| Part One.Preparation and Properties for the Chemical Crosslinking Scaffold of Methacrylic Anhydride Modified Gelatin-Bioglass[Objective] To prepare a chemical crosslinking scaffold photopolymerized by methacrylic anhydride(MA)-gelatin(Gel)and mesoporous bioactive glass nanoparticles(MBGNs),and to study its physical-chemical and biological characteristics.[Methods] The Gelwas modified by MA to obtain the GelMA solution.The MBGNs were prepared by using the tetraethyl orthosilicate(TEOS),the triethyl phosphate(TEP)and the calcium nitrate tetrahydrate(CNT)as raw materials,the cetyltrimetylammonium bromide(CTAB)as template agent,and the Tris-HCl buffer solution(p H=8)as catalyzer.The silane coupling agent(APTES)was used to modify MBGNs with active amino group.The chemical crosslinking agents including N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS)were added into the 15%(w/v)GelMA solution and mixed uniformly.Then the APTES-MBGNs were added into the mixture solution(EDC,NHS and GelMA)to obtain the GelMA-MBGNs granules.These granules were dissolved into 1 wt% Irgacure 2959 photoinitiator,and mixed with 15%(w/v)GelMA solutions to obtain the GelMA-MBGNs scaffolds with different mass fraction of MBGNs under UV irradiation photopolymerization.In addition,MBGNs were mixed with 15%(w/v)GelMA solution to obtain the GelMA/MBGNs physical blending scaffold.The micro-morphology of the scaffolds were evaluated by the scanning electron microscopy(SEM)and the transmission electron microscopy(TEM),structural changes by the fourier transformation infrared spectrum(FTIR),stability by the weight loss analysis,hydrophilicity by the water absorption test,compressive strength and modulus by the biomechanics test,and bioactivity by the formation of carbonate hydroxyapatite(HCA)on the simulated mineralized body fluid(SBF).[Results] GelMA had sponge-like structure,and uniform and interconnected pores with a mean diameter of 150±20 μm under SEM.MBGNs displayed spherical particles with uniform size(250±30 nm),good dispersion and no obvious agglomeration.Both scaffolds demonstrated as 3D composite structure,rough surface,and uniform and interconnected pores,of which the MBGNs particles were embedded uniformly in the GelMA pores.Statistical differences were not detected on the pore size among the GelMA-MBGNs,GelMA/MBGNs and GelMA scaffolds with the same mass fraction of MBGNs(p>0.05).With the mass fraction of MBGNs increasing,no significant difference in pore size was detected between the GelMA-MBGNs and the GelMA/MBGNs(p>0.05).The FTIR indicated the special chemical bond between GelMA and MBGNs.After 10 days,the weight loss was significantly greater in the GelMA/MBGNs group than in the GelMA-MBGNs group at the same time point(p<0.05).With the time going,both the scaffolds had a decline tendency in weight loss,whereas the GelMA-MBGNs declined moderately.At the same time point,the water absorption was significantly greater in the GelMA/MBGNs group than in the GelMA-MBGNs group with the same mass fraction(p<0.05).With the time going,the water absorption in both groups increased(all p<0.05).With the mass fraction of MBGNs increasing,the water absorption in both scaffolds decreased(all p<0.05),of which the GelMA-MBGNs had a slight decline.Both compressive strength and modulus in the GelMAMBGNs group with the same mass fraction of MBGNs were higher than in the GelMA/MBGNs group,but only the scaffold with MBGNs 3 wt% had a significance(p<0.05).The extent of HCA formation in the GelMA-MBGNs group was comparable with that in the GelMA/MBGNs group.With the mass fraction of MBGNs increasing,extent of HCA formation increased.[Conclusion] The structural stability,hydrophilicity and biomechnics properties of the chemical crosslinking GelMA-MBGNs scaffold are superior to the physical blending GelMA/MBGNs scaffold.When the mass fraction of MBGNs is 3 wt%,the GelMA-MBGNs scaffold has the best compressive strength and modulus.The bioactivity of the GelMA-MBGNs is comparable with the GelMA/MBGNs scaffold.Part Two.Preparation and Properties for the Bioactive GelMA-MBGNs/rhBMP-2[Objective] To prepare the GelMA-MBGNs scaffold as a controlled release carrier for rhBMP-2,and to study its physical-chemical and biological properties[Methods] The cylinder scaffold of GelMA-MBGNs(5 mm in diameter and 2 mm in height)with 3 wt% MBGNs was manufactured.Then the scaffold was immersed in 5 μg/ml rhBMP-2 solution,and underwent incubation,evacuation and lyophilization sequentially.GelMA/rhBMP-2 was prepared by the same method as a control.The micro-morphology of the biomaterials was evaluated by SEM.Then the release characteristics of rhBMP-2 in vitro,the degradation capacity,the release characteristics of calcium,phosphate and silicon ions,and biomineralization on SBF were assessed.[Results] The GelMA-MBGNs/rhBMP-2 displayed as white and ductile solid appearance.GelMA/rhBMP-2 demonstrated as semi-transparent and tremellose cylinder.The GelMA-MBGNs /rhBMP-2 with a pore size of 150±20 μm demonstrated uniform and interconnected pores,where the MBGNs particles were embedded uniformly under SEM.The GelMA/rhBMP-2 had sponge-like structure,and uniform and interconnected pores with a mean diameter of 150±20 μm.Both the materials demonstrated biphasic release characteristics,namely early explosive and late sustained release.The GelMA-MBGNs/rhBMP-2 released rhBMP-2 in a relatively milder and longer manner compared with the GelMA/rhBMP-2.Both the materials experienced a similar first fast and slow degradation in vitro,but the GelMA-MBGNs/rhBMP-2 had a relatively slow velocity.The GelMA-MBGNs/rhBMP-2,GelMA-MBGNs and MBGNs had similar release characteristics of phosphate and silicon ions,whereas the former two displayed a moderate release rate and volume.Concentrations of calcium ions fluctuated within a certain range in both the GelMA-MBGNs/rhBMP-2 and the GelMA-MBGNs groups,whereas a sharp first and slow descent was observed in the MBGNs group.The HCA formation capacity of the GelMA-MBGNs/rhBMP-2 was similar to the GelMA-MBGNs,which were superior to the GelMA/rhBMP-2,with more HCA coverage and thickness.[Conclusion] GelMA-MBGNs/rhBMP-2 demonstrates as biphasic stage release characteristics,which decreases the initial explosive and prolongs the late sustaining release.The release characteristics of silicon and phosphate ions of the GelMA-MBGNs/rhBMP-2 are similar to the MBGNs but with a slower velocity.HCA formation capacity of the GelMA-MBGNs/rhBMP-2 is greater than the GelMA/rhBMP-2.Part Three.Effects and Mechanisms of Osteogenesis by the GelMA-MBGNs/rhBMP-2 in Vitro[Objective] To explore the capacity in promoting BMSCs adhesion,proliferation and differentiation by the GelMA-MBGNs/rhBMP-2 in vitro[Methods] The SD rats’ BMSCs were isolated by the density gradient centrifugation method and indentificated by the flow cytometry(FAC).Then the cells underwent primary and passage culture.The morphological characteristics of BMSCs were observed and the growth curves were described.The GelMA-MBGNs/rhBMP-2,GelMA-MBGNs and GelMA were classified as experimental groups and no graft as the blank control group.The third generation of BMSCs suspension was inoculated on the materials with a certain density.At different time points,the adhesion and spreading of BMSCs was observed by SEM.The cell skeleton was stained by phalloidin marked with FITC and nucleus by DAPI.The cell proliferation status was evaluated by CCK-8 method and the survival status by Dead/Alive cell fluorescence staining.Then these materials were placed into the upper chamber of 6-well Transwell plate(pore size,8 μm)and BMSCs with a density of 3×105 into the lower chamber.The ALP staining and its bioactivity,the mineralized nodule staining and calcium content,the expression of ALP m RNA,Runx2 m RNA,Col-Ⅰ m RNA,OPN m RNA and OCN m RNA by q RT-PCR,and the OCN immunofluorescence staining were evaluated.[Results] The third generation of BMSCs demonstrated long spindle shape with a vortex-like growth.Positive markers of CD90 and CD105 were expressed as 94.18% and 92.33%,respectively,while the negative markers of CD11 b and CD34 were 5.74% and 1.77%,respectively.The passage cells growth experienced incubation,logarithmic and plateau stages,of which the growth curve demonstrated as ‘S’ shape.The SEM indicated excellent and well adhesion and spreading of cells on all three materials’ surface to some extent,of which the adhesion and spreading area increased with the time being.The cytoskeleton and nucleus staining also confirmed the excellent cell adhesion and spreading status.There was no significant difference in OD value by CCK-8 method among all groups at 1 d(p>0.05).At 3 d,7 d and 9 d,OD values in the GelMA-MBGNs/rhBMP-2 group were greater than in the GelMA-MBGNs group(all p<0.05),which were higher than in the GelMA group(all p<0.05).With the time being,OD values in all groups increased(all p<0.05).At the same time point,both living cells percent(Plive)and living cells density(Dlive)were similar among all groups(all p>0.05),of which the values didn’t change with the time going(all p>0.05).The GelMA-MBGNs/rhBMP-2 group demonstrated the earliest and strongest positive ALP staining.ALP activity in GelMA-MBGNs/rhBMP-2 group was greater than GelMA-MBGNs group and GelMA group at 14 d(all p<0.05),but no significant difference was detected between the latter two groups(p>0.05).With the time being,ALP activities in all groups increased(all p<0.05).The GelMA-MBGNs/rhBMP-2 group showed the earliest and strongest positive mineralized nodules staining.At 7 d,calcium content in the GelMA-MBGNs/ rhBMP-2 group was greater than in the GelMA-MBGNs group(p<0.05),which was higher than in the GelMA group(p<0.05).At 21 d,calcium content in the GelMA-MBGNs/rhBMP-2 group was greater than in the GelMA-MBGNs group and the GelMA group(p<0.05),but no significant difference was found between the latter two groups(p>0.05).With the time going,calcium contents in all the materials’ group increased(all p<0.05).The ALP m RNA and Runx2 m RNA expressions in the GelMAMBGNs/rhBMP-2 group were greater than in the GelMA-MBGNs and GelMA groups at 3,7 and 14 d(all p<0.05).The Col-I m RNA expression in the GelMA-MBGNs/rhBMP-2 group was higher than in the GelMA-MBGNs and GelMA groups at 3 and 7 d(all p<0.05).OPN m RNA and OCN m RNA in the GelMA-MBGNs/rhBMP-2 group were expressed greater than in the GelMA group at 7 and 14 d(all p<0.05).The GelMA-MBGNs/rhBMP-2 group demonstrated the earliest and strongest positive of OCN immunofluorescence staining.[Conclusion] The adhesion,spreading,proliferation and osteogenic differentiation of BMSCs are significantly promoted by the GelMA-MBGNs/rhBMP-2.Part Four.Repair of Bone Defects by the GelMA-MBGNs/rhBMP-2 in vivo[Objective] To evaluate the capacity of osteogenesis and angiogenesis by the GelMA-MBGNs/rhBMP-2 in vivo,and the feasibility on healing skull bone defects in rats.[Methods] Twenty-four SD rats were included to develop the bilateral critical size skull defects with a diameter of 5 mm.These animals were randomly divided into four groups according to the grafts: the blank control group,the GelMA group,the GelMA-MBGNs group and the GelMA-MBGNs/rhBMP-2 group.The rats were sacrificed at 4 and 8 weeks to perform the following examinations sequentially: gross observation,micro-CT scanning,and histology including HE staining,Masson trichrome staining,and Col-Ⅰand CD31 immunohistochemical staining.[Results] All animals had normal mental state,activity,gait,diet,stools and urine postoperatively.No redness,swelling,or skin necrosis or ulceration was observed.At 4 weeks,the trabecular bone volume fraction(BV/TV,%)in the GelMA-MBGNs/rhBMP-2 group was greater than in the GelMA-MBGNs group,and BV/TV in the GelMA-MBGNs group was higher than in the GelMA group,but no significant difference were detected(all p>0.05);whereas the BV/TV in the GelMA-MBGNs/rhBMP-2 group was greater than in the GelMA group(p<0.05).At 8 weeks,BV/TV from high to low were as GelMA-MBGNs/rhBMP-2 group,GelMA-MBGNs group and GelMA group,respectively(p<0.05).With the time going,BV/TV in all the three materials’ groups increased(all p<0.05).Histology(HE and Masson staining)showed the GelMA-MBGNs/rhBMP-2 had the best capacity in new bone and blood vessels formation.At 4 weeks,the percentage of new bone area(semi-quantitative analysis according to HE staining)in the GelMA-MBGNs/rhBMP-2 group was greater than in the GelMA-MBGNs group,but no significant difference was detected(p>0.05).However,the percentages of new bone area in both the GelMA-MBGNs/rhBMP-2 group and the GelMA-MBGNs group were greater than in the GelMA group(all p<0.05).At 8 weeks,the new bone formation from high to low was the GelMA-MBGNs/rhBMP-2 group,the GelMA-MBGNs group and the GelMA group,respectively(p<0.05).With the time going,the new bone formation areas increased in all materials’ groups(all p<0.05).At 4 weeks,neovascular formation(according to CD31 immunohistochemical staining)in the GelMA-MBGNs/rhBMP-2 group was greater than in the GelMA-MBGNs group,and the GelMA-MBGNs group was higher than in the GelMA group,but no significant difference was detected(p>0.05).Neovascular formation in the GelMA-MBGNs/rhBMP-2 group was greater than in the GelMA group(p<0.05).At 8 weeks,neovascular formation in the GelMA-MBGNs/ rhBMP-2 group was greater than in the GelMA-MBGNs group,but no significant difference was detected(p>0.05).However,neovascular formation in the GelMA-MBGNs/rhBMP-2 group and the GelMA-MBGNs group were greater than in the GelMA group(all p<0.05).Compared with at 4 weeks,neovascular formation decreased in all materials’ groups at 8 weeks(all p<0.05).[Conclusion] GelMA-MBGNs/rhBMP-2 promotes osteogenesis and angiogenesis significantly in vivo,and achieves high healing rates in skull bone defects within a short period of time. |
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