Hydroxyapatite (HA) is a biologically active ceramic used in surgery to replace bone. While HA promotes bone growth, it suffers from weak mechanical properties and does not possess any antibacterial property. Multi walled carbon nanotubes (MWCNTs), as one of the strongest and stiffest materials, have the potential to strengthen and toughen HA, thus expanding the range of clinical uses for the material. Furthermore, Silver nanoparticles (Ag NPs) can be decorated to sidewalls of the MWCNTs which could be released over a period of time to prevent infection following surgery. This work sought to develop and characterise Ag NPs- MWCNTs – HA composites in four main areas: 1) production and characterisation of the composite, 2) evaluation of mechanical properties, 3) investigation of antimicrobial property and 4) assessment of biological response to in vitro cell culture. Pristine (p-MWCNTs) and acid treated MWCNTs (f-MWCNTs) were decorated with Ag NPs. In the presence of 0.5 wt % Ag NPs-MWCNTs, HA was precipitated by the wet precipitation method in the presence of either poly vinyl alcohol (PVA) or Hexadecyl trimethyl ammonium bromide (HTAB) as the surfactant. Composites were characterised using various techniques and the diameteral tensile strength and compressive strength of the composites were measured. The antibacterial effect of these composites was investigated against clinically relevant microbe, Staphylococcus aureus. To determine the ability of the HOB cells to differentiate and mineralize in the presence of the composite, HOB cells were cultured on the composites for 21 days. Gene expression studies was performed along with the biochemical assays and scanning electron microscopy was used for qualitative analysis. Pure HA was used as control in all the studies. The study revealed that both the MWCNTs and surfactants play a crucial role in the nucleation and growth of the HA. XRD and FTIR characterisation revealed that HA was the primary phase in all the synthesised powders. Composites made with f-MWCNTs were found to have better dispersion and better interaction with the HA compared to composites with p-MWCNTs. Although mechanical strength was improved in all the composites, p-MWCNTs composites exhibiting maximum strength. Antibacterial studies showed 80% bacterial reduction in the treatment composites compared to pure HA. The biocompatibility study showed reduced activity of the HOB cells, however, no significant difference was observed between the control and the treatments. This systematic study of the synthesis and properties of the Ag NPs- MWCNTs-HA composites has resulted in improved understanding of the production and processing of these materials and the effect of MWCNTs and silver nanoparticles on primary human osteoblast cells. Additionally, it has yielded interesting biocompatibility result favouring the use of MWCNTs in the development of implants. There is potential to translate Ag NPs-MWCNTs-HA composites into clinically approved product.

This research investigates the effects of the human body on the mechanical, chemical, and morphological properties of the surface of internal fixation devices. Stainless steel and titanium devices that had failed were provided from the Shandong Provincial Hospital in China, along with controls: implants that had never been used. Comparative study was conducted by evaluating properties of these implants before and after implanting. The first part of the research was simulation, and a model of the human femur was analyzed in Solidworks. The stress analysis software simulated the stress distribution, the strain distribution, and the deformation pattern. Two cases were simulated: walking and car accident. The simulations showed the points of highest stress and led to the analysis of the implants that were used in those regions. The next part of the research was to experimentally examine the properties and behavior of materials. Test samples fell into one of three categories: stainless steel femur implant, stainless steel tibia implant, and titanium femur implant. Material properties were characterized and effects of the human body on each of these groups were studied. Hardness was measured using Vickers hardness indentation. Surface roughness was analyzed using light interferometric technique. Potentiodynamic polarization analysis was performed to evaluate corrosive behavior before and after implanting. Scratch tests were conducted to evaluate wear resistance and the microstructure was analyzed to further understand the morphological changes that occurred of implanted samples. Results showed that the human body generally degraded the material properties of the stainless steel femur implant. There were no measurable effects of the same on stainless steel tibia and on titanium alloy.

No description available.

The cement line is the first mineralized matrix deposited on an implant surface during contact osteogenesis forming the bone/implant interface. The hypothesis underlying the present project was that non-collagenous cement line proteins must be deposited into the submicron undercuts on substrate surfaces prior mineralization. In vitro osteogenic cultures were used to grow bone nodules on Thermanox® coverslips modified with calcium phosphate nanocrystals, creating an undercutted surface. Electron microscopy was used to observe cement line formation. BSP immunogold labelling was used to determine if the cement line organic matrix is deposited within undercuts prior mineralization. The results showed the deposited bone nodules, and on test coverslips the deposited cement line was thicker and evenly distributed than control. Furthermore, positive BSP labelling was found within the undercuts prior to cement line mineralization. Thus, it can be concluded that cement line proteins are deposited into submicron undercuts on substrate surfaces prior to mineralization.

The cement line is the first mineralized matrix deposited on an implant surface during contact osteogenesis forming the bone/implant interface. The hypothesis underlying the present project was that non-collagenous cement line proteins must be deposited into the submicron undercuts on substrate surfaces prior mineralization. In vitro osteogenic cultures were used to grow bone nodules on Thermanox® coverslips modified with calcium phosphate nanocrystals, creating an undercutted surface. Electron microscopy was used to observe cement line formation. BSP immunogold labelling was used to determine if the cement line organic matrix is deposited within undercuts prior mineralization. The results showed the deposited bone nodules, and on test coverslips the deposited cement line was thicker and evenly distributed than control. Furthermore, positive BSP labelling was found within the undercuts prior to cement line mineralization. Thus, it can be concluded that cement line proteins are deposited into submicron undercuts on substrate surfaces prior to mineralization.

Bone implants procedures involve millions of people every year worldwide. One of the main factors determining implant success is related to the ability of the prostheses to osseointegrate, i.e. to create a structural and functional connection with the living bone. Titanium and titanium alloys are widely used biomaterials for bone implants, due to their superior biocompatibility and corrosion resistance, suitable mechanical properties, and natural ability to osseointegrate. To further enhance the inherent tendency of this class of materials to bond with the host bone tissue, the surface of Ti-based implant is often modified to improve cell responses in terms of adhesion, proliferation and differentiation, all factors contributing to successful osseointegration. In particular, surface topography, both at the micro- and nanoscale, can enhance the implant-living bone interaction. Herein, a possible surface modification strategy aimed at the creation of a dual-scale topography on two different titanium alloys, Ti-6Al-4V and Ti-5Al-5Mo-5V-3Cr, is presented. Dual-scale topography was obtained by electrochemically anodizing samples manufactured by selective laser melting to combine their intrinsic microtopography with the nanotopography offered by titanium dioxide nanotubes (TNTs) generated by anodization. Characterization of the as-printed and as-anodized samples was performed to evaluate parameters of significance in the context of osseointegration. Concerning wettability, it was observed that surfaces with TNTs exhibited high hydrophilicity. The influence of the anodization process parameters on TNTs morphology was examined, and linear dependence of the nanotube diameter on the voltage was identified. Annealing of the as-anodized samples showed that anatase was produced, while preserving the nanotube integrity. Preliminary studies to assess the bioactive properties of the samples showed the spreading of bone-like cells on these substrates and the deposition of mineral during simulated body fluid testing. Therefore, both studies provided promising results to corroborate the hypothesis that dual-scale topography could potentially improve osseointegration. / Thesis / Master of Applied Science (MASc) / Bone implants are often made of titanium-based materials, which, despite their suitable properties, may not sufficiently bond with the living bone tissue. This can lead to implant loosening and failure. To produce customized implants, additive manufacturing, or 3D-printing, can be employed. However, these surfaces require substantial post-processing to produce features capable of promoting bone integration. In this work, a dual-scale surface topography to combine the advantages of both micro- and nanoscale roughness was created using electrochemical anodization on 3D-printed titanium alloy substrates. Preliminary physical, chemical, and biological characterizations suggest that the creation of titania nanotubes on the 3D-printed surfaces of Ti-6Al-4V and Ti-5Al-5Mo-5V-3Cr could improve their ability to bond with bone.

The thesis is divided into three main parts. The first section summarizes the theory of the issue. Here we are unified theoretical information about the various possibilities of different approaches. The result of this part is therefore a general summary of theoretical possible procedures of creation bone implant, where each are listed the advantages and disadvantages. The theoretical part also contains information that ultimately, in practice, the author did not use. But his idea was to create a comprehensive look at the issue from several angles. The second part uses theoretical knowledge from the previous set of information as a basis for defining the steps required to successfully manage the issues of bone 3D printing. The third part will follow the guidelines of both previous and focuses on practical making bones and subsequent evaluation method chosen. There are discussed various steps that led to the final conclusion, making bones and work is then focused on the evaluation of the success of selected procedures and recommendations for future action.

Final publication is available at http://link.springer.com/article/10.1007%2Fs10856-015-5612-2 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19566号 / 医博第4073号 / 新制||医||1013(附属図書館) / 32602 / 京都大学大学院医学研究科医学専攻 / (主査)教授 安達 泰治, 教授 戸口田 淳也, 教授 鈴木 茂彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Metallic bone implant devices are commonly used to tackle a wide array of bone failures in human patients. The success of such implants relies on the biomechanical and functional bonding between the living bone tissue and implant, a process defined as osseointegration. However, the mechanism of osseointegration is still under debate in the scientific community. One efficient method to help understand this complex process is to characterize the interface between human bones and implant devices after the osseointegration has been established, while another approach is to visualize mineralization in real-time under simulated body conditions. Both of these approaches to understand mineralization have been explored in this thesis. Firstly, due to the inhomogeneous nature of bone and complex topography of implant surfaces, a suitable sample geometry for three-dimensional (3D) characterization was required to fully understand osseointegration. Electron tomography has been proven as an efficient technique to visualize the nanoscale topography of bone-implant interface in 3D. However, resulting from the thickness and shadowing effects of conventional transmission electron microscope (TEM) lamellae at high tilt angles and the limited tilt-range of TEM holders, “missing wedge” artifacts limit the resolution of final reconstructions. In Chapter 3, the exploration of a novel sample geometry to explore osseointegration is reported. Here, on-axis electron tomography based on a needle-shaped sample was applied to solve the problem of the “missing wedge”. This resulted in a near artifact-free 3D visualization of the structure of human bone and laser-modified titanium implant, showing bone growth into the nanotopographies of the implant surface and contributing to the evolution of the definition of osseointegration towards nano-osseointegration. One of the key issues regarding the mechanism of osseointegration that remains is that of the chemical structure at the implant interface, namely distribution of calcium-based and carbon-based components at the interface and their origins. Thus, the second objective of this thesis aimed to push characterization techniques further to four dimensions (4D), by incorporating chemical information as the fourth dimension after the spatial X,Y,Z coordinates. In Chapter 4, correlative 4D characterization techniques including electron energy-loss spectroscopy (EELS) tomography and atom probe tomography (APT) and other spectroscopy techniques were used to probe the nanoscale chemical structure of the bone-implant interface. This work uncovered a transitional biointerphase at the bone-implant interface, consisting of morphological and chemical differences compared to bone away from the interface. Also, a TiN layer between the surface oxide and bulk metal was identified in the laser-modified commercial dental implant. Both findings have implications for the immediate and long-term osseointegration. Since bone formation at the implant interface is a dynamic process, which includes calcium phosphates (CaP) biomineralization as a basis of these reactions, the third objective of this work focused on exploring real-time mineralization processes. Liquid-phase transmission electron microscopy (LP-TEM) is a promising technique to enable real-time imaging with nanoscale spatial resolution and sufficient temporal resolution. In Chapter 5, by using this technique, we present the first real-time imaging of CaP nucleation and growth, which is a direct evidence to demonstrate that CaP mineralization occurs by particle attachment. Overall, this thesis has applied state-of-the-art advanced microscopy techniques to enhance the knowledge and understanding of osseointegration mechanisms by investigating established biointerfaces and real-time mineralization. The developed correlative 4D tomography workflow is transferable to study other interfacial applications in materials science and biological systems, while the LP-TEM work forms a basis for further mineralization research. / Thesis / Doctor of Philosophy (PhD)

Magnesium composites have the potential to be used within the medical setting as a material, particularly for bone implants. Their potential comes from their possession of biodegradability characteristics and material properties that resemble the cortical bone. The biodegradability of the magnesium biomaterials can reduce the need for a second surgery to remove implants when a level of bone regeneration is reached to be self-sufficient, therefore removing the dependency on the implant. However, magnesium in its naturally occurring state demonstrates high corrosivity and degradation when simulated in a biological context. We investigate a magnesium composite (magnesium-bioglass) by testing biodegradation and mechanical properties, evaluating the differences in properties when compared to the mechanical properties of pure magnesium, and analyzing scanning electron microscopy results applied to samples immersed in a solution to simulate the in vivo setting. Through the various modes of fabrication of the magnesium composites, increased bioactivity can be measured. The results support the potential of using the bioactive magnesium-bioglass composites for orthopedic implants.