Fabrication And Characterization Of Nanoyarn Reinforced Three-Dimensional Nanofibrous Scaffold And Heparin Loaded Nanofibrous Scaffold For A Novel Small-Diameter Endovascular Covered Stent For Aneurysm Therapy

Tissue engineering scaffold which provide the provisional structural and anchoring support for the seeded cells plays an essential role in successful tissue regeneration and organ reconstruction. Therefore, the design of tissue engineering scaffold has become an important field in biomaterials and regenerative medical research. A desirable tissue engineering scaffold should not only own favorable biocompatible to support cell attachment and proliferation and porous architecture to promote the ingressing of cells but also have enough mechanical strength in the process of tissue regeneration. The electrospun nanofibrous scaffold (ENS) owning high porosity and interconnected porous architecture could mimic the construction of extracellular matix (ECM), thereby facilitate cell adhesion and proliferation. However, the structure of ENS may be damaged in the process of physical manipulation and tissue regeneration due to the break of single ultra-thin fibers, which may impede the growth of regeneration tissue and be unfavorable for functional recovery. On the other hand, monolayer growth pattern of cells on the surface of traditional ENS would limit its application in tissue regeneration.Here, we modified the typical process of electrospinning and succeeded to fabricate nanoyarns bundled by poly L-lactic acid nanofibers (PLLA) via a rotating funnel. Subsequently, the electrospun nanofibers and continuous nanoyarns were collected by a rotating mandrel in the meantime and eventually formed the nanoyarns reinforced three-dimensional nanofibrous scaffolds (NRS). Scanning electron microscopy (SEM) images revealed the nanoyarns with rough surface were bundled by aligned nanofibers and the scaffold was composed of random nanofibers and aligned nanoyarns. The NRS still keeping the ECM-like structure possed high surface-to-volume ratio and interconnected porous architecture. The result of water contact angle test (WCA) and apparent density test respectively showed NRS had a rougher surface compared with that of ENS and the porosity of NRS was higher than that of ENS. X-ray diffraction (XRD) and differential scanning calorimetry analysis (DSC) cleared the crystal structure of PLLA and thermodynamics of NRS had been changed due to the fabrication of the nanoyarn. Stress-strain testing demonstrated that the NRS had excellent mechanical properties. The degradation of ENS and NRS in vitro were studied in phosphate buffered saline (PBS) at37℃, no obvious change occurred in process of the degradation according to the SEM images, which suggested the degradation rate of ENS and NRS were very slow, which had been demonstrated by the result of loss weigh and pH changes of PBS solutions. However, the molecule weigh would decrease clearly according to the result of gel permeation chromatography (GPC). The stress of NRS parallel to nanoyarns was better than that of ENS with the degradation in PBS. Pig iliac endothelial cells (PIECs) and mouse fibroblast cells (L929) were utilized to examine the biocompatibility of the NRS and ENS in vitro. MTT assay showed the NRS could support cell adhesion and proliferation better than ENS. SEM images confirmed that the cells planted on NRS exhibited extremely stretched phenotypes and spread out well on the nanoyarn scaffolds. Histological analysis (H&E staining) illustrated that the cells penetrated into the inner parts of the NRS, while on the nanofibrous scaffolds, the cells were restricted to proliferate on the surface.Aneurysm with high mortality and disability is a common angiocardiopathy. Endovascular treatment has become the preferred choice for craniocervical vascular diseases such as aneurysm with the development of medical technology. However, similar to the small-diameter vascular regeneration and reconstruction, the acute thrombosis and intimal hyperplasia also occurs in the treatment with covered stent, which delayed the development of small-diameter edovascular treatment. Thus, a novel anticoagulant covered stent is needed to avoid these disappointing long term results. In general, the localized delivery of heparin to the site could effectively avoid the formation of acute thrombus. So, we tried to fabricate a novel heparin loaded covered stent.To avoid the co-axial spinning nozzle used in electrospinning, emulsion electrospinning technique has been developed to fabricate the heparin loaded poly(1-lactide-co-caprolactone) P(LLA-CL) core-shell nanofibers and formed heparin loaded P(LLA-CL) nanofibrous scaffold (PLCL-Hep-NS) in this study. SEM images illustrated the heparin loaded nanofibrous matrix had nanofibrous structure. Transmission electron microscopy (TEM) images revealed that PLCL-Hep-NS had a novel multi-cores inner-structure, which was different from conventional core-shell structure of coaxial nanofibers. Fluorescence microscopy (FM) images demonstrated the cores containing water-soluble FITC had been incorporated into nanofibers and well distributed in each part of PLCL-Hep-NS. Fourier transform infrared spectroscopy (FTIR) demonstrated that heparin had been loaded into the PLCL-Hep-NS. XRD, DSC and thermogravimetry analysis (Tg) demonstrated the crystal structure and thermodynamics of P(LLA-CL) had not been transformed in the process of electrospinning. WCA confirmed the PLCL-Hep-NS was of good hydrophilicity with the addition of Span80and heparin. Stress-strain testing demonstrated that the PLCL-Hep-NS had a comparable mechanical properties compared with P(LLA-CL) nanofibrous scaffold (PLCL-NS). The scaffolds incorporated with heparin showed better blood compatibility compared by PLCL-NS. However, PIECs and L929were utilized to examine the biocompatibility of PLCL-Hep-NS, MTT assay showed PLCL-Hep-NS could not support cell proliferation better than PLCL-NS. The endovascular metallic stent covered with PLCL-Hep-NS was fabricated for aneurysm treatment in small-diameter artery. The aneurysm was immediately obliterated after stenting. Angiogram at14days follow-up showed that the aneurysm was still invisible and the parent artery was patent without stenosis.