Effect Of IGF-1 On Proliferation And Differentiation Of Human Dental Pulp Cells In Vitro And In Vivo

Objective: The aim of this study is to explore the human dental pulp cell' proliferation and differentiation under the action of insulin-like growth factor 1(IGF-1)in vitro and in vivo of nude mice.Methods: Healthy teeth which were extracted freshly for orthodontic reasons from young adults were collected.The tissue block and enzymatic digestion method was used to isolate and culture primary human dental pulp cells(HDPCs).Cells were detached with 0.25% trypsin when they reached 80% confluence and subcultured to forth generation.Only the cells from the forth generation were used in the further experiment.1 The effect of IGF-1 on proliferation of HDPCs in vitro: Cells were seeded four plates at a density of 2×10~4cells per well in a 96-well plate,according to 5 line×5 column.The cells were cultured 24 hours,then minimum essential medium(DMEM)were taken out.Each column is used as a group.The cells in experimental group were cultured with DMEM with 2% serum concentration and 50,75,100ngIGF-1/ml respectively.The cells in the control group were cultured with DMEM with 2% serum concentration.The culture medium was refreshed every 3 days.The cells were incubated for 1,3,5 and 7 days,and then proliferation of the cells was detected in vitro.As follows,the cells treated with 20?l of MTT for 4 hour,MTT solution was removed and replaced with 150 ?L DMSO.The absorbance was read at 490 nm with an automatic enzyme-linked immunosorbent assay reader.2 Proliferation and differentiation of human dental pulp cells in nude mice: 1)human dental pulp cells were seeded into ADM: We cut ADM into 1cm*1cm*1cm shape,then them were put into a 48-well plate and added 100 microlitre fetal bovine serum(FBS)in a biological safety cabinet.The 48-well plate was placed in the carbon dioxide incubator and the FBS was taken out after 24 hours.The cells suspension of the forth passage human dental pulp cells were prepared at a density of 2×10~5 cells per milliliter.The human dental pulp cells were dibbled on ADM,standed for a half an hour,then one milliliter mineralizing solution was added and cultured for three days.2)ADM-cells transplanted into nude mice: The four week old nude mice were selected and the cuts on the left and right back skin of the nude mice under the sterial condition were made.The wound is about one centimiter long.The ADM-cells complex was transplanted into the left back subcutaneous tissue of the nude mice and the wound was sutured.The left sides were used as the experimental sides.The ADM was transplanted into the right back subcutaneous tissue of nude mice and the wound was sutured.The right sides were used as the control sides.After these nude mice were breeded for two weeks,the half of these nude mice were executed with the methed of an overdose anesthetic,and another half of these nude mice were executed with the same method after four weeks.The transplants were removed immediately,partly was used for prepareing frozen sections and partely was put in 4% glutaraldehyde solution for ultrathin sections.HE staining,alizarin red staining and Masson trichromatic dyeing were made for these frozen sections,then were observed by optical microscope.These ultrathin sections were observed by transmission electron microscope.3)Statistics were calculated using SPSS 13.0.Results were expressed as the mean±standard deviation.Differences for statistical significances between groups were tested using OneWay ANOVA.Comparisons between means were assessed using S.N.K.P value of < 0.05 was considered to be significant.Results:1 Human dental pulp cells were isolated and cultured in vitro successfully by the method of tissue explant-collagenase digestion.Immunoc-ytochemistry staining showed that hDPCs are positive for vimentin and negative for cytokeratin.The cells are identified mesenchymal origin.2 The 50–100ng/ml of IGF-1 promotes proliferation of HDPCs.When cells were cultured 5 days or 7 days,IGF-1 at 50-100ng/ml enhanced proliferation of HDPCs compared with the control groups(P<0.05).IGF-1 at 100ng/ml presented the highest activity of proliferation among all groups(P<0.01).3 ADM-human dental pulp cells were transplanted into nude mice in vivo and cultivated the nude mice two weeks and four weeks respectively.The transplants were taken out and frozen sections preparated partly.The fibroblasts of the specimens were observed by HE staining,obvious mineralized nodules were found by alizarin red staining,and sight of blue collagen fibers were cought by Massion staining.4 The transplants were taken out after cultivating two weeks and four weeks separately and preparated ultrathin sections.The specimens showed many mitochondria,golgi complex in cells and a large number of collagen fibers.Conclusions:1 The HDPCs are successfully cultivated by the method of tissue explantcollagenase digestion.They are indentified as mesenchymal origin by immunohistochemistry staining.The study selected the forth passages HDPCs which had higher cell proliferation and well cell morphology.The forth passages HDPCs meet the requirement of the experiment.2 IGF-1 of 50-100ng/ml can stimulate the proliferation of HDPCs in a concentration-dependent manner.Furthermore,100ng/ml IGF-1 is the optimal concentration and is used for subsequent experiments.3 ADM-human dental pulp cells were cultivated in vivo of nude mice,then frozen section and ultrathin section were preparaed.The frozen section was observed by optical microscope,the ultrathin section was observed by transmission electron microscope,and confirmed IGF-1can promote human dental pulp cells' proliferation and differentiation in nude mice in vivo.