Preparation And Characterizations Of Conductive Polyorganophosphazene-based Biomaterials For Bone Regeneration

The increasing incidences of bone defects,which may result from traumas,inflammations,bacterial infections,encourage researchers to develop multi-functional materials in order to promote bone repairs.Though auto-and allo-grafts are deemed as gold standards clinically,the resource stability and donor morbidity restrict their applications.Currently,the available synthetic polymeric materials in constructing bone tissue engineering scaffolds are the biodegradable aliphatic polyesters such as poly(L-lactic acid)(PLLA),polycaprolactone(PCL),poly(lactic-coglycolic acid)(PLGA)etc..However,the limited bioactivity and osteocompatibility of these polyesters confine their broad usages in promoting bone regeneration.Corresponding modifications such as integrating with bioactive components(e.g.growth factors,metallic ions)are usually applicable to improve their osteoconductivity and osteoinductivity.To prepare such kind of composite scaffolds,the laying hinderance is that how to ensure the stability and bioactivity of those incorporated bioactive components.In this thesis,focusing on osteocompatible and biodegradable polyorganophosphazenes,a series of conductive materials were designed as electrophysiological scaffolding biomaterials,and evaluated if they were superior to polyesters in inducing osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs),and promoting bone regeneration.The researches included for four parts:(1)Conductive composite films were prepared by blending the amino acid ethyl ester co-substituted polyphosphazene(PAGP)with carbon nanotubes(CNTs).By applying electrical stimulation(ES)to mimic the in vivo electrophysiological microenvironment,the results of cell culture were to show if the combination of conductive substrates and ES had a synergistic promotion on the osteogenic differentiation of BMSCs.(2)A biodegradable conductive polyorganophosphazene(PATGP)was synthesized by introducing aniline tetramer(AT)and glycine ethyl ester as side groups,and the PATGP was then shaped into microspheres for cell culture and in vivo implantation,using rat calvarial bone defect model in order to evaluate PATGP-microspheres' promotion effect on osteogenesis.(3)Conductive PATGP microspheres were then coated with polydopamine(PDA)and subsequently loaded with silver nanoparticles(AgNPs)to be endowed with antibacterial performance.These microspheres and S.aureus were co-implanted into rat calvarial bone defects to evaluate the in vivo antibacterial performance and promotion for neo-bone formation.(4)PATGP/PLGA composite nanofibers were fabricated via blend or coaxial electrospinning.The performances of these two types conductive composite nanofibers(e.g.blend,core-shell groups)for bone regeneration were studied by cell culture and rat distal femur defect models.The main results of these studies were listed as follows:(1)Conductive composite films PAGP/CNT were prepared by blending non-conductive PAGP with 3 wt.%CNT via solution-casting and solvent evaporation.For cell culture studies,conductive PLLA/CNT composite films were prepared similarly and set as controls.To avoid the different cell affinity of the two polymers with concerns of causing uncertainties,both PAGP/CNT and PLLA/CNT composite films were coated with PDA.During cell assays,it was found that BMSCs on the PAGP/CNT/PDA films showed higher proliferation rates and osteogenic differentiation potentials than those on the PLLA/CNT/PDA films.Under the optimized ES parameters(1.5 V,2 h per day),biological activities of BMSCs on the PAGP/CNT/PDA films were further improved in comparison with other cases.These results demonstrated that the combination of conductive polyphosphazene substrates and an appropriate ES showed a synergistic promotion effect on the osteogenic differentiation of BMSCs.(2)A biodegradable conductive PATGP was synthesized by substituting AT and glycine ethyl ester onto the polyphosphazene backbone as functional pendant groups.To facilitate cell culture and in vivo implantation,the polymer was shaped into microspheres(approximately 100 ?m in diameter)via emulsification method,PLGA and PAGP microspheres were prepared as controls.Corresponding assays including conductivity,degradability,reactive oxygen species(ROS)scavenging ability,cytocompatibility and osteocompatibility were carried out.Among all the microspheres,the conductive PATGP microspheres showed the significant antioxidant performance,and the highest capacity to promote the growth and osteogenic differentiation of BMSCs.By implanting the microspheres into the rat defect,it was the PATGP microspheres showing the best promotion on new bone formations,benefiting from the facts that they were able to eliminate and scavenge the oxidative stress in the defected areas,and provide the electrophysiology feature similar to native bone tissues.(3)Based on the previous PATGP microspheres,subsequent PDA coating and AgNPs loading were conducted to endow the electroactive microspheres with antibacterial activity,which were then co-implanted into rat calvarial defects with S.aureus to evaluate their capacity in treating infectious bone defects.Via antibacterial and cytocompatible assays,it was determined that AgNPs-loaded PATGP microspheres could achieve sufficient capacity to kill S.aureus,while showing no adverse effect on cell viability at an optimized AgNPs loading.The AgNPs-loaded PATGP microspheres also showed obvious antioxidant activity and efficient osteocompatibility to enhance the osteogenic differentiation of BMSCs.Using PATGP with PDA coating and AgNPs-loaded PAGP microspheres as controls,the results of in vivo tests demonstrated that AgNPs-loaded PATGP microspheres displayed superiority in antibacterial efficiency and acceleration in bone regeneration.(4)PATGP/PLGA composite nanofibers were fabricated to take advantages of the two different types of polymers.Blending electrospinning was applied to obtain PATGP/PLGA blend nanofibers,while coaxial electrospinning was applied to produce PLGA(core)-PATGP(shell)nanofibers.The effects of these two composite nanofibers on BMSCs proliferation and differentiation as well as ROS scavenging efficiency were studied.Finally,the nanofibrous mats were inserted into rat distal femur defects,their antioxidant performance and neo-bone formation were estimated and compared in terms of early inflammation inhibition at 1-week,neo-bone and collagen formation at 12-week.The results showed that both types of PATGP/PLGA composite nanofibers could inhibit inflammation and enhance osteogenesis significantly,among the groups,the smallest neutrophil numbers,the most collagen depositions and the highest volumes of bone formation were detected in the group of the core-shell structured fibrous mats.Taken together,conductive and non-conductive biodegradable polyphosphazene materials could be synthesized.Compared with conventional polyesters,polyorganophosphazenes own inherent osteocompatibility due to their phosphorus and nitrogen-rich feature,which can be further functionalized for enhancing bone regeneration by introducing different side groups.Since bone regeneration is complex and faced with events including oxidative stress,inflammations,bacterial infections,material-related symptoms such as protein adsorption and biocompatibility,which all have significant impacts on bone tissue remodellings and function reconstructions.To meet the goal,the development of biodegradable polyphosphazenes with biodegradability,antioxidant and antibacterial activity,electroactivity and osteocompatibility undoubtedly showed advantages in regulating these important phases in relation to bone regeneration,indicating their promising applications in the field of bone tissue engineering.