| To remain the activity of dental pulp is one important goal of endodontic biological research and therapeutics. By the development of some related subjects such as molecular biology, tissue engineering and cytobiology, the regeneration of dentin-pulp complex has shown great prospects.In this study, we examined the cell differentiation potential of cultured human dental pulp cells(hDPCs) grown in pulp cavity in vitro, and studied the effects of EDTA extracted dentin matrix proteins(EDMPs) on the proliferation and differentiation activity of hDPCs. We hope that through these studies, we can provide any experimental basis for the development of tissue-engineered pulp. Part I : Culture of human dental pulp cells in vitroMethods: Pulp tissues were gently separated from human premolars which were extracted due to orthodontic treatment(18～30 years old), then cultured by the combined use of explants technique and enzymatic separation method. Hematoxylin-eosin(HE) staining was used to observe the morphology of hDPCs after the cultured cells were fixed, cell origin was detected by immunocytochemical staining. To measure the cell growth curve, the cultured cells were counted for continuous 8 days with a hemacytometer.Results: Cultured hDPCs were fibroblast-like cells, the expression of Vimentin was positive while Keratin was negative. The cell growth curve was approximately "s" shape, logarithmic growth time was the 3～7th day after inoculation, the population doubling time(PDt) was 27.6 h.Conclusion: The cultured cells were identified as hDPCs, and grew stably in vitro, could be used for next step of our experiment.Part II: A study of growth and differentiation potential of human dental pulp cells grown in pulp cavity in vitroMethods: Extracted human non-caries posterior teeth were opened, followed by remove the predentin. Then the teeth were treated by 10% ethylene diamine triacetic acid(EDTA), 19% citric acid and 75% alcohol before hDPCs were inoculated into the pulp cavity. 2 weeks later, thesamples were fixed, decalcified, embedded, sliced, stained and thenobserved by microscope .Results: The dental pulp cells grew well on dentin surface and some ofthem exhibited 2 or 3 cytoplasmic processes extending into dentinaltubule, establishing an odontoblast-like morphology on dentin surface invitro.Conclusion: Our data demonstrated the differentiation of cells withodontoblastic morphology on existing dentin, suggesting that isolatedhDPCs could differentiate into odontoblasts on dentin in vitro.Part III: Effects of dentin matrix protein on the proliferation and differentiation activity of cultured humandental pulp cellsMethods: The well-grown hDPCs of 3～5 passages were made into single cell suspension and inoculated into 96 holes plates, culture medium with EDMPs, mineralized condition medium and common culture medium were separately added in to the plates. The proliferation of hDPCs was measured with methyl-thiazolyl-tetrazolium(MTT) assay for continuous 8 days. Another single cell suspension was inoculated into 25 mL culture flasks, culture medium with 1 ug/mL EDMPs or 10 ug/mL EDMPs, mineralized condition medium and common culture medium were separately added in to the culture flasks. After 3 weeks, the formation of mineralized nodules was detected with alizarin red stain.Results: The results of MTT assay showed that the optical density value of EDMPs group was the highest, and there was significant difference among the three groups. The results of mineralization induction showed that at day 12, mineralized nodules had formed in groups of 1 ug/mL EDMPs and 10 ug/mL EDMPs. After 3 weeks, alizarin red staining showed that groups of 1 ug/mL EDMPs, 10 ug/mL EDMPs had formed larger red mineralized nodules.Conclusion: EDMPs had significant influence on the proliferation and differentiation of hDPCs, and would play an important role in dental tissue engineering.
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