Fabrication And Evaluation Of Bioactive Nanofibrous Scaffold By Optimized Electrospinning Strategies For Bone Regeneration

Over the last few decades,Bone Tissue Engineering（BTE）research has sparked progress with new materials.Nanostructured scaffolds have been discovered to be more efficient for bone regeneration than macro/micro-sized scaffolds because they sufficiently permit cell adhesion,proliferation,and chemical transformation.Nanofibrous scaffolds mimicking artificial extracellular matrices provide a natural environment for tissue regeneration owing to their large surface area,high porosity,and appreciable drug-loading capacity.Electrospun nanofibrous scaffolds have demonstrated promising potential in bone tissue regeneration using a variety of nanomaterials.Electrospinning is a versatile method for the fabrication of précised nanofibrous materials for biomedical applications including tissue engineering and drug delivery.A scaffold material’s impact on osteoblast is equally as important as its impact on osteoclasts when considering tissue engineering for bone defect repair.Biomaterials consist of biodegradable polymers and bioactive ceramics,are suitable for BTE.Hydroxyapatite（HA）is the main mineral found in bones and one of the most studied biomaterials.Alendronate（ALN）is bisphosphonate family and is capable of inhibiting osteoclastic bone resorption as well as inducing mineralization of the newly formed bone by osteoblasts.PVP,PVA,PCL,and PLGA（P）are biocompatible and biodegradable polymers.Polydopamine（PDA）coating on the surface of biomaterials has shown advantageous properties such as biocompatibility,enhanced mechanical strength,cell adhesion,proliferation,and osteogenic properties.This thesis mainly includes the following three parts of research content:1.The first study aimed to fabricate the nanofiber by traditional electrospinning technique and investigate how the process parameters and polymer/solvent combinations influence the properties of electrospun PVP/PVA nanofibers using design of experiments.The solvents used for electrospinning of PVP/PVA nanofibers were ethanol and 90%acetic acid,optimized with central composite design（CCD）via Design Expert software.Nanofibers were systematically analyzed for morphology,mechanical strength,swelling ability,contact angle,crystallinity,thermal properties,and surface morphology,as well as in-vivo tissue response.SEM images confirmed its diameter within 150-400 nm,tensile strength of PVP/PVA’s in acetic acid and ethanol strengths were 18.3 and 13.1MPa,respectively.XRD data revealed the amorphous nature of PVP/PVA nanofiber.FTIR analysis confirmed solvent-independent interactions.Contact angles of PVP/PVA nanofiber in acetic acid and ethanol（NF2 and NF35）revealed their hydrophilicity（67.89°and 58.31°,respectively）.Swelling and biodegradability studies displayed extended water retention and two-week biodegradation,suitable for sustained and controlled release requirements.Subcutaneous rat implantation enabled in-vivo appraisal of tissue response and month-long biodegradation,showing tissue biocompatibility which was confirmed by histological analysis of the tissue surrounding the implanted nanofibers.Additionally,hemocompatibility and cytotoxicity studies revealed the non-toxicity of fabricated nanomaterial on cells.The study’s findings reveal how electrospinning parameters and polymer/solvent combinations influence nanofiber characteristics,offering insights for tissue engineering and guiding the creation of customizable biodegradable materials,thus contributing significantly to biomaterials and nanotechnology for biomedical applications.2.Electrospun composite nanofiber scaffolds are well known for its bone and tissue regeneration applications.In the second part of study,HA was synthesized and characterized for their particle size,phase,and purity using SEM,EDS,XRD,and FTIR analysis.The HA particles were incorporated in the PVP/PVA optimized blend solution to fabricate polymer-ceramic nanocomposite.This research focused on the development of a multifunctional PVP/PVA nanofiber composite scaffold enriched with HA and ALN by a novel electrospinning technique and investigated its physiochemical as well as bone regeneration potential.The composite scaffolds were characterized for its mechanical strength,thermal stability,surface morphology,swelling and biodegradable behaviour,FTIR analysis,contact angle,crystallinity,as well as in-vitro cell line studies.The results obtained from particle size,zeta potential,SEM,and EDS analysis of HA nanoparticles confirmed its successful fabrication.SEM analysis verified nanofiber diameters of 200-250 nm,while EDS analysis confirmed successful HA and ALN incorporation into scaffold.XRD and TGA analyses revealed the amorphous and thermally stable nature of the nanofiber composite scaffold.FTIR and contact angles indicated no drug-polymer interactions and hydrophilicity.Swelling and biodegradability results supported appropriate water uptake and in-vitro degradation for tissue regeneration.Addition of HA into the nanofiber scaffold enhanced the physiochemical properties.Hemocompatibility and cytotoxicity studies revealed the non-toxicity of fabricated nanomaterial on red blood cells（RBCs）and MC3T3-E1 bone cell lines,respectively.Live/dead assay proved its non-toxic behavior.SEM photomicrographs showed adequate cell adhesion and enhanced cell proliferation,which was further assessed by confocal laser scanning microscopy（CLSM）by culturing MC3T3-E1 cells on nanofiber composite scaffolds.Furthermore,Alkaline phosphatase（ALP）and tartrate-resistant acid phosphatase（TRAP）demonstrated that HA-ALN-loaded nanofibers effectively promoted osteoblast formation and osteoclast inhibition.These findings suggest the potential application of HA and ALN-loaded PVP/PVA-ALN-HA nanofiber composite scaffold for BTE.3.The novelty of third study explored to create a dual drug release system that allows for time-controlled release to facilitate bone regeneration.We fabricated surface-functionalized core-shell ALN-PCL/PHA nanofibers using coaxial electrospinning.The surface of the nanofiber construct was functionalized with mussel-derived PDA to induce hydrophilicity and enhance cell interactions.Surface characterizations confirmed the successful synthesis of PDA@ALN-PCL/PHA nanofibers endowed with excellent mechanical strength（20.02±0.13 MPa）and hydrophilicity（22.56°）,as well as the sustained sequential release of ALN and calcium ions(Ca²⁺).In vitro experiments demonstrated that PDA-coated core-shell ALN-HA scaffolds possessed satisfactory cytocompatibility,effectively promoting cell adhesion,spreading,proliferation,ALP,ARS,osteoblast,and osteogenesis-related genes.In addition to osteogenesis,the engineered scaffolds also significantly reduced osteoclastogenesis,and the expression of genes related to osteoclast formation,such as TRAP,has decreased.In a rat skull defect model,bone was implanted and covered with a PDA@ALN-PCL/PHA membrane.After 12 weeks,we observed that the nanofiber scaffold had a bone-guided regeneration effect.Micro-computed tomography,histological,and immunohistochemical analyses revealed that the PDA-coated core-shell ALN-HA scaffolds possessed excellent osteogenesis-inducing and osteoclastogenesis-inhibiting effects.These results demonstrate that time-controlled release enabled by the core-shell nanofiber assembly and bio-inspired surface modification of the PDA@ALN-PCL/PHA provides a promising strategy to facilitate bone healing.