A Study Of Human Osteoprotegrin Gene Modified Tissue-engineered Compound On Healing Of Autologous Periodontal Tissue Defects

IntroductionChronic periodontal disease is an infection disease that affects the supporting tissuesurrounding the tooth, which includes periodontal ligament, alveolar bone andcementum. Traditional periodontal treatment methods such as scaling and rootplanning can only arrest disease progress; however the loss of supporting tissue couldhardly be recovered.Osteoprotegerin (OPG), a member of the tumour necrosis factor (TNF) receptorsuperfamily, contributes determinatively to the bone remodelling by inhibiting therecruitment, proliferation and activation of osteoclasts. The aim of the study was toexplore whether autologous implantation of human OPG(hOPG) gene modifiedperiodontal ligament cells(PDLCs)-collagen membrane complex could enhance tissuerepair in the experimental class Ⅱ furcation defects in beagle dogs.Methods and resultsFirstly, we established optimal culture condition for PDLCs.Human PDLCs (hPDLCs) were cultured in α-MEM,DMEM-low glucose andRPMI1640medium respectively in vitro with the modified tissue block culturemethod. Increased proliferation was observed in α-MEM cultured cells and increaseddifferentiation was found in DMEM-low glucose cultured cells.Secondly, we evaluate hOPG gene tranfection efficiency.PDLCs of beagle dogs were isolated and cultured as above, and the cultured cellsshowed characteristics of mesoderm by immunocytochemistry. Then, adenovirusvector carried hOPG gene was structured in vitro and transfected into canine PDLCs.More than80per cent transfected cells positively expressed the green fluoresceneprotein under fluorescence microscope and by flow cytometry. Effective expression of hOPG was also observed by real time PCR, ELISA and Western Blot, which provedthe successful transfection of hOPG into canine PDLCs.Thirdly, we compared the difference in the proliferation and osteogeniccapability between the blank and transfected canine PDLCs.No obvious alteration in cell morphology was observed under optical andfluorescent microscope. No significant difference in the proliferation, cell cycle andapoptosis was observed by MTT and flow cytometry. Increased mineralization noduleformation was observed in transfected cells group using Von Kossa staining, showingincreased osteogenic capability in transfected cells. Levels of alkaline phosphatase,osteocalcin and bone sialoprotein were significantly increased. And the concentrationof hOPG was gradually increased in12days after transfection, while the ratio ofcRANKL/cOPG showed downward tendency.Then, we evaluated whether hOPG transfected PDLCs could form a clinicapplicable complex with BME-10X collagen membrane scaffold in vitro and invivo.Large numbers of PDLCs adhered to the scaffold under confocal laser scanningmicroscope, which also exhibited good stretch and growth. Furthermore each layer ofthe collagen membrane were attached by different number of PDLCs by layeredscanning, thus proved the biocompatibility of collagen scaffold in vitro.The constructed collagen-PDLCs complex was then implanted into subcutaneoustissue of nude mice. After6and12weeks, animals were sacrificed and theregeneration of tissue-engineered compound was evaluated by X-ray and histology.Osteoid tissue was found in hOPG transfected group under the subcutaneous tissue ofnude mice.Lastly,we evaluated whether autologous implantation of hOPG modifiedperiodontal ligament cells could enhance tissue regeneration in periodontalfurcation defects in dogs.A total of36experimental class Ⅱ furcation defects were prepared bilaterally onmandibular premolars in six beagle dogs. hOPG transfected PDLCs-collagen scaffoldor Ad-Null transfected PDLCs-scaffold or non-transfected PDLCs-scaffold complex were autologously implanted into the furcation defects, and then covered with e-PTFEmembranes. Blank collagen membranes without cells were used as control.Periodontal repair was monitored by X-ray on6weeks and12weeks after operation.The animals were sacrificed at12weeks after operation. Periodontal regeneration wasevaluated histomorphometrically. Various extent of periodontal tissue regenerationwas observed in all groups. Significantly more cementum and alveolar boneregeneration was observed in hOPG transfected PDLCs-scaffold group, than inAd-Null transfected PDLCs-scaffold group, non-transfected PDLCs-scaffold groupand blank collagen scaffold group.Conclusions1) Selecting an appropriate culture medium was essential inpromoting cell growth, proliferation and differentiation.2) hOPG transfectionpromoted the differentiation of PDLCs into osteoblasts in vitro. hOPG gene modifiedPDLCs may serve as an ideal cell source for periodontal tissue engineering.3) hOPGtransfected PDLCs grew well on BME-10X in vitro, and hOPG transfectedPDLCs-collagen membrane showed more prominent osteogenic potential in vivo.4)hOPG gene transfected PDLCs enhanced both cementum and alveolar boneregeneration, showing potential application in periodontal diseases treatment.