| Up-to-date dentists still face a challenge saving patients’ pulp without root canal therapy when treating pulp disease.In the tooth, there are four major components: enamel, cementum, dentin, and pulp. The rigid dentin provides powerful protection for dental pulp and support for enamel. Caries, trauma, abrasion and other damage can cause tooth hard tissue defects and pulpitis. One of the main reaction of odontoblasts on dental pulp damage is to form tertiary dentin and then to protect the pulp from further damage. Therefore, how to promote the formation of tertiary dentin and save the vitality of pulp is an important scientific problems.The tertiary dentin formation include the migration of dental pulp stem cell, the proliferation and differentiation of the odontoblasts, and the secretion of dentin matrix. The differentiation of dental pulp stem cells to the odontoblasts is an important process in reparative dentine formation. A variety of factors, cell factors and signaling pathways involve in and control the process. Growth factors can promote the formation of tertiary dentin by promoting the proliferation, differentiation and function of odontoblasts. Studies have showed that VEGF can increase the Runx2 protein by preventing Runx2 protein degradation contributing to Dspp expression.VEGF, the best-characterized angiogenic factor, with endothelial cell specific mitogen activity,can promote the angiogenesis and increase the permeability of vessels in vivo. Therefore, the application of VEGF may contribute to the tertiary dentin formation by promoting the proliferation and differentiation of odontoblasts and then protect the pulp. Bone morphogenetic protein 2(BMP2), belongs to the TGF-β superfamily of proteins. BMP2 is an autocrine secretory protein. BMP2 can promote odontoblastic differentiation and reparative dentin formation through the Smad 1/5, p38 a MAPK and other signaling pathways. Odontoblasts can produce and secrete BMP2 which directly affects the secreting and the adjacent odontoblasts to stimulate proliferation and differentiation of odontoblasts, resulting in dentin formation. Studies have been done to form the tertiary dentin by promoting BMP2 protein expression or applying exogenous BMP2 protein.But BMP2 protein is expensive. The application of viral vector carrying BMP2 gene may promote the functions of odontoblasts by BMP signaling pathways and form the tertiary dentin to repair the pulp damage.For the role of tertiary dentin in pulp disease treatment, according to the effects of VEGF and BMP2 on dental pulp stem cells and the proliferation and differentiation of odontoblasts, based on the osteogenesis study of adenovirus mediated VEGF and BMP2, our research was proposed. This will build the foundation treatment of tooth pulp disease for clinical application of VEGF and BMP2.First of all, we isolated and cultured dental pulp cells from normal human exfoliated deciduous incisors(h DPCs). The third generations of dental pulp cells were used in all experiments. The h DPCs were cultured for 5 weeks in an adipogenic induction media containing 0.5 m M isobutylmethylxanthine, 1 μM dexamethasone, 10 mg/L insulin, and 200 μM indomethacin and then stained with Oil Red O. The positive lipid droplet could be observed. In the mineralized induced medium, the cells also could be observed positive by alizarin red staining. This showed the cultured dental pulp cells contained stem cells and had ability of differentiation.The third passage cells were transduced with recombinant adenovirus vector encoded with enhanced green fluorescent protein(EGFP), Ad CMV-EGFP, at 0, 400, 800 or 1000 particles/cell. The transduced efficiency of adenoviral vector was increased from 0 particle/cell to 1000 particles/cell without affecting the proliferation of h DPCs. Considering the toxicity of adenovirus, the h DPCs were transduced with Ad CMV-EGFP at 1000 particles/cell. About 70% of h DPCs were EGFP-positive on day 3. Finally, VEGF and BMP2 expression was increased after the h DPCs were transduced with Ad CMV-h VEGF at 1000 particles/cell. This indicates that adenoviral vector can be used to transduce h VEGF and h BMP2 into these cells.After transduced with adenoviral vector at 1000 particles/cell and cultured in the mineralized induced medium(containing 10 m M/L sodium β-glycerol phosphate, 50 mg/L L-ascorbic acid, and 10-8 M/L dexamethasone), the comparison of the effects of h VEGF or h BMP2 on h DPCs with EGFP was conducted in a series of cellular and molecular aspects,respectively. Data demonstrated that calcium deposition occurred in the h DPCs, whereas the Ad CMV-h VEGF or Ad CMV-h BMP2 treated group had significantly more Alizarin red S positive cells or mineralized nodule on days 14 and 28 compared to the Ad CMV-EGFP control group. The experimental group also increased ALP activity after 7 days post-culture(P<0.05). RT-QPCR assays show that h VEGF or h BMP2 significantly increased the gene expressions of osteogenic/odontogenic gene markers compared to Ad CMV-EGFP group(P<0.05). The Ad CMV-h VEGF group increased on days 7 is more significant, while Ad CMV-h BMP2 is more significant on days 14.We prepared a rat model to evaluate the tertiary dentin in vivo. The formation of tertiary dentin was observed using histological and immunohistochemical methods after Ad CMV-h VEGF or Ad CMV-h BMP2 adenovirus carrier applied on the surface of prepared cavities. Data from in vivo assays indicated that h VEGF and h BMP2 enhanced pulp cell proliferation and neovascularization,and dramatically increased formation of tertiary dentin in dental pulp.The innovation of this research is to study the effects of h VEGF and h BMP2 transduction mediated by adenovirus vector on the formation of tertiary dentin and the dentinogenic differentiation of h DPCs. We study the feasibility of gene transduction into h DPCs with adenovirus vector and the influence of h VEGF or h BMP2 transduction on the function of h DPCs. Our in vitro and in vivo data suggest that Ad CMV-h VEGF and Ad CMV-h BMP2 have potential clinical application and provides solid evidence to allow us to move to the next level in treating dental pulpitis. |
