Electrospun Fibers With Loading Of Plasmid DNA/Calcium Phosphate Nanoparticles As Potential Scaffolds For Vascular Tissuse Engineering

Electrospun polymeric fibers with uniform and continuous diameters down to a nanoscale dimension, closely mimic the size scale of fibrous proteins found in natural extracellular matrix (ECM), and have a very high fraction of surface with porous construction available to interact with cells. The electrospun nanofibrous scaffold mediated bioactive macromolecules delivery shows potentials as inductive tissue engineering scaffolds, which can not only support the cell proliferation and migration, but also maintain the cell functions and phenotype-specific activities, enhance the ECM secretion, and promote the tissue regeneration. On the other hand, vascularization is a capital challenges in tissues engineering, associating with regenerated tissues supplied with enough nutrients, which is the key issues of getting fully vascularized in engineered tissues. In this study, plasmid DNA (pDNA) encoding bFGF and VEGF were encapsulated into calcium phosphate nanoparticles, and electrospun fibers encapsulated pDNA/calcium phosphate composite nanoparticles were prepared, which were investigated to promote the regeneration of blood vessels.Reverse microemulsion was used in this study to synthesize calcium phosphate/pDNA nanoparticles. The average size of nanoparticles was110nm, and the entrapment efficiency was over95%. Integral structure of the pDNA was protected from digesting by DNase for calcium phosphate/pDNA composite nanoparticles, and the nanoparticles can controlled the release of pDNA. The cell experiments showed a high level of cell viability and transfection efficiency on HUVECs and SMCs, which showed the advantages as a gene vector.Electrospun fibers with encapsulated composite nanoparticles containing pDNA encoding VEGF and bFGF or both of them were prepared by electrospining, which showed regular morphology, and composite nanoparticles were encapsulated into fibers by laser scanning confocal microscope (LSCM). Biodegradable electrospun fibers with loadings of calcium phosphate/pDNA nanoparticles remained a sustained release of pDNA for around4wk, consistent with the periodicity of vascularization in vitro release, when polyethylene glycol (PEG)of2kDa was added to polymeric solution with the ratio of10%. Human umbilical vein endothelial cells (HUVEC) and smooth muscle cells (SMC) were seeded on pDNA-loaded fibrous scaffolds to investigate the capabilities of promoting cell proliferation, transfection and ECM secretion. The results indicated the promotions of cell adhesion and proliferation and ECM secretion by the autocrine of growth factors, compared to pDNA-infiltrated fibrous mats. An effective cell transfection and target protein expression proceeded for over28days. The pDNA-loaded fibrous scaffolds were subcutaneously implanted into rats to study their angiogenesis via macroscopic observation, hematoxylin-eosin staining and immunohistochemical staining. The results showed that the pDNA-encapsulated fibrous scaffolds could effectively enhance the expression of CD31, collagen IV and a-smooth muscle actin. After4weeks implantion, the result of microvessel density was3times more than pDNA-infiltrated fibrous mats, demonstrating the potential application as scaffolds in vascular tissue engineering.