Construction Of Periodontal Supportive Tissues By Dental Pulp Stem Cells And PGA Scaffolds On TDM

ObjectiveIn this study,the canine dental pulp stem cells(cDPSCs)and periodontal ligament stem cells(cPDLSCs)were isolated,cultured,identified and compared by the stem cells characteristics in vitro.Both cells were combined with PGA scaffolds and TDM respectively,then ectopically transplanted into the kidney capsule of beagle dogs.The formation of periodontal tissue-like structure around TDM was observed and compared.Methods1.cDPSCs and cPDLSCs were isolated and cultured by enzyme digestion.The cell morphology was observed by inverted microscope.The cell clone formation rate and CCK8 assay were used to evaluate the proliferation ability of them.Cell sources and surface markers were identified by cell immunocytochemistry and flow cytometry.Osteogenic,adipogenic and chondrogenic induction potentials of them were test.2.cDPSCs and cPDLSCs were cultured into the complex of PGA scaffolds and TDM respectively.After 2 weeks of static mechanical stimulation,the biocompatibility of cell-scaffolds were observed under inverted microscope.3.cDPSCs were transfected with lentivirus-mediated green fluorescent protein(GFP),the morphology,growth and expression intensity of GFP were observed under inverted fluorescence.Similar to method 2,cDPSCs were inoculated into the PGA scaffolds and TDM for 2 weeks of static stress.4.2 weeks later,the cDPSCs-PGA-TDM,cPDLSCs-PGA-TDM,PGA-TDM(control group)and cDPSCs-GFP-PGA-TDM were ectopically transplanted into the kidney capsule of beagle dogs.After 8 weeks transplantation,samples were harvested.Routine fixation,decalcification,embedding and sectioning were performed.HE and Azan staining was used to observe the formation of periodontal tissues around TDM.We analyzed the protein expression of CEMP1,COL-I,and PN using immunohistochemistry to determine whether the tissue surrounding TDM was cementum/periodontal ligament-like structures.Immunofluorescence GFP further detected whether the tissue derived from our implanted cells.Results1.The primary cultures of cDPSCs and cPDLSCs were clone-like growth.Most of them were short spindle and had a small volume.However,the growth rate of cPDLSCs was slower than that of cDPSCs,so as longer culture period and relatively fewer colonies.The colony formation rate showed that cDPSCs(7.3±1.2)% wassignificantly higher than that of the cPDLSCs(3.4±0.4)%(P<0.05).Moreover,the colony area formed by cDPSCs was larger and the number of cells was more.The proliferation curve of them were in accordance with the logarithmic growth curve,showing a clear " S " shape,and the proliferation of cDPSCs was stronger than that of cPDLSCs.Immunocytochemistry showed that both cells positively expressed Vimentin,but not Keratin.At the same time,they were positive for STRO-1,CD146,CD105,CD90,while negative for CD45.Both cDPSCs and cPDLSCs had multipotent capability.Quantitative analysis of osteogenesis showed that the formation of mineralized nodules in cPDLSCs(2.62±0.06)was higher than that in cDPSCs(2.11±0.07),indicating that cPDLSCs had greater osteogenic differentiation potential than cDPSCs(P<0.05).2.The cells were seeded on PGA-TDM composite scaffolds for a week.Under inverted microscope,there were more cells attachment,multiple growth and growth well on the edge of TDM.The cells adhered well to the PGA scaffold and stretched along the PGA fibers,secreting abundant extracellular matrix that formed a network-like structure.3.cDPSCs were transfected with lentivirus-mediated GFP for 72 h.Under inverted fluorescence microscope,the GFP positive rate was approximately70 %-80 %,the cell morphology and proliferation capacity were not obviously changed.4.After 8 weeks transplantation of kidney capsule,histological observation showed that uneven mineral deposits,parallel / oblique fibrous tissues were generated in both cDPSCs and cPDLSCs groups,similar to cementum/periodontal ligament-like structures.While a thin and loose fibrous tissue was found in the control group.Positive staining of CEMP1,COL-I and PN in the tissues surrounding TDM were detected by immunohistochemistry,indicating that cementum/periodontal ligament-like tissue could be formed in both cDPSCs and cPDLSCs groups.Immunoflurescence GFP detected that the expression of tissue was positive,which proved that the surrounding tissue derived from our implanted cells.Conclusion1.cDPSCs and cPDLSCs isolated and cultured in this study possessed the characteristics of mesenchymal stem cell.2.The PGA-TDM composite scaffolds had good biocompatibility for the inoculated cells.3.In the ectopic periodontal tissue regeneration model of beagle dog,both cDPSCs and cPDLSCs can form obvious cementum/periodontal ligament-like structures.