The Effects Of Substance P On The Biological Characteristics Of Cultured Human Dental Pulp Cells In Vitro

With the development of society, the accidental exposure of dental pulp isincreasing gradually, the effects of traditional pulp capping are oftenunsatisfactory, in recent years, more and more scholars focus on the dentalpulp cells, in order to providing an experimental basis for clinical researchfrom the cellular level.Objective: The aim of the experiment is to use the method of tissueexplant-collagenase digestion to obtain dental pulp cells which maintained thestem cell characteristics, to study the effects of substance P on proliferationactivity, differentiation activity and the ultrastructure of human dental pulpcells (HDPCs) in vitro, to investigate the effects and the action mechanism ofsubstance P on biological behaviour of HDPCs, and to provide a new way ofprotecting dental pulp in clinical.Methods:1The primary culture and identification of HDPCsPulp samples were obtained from healthy，caries-free impacted thirdmolars or premolar for orthodontic purposes，after cleaning the teeth surfacethe pulp tissue was removed under the sterile conditions and culturedaccording to the tissue explant-collagenase digestion method. The culturedhuman dental pulp cells were passaged in situ for the firsrt time，and thenwhen the cells reached80%of the culture wells’ area, they were passaged at1:2or1:3split ratio. The third generation human dental pulp cells wereinoculated in slides and stained by immunohistochemistry and HE to identifythe cell source and cell morphology by microscopy.2The detection of HDPCs’ growth curveGeneration3HDPCs were inoculated in24-well plates at2×104cells/well, then5wells were trypsinized random for cell count everyday since the second day after inoculation, averaged and lasted8days. The HDPCs’ growthcurve was based on the average.3The effects of substance P on the proliferation activity of HDPCsGeneration4HDPCs in logarithmic phase were trypsinized and madeinto cell suspension. Then the cell suspension was planted in five96-wellmicroplates with2×104cells/ml and200μl for each well. The supernatant fluidwas abandoned after24h, the cells were divided into5experimental groupsand1control group, and a blank control group was added. The experimentalgroup:200μl substance P was added (whose terminal concentration was10-5,10-6,10-7,10-8,10-9mol/L respectively). Substance P of the terminalconcentrations was made with DMEM containing1%FBS. The control group:only200μl DMEM containing1%FBS was added. At1、2、3、4and5d theeffects of substance P on HDPCs’ proliferation were detected by CCK-8method, the OD values （A450）were tested by microplate reader in450nm.4The effects of substance P on the differentiation activity of HDPCs4.1The effects of substance P on the alkaline phosphates(ALP)activity ofHDPCsGrouping and culture conditions as above, at1、3、5and7d took out of a96-well microplate, abandoned supernatant fluid and cleaned3times by PBSbuffer, then blotted the PBS buffer, added50μl0.1%TritonX-100, and took itinto4℃refrigerator overnight at last. If no complete cell structure wasobserved, added alkaline phosphatase substrate under the instructions of thealkaline phosphatase kit,37℃for30min. Finally, added0.2mol/L NaOHinto each well to stop the reaction and tested the OD values（A410） bymicroplate reader in410nm.4.2The effects of substance P on the dentin sialophosphoprotein(DSPP）mRNA of HDPCsGeneration4HDPCs in logarithmic phase were trypsinized and madeinto cell suspension. Then the cell suspension was planted in six culture flaskswith104cells/ml and3ml for each culture flask. The six culture flasks weredivided into3control groups and3experimental groups. The control group was added3ml DMEM containing1%FBS. The experimental group wasadded3ml substance P of10-7mol/L, which was made with DMEM containing1%FBS. The effects of substance P on HDPCs’ DSPP mRNA were tested:took out of a control culture flask and a experimental culture flask at7d、14dand21d respectively, abandoned supernatant fluid and cleaned3times by PBSbuffer，added1ml Trizol to make the cells completely lysed, got the total RNAaccording to the instructions of kit, detected DSPP mRNA by RT-PCR. Theprocedure was repeated three times.5The effects of substance P on the ultrastructure of HDPCsGeneration4HDPCs in logarithmic phase were trypsinized and madeinto cell suspension. Then the cell suspension was planted in six culture flaskswith2×104cells/ml and3ml for each culture flask. The six culture flasks weredivided into3control groups and3experimental groups.3ml DMEMcontaining1%FBS was added in the control group.3ml substance P of10-7mol/L was added in the experimental group, which was made with DMEMcontaining1%FBS. After48h of culture, cells in both groups were washed,fixed2hours by2.5%glutaraldehyde, dealt with normal method for TEMobservation.6Statistical analysisAll data were statistically analyzed utilizing SPSS13.0software byone-way analysis of variance, and S-N-K test for comparison between twogroups. All data were expressed as mean±se. A probability value of P <0.05was considered statistically significant.Results:1Biological characteristics of human dental pulp cells cultured in vitroIt can be successfully cultivated fibroblast-like cells by the method oftissue explant-collagenase digestion, the cells which were round or ellipticalnucleus with clear nucleolus were observed, the cell body was plump and fullof abundant cytoplasm. The HDPCs’ growth curve was “S” style. The culturedcells showed negative staining for waveform fibroin antibody and positivestaining for cytokeratin antibody. It met the requirements of the experiment. 2The effects of substance P on the proliferation activity anddifferentiation activity of HDPCsSubstance P in the concentration of10-9mol/L~10-5mol/L couldstimulate the growth proliferation activity （P＜0.05）, the optimalconcentration was10-6mol/L; raise ALP activity（P＜0.05）, the optimalconcentration was10-7mol/L; RT-PCR results showed that at7d、14d and21d,the experimental group express more DSPP mRNA than the control group.3The effects of substance P on the ultrastructure of HDPCsTEM results revealed that after cultured with substance P the roughendoplasmic reticulum expanded significantly and there were moremitochondrion comparing with the control group. It can be proved thatsubstance P could enhance the synthesis and secretion function of HDPCs.Conclusion:1The cultured HDPCs by the method of tissue explant-collagenasedigestion have the characteristics of stem cells, they have good proliferationand differentiation ability. It can provide a reliable cell source for the study ofdental pulp.2The substance P could stimulate the proliferation and differentiationactivity and enhance the synthesis and secretion function of HDPCs. It can beproved that substance P plays a positive role in the regulation of HDPCs’proliferation and differentiation process. When the dental pulp was damaged,the repair and healing process may depend on substance P.