Confocal Microscope Analysis Of Human Dpmc Behavior During Osteogenesis

Objectives: The aim of this study was to investigate the in vitro behaviour of osteoblast-like cells derived obtained from human dental papilla mesenchymal cells (DPMCs) after in vitro osteogenic stimulation.
Methods: Dental papillae were obtained from impacted third molars of healthy young patients. Mesenchymal stem cells were isolated from dental papillae by tissue digestion. Then, cells were sorted by flow cytometer using monoclonal antibody for CD29, CD90, CD146 and CD166, FITC- or PE-marked. DPMCs were cultured in αMEM and at the third passage a quantity of 1,5 x106 cells were cultured in osteogenic medium for 3 weeks. Cells were investigated by RT-PCR and Western Blot for osteogenic markers. Light microscope, after Alizarin Red staining, and confocal microscope analyses were performed at established times to appreciate extracellular matrix proliferation and mineralization.

Results: At confocal microscope evaluation, differentiated cells seemed to have a typical osteoblastic cell morphostructure with remarkable biologic activity, as demonstrated by intracytoplasmatic markers for mRNA. Moreover, the presence of mineralized nodules were detected by Alizarin red staining, attesting the production of mineralized extracellular matrix by these cells after osteogenic differentiation.

Conclusions: Dental papillae could be considered a useful source of undifferentiated mesenchymal cells. DPMCs are demonstrated to be able to in vitro differentiate into osteoblastic-like cells and to produce a thick mineralized net, indicating they could be successful used in bone tissue engineering.