Fabrication And Characterization Of NAC-loaded PLGA Control-released Electrospinning Scaffolding System & Case Report

Biodegradable PLGA polymer is choose as bone engineering scaffold in this study,and MSNs is used as nanocarrier for the loading of the small molecule NAC,which is antioxidantive and osteogenic.r BMSCs are used as stem cell.A biomaterial scaffold with separate compartments for the promotion of osteogenesis is synthesized.Biocompatibility,mechanical properties cell proliferation on scaffold and oseteopromotive property of scaffold are analyzed.Objective: This study aimed to develop a unique osteo-promotive NAC-loaded PLGA electrospun scaffold with separate compartments,which can release NAC both in burst release and controlled release.The physical and chemical properties,biocompatibility and osteogenic potential of scaffolds were evaluated.Experiment ?.The synthesis and characterization of MSNs and NAC-loaded MSNsMethod: CTAB and Na OH(2 M)were added to distilled water.The solution was heated and after 4 hours of stirring TEOS was added dropwise to the solution.The sample was washed with distilled water and ethanol after reaction and then collected through centrifugation at 10,000 rpm.The surfactant was removed through three extraction cycles in a mixture of HCl and ethanol at 90°C.MSNs were obtained through centrifugation and then freeze-dried for 12 hours.The morphologies of MSNs were observed through scanning electron microscopy(SEM)and transmission electron microscopy(TEM).In loading NAC onto MSNs,MSNs was immersed in NAC solution.After 48 hours of stirring in the dark,NAC-loaded MSNs(NAC@ MSN)were collected through centrifugation and then freeze-dried.The amount of NAC loaded onto MSNs was measured through thermogravimetric analysis(TGA)and Fourier transform infrared spectroscopy(FTIR).The drug-loading rate and chemical contents of NAC@MSN were confirmed through TGA and Fourier transform infrared spectroscopy.Results: SEM and TEM observations reveal that the sizes and morphologies of spherical MSNs have highly uniformity.Extensive pore structures can be viewed on the surfaces of MSNs.The FTIR spectra of NAC@MSNs exhibit characteristic peaks of NAC beside the characteristic peaks of MSNs.The TGA thermograms suggesting that an amount of NAC,~12.67 wt%,is loaded onto MSN.Experiment ?.The fabrication and physical analysis of PNNM scaffoldMethod: preparation of 4 group of scaffolds.1.Control group(Pure PLGA scaffold): PLGA were added to a mixture solvent consist of DMF and THF and then dispersed with magnetic stirring;2.PN scaffold(PLGA scaffold with free NAC): NAC were added to the mixture solvent and PLGA was added to the solution and then dispersed;3.PNM scaffold(PLGA scaffold with NAC@MSN): NAC@MSN were added to the mixture solvent and PLGA was added to the solution and then dispersed;4.PNNM scaffold(PLGA scaffold with free NAC and NAC@MSN): NAC and NAC@MSN were added to the mixture solvent and PLGA was added to the solution,and then the suspension was stirred until homogeneous.4 group of suspensions was electrospun at room temperature under the relative humidity of 50%.The applied voltage was controlled at 20 k V and the feeding rate of syringe pump was fixed at 2 m L/h.The distance between the needle and the collector was 15 cm.All of the groups of electrospun scaffolds were vacuum-dried for to remove the residual solvent and observed under SEM and TEM.The swelling rate,water contact angle,mechanical properties and degradation were tested and analyzed.Results: The surfaces of the pure PLGA and PN fibers appear smooth shown in SEM images,and the fibers of PNM and PNNM appear rough because of the bead-like structures presented on the surfaces of the fibers.The TEM images show that NAC@MSNs are homogeneously encapsulated within the electrospun PNM and PNNM fibers.The swelling ratios of all of the scaffolds are not significantly different.The contact angle value of the PNNM scaffold is lower than that of other scaffolds which means better wettability and hydrophilicity of the fibers.The PNNM scaffold displayed the highest harness and elastic modulus.After immersed in water for 21 days,the PLGA and PN fibers are fragmented,and some fibers are fused with one another,more pores are formed on the surface of PNM and PNNM fibers.Experiment ?.The kinetics of drug,release and biocompatibility and osteogenic potential of scaffolds were evaluatedMethod: 1.PN,PNM,and PNNM scaffold samples(20 mg,n=3)were immersed in 2 m L of distilled deionized water and cumulative NAC release from the scaffolds was detected through ultraviolet spectrophotometry at 24,72,120,144,168,216,and 264 hours;Other groups of samples(20 mg,n=3)were immersed in water and cumulative Si release was measured at 6,12,18,and 24 days through inductively coupled plasma–atomic emission spectroscopy(ICP–AES).2.The samples of PLGA,PN,PNM,and PNNM electrospun scaffolds were cut into 0.5×0.5 cm mats(n=5)and sterilized under UV;r BMSCs were seeded onto the scaffolds in 24-well plates and cultured.The samples were stained in the live/dead staining assay and phalloidin staining and visualized under confocal laser scanning microscopy(CLSM)3.The samples of PLGA,PN,PNM,and PNNM electrospun scaffolds were cut into 1×1 cm mats(n=2)and sterilized.r BMSCs were seeded onto the scaffolds in.96-well plates and were cultured for MTT test.4.The samples of PLGA,PN,PNM,and PNNM electrospun scaffolds were cut into 2×2 cm mats(n=2)and sterilized.r BMSCs were cultured and then prepared for q RT-PCR;and the samples were cut into mats(n=2)and sterilized.r BMSCs were seeded onto the sample in 24-well plates and were cultured.The mineralization nodules were stained with alizarin red and semi-quantitative analyzed then.Result:The release profile of NAC shows PN groups display an initial burst release while PNM scaffold displays controlled releases,and the PNNM group exhibits a biphasic pattern characterized by an initial burst-release stage and continuous release stage;Si is gradually and continuously released over a long period from PNM and PNNM sacffolds.Live/dead staining results reveal all of the prepared scaffolds are biocompatible and phalloidin staining results show that the cells can attach and spread on all of the PLGA scaffolds.In line with the MTT results,cell activity is significantly higher in the PNNM groups than other group.The m RNA expression levels,the alizarin red staining and quantitative analysis shows compared other groups of scaffold,PNNM scaffold possess the best capacity to induction of osteogenesis.Conclusion:1.After react and remove the solvent,dispersed and highly uniform spherical MSNs were acquired.Pore structures can be viewed on the surfaces of MSNs.The result of FTIR spectra and TGA thermogram proved that NAC is loaded at the amount of ~12.67 wt.% onto MSNs.2.The surfaces of the pure PLGA and PN fibers appear smooth and the fibers of PNM and PNNM appear rough with bead-like structures on the surfaces,which suggest MSN@NAC are homogeneously encapsulated within the fibers.PNNM scaffold displays highest wettability,highest harness and elastic modulus.After degradation,PNNM fibers are more stable than PLGA and PN fibers.3.PNNM group exhibits a biphasic pattern both in burst-release and controlled-release.Live/dead and phalloidin staining results reveal cytoactivity,cells attachment and spread on all scaffolds has no differences.MTT results shows cell activity is significantly higher in the PNNM groups.The m RNA expression levels,the alizarin red staining and quantitative analysis reveals PNNM scaffold possess the best capacity to induction of osteogenesis compared with other scaffolds.