Dissertation
Dissertation > Medicine, health > Basic Medical > Medical science in general > Biomedical Engineering > General issues > Biomaterial

Biomimetic Artificial Bone Based on Nano-hydroxyapatite/Collagen Preparation and Property Research

Author XuNing
Tutor YeXiaoJian
School Second Military Medical University
Course Surgery
Keywords nano-hydroxyapatite collagen biomimetic rapid prototyping artificial bone scaffolds
CLC R318.08
Type Master's thesis
Year 2013
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ObjectIn recent years,with the growing demands of bone implant materials and sideeffects of autologous bone implant,developing bone implant materials with the similarcharacters of natural bone become increasingly urgent. At present, internationally bonesubstitute materials researches focus on biological ceramic as the representative ofinorganic biomaterial (hydroxyapatite, tricalcium phosphate, biological glass), polymer(collagen and high molecular compound) and hydroxyapatite (HA)/collagen compositematerials. Hydroxyapatite and calcium phosphate become hot research area because theyhave proportion of Ca, P, good biocompatibility, bone induction activity and biodegradablelike normal human bone tissue,but biological ceramic has some defects such as greatbrittleness, small toughness, and higher elastic modulus,while pure collagen and organicpolymer biomaterials have high toughness, the poor mechanical character is difficult tomeet the requirements of bone reconstruction. The development of inorganic/organiccomposite biomaterials by material modification, structural design and interfacecharacteristics research is one of the effective ways to solve the problem that biologicalmaterials is difficult to meet the clinical applications.Therefore research of nHA/COL/polymer composites which assemble organizational structure and function has become hotresearch area of artificial bone substitute materials.Through study on osteoblast mechanism deeply, it has been realized that the naturalbone micro-composition is nano-hydroxyapatite whiskers and type I collagen fibersconnected with specific arrangement and bonding. At present, nHA/COL particles whichhas natural bone like microstructure can be synthesized in the laboratory, thereby achievedthat the nano-hydroxyapatite and collagen fibers have ordered structure in3D. The orderedstructure is the basis of natural bone biomechanical properties. Nano-hydroxyapatitewhiskers and type I collagen in the micro-particles have the similar composition to naturalbone,but since it does not have macroscopic morphology and function is not be used onclinical. The main drawback are poor degradation and mechanical character, can not beused for function reconstruction, the shape and internal structure of the artificial bone cannot be controlled, can not be molded as needed,and it is difficult to use in a wider rangeand the need to shape the bone defect repair. To sum up,this experiment is proposed collection the data of natural bone structure byCT scanning,then achieved from design to manufacturing automation and control.So as torealize the integration and systematic from synthesis of nano-hydroxyapatite/type Icollagen micro-particles to prepared biomimetic artificial bone. To overcome thedisadvantages of traditional artificial bone preparation method,in order to artificial bone inthe proportion of material composition, porosity, shape, and biomechanical propertiessimilar to the natural bone. Truly biomimetic preparation of artificial bone material.MethodThe first part of the experiment: Preparation of nHA/COL composite characterizationand biological performance research. Preparation of nHA/COL by self-assembly methodcomposite with CaCl2, NaH2PO4and NaOH solution. Transmission electron microscopy(TEM) observed nHA/COL composite material grain size and microstructure. X-raydiffraction (XRD) analyse nHA/COL composite material phase and crystallinity, andcompare with natural bone and nano-hydroxyapatite. Analysis nHA/COL compositeinfrared spectroscopy by Fourier transform infrared spectroscopy (FTIR), and comparativewith collagen,nHA and natural bone. nHA/COL composite materials cytology toxicitywas evaluated using mouse MC3T3-E1cells by MTT assay.The second part of the experiment: Preparation and properties research of3Dbiomimetic artificial bone scaffolds. Polycaprolactone (PCL) as a binder, nHA/COLpowder as raw material, pre-mixed in accordance with a mass ratio of7:3to obtain nHA/COL/PCL composite material. CT scan goat femur, Mimic software3D reconstruction toobtain3D electronic model. nHA/COL/PCL composite materials,as raw materials,manufacture three dimensional biomimetic human bone scaffolds under the control of3Delectronic model data by Fused Deposition Modeling equipment (FDM). Scanning electronmicroscopy (SEM) observed surface morphology and microstructure of the3D biomimetichuman bone scaffolds. Determination of porosity of3D biomimetic human bone scaffoldsby the liquid displacement method. Mechanical performance of3D biomimetic artificialbone scaffolds were analyzed by universal mechanical machine. Calculating the largestcompressive strength and elastic modulus,and compared with BAM artificial bone. CCK-8method to detect3D biomimetic artificial bone scaffolds of MC3T3E1-cellproliferation. The third part of the experiment: The experimental research of3D biomimeticartificial bone scaffolds to repair goats femur defect. Made a1.5cm defect,keep side ofcortical bone,on goats femoral by surgery. In experimental group implant3D biomimeticartificial bone scaffolds,the control group implant BAM artificial bone.12weeks later,observed by X-ray and histological.ResultsThe first part of the experiment: Through the analysis of physical and chemicalproperties of nHA/COL complex we can see that the shape of nHA grain change fromrodlike to needlelike because of mixing the collagen.Longer length,less width and length todiameter ratio increases significantly,but the grain size is still in the nanometer scale. nHAgrain presents a densely crisscross patchy distribution. The above phenomenon isbecause in the structure of mineralized tissues are derived from biological macromolecularself-assembly of neat, collagen as a template to control the crystallization of nHA, growth,size and morphology. XRD analysis demonstrate the collagen to join did not change thecrystal phase of nHA,at the same time (002) crystal plane diffraction peak is more close tothe diffraction peak of natural bone.It is demonstrate that collagen has induced nHA (002)preferred orientation of the face, and make the nHA/COL complexes on the phase is moreclose to natural bone. FTIR analysis demonstrate that in nHA/COL complex samplesappeared the characteristics absorption peak of nHA OH,PO4groups and absorption peakof protein amide Ⅰ, Ⅱ, Ⅲ,while the absorption peak of protein amide has red shift tohigh waves. This phenomenon show that collagen and HA happened adsorption andmolecular chain chemical rather than purely physical mix. The cytotoxicity of nHA/COLcompounds were tested by MTT method, the compound of the cytotoxicity at the level of0-1. May be due to self-assembly method in the preparation of compounds in the processof purification is not completely.But even so, complex cell toxicity is still in line with theISO10993(GB/T16886) standard. The condition of MC3T3-E1cells growth on the surfaceof material shows that compound has obvious enhanced adhesion effect of cells, and doesnot change the cell morphology, no toxic action, in conformity with the cell toxicity testresults.The second part of the experiment: Using3D rapid prototyping technologymanufacture goat femur length1.5cm biomimetic artificial bone scaffolds with corticalbone, cancellous bone and marrow cavity structure.The appearance of artificial bone scoffolds are fit with bone defect. Observing transverse and longitudinal section of thesurface morphology of artificial bone scaffolds by SEM,we can see that the scaffolds hasgood aperture connectivity and deep penetration to the scaffolds. The porosity of3Dbiomimetic artificial bone scaffolds is71.18~75.05%,compressive strength is15.43MPa,between cortical bone and cancellous bone,additionally its appearance inconformity with the bone defect site, conducive to the implant.So it is better than BAMartificial bone for weight bearing bone repair material. Cell proliferation experimentdemonstrate3D scaffolds absorbance value (OD value) is greater than the control group atall time points, but less than BAM group. nHA/COL played a significant role in theperformance of proliferation,but due to the3D scaffold porosity is lower than the BAMartificial bone and its proliferation role lower than BAM artificial bone. Under theexisting technical conditions and preparation technology, it’s hard to do porosity,mechanical properties and biological properties of both.According to the different placedifferent demands on the properties of scaffold materials, to adjust the3D scaffoldporosity in order to meet the requirements of different parts repair.The third part of the experiment:3D biomimetic artificial bone scaffolds have thesame appearance of bone defect,it is convenient for individualized manufacture biomimeticscaffolds according to different bone defects,meanwhile providing a method for repairingsome irregular structure bone defects.X-ray observed that callus shape of experiment groupare more regulation than control group,it is better fit for bone defect,because biomimeticscaffolds provide a fromwork for moulding of callus.3D biomimetic scaffolds canbasically meet the demand of weight-bearing bone repair, and it has a larger advantage inthe fine structure of bone repair. Histological biopsy confirmed the3D scaffolds has apromoting effect of bone repair.In the histological biopsy of experiment can find bonetrabecula and haversian canal,it illustrate the new bone have the similar microstructurewith natural boneConclusionnHA/COL compound prepared by self-assembly have similar microstructure withnatural bone,between collagen and nHA emerge chemical bond, the grain size in thenanometer scale,no cytotoxicity, cells grow on the surface in good condition.3Dbiomimetic artificial bone scaffolds have cortical bone, cancellous bone and marrow cavitystructure,the appearance is fit for goats femur defects.It has good connectivity, porosity, mechanical properties,and biological properties.The further animal experiment repaired thegoat femur bone defects successfully,meanwhile it has large adventage in the aspect oftransplant, moulding of callus,and the structure of osteogenesis.To sum up, the3Dbiomimetic artificial bone scaffolds which manufactured by the method of3D rapidprototyping technology and based on nHA/COL has good mechanical properties andbiological properties.It has similar microcosmic,macroscopic structure with natural boneand great potential of clinical application

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