| ObjectiveTo construct the sequential release system of rhBMP-2and bFGF using the sustainedrelease effect of chitosan nanoparticles, and then combined to the antigen-extractedxenogeneic cancellous bone (AXCB) which was transplanted in mice fibular muscle bagfor ectopic osteogenesis effect research. And to learn of the difference of ectopicosteogenesis effect by rhBMP-2and bFGF in different delivery order. Finaly, to provethe most ideal design of rhBMP-2and bFGF sequential release system and to providetheoretical and experimental basis for the clinical application and further study in future.MethodsExperiment1: To construct CS/rhBMP-2/bFGF sequential delivery system ofrhBMP-2molecules and bFGF molecules with chitosan nanoparticles, and then coatedonto the surface of the Antigen-extracted xenogeneic cancellous bone, and then usescanning electron microscope for morphology observation, proportion method forporosity detection, modulus of compression test for biomechanics;Experiment2: To determine the drug loading capacity of the material buy theencapsulation efficiency and drug loading, and to calculate drug release duration,depicting the release curve by in vitro release experiment, verifying the successfulpreparation of composite bone graft material.Experiment3:64SD mices of about30g weight were selected and randomlydivided into experimental groups and control groups, the experimental groups weredivided into6groups according to different sequential release order: group A: AXCB+rhBMP-2/bFGF, group B: AXCB+CS/rhBMP-2/bFGF, group C: AXCB+rhBMP-2/CS/bFGF, group D: AXCB+bFGF/CS/rhBMP-2, group E: AXCB+CS/rhBMP-2, and group F: AXCB+CS/bFGF; the control groups were divided into twogroups: the blank control group G:AXCB+CS and negative control group H: withoutany implantation. At two time points of2weeks and4weeks, animal specimens wereobtained and observed by gross examination and weighing, μCT observation of the three-dimensional microstructure and parameter measurement, bone calcium contentdetection, histological staining (HE staining, alizarin red staining) and histochemicalstaining (ALP staining and immunohistochemical staining of CD34). All the dataobtained from the experiment were analysed by application of SPSS13.0software, usingsingle factor variance completely random design analysis, multiple sample meanvariance SNK-q test methods for statistical analysis of data, setting the test the levelofα=0.05and P<0.05as the statistics significance difference.ResultsExperiment1: preparation and characterization of AXCB/CS/rhBMP/bFGFsequential release system1.1SEM results showed that the antigen-extracted xenogeneic cancellous bone waswith a porous structure, and CS/rhBMP-2/bFGF release system of about100nmgranular structure was adsorbed on the surface of the bone trabecula;1.2Porosity results displayed the antigen-extracted porcine cancellous bone was withup to85%of the porosity, and adsorption of growth factor nanoparticles made nodifference of the porosity, which were maintained at between85%-86%;1.3Mechanical properties results showed that compression modulus of antigen porcinecancellous bone was13.44MPa, and adsorption of growth factor nanoparticlesmade no difference of the compression modulus, which were maintained atbetween12MPa-14MPa, which could meet the requirements for bone tissueengineering implant material strength;Experiment2: Study on drug loaded and release of AXCB/CS/rhBMP/bFGFsequential release system2.1Entrapment efficiency of rhBMP-2and bFGF of groups of materials were relativelyapproximate, which were above55%and60%; and the drug loading amount ofrhBMP-2and bFGF of groups of material were relatively approximate too,respectively in22‰and0.012‰;2.2Release curves by in vitro release test described that, group A: the accumulativerelease rate of rhBMP was reaching80%in9days respectively, and13days for bFG to reach F70%cumulative release rate; group B: rhBMP-2and bFGF weresustained release up to23rd day; group C: the accumulative release rate of rhBMPwas reaching80%in9days, while the bFGF started to release at8th day, and thensustained release up to70%to26th day; group D: the accumulative release rate ofbFGF was reaching70%in13days, while the rhBMP-2started to release at7th day,and then sustained release up to70%to28th day. group E: rhBMP-2sustainedrelease up to24th day when the cumulative release rate reached80%; group F:bFGF sustained release up to22nd day when the cumulative release rate reached70%.Experiment3: Study on ectopic osteogenesis effect of AXCB/CS/rhBMP/bFGFsequential release system3.1General observation and weighing results showed that weight of group A-2, B-2,C-2, A-4, B-4, C-4, D-4, E-4material were significantly higher than thepreoperative material (P<0.05); At2weeks, the weight of group A was higher thanthat in other groups (P<0.05); At4weeks, the weight of group B was higher thanthat in other groups (P<0.05weight); and the material weight of group B and E at4weeks were higher than that of2weeks (P<0.05);3.2Results of calcium test displayed that the material calcium concentration at2weekswas group A, C> group B> group D, E, F, G (P<0.05), and the maximum calciumconcentration was of group A (348.5±9.66mg/dL); at4weeks, group B> group A,C, D, E> group F, G(P<0.05); and calcium concentration of group was the most(414.7±12.03mg/dL). At the same time, calcium concentration of group B, D, Eat4weeks were higher than that of2weeks (P<0.05), respectively higher123.1mg/dL,78.2mg/dL,88.3mg/dL;3.3Analysis of the results of μCT scan and bone parameter displayed that bone volume,bone surface and bone volume fraction of all the implanted antigen porcinecancellous bone were approximate(P>0.05); and the bone mineral density analysisshowed that comparison results of each groups at2weeks was group A, C, E>group B>group D, F, G(P<0.05), and maximum in group A(307.09±8.27mg/cc);at4weeks was group B> group A, C, D, E> group F, G(P<0.05), and maximum in group B(367.52±11.64mg/cc); At the same time, bone density of group A, B, D, Eat4weeks were higher than that at2weeks (P<0.05), respectively higher32.55mg/cc,89.06mg/cc,38.18mg/cc,29.57mg/cc; Trabecular thickness analysisshowed that comparison results of each groups at2weeks was group A, E>groupB, C> group D F, G(P<0.05), maximum in group A (95.33±7.28μ m); at4weekswas group B, C>group A, D> group E, F, G(P<0.05), and maximum in groupB(126.17±11.36μ m); At the same time, trabecular thickness of group A, B, C, Dat4weeks were higher than that of2weeks (P<0.05), respectively, higher than14.51μm,39.75μm,23.15μm,22.49μm;3.4H.E. staining results showed that large area of calcified cartilage and hematopoietictissue were obviously formed in group A, B, C, D, E, the translucent chondrocytewas visible on the newborn calcified cartilage and osteoblasts lining in cartilageedge, and the red cell filling hematopoietic tissue was visible between cartilage,new heterogeneous trabecular bone and soft tissue, including hyperchromatic cellsof hematopoietic cells and red cells of red blood cells.3.5Alkaline phosphatase immunohistochemical staining results showed positiveexpression of ALP mainly located in the peripheral areas of cartilage and expressedin cartilage cell cytolymph of all implants, the positive ALP expression of groupA-2, A-4, B-4, C-4, D-4, E-4were higher than that of the other groups;3.6The mouse position CD34immunohistochemical staining results showed thepositive expression of CD34mainly expressed in vascular endothelial cellmembrane and cytoplasm of all implants, the positive expression of CD34in groupA-2, A-4, B-4, C-4, D-4, F-4were higher than that of the other groups.ConclusionThis study had successfully completed the preparation, characterization, in vitrorelease and in vivo osteogenic efficiency research on AXCB/CS/rhBMP-2/bFGFsequential release system. From the experimental results it could be seen that theAXCB/CS/rhBMP-2/bFGF sequential release system prepared had ideal porousstructure, biomechanical properties which could meet the requirements of bone tissue engineering, and nano particle of growth factors coated with chitosan successfullyadsorbed on the trabecular bone surface, and there was no difference between theamount of rhBMP-2and bFGF groups in the material and the total mass transplantationmaterial ratio; in vitro release experiment confirmed that AXCB/CS/rhBMP-2/bFGFsequential delivery system was prepared successfully, rhBMP-2and bFGF in eachmaterial were sequential released in accordance with the experimental design. At thesame time, the animal experiment results showed that at2weeks,the group of rhBMP-2and bFGF burst release at the same time was with the highest osteogenic efficiency; at4weeks, the group of rhBMP-2and bFGF sustained-releasebone at the same time waswith the highest osteogenic efficiency; In view of the cycle time of new bone formationand mineralization, we concluded that the group of rhBMP-2and bFGFsustained-release at the same time was the most ideal drug release way, had the besteffect in inducing osteogenesis for antigen-extracted porcine cancellous bone allograft. |
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