| PartⅠThe assessment on proliferation and osteogenic differentiation abilities of MC3T3-E1 cells in vitro using BMP-2-related peptide P28Objective By comparing the effects of P28 and rhBMP-2 on the proliferation and osteogenic differentiation of MC3T3-E1 cells,to assess the osteogenic activity of P28.Methods The MC3T3-E1 cells were cultured in medium containing P28 or rhBMP-2,and the Control without any growth factor.Cell proliferation ability was measured by Calcein-AM staining after 1,3 and 5 days of culture,and MTT assay after 1,3,5 and 7 days of culture.ALP activity was measured by ALP staining and ALP kit after 7 and 14 days of culture.Calcium nodules were observed by Alizarin red staining after 14 and 21 days of culture.The osteogenic differentiation related genes of Runx2,OCN and Col I were measured by Real-Time PCR,and the osteogenic differentiation related proteins of Runx2,p-Smad 1/5/8,OCN and Col I were measured by Western blot after 1,3 and 7 days of culture.Results The Calcein-AM staining and MTT assay results showed that the MC3T3-E1 cells proliferation ability of P28 group and rhBMP-2 group were significantly higher than Control(P<0.05),and there was no significant difference between P28 group and rhBMP-2 group(P>0.05).The results of ALP activity and Alizarin red staining showed that the P28 had similar osteoinductive to rhBMP-2,and both of P28 group and rhBMP-2 group had significant difference between with Control(P<0.05).The Real-Time PCR and Western blot results also showed that P28 and rhBMP-2 played similar osteoinductive by regulating the Smad pathway.Conclusion P28 had similar osteoinductive to rhBMP-2 in promoting proliferation and osteogenic differentiation of MC3T3-E1 cells.Part ⅡA study on the preparation and physic-chemical property of true bone ceramics/mesoporous silica nanoparticles/P28,and its controlled release in vitroObjective To investigate the preparing method and physic-chemical property of true bone ceramics/mesoporous silica nanoparticles/P28,and evaluated its release kinetics in vitro.Methods The mesoporous silica nanoparticles(MSN)were prepared,and MSN were detected by transmission electron microscope(TEM),Brunauer-Emmett-Teller(BET)method and Barrett-Joyner-Halenda(BJH)mothod.According to the different ratio of MSN:P28 quality(1:1.5,1:2,1:2.5,1:3,1:3.5,1:4)to determine the optimum dosage ratio between P28 and MSN.True bone ceramics(TBC)and TBC/MSN scaffold materials were prepared,and the surface morphology and physic-chemical property of them were investigated by three-dimensional video microscope,scanning electron microscope(SEM)and energy dispersive spectrometer(EDS).TBC and TBC/MSN were loaded with 3 mg P28,and the release kinetics of TBC/P28 and TBC/MSN/P28 were measured and compared at 1,2,3,5,7,10,12,14,18,and 30 days.Results The particle size of MSN was mainly distributed in 180 ± 30 nm,the specific surface area was 702.7 m2/g,the pore size was about 4 nm,and the pore volume was about 0.8 cm3/g.Thus,MSN had good mesoporous structure and sufficient drug loading space.P28:MSN = 1:3 as a suitable dosage proportion of drug loading,encapsulation efficiency reached 81.88 ± 4.44%.The results of three-dimensional video microscope,SEM and EDS showed that MSNs were successfully attached to the interior of the TBC,and had a uniform distribution.Moreover,MSNs did not have a greater impact on the pore structure of TBC.The drug loading rate of P28 in TBC and TBC/MSN was about 80%and 98%respectively,and the release kinetics of P28 in TBC/MSN was slower than that in TBC(P<0.05).Conclusion MSN is a kind of satisfactory carrier material for P28,which can be combined satisfactory with TBC scaffold,and TBC/MSN had slower release of P28 in vitro.Part ⅢAdhesion,proliferation and osteogenic differentiation of MC3T3-E1 cells on the TBC/MSN loaded with P28Objective By comparing the adhesion,proliferation and osteogenic differentiation of MC3T3-E1 cells on TBC,TBC/MSN,TBC/P28 and TBC/MSN/P28 in vitro,to evaluate the ability of TBC/MSN/P28 to induce osteogenic differentiation.Methods TBC and TBC/MSN were loaded with 3 mg P28 respectively,and the study included four kinds of material:TBC,TBC/MSN,TBC/P28 and TBC/MSN/P28.The MC3T3-E1 cells were cultured on the TBC,TBC/MSN,TBC/P28 and TBC/MSN/P28 scaffold materials,and the adhesion rate of MC3T3-E1 cells were measured after 24 hours of culture.The cells were stained with Calcein-AM and PI after 3 days of culture,and the viability and distribution were observed by confocal laser scanning microscope.The proliferative ability was measured by MTT assay after 2,4,6 and 8 days of culture.The ALP activity was measured by ALP kit after 5,10 and 15 days of culture.Results The adhesion rate of TBC/P28 and TBC/MSN/P28 group was higher than the TBC and TBC/FMSN group(P<0.05),and there was no significant difference between TBCfP28 and TBC/MSN/P28 group(P>0.05).The confocal laser scanning microscope showed that the viability and distribution of TBC/P28 and TBC/MSN/P28 group were better than TBC and TBC/MSN group,and there were few dead cells in four groups.The MTT assay results showed that the proliferation ability of TBC/P28 and TBC/MSN/P28 group was higher than the TBC and TBC/MSN group in the whole culture period(P<0.05),and TBC/MSN/P28 group was higher than TBC/P28 group after 6 and 8 days of culture(P<0.05).The results of ALP activity showed that TBC/P28 and TBC/MSN/P28 group was higher than the TBC and TBC/MSN group in the whole culture period(P<0.05),and TBC/MSN/P28 group was higher than TBC/P28 group after 15 days of culture(P<0.05).Conclusion The four materials had good biocompatibility,and the adhesion,proliferation and osteogenic differentiation abilities of TBC/P28 and TBC/MSN/P28 were better than TBC and TBC/MSN.The sustained release properties of TBC/MSN/P28 can promote the P28 to play biological activity.PartⅣRepair of rabbit radial critical bone defect using TBC/MSN loaded with P28Objective To evaluate the ability of TBC/MSN/P28 to repair the radial critical bone defect in rabbits,and explored the ability of the composite material to repair large bone defects in vivo.Methods 20 New Zealand rabbits were randomly divided into four groups:TBC group,TBC/MSN group,TBC/P28 group and TBC/MSN/P28 group.A 15mm length critical bone defect was prepared in bilateral radius of rabbits,and the corresponding material was filled in the defect.After 6 and 12 weeks of operation,to evaluate the bone defect repair of four groups by the X ray,three-dimensional CT(3D-CT)and histological examination(HE and Masson staining).Results The results of gross observation,X ray and 3D-CT showed that there was no obvious new bone formation in TBC group after 6 and 12 weeks of operation.The findings of TBC/MSN group were similar to TBC group.In TBC/P28 and TBC/MSN/P28 group,there was small amount of callus covered the surface of the material at 6 weeks after operation.At 12 weeks,there was a large number of callus covered the surface of the TBC/P28 and TBC/MSN/P28 materials,and the materials were firmly connected with the host bone.In addition,the material had a certain degree of degradation.The results of histological examination showed that TBC group had no obvious new bone tissue at 6 weeks,and only a small amount of new bone tissue was observed at 12 weeks.The findings of TBC/MSN group were similar to TBC group.At 6 weeks,there were a large number of new bone tissues in TBC/P28 and TBC/MSN/P28 group,and the area of new bone tissue was significantly more than that of TBC and TBC/MSN groups at 6 and 12 weeks(P<0.05).At 12 weeks,the new bone tissues of TBC/P28 and TBC/MSN/P28 group were further increased,and further matured into lamellar bone.And the area of new bone tissue in TBC/MSN/P28 group was more than that in TBC/P28 group(P<0.05).Conclusion TBC/MSN/P28 could sustain release the BMP-2-related peptide P28 which has the ability of osteoinductive,and it is an ideal repair complex material for bone tissue engineering. |
PDF Full Text Request