Fabrication Of PHBV-based Nano-composited Scaffold And Its Application For Bone Tissue Engineering

Bone tissue is prone to be injured easily because of its feature and function.Every year,millions of people suffer from bone fracture and bone defect caused by trauma,tumor,infection and bone disease.However,desirable method to regenerate bone has not been developed,which becomes a significant problem in clinical treatment.Recently,the appearance of bone tissue engineering offers a hopeful way to dissolve the above challenge.An appropriate bone tissue scaffold should mimic the natural extracellular matrix(ECM)not only about the elements and structure but also the properties.Nanofibers attract many researchers' attention owing to its fine diameter,high surface area,good flexibility and strong mechanical properties.Especially,diverse nanofibrous scaffolds fabricated by electrospinning technique are preferred since the method is easy to operate and control with high production efficiency.In addition,coaxial electrospinning is able to prepare nanofibers with core-sheath structure,which broadens the application of such technique.In this report,versatile nanofibrous composites based on poly-3-hydroxybutyrate-co-3-hydroxyvalerate(PHBV)were manufactured by electrospinning and coaxial electrospinning technique for the application of bone tissue renovation and the treatment of bone disease.A series of natural polymers such as polyaspartic acid(PAA),chitosan(CTS)and hyaluronic acid(Hy A)were contained to improve the surface affinity of scaffolds.The inorganic hydroxyapatite(HAp)and perovskite(Na2La2Ti O3O10,NLT)were also involved to increase the osteoconductivity and osteoinductivity.In the meantime,different kinds of drugs were encapsulated in the core of coaxial electrospun nanofibers.The influence of nano-composites to osteoblasts growth and bone tissue remodeling were determined by systematic and comprehensive evaluation,including the deposition of HAp on nanofibers surface,the content of HAp in the scaffolds,the synergistic effect between inorganics and natural polymers,as well as the piezoelectricity of perovskite.Firstly,dipping method and hybrid electrospinning were both utilized to fabricate PHBV-based nanofibrous scaffolds which contained n HA particles with different morphology.For the diddping method,the deposition and structure would be impacted by dipping time and dipping circles.The accurate procedure and final deposite contents were difficult to control.However,uniform and beadless nanofibers containing HAp were fabricated by simple hybrid electrospinning.And all the results showed that PHBV-based nanofibrous scaffolds containing PAA and hydroxyapatite might enhance osteoblasts proliferation and mineralization.Considering two kinds of hydroxyapatite involved on or in the scaffolds,the second method can control the percentage of inorganics and avoid blocking the pores of nanofiber mats,which are better for cells necessary breath and growth.On the bases of above results,we directly chose hybrid electrospinning technique to produce composited PHBV-based nanofibers,namely PHBV/CTS,PHBV/CTS/HA4 and PHBV/CTS/HA8,respectively.All the scaffolds were detected by various detections and in vitro evaluation.It was found that,scaffolds with chitosan and HA possessed higher wettability.Comparing to PHBV/CTS/HA4 nanofiber,the interaction between chitosan molecular and HA lead to better tensile mechanical properties for PHBV/CTS/HA8 nanofibers.The results of SBF indicated that plenty of calcium-phosphate compound appeared in the scaffolds of PHBV/CTS/HA since calcium ion from HA provided numerous crystallization nucleus and accelerated the process of crystallization.Higher percentage of HA,more and bigger crystal deposition on the surface of nanofibrous scaffolds.Thus,better bioactivity of the scaffolds was the result of loading more HA.Finally in this chapter,the analysis of cells proliferation and protein expression demonstated that PHBV/CTS/HA8 scaffolds had best biological activity and osteogenic ability.For the purpose of efficient treatment of bone diseases,we designed coaxial electrospinning nanofibers entrapping special drugs inside and tried to achieve sustained drug release.However,the standard “core-sheath” nanostructure is essential for release behavior.In this chapter,we focused on the theoretical exploration of single coaxial jet and the formation of core-sheath structure in the light of previous research about traditional electrospinning.(1)Theoretically analyzing the infinitesimal coaxial jet stretching motion under the conditions of “sheath driving” and “core driving”.(2)According to the conservation of mass,charge and momentum,establishing the motion model of coaxial jet,like finding the proportional relation between core diameter,sheath diameter and the axial distance(1~;2~)under two conditions.(3)Based on the theoretical derivation of jet moving,analyzing the condition under which core-sheath structure can be formed stably.(4)Intercepting the jet from the top of Taylor cone and observing the fiber diameter and morphology along with the axial distance.We analyzed the influence of different flow rates on the Taylor cone and the length of straight jet.(5)Exporing the appropriate parameters to fabricate PHBV-based nanofibers with steady core-sheath structure under theoretical guidance.Preparing PHBV-based coaxial electrospun nanofirou scaffolds loading NLT in the sheath and encapsulating Alendronate(Aln)in the core under the theoretical guidance.The addition of NLT and Aln impacted the thermal mechanical properties significantly,which slowed the melting rate of nanofibers,and decreased the recrystallization temperature and rate.What else,the attendance of inorganics and drug reduced the decomposition temperature of scaffolds and slow down the process of decomposition.The in vitro release curve of Aln illustrated that nanofibers containing NLT remitted the drug burst release and obtained uniform release behavior.One hypothesis was drawn based on the results that such core-sheath electrospun nanofibers were proper for drug carrier and porential for bone tissue regeneration.Bone is a kind of inartificial piezoelectrics,which can transform the external and internal load into electrical signal,followting the process of mechanical stimulation--potential change--motivate active substances--reformation of tissue.Obviously,the proper piezoelectric effect of perovskite with the typical molecular structure of ABX3 is extraordinary semblable to the function of natural bone.Here,we fabricated PHBV-based coaxial electrospun nanofibrous scaffolds loading NLT and HAp in the sheath as well as entrapping icariin(ica)in the core.Comparing the different testing results of PHBV/NLT-Hy A/ica and PHBV/HAp-Hy A/ica nanofibers,including the morphology,wettability,mechanical properties,in vitro drug release behavor,degradation feature and cell culutre,we found that Hy A,NLT and HAp promoted cells adhesion,spreading,proliferation and mineralization.Furthermore,PHBV/NLT-Hy A/ica coaxial nanofibrous scaffold was noncytotoxic to osteoblasts,but contributed to cell growth.Nanofibrous scaffolds electrospun by traditional or coaxial electrospinning could biomimic the natural extracellular matrix on both micro-nano structure and size.It makes a big difference that applying electrospun nano-composites to bone tissue reformation and evaluating theirs biocompatibility.In conclusion,the innovation of this thesis is consisted of three parts.Firstly,the systematical analysis about HA morphology and contents to the properties of scaffolds was obtained.Secondly,we fully explored the formation of stable and standard core-sheath structure about single coaxial fiber.Thirdly,we evaluated the potential application of NLT-loaded coaxial nano-composite for bone tissue reparation.Finally,both experimental detections and theoretical analysis ignited the light of fabricating multi-structured nano-composites and enlarging their utilization in tissue engineering.