| Part ?:Preparation and Properties for the SF-HAp/SDF-BMP ScaffoldObjective:To prepare a silk fibroin(SF)/nano-hydroxyapatite(n HAp)scaffold which can release SDF-1 and BMP-2 in a sequential and controlled manner,and to study its physical-chemical characteristics.Methods:The SF microspheres were fabricated using the laminar jet break-up method.The SF solution with concentrations at 2%,4%,6%,and 8%was used to make microspheres,and the appropriate concentration was determined through SEM and particle size analysis.The SF-HAp suspension was prepared by the method of ultrasonic oscillation and magnetic stirring,with a mass ratio of n HAp to SF at 1:2,1:4,1:10 and 1:20,respectively.After observing the solid liquid separation,the appropriate mass ratio of n HAp to SF was determined.The scaffold was prepared by freeze-drying and methanol soaking.SDF-1 and/or BMP-2 were loaded in each scaffold by means of physical adsorption and microsphere encapsulation.Then the scaffolds were divided into six groups,Control,SDF-1,BMP-2(P),BMP-2(E),S+B(P)and S+B(E)scaffold.The morphology of the scaffolds were evaluated by SEM,particle size of the microspheres and the pore size of the scaffolds were analyzed by Nano Mesurer software structural changes,structural changes by FTIR,porosity by liquid displacement method,hydrophilicity by the water absorption test,compressive strength and modulus by the biomechanics test.Finally,the release of SDF-1 and BMP-2 from the scaffold in vitro was quantified via ELISA at 3 h,12h,24 h,4 d,7 d,10 d,13 d,19 d,25 d,31 d and 34 d.Results:Through SEM identification and particle size analysis screening,the SF solution with concentrations at 6%was used to make microspheres.The particle size of SF microspheres and the BMP-2 loaded SF microspheres was 127±29 and 131±33?m(P>0.05),respectively.After screening,the mass ratio of n HAp to SF at 1:20 was determined.SEM showed the whole body of the scaffold was spongiform,and the surface and cross section were honeycomb.Particle size analysis indicated the pore size of the scaffold without microspheres was 124±42?m while the scaffold with microspheres was85±25?m(P<0.05).The FTIR spectra of pure SF,SF/n HAp and SF/n HAp scaffold loaded with BMP-2 and/or SDF-1 indicated no obvious difference.The peaks at 1650 cm-1and 1516 cm-1 correspond to the SF.The existence of n HAp in the SF scaffold is confirmed by the appearance of a peak at 1025 cm-1.SDF-1 and/or BMP-2 have no obvious effect on the peak.As for the porosity,it was 49.3±7.4%in Control group,there was no significant difference with the scaffold without microsphere(SDF-1?BMP-2(P)and S+B(P)group).However,the porosity of the scaffold with microsphere decreased to 41.2±6.9%(BMP-2(E)group)and 40.7±9.2%(S+B(E)group),respectively(p<0.05).With regard for water absorption rate,there was no difference among the six groups(P>0.05).Biomechanical test confirmed that the compressive modulus was around 12 k Pa,there was also no significant difference among the six groups(P>0.05).The release profiles in vitro were investigated using the corresponding ELISA kits.In the SF-HAp/SDF-BMP scaffold at 1 d,there was a burst release with 46.8±5.4%of the total SDF-1 released was identified;and then 84.7±3.3% of the SDF-1 released after 7 days.Comparatively,21.1±1.7%of the total BMP-2 released from the scaffolds the first day,the BMP-2 was released for more than four weeks,and the cumulative release was 74.4±1.1%.Conclusions:The SF-HAp/SDF-BMP scaffold had appropriate pore size,porosity,hydrophilicity,and mechanical property.The scaffold was able to release SDF-1 and BMP-2 in a sequential manner.Part ?: Effects of promoting BMSCs migration and osteogenic differentiation by the SF-HAp/SDF-BMP scaffold in vitroObjective: To explore the capacity in promoting BMSCs migration,adhesion,proliferation and differentiation by the SF-HAp/SDF-BMP scaffold in vitro.Methods: The BMSCs of SD rat were isolated with the density gradient centrifugation method.The BMSCs' phenotype was identified by flow cytometry and immunofluorescence staining.Multi-differentiation potential of BMSCs were performed through induced differentiation for osteogenesis,adipogenesis and chondrogenesis.Thescaffolds were divided into six groups as before,Control,SDF-1,BMP-2(P),BMP-2(E),S+B(P)and S+B(E)scaffold.The third generation of BMSCs were applied to detect the biocompatibility of scaffolds.The cells were seeded on the scaffolds directly,and the morphology of BMSCs was investigated using SEM after culture for 1,5 and 9 d.The proliferation of the cells on the scaffold was evaluated via a CCK-8 assay at 1,3,5,7 and 9 d.The ability of the scaffolds to promote the cells migration in vitro was assessed using the Transwell system.Transwell plate was used to co-culture the cells with the scaffolds indirectly.The cell nucleus and skeleton were stained by DAPI and FITC-phalloidin,respectively.The ability of scaffolds to promote osteogenesis was assessed.The ALP activity,calcium content and osteogenesis marker of OCN of the BMSCs culture with scaffolds were assessed via ALP staining,Alizarin Red staining,and immunofluorescence staining,respectively.To further affirm the effects of the scaffolds on BMSCs' osteogenic differentiation,the expression of osteogenic genes such as ALP,Run X2,OPN and OCN,was determined by RT-q PCR at 3,7 and 14 d.Results: The surface molecular profile of BMSCs was confirmed via a flow cytometry assay.Positive markers of CD44,CD90 and CD29 were expressed as 96.42%,94.14% and 86.10%,while the negative markers of CD11 b,CD45 and MHC-? were 2.33%,0.87% and 1.32%,respectively.CXCR4 was identified both intracellular(39.86%)and extracellular(15.07%)expression.BMSCs was able to differentiate into the osteoblast,adipocyte and chondrocyte respectively after they were cultured with specific inducer.SEM demonstrated that BMSCs could adhere to the surfaces of all scaffolds.CCK-8 assay confirmed that BMSCs exhibited a good proliferating ability on the scaffolds loaded with BMP-2,especially S+B(E)and BMP-2(E)group,there was significant difference compared with Control group(P<0.01).Cell migration across the transwell membrane was identified as a result of the released SDF-1 or BMP-2 from the scaffolds.The number of recruited BMSCs in the groups that contained SDF-1 were significantly increased compared with Control group or the scaffold loaded with BMP-2 alone(P<0.01),while the number of recruited BMSCs in the three groups with SDF-1 were without difference(P>0.05).5 days after cultured with the scaffolds,the cytoskeleton and nucleus staining confirmed BMSCs exhibited better proliferation and apparent viability in the scaffold with SDF-1+BMP-2 than the other scaffolds.7 days later,ALP staining demonstrated that all the scaffolds contained BMP-2 exhibited higher ALP activity compared with the scaffoldswithout BMP-2,however,there was no remarkable difference among the four groups.21 days later,the S+B(E)and BMP-2(E)group exhibited an obvious increase in the calcium content and OCN content when compared to S+ B(P)and BMP-2(P)group;S+ B(E)group expressed the highest level among all groups.The expression of ALP,Run X2,OPN and OCN m RNA was quantified at 3,7 and 14 d.The expression patterns of ALP and Run X2 were similar.The scaffolds contained BMP-2 demonstrated an up-regulated expression of the two genes compared with Control scaffold.Among the four groups,S+B(E)group exhibited the highest level both at 7 d and 14 d.As for OPN and OCN,they also had a similar expression pattern.At 3 d,there was no obvious difference among the six groups.However,at 7 d and 14 d,the scaffolds contained BMP-2 were identified an up-regulated expression of both OPN and OCN.The level of OPN and OCN expression was in the order of S+ B(E)> BMP-2(E)> S +B(P)> BMP-2(P),and there was a significant difference between S+ B(E)and BMP-2(E)group(p < 0.05).Conclusions: The SF-HAp/SDF-BMP scaffold established well biocompatibility.It recruited BMSCs in vitro and promoted the cells adhesion,proliferation,and osteogenic differentiation significantly.Part ?: Effects of BMSCs recruitment and bone regenertion by the SF-HAp/SDF-BMP scaffold in vivoObjective: To assess the ability of SF-HAp/SDF-BMP scaffold in promoting BMSCs recruitment and bone regeneration in vivo.Methods: The lentivirus vector containing firefly luciferase reporter gene(Lenti-Luc)was employed.It was applied to infect BMSCs in order to mark the target cells(Luc-BMSCs).After the cells adhered to the Petri plate,different multiplicity of infection(MOI)(1?2?5?10?15?20?25?30?50 and 80)was applied to infect BMSCs.The bioluminescence imaging was performed to determine the best MOI in vitro.SDF-1 was selected to be the positive drug,Luc-BMSCs was injected into the body via tail vein of the rat central calvarial defect model.The bioluminescence imaging in vivo was performed to ascertain the cell concentration and cell migratory route.18 SD rats were divided into six groups and were anesthetized 3 days after the creation of a calvarial defect model.Each rat was injected with Luc-BMSCs,after injection at 30 min,1 d,3 d,7 d and 14 d thebioluminescence imaging in vivo was obtained to record the cell migration route,homing and proliferation on the scaffold.The rat 5.0 mm bilateral calvarial defect model was established with six groups of scaffolds.After 8 and 12 weeks post-surgery,the calvarial specimen was harvested for ?-CT imaging and histological analysis to evaluate the new bone formation,angiogenesis and scaffold degradation.Results: The concentration of Lenti-Luc was 650 ng/?L,OD260/280 was 1.9.The best MOI of Luc-BMSCs was 50.The appropriate concentration of cells was determined 1.0×106 by the bioluminescence imaging in vivo 3 days after cell injection.It was also found that Luc-BMSCs were mainly in the lungs and kidneys 12 h after injection.At 30 min and 1 d,the Luc-BMSCs were mainly retained in the lungs for all groups.As the time increased to 3 d and 7 d,the luminescence intensity decreased in the lung,while it increased at the calvarial defect region for all groups,particularly for the three groups contained SDF-1,however,there was no significant difference among the three groups(p>0.05).At 14 d,a significant decrease in the intensity of the signal in the calvarial defect region was found for all groups,the groups with SDF-1 continued to exhibit a higher intensity than the other three groups,meanwhile,the strongest signal was identified in S+B(E)group.The implant area and new bone formation in the calvarial defect region was detected with ?-CT.The images indicated the bone volume at 12 w was increased compared with 8 w.New bone formation in BMP-2(E)and S+B(E)group was more than the other four scaffolds at both 8 and 12 w.The bone content was the highest in S+B(E)group at 12 w,the BV/TV was 31.21±3.50% and 53.78±9.84% in BMP-2(E)and S+B(E)group(p < 0.05),respectively.Histological results indicated that there was a thicker bone matrix,more new blood vessels and less remnant scaffold in S+B(E)group compared with the other groups.The formation of new bone was with typical structure in S+B(E)group at 12 w.Conclusions: The SF-HAp/SDF-BMP scaffold which can release SDF-1 and BMP-2 in a sequential manner had a good ability to recruit Luc-BMSCs in vivo.It also demonstrated an excellent capacity to promote new bone formation which was compatible with scaffold degradation. |
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