Fabrication And Characteration Of PEGFP-N1-BMP-2/PLGA Core-shell Nanofibers By Coaxial Electrospinning

Gene activated matrix（GAM） is a kind of local release system for gene by combining biological scaffold materials with plasmid DNA carrier. GAM can provide a certain mechanical strength and biodegradable porous scaffolds for cell growth, which can provide space support for seed cells during the tissue engineering process. It can also transfect seeded cells intentionally by carrying genes, such as plasmid DNA, a mixture of gene plasmid and transfection reagents, directly with stent materials. Finally, it can promote tissue regeneration by secreting growth factors which are beneficial to tissue reparation and inducing specifically the directional differentiation of seeded cells. Electrospinning is a kind of simple method to prepare nanofibers. The nanofibrous membrane prepared by electrospinning has the advantages of high specific surface area, large porosity, which are similar to the structure of natural extracellular matrix and are beneficial to cell adhesion, growth and reproduction. For traditional electrospinning, plasmid needs to be mixed with biodegradable polymer materials. As a result, plasmid would inevitably contact with organic solvent in electrospinning process, which may cause a certain influence to the activity of plasmid. In addition, blended to nanofibrous scaffold directly would result in a burst release of genes.In the present study, coaxial electrospinning was chosen to prepare core/shell nanofibers consists of poly lactic-co-glycolic acid（PLGA） and plasmid microspheres loaded with Bone Morphogenetic Protein 2（BMP-2）, aiming at the preparation of a new GAM possessing both good biocompatibility and controlled-release effect.Objectives: The purposes of this study are to prepare PLGA and BMP-2 plasmid core/shell nanofibers by coaxial electrospinning, to characterize its microstructure, physical and chemical properties, and biocompatibility through being co-cultured with the human periodontal ligament stem cells, and to provide a research foundation for further application of GAM by measuring the transfection efficiency and BMP-2 expression..Contents: 1. Restriction enzyme digestion of carrier, amplification of purpose fragment, construction of p EGFP-N1-BMP-2 recombinant plasmid by PCR and sequencing identification, amplification and extraction of recombinant plasmid.2. Preparation of PEI/PLGA coaxial nanofiber, determination of parameters suitable for coaxial electrospinning. Preparation of p EGFP-N1-BMP-2/PLGA coaxial nanofibers, characteration of fibrous microstructure, physical and chemical and degraded performance.3. Culture of the original periodontal ligament stem cells, morphology evaluation by HE examination, identification of surface molecular markers of stem cell by flow cytometry, confirmation of multi-directional differentiation ability. Biocompatibility evaluation by co-cultivating. The periodontal ligament stem cells with p EGFP-N1-BMP-2/PLGA fibers, detection of its transfection efficiency by laser scanning confocal microscopy, and evaluation of the transfection efficiency and BMP-2 expression.Results: 1. Linearized carrier of 4.7 Kbp was obtained after restriction enzyme digestion; targeted gene of 1191 bp was obtained by PCR; recombinant plasmid was obtained After connecting the linearized carrier and targeted gene; gene fragments of about 1.4 Kbp was amplified by PCR, and the result of sequencing is consistent with the transcript of h BMP-2（NM₀01200） in Gene Bank.2. Diameter of PEI/PLGA nanofiber prepared by coaxial electrospinning is bigger than that of PLGA fiber, and the coaxial structure can be observed under confocal laser scanning microscopy. But its ultimate strength and elastic modulus are smaller than that of PLGA fiber. Surface smoothness of p EGFP-N1-BMP-2/PLGA coaxial electrospinning fibers is worse than that of p EGFP-N1-BMP-2/PLGA blended fiber and PLGA fiber. Plasmid is distributed in the middle of the fiber under TEM. There is no difference in porosity among the three groups, while the contact angle of p EGFP-N1-BMP-2/PLGA coaxial electrospinning fibers locates between that of BMP-2 / PLGA blended fibers and PLGA fibers, and its bibulous rate is lower than that of p EGFP-N1-BMP-2/PLGA blended fibers, either is its mechanical strength. However, its degradation is more smoothly than that of the blended one.3. Periodontal ligament stem cells were isolated successfully by enzyme digestion and limited dilution method, indicating certain clone formation ability. They meet the features of stem cell after Flow cytometry analysis, namely the multipotential differentiation ability. Human periodontal ligament stem cells stretched fully and grew well on the surface of the p EGFP-N1-BMP-2/PLGA coaxial fibrous membrane. MTT test showed that p EGFP-N1-BMP-2/PLGA coaxial fiber membrane has better biocompatibility than p EGFP-N1-BMP-2/PLGA blended fiber membrane. The green fluorescent protein can be observed by confocal laser scanning microscopy in all the group, while the fluorescence reduces（or degrades） faster in p EGFP-N1-BMP-2/PLGA blended fiber membrane group than in the coaxial group. Flow cytometry instrument showed the transfection efficiency of coaxial p EGFP-N1-BMP-2/PLGA group is higher than that of blended p EGFP-N1-BMP-2/PLGA group. BMP-2 expression of blended p EGFP-N1-BMP-2/PLGA fiber is generally lower than that of coaxial p EGFP-N1-BMP-2/PLGA fiber, except for the four-hour group.