The Study Of Using PCL/PVA/pNSR32 Electrospinning Nanomaterials As Tendon Tissue Engineering Scaffold

As polycaprolactone (PCL) has good biocompatibility, biodegradability, mechanical properties, easy processing of drugs, and the degradation product of the material in the body isn't harmful and so on, it has been widely used in the tissue engineering.However, due to lack of cell affinity sites on the PCL surface and the material's degradation is low, the modify of PCL is necessary to apply in the tendon tissue engineering.We have transferred the genes of spider dragline silk protein into the prokaryotic expression system of E. coli and introduce the arginine-glycine-asparticacid(RGD) tripeptide sequences into it at the same time to built a engineered bacteria named pNSR32/BL21 (DE3).Then we produced the RGD-recombinant spider silk protein named pNSR32(Mw:108kD) in mass through high-density fermentation. Due to the introduction of Arg-Gly-Asp tripeptide (RGD) to pNSR32, the recombinant spider silk can promote the adhesion and proliferation of the cells,which make up the shortcomings of PCL material.Polyvinyl alcohol-124 (PVA-124) is a water-soluble polymer materials,with relatively good mechanical properties, it has been utilized as a medical material.Due to PVA-124 is Hydrolyzed, it can improve the hydrophilic degradation of PCL.Electrospinning is a kind of technology used to make nano fiber, which has been widely applied in textile, medical, military, electronic and energy fields. Because the morphology of nano-fiber is similar with extracellular matrix (ECM),electrospinning has a potential prospect in tissue engineering.This study was base on the previous studies of our laboratory, we made the PCL/PVA/pNSR32 nanofiber material by electrospinning technology. In order to find out the best proportion of PCL and PVA and the best of volume pNSR32 which can produce the best nanofiber material, the spin materials was observed by scanning electron microscopy (SEM).At the same time we optimized the process parameters of the spinning. By IR analysis, the exsitant condition of the three components of the nano-materials was studied. At last, we measured the porosity rates of scaffold materials with Pycnometer.Results:①Under the voltage of 80kV, extrusion rate of 5mL/h, accept distance 20cm, temperature 45℃, PCL:PVA= 2:1 in the formic acid solvent system, add 5% RGD-pNSR32, diameter of the fibers in the nanofiber material is the most uniform. After studying,we find out that PCL,PVA and pNSR32 existed in the nanofiber with a simple physical form,the porosity of the PCL/PVA& PCL/PVA/pNSR32 nanofibral materials is at high level of 90.2% and 88.6% respectively.②In order to evaluate the degration of The PCL, PCL/PV PCL/PVA/pNSR32A, PCL/PVA/pNSR32 nanofibral materials,we impregnated the three kinds of electric spinning materials with the pH= 7.4 PBS solution and 0.1%(w%)lipase solution. After 4 months' test,the results show that, the degradation rates of PCL/PVA and PCL/PVA/pNSR32 composite materials are much better than PCL electric spinning material. It means the existance of PVA can effectively improve the material degradation rate.③The mouse fibroblast cells named NIH-3T3 were cultured with the extracted mediums of PCL/PVA and PCL/PVA/pNSR32 materials, then we evaluated cytotoxicity of the two materials through the MTT test according cytotoxicity grading standard of biological materials.The cells were cultured with treated PCL/PVA and PCL/PVA/pNSR32 materials directly.7 days later, the situation of the NIH-3T3 cells on the materials were observed.with SEM and HE staining. Used MTT test to analysed the proliferation of the cells. The results showed that the cytotoxicity of the two materials are qualified; After 7 days of culture, we found the NIH-3T3 cells adhere to both the surfaces of PCL/PVA and PCL/PVA/pNSR32; the MTT test showed that the proliferation of the NIH-3T3 cells on the PCL/PVA/pNSR32 surface is significantly better than on the PCL/PVA surface, because the RGD tripeptide in the pNSR32 can promote cell adhesion and proliferation.④Collagen (medical level), PCL/PVA/pNSR32 and PCL/PVA were implanted into SD rat's bodies,3d,7d,14d and 30d after the operation,the samples was drew out to make paraffin respectively,The paraffins were observed and analysised afeter HE staining to evaluate oganizational compatibility of the two materials, The results showed that, the organizational compatibility of PCL/PVA/pNSR32 composite scaffold are greatly improved compared to PCL/PVA scaffold, it can accelerate the healing of the wound effectively and reduce the inflammation of the organization around the materials. After 30 days inflammatory cells in the PCL/PVA/pNSR32 group has almost disappeared,and the inflammation subsided,There are a large number of tissue cells surrounding the material which is the same to the blank control group, the inflammatory response is under Grade I, and the wound healed completely. The histocompatibility order of each groups:collagen>PCL/PVA/pNSR32 scaffold>PCL/PVA scaffold.We believe that the PCL/PVA/pNSR32 scaffold can be applied in tendon tissue engineering because of it's compatibility.⑤The PCL/PVA/pNSR32 electrospinning scaffolds were made into bars, then we knitted 3 bars together into artificial tendon(length:30mm,Φ1.48mm) just like ponytail. Then mechanical propeties the artificial tendons and PCL/PVA/pNSR32 electrospinning scaffolds were tested, the tensile strengths and elongations at break were calculated.The results show that the tensile strength and elongation at break are increased markedly after PCL/PVA/pNSR32 electrospinning scaffolds was made into artificial tendon. the PCL/PVA/pNSR32 material is expected to used in the tendon tissue engineering in the future after further research and improvement.