[Relevant Parameters of Optic Nerve Analysis from Spectral Domain OCT for Glaucoma Diagnostics]. / Relevante Parameter der Papillenanalyse mittels Spectral-Domain-OCT zur Glaukomdiagnostik.

BACKGROUND: In early diagnosis and follow-up of patients with glaucoma anatomic-diagnostic examinations have become more important in addition to static perimetry. Patients with open angle glaucoma suffer a slow visual field loss due to the loss of ganglion cells, which these examinations could detect earlier than perimetry can. METHODS: Parameters of the optical coherence tomography (OCT) were analysed in 89 patients (175 eyes) with advanced open angle glaucoma. In a prospective study, the functional findings from static perimetry (HFA-II, 24-2-programme) and the anatomic parameters of optic nerve analysis with SD-OCT (Cirrus-OCT) were evaluated. RESULTS: The results showed a mean deviation (MD) in perimetry of - 8.31 ± 9.76 dB and in the analysis of the optic nerve head of 71.93 ± 15.86 µm retinal nerve fibre layer (RNFL) as well as 85.54 ± 28.2 µm RNFL for the inferior quadrant, 0.95 ± 0.46 mm(2) for the rim area and 0.69 ± 0.18 for the vertical CD ratio. There was a significant correlation (p < 0.05) between MD und RNFL (r = 0.603), as well as RNFL of the inferior quadrant (r = 0.620), rim area (r = 0.552) and average CD ratio (r = - 0.551). The best correlation for the optical nerve head analysis was found between MD and vertical CD ratio (r = - 0.568). CONCLUSIONS: There was a good correlation between functional and anatomic parameters in perimetry and OCT. In particular, the mean and inferior retinal nerve fibre layer thickness, the rim area, and the vertical CD ratio revealed to be significant parameters in glaucomatous eyes.

Amelogenin is a cell adhesion protein.

Amelogenin, the major protein component of tooth enamel, is shown to be a cell adhesion protein. Since it had been shown that an amelogenin-containing preparation, Emdogain, possessed cell-adhesive activity, we tested the hypothesis that amelogenin was responsible for cell-adhesive activity. Recombinant amelogenin was found to promote adhesion at less than 15 micro g/60-mm plate and requires divalent cations for activity. While we found that amelogenin does not bind to collagen or heparin under physiological conditions, it was demonstrated previously that amelogenin does bind to hydroxyapatite. The cell-adhesive activity of amelogenin may play a role in development and may provide a partial explanation for the therapeutic effects of Emdogain in periodontal regeneration.

In vitro wound healing responses to enamel matrix derivative.

BACKGROUND: Enamel matrix derivative (EMD) contains a variety of hydrophobic enamel matrix proteins and is extracted from developing embryonal enamel of porcine origin. EMD has been associated with the formation of acellular cementum and it has been found to stimulate periodontal regeneration. The present study was established to investigate the influence of EMD on human periodontal ligament (PDL) cells, gingival fibroblasts (GF), and osteosarcoma (MG-63) cells on wound-fill rates using an in vitro wound model. METHODS: Wounds were created by making 3 mm incisions in cell monolayers across the length of tissue culture plates. The wounded PDL, GF, and MG-63 cell monolayers were treated with media containing EMD over a concentration range of 5 to 100 microg/ml, platelet-derived growth factor (PDGF-BB) at 20 ng/ml as a positive control and insulin-like growth factor (IGF-I) at 100 ng/ml as a negative control. PDL cell wounded monolayers also were treated in EMD coated tissue culture plates. After an incubation period (up to 9 days), the cells were fixed and stained and cellular fill was measured across the width of the wound by computer-assisted histomorphometry. RESULTS: When PDL, GF, and MG-63 cells were exposed to EMD in culture medium, an enhanced wound-fill was observed for all cells compared to untreated conditions. At early time points, PDL wound-fill rates in the presence of EMD were statistically greater than the rates of GF and MG-63 treated with EMD (P<0.001). There were no significant differences in wound-fill rates of PDL cells treated with EMD in medium versus EMD coated on culture plates. At days 3 and 6 post-wounding, PDL cells showed a significantly greater response to EMD than to PDGF-BB (P <0.001). EMD also had a greater effect on GF wound-fill rates than PDGF-BB at days 6 and 9. MG-63 cells were less responsive to PDGF-BB and EMD than PDL cells and GF. All 3 cell types treated with IGF-I showed no significant increase of wound-fill rates. CONCLUSION: The present data support the concept that clinical application of enamel matrix derivative may enhance periodontal wound regeneration by specifically modifying periodontal ligament cell proliferation and migration.

Development of an in vitro wound healing model for periodontal cells.

BACKGROUND: Periodontal wound healing and regeneration are influenced by a multitude of factors. While many in vitro investigations have compared the proliferation of periodontal ligament (PDL) cells and gingival fibroblasts (GF), there are no reports directly comparing the abilities of these 2 cell types to fill a wound site. As such, the goals of this research were: 1) to develop an in vitro model of wound healing which would allow for the investigation of the biologic basis of periodontal wound healing and regeneration and 2) to compare the rates of PDL cells and GF to fill an in vitro wound site. METHODS: Using both human PDL cells and GF confluent cultures, in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer. Wounded cultures were then incubated for time periods up to 12 days in media containing fetal bovine serum (FBS) concentrations (0, 0.1, 1, 5, 10, and 20%) as appropriate for each experiment. Slides were fixed, stained, and cells quantified within the wound boundaries by computer-assisted histomorphometry. The effect of wounding a cell layer was determined by comparing wounded cells as described above with a cell layer margin created without physically disrupting the cell layer. RESULTS: The in vitro model for periodontal wound healing established in this study showed that GF fill in the wound site at a significantly (P <0.0025) faster rate than PDL cells over 12 days of healing. In addition, PDL cells and GF were found to have unique concentration-dependent responses to FBS (P<0.0025). It was also shown that wounding resulted in a significant delay (P <0.01) in the initial healing response of an in vitro wound. CONCLUSION: This in vitro model demonstrated that the characteristics of wound healing are dependent on cell type, disruption (wounding) of the cell layer, and serum concentration. In addition, this model has incorporated both proliferation and migration to provide the first direct evidence demonstrating GF has a significantly greater ability to fill a wound site than PDL cells. This in vitro model may be utilized in future investigations of the biologic basis of periodontal wound healing.

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 interleukin-1 beta on matrix metalloproteinase-3 levels in human periodontal ligament cells.

MATRIX METALLOPROTEINASE-3 (MMP-3), or stromelysin-1, is an enzyme responsible for the degradation of a wide range of extracellular matrix proteins. Increases in MMP-3 activity have been found in several chronic inflammatory diseases, and this increased activity is thought to be mediated by interleukin-1 beta (IL-1 beta). Because IL-1 beta has been strongly associated with inflammatory periodontal disease, the purpose of this in vitro study was to investigate the role of IL-1 beta on the regulation of MMP-3 levels in cells derived from the human periodontal ligament (PDL). Human PDL cell cultures were treated with IL-1 beta at varying concentrations (0.01-1.0 ng/ml) for 24 hour prior to analysis at either transcript or protein levels. Following the isolation of total RNA, the relative levels of MMP-3 mRNA were determined using reverse transcription-polymerase chain reaction (RT-PCR) with 32P-end-labeled primers. Immunocytochemical detection of MMP-3 protein was performed using polyclonal antibodies to human MMP-3. The results of RT-PCR analysis demonstrated a concentration-dependent increase in MMP-3 mRNA expression, with IL-1 beta treatments of 0.1 and 1.0 ng/ml significantly (P < 0.01) increased over those cells not treated with IL-1 beta. This increase in mRNA expression was paralleled by significant (P < 0.001) changes at the protein level, with an average of 27.6% of the cells stained positive for MMP-3 following IL-1 beta treatment (1.0 ng/ml), compared with control cells showing no positive staining for MMP-3. In conclusion, the results of this study demonstrate that IL-1 beta upregulates MMP-3 in human PDL cells on both an mRNA and a protein level. These findings suggest possibly important roles for IL-1 beta and MMP-3 in both normal turnover and maintenance of the PDL and in the connective tissue degradation associated with periodontal disease.

Development and characterization of a transformed human periodontal ligament cell line.

Periodontal ligament (PDL) cells are thought to be important for establishing and maintaining a stable interface between bone and teeth. In addition, PDL cells are thought to play critical roles in both the pathogenesis of periodontal disease and the regeneration of periodontal ligament tissues. The purpose of this study was to develop a continuous or stable human PDL cell line as an in vitro model for the investigation of cellular mechanisms involved in periodontal regeneration and destruction. Human PDL cells, derived from a primary cell culture, were transfected with simian virus 40 (SV40) T antigen-containing virus with a neomycin resistance gene. The transformed cells expressed the SV40 T antigen mRNA as assayed by reverse transcription polymerase chain reaction (RT-PCR). This cell line was also characterized for morphological changes and growth characteristics compared to primary PDL cell cultures. The transformed cells were shown to form a multilayer pattern and distinct colonies on tissue culture surfaces. However, no colony formation was found in soft agar. The transformed PDL cell line was found to have a greater rate of proliferation in 10% fetal bovine serum than primary culture, and continued to proliferate in low serum concentrations capable of producing quiescence in primary cells. Interleukin-1 beta (IL-1 beta) was shown to produce a 7-fold elevation in collagenase (MMP-1) mRNA levels, consistent with primary PDL cells. In addition, IL-1 beta was shown to produce a decrease in alkaline phosphatase activity in a concentration-dependent manner. The transformed cell line has been maintained for over 30 generations of cell culture. In conclusion, a stable human PDL cell line has been established to serve as a model for future in vitro investigations into periodontal pathogenic mechanisms and to evaluate therapies directed at the regeneration of periodontal ligament.