Effects of enamel matrix protein application on the viability, proliferation, and attachment of human periodontal ligament fibroblasts to diseased root surfaces in vitro.

OBJECTIVES: The purpose of this research was to examine the influence of enamel matrix proteins (EMP) on the viability, proliferation, and attachment of periodontal ligament fibroblasts (PDLF) to diseased root surfaces. MATERIALS AND METHODS: Primary cell cultures of PDFL were obtained from clinically healthy third molars or premolar teeth. Viability and proliferation rates were carried out over a 10-day period. A total of 80,000 cells were plated in 24-well plates followed by EMEM with 10% FBS (positive control) and EMEM plus EMP at 25, 50, 75, and 100 micro g/ml. Cells were harvested on days 1, 3, 5, 7, and 10 and viability was performed utilizing an MTS assay. PDLF proliferation rates were assessed by a CyQUANT GR dye assay. SEM analysis was used to examine the qualitative effects of cellular attachment to diseased root surfaces following EMP compared to nontreated controls. RESULTS: The results indicated that viability was negatively affected for higher doses over time while lower doses displayed viability effects similar to control. Proliferation, however, appeared to be ameliorated following exposure to EMP. The SEM analysis suggests that cellular attachment to diseased dentin was enhanced following EMP application. CONCLUSION: These in vitro studies support the concept that EMP may act as a suitable matrix for PDLF.

Interleukin-1beta regulation of adhesion molecules on human gingival and periodontal ligament fibroblasts.

BACKGROUND: Adhesion molecules have been implicated in the pathogenesis of rheumatoid arthritis and may also play a role in the pathogenesis of periodontal disease by promoting the recruitment and retention of leukocytes in gingival tissue. METHODS: The aim of the present study was to evaluate the capacity of interleukin-1beta (IL-1beta) to regulate adhesion molecule expression on clinically healthy human gingival (HGF) and periodontal ligament (PDL) fibroblasts. The HGF (n = 6) and PDL (n = 3) fibroblasts were treated with 1.0 ng/ml of IL-1beta for 24 hours and then incubated with primary intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1) antibodies followed by FITC-conjugated secondary antibodies. The expression of ICAM-1 and VCAM-1 was measured by immunofluorescence flow cytometry. RESULTS: The levels of ICAM-1 expression in IL-1beta treated HGF and PDL fibroblasts were statistically significant (P < or = 0.05) compared to normal untreated controls using log-transformed data and 3-way analysis of variance. Both cells expressed VCAM-1 after IL-1beta treatment, but the levels were not statistically different from controls. CONCLUSIONS: This study demonstrated that IL-1beta upregulated ICAM-1 expression in both HGF and PDL fibroblasts. Even though the level of VCAM-1 was not statistically different from both HGF and PDL fibroblasts treated with IL-1beta compared to controls, both cells do express the VCAM-1 molecules. These results suggest that ICAM-1 and VCAM-1 might be involved in the pathogenesis of periodontal disease.

Connexin-43 expression in oral-derived human osteoblasts after transforming growth factor-beta and prostaglandin E2 exposure.

Dental implant placement stimulates a response in the supporting tissue; the response involves bone remodeling and release of wound-healing factors, including cytokines. Important factors such as transforming growth factor-beta (TGF-beta), which promotes matrix synthesis, and prostaglandin E2 (PGE2), a mediator of inflammation, have the potential to alter the communication between bone cells and interfere with implant site healing. Cells responsible for the formation of bone are interconnected to form a multicellular network. Cell-to-cell communication in this network occurs in part via gap junctions. In bone cells, the predominant gap junction protein is connexin-43. TGF-beta is a growth modulator produced by osteoblasts and released from the matrix in response to resorption and may influence the progression of periodontal disease. TGF-beta also promotes the synthesis of extracellular matrix proteins such as collagen, fibronectin, and adhesion molecules. PGE2 is a mediator of inflammation produced in response to periodontal pathogens. PGE2 levels in the gingival sulcular fluid have been correlated with attachment loss and bone resorption. The relationship between these factors and connexin-43 is unclear. Oral-derived (alveolar) bone was used because the phenotype of bone can differ between species and between different sites in the body. For our studies, explants of human osteoblasts were cultured on eight well plates and characterized by their expression of osteocalcin, osteonectin, alkaline phosphatase, type 1 collagen, and connexin-43. Cells were grown to near confluence on 12 well plates in 20% fetal bovine serum (FBS) Dulbecco modified Eagle medium (DMEM) and then cultured for 24 hours in 0.5% FBS DMEM before exposure to either 1, 5, or 10 ng/mL of TGF-beta in serum-free DMEM for 12 or 24 hours or to 20, 80, or 300 ng/mL of PGE2 in serum-free DMEM for 12 or 24 hours. After incubation, cells were removed from plates by scraping and assayed for connexin-43 protein, first by Western blot to confirm the specificity of the anti-connexin-43 antibody and then by slot blot analysis for quantitative comparison of connexin-43 expression. Our studies showed no significant changes in connexin-43 expression in response to either factor. These studies suggest that exogenous TGF-beta and PGE2 do not alter connexin-43 expression.

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

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

Determination of periodontal ligament cell viability in long shelf-life milk.

The purpose of this study was to determine the ability of long shelf-life milk to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability on avulsed teeth. PDL cells were plated onto 24-well culture plates and allowed to attach for 24 h. Minimal Essential Medium was replaced with regular pasteurized milk (refrigerated milk), long shelf-life milk (Parmalat), or Save-A-Tooth. Tap water served as the negative control, and Minimal Essential Medium served as the positive control. The tissue culture plates were incubated at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined using a cell proliferation assay (CellTiter 96 AQ Assay) and absorbance read at 490 nm. ANOVA indicated that all media performed significantly better than tap water at all time periods. At 8 h, PDL cell viability in regular pasteurized milk and long shelf-life milk were significantly greater than in Save-A-Tooth (p < or = 0.001). There was no significant difference between regular pasteurized milk and long shelf-life milk at any time period. These results suggest that long shelf-life milk, which has the advantage of not requiring refrigeration, is as effective a storage medium for avulsed teeth as regular pasteurized milk and more effective than Save-A-Tooth.

A relationship between proteinase activity and clinical parameters in the treatment of periodontal disease.

The objective of this research was to determine the effectiveness of a biochemical assay which measures proteolytic enzyme activity in gingival crevicular fluid (GCF) and to relate this enzyme activity to clinical parameters traditionally utilized for periodontitis detection. A clinical trial was conducted on 8 periodontitis subjects with > or =4 sites exhibiting a loss of attachment of > or =5 mm and probing depths of > or =5 mm with bleeding on probing. On each subject, a plaque index was performed, followed by GCF sampling at those sites which exhibited a loss of attachment and probing depths. GCF was analyzed for activity against benzoyl-L-arginine-p-nitroanilide in the presence (BAPNA w/gly-gly) and the absence (BAPNA w/o gly-gly) of glycyl-glycine and against MeOSuc-Ala-Ala-Pro-Val-pNA and Suc-Ala-Ala-Pro-Phe-pNA for neutrophil serine proteinases activity (elastase and cathepsin G, respectively). Subsequently, a gingival index was performed, attachment levels and probing depths were recorded using a constant force probe with bleeding on probing being noted. A split-mouth design was employed and half mouths were randomly assigned to the following treatment groups: group A, half of the mouth received scaling/root planing and polishing: group B, half of the mouth received no treatment (control). Subjects were treated, then instructed on toothbrushing and interdental cleaning. After 4 weeks, subjects returned to receive a plaque index; GCF sampling, gingival index, attachment levels, probing depths and bleeding on probing as described above. Using a paired Student t-test, the findings suggest that BAPNA w/gly-gly was significantly less in treatment sites than in non-treated control sites (p=0.05). No such correlation was found for other activities, including neutrophil serine proteinases which were shown to occur in GCF in free, proteolytically active forms. In addition, significant treatment effects were detected for probing depths (p= 0.03) which reduced by 1.3 mm and attachment levels (p=0.02) which gained 0.7 mm. The reduction of P. gingivalis from treated periodontitis sites as detected by a significant decrease in BAPNA w/ gly-gly may prove to be a valuable marker for periodontal disease activity.

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.

An isolation and in vitro culturing method for human intraoral bone cells derived from dental implant preparation sites.

In dental implantology, the biocompatibility of the osseous tissue to the implant surface and to local environmental factors plays an important role in the process of healing. Bone cells derived from intraoral osseous tissue proves to be an important source of the osteoprogenitor cells required for healing of the periodontium around implants. Historically, the rat calvaria model has been employed to study the effects of various dental treatments on bone in vitro. However, there are morphological and functional differences which exist between bone cells derived from rat calvaria and human intraoral osseous tissue that impose certain limitations on the usefulness of the rat calvaria model for dental implant applications. Therefore, an in vitro culturing method for the isolation, growth and maintenance of human intraoral bone cell cultures derived from osseous tissues is truly warranted. In addition, a method for the accurate characterization of these bone cells as osteoblasts is also vital. The specific objective of this study was to establish isolation and in vitro culturing methods utilizing human intraoral bone cells derived from dental implant preparation sites. This paper describes techniques for the harvesting of human bone cells from the intraoral derived osseous tissues and discuss the procedures for maintaining the primary intraoral bone cell culture. In addition, our studies utilize established protocols for the characterization of these cells as osteoblasts by means of alkaline phosphatase activity determination, identification of cellular osteonectin and osteocalcin antigens, establishing the presence of cells expressing type I collagen and determining the ability of cells to produce calcifications. The utilization of intraoral osseous tissue may prove useful for future dental implant research by providing an in vitro model system more closely related to conditions encountered clinically.

A comparison of 2 patient populations using fractal analysis.

This study was undertaken to demonstrate that the fractal dimensions calculated using digitized non-standardized, clinical radiographs of mandibular alveolar bone from a population of patients diagnosed with periodontitis are statistically different from fractal dimensions calculated from another population diagnosed as having gingivitis or healthy gingiva. The fractal dimension was calculated using a public domain fractal analysis program distributed by the National Institutes of Health (NIH). Fractal dimensions were calculated from digitized clinical radiographs for 29 patients diagnosed with healthy gingiva and/or gingivitis and 32 patients diagnosed with periodontitis and compared. To estimate the reproducibility of the technique, we recalculated the fractal dimension from images of the gingivitis patients 3 months after the original calculations and compared them to the originals. A 2 sample, 2-tailed Student t test showed the gingivitis data group to be different from the periodontitis data group (P = 0.0012). The original gingivitis and repeat gingivitis groups fractal dimension calculation were the same and analysis showed the two data sets were not significantly different (P = 0.99). We found that: 1) fractal dimensions could be used to distinguish between gingivitis and periodontitis patient groups; 2) fractal dimensions could be calculated from non-standardized clinical radiographs; and 3) fractal dimensions for gingivitis patients were reproducible over a 3-month period.

Morphologic operations used to distinguish between two patient populations differing in periodontal health.

OBJECTIVES: This study was conducted to determine whether morphologic operation procedures applied to digitized, non-standardized, clinical radiographs of mandibular alveolar bone could be used to distinguish between a population of patients diagnosed with periodontitis and a population of patients either diagnosed with gingivitis or having healthy gingivae. STUDY DESIGN: Two groups, one consisting of 29 patients who either had healthy gingivae or had been diagnosed with gingivitis and the other consisting of 32 patients who had been diagnosed with periodontitis, were compared. Pre-existing clinical radiographs were digitized, and for each patient three to six regions of interest were placed on an image of the mandibular posterior region of the interdental bone. The regions of interest were processed under two morphologic-operations protocols, and a mean density (referred to as an MO number) was calculated for each patient. With paired t-tests, the resulting MO numbers for the two groups were compared. RESULTS: The two populations were statistically different (p < 0.05). CONCLUSION: The results of this study indicate that morphologic operations have the potential to differentiate between patient groups differing in periodontal health.

Comparison of various transport media on human periodontal ligament cell viability.

The purpose of this study was to determine the ability of various solutions to maintain human periodontal ligament cell (PDL) viability in vitro. PDL cells were obtained from extracted third molars and premolars of healthy individuals. These cells were placed into 24-well culture plates containing milk, Save-A-Tooth, Save-A-Tooth supplemented with platelet-derived growth factor-BB (PDGF), or Gatorade at a concentration of approximately 80,000/well. Cells left dry served as negative controls, and cells placed in Eagles' Minimal Essential Medium served as positive controls. At 1, 2, 4, 8, and 12 h, cell viability was evaluated using an MTS assay and an ELISA plate reader to determine optical density. ANOVA and Student-Newman-Keuls tests indicated that milk and Save-A-Tooth with PDGF are suitable as transport medium for avulsed teeth and that the addition of PDGF to Save-A-Tooth may enhance its ability to maintain PDL cell viability. They also suggests that Gatorade would be unsuitable as a transport medium.

Porous polysulfone coated with platelet-derived growth factor-BB stimulates proliferation of human periodontal ligament fibroblasts.

The purpose of this study was to investigate if the treatment of porous polysulfone (PPSF) with various concentrations of platelet-derived growth factor-BB (PDGF-BB) would stimulate the proliferation of the adherent human periodontal ligament fibroblasts (HPDLF) in culture. Sterilized PPSF cylinders were immersed in an Eagle's minimum essential medium supplemented with 0.5% fetal bovine serum and 1% penicillin/streptomycin containing either 0, 10, 20, or 50 ng/ml of PDGF-BB for 24 hours. After 24 hours, the PPSF cylinders were removed and allowed to dry then placed in culture plates for each time point. Pooled HPDLF (8 x 10(4)) and 3H-thymidine in medium were pipetted into each well to cover the treated and control PPSF cylinders and plates were then incubated. At 1, 4, and 10 days the PPSF cylinders were removed and macromolecular precipitation was performed. Incorporation of 3H-thymidine was measured and a 2-way ANOVA with repeated measures was performed. In addition, determination of binding and release was performed using I125-PDGF-BB treated PPSF at 0, 2, 12, and 24 hours, and at 4 and 10 days. Results showed that the effects on HPDLF were significant for dose (P = 0.0012; F = 5.74) and time (P = 0.0001; F = 40.83). At 4 days, the percent increases above the control for the doses 10, 20, and 50 ng/ml were 192%, 310%, and 162% respectively. In conclusion, our findings suggest that treating PPSF with PDGF-BB results in a significant increase in the proliferation of HPDLF cells adherent to PPSF.

Human periodontal ligament and gingival fibroblast response to TGF-beta 1 stimulation.

The purpose of this study was to measure the time-sequence response of RNA and protein synthesis to transforming growth factor-beta 1 (TGF-beta 1) by human periodontal ligament (HPDLF) and gingival (HGF) fibroblasts in culture. HPDLF and HGF were cultured from explants of healthy gingival tissue and freshly extracted teeth. Cultures of 8 x 10(4) cells/ml were exposed to medium containing 3H-uridine and 35S-methionine with TGF-beta 1 at concentrations from 10(-9) M to 10(-21) M, or control medium, for up to 60 hours in order to assess RNA and protein synthesis. Protein concentrations of comparable cultures were also assayed colorimetrically. Results were reported as specific activity (CPM/microgram protein). The results indicate that 10(-9) M TGF-beta 1 treated cultures showed a significant increase in RNA synthesis by HPDLF and HGF over time, as compared to the control cultures. HPDLF showed a significant increase in protein synthesis over time while that by HGF was not significant as compared to the control cultures. Lower concentrations of TGF-beta 1 demonstrated no significant differences from control. Results suggest that the effects of TGF-beta 1 on HPDLF and HGF are both time and dose dependent, with 10(-9) M TGF-beta 1 providing the best response of those concentrations tested. These findings support the concept that TGF-beta 1 may play a role in periodontal regeneration due to its ability to promote fibroblast RNA and protein synthesis. The results also demonstrate that although these two cells types appear morphologically similar, they exhibit distinct biological responses to growth factors such as TGF-beta 1.