Hydroxyapatite Nanocrystal-based Functional Materials Preparation And Applications In Bone Tissue Engineering

Hydroxyapatite(HAp)is the main inorganic component of human bones and teeth.Compared with other bone tissue repairing biomaterials,human body homologous HAp materials can better promote bone tissue regeneration and repair.Meanwhile,these materials can hardly bring side effects.Therefore,HAp-based materials were widely used in bone tissue engineering.The unique advantages of nanomaterials,such as more surface defects,large specific surface area,and high surface roughness,have expanded the application of materials in tissue engineering.Compared with bulk materials,calcium phosphate nanomaterials have a more positive effect on cell adhesion,proliferation,differentiation,bone healing and induction.Therefore,the regulation and optimization of HAp nanostructures play an important role in the application of HAp-based biomaterials in medical engineering.Although the optimal regulation of HAp nanocrystalline morphology has been extensively studied,more effort still is needed in the controllable preparation and performance optimization of the morphology.And,studying the interaction between materials and cells by fluorescence is very important to bone tissue engineering.In addition,nanostructures have a significant impact on stem cell differentiation.Osteogenic differentiation of stem cells can be promoted to a large extent by relying on the synergistic effect of suitable nanostructures,and bone healing can be further promoted.With the development of society and increasing requirements,a single type of material is often difficult to meet the needs of tissue repair.Composite materials often have the combined advantages of two or more materials to achieve optimal and purposeful tissue repair.Therefore,composite materials have become a research hotspot in tissue engineering field.Based on the structural design and performance optimization of HAp nanocomposites,HAp can be endowed with more functions to fully satisfy specific tissue repair requirments in complex environments.Traditional HAp composites usually were simply mixed with orther materials,losing the possibility to obtain the best performance,and failing to give full play to the advantages that materials should have.Therefore,resorting to the reasonable design of the structure,the HAp composite materials can possess more functions and achieve the purpose of accurate repair in bone tissue engineering.Based on the above problems,this thesis chooses nano-HAp as the research object,and aims to optimize the morphology and structure of HAp nanocrystals by adjusting parameters,and prepare HAp nanocrystals with excellent properties.Through in-depth understanding of the interaction between materials composition/structure and cells,HAp nanomaterials with different composition and structures have been designed and synthesized,following to study the effects of composition,structure and surface interface state on cells behaviors,and achieve different biological applications.Meanwhile,based on the design of material functions,multifunctional HAp-based composite materials have been prepared by combining with other materials,following to obtain more biological applications.This thesis mainly includes the following four aspects:(1)HAp-based fluorescent nanocrystals for cell imaging and high-efficiency drug delivery.Two kinds of fluorescent HAp-based nanomaterials with different luminescence mechanisms were prepared by a modified solvothermal method.Eu/Tb FAp(fluorinated HAp)nanorods have a uniform morphology with the length of?200 nm.Through partial energy transfer between Tb ions and Eu ions,Eu/Tb FAp nanorods can simultaneously emit green fluorescence and red fluorescence under a single excitation wavelength(488 nm).By receiving different wavelength range,cells can be imaged by two-color fluorescence,which is convenient to image and track cells in the complex environment.The endocytosis process of the nanorods by the cells can be observed by the fluorescence visualization.In addition,rare earth-amino acid fluorescent nanocrystals were used to confirm the safety of rare earth elements in cells.The results show that the rare earth elements(Tb or Eu)almost have no obvious toxicity to cells.Therefore,Eu/Tb FAp nanorods will not cause significant side-effects on the cells after degradation.The CQD-HAp composite nanorods are approximately 100 nm in length and approximately 15 nm in width.After binding to HAp,the surface functional groups of CQDs are protected to a certain extent,so that CQD-HAp hybrid nanorods have a longer fluorescence lifetime than pure CQDs.CQD-HAp composite nanorods has a large number of mesopores on the surface,and have strong adsorption capacity for Dox,which can be used as a good drug carrier.At the same time,Dox-CQD-HAp hydrid nanorods possess higher lethality to HeLa cells than the pure Dox with equivalent concentration.Two kinds of fluorescent nanorods have high cytocompatibility and fluorescence persistence,and can be used to label and track cells for long period.Excellent fluorescence performance extends the applications of HAp nanorods in cell imaging and tracking,visual observation of endocytosis and drug delivery and release,and provides an important reference for exploring the interaction between cell behaviors and nanomaterials.(2)One-dimensional hydroxyapatite nanostructures with tunable length for efficient stem cell osteogenic differentiation regulation.1D HAp nanocrystals with different length were synthesized by a solvothermal method.The effects of different nanostructur constructed substrate topography coatings on stem cell morphology,extension,activity and differentiation were investigated.In the same reaction system,changing the solvent ratio(ethanol/water)can modulate the length of the 1D HAp nanocrystals.The prepared 1D HAp nanocrystals have the same surface properties and crystal growth direction,which provides a guarantee for the study of the influence of nanostructures on stem cell behavior Different 1D HAp nanostructures have different effects on cell morphology,adhesion,stretch and pseudopod state,and even have a significant regulatory effect on cell viability.Cells have good activity on short nanowires and nanorod substrates.However,cell activity is very low on long HAp nanowire substrates.Under the culture condition without any induction factor,real-time quantitative polymerase chain reaction(q-PCR)confirmed that HAp short nanowire can significantly promote the expression of Runx2,osteopontin(OPN)and osteocalcin(OCN)gene levels.Alkaline phosphatase(ALP)test and immunofluorescence staining results also confirmed that HAp short nanowires can significantly promote the expression of ALP,OPN and OCN proteins.Therefore,the HAp short nanowires coating can effectively promote the osteogenic differentiation of stem cells only by the nanostructure mediated stimulation.The HAp nanorods coating can slightly promote the differentiation of stem cells.While the HAp long nanowires have a certain inhibitory effect on the adhesion and extension of stem cells.The osteogenic differentiation of stem cells only relies on HAp mediated nanostructures without any inducing factor,which is of great significance for stem cell engineering and bone tissue engineering,and also has certain guiding significance for the design of surface coatings of other biological materials.(3)Hydroxyapatite nanobelt/polylactic acid Janus membrane with osteoinduction/barrier dual functions for precise bone defect repair.Through the rational design of the structure,HAp/PLA Janus membranes with osteoinductive and barrier dual functions were prepared by a simple and effective method.The Janus composite membranes not only prevent postoperative adhesion after tissue repair,but also effectively promote bone regeneration,which brings great advantages in accurate repair of bone tissue.Firstly,HAp nanowires with?20 ?m length were prepared according to the previous method.Then,HAp nanowires paper was prepared by suction filtration.In the Janus membranes preparation process,the depth of diffusion can be controlled by diffusion-evaporation process.PLA film formed on one side of the HAp nanowire after diffusion-evaporation.After compaction,flat HAp sdie can promote cells adheresion and growth.The preparation process of HAp/PLA Janus membrane is simple,low cost,and easy to be practically promoted and applied.By utilizing the hydrophilicity of HAp and the hydrophobicity of PLA,the HAp/PLA Janus membrane is imparted with different properties:HAp side can promote cell adhesion and osteogenic differentiation,and PLA side prevents cell adhesion and growth.CCK-8 cells activity assay and live/dead cells staining showed that the cells only grew and adhered to the HAp surface of the Janus membrane,and HAp side can promote the osteogenic differentiation of stem cells.On PLA side,the cells could not adhere and survive,thus acting as a barrier.It was confirmed by q-PCR that the Janus membrane HAp side promoted the expression of Runx2,osteopontin(OPN)and osteocalcin(OCN)in gene levels.ALP test and immunofluorescence staining results also confirmed that HAp side can promote the expression of ALP,OPN and OCN in proteins level.In vivo experiments results have shown that the HAp side of the HAp/PLA Janus membrane can effectively promote bone regeneration,while the PLA surface can prevent postoperative adhesion between the new bone and surrounding soft tissue.Therefore,the structurally optimized HAp/PLA Janus membrane will actively promote and guide the accurate repair of bone tissue.(4)HAp nanowire/polylactic acid Janus membrane loaded copper-cysteine nanoparticles for postoperative tissue repair of bone cancer.Copper-cysteine nanoparticles(Cu-Cys NPs)were successfully prepared by coordination interaction.As a novel nanoformulation,Cu-Cys NPs can be produced on a large scale and effectively kill cancer cells and inhibit tumor growth.Cu-Cys NPs are composed of amino acids and copper ions.Therefore,they do not cause side effects to the body after degradation.Cu-Cys NPs can react with intracellular GSH to produce Cu+,then Cu+ and H2O2 undergo a Fenton-like reaction to produce a highly aggressive hydroxyl radical(·OH),which causes apoptosis of cancer cells.Cu-Cys NPs and intracellular GSH/H2O2 undergo cascade chemical kinetic reactions,which can consume the reducing substance GSH,bring high lethality to cancer cells.In addition,animal experiments have shown that Cu-Cys NPs can effectively inhibit the growth of drug-resistant tumors and will not cause systemic toxicity in vivo.By loading Cu-Cys NPs on HAp nanowires,Cu-Cys NPs/HAp/PLA multifunctional composite membrane was obtained according to the preparation process of the HAp/PLA Janus membrane.The HAp side of the composite membrane is highly lethal to cancer cells.Therefore,the multifunctional composite membrane can accurately repair bone defects caused by postoperative bone cancer,and meanwhile kill cancer cells that may remain following cancer relapse inhibition.Therefore,the multifunctional composite membrane will have a wide range of applications in the repair of tissue defects caused by bone cancer,and also provide guiding significance for the repair of other tissue defects caused by other cancers.In summary,this thesis has prepared performance-optimized HAp nanocrystals and novel structured composites based on the HAp nanocrystals,and studied their properties and materials in-depth research.Fluorescent HAp nanocrystals can visualize endocytosis as well as drug delivery and release,and observe nanoparticle-cells interactions.The nano-topography constructed by different HAp nanostructures can significantly promote the osteogenic differentiation of stem cells without the assistance of inducing factors,which provides great convenience for the practical application of HAp materials in bone tissue engineering.The well-designed osteoinductive/barrier dual-function HAp nanowire/polylactic acid Janus membrane provides a new solution for the accurate repair of bone tissue.In addition,by loading Cu-Cys NPs on HAp nanowires,Cu-Cys NPs/HAp/PLA multifunctional composite membranes have been prepared for the repair of bone cancer postoperative caused bone tissue defects to inhibit cancer recurrence.