Osteogenic Potential of Pulp Cells ex vivo and in vivo

Objectives: Dental pulp is considered a promising source of mesenchymal stem cells for tissue engineering and repair. The aim of this study was to investigate the osteogenic potential of human pulp cells upon ex vivo differentiation and in vivo application in an animal model of tibia fracture. Methods: Pulp cells from human immature impacted third molars were expanded in culture. For the ex vivo study, cells were treated with osteoinductive factors for 28 days and were weekly tested for calcium depositions and expression of dentinoblastic and osteoblastic markers. For the in vivo study, non-induced cultured pulp cells were applied directly to the area of a bone fracture in the tibia of adult male Wistar rats (n=6). Animals of the control group (n=5) did not receive any cells. Postorperatively, rats were followed radiographically and killed 8 weeks later. Tibia were paraformaldehyde fixed, decalcified, paraffin embedded, stained with heamatoxylene and eosin and tested for alkaline phosphatase activity. Results: Upon ex vivo induction, cells adopted an osteoblast-like phenotype over time; Alizarin red staining detected calcium depositions after three weeks of induction while the levels of osteogenic markers Runx2 and osteonectin increased from the 2^nd and 3^rd week, respectively. At the same time, the levels of dentin sialoprotein were gradually decreased. In the in vivo model, the fractures of the cell-treated group demonstrated a bigger and, radiographically and histologically, more homogenous callus formation. Cell-treated animals also showed a faster fracture union and less fibular nonunions compared to controls. Alkaline phosphatase activity was considerably increased in the callus formed in cell-treated animals. Conclusions: This study showed that pulp cells, used as a mixed population, can be induced to acquire an osteoblast-like phenotype and seem to accelerate the ossification and fracture union in an in vivo animal model.