Preparation And Performance Study Of Double-layer Dual-orientation Biomimetic Vascular Tissue Engineering Scaffold

With the development of social economy and the improvement of people's living standards,cardiovascular disease has become a huge obstacle to human health.At present,large and medium-diameter artificial blood vessels have been used in clinics and have achieved good results.Small-diameter artificial blood vessels are prone to cause thrombosis and restenosis.The low long-term patency rate limits its clinical application.A tissue engineering scaffold mimicking natural vascular extracellular composition and structure can be constructed by the combination of applied materials science and biological science,while conducting tissue engineering co-culture with vascular cells in vitro can reduce the immunogenicity of the graft,prevent the thrombosis and improve the long-term transplant.Which make the tissue-engineered blood vessels constructed in vitro a great potential artificial replacement for severely diseased blood vessels.The extracellular matrix(ECM)of the natural blood vessel wall is a three-dimensional nanofiber network composed of collagen and elastin,on which cells exhibit multi-layer and dual-oriented growth.The artificially constructed vascular tissue engineering scaffold needs to have suitable mechanical properties,biocompatibility,degradation properties and ECM-like structures.Ultrafine fibers in the range of micrometers to nanometers can be prepared by electrospinning,which can well mimic the ECM structure and function of natural blood vessel.In this paper,a dual-oriented/bilayered tubular small diameter polymer composite nanofiber scaffold was firstly prepared by electrospinning,and the physical and chemical properties and the tissue engineering process of the scaffold were studied.By studying the hydrophilicity and mechanical properties of different polymer fiber membranes,it was finally determined that the tubular scaffolds were prepared by using the composite materials of polycaprolactone(PCL):polylactic acid glycolic acid copolymer(PLGA):gelatin(GE)mass ratio=1:1:1.In order to mimic the structure and function of natural blood vessels as much as possible,we modified the electrospinning device and designed a two-step electrospinning method to prepare a double-layer bidirectional tubular small-diameter vessel scaffold.By characterizing the morphology of the electrospun nanofibers and the structural characteristics of the tubular scaffolds,it was confirmed that the tubular scaffolds have the structural characteristics of functional cell-oriented arrangement growth in natural blood vessels.Secondly,the mechanical properties,hydrophilicity and in vitro degradation of the scaffolds were studied and analyzed.The ultimate tensile stress of the scaffolds was 2.04±0.15MPa,the elongation at break was 342.48±4.13%,and the fracture pressure of the scaffolds reached 111.03±6.03 kPa under wet conditions;The scaffolds is completely hydrophilic,and the contact angle changes to 0 when the water drops are on it for 5 seconds.At week 4,significant degradation of the scaffold had occurred,and the weight of the scaffold degraded to 49 ±2%of the initial weight at the 12th week.After in vitro inoculation of smooth muscle cells(SMCs)and endothelial cells(ECs),it was found that the scaffold had a good promoting effect on the proliferation and growth of cells,and the orientation structure of fibers played a good contact guiding effect on the growth of cells,the cells grown on it exhibit the same orientation and growth as the fibers;Scanning electron microscopy(SEM)observed that the pseudopodia of cells firmly grasped or crossed a single fiber and had a tendency to migrate and grow into the fiber membrane.After staining the cytoskeleton,it was found that the cytoskeleton grew along the orientation of the fibers,and the cells presented a spindle structure.These results indicate that the composite material PCL:PLGA:GE=1:1:1(PPG is used to represent the composite material)has good cellular activity and biocompatibility.Finally,we designed and prepared a bionic natural vascular physiological microenvironment with dual-circulation dynamic perfusion system,and performed in vitro tissue engineering of the tubular scaffold for 4 weeks.HE staining results showed that the three-dimensional tubular scaffold presented a loose porous structure,and abundant light-pink stained collagen fibers were observed,forming a structure like natural vessel wall ECM,which was conducive to the migration and growth of subsequent cells to the scaffold.In a word,dual-oriented/bilayered nanofiber scaffolds with functional cell-oriented arrangement structure of natural vascular wall were successfully prepared by improving the electrospinning method.The results show that the tubular scaffold can meet the mechanical,hydrophilic,biocompatible and in vitro degradation requirements of vascular tissue engineering scaffolds.The orienting fibers can provide contact guidance to the cells growing on them,and the pore size of the orienting fibers is enough to support the migration and growth of vascular SMCs into the scaffold.Tissue engineering results show that it has a loose and porous structure and abundant light pink collagen fiber,which indicates the formation of a natural vascular-like ECM structure.