| Background:Orthopedic surgeons repair bone defects using a varied range of materials,such as autografts,allografts and artificial bone graft replacements,etc.Autologous cancellous bone grafts,due to their osteogenic,osteoinductive and osteoconductive properties,remain the gold standard for transplantation[1].However,they have limitations,such as limited supply,significant clinical morbidity,including prolonged hospitalization and recovery time,increased risk of infection and surgical complications,etc.[2].Allografts can be used as an alternative,but they have the risk of transmission of pathogenicviruses,host immune reaction and infection[1].With an increasing demand for traditional bone grafts and decreasing supply,tissue engineering technologies are being developed to provide suitable alternatives for clinical use.Bone tissue engineering is a complex and rapidly developing field,which combines biomimetic scaffolds,osteoinductive molecules and osteogenic cells to achieve regeneration following the acquisition of major defect bones or other pathologies[3].Mesenchymal stem cells(MSCs)are multipotent cells with osteogenic potential that can be isolated from a variety of tissues[4].Scaffolds provide the three-dimensional microenvironment during the tissue engineering of bone and induce cell adhesion,proliferation and differentiation to form extracellular matrix(ECM)[5,6].An optimized biomimetic scaffold should deliver bioactive stimulation and simultaneously direct the osteogenic differentiation of MSCs.Based on this requirement,osteoinductive growth factors and osteoconductive carrier materials were combined to fabricate bone forming scaffolds with controlled growth factor delivery.Among bone growth factors,bone morphogenetic proteins(BMPs)are regarded as the most effective in formating bone tissue during the development of embryos,in growth,healing and throughout adulthood[7,8].BMPs are widely utilized as osteogenic induction factors in clinical bone repair(e.g.bone nonunion and spinal fusion).Some products that incorporate recombinant BMPs(rh BMPs)have been approved by the FDA for orthopedic surgery[9].However,due to the complications and limitations of rh BMPs in clinical applications,such as heterotopic bone formation and the costs associated with the high doses required for treatment,its clinical use has been seriously affected [10,11].Dexamethasone is a synthetic glucocorticoid,which can stimulate the proliferation and osteogenic differentiation of MSCs in vitro,in association with ?-glycerophosphate and ascorbic acid[12].Combining BMP-2 and DEX into a biomaterial scaffolds can improve osteogenic differentiation in bone tissue engineering.In addition,in order to retain an effective concentration,extend reaction time and reduce the risk of growth factors becoming exhausted,their release from a biomaterial scaffold must be controlled for bone tissue engineering applications[13-15].Many methods for spinning fibrous scaffolds comprising micro or nano-sized fibers have so far been described,which control the shape,surface characteristics and composition of a single fiber,the most common of which is electrospinning.Two and three-dimensional scaffolds prepared using these fibers can transmit chemical and physical signals that regulate cellular behavior,including cell adhesion,proliferation,differentiation and extracellular matrix(ECM)formation[16].The major advance in electrospinning has been coaxial electrospinning,where two concentric layers can be formed,with appropriate characteristics[17].The benefit of coaxial spinning is the ability to isolate various stimulants in different compartments and adjust their release dynamics by altering the thickness and location of the fibers.For example,the inner core layer could contain the therapeutic biomolecules and a second layer which is oriented as coaxial fibers,modulating the biological and mechanical properties of the device[18].Coaxial electrospinning technology overcomes the limitations of traditional drug delivery techniques.This drug delivery method has aroused great interest for use in many biomedical applications.Clinical use of biomaterials demands appropriate levels of biocompatibility with low toxicity,often lacking in artificial materials.Instead,biological materials from plants tend to be more biocompatible,have little or no toxicity,are cheap,sustainable and biodegradable.Interest in such proteins in controlled and targeted drug delivery and tissue engineering is increasing exponentially[19].Zein is a major protein extracted from corn endosperm and capable of self-assembly into various structures including microspheres,bicontinuous sponges,films and fibers[20].Sixty % of the amino acids in Zein are hydrophobic,rendering the protein amphiphilic.As a natural biomaterial,Zein has advantages over manufactured synthetic polymers,such as high levels of biodegradability,low toxicity and excellent biocompatibility.For this reason,Zein has been explored as a vehicle in drug delivery,food packaging and tissue regeneration applications[21].Nanofibers fabricated by electrospinning simulate the extracellular matrix of bone cells and so researchers have taken a keen interest in them for regenerating bone tissue.Scaffolds possessing osteoinductivity and osteoconductivity are very important for bone tissue engineering applications.Objective:A dual-drug-loaded core-shell scaffold was designed for bone tissue engineering.The aim of this study was to fabricate novel core-shell nanofibers from the natural biomaterial-Zein,having the capability to undergo the controlled release of BMP-2 and DEX so as to promote osteogenic expression of MSCs for bone tissue engineering,and explore the potential of natural biological materials as the raw materials in the tissue engineering of bone.Methods:1.Preparation of core-shell electrospun scaffolds loaded with BMP-2 and dexamethasone: Initially Zein,PLLA,Zein-DEX and PLLA-BMP-2 were dissolved in HFIP.The solutions were stirred magnetically for 4 h at room temperature.PLLA-BMP-2 solution was prepared by dissolving rh BMP-2(48 ?g/m L,stabilized by 1% BSA)into PLLA solution before electrospinning.In this experiment,the nanofiber mats were produced by coaxial electrospinning,using the same mass ratio of Zein or Zein-DEX to PLLA or PLLA-BMP-2(3:1).The coaxial spray nozzle comprised two concentric stainless-steel needles.PLLA or PLLA-BMP-2 solution was fed through aninner needle at a flow rate of 0.006 m L/min and Zein or Zein-DEX solution was delivered through the external nozzles at a flow rate of 0.008 m L/min using a 12 cm working distance and an applied voltage of 15 k V.The electrospun scaffolds were placed in a vacuum drying oven at room temperature for 7 days.2.Environmental scanning electron microscopy(ESEM): The morphological characteristics of the nanofibers were observed using ESEM(XL-30,FEI Co.,Ltd.,USA)after the nanofiber membranes had beensputter-coated with gold for 90 s at an accelerating voltage of 10 k V.Nano Measure software v1.2(Fudan University,China)was used to measure fiber diameter and their distribution in the nanofiber membrane.3.Transmission electron microscopy(TEM): The core-shell structure of the nanofibers was observed by transmission electron microscopy(TEM,H-7650,Japan Hitachi Co.,Ltd.,Japan)at an accelerating voltage of 100 k V.The observed fiber-film samples were placed onto carbon-coated Cu grids with 400 mesh support.4.Water contact angle(WCA): Water contact angle was measured at room temperature with a bespoke instrument(XGCAMA,Xuanyi Industrial Equipment Co.Ltd.,Shanghai,China).A drop of deionized waterwas placed onto a flattened piece of fiber membrane and the contact angle measured immediately and after 10,20 and 30 s.The mean of 5 contact angle readings was determined for each sample.5.In vitro drug release: Each nanofiber mat was placed into a 10 m L tube and 2m L phosphate buffer solution(PBS,p H 7.4)added then shaken at 37 °C at 100 rpm for 21 days in an oscillating water bath.At various time intervals the PBS was collected and replaced with an equal quantity of fresh PBS.The collected PBS was centrifuged at 3000 rpm(1000×g)for 15 min and the supernatant collected and frozen at-80 °C until analyzed.A rh BMP-2 ELISA kit was used to quantify the concentration of rh BMP-2 in the extract medium by measuring adsorption at 450 nm using a microplate reader(Bioradimark 14071)according to the manufacturer's instructions.The concentration of released DEX was determined using high performanceliquid chromatography(HPLC,LC-2010 AHT,Japan Shimadzu Co.,Ltd.,Japan)at 240 nm by reference to a pre-established DEX standard curve[22].6.Cell culture: MSCs were obtained by aspiration of bone marrow from 3 week-old male Sprague Dawley rats and culture of the whole marrow[23].Purified MSCs were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12(DF12,Sigma)supplemented with 10% fetal bovine serum(Hyclone)and cultured until 80% confluent.P2 cells were used for analysis and were removed from culture by trypsinization then counted with a hemocytometer.Fifteen mm diameter coverslips that were coated with electrospun nanofiberswere placed in each well of a 24-well plate,ensuring that they were fully inserted using an instrument with an appropriately-sized stainless-steel ring.The fibers were sterilized with an ultraviolet lamp for 2 h on each side then 2×104 MSCs were seeded in each well(n=6)and low glucose DMEM(LG,Sigma)added.The culture medium was changed every 3 days.The negative control comprised glass coverslips with normal culture medium.Positive controls were Zein/PLLA-coated coverslips with osteogenic induction culture medium(low glucose DMEM supplemented with 10 mmol/L glycerol phosphate disodium salt hydrate,0.1 ?mol/L dexamethasone,50 ?mol/L L-ascorbic acid sodium salt).7.Cell adhesion and viability assay7.1 Cell adhesion on materials evaluated using SEM: The adhesion,motility and morphology of MSCs cultured on Zein/PLLA,Zein-DEX/PLLA-BMP-2 and virgin coverslips were observed by scanning electron microscopy(SEM,S-3400 N Japan Hitachi Co.,Ltd.,Tokyo,Japan).Samples were removed from culture after 12 hours or 7 days and washed 3 times with PBS then fixed with 4% glutaraldehyde for 2 hours.They were then dehydrated and in an ascending ethanol gradient then frozen and freeze-dried overnight.7.2 Cell adhesion on materials loaded with 5-FITC conjugated rh BMP-2 observed using CLSM: In order to verify that rh BMP-2 was successfully loaded into the coaxial electrospun nanofiber and to observe MSC adhesion and differences in morphology on different materials,the core aqueous solution was formulated with 5-FITC-conjugated rh BMP-2 when preparing the nanomaterials.Furthermore,a yellow-orange fluorescent dye(PKH26)that stains the lipid regions of the cytoskeleton was utilized.One m L of the cell suspension containing 2×104 viable cells was seeded into 24-well plates containing five different groups of scaffolds(NC,Zein/PLLA,Zein-DEX/PLLA,Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2).After 36 hours,the MSCs were stained with 4',6'-diamidino-2-phenylindole(DAPI)for 30 minutes then observed using a Confocal Laser Scanning Microscopy(CLSM,Leica TCS SP8,Germany)and images analyzed using Leica Microsystems software.8.Evaluation of cell proliferation using CCK-8 assay: Evaluation of the cytotoxicity of electrospun scaffolds and their effect on MSC proliferation were detected using a CCK-8 assay.The scaffolds were placed into 24-well plates and cells seeded with 2.0×104 viable cells.At specific time points(1,3,5 and 7days),200 ?L of CCK-8 reagent were added to each well and incubated for 1 hour at 37°Cprior to transfer to the wells of a 96-well plate.Optical density(OD)was measured at 450 nm using a microplate reader(Bioradimark 14071).9.Osteogenic differentiation of MSCs9.1 Alkaline phosphatase(ALP)activity and quantitative assay: Alkaline phosphatase(ALP)is an early marker of osteoblast differentiation and mineralization.In this study it was stained using the Gomori staining method.Five,10,15 and 20 days after cell seeding,the scaffolds were fixed with 70% alcohol for 10 min,rinsed three times with double distilled water then the Gomori reagent was added at room temperature.Protein concentration was estimated from a standard curve according to the manufacturer's instructions allowing quantitative analysis of ALP expression.9.2 In vitro mineral deposition Alizarin red staining and quantitative assay: Calcium mineralization that was induced by the different electrospun scaffolds was stained with Alizarin Red S(ARS).After 7,14 and 21 days,the samples were washed three times with PBS,fixed in 4% paraformaldehyde for 30 min at 4°C then immersed in 1% ARS solution for 45 min at room temperature.Mineralization was visualized using an optical microscope.The calcium mineralization products of ARS were dissolved with 10% cetylpyridinium chloride(Sigma-Aldrich,USA)in water for 10 min at 37°C.The absorbance of the ARS extracts was measured at 562 nm with a microplate reader.9.3 Immunofluorescence staining of Osteogenic proteins(RUNX-2+CD90,OPN+CD90): Osteogenic differentiation of MSCs was confirmed using immunofluorescence staining by employing the MSC-specific marker CD90 combined with the osteoblast-specific markers Osteopontin(OPN)and runt-related transcription factor 2(RUNX2).The MSCs on the scaffolds were simultaneously incubated with mouse anti-CD90 primary monoclonal antibody(1:1000 dilution in 5% BSA,ab225,Abcam,Cambridge,UK)and either rabbit monoclonal anti-OPN primary antibody(1:100 dilution in 5% BSA,ab91655 Abcam,Cambridge,UK)or rabbit monoclonal anti-RUNX2 primary antibody(1:1600 dilution in 5% BSA,#12556,CST,USA).After washing with PBS,the samples were incubated for 1h with one of two secondary antibodies at room temperature:(Alexa Fluor 488 donkey anti-mouse Ig G,1:1000 dilution,Invitrogen Co.,Carlsbad,CA,USA)or(Alexa Fluor 568 donkey anti-rabbit Ig G,1:1000 dilution,Invitrogen Co.,Carlsbad,CA,USA).The scaffolds were washed three times with PBS for 5 min to remove excess stain.Finally,the cells were incubated with DAPI(Invitrogen Corp,Carlsbad,CA,USA)at a dilution of 1:1000 for 30 min.The samples were then removed from the 24-well plates and mounted on glass slides using mounting medium prior to observation using CLSM(Leica TCS SP8,Germany)and analysis using Leica Microsystems CMS Gmb H software.10.Statistical analysis: All quantitative results were presented as mean ± SD.Statistical analyses were performed using SPSS 13.0 software.Differences between groups were considered to be significant if p< 0.05.Rusults:1.It is generally considered important to simulate the natural ECM scaffold structure,such as fiber diameter,porosity and surface morphology when fabricating a scaffold for engineeringa tissue[24].The scaffolds were continuous,uniform and smooth with interconnected pores between the nanofibers.Incorporation of the therapeutic molecules increased the diameter of the fibers.The Zein/PLLA nanofibers had the smallest diameter(305 ± 73nm)while Zein-DEX/PLLA(389 ± 103nm)fibers prepared by coaxial electrospinninghad the largest diameter.Zein/PLLA-BMP-2(312 ± 76 nm)and Zein-DEX/PLLA-BMP-2(335 ± 69 nm)nanofibers were intermediate in size.TEM images of the coaxial Zein/PLLA and Zein-DEX/PLLA-BMP-2 nanofibers that the two materials prepared by coaxial electrospinning have a continuous core-shell structure.The boundary between core and shell structure in the Zein/PLLA coaxial nanofibers is very clear.However,the boundary became blurred after the addition of the bioactive molecules and the thickness ratio of inner to outer layer became larger.2.Effects of electrospinning components on surface hydrophobicity of nanofibrous scaffolds: The hydrophilicity of scaffolds plays a key role in determining their overall performance in tissue engineering applications.This was determined using water contact angle(WCA)equipment.The WCA of Zein/PLLA and Zein-DEX/PLLA were approximately 130.4 ± 2.4° and 131.1 ± 2.3°,respectively.Following initial contact with these surfaces,the water droplet maintained a spherical shape after 30 s,indicating that the nanofiber mats remained hydrophobic,DEX in the nanofiber having no influence on the hydrophilicity of the nanofiber scaffolds.Furthermore,the WCAs of the Zein/PLLA(124.8 ± 1.6°)and Zein-DEX/PLLA(128.3 ± 2.6°)nanofibers after 30 s were much higher than that of Zein/PLLA-BMP-2(93.2 ± 2.1°)and Zein-DEX/PLLA-BMP-2(76.4 ± 1.9°),which had decreased,their surfaces becoming more hydrophilic.Zein is a hydrophobic protein,however,pure Zein nanofiber mats resulted in poor morphological stability with a lower WCA,tending to collapse into a film[25].In the case of Zein/PLLA coaxial electrospun fibers,the nanofiber scaffolds remained hydrophobic because of the good morphological stability of the core PLLA structure when water contacted the core structure.3.In vitro drug release: The release of BMP-2 and DEX from Zein-DEX/PLLA,Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2 nanofiber scaffolds was shown in study.Each drug was released faster from the nanofibers when incorporated individually compared with the dual-drug-loaded nanofibers.DEX demonstrated an early rapid release from the nanofibers because of dissolution or phase separation due to DEX being located in the outer layer of the nanofibers.Conversely,BMP-2 demonstrated mild,slow release similar to zero-order release kinetics due to protection from the outer shell acting as a barrier,suggesting that in terms of continuous release of BMP-2,the core-shell structure performed better than single electrospun nanofibers[26].More meaningfully,the dual-drug-loaded system demonstrated a sequential release pattern,where DEX was released quickly and in large quantities for the first three days,during which the release of BMP-2 was at a low level,gradually increasing to a higher and sustained level.The drug release occurred in three stages(Stages I,II,III)referring to an early rapid release,decreased release then followed by sustained release.There was an early rapid release of DEX(within 12 h)from the Zein-DEX/PLLA-BMP-2 and Zein-DEX/PLLA nanofibrous mats during stage I,as shown in Fig.3A,with cumulative release of 37.8% and 52.5%,respectively.From 12 to 72h(stage II)a decreasing rate of release was observed with 53.5% and 70.7% released,respectively.The cumulative DEX release from the Zein-DEX/PLLA was significantly higher than from Zein-DEX/PLLA-BMP-2.After 72 h,70.8% and 81.1% of the total DEX had been released from Zein-DEX/PLLA-BMP-2 and Zein-DEX/PLLA nanofiber mats,respectively,the rate of release slowing to a sustained rate.Furthermore,the release profiles of BMP-2 from Zein-DEX/PLLA-BMP-2 and Zein/PLLA-BMP-2.The BMP-2 release profiles were initially slow with steady growth within the first 6 h(5.3% and 6.7% released,respectively),without an initial burst release(Fig.3A,stage I).Subsequently,BMP-2 release became stable and sustained(stage II).After 7 days the release increased,followed by constant release(stage III).The quantity of BMP-2 released from Zein-DEX/PLLA-BMP-2 and Zein/PLLA-BMP-2 reached 40.5% and 53.7% after 21 days,respectively,the release from Zein-DEX/PLLA-BMP-2 being slower.The difference was attributed to the slightly higher hydrophobicity of the Zein-DEX/PLLA-BMP-2 due to the addition of hydrophobic DEX[27].The release results demonstrated that approximately 45-55% of the loaded BMP-2 remained within the scaffolds for future release after 21 days of continuous release.4.Cell adhesion and viability analysisSEM images showed that Zein/PLLA and Zein-DEX/PLLA-BMP-2 nanofiber scaffolds 12 hours after inoculation with MSCs demonstrated very good cell adhesion.In particular,the MSCs adhered onto Zein-DEX/PLLA-BMP-2 had already stretched along the nanofiberscaffold in all directions.Magnified SEM images show that they were tightly anchored along the fibers and exhibited extensive cellular pseudopods projecting along the direction of the fibers.After 7 days culture,the MSCs on the Zein/PLLA and Zein-DEX/PLLA-BMP-2 appear to be larger and flatter than on the NC,however,there were more cells which were larger that were growing on the Zein-DEX/PLLA-BMP-2 nanofiber scaffold.The proliferation and viability of MSCs on nanofiber scaffolds was evaluated by CCK-8 assay after 1,3,5 and 7 days.One day after seeding,the cells on Zein-DEX/PLLA-BMP-2 were morphologically similar to those on the NC.Because the burst release of DEX resulted in slight toxicity,the cellular activity and proliferation of cells cultured on Zein-DEX/PLLA nanofibers was lower than other scaffolds after 1 and 3 days.However,cell proliferation on the Zein-DEX/PLLA and Zein-DEX/PLLA-BMP-2 was significant after 5 and 7 days,and the rate of release of DEX at these time points was considered to be the appropriate rate for accelerating cell proliferation.In addition,the proliferation of cells on Zein-DEX/PLLA-BMP-2 was greater than on Zein-DEX/PLLA.Immunocytochemical images of MSCs on nanofiberscaffolds showing morphology and proliferation after 36 hours of culture were shown.The cells had grown and were well spread on the nanofibers.However,the numbers of cells on Zein/PLLA and Zein-DEX/PLLA nanofibers were lower than on the Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2 nanofibers.The MSCs cultured on the nanofiber scaffolds were thinner than those on the NC and had a polygonal morphology,demonstrating good adhesion to the scaffolds and clustered growth.Furthermore,the Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2 nanofibers loaded with 5-FITC-BMP-2 emitted green fluorescence,demonstratingthat BMP-2 could be successfully loaded into the core-shell structure nanofibers.5.Osteogenic differentiation analysis5.1 Alkaline phosphatase(ALP)activity and quantitative analysis: ALP activity is a marker of osteogenic differentiation and plays an important role in the formation of mineral deposits.Study showed that 10 days after the start of cell culture,ALP began to be observed in the cells on Zein/PLLA-BMP-2,Zein-DEX/PLLA-BMP-2 and PC scaffolds,but not clearly on the others.ALP activity increased in all test groups over time.Significantly increased ALP activity was observed in Zein-DEX/PLLA,Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2 which were loaded with BMP-2 and/or DEX compared with Zein/PLLA or NC.In addition,the ALP activity of cells on Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2 was significantly higher after 15 and 20 days than on Zein-DEX/PLLA.Increased activity was caused by the continuous release of BMP-2 from the nanofibers resulting in a higher concentration of osteogenic differentiation factors in the medium.More significantly,large,dense nodules high in ALP activity were observed on the Zein-DEX/PLLA-BMP-2 scaffold.5.2 Analysis of mineralization of differentiated osteogenic cells: Calcium deposition and calcium nodules were further characterized through alizarin red staining(ARS).Quantitative data demonstrated significant differences between every group compared with the negative control(*p ? 0.05).Zein-DEX/PLLA,Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2 scaffolds loaded with BMP-2 or DEX and the PC demonstrate clear positive staining,whereas both Zein/PLLA and the NC showed almost no positive staining.More importantly,Zein-DEX/PLLA-BMP-2 scaffold double-loaded with BMP-2 and DEX showed the greatest positive staining,demonstrating the synergistic effect of BMP-2 and DEX combined with the intrinsic ability of the fiber surface topography which provided osteoinduction.5.3 Analysis of osteogenic proteins from differentiated osteogenic cells: R UNX2 and OPN are bone specific proteins related to bone formation.RUNX2 is crucial for the maturation of osteoblasts and is involved in osteoblast differe ntiation and skeletal morphogenesis[28].OPN is involved in the attachment of ce lls to mineralized bone matrix,binding hydroxyapatite with high affinity after s ecretion.It is involved in the regulation of normal mineralization within the ex tracellular matrices of bones and teeth[29,30].Immunofluorescence images of RU NX2 and OPN 14 days after MSC seeding demonstrated that the cells expresse d the MSC-specific marker CD90.These cells differentiated along the osteogeni c lineage to express RUNX2 and OPN proteins in addition to CD90.These results confirm the osteogenic differentiation of MSCs after culture on Zein-DE X/PLLA,Zein/PLLA-BMP-2,Zein-DEX/PLLA-BMP-2 scaffolds and the PC b y expressing both CD90 and RUNX2 in addition to CD90 and OPN.Very littl e expression of RUNX2 or OPN was observed on the NC or Zein/PLLA.Mor e RUNX2 and OPN was expressed on Zein/PLLA-BMP-2 and Zein-DEX/PLLA-BMP-2 scaffolds because of the incorporation of BMP-2 into the scaffolds,a t rend that was consistent with the results of ALP expression.DEX and BMP-2 utilize different mechanisms to accelerate bone repair,but they also exert a syn ergistic effect.DEX can modulate the effect of BMP-2 on stem cell differentiat ion[31-33].Moreover,cells cultured on Zein-DEX/PLLA-BMP-2 scaffolds exhibite d the characteristic cuboidal morphology of osteoblasts and expressed RUNX2 and OPN representing complete osteogenic differentiation.Conclusion:1.In this study,we fabricated novel Zein/PLLA nanofibers preloaded with BMP-2 and DEX by coaxial electrospinning for dual controlled release for bone tissue engineering applications.2.This drug-delivery system showed a sequential release pattern.Locating DEX in the shell layer resulted in burst release during the initial stage.However,incorporation of BMP-2 that had been dissolved in BSA solution into the core of the nanofibers,the release was slow with a steadily increasing profile.3.Cell adhesion and viability analysis demonstrated that cell proliferation was more pronounced on the Zein/PLLA coaxial electrospun nanofibers with dual controlled-delivery of DEX and BMP-2.4.Osteogenic differentiation analysis showed that by combining BMP-2 with DEX,the Zein-DEX/PLLA-BMP-2 nanofiber scaffold induced significantly greater ALP activity and ECM mineral deposition and was superior to the PC.The synergistic effects of BMP-2 and DEX combined with the intrinsic ability of the surface topography of Zein-DEX/PLLA-BMP-2 nanofiber scaffold provided rapid and effective osteoinduction.5.The cells cultured on Zein-DEX/PLLA-BMP-2 scaffolds exhibited the characteristic cuboidal morphology of osteoblasts and expressed RUNX2 and OPN representing complete osteogenic differentiation.6.These results establish that the controlled dual release of BMP-2 and DEX by Zein/PLLA core-shell electrospunscaffolds stimulates not only early cell adhesion but also osteogenic protein expression during differentiation and are therefore suitable for use in bone tissue engineering applications. |
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