The Preliminary Study On The Cell Biology Of Dental Pulp Mesenchymal Cells From Advanced Chronic Periodontitis

Obiective Isolate and culture the inflamed dental pulp mesenchymal cells(i DPCs).from extracted teeth of advanced chronic periodontitis patients in clinic.The control group is the dental pulp mesenchymal cells(DPCs)which derived from the healthy teeth because of impaction.The cell characteristics,including proliferation,the ability to keep activity seeding on biological materials,capability to form cell sheet,osteoblastic differentiation were all evaluated in vitro.This study is the preliminary study on the biological characteristics of DPCs from advanced chronic periodontitis.Methods Involving subjects were recruited from outpatient clinics of the advanced chronic periodontitis who accept tooth extraction.Healthy pulp tissues were collected from the healthy wisdom teeth extracted because of impaction.We isolate and culture the DPCs using the method of tissue block.After the success of the primary cell culture,subculture the cells by trypsin digestion according to the conventional and the P3-P4 cells were used in research.A series(0,10.0,20.0,and 40.0?g/ml)doses of vitamin C were added to the culture medium for studying the effects of vitamin C on cell proliferation.Approximately 1× 10~6/ml DPCs suspension were prepared and were seeded onto Bio-Oss scaffolds.The complex were incubated for 7 days before harvested for scanning electron microscope(SEM).Prior to the cell sheet culture,a series of response experiment was accomplished to test the optimal dose of vitamin C.Prepare the cell sheets using the optimal concentrations of vitamin C.Observed the cell sheets by hematoxylin and eosin(H&E)staining and scanning electron microscope(SEM).The cells undergoing osteogenic differentiation stimulation and then quantify mineralized nodule formation,take alkaline phosphatase(ALP)activity analysis,gene expression analysis respectively.Results A total of 25 subjects received tooth extraction due to severe chronic periodontitis were included,and 47 teeth were collected for cell culturing.Healthy wisdom teeth were collected from 3 patients used as a control.The success rate of cell culture is 34/47(72%)and 100% respectively.Primary cells generally migrated out of the dental pulp tissues derived of severe chronic periodontitis on day 7-14 and the control group was on day 3-5.There was no significant difference in cell proliferation observed between the two groups by MTT assay.The vitamin C shows a biphasic effect in a dose-dependent manner.The cells of 20.0 ?g/ml vitamin C groups demonstrated a significantly higher proliferative rate(P<0.05)while the cells of 40.0?g/ml vitamin C groups presented a lower proliferative rate(P<0.05).When the cells became subconfluent after 2–3 days in culture,the optimal concentrations of vitamin C was added to the complete medium for the duration of the culture time and successfully form cell sheets.The morphology of the whole cell sheet was observed under inverted microscope,(H&E)staining and SEM.The cells extended regularly and contacted each other tightly.There was no difference between cell sheets of H-DPSCs and i-DPSCs in terms of cell sheet structure.SEM micrographs revealed that dental pulp cells were exposed on the surface of Bio-Oss with good shape.Both groups of the DPCs exhibited osteogenic potential and produced extracellular mineral deposits that were positively stained with Alizarin Red S.The ALP activity analysis showed that DPCs from both groups showed increased mineralization over the time course of the experiment.q RT-PCR analysis was performed.m RNA for IL-1?,IL-6,IL-8 and TNF-? was detected in DPCs.A significantly higher level of IL-1?,IL-6,IL-8,and TNF-? m RNA was detected in the DPCs isolated from the teeth of chronic severe periodontitis than the normal teeth(P<0.001).q RT-PCR analysis further indicated increased expression of ALP,BMP2,OCN and RUNX2 in the h DPCs and i DPCs populations over the time course of osteogenic induction(P<0.05).The i DPCs showed lower expression of osteoblastic gene m RNA compared with the h DPCs during the whole time of osteogenic induction(P<0.05).Conclusion Our data demonstrate the existence of functional DPCs in the dental pulp of extremely loose teeth caused by chronic periodontal inflammation.There is no obvious change on the characteristics of proliferative ability,grown on biological materials,cell sheet formation that can be attributed to periodontal inflammation.Further,both of the DPCs isolated from periodontally affected teeth and normal teeth showed osteoblastic differentiation ability in vitro,however chronic periodontal inflammation down-regulated the ability to undergo osteoblastic differentiation.