A Preliminary Study Of Using RDFCs Cell Sheets Co-Cultured With RBMSCs In Three Dimensional Scaffold To Promote Periodontal Tissue Regeneration

Objective:This study was to discuss the feasibility that combined rDFCs cell sheets and co-cultured BMSCs’paracrine effect applied to periodontal tissue reconstruction.Methods:1. Isolation, culture and identification of rats’dental follicle cells and bone marrow mesenchymal stem cells2. Construction and assay of rDFCs cell sheets When rDFCs reached the 3rd passage, they were seeded into temperature-responsive culture dishes. And then, they were cultured with complete culture solution containing 80μg/ml ascorbic acid phosphate. After incubation for 2 weeks, the cell sheets were observed by inverted microscope and HE staining.3. Construction and assay of the complexes of rBMSCs and nHA-PLGARats’BMSCs, which was in the 3rd passage and in good condition, were seeded on nHA-PLGA. After incubation for 6 days, the complexes were observed by scanning electron microscope.4. Transplantation of rDFCs cell sheets and the complexesThe transplants were divided into three groups:group 1:dentin slice and nHA-PLGA; group 2:rDFCs cell sheets、dentin slice and nHA-PLGA; group 3:rDFCs cell sheets、dentin slice and the complex of BMSCs and nHA-PLGA. In group 2 and group 3, the nHA-PLGA materials were wrapped with rDFCs cell sheets, then bound together with dentin slice, and then transplanted those compounds into nude mice subcutaneously. Eight weeks after transplantation, the results were identified by Masson and HE staining.Results:1. Rats’dental follicle cells and bone marrow mesenchymal stem cells were obtained and identified successfully.2. The HE staining result of rDFCs cell sheets showed that they had a certain thickness, including two to five layers cells,and that they were rich in cell matrix secrtion.3. The scanning electron microscope results of the complex of rBMSCs and nHA-PLGA showed that rBMSCs can stick in the nHA-PLGA scaffolds materials to grow. So the nHA-PLGA scaffold material is a kind of good scaffold material that with excellent biocompatibility.4. The samples were collected after they were transplanted into nude mice subcutaneously 8 weeks later. The HE and Masson staining showed that in group 3, there were some periodontal ligament like tissue between dentin slice and nHA-PLGA and some bone-like tissue on the side of scaffold; that in group 2, there were also some periodontal ligament like tissue, but no bone like tissue; that in group 1, there were no periodontal ligament like and bone-like tissue.Conclusion:That using rDFCs cell sheet which were wrapping the complexes of rBMSCs and scaffold material can induce periodontal ligament like and bone like tissue. So rDFCs cell sheets, combined with rBMSCs’paracrine effect, which were co-cultured derectly with rBMSCs in 3D scaffold material, could enhance the capacity of periodontal tissue regeneration effectively. So it provided a new train of thought for periodontal tissue regeneration.