Influence of Serum Ti-Dental-Monomaterial-Eluates in Comparison to Precious-Dental-Alloy-Eluates on Primary Human CD4⁺-Lymphocyte-Migration.

Active cellular locomotion is a feature of diverse T cell types. Using a 3-D collagen matrix migration model in combination with computer-assisted cell tracking for reconstruction of migration paths and confocal microscopy, we investigated the locomotion behavior of CD4+ lymphocytes governing cell-dental material interactions due to the influence of different dental alloys. We tested the hypothesis of whether changes of lomotory behavior after induction of chemotaxis could be ascribed to changes of the regulatory signal transduction of migration. We did this on the basis of the trading that spontaneous locomotion of T lymphocytes was regulated by PTK activity and was distinct from a second, protein kinase C (PKC)-dependent type of migration inducible by PKC-activating phorbol ester due to different serum eluates of dental alloys. Material and methods: Human peripheral CD4+ cells were isolated from heparinized blood of healthy donors by density-gradient, centrifugation using Ficoll-Hypaque. CD4+ cells were positively selected using immunomagnetic beads coated with mouse anti-human CD4+mAb. Subsequently, cell-bound beads were detached using polyclonal anti-mouse Fab antibodies. Purified CD4+ cells were > 98% Cd3 and 96.99% CD4+, respectively, as detected by flow cytometry. More than 98% of the cells were viable, as assessed by propidium iodide staining and flow cytometry. For collagen lattices preparations 2,5 x 105 cells were mixed with 100µl of buffered collagen solution (pH 7,4) containing 1,67 mg/ml collagen type I in minimal essential Eagle's medium. Locomotion of CD4+ lymphocytes suspended in type I collagen gels was recorded using time-lapse video-microscopy. Paths of 30 randomly selected locomoting cells over a period of two hours were digitized, reconstructed and quantitatively analysed. A dental alloy free assay served as a control. We evaluated two different quantitative parameters using Ti-dental monomaterials in comparison to precious dental alloys: (1) the average percentage of CD4+ cells moving and (2) the velocity of the migrating CD4±cells due to the influence of serum dental material eluates. Results: We could show a reduction of average percentage of CD4+ cells migration in the presence of precious alloys (25,6%5,8 for 'high-precious-alloys', 53,1%5,6 for 'reduced-precious-alloys', 28%5,8 for palladium-based alloys) in comparison to the non-reduction of the average migration in the Ti-group. Concerning the velocity the same deminishing tendency could be seen for precious and palladium-based alloys (range from 1,65µm/min2,0 up to 3,0µm/min2,4) in comparison to the highly biocompatible results of Ti-monomaterials (4,8µm/min2,4). Discussion: We presume that the CD4+ cells are migrating in a 3D collagen matrix migration model in a 'random-walk' fashion influenced by the components of serum dental material eluates. Further the developed test could be used as an indicator for biocompatibility of different dental materials. Conclusion: The results of our newly developed test showed a higher biocompatibility of Ti-monomaterials in comparison to precious dental alloys. Keywords: CD4+ lymphocyte, migration, titanium, dental alloy