Cytotoxicity testing with three-dimensional cultures of transfected pulp-derived cells.

SV40 large T-antigen-transfected bovine pulp-derived cells were grown three-dimensionally on polyamide meshes. For optimal cell growth, various cell numbers and mesh coatings were tested. Next the three-dimensional cultures were used in a dentin barrier test device, and the system was evaluated by testing a set of dental filling materials. After 24 hr exposure with or without perfusion of the pulpal part of the test device, cell survival was evaluated using an MTT assay. In all experiments pulp-derived cells transfected with SV40 large T-antigen grew three-dimensionally on polyamide meshes and showed growth kinetics similar to those on cell culture plates with lag, log, and plateau phases (reached after about 14 days of incubation). Cross-sections of the three-dimensional cell cultures revealed about 15 to 20 cell layers. In vitro cytotoxicity tests resulted in cell survival rates which are in good agreement with in vivo data and with results obtained from cytotoxicity tests with three-dimensional cultures of human foreskin fibroblasts.

Dentin barrier test with transfected bovine pulp-derived cells.

Growth kinetics of SV40 large T-antigen-transfected bovine pulp-derived cells on dentin were investigated. These cells were used in a dentin barrier test device, and the system was evaluated by testing a set of dental filling materials. Cells (120 cells/mm2) were seeded on dentin slices and incubated for up to 21 days. Cell proliferation was recorded using MTT assay. For cytotoxicity tests 3500 cells/mm2 were seeded on dentin discs, which were then incorporated into the dentin barrier test device. After 72 h preincubation test materials were applied. After a 24 h exposure with or without perfusion of the pulpal part of the test device, cell survival was evaluated using MTT assay. The cells revealed similar growth kinetics on dentin slices and on tissue culture plates. In cytotoxicity tests the cells were more sensitive toward the test materials than previously used three-dimensional cultures of human foreskin fibroblasts and as anticipated from clinical experience. Further improvement is expected by using three-dimensional cultures of pulp-derived cells.

An in vitro pulp chamber with three-dimensional cell cultures.

To better simulate the in vivo situation, a three-dimensional fibroblast cell culture was introduced into an in vitro pulp chamber model. The system was evaluated by testing a series of dental filling materials. After a 24-h exposure with (0.3 or 5 ml/h) and without perfusion of the pulp chamber, the tissues were subjected to a routine MTT assay. Zinc phosphate cement, conventional glass ionomer cements, a silicone impression material, and zinc oxide-eugenol did not influence cell viability, compared with untreated controls; but, a light-curing glass ionomer cement significantly reduced cell survival. Perfusion of the chambers did not significantly influence the results, but perfusion conditions of 5 ml/h lead to a general decrease of cell vitality. The three-dimensional cell culture system in an in vitro pulp chamber seems to be a substantial improvement, because zinc oxide-eugenol does not evoke a cellular reaction (as is the case in vivo), and the test system is sensitive enough to detect other toxicants.

Influence of metals on IL-6 release in vitro.

Certain dental alloys have been claimed to cause gingival and periodontal inflammation. However, little information is available on the molecules mediating the mechanism of such an effect. Recently, a three-dimensional cell culture system consisting of human fibroblasts and keratinocytes has been introduced for evaluating the irritancy of cosmetic products, including the analysis of inflammatory mediators. In the present study the influence of pure metals and a high noble dental cast alloy upon cell viability and the synthesis of the proinflammatory mediator interleukin-6 was recorded in this in vitro skin equivalent model. The cultures were exposed to test specimens fabricated from copper, nickel, cobalt, zinc, palladium, tin, indium, a high noble cast alloy and a dental ceramic. Cell vitality was reduced after a 24 h exposure to copper (14-25% of untreated controls), cobalt (60%), zinc (63%), indium (85%), nickel (87%), and the heat treated and not heat treated high noble cast alloy (87%/90%). Dental ceramic, palladium and tin did not influence cell viability. Increased IL-6 levels were observed in cultures exposed to copper (5-19-fold compared to untreated controls), zinc (16-fold), cobalt (12-fold), nickel (10-fold) and palladium (4-fold). Other materials tested produced IL-6 levels comparable to those of untreated controls. Our findings suggest that metal ions are involved in proinflammatory activity at low toxicity and non-toxic levels as assessed by different biological endpoints.

A heterologous in vitro coculture system to study interaction between human bladder cancer cells and fibroblasts.

Three-dimensional multicellular spheroids of two fibroblast cell lines (WI-38 and N1) and two differently differentiated bladder carcinoma cell lines (RT4 and J82) were used for cocultures of multicellular tumor spheroids with multicellular spheroids of fibroblasts. The aim of the study was the establishment and characterization of a standardized three-dimensional model for studies of tumor cell-fibroblast interaction as one aspect of tumor-stromal cell interactions of in vivo tumor tissue. Interaction of multicellular spheroids of both fibroblast cell types was analyzed by staining with antibodies against cytokeratin, vimentin and different extracellular matrix molecules. Further, proliferation assessment and phenotypic characterization of the cocultures are presented. Interactions varied with tumor cell type and fibroblast cell type, reflecting intrinsic properties of tumor cells and fibroblasts. The coculture of tumor cells with N1 reflected the in vivo situation the closest, since invasive properties of J82 as well as noninvasive properties of RT4 were characteristics seen in coculture.

The effect of low-frequency whole-body vibration under different visual conditions on auditory evoked potentials.

Auditory evoked brain potentials (AEP) were recorded from 9 healthy males during sinusoidal whole-body vibration (WBV) in the longitudinal (+/- az) direction with 0.6 Hz, 1.85 ms-2rms (F1), 1.01 Hz, 4.27 ms-2rms (F2) and without WBV (F3) under 3 visual conditions--homogeneous bright visual field (B), normal vision (N), and complete darkness (D). The sequences of the different experimental conditions were arranged according to a 9 X 9 Latin Square design. A subtraction technique was used to eliminate vibration-synchronous activity from the EEG. The N1 and N1P2 amplitudes decreased during F1 and F2, compared to F3. The latencies of N1 and P2 increased during F1 and F2. The effects of F1 and F2 did not differ. The visual conditions exhibited no systematic effect on the AEP. The results suggest (1) F1 and F2 to be equivalent exposure conditions and (2) the dominance of vestibular-auditory interactions, compared with visual-auditory ones.

Changes in auditory evoked brain potentials during ultra-low frequency whole-body vibration of man or of his visual surround.

Auditory evoked brain potentials (AEP) were recorded from nine healthy male subjects during three types of condition: A - subject and visual field stationary; B - subject vibrated (z-axis, 0.6 Hz, 1.85 ms-2 rms), visual field stationary; C - subject stationary, visual field vibrated (as for B). The visual surround was confined to a checkerboard pattern in front of the subject. Auditory stimuli (1000 Hz, 86 dB, interstimulus interval 7 s) were delivered via headphones to evoke AEP. Vibration-synchronous activity in the EEG was eliminated by a subtraction technique. In comparison with condition A, conditions B and C caused an attenuation of P2 and N1P2 components of AEP together with an increased latency of N1. Effects of conditions B and C did not differ. Direct vestibular stimulation and mechanisms specific for whole-body vibration were rejected as modes of action. The AEP-changes and the subjective evaluation of experimental conditions, arousal and performance, as well as symptoms of kinetosis (motion sickness) suggest a sensory mismatch, leading to a "latent kinetosis" with de-arousal, as the dominating mechanism by which the processing of information was affected. This suggestion was supported by an additional pilot study. Under real working conditions a similar effect can be expected during relative motion between the driver and his visual surround, i.e. even with perfect vibro-isolation of the driver's seat.

[The effect of low-frequency whole-body vibration on the vestibular apparatus]. / Einfluss niederfrequenter Ganzkörpervibration auf den Vestibularapparat.

For the first time, effects of very low-frequency whole-body vibration (WBV) on the functional state of the vestibular system were examined by means of electronystagmography (ENG) which is considered as a sensitive and adequate method. During vertical WBV-exposure with 0.6 Hz and 1.87 ms-2 rms for 50 minutes, a vertical nystagmus was observed. The results suggest the applicability of the ENG under laboratory conditions with WBV-exposure as a method for the assessment of the vestibular function. Changes of ENG amplitudes occurred with longer duration of WBV-exposure; they were different between subjects.