Construction Of Canine Periosteal Cells Sheet In Vitro

PartⅠ:In vitro multidirectional differentiation potential of periosteal cells Objective To culture canine periosteal cells in vitro and evaluate it’smulti-differentiations potency.Methods Periosteal cells(PCs)were obtained from Beagle dogs’ periosteum explant cultures,and the ability of cell cloning was detected by crystal violet staining.Alkaline phosphatase(ALP)staining,Alizarin red S(ARS)staining,Oil red O staining and Alcian blue staining were performed to detect the osteogenic,adipogenic and chondrogenic differentiation of PCs.Results PCs were successfully isolated and cultured with the manifestation that the single cell grew in clony.ALP staining of PCs was obvious after culturing in osteogenic differentiation medium for 7 days.After 21 days’ osteogenic induction,PCs demonstrated mineralized nodules in the culture,as examined by ARS staining.Oil red O positive lipid droplet could be observed after adipogenic induction for 21 days.After 21 days’ chondrogenic induction,alcian blue staining were positive.Conclusions PCs can be effectively cultured from periosteum explant cultures and have the potential of multi-directional differentiation.Part Ⅱ: Construction of periosteal cell sheet in vitroObjective To construct canine periosteal cell sheet in vitro and observe it’s microstructure.Methods PCs sheets were constructed by adding vitamin C to the culture medium.HE and toluidine blue staining,scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were employed for morphological and ultrastructure observation of the sheets.Results PCs sheets were successfully constructed with the appearance of milky white and translucent film-like structure.HE and Masson staining showed PCs sheets contained multiple layers of cells,secreted a rich extracellular matrix(ECM)and formed a tight network of collagen fibers.SEM and TEM examination revealed that vitamin C induced PCs sheets preserved intercellular junctions with ECM.Conclusions Vitamin C can induce PCs to construct cell sheet.PCs sheet is made up of vigorous cells and endogenous ECM,which is expected to be used in periodontal tissue engineerig.Part Ⅲ:Allogeneic transplantation of periosteal cells sheets in a canine model of2-wall periodontal defectsObjective To evaluate the regenerative potential of the cell sheet therpy combining allogenic PCs sheet with in β-tricalcium phosphate(β-TCP)on periodontal wound healing in intrabony defects in canine.Methods Periodontal two-wall intrabony defects(3mm×3mm×5mm)were surgically created on mesial side of mandibular second,third and fourth premolars bilaterally in 5 beagle dogs.The 30 defects were randomly assigned to one of the following treatments:group A,PC sheet bound to β-TCP+collagen membrane;group B,β-TCP+collagen membrane;group C,non-grafted.Record the probing depth,clinical attachment level and positive rate of bleeding on probing at the time of normal condition,pre-transplantation surgery(one month after experimental periodontitis induced).At 8 weeks after surgery,the animals were euthanized for micro CT and histologic evaluation.Result The Histological images showed that new periodontal attachment could be obtained in both group A and B.However,more complete periodontal regeneration including periodontal ligament-cementum complex structure and alveolar bone was observed in group A than group B.Conclusions These fndings indicate that PC sheet combined with β-TCP could promote periodontal regeneration in a canine model of 2-wall periodontal defects.