The Effect Of Growth Factor On Dental Implant-Bone Osseointegration And Construction Of Transfer Gene Plasmid

Part ⅠThe influence of growth factor on implant-boneosseointegrationExperiment Ⅰ: the influence of rhBMP-2 on the implant-bone osseointegration: biomechanical analysis and CLSM observationThis study measured the implant-bone bonding strength by pull-out testing and observed the new bone formation at different period by CLSM. The aim was to investigate the effect of rhBMP-2 on osseointegration of bone-implant interface. 32 implants were coated with polylactic acid (PLA) and divided into A and B groups averagely. Group A was applied with 1.0mg rhBMP-2; group B without rhBMP-2, just as the control. 8 rabbits were used and two implant osteotomies were drilled on the femur bilaterally, and in which four implants including 2 group A and 2 group B were randomly placed. At the 4-week after implantation, 20mg calcein green/kg body weight was administratered i.v, at 8-week, 20mg alizarin/kg body weight was administered i.v, and at 12-week, the animals were killed. 16 bone-implant blocks including 8 group A and 8 group B were measured thebonding strength of implant-bone interface by using pull-out testing and the extracted implants were prepared for SEM observation. The other blocks were embedded in MMA (methylmethacrylate, MMA) and sectioned, then analyzed the percent of marked bone surrounding implant by CLSM. Results: the pull-out strength of group A (36.50+2.02)N implant-bone block were greater than that of group B(27.63 + 1.31) N (P<0.05). SEM showed there was more calcified substance on the implant surface of group A than group B. There was more yellow-green marked bone around group A implants(6.53 + 0.46)% than group B (4.96+0.37)% (P<0.05) and the red marked bone of group A and group B were (8.26+0.57) % and( 5.55+0.37) % respectively, group A was more than group B (P<0.05), the total marked bone of group A (14.79+0.93)% was also more than that of group B (10.51+0.66)% (F<0.05) .From these result, we could conclude that rhBMP-2 could accelerate new bone formation around dental implant and increase the binding force of bone-implant interface as well. rhBMP-2 improves not only the quantity but also the quality of osseointegration of bone-implant interface.Experiment II The influence of combination of rhBMP-2/rhbFGF and rhBMP-2/rhIGF-I on implant-bone osseointegrationThis study aimed to study the influence of recombination rhBMP-2/rhbFGF and rhBMP-2/rhIGF-I on dental implant-bone osseointegration. 64 implants were coated with PLA and divided into 4 groups averagely. Group I was applied with 1.0mg rhBMP-2 and 200 u g rhbFGF; group II with 1.0mg rhBMP-2 and 250 u grhIGF-I; group III with 1.0mg rhBMP-2; group IV without any growth factors. 16 rabbits were used and two osteotomies were drilled on each femur, in which four groups were randomly placed. On the 4-week after implant, 20 mg calcein green/kg body weight was injected i.v, at 8-week, 20mg alizarin/kg body weight was injected also i.v, and at 12-week, the animals were killed. The block of bone with implant was embedded in MMA and sectioned, then measured the percent of marked bone by CLSM. There were more yellow-green, red color bone and the total marked bones in rhBMP-2 applied groups than these in non-applied group (P<0.05); and no difference between combination rhBMP-2/rhbFGF and rhBMP-2/rhIGF-I groups. The marked bones of group II were greater than these of group III (P<0.05); the red color bones and the total marked bones in group I were more than group III (P<0.05), but the yellow-green color bones in these two groups had no different. Conclusions: RhBMP-2 could increase new bone formation and it acted synergistically with rhbFGF and rhIGF-I to improve implant-bone osseointegration.Part II the construction and expression of pEGFP-PDGF-B.Experiment HI the construction and identification of eukaryotic expression plasmid pEGFP-PDGF-B.The Aim was to construct the enkaryotic expression plasmid pEGFP-PDGF-B The up and down primers were designed according to the imformation of human PDGF-B (Genbank No X02811), and EcoRI and BamH I sites were inserted. Theup primer was 5'- ggaattccatgaatcgctgctgggcgct-3', and the down primer was 5'-cgcgga tccctaggctccaagggtctcctt-3'. pSV7d-PDGF-B as template, PCR was proceeded by 30 cycles of 45 sec at 94°C, lmin at 58°C and lmin at 72°C using Pfu polymerase. After the resulting PCR product and pEGFP-Cl were digested with EcoRI and BamH I, they were linked by T4 DNA enzyme. The recombination was chosed by PCR and identified by enzyme and the sequence was mearued. The result shown the PCR product was single band and the length was 0.7kb. And the recombination was enzymed and two bands were shown. The sequence and the blast results shown the construction were true.Experiment IV the expression and location of green fluorescent protein (GFP)- PDGF-B fused proteinThe aim was investigate the expression and location of GFP-PDGF-B fused protein Mammalian cells CHO was transiently transfected with pEGFP-PDGF-B, pEGFP-Cl as control. The green fluorescence was observed by fluorescent microscope and CLSM. Proteins were extracted from the nuclear, cytoplasm and supernate of cells; PDGF-BB was measured by Western blot method. The result showed green fluorescence as grain and round mass concentrated in the cytoplasm of pEGFP-PDGF-B transfected cells, only little in the nucle, and at the late period, lots of fluorescence in the supernate. There were amount of fluorescence in the whole cell transfected by pEGFP-Cl, and no fluorescence in non-transfected cell; GFP-PDGF-BB fused protein could be detected in the pEGFP-PDGF-B transfected cells cytoplasm and supernate though Western blot. GFP-PDGF-BB could be synthetized and expressed in mammalian cells, and could be secreted outof the cell.Experiment V the culture of human periodontal ligament cells (PDLC) and transfected with plasmid pEGFP- PDGF-BThe aim was investigate the effect of pEGFP-PDGF-B transfected human PDLC. Human PDLC was cultured in vitro, and transiently transfected with pEGFP-PDGF-B, pEGFP-Cl as control. The green fluorescence was observed by fluorescent microscope after 24h. Several cells showed green fluorescence in pEGFP-PDGF-B and pEGFP-Cl transfected group, no fluorescence in non-transfected cell. Green fluorescence in pEGFP-Cl transfected cells was massed the whole cell and the cell's shape had no change. Green fluorescence in pEGFP-PDGF-B transfected cells was only expressed in the cytoplasm, the cell's shape turned into round and larger than the cell without green fluorescence, and the process was shown. Maybe pEGFP-PDGF-B induced the PDLC to differentiate the osteoblast or cementoblast. While the efficiency was low, we could not detect furthermore.