Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-1 beta, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1 beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TCF-beta1 and IL-1 beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TCF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the nation of distinct functions for these cell-surface proteoglycans. (C) 2001 Wiley-Liss, inc.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis: of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

Differential Production of Macrophage Inflammatory Protein-1 alpha, Stromal-Derived Factor-1, and IL-6 by Human Cultured Periodontal Ligament and Gingival Fibroblasts Challenged With Lipopolysaccharide From P. gingivalis

Background: Fibroblasts are considered important cells in periodontitis. When challenged by different agents, they respond through the release of cytokines that participate in the inflammatory process. The aim of this study is to evaluate and compare the expression and production of macrophage inflammatory protein (MIP)-1 alpha, stromal-derived factor (SDF)-1, and interleukin (IL)-6 by human cultured periodontal ligament and gingival fibroblasts challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Methods: Fibroblasts were cultured from biopsies of gingival tissue and periodontal ligament of the same donors and used on the fourth passage. After confluence in 24-well plates, the culture medium alone (control) or with 0.1 to 10 mu g/ml of LPS from P. gingivalis was added to the wells, and after 1, 6, and 24 hours, the supernatant and the cells were collected and analyzed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results: MIP-1 alpha, SDF-1, and IL-6 protein production was significantly greater in gingival fibroblasts compared to periodontal ligament fibroblasts. IL-6 was upregulated in a time-dependent manner, mainly in gingival fibroblasts (P<0.05), which secreted more MIP-1 alpha in the lowest concentration of LPS used (0.1 mu g/ml). In contrast, a basal production of SDF-1 that was inhibited with the increase of LPS concentration was detected, especially after 24 hours (P<0.05). Conclusion: The distinct ability of the gingival and periodontal ligament fibroblasts to secrete MIP-1 alpha, SDF-1, and IL-6 emphasizes that these cells may differently contribute to the balance of cytokines in the LPS-challenged periodontium. J Periodontol 2010;81:310-317.

A fluorometric microassay for histamine release from human gingival mast cells

Mast cells are important effector cells of the immune system. We describe a rapid and inexpensive microassay to determine histamine release from human gingival mast cells. The assay is based on the coupling of histamine with o-phthalaldehyde (OPT) at a highly alkaline pH to form a fluorescent product. Using this assay with a sample volume of 10 mul/well in a 384 black well microplate, the histamine detection limit was 0.031 mug/ml. The human mast cell line (HMC-1) and fresh mast cells isolated from human gingival tissue (n = 10) were stimulated with substance P, anti-IgE or calcium ionophore A23187, Calcium ionophore significantly increased histamine release from HMC-1 cells and gingival mast cells (p < 0.05). This microassay will facilitate the study of mast cell histamine release in diseased oral mucosa.

Effect of laser phototherapy on the release of fibroblast growth factors by human gingival fibroblasts

The effects of laser phototherapy on the release of growth factors by human gingival fibroblasts were studied in vitro. Cells from a primary culture were irradiated twice (6 h interval), with continuous diode laser [gallium-aluminum-arsenium (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP),_660 nm] in punctual and contact mode, 40 mW, spot size 0.042 cm(2), 3 J/cm(2) and 5 J/cm(2) (3 s and 5 s, respectively). Positive [10% fetal bovine serum (FBS)] and negative (1%FBS) controls were not irradiated. Production of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) was quantified by enzyme-linked immunosorbent assay (ELISA). The data were statistically compared by analysis of variance (ANOVA) followed by Tukey`s test (P a parts per thousand currency signaEuro parts per thousand 0.05). The characterization of the cell line indicated a mesenchymal nature. KGF release was similar in all groups, while that of bFGF was significantly greater (1.49-times) in groups treated with infra-red laser. It was concluded that increased production of bFGF could be one of the mechanisms by which infra-red laser stimulates wound healing.

Substantivity of chlorhexidine to human dentin

Objectives. To better comprehend the role of CHX in the preservation of resin-dentin bonds, this study investigated the substantivity of CHX to human dentin. Material and methods. Dentin disks (n = 45) were obtained from the mid-coronal portion of human third molars. One-third of dentin disks were kept mineralized (MD), while the other two-thirds had one of the surfaces partially demineralized with 37% phosphoric acid for 15 s (PDD) or they were totally demineralized with 10% phosphoric acid (TDD). Disks of hydroxyapatite (HA) were also prepared. Specimens were treated with: (1) 10 mu L of distilled water (controls), (2) 10 mu L of 0.2% chlorhexidine diacetate (0.2% CHX) or (3) 10 mu L of 2% chlorhexidine diacetate (2% CHX). Then, they were incubated in 1 mL of PBS (pH 7.4, 37 degrees C). Substantivity was evaluated as a function of the CHX-applied dose after: 0.5 h, 1 h, 3 h, 6 h, 24 h, 168 h (1 week), 672 h (4 weeks) and 1344 h (8 weeks) of incubation. CHX concentration in eluates was spectrophotometrically analyzed at 260 nm. Results. Significant amounts of CHX remained retained in dentin substrates (MD, PPD or TDD), independent on the CHX-applied dose or time of incubation (p < 0.05). High amounts of retained CHX onto HA were observed only for specimens treated with the highest concentration of CHX (2%) (p < 0.05). Conclusion. The outstanding substantivity of CHX to dentin and its reported effect on the inhibition of dentinal proteases may explain why CHX can prolong the durability of resin-dentin bonds. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Human susceptibility to Schistosoma japonicum in China correlates with antibody isotypes to native antigens

Antibody isotypic responses (IgE, IgA, IgG1, IgG2, IgG3 and IgG4) to Schistosoma japonicum antigens-adult worm (AWA), soluble egg (SEA) and the recombinant proteins TEG (22.6-kDa tegumental antigen, Sj22) and PMY (paramyosin, Sj97)-were measured (in 1998) in a cohort of 179 Chinese subjects 2 years post-treatment. Subjects in the highest intensity re-infection group (> 100 eggs per gram faeces) had significantly higher levels of IgG1 and IgG4 against AWA. Analysis of IgG4/IgE ratios for AWA and SEA linked IgG4 excess to re-infection and IgE excess to non-re-infection. Two years after chemotherapeutic cure, 29 subjects, who were re-infected or never infected but highly water-exposed, were classified as epidemiologically susceptible (n = 15) or epidemiologically insusceptible to infection (n = 14). IgG4 levels against native antigens (AWA and SEA) were higher in susceptibles and IgE levels were higher in insusceptibles but antibody responses to the recombinant proteins (PMY and TEG) showed no clear pattern or difference between susceptibility groups. These and earlier findings provide evidence that immunity develops against schistosomiasis japonica in China and that susceptibility/resistance correlates with antibody isotypes against native schistosome antigens.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

The mm of this work was to evaluate the biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane to be used in guided tissue regeneration (GTR) Fibroblasts from human periodontal ligament (hPDLF) and keratinocytes (SCC9) were plated on P(VDF-TrFE)/BT and polytetrafluorethylene membranes at a cell density of 20.000 cells well(-1) and Cultured for up to 21 days Cell morphology, adhesion and proliferation were evaluated in hPDLF and keratinocytes, while total protein content and alkaline phosphatase (ALP) activity were assayed only for hPDLF Using a higher cell density. real-time polymerase chain reaction (PCR) was performed to assess the expression of typical genes of hPDLF, such as periostin, PDLs17, S100A4 and fibromodulin, and key phenotypic markers of keratinocytes, including involucrin, keratins 1. 10 and 14 Expression of the apoptotic genes bax, bcl-2 and Survivin was evaluated for both cultures hPDLF adhered and spread more oil P(VDF-TrFE)/BT, whereas keratinocytes showed a round shape on both membranes. hPDLF adhesion was greater oil P(VDF-TrFE)/BT at 2 and 4 h, while keratinocyte adhesion was similar for both membranes. Whereas proliferation was significantly higher for hPDLF on P(VDF-TrFE)/BT at days 1 and 7. no signs of keratinocyte proliferation could be noticed for both membranes Total protein content was greater on P(VDF-TrFE)/BT at 7, 14 and 21 days, and higher levels of ALP activity were observed oil P(VDF-TrFE)/BT at 21 days. Real-time PCR revealed higher expression of phenotypic markers of hPDLF and keratinocytes as well as greater expression of apoptotic genes in cultures grown on P(VDF-TrFE)/BT. These results indicate that, by favoring hPDLF adhesion. spreading. proliferation and typical mRNA expression, P(VDF-TrFE)/BT membrane should be considered an advantageous alternative for GTR (C) 2009 Acta Materialia Inc Published by Elsevier Ltd All rights reserved

In Vitro Proliferation and Osteoblastic Phenotype Expression of Cells Derived From Human Vertebral Lamina and Iliac Crest

Study Design. Osteoblastic cells derived from vertebral lamina and iliac crest were isolated and cultured under the same conditions (osteogenic medium, pH, temperature, and CO(2) levels). Objective. To compare proliferation and expression of osteoblastic phenotype of cells derived from vertebral lamina and iliac grafting. Summary of Background Data. Many factors play a role in the success of bone graft in spinal fusion including osteoblastic cell population. Two common sources of graft are vertebral lamina and iliac crest, however, differences in proliferation and osteoblastic phenotype expression between cells from these sites have not been investigated. Methods. Cells obtained from cancellous bone of both vertebral lamina and iliac crest were cultured and proliferation was evaluated by direct cell counting and viability detected by Trypan blue. Alkaline phosphatase (ALP) activity was evaluated by thymolphthalein release from thymolphthalein monophosphate and matrix mineralization by staining with alizarin red S. Gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, osteoprotegerin, and receptor activator of NF-kB ligand was analyzed by real-time PCR. All comparisons were donor-matched. Results. Proliferation was greater at days 7 and 10 in cells from vertebral lamina compared with ones from iliac crest without difference in cell viability. ALP activity was higher in cells from vertebral lamina compared with cells from iliac crest at days 7 and 10. At 21 days, mineralized matrix was higher in cells derived from vertebral lamina than from iliac crest. At day 7, gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, receptor activator of NF-kB ligand, and osteoprotegerin was higher in cells derived from vertebral lamina compared with iliac crest. Conclusion. Cell proliferation and osteoblastic phenotype development in cells derived from cancellous bone were more exuberant in cultures of vertebral lamina than of iliac crest.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

Human osteoblastic cell response to a Ca- and P-enriched titanium surface obtained by anodization

The aim of the present study was to evaluate the in vitro osteogenic potential of subcultured human osteoblastic cells derived from alveolar bone on a titanium (Ti) surface produced by an anodized alkali treatment (BSP-AK). Primary osteoblastic cells were subcultured on BSP-AK and machined Ti discs (control) and grown for periods of up to 21 days under osteogenic conditions. Morphologic and biochemical methods were used to assess important parameters of in vitro bone-like tissue formation. Although no major differences were observed between the BSP-AK and the control Ti surface in terms of cell attachment and mineralized matrix formation, a significant increase in cell population, ALP activity, and collagen content was detected in cultures on BSP-AK surface. Our results demonstrate that human osteoblastic cells are sensitive to the BSP-AK-modified Ti surface during the transitional stage between the end of the proliferative phase and the onset of the differentiation /matrix maturation ones. Together with the good mechanical properties exhibited by the Ca- and P- coating, our findings suggest that BSP-AK treatment could be useful for the development of a new surface for dental and orthopedic implants. (c) 2008 Wiley Periodicals, Inc.J Biomed Mater Res 88A: 841-848, 2009

Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface

The aim of this study was to evaluate the development of the osteoblastic phenotype in human alveolar bone-derived cells grown on collagen type I-coated titanium (Ti) surface (Col-Ti) obtained by plasma deposition acrylic acid grafting compared with machined Ti (M-Ti). Osteoblastic cells were cultured until subconfluence and subcultured on Col-Ti and M-Ti for periods of up to 21 days. Cultures grown on Col-Ti and M-Ti exhibited similar cell morphology. Cell adhesion, total protein content, and alkaline phosphatase (ALP) activity were not affected by Ti surface modification in all evaluated periods. Growth analyses indicated that there were significantly more cells in cultures grown on Col-Ti at day 3. Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG) mRNA expression of cells subcultured on Col-Ti was higher, whereas collagen type I (COL) was lower compared with M-Ti. Ti surface modification neither affected the osteocalcin (OC), ALP and receptor activator of NF-kappa B ligand (RANKL) mRNA expression nor the calcium content extracted from mineralized matrix. These results demonstrated that Col-Ti favours cell growth during the proliferative phase (day 3) and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase (day 14), suggesting that this Ti surface modification may affect the processes of bone healing and remodelling. To cite this article:Assis AF, Beloti MM, Crippa GE, de Oliveira PT, Morra M, Rosa AL. Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface.Clin. Oral Impl. Res. 20, 2009; 240-246.doi: 10.1111/j.1600-0501.2008.01641.x.

Human Alveolar Bone-Derived Cell-Culture Behaviour on Biodegradable Poly(L-lactic Acid)

Poly(L-lactic acid) (PLA) is a polymer of great technological interest, whose excellent mechanical properties, thermal plasticity and bioresorbability render it potentially useful for environmental applications, as a biodegradable plastic and as a biocompatible material in biomedicine. The interactions between an implant material surface and host cells play central roles in the integration, biological performance and clinical success of implanted biomedical devices. Osteoblasts from human alveolar bone were chosen to investigate the cell behaviour when in contact with PLA discs. Cell morphology and adhesion through osteopontin (OPN) and fibronectin (FN) expression were evaluated in the initial osteogenesis, as well as cell proliferation, alkaline phosphatase activity and bone nodule formation. It was shown that the polymer favoured cell attachment. Cell proliferation increased until 21 days but in a smaller rate when compared to the control group. On the other hand, ALP activity and bone mineralization were not enhanced by the polymer. It is suggested that this polymer favours cell adhesion in the early osteogenesis in vitro, but it does not enhance differentiation and mineralization. (C) Koninklijke Brill NV, Leiden, 2009

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain

Eag1 (K(v)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K+ channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.