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Primary cells in culture have a limited capacity to divide and soon reach a non-proliferative state. This cellular senescence limits the investigation of cells derived from human pulp concerning cellular pathways, gene regulation, mechanisms of dentin formation, or responses to material exposure. To overcome this problem, primary human pulp-derived cells were established and transfected with a plasmid containing coding sequences of Simian Virus 40 (SV40) large T-antigen. This resulted in the establishment of several cell clones showing an extension of life span. Expression of T-antigen transcripts and protein was verified by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Primary human pulp cells were cultured until senescence (i.e. up to passage 7) and transfected cells could be cultured to passage 18 after transfection, when a cellular crisis with massive cell death occurred. One clone escaped from crisis and has been maintained in culture for 55 wk. Experiments were performed to characterize transfected cells in comparison to primary cells. Cell morphology and proliferation were analyzed, and expression of cell-specific gene transcripts and proteins (including collagen types I and III, alkaline phosphatase, bone sialoprotein, osteocalcin, and dentin sialophosphoprotein and dentin matrix protein I) was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining many of the phenotypic characteristics observed in primary cells.

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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.

This investigation evaluated the performance of a resin-modified glass ionomer, a compomer, and a bonding system/composite combination for the restoration of cervical erosion lesions without cavity preparation. Forty-eight lesions (11 patients) were restored with a bonding agent/composite combination [Prime & Bond 2.1/PrismaTPH (P & B/TPH); DeTrey/Dentsply], a compomer (Dyract; DeTrey/Dentsply), or a light-curing glass ionomer (Fuji II LC; Fuji). The materials were randomly assigned to the patients in triplets. No cavity preparation was performed. The procedures strictly followed the manufacturers' instructions. The restorations were evaluated clinically, using modified USPHS criteria, and by quantitative scanning electron microscope (SEM) analysis, at baseline and 12 months. The clinical data were statistically evaluated with the Pearson chi-square test, the SEM data (criterion gap formation) were analyzed with the Mann-Whitney U-test and error rates method. Clinically, two restorations could not be evaluated. One Dyract restoration failed. With respect to marginal discoloration, recurrent caries and contour, no significant differences could be found between the materials. The surface texture of P & B/TPH and Dyract was significantly better than that of Fuji II LC at baseline and 12 months. Compared to P & B/TPH and Fuji II LC, Dyract revealed a significant decrease in marginal integrity between baseline and 12 months. In SEM analysis, gap formation was determined as follows: baseline, enamel interface: 4% Dyract= 4% Fuji >2% P & B/TPH and dentin interface: 11% Dyract >9% P & B/TPH >2% Fuji; 12 months, enamel interface: 15% Dyract >4% Fuji >3% P & B/TPH and dentin interface: 11% P & B/TPH >6% Fuji >5% Dyract. The error rates method revealed no significant differences, in general, between the three materials with regard to gap formation. In conclusion, the restorations of erosion lesions with different classes of adhesive materials were well retained after 12 months. None of the materials studied revealed superiority over the other materials. All materials revealed shortcomings with respect to either surface texture, marginal integrity or color stability clinically and for all materials gap formation was recorded in the SEM evaluation.