-A,-B class I H chains in JEG-3. JAR was found to be devoid of detectable class I H chains but contained beta 2-microglobulin and calreticulin. However, calreticulin-beta 2-microglobulin complexes were not detected in JAR. Calreticulin and class I were apparently co-localized within the endoplasmic reticulum of JEG-3 cells whereas only class I was expressed at the cell surface. These studies demonstrate that calreticulin is associated with non-HLA-A,-B class I products in human choriocarcinoma cells.     

Hyporesponsiveness of inflamed human gingival fibroblasts from patients with chronic periodontal diseases against cell surface components of Porphyromonas gingivalis

Guidelines for conducting forensic psychiatric consultations and evaluations have not been clearly established. The authors offer and discuss such guidelines, which are based upon the boundary guidelines in general psychiatric practice, ethics principles in general psychiatry, ethics principles in forensic psychiatry, and the relevant case and statutory law. These guidelines are intended to assist the psychiatrist in appropriately conducting forensic evaluations whether in litigation or administrative proceedings.     

Effects of extracellular matrix constituents on the attachment of human oral epithelial cells at the titanium surface

This in vitro study attempts to delineate the role of extracellular matrix (ECM) constituents at the epithelial tissue-implant interface. To know which ECM constituents have a beneficial influence on the behavior of epithelial cells, the attachment, proliferation, morphologic pattern, and differentiation or cytoskeletal organization of human oral epithelial cells on ECM-coated (type IV collagen, fibronectin, type I collagen, laminin, and vitronectin) and noncoated titanium surface have been evaluated and compared. In each experiment comparing commercially pure titanium and oxygen plasma-cleaned titanium, the same ECM constituents were used. In this study, type IV collagen could provide an excellent substratum for epithelial cell attachment on titanium surface, but vitronectin-coated titanium revealed lower effectiveness for attachment of epithelial cells than noncoated titanium. These results suggested that type IV collagen could be used as a means for obtaining good epithelial seal, whereas vitronectin could be used to restrain the attachment of epithelium to dental implants.     

Differentiation of periodontal ligament fibroblasts into osteoblasts during socket healing after tooth extraction in the rat

In mammals, a large proportion of the bulbospinal 5-hydroxytryptamine (5-HT) neurons also contain neuropeptides, such as substance P (SP) and galanin (GAL). To examine whether a similar coexistence occurs in an amphibian, an immunofluorescence double-labelling technique was employed on sections of the Xenopus laevis spinal cord. Antisera raised against SP, GAL, enkephalin (ENK), corticotropin-releasing factor (CRF), calcitonin gene-related peptide (CGRP), and cholecystokinin (CCK) produced a labelling of fibers at all rostrocaudal levels of the spinal cord, with the highest fiber densities for SP and ENK and intermediate densities for GAL, CCK, and CGRP, while CRF-immunoreactive fibers were barely detectable in intact animals. 5-HT-immunoreactive fibers were widely distributed in the spinal cord, and they often occurred in the vicinity of different types of peptide-immunoreactive fibers. However, no coexistence between 5-HT and the different peptide immunoreactivities could be detected, although SP and GAL immunoreactivities were sometimes found to be colocalized in the same fiber. Similar negative results were obtained when 5-HT+SP- and 5-HT+GAL-labelled sections were examined in single focal planes with a confocal microscope. After a spinal transection, (survival period 6 weeks to 4 months), almost all 5-HT-immunoreactive fibers below the lesion were lost, and a build-up of immunoreactive material occurred in fibers just rostral to the cut. In contrast, no significant loss of peptide-immunoreactive fibers occurred, although some swollen SP-, GAL-, ENK-, CRF-, and CCK-immunoreactive fibers were present rostral to the cut. The distribution of swollen peptide-immunoreactive fibers did not overlap with that of the swollen 5-HT-immunoreactive fibers. Although negative immunohistochemical data must be interpreted with caution, in conjunction with previous studies (Brodin et al. [1988] J. Comp. Neurol. 271:1-18; Sakamoto and Atsumi [1991] Cell Tissue Res. 264:221-230), the present results indicate that bulbospinal 5-HT neurons in nonmammalian vertebrates cocontain neuropeptides to a lesser extent than in mammals.     

Glucose modulates growth of gingival fibroblasts and periodontal ligament cells: correlation with expression of basic fibroblast growth factor

Diabetes mellitus is a systemic disease with profound effects on oral health and periodontal wound healing. Uncontrolled diabetes adversely affects surgical wound healing and is often associated with abnormal proliferation of fibroblasts, excessive angiogenesis and poor bone regeneration. Human gingival fibroblasts and periodontal ligament cells from both diabetics and non-diabetics were evaluated for growth responses following culture in 20 mM glucose, a concentration compatible with blood glucose levels in uncontrolled diabetics. Gingival fibroblasts derived from 9 non-diabetic patients and 3 insulin-dependent diabetics either proliferated or showed little change of growth in elevated glucose. Enhanced proliferation was observed following 1 wk of culture in glucose. Growth of periodontal ligament cells from 5 non-diabetic patients was inhibited by 20 mM glucose. Fibroblasts that were markedly growth stimulated were probed for expression of basic fibroblast growth factor (bFGF) using a reverse-transcribed polymerase chain reaction (RT-PCR). Results indicate that fibroblasts exhibiting the greatest increase in growth in response to high glucose also exhibited increased expression of bFGF. No changes were observed in mRNA expression for platelet-derived growth factor-AA, platelet-derived growth factor-BB, insulin-like growth factor and transforming growth factor-beta 1. Mitogenic effects induced by the cytosol of fibroblasts exhibiting increases of growth in 20 mM glucose were abrogated by neutralizing antibodies to bFGF. In addition, some periodontal ligament cells that were growth inhibited by high glucose had reduced expression of bFGF. These data suggest that bFGF may play a role in the abnormal wound healing associated with periodontal surgery of uncontrolled diabetics.     

Effect of different magnitudes of tension force on prostaglandin E2 production by human periodontal ligament cells

Periodontal ligament (PDL) cells are known to produce prostaglandin E2 (PGE2) in response to mechanical stress. However, the rate of PGE2 production from PDL cells in response to different magnitudes of tension forces has not been examined. This study, therefore, was undertaken to determine the effect of different magnitudes of tension forces on PGE2 production and inositol trisphosphate (IP3) levels in PDL cells in vitro. Human PDL cells were cultured on flexible-bottomed plates and placed on a Flexercell strain unit. Cells were flexed at six cycles (5-s strain, 5-s relaxation) at six steps of tension force (9, 12, 15, 18, 21, 24% increase in surface area) for 5 days. PGE2 production and IP3 levels were determined by radioimmunoassay. There was a 6- and 25-fold increase in the rate of PGE2 production by cells exposed to low (9%) and high (24%) tension forces, respectively, and these increases were tension force-dependent. Tension force also induced increases in the intracellular levels of IP3 that did not seem to be directly related to the production of PGE2. The different rates of PGE2 production by PDL cells in response to different magnitudes of mechanical stress may be of importance in PDL and alveolar bone metabolism.     

Synthetic integrin-binding peptides promote adhesion and proliferation of human periodontal ligament cells in vitro

Periodontal ligament (PDL) cells have been shown to express several integrins (alphav, alpha5, beta1, beta3) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [3H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands for the human PDL cells, and that they could provide a basis for the development of new strategies aimed at the regeneration of the periodontium.     

Induction of COX-2 expression by mechanical tension force in human periodontal ligament cells

Spectral EEG powers were compared in 4 frequency ranges (8-13, 15-25, 25-35, and 35-45 Hz) in a group of 20 subjects during the performance of tasks requiring mental rotation of two- and three-dimensional objects. Only those EEG segments corresponding to tasks with identical solution times were analyzed. The spectral powers of oscillations in the alpha range were higher in control conditions than during task performance. Power in the frequency range 15-45 Hz was greater during task performance than in control conditions; this supports the concept that alpha rhythm desynchronization accompanies the synchronization of higher-frequency EEG rhythms. Frequency power during task performance with two-dimensional objects was greater than that during tasks with three-dimensional objects. Since the angle of rotation between two-dimensional objects was greater than that between three-dimensional objects, this factor, rather than the depth of the perceived space, increased the level of cortical activation. In all experimental situations, power at frequencies of 15-45 Hz was significantly greater in the occipital regions than any other regions, reflecting the visual modality of the stimulus. Particular changes were noted in the gamma range (35-45 Hz), where power in the first second of task performance was significantly higher than in the second second; this may provide evidence that this range is more closely associated with perception and recognition processes than with mental transformation of the image.     

PDGF-alpha receptor subunit expression down-regulated by IL-1beta in human periodontal ligament cells

The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.     

Effects of polyelectrolyte complex (PEC) on human periodontal ligament fibroblast (HPLF) function. I. Three-dimensional structure of HPLF cultured on PEC

The functional effect of activating Ca2+-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca2+-permeable nAChRs or with voltage steps to activate voltage-dependent Ca2+ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at -80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd2+ to block voltage-activated Ca2+ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca2+ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca2+ channels alone. Our data suggest that Ca2+ influx via nAChRs affects Ca2+-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca2+ entry pathway underlying Ca2+-dependent cellular processes such as exocytosis.