Preparation Of Polypyrole/Silk Fibroin Conductive Composite Scaffolds And Its Effects On Nerve Regeneration

The repair of peripheral nerve defects still depends on tissue-engineered nerve grafts.There is a large gap between the tissue-engineered nerve grafts and autologous nerve graft.It is urgent to solve the problem that selecting one kind of material to promote the peripheral nerve regeneration more effectively,and enhance the specific different nerve fiber directed to the targeted tissues more accurately.The peripheral nervous systems are composite materials with hierarchical structure and electrophysiological property.Thus,in this study,silk was used as raw material,PPy/SF conductive composite scaffold was fabricated by employing 3D bio-printing and electrospinning together.The physical and chemical properties and biological activity of PPy/SF composite scaffolds were evaluated in vitro.In addition,electrical stimulation and the PPy/SF composite scaffolds ware combined to investigate whether PPy/SF conductive composite scaffold was suitable for neural tissue application.1.Fabrication of conductive composite scaffolds:The stability and mechanical properties were used to test PPy/SF conductive composite scaffold with different structure and biomaterials.Then the proper structure and biomaterials were selected to fabricate PPy/SF conductive composite scaffold.The effects of different degumming methods on different sources of silk were analyzed,according to the degumming detection,apparent viscosity measurement,transmission electron microscopy（TEM）,and iTARAQ quantitative analysis.3D printing was applied to fabricate oriented silk fibroin fibers as the inner core and polymerization reaction of polypyrrole to form the shell.Considering the effect of various reaction factors on the conductivity of PPy/SF,sucn as reaction time、reaction temperature and the ratio of FeCl₃ and pyrrole,the optimum polymerization reaction conditions of PPy/SF was obtained.The inner and outer layers of PPy/SF conductive electrospining composite scaffolds were prepared by electrospinning.Effects of solution concentration,flow rate and rotating speed on the morphology and size of electrospun nanofiber were studied by means of optical microscope and scanning electron microscope（SEM）.And the optimum electrospining parameters of aligned nanofiber ware obtained.The conductive fiber with"Shell-Core"structure has better stability and can enhance the mechanical tensile properties of composite scaffold.The preferred silk fibroin and polypyrrole combination to fabricate"Shell-Core"structure for they have higher biological stability and long-term maintenance of electrical activity.We selected the natural silkworm from Zhejiang and boiled three times in 0.05%Na₂CO₃ solution for degumming,according to the structural integrity and protein structure.Due to the proper conductivity of PPy/SF conductive fibers,the optimum polymerization reaction conditions were obtained:FeCl₃ as oxidant,the ratio of fixed FeCl₃ to pyrrole was 1:1,the polymerization temperature was 0°C,and the polymerization time was 12h.The optimal parameters were:concentration:10%,flow rate:0.1 mL/h,voltage:21 kV,rotating speed:2000 rpm.There is no beads,the diameter of the nanofibers is smaller and the most of the nanofibers show the tendency in this way.2.Characterization of conductive composite scaffolds:PPy/SF electrospun conductive composite scaffolds with different diameters and spacing can be obtained by changing the 3D printing pressure,speed,needle and spacing,and the PPy/SF conductive fibers without electrospining SF fibers were used as control.Morphological observation was used to observe the morphology.And the conductivity test,stability test,infrared spectroscopic analysis,contact angle and the degradability in PBS and protease were performed to analyze the physicochemical properties of two different groups of scaffolds.Morphological observation shows that the inner layer has the orientation structure and the outer layer is composed of electrospinning silk protein nanofibers.Stability tests suggested that the stability of composite PPy/SF scaffolds were superior to most aligned PPy/SF scaffolds,the electrospun coated scaffolds can maintain their topography integrity more than 50%until 4 week,but the aligned PPy/SF scaffolds peeled off in 10 day.The conductivity results showed that the deposition of PPy contributes to the formation of a conductive polymer in the SF matrix.The electrical conductivity was in the region of1×10^-5–1×10^-3 S/cm proportional to the size of aligned silk diameter.In addition,the thin layer of nanofiber coating has little effect on the conductivity of the PPy/SF conductive fiber.The infrared spectral analysis found that PPy was successfully incorporated into SF matrix and changed the molecular structure of PPy to some extent.The contact angle measurement of composite PPy/SF scaffolds showed good hydrophilicity for their contact angle were less than90°.In vitro degradation results indicated that the weight loss ratio of PPy/SF conductive electrospun composite scaffolds was 55%for 4 week.Thus the PPy/SF conductive electrospun composite scaffolds could be degraded in the protease XIV solution.3.Biological properties of conductive composite scaffolds:L929 cells were used to determine the in vitro cytotoxicity of the extracts of PPy/SF electrospun conductive composite scaffolds.The Schwann cells were used to evaluate the biological function of PPy/SF electrospun conductive composite scaffolds with different sizes.After cocultured for different time,the activity of Schwann cells on PPy/SF electrospun conductive composite membrane was measured by cell counting kit 8（CCK8）.The proliferation activity of Schwann cells was tested by EDU,and the morphology of Schwann cells was observed by scanning electron microscope and immunohistochemistry.The dorsal root ganglion（DRG）and DRG neuron cell was also used to evaluate the biological function of PPy/SF electrospun conductive composite scaffolds with different sizes.The morphology of DRG cells was observed by optical microscope,and the axon growth was evaluated by immunohistochemistry.The effects of different sizes of PPy/SF electrospun conductive composite scaffolds on nerve cell vitality were analyzed.The results show that the cellular viability decreased as the diameter of composite PPy/SF scaffold（d1）increased,it was still almost non-toxic and shows a good effect on the growth of L929 cells.Schwann cells can be attached to the surface of the scaffold to maintain their normal configuration-full,spindly and smooth,intercellular interconnections,and proliferate well on all composite PPy/SF scaffolds.The general trend showed that as the diameter increased,the cell viability and propagating rate was declining.At the same time,conductive composite tissue engineering scaffold facilitates the adhesion and growth of DRG,and it can promote the extension and growth of axons.The results show that the conductive composite scaffold has good biocompatibility with the peripheral nerve tissue and cells,and it is found that the material with the optimized conductive polymer（a1′and b1′）is the proper candidate for the application of biomaterials.4.The combination of conductive composite scaffolds and electrical stimulation promote the growth of neural cells:The PPy/SF electrospun conductive composite scaffolds were combined with electrical stimulation,and the proper electrical stimulation parameters for Schwann cells and DRG neuron cells were selected according to the apoptosis assessment.The effects of PPy/SF electrospun conductive composite scaffolds combined with electrical stimulation on the co-cultured Schwann cells were assessed.The morphology of Schwann cells was observed by optical microscope and immunohistochemistry.The cell viability of Schwann cells on PPy/SF electrospun conductive composite membrane was measured by MTT assay.The proliferation activity of Schwann cells was tested by EDU,the cell migration was observed by the migration chamber,and the gene expression of neurotrophic factors was detected by realtime PCR.The effects of PPy/SF electrospun conductive composite scaffolds combined with electrical stimulation on the co-cultured DRG neuron cells were assessed.The morphology of DRG cells was observed by optical microscope,and the axon growth was evaluated by quantitative analysis of neurite alignment and orientation.The optimal electrical stimulation parameters for Schwann cells were:100 mV/mm,4 h for the co-cultured Schwann cells with little apoptosis and have better cell viability.And the optimal electrical stimulation parameters of DRG neurons were:5 V,10 Hz,30 minutes due to the intact structure and little apoptosis.The results showed that the electrically-conductive scaffold combined with electrical stimulation can not only induce the arrangement and extension of Schwann cells through the surface of the scaffold-oriented structure,but also promote the proliferation of Schwann cells and the expression of the nerve growth factor gene.In addition,the combined effect of this electrically conductive material combined with electrical stimulation patterns also greatly promoted the neurite extension of DRG/DRG neuronal cells in highly uniform directionality.The comprehensive results show that these electrically conductive scaffolds combined with electrical stimulation are more conducive to control the directional growth and differentiation of cells,promote the expression of NGF and BDNF gene expression.And these electrically conductive scaffolds combined with electrical stimulation significantly also promote the neurite extension and improve the neurite alignment behavior of DRG neuron cells.This paper reports on the development of composite scaffold with aligned conduction and conductivity prepared by combing 3D bioprinting and electrospining.The physicochemical assessment showed improved performance of stability,mechanical property and electroactivity of conductive composite scaffolds.The in vitro cytotoxicity tests and biological function assessment demonstrate that the conductive composite scaffolds show good compatibility,and the combination with electrical stimulation could promote the growth of neural cells.The research of this topic provides a new method and idea for the construction of artificial nerve grafts,extends the function and application of the fiber based material,and provides a preliminary research basis for the application of new conductive materials in the field of tissue engineering,also provides assistance for subsequent research and application.