Biological Impacts Of SPIO On Two Kinds Of Stem Cells And Study Of Cell Migration Using Magnetic Targeting

Background and aimCaries and periodontal disease are common diseases in clinical practice,which often result in pulp necrosis and periodontal defect.However,current therapy is impossible to realize the physiological and functional regeneration of dental pulp and periodontal tissue in a real sense.Therefore,the dental pulp and periodontal tissue regeneration is still a hot-spot topic in current stomatology study.With the continuous development of tissue engineering,exogenous stem cells implantation and autologous stem cells homing therapy have been widely applied in many tissue and organ regeneration studies.However,because of the particularity and complexity of anatomical structure of tooth and periodontal tissue,it is hard to deliver sufficient stem cells to the pulp chamber or periodontal defect.Meanwhile,it is also difficult to tracing the implanted stem cells in vivo during therapy period.These difficulties have brought enormous challenge for the in-situ regeneration of dental pulp and periodontal tissue.The recently emerged Magnetic targeting strategy can significantly enhance the delivery and retention of transplanted therapeutic cells within a target organ.At the same time,the successful delivery can be confirmed using clinically applied MRI analysis.So,the two complement each other and indicate a new research direction for dental pulp and periodontal regeneration.In the first part of present study,we characterized the labeling and loading properties of MIRB on human dental pulp stem cells(h DPSCs).Meanwhile,the cell proliferation,apoptosis,and odonto-/osteogenic differentiation were qualitatively and quantitatively analyzed.Furthermore,we established a dental pulp ectopic regeneration model using h DPSCs cell sheet and immunodeficiency mice and then assessed the potential for imaging and monitoring of transplanted cell sheet by MRI in vivo.In the second and third part,we first determined whether biological functions(viability,proliferation,and differentiation)of rat bone marrow mesenchymal stem cells(r BMMSCs)are affected by SPIO-labeling in vitro.We then investigated whether the migration of SPIO-labeled r BMMSCs could be enhanced using a magnetic field in vitro.Next,in vivo studies were performed to determine the effects of magnetic force on the targeted delivery or retention of SPIO-labeled r BMMSCs to the periodontal defect zone in rat surgical periodontal defect model.Our study realized cell-tracking in vivo using MRI system and also enriched the application of Magnetic targeting in periodontal regeneration,which would provide some supports for using Magnetic targeting for dental pulp and periodontal regeneration in the future.The results are as follows:Part 1 The biological impacts of MIRB-labeling on h DPSCs and MRI imaging of MIRB-labeled h DPSCsin vitro and in vivo1 Isolation and culture of h DPSCsHDPSCs were obtained through single clone method from adult impacted teeth.The stem cell property was identified from detection of multi-lineage differentiation and cell surface antigen markers.2 MIRB h DPSCs loading characterizationHDPSCs were labeled by 12.5-100g Fe/m L SPIO(MIRB),Laser confocal microscopy,Prussian blue staining and Transmission electron microscope were separately performed to observe the localization of SPIO particles within the cytoplasm of h DPSCs,intracellular iron content was also detected using spectrophotometer.Results showed that h DPSCs could be efficiently labeled by MIRB without using any transfection agent,MIRB labeling had no effect on morphology of cells and intracellular iron content increased with the concentration of MIRB.3 Biological impacts of MIRB on h DPSCsTrypan blue exclusion experiments,CCK8,flow cytometry and osteogenic induction were carried out to detect the biological impacts of MIRB on cell viability,proliferation,stem cell surface antigen expression,cell cycle and apoptosis and osteogenic differentiation of h DPSCs.Results showed that 12.5-50g/m L MIRB had no side effect on cell viability and could enhance cell proliferation,but 100g/m L MIRB had toxic effect on h DPSCs.12.5g/m L MIRB could accelerate cell cycle and have no effect on cell apoptosis;12.5-50g/m L MIRB had no effect on osteogenic differentiation of h DPSCs.So the optimum labeling concentration was 12.5g/m L.4 MRI imaging of MIRB-labeled h DPSCs in vitro and in vivoMRI was used to detect h DPSCs cell pellet in vitro and cell sheet/root fragment complex in vivo.In vitro study showed that 1 × 106 cells labeled with various concentrations of MIRB(12.5g/m L–100g/m L)could be visualized and the signal intensity increased with increasing concentrations of MIRB.However,the low signal region of 1 ×105 cells labeled with 12.5g/m L was not quite obvious,but cell imaging in other groups could be easily identified.In vivo study showed that MIRB-labeled cell sheet showed low signal region and the area of low signal region became smaller with the time.Histological examination showed that the number of blue staining cells decreased with the time which was in accordance with the MRI result.Part 2 Biological impacts of SPIO(Resovist?)labeling on r BMMSCs1 Isolation and culture of r BMMSCsThe whole bone marrow adherent method was used to obtain rat bone marrow stem cells(r BMMSCs).Multi-lineage differentiation and cell surface antigen markers were detected to identify its stem cell property.2 Resovist r BMMSCs loading characterization25-100g/m L Resovist was used to label r BMMSCs.Prussian blue staining and Transmission electron microscope were separately performed to observe the localization of SPIO particles within the cytoplasm of r BMMSCs.3 Biological impacts of Resovist labeling on rBMMSCsTrypan blue exclusion experiments,CCK8,flow cytometry and osteogenic induction were carried out to detect the biological impacts of Resovist on cell viability,proliferation,cell cycle and apoptosis and osteogenic differentiation of r BMMSCs.Results showed that 25-50g/m L Resovist had no effect on cell viability but 100g/m L showed cytotoxicity;25g/m L had no effect on cell proliferation,cell cycle or apoptosis but 50-100g/m L Resovist could inhibit cell proliferation,cell cycle and promote cell apoptosis.These results indicated 25g/m L could be a proper concentration of Resovist on labeling r BMMSCs.Meanwhile,25g/m L Resovist could enhance osteogenic differentiation of r BMMSCs.Part 3 Study of the migration and engraftment of SPIO-labeled r BMMSCs in vitro and in vivo1 Study of the migration of SPIO-labeled r BMMSCs under magnetic field in vitroTo assess migration in vitro,Transwell assay showed that magnetic field could obviously promote the migration of SPIO-labeled r BMMSCs.Furthermore,after seeding the SPIO-labeled r BMMSCs into a dish at the outside bottom of which was adhered with a magnet,the migration was also observed and result showed that SPIO-labeled cells located around the magnet as a circle 12 h after seeding.2 Study of the migration or retention of SPIO-labeled r BMMSCs under magnetic field in vivoTo investigate targeted delivery of r BMMSCs in vivo,rat periodontal defect modal was made and then rats were separated into four groups: intravenous injection of SPIO-labeled r BMMSCs only(IN),intravenous infusion of SPIO-labeled r BMMSCs with magnet exposure(INM),local injection of SPIO-labeled r BMMSCs only(LO),local injection of SPIO-labeled r BMMSCs with magnet exposure(LOM).Twenty-four hours after periodontal defect was made,labeled r BMMSCs were injected intravenously or locally.Rats were sacrificed 3d,5d and 7d after cell injection and Prussian blue-positive r BMMSCs were measured in alveolar bone and viscera.Results showed that intravenously delivered SPIO-labeled r BMMSCs could not be targeted delivered in periodontal defect by magnetic field in vivo,most r BMMSCs were engulfed by splenic macrophages.But seven days after periodontal defect,LOM rats had a large number of Prussian blue-positive r BMMSCs accumulated around the periodontal defect zone.Conclusions1.The first part of this study showed that the newest commercially available SPIO(MIRB)can efficiently label h DPSCs without any transfection agent.12.5–50g/m L MIRB is a safe range for labeling h DPSCs.MIRB labeled h DPSCs cell can be visualized by MRI in vitro and in vivo.These data demonstrate that MIRB is a promising candidate for h DPSCs tracking in h DPSCs based dental pulp regeneration therapy.2.The second part of this study demonstrated that Resovist,produced by Schering Company,can also efficiently label r BMMSCs without any transfection agent.25g/m L SPIO(Resovist)did not influence the viability or proliferation of r BMMSCs,but 100g/m L SPIO obviously inhibited the viability and proliferation of r BMMSCs.Meanwhile,25g/m L SPIO labeling could improve the osteogenic differentiation of r BMMSCs in vitro.The mechanism needs further investigation in the future.3.The third part of this study showed that,magnetic migration could be simulated perfectly in magnetic field in vitro.Meanwhile,our study firstly brought magnetic targeting strategy into periodontal regeneration therapy.Results suggest that using magnetic field may be useful for improving the efficacy of retention and long term engraftment of stem cells in stem cell based therapy in periodontal regeneration.But long distance migration by magnetic targeting through intravenous injection did not get ideal results.Meanwhile,whether or not the transplanted stem cells within periodontal defect can proliferate,differentiate into new osteocytes and participate in alveolar bone regeneration and reconstruction still need further in-depth study in the future.