Construction Of Dental Follicle Stem Cell Sheet And Its Biological Characteristics In Beagle Dogs

Periodontal disease, acute or chronic, can lead to the destruction of tooth-supporting structures of the periodontium such as alveolar bone, cementum, the periodontal ligament, and gingiva. Thus, the regeneration of alveolar bone becomes a ultimate goal for clinicians to repair periodontal defects.For various regenerative procedures of periodontium, tested and evaluated in the last two decades, the desired results have not being achieved, including surgical procedures, occlusive barrier membranes, a variety of bone grafts, and osteoconductive bio materials.However, these conventional therapies for periodontal tissue regeneration have showed limited outcomes. With the rapid development of cell sheet technology, the cell sheet has become one of the hotspots of tissue engineering, bringing new vitality and energy for the management of periodontal defects.Dental follicle tissues are composed of progenitor cells, capable to form osteoblasts of the alveolar bone, periodontal ligament-fibroblasts and cementoblast. It is known that dental follicle stem cells (DFSCs) isolated from dental follicle are capable of differentiation toward osteoblasts and establish the alveolar bone. However, dental follicle stem cells (DFSCs) are an important candidate source to treat critical-size periodontal detects, with the addedadvantage of being easily harvested from extracted impacted human wisdomteeth.Objective： To identify cell surface CD marker and verify the ability ofproliferation and differentiation of dental follicle stem cells (DFSCs) in beagledog which were isolated from the canine dental germ extracted. And to constructdental follicle cell sheet and examine the biological characteristics of DFSCssheet.Methods：DFSCs were obtained by collagenase Ⅰ digestion of beagle dogcanine dental germ. The3rd passage DFSCs were applied to do the experiments:Colony-forming, flow cytometry, et al. After identification, DFSCs weresubcultured to construct DFSCs sheet. Cell sheet was observed by invertedmicroscope, HE staining and scanning electron microscope (SEM). Afterincubation for10days, DFSCs sheets were induced in adipogenic inducingmedium and in osteogenic medium for14days separately. Oil red staining andAlizarin red staining was applied to examine adipogenic and osteogenicinduction.Results：DFSCs are mesenchymal cells with typical spindle shape morphologyand these DFSCs were the dominant cell population at the3rd in vitro passage.Colony-forming assay showed about5.1%DFSCs colony formation. DFSCswere positive for CD29and CD44, but were negative for CD34. MTT manifestedthe growth and proliferation was good. Cell cycle testing resulted: G1=87.1%,G2=5.54%. DFSCs sheets were constructed successfully. Under invertedmicroscope, they were found that DFSCs grew in multilayer. By scanningelectron microscopy, they can be seen that DFSCs expanded adequately andextracellular matrix (ECM) was clear and numerous. Oil red staining and alizarin red staining both demonstrated positive reactions in DFSCs sheet after induction.Conclusion：It suggested that DFSCs cell sheet may be constructed successfullyand has a strong bone-forming ability. It provides us a good opportunity thatDFSCs cell sheet has a potential to regenerate the periodontal defect.