| Part1: The study on the osteoinductivity of bone targeted peptide on MC3T3-E1 cell in vitroObjective: To evaluate the osteoinductivity of bone targeted peptide in vitroMethod: With Alkaline Phosphatase Staining and Activity,real time-PCR and Western blot,to investigate the osteoinductivity of MC3T3-E1 cell bone induced by bone targeted peptide.Results: 1.Bone targeted peptide promoted alkaline phosphatase activity of MC3T3-E1 cells(P<0.05).2.ALP,COL-1 and Runx2 of MC3T3-E1 cells gene expression and protein function were regulated up induced with targeted peptide(P<0.05)Conclusion: Bone targeted peptide showed good osteoinductivity.Part2: The study on the manufacture of bone targeted peptide / true bone ceramic(TBC) and the physicochemical property of TBCObjective: To investigate the physicochemical property of TBC and the affinity to TBC for bone targeted peptide.Method: 1.The TBC scaffold were sterilizated by 60 Co,and determined by Fourier transform infrared spectroscopy(FTIR)and Scanning electron microscope(SEM).2.The TBC(0.5cm×0.5cm×0.5cm)was immersed in 100?g/ml liquid with peptide,bonetargeted peptide and rh BMP-2 repectively,the points of soak time are 10 mins,20min,30 min,60min,90 min,120min,150 min,and 180 min,then,measure the concerntration of the three kinds liquid by Bicinchoninc acid assay(BCA),and figure out the adherence rates of all the TBC by formula.3.The TBC was immersed in 100?g/ml liquid with peptide,bonetargeted peptide and rh BMP-2 with fluorescence labeling respectively.120 mins later,put them into the freezer dryer for 12 hours,then observe them by fluorescence microscope.4.The TBC combined with bone-targeted peptide,peptide and rh BMP-2 was put into germfree cryogenic vials with 2ml germfree PBS liquid,then wobble these cryogenic vials continuously,then determine the amount of release of the peptide after 1days,2days,3days,4days,5days,6days,7days,8days,9days,10 days,12days,14 days,17days,21 days,28days repectively.Draw the curve of the accumulated amount of release.Results: 1.The hydroxyapatite(Ca10(PO4)6(OH)2)was observed on the surface of the TBC by XRD,FTIR,and SEM,the diameter of the diameter of the microvoids ranged from 220?m to 650?m,the poriness was 84.4±0.3%,the maximum load and maximum intensity of pressure were 65 N and 2.6Mpa respectively.2.More bone-targeted peptide was adhered on the TBC than peptide and rh BMP-2.3.The adherence curve graphy revealed that bonetargeted peptide,peptide and rh BMP-2 could adhere to the sintered bone in 30 min quickly,while peptide and rh BMP-2 reached the adhere peak value.4.The cumulative release curve graphy showed that bone-targetd peptide gradually release from the TBC scaffold.Conclusion: TBC maintain the physicochemical property and architecture of bone as an excellent scaffold.The bone-targeted peptide were higher affinity to TBC in vitro,and the bone-targeted peptide with TBC had the ability of slow-release.Part3 The study on the biocompatibility and osteoinductivity of the TBC with bone-targeted peptideObjective: To investigate the biocompatibility and osteoinductive activity of the TBC with bone-targeted peptide,so as to provide the evidences for the application of the TBC with bone-targeted peptide.Method: 1.The TBC with bone-targeted peptide was sealed in a 37? calorstat with the culture medium according to the proportion of 0.2g/ml,prepare the leaching liquor after 24 h.Culture the L929 cells in the 0.64% phenol solution?culture medium?and 100%?75%?50%?25% leaching liquor,observe the cell growth and proliferation after 24h?48h and 72 h,respectively.2.20 rats were randomly divided into two groups,the TBC(0.5cm×0.5cm×0.5cm)with bone-targeted peptide was implanted subcutaneously under anesthesia.All rats were observed after the surgery and euthanasized for HE staining after 12 weeks.3.MC3T3-E1 cells were inoculated in the the TBC with bone-targeted peptide?the TBC with peptide and TBC with rh BMP-2.Observe the growth of the cells by SEM,and measure the activity of ALP of the cells by MTT.4.8 mices were randomly divided into two groups: the bone-targeted peptide group(n=2)?the peptide group(n=2)?the rh BMP-2 group(n=2),and the TBC group(n=2).The TBC with bone-targeted peptide?the TBC with peptide?the TBC with rh BMP-2,and the TBC was respectively implanted into the spatium intermusculare of the quadriceps femoris of the mices,Observe the postoperative generalcondition,after 4 weeks,all mices were euthanasized.Results: 1.The study on cytotoxicity revealed that all L929 cells were in good growth,had no cytotoxicity.The rate of proliferation of the cells in each leaching liquor had no statistical difference with the control group.The stage of the cytotoxicity was I stage.2.The study on the subcutaneous implantation revealed that the experimental group had no statistical difference with the control group in all aspects.3.The MT3T3-E1 cells in the the TBC with bone-related peptide and TBC with rh BMP-2 were in better growth than the cells in TBC with bone-targeted peptide,and the activity of ALP was superior to the cells in the TBC with bone-targeted peptide.4.In the TBC group,the materials were surrounded by the fiber texture.In the bone-targeted group,new bone was regenerate into the scaffold,whereas,in the rh BMP-2 group,the new bone was regenerate on the interface between the material and muscle.Concusions: There was no cytotoxicity in the TBC with bone-targeted peptide group and the MT3T3-E1 cells show good growth on the TBC surface.The TBC with bonetargeted peptide showed excellent biocompatibility and osteoinductive ability.Part4 The study on the repair of the rat calvarial defect using TBC with bone-targeted peptideObjective: To investigate the relevant factors for the establishing of the rat calvarial defect,and to evaluate the efficancy of the TBC with bone-targeted peptide for enhancing healing of rat calvarial defect,so as to explore the feasibility of thire applications.Method: To measure the transverse diameter and the anteroposterior diameter of the calvarial bone of the rats.50 adult male rats were randomly divieded into two groups: the 5mm defect group(n=40)and the 8mm defect group(n=10),the 5mm defect group were divided into four groups: the dural PTFE group;the pericranium PTFE group;the double PTFE group;and the control group.Rats were euthanized at 8 weeks and 12 weeks after the operation respectively,evaluate the repire of the rat calvarial defect with Microcomputed tomography and histological analyses.The TBC and TBC with bone-targeted peptide/ peptide/ rh BMP-2 were respectively implanted into the defects.Rats were euthanasized at 4 weeks and 8 weeks after the operation,and the Micro-computed tomography and histological analyses were used to evalue the repair of the defects.Results: 1.The parietal bone of the 10 male SD rats was measured using callipers: the transverse diameter was 5.13±0.12 mm(mean ± SD)and the anteroposterior diameter was 7.44±0.44 mm.2.The BV-NFB and BV/TV were the highest in the control group at 8 weeks(61.6 ± 7.4%)and 12 weeks(90.6 ± 4.1%)compared to the double PTFE group at 8 weeks(10.6 ± 3.4%)and 12 weeks(17.4 ± 5.7%).The H E staining showed that there was a lot of fiber connective tissue and little new bone in the double shiled group,however,there was a lot of new regenerated bone in the control group.3.There was no statistical difference about the BV/TV between the 5mm defect group and the 8mm defect group.But the blood loss,operation time,and mortality rates are much higher in the 8-mm group than in the 5-mm group.4.Rats were euthanized at 4 and 8 weeks after the surgery,and Micro-computed tomography consistently revealed that there was no statistical difference between the TBC with bone-targeted peptide and the TBC with rh BMP-2.The BV/TV were the lowest in the TBC group.5.The HE staining revealed that there was a little new regenerated bone in the TBC group,whereas,there was more new regenerated bone in the TBC with bone-targeted peptide/ peptide/ rh BMP-2 group.A lot of new regenerated bone were observed around the scaffold material and on the margin of defect in the TBC with bone-targeted peptide/ rh BMP-2 group.6.Positive immunohistochemical staining for alkaline phosphatase and osteonectin was observed in the cells and matrix.Immunostaining for ALP revealed intense staining in the bone-targeted group at 4 weeks compared with the TBC group.And Immunostaining for Osteonectin revealed strongly staining in the bone-targeted group and rh BMP-2 group.Conclusions: There was not significantly different between the 8-mm group and the 5-mm group about the percentage of the BV-NFB,when the dura mater and periosteum were both shielded.And the TBC with bone-targeted peptide can enhance the healing of the rat calvarial defect.Part5 The study on the repair of the cancellouse bone defect of the rabbit femoral condyle using the TBC bone-targeted peptideObjective: To investigate the efficancy of the TBC with bone-targeted peptide for enhancing healing of the cancellouse bone defect of the rabbit femoral condyle,so as to explore the feasibility of thire applications.Method: 20 adult New Zealand White rabbits were anesthetised,and to build a cancellouse bone defect with the diameter of 6mm and 10 mm deep.The TBC and TBC with bone-targeted peptide/ peptide/ rh BMP-2 were respectively implanted into the defects.Rats were euthanasized at 4 weeks and 8 weeks after the operation,and the Micro-computed tomography and histological analyses were used to evalue the repair of the defects.Results: 1.Rabbits were euthanized at 4 and 8 weeks after the surgery,and Microcomputed tomography consistently revealed that there was no statistical difference between the TBC with bone-targeted peptide and the TBC with rh BMP-2.The BV/TV were the lowest in the TBC group.2.The HE staining revealed that there was a little new regenerated bone in the TBC group,whereas,there was more new regenerated bone in the TBC with bone-targeted peptide/ peptide/ rh BMP-2 group.A lot of new regenerated bone were observed around the scaffold material and on the margin of defect in the TBC with bonetargeted peptide/ rh BMP-2 group.Some lamellar bone and blood vessel were observed around the TBC in the TBC with rh BMP-2 group and the TBC with peptide group.There was a lot of new regenerated bone on the interface between the material and parasitifer in these groups.In the Lane-Sandhu scores,there was no statistical difference between the TBC with rh BMP-2 group and the TBC with bone-targeted peptide,which were both higher than the TBC with peptide group,whereas the TBC group was lowest.Conclusions: The TBC with bone-targeted peptide can enhance the healing of the cancellouse bone defect of the rabbit femoral condyle... |
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