| Objective: Porphyromonas endodontalis(P.endodontalis)is a core pathogen in the infected dental pulp and periapical tissues.Pulpal and periapical diseases affect the continuous root development in immature permanent teeth.As an essential source of odontoblasts,stem cells from the apical papilla(SCAP)form the physiological basis for root formation and regeneration.Therefore,vital SCAP are important for maintaining the growth and development of immature permanent teeth after bacterial infection of the root canal system.In this study,P.endodontalis was co-cultured with SCAP to construct an in vitro model.This study aimed to observe the effect of P.endodontalis on the proliferation,apoptosis,and cell ultrastructure of SCAP,and to preliminarily explore the biological mechanism by which the roots of young permanent teeth retain their developmental potential after pulp and periapical tissue infection,providing an experimental and theoretical basis for vital pulp therapy in young permanent teeth with pulp inflammation.Methods:1.SCAP of primary teeth were isolated and extracted using enzymatic digestion.Osteogenic and adipogenic differentiation experiments were conducted to determine the pluripotency of SCAP.The expression of mesenchymal and hematopoietic stemcell surface markers was detected using flow cytometry.2.The international standard strain P endodontalis ATCC 35406 was cultivated under anaerobic conditions.After Gram staining,the bacteria were observed and photographed under the microscope.3.P endodontalis and SCAP or VECs were co-cultured in a cell incubator to construct viable bacterial infection cell models with different multiplicities of infection(MOI)(0,10,100,and 500)and infection durations(3,6,12,and 24 h).With normal cultured cells as control,the cell proliferation under different MOI(0,10,and 100)and infection durations(3,6,12,and 24 h)was determined using the MTT and Ki-67 assay.Compare the relative proliferation rate of SCAP and VECs infected by P.endodontalis(MOI = 100).4.After SCAP and VECs were infected with P.endodontalis(MOI = 100)for six hours,the apoptosis rate was determined using flow cytometry.5.Transmission electron microscopy was used to observe the intracellular ultrastructure and bacterial status inside and outside the cells after six hours of incubation with P.endodontalis(MOI = 100).6.The experimental data were analyzed using SPSS version 22.0.One-way analysis of variance was used for intragroup comparisons.The LSD-t test was used for intergroup comparisons between the two samples.Statistical significance was set at P< 0.05.Results:1.Cultured primary-tooth SCAP appeared as fusiform fibroblasts.Mineralized nodules were observed after four weeks of osteogenesis induction,and Alizarin red S staining was positive.The formation of lipid droplets was observed four weeks after lipogenic induction in vitro,and Oil Red O staining was positive.The mesenchymal stem-cell surface markers CD73,CD90,and CD105 were expressed,whereas hematopoietic stem-cell surface markers CD31,CD34,and CD45 were not expressed.2.P.endodontalis ATCC 35406 produced smooth black colonies after resuscitation and red rods after Gram staining.3.MTT assay results showed that compared with SCAP under normal culture,pretreatment with P.endodontalis(MOI = 10)for 3,6,12,and 24 h had no significant effect on the proliferation of SCAP(P > 0.05).At MOI = 100,compared with SCAP under normal culture,pretreatment with P.endodontalis for 3 or 6 h had no significant effect on the proliferation of SCAP(P > 0.05).After 12 or 24 h of pretreatment with P.endodontalis,the proliferation ability of SCAP decreased,and the difference was statistically significant(P < 0.05).The relative proliferation rate of SCAP was significantly higher than that of VECs at 1 d,3 d and 5 d after pretreatment with P.endodontalis(MOI = 100)for 6 h,and the difference were statistically significant(1d : t =-4.452,P = 0.011;3 d : t =-4.276,P = 0.013;5 d : t =-3.312,P = 0.030).Ki-67 assay results showed that the proportion of Ki-67 stained positive SCAP was significantly higher than that of Ki-67 stained positive VECs,after pretreatment with P.endodontalis(MOI = 100)for 6 h,and the difference were statistically significant(t= 4.014,P = 0.016).4.Flow cytometry results showed that the early apoptosis rate of SCAP was lower than that of VECs after P.endodontalis infection for 6 h,and the difference was statistically significant(t =-3.922,P = 0.017),and the late apoptosis rate of SCAP was also lower than that of VECs after P.endodontalis infection for 6 h,and the difference was statistically significant(t =-3.533,P = 0.024).5.Transmission electron microscopy,revealed that after six hours of P.endodontalis infection(MOI = 100),the cell morphology of SCAP did not change significantly.In most cells,the P.endodontalis was in a single free or multiple cell aggregation state,the capsule was broken,and seemed to be dissolved and destroyed,and some bacteria were surrounded by vesicles.Most mitochondria in SCAP showed a complete structure;few mitochondria in the local internal ridge structure were blurred and slightly swollen.After six hours of P.endodontalis infection(MOI = 100),the cell morphology of VECs did not change significantly.However,in the VECs group infected with P.endodontalis,bacteria could be seen in individual cells,the cell structure was incomplete,most mitochondria in the cells were damaged or dissolved,and morphological swelling was obvious,suggesting that VECs showed more serious mitochondrial damage than SCAP.Conclusions: In vitro,the short-time pretreatment(3 h or 6 h)with P.endodontalis(multiplicity of infection was 100)had no significant effect on the proliferation rate of SCAP,while long-time pretreatment(12 h or 24 h)with P.endodontalis decreased the proliferation rate of SCAP.After six hours of P.endodontalis infection,SCAP showed stronger cell proliferation capacity and resistance to apoptosis,and less serious mitochondrial damage.The results of this in vitro study suggest that SCAP have better cell viability under a certain degree and period of bacterial infection in the root canal system,which might be an important biological basis for physiological root development after pulp-preservation treatment in young permanent teeth with pulpal inflammation. |
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