A Study On The Implantation Of Autologous Growth Factors And/or PDLFs To Canine Artificial Furcation Defects

Objective: To evaluate the effects of PRP on the proliferation of periodontal ligament fibroblasts(PDLFs) and the implantation of autologous growth factors and/or PDLFs to canine artificial furcation defects. Methods 1. The periodontal ligament tissues attached to the middle third of the root surface of the canine incisor teeth were gently curetted ,removed ,cut into small pieces,and plated onto 6-well plate with cover slip in place of the dry anchoring traditionally. When the cells surrounding the tissue explants were confluent.they were subcultured with 0.25% trypsin and 0.1%EDTA and transferred to a tissue culture flask .Cell morphology and growth pattern were observed. MTT colorimetric assay, Von Kossa staining were used to determine their biological characteristics. 2. Platelet-rich plasma was isolated by two-step centrifugation (lOOOrprm 15min; 3000rpm, 8min) from 10 adult dogs. Primary cell cultures of PDLFs were obtained from healthy third incision teeth.2 X 104cells/well were incubated in a 96-well plate in Alpha-MEM medium containing 15% calf serum for 24 hours. The medium was then removed and replaced with Alpha-MEM medium without CS and incubated for a further 48h to quiesce the cells. Then the medium was dicarded and cells were exposed to 5,10,20,30 and 50% PRP in 100ul Alpha-MEM medium for 24h. Control group was cells in serum-free Alpha-MEM medium .Cell proliferation analysis was performed utilizing MTT assay . 3. A total of 30 experimental class II furcation defects were surgically treated with either a combination of platelet-rich plasma/Bio-OSS/Guided tissue regeneration or GTR.The animals were sacrificed at 12 weeks postsurgery for histologic and histometric analysis. Results 1. The success rate of primary culture of canine PDLFs was upto about 89%. The PDLFs identified by positive stain for vimentin and negative for keratin can produce mineral-like nodules in vitro. 2. This two-step centrifugation method sequestered and concentrated platelets to a level over four times baseline whole blood . Cell proliferation was enhanced by addition of 5,10,20,30,50% PRP supernatant to the cell cultures in comparison to cells in basal medium.At the dose of 5,10,20,30% PRP promoted PDLFs growth in a dose-dependant manner. 3. The results of animal experimental showed that the PRP/Biooss/GTR, PDLFs/GTR, PRP/Biooss/PDLFs/GTR groups presented with significantly greater periodontal regeneration than the GTR group and there were no significant differences between them.The amount of new alveolar bone formation,new cementum formation,new periodontal ligament tissue formation in the GTR group is 0.52+/-0.21 mm,0.8+/-0.13 mm,1.9+/-0.10mm,respectively.The PRP/Bio-Oss/GTR group: 1.36+/-0.17 mm,1.92+/-0.18 mm,2.62+/-0.16mm;the PRP/Bio-Oss/cells/GTR group: 1.42+/-0.22 mm,2.07+/-0.19 mm,2.68+/-0.20mm;the cells/GTR group: 1.39+/-0.19 mm,1.82+/_ 0.16 mm,2.55+/-0.12mm. Conclusions A novel, convenient method to obtain PDLFs was established.PDLFs in this culture system kept their phenotype in vitro, indicating that they have potential as seed cells in periodontal tissue engineering. A convenient two-step centrifugation method of concentrating platelets was also established. PRP produced by this technique can augment PDLFs proliferation.Last, It was concluded that the implantation of PRP-BioOss and/or PDLFs combined with GTR is an effective modality of regenerative treatment for class II furcation defects.