Research On Recombinant RGD-spider Silk Protein Composite Scaffold Materials

As a biomaterial of excellent properties, the spider silk protein has sparked prospect in biomedical applications such as tissue engineer tendon and ligament. However, there are relatively few studies except for some reports from abroad and those from our laboratory.With technologies of biology and physical chemistry, recombinant spider silk which contains peptides RGD could be well composited with synthetic polymers such as PVA and PCL. The porosity scaffolds with pNSR16/PVA and pNSR16/PCL could be produced respectively with Freeze-drying/Particle-leaching Method. Besides, nanofibers with the same materials could be prepared by electrospining. The morphology and structure of blend scaffolds were examined by scanning electron microscopy(SEM) and Fourier transform infrared spectroscopy(FTIR). The biological properties were also detected here.The results of FTIR show the comformation of pNSR16 would transit to random coil after dissolving in formic acid. However, it would transit toβ-sheet in groups with LiBr-dealing or alcohol-dealing. The same effect would be achieved by heating. In the groups where recombinant spider silk mixed with synthetic polymers, it was found that the comformation of pNSR16 could be transited toβ-sheet from random coil after blending with PVA or Chitosan.The results of mechanic characterization show that the mechanic property of pNSR16 scaffolds can be improved when blended with PVA or electrospinning.The optimal protocol of freeze-drying/particle-leaching method for preparing pNSR16/PVA porosity scaffolds is:(1) using NaCl particulates(150-250μm) as pore-forming material and blending with pNSR16/PVA (100/4, w/w) at ratio 1/1.(2) freeze drying at -80℃for 130 min, then leaching the salt with alcohol(65-75%).The scaffolds prepared according to the protocol are highly porous. They are oflarger pore size, ranging from 100μm to 200μm. The well distributed, interconnected and open pore wall structure is critical for cell cultivation.An optimal electrospinning condition was obtained in producing uniform cylindrical nanofibers. It was as follows: temperature 45℃, concentration 15%, voltage 80 kv, distance 20 cm, solution flow rate 5 ml/h. The results show that the fiber diameter tends to increase with the concentration of electrospun solution, the nozzle-to-ground distance, the applied voltage and the solution flow rate. The fibers will adhere together after being dealt with alcohol.According to the same protocol of freeze-drying/particle-leaching method mentioned above, the pNSR16/PCL scaffolds with pore size larger than 100μm can be prepared by using pNSR16/PCL acid solution with concentration 30% and NaCl with particle diameter 150-250μm. Although the pores of scaffolds do not well distributed, the pNSR16/PCL scoffolds are suitable for cell cultivation.For electrospinning of pNSR16/PCL acid solution, an optimal condition was obtained in producing uniform cylindrical nanofibers. It was as follows: temperature 45℃, concentration 30%, voltage 80 kv, distance 20 cm, solution flow rate 5 ml/h. The results show that the fiber diameter tends to decrease with the applied voltage and the nozzle-to-ground distance when the concentration is 30%, the ratio of pNSR16/PCL is 5/100. As solution flow rate increases, the average fiber diameter increases steadily. However, the fibers will adhere together and the diameter is uneven when the voltage or solution flow rate is too high.The degradation behavior of pNSR16/PVA scaffolds in PBS buffer, PBS buffer with elastase or chymotase or trypase was analyzed. It was found that, by adapting the ratio of pNSR16/PVA, the degrading rate was in control. Cyto-compatibility of the porous scaffolds of pNSR16/PVA was investigated by cytotoxicity determination of the extract of the scaffolds. According to the evaluation criterion in ISO10993, cytotoxicity of the extract of the pNSR16/PCL scaffolds is of rank I, so the scaffolds are qualified. Preparation of Cell-scaffold constructs with Caco-2 cells and NIH-3T3 cells indicates that pNSR16/PVA scaffolds have good cyto-compatibility.The porosity of pNSR16/PVA electrospun mats is 84.85%, the absorption capacities 4.381g/g, the ratio of amount osmo-solution between interfibrous and intrafibrous 6.140g/g, while those of pNSR16/PCL electrospun mats are 86.47%, 4.794g/g and 14.58g/g. All the above parameters are higher than those of cast film prepared with same compositions. Cyto-compatibility of the porous complex scaffold of pNSR16/PCL was investigated by cytotoxicity determination of the extract of the scaffolds and preparation of cell-scaffold constructs. According to the evaluation criterion in ISO10993, cytotoxicity of the extract of the pNSR16/PCL scaffolds is of rank I, so the scaffolds are qualified. Results show that the pNSR16/PCL scaffolds promote the adhesion and propagation of NIH 3T3, namely, the scaffolds have good cyto-compatibility. Compared with the porosity scaffolds preparing by freeze-drying/particle-leaching method, electrospun membranes could induce the cell to adhere and proliferate.