An Experimental Study On Repair Of Defects In Periodontal Tissue With Tissue Engineered Composites With Overexpression Of HVEGF And HBD3 Genes

Objective:Beagle canine periodontal ligament cells(PDLCs)were transfected with the adenoviral vector AdV4-hBD-3-GFP and the lentiviral vector LentiV5-hVEGF165GFP,then inoculated onto Bio-Gide collagen membrane.On the membrane,a tissueengineered composite was formed.This composite was co-implanted in a bifurcated region of a Beagle dog with a lithium-containing silicate(LCS)bioceramics to explore the ability of genetically modified tissue-engineered complexes to repair defects in the periodontal tissue under the combined action of hBD-3 and VEGF.Methods:1.Beagle canine PDLCs were isolated and cultured in vitro by tissue block method.Cellular sources were identified by routine immunohistochemistry.2.The virus vector carrying hBD-3 gene and hVEGF165 gene was constructed in vitro and transfected into beagle PDLCs.The transcription and expression of hBD-3 and VEGF was detected by fluorescence microscopy,flow cytometry,Real-Time PCR and Western Blot.3.After in vitro transfection of beagle PDLCs with hBD-3 gene and hVEGF165 gene,the morphological changes of cells were observed under fluorescence microscope,cell proliferation was detected by CCK-8.The concentration of VEGF and hBD-3 in the cell supernatant was detected by ELISA at 3 days and 5 days after transfection,and the concentration of VEGF and OPN in the cell supernatant was detected within 3 weeks after osteogenic induction.At 6 and 12 hours after infection with 1 ?g/ml E.coli-LPS,the concentration of inflammatory factors in the culture supernatant of each group was measured.Real-time PCR and Western Blot were used to detect the expression of osteogenic markers of ALP,OPN,Col I and Runx2 in osteoblastic differentiation at early stage after transfection.Alkaline phosphatase and alizarin red staining were used to observe osteogenic induction of osteogenesis.4.To examine the cementogenic/osteogenic differentiation of PDLCs after cultured in different concentrations of lithium-containing silicate(LCS)bioceramics extracts in DMEM medium,including periodontal regeneration-related genes and protein expression(ALP,OPN,OCN,BSP,CAP,CEMP1,VEGF,RUNX2)and Wnt/?-catenin,ERK signaling pathway-associated genes and protein expression(wnt3a,?-catenin,pp65,MMP2,FN and col ?).Meanwhile,these genes and protein expression were detected after the inhibition of these two signaling pathways.5.Establishment of a model of chronic class ? furcation defects bilaterally on mandibular premolars in Beagle dogs.6.The beagle canine PDLCs transfected with the hBD-3 gene and the hVEGF165 gene were composited with the Bio-Gide collagen membrane.The composites were observed by scanning electron microscopy and confocal laser scanning microscopy.At the same time,the complexes combined with lithiumcontaining silicate bioceramics were co-implanted into the chronic class ? furcation defects of Beagle dogs.The animals were sacrificed at 12 weeks after surgery.The CBCT was taken and the teeth and alveolar bone were taken from the test area.Paraffin sections were prepared and histological observations were performed.Results:1.Successful isolation and cultivation of Beagle PDLCs.2.The transfection of PDLCs was successfully performed by the recombinant adenovirus AdV4-hBD-3GFP and LentiV5-hVEGF165 lentiviral vector.Transfected cells emit strong green fluorescence.Real-Time PCR and Western Blot showed that hBD3 and VEGF can be efficiently expressed.3.There was no significant change in the morphology of PDLCs after transfection with hBD3 and VEGF,and there was no obvious change in proliferation.The secretion of VEGF in the supernatant of transfected VEGF group was higher than that in other groups.The secretion of hBD3 in the supernatant of transfected hBD3 group was higher than that in other groups.The transfection of hBD3 and VEGF group resulted in higher OPN secretion in the late and late osteogenic induction.At the same time,transfection of hBD3 cells can effectively inhibit the inflammatory response induced by E.coli-LPS.Transfection of hBD3 and VEGF groups increased the expression of osteogenic markers of ALP,OPN,ColI,and Runx2 in the early stage of osteogenic induction,and promoted the formation of calcified nodules in the middle and later stages.4.A certain concentration of LCS extracts could significantly stimulate cementogenic/osteogenic differentiation,LCS bioceramics may possess the capacity to trigger the activation of the Wnt/?-catenin and ERK signaling pathway.5.Successfully constructed the model of chronic class II furcation defects bilaterally on mandibular premolars in Beagle dogs.6.The virus transfected cells adhered and stretched in the collagen membrane and grew well.Under the laser confocal microscope,cells emitting green fluorescence were observed to adhere to the collagen membrane.In the periodontal regeneration experiment of chronic root furcation involvement in Beagle dogs,the results of three-dimensional reconstruction of CBCT and related parameters,combined with the results of histological section showed that PDLCs with hVEGF,hBD3 gene overexpression on Bio-gide collagen membrane plus LCS bioceramics can promote the repair of beagle root furcation involvement.Conclusion:Viral vector-mediated hBD3 and VEGF and complex LCS bioceramics have a synergistic effect in the repair of periodontal tissue defects caused by periodontitis,it will provide some reference for the future application of genetically modified tissue engineering technology in the periodontal field.