Bovine dental papilla-derived cells immortalized with HPV 18 E6/E7.
In vitro investigations of cell-specific metabolism and cell interactions as well as biocompatibility studies are often hampered by the limited lifespan of primary cells originating from target tissues like the oral mucosa, gingiva or pulp. Pulp cells, as do other primary cells, undergo senescence after several passages in vitro. However, senescence can be overcome by transfection of primary cells with oncogenes like the HPV 18 (human papillomavirus 18) E6/E7 oncogene, resulting in immortalized cell lines. The purpose of our study was to establish and preliminary characterize an immortalized bovine dental papilla-derived cell line by transfection with HPV 18 E6/E7 for future use in biocompatibility testing of dental materials. First passage dental papilla-derived cells from molar tooth germs of 6-month-old calves were transfected by electroporation with pUC18 LCR E6/E7 coding for the HPV 18 E6/E7 oncogene. Cells underwent crisis after 5 wk in culture. Distinct cell colonies arose after about 9 wk. Cells were cloned by single cell dilution in passage 15 and 17. Out of three cell clones maintained in culture, two cell clones showed cell death after 28 and 30 wk, respectively. One cell clone overcame a second crisis after 38 wk and was maintained in culture until passage 75. Stable gene expression of HPV 18 E6/E7 oncogenes was verified by polymerase chain reaction (PCR) and immunohistochemistry. Reverse transcription (RT)-PCR revealed that the established cell line (passage 50) expresses procollagen type I, alkaline phosphatase and osteocalcin. This suggests that the immortalization with HPV 18 E6/E7 results in a cell line, which maintained phenotypic characteristics of odontoblast-like cells.
Thonemann
B
B
Department of Operative Dentistry and Periodontology, University of Regensburg, Germany. birger.thonemann@klinik.uni-regensburg.de
Schmalz
G
G
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D002417:Cattle; D002461:Cell Line, Transformed / Q000378:metabolism; D003771:Dental Papilla / Q000166:cytology*; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D007150:Immunohistochemistry; D009857:Oncogenes; D015675:Osteocalcin / Q000096:biosynthesis; D027383:Papillomaviridae / Q000235:genetics*; D011347:Procollagen / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D011347:Procollagen; D015675:Osteocalcin; D000469:Alkaline Phosphatase