Preparation Of Conductive Composites And Application In Tissue Engineering Under Electrical Stimulation

The loss or functional impairment of tissues and organs,as well as injuries caused by accidents and degenerative diseases are the major health challenges of human health.The emergence of tissue engineering provides a reference solution with strong adaptability and less autologous damage.As a place where cells adhere and grow in tissue engineering,the construction of functional bio-scaffolds is a necessary guarantee for the repair and regeneration.Under the synergistic effect of external electrical stimulation(ES)and endogenous electric field,it can regulate and mediate cell behavior,i.e.,cell adhesion,growth,proliferation,and differentiation.Due to the stable electrical activity and good biocompatibility of conducting polymers,conductive composite bio-scaffolds have become potential candidates in tissue engineering and regenerative medicine.In this paper,from the perspective of the preparation and design of conductive composite scaffold,two biological scaffolds,conductive degradable nanofibers and flexible interdigital electrode film,were synthesized and prepared.Besides,without growth factors,the cells growth,proliferation,and differentiation with electrical stimulation parameters(such as,electric field strength and stimulation time)was evaluated.The main experimental contents and conclusions are as follows:(1)As the substrate,the mechanical properties and porosity of electrospinning poly lactic acid(PLA)nanofibers are essential to meet the needs in vivo.Due to the hydrophobicity of PLA,it’s necessary to select optimal the surface treatment by evaluating cell growth and adhesion on the surface of the polyaniline/polylactic acid(PANI/PLA)nanofibers.15wt%PLA can meet the requirements of the mechanics and porosity of biological scaffold.After being plasma treated,the PANI/PLA nanofibers obtained by in-situ polyaniline polymerization showed good wettability,thus improving cell adhesion and growth.(2)It can’t exhibit its own electrical activity because of weakened electrical conductivity of PANI in physiological environment.During the in-situ polymerization of PANI/PLA nanofibers,three inorganic acids(hydrochloric acid,sulfuric acid and perchloric acid)were selected as dopants to regulate the surface morphology of polyaniline,and to evaluate the effects of surface morphology on cell behaviors.The increase of surface roughness of PANI/PLA nanofibers is beneficial to the wettability.The larger roughness provides more contact sites for cell adhesion,which increases cell activity and shows excellent biocompatibility.However,the PANI doped with inorganic acid is unstable and easy to dedoping.The influence of the biocompatibility of PANI with surface morphologies obtained with different dopants of organic acid(tartaric acid)was investigated.Tartaric acid doped PANI is attached to the surface of PLA nanofibers.Nano-fibrous PANI has a significantly better biocompatibility than nanoparticle PANI,and PANI with a hollow tubular structure has the best biocompatibility(3)Considering the electrical activity in the body fluid environment,polypyrrole(PPy),which is not sensitive to pH,is selected as the conductive substrate.By introducing reduced graphene oxide(rGO),the PPy could be coated continuously and densely on the surface of the polylactic acid/reduced graphene oxide(PLA/rGO)nanofibers.The obtained PLA/rGO/PPy composite showed continuous and stable electrical conductivity.On this basis,the regeneration and differentiation of nerve cells attached to the surface of composite nanofibers by different electrical stimulation was observed.The incorporation of rGO increased the conductivity of PLA/rGO/PPy composite nanofibers.When the content of rGO was 3.5%,the conductivity of PLA/rGO/PPy was the maximum 1.46x10-1 S/cm,showing good conductivity.When electric fields of 0,100,400 and 700 mV/cm were applied to PC-12 cells seeded on the surface of PLA/rGO3.5/PPy composite nanofiers,the adhesion,growth,and proliferation increased first with the increase of the electric field.The expression level is the highest at 400 mV/cm,and the neurite growth manifest a similar tendency as well.The above results could be explained by the variation of protein adsorption.(4)Given the limitations of conductive nanofibers and side effects in the actual application in vivo,the graphene/polypyrrole interdigital electrode composite conductive film based on flexible polyimide(PI)was performed to overcome the disadvantage.The laser-induced graphene(LIG)electrode was obtained by laser etching on the surface of the flexible PI film,and the PPy was deposited on the graphene surface by electrochemical polymerization to obtain the LIG/PPy interdigital electrode composite film.The thickness of deposited PPy and the electrical stimulation time was explored through the effect on the regeneration and differentiation of PC-12 cells.The surface of the LIG after laser etching presented an obvious 3D porous structure interconnected framework.The longer the electrochemical deposition time,the greater the conductivity of LIG/PPy.Under 400mV/cm electrical stimulation,the proliferation of PC-12 cells on the LIG/PPy surface is mainly affected by the electric field,indicating that electrical stimulation is field effect.When electrical stimulation was applied for different times under 400mV/cm,the MTT value of PC-12 cells on the LIG/PPy surface increased with time.However,the increasement of MTT value appeared slow after 4 h.Without nerve growth factor(NGF),the differentiation and proliferation of PC-12 cells could be appreciably improved under electrical stimulation.Increasing the time,the nerve phenotype enhanced significantly under electrical stimulation.whereas the increase in neural phenotype was limited after 8h and 12h.As the duration of electrical stimulation increases,the protein adsorption gradually increased,especially after 4h and 8h,the increasement of protein adsorption gradually slowed down.When the stimulation time was extended to 12h,the amount of protein adsorption even showed a downward trend.