The success of orthopaedic implants fixed in the skeletal system using bone ingrowth into porous surfaces is critically dependent on the extent and quality of the initial bone ingrowth and the subsequent long-term maintenance of the bone within the porous structure. Biologically-significant elements (Ca, Mg, Mn) were incorporated at concentrations up to 5 mol% in solid solution in yttia-stabilised tetragonal zirconia polycrystal (3YTZP), whilst controlling microstructure and phase composition, to investigate the effect of ceramic chemistry on cellular behaviour in vitro and bone ingrowth into porous structures in vivo. Cellular attachment, proliferation, and migration on the ceramics were investigated using in vitro assays using fibroblasts. Cells were able to adhere strongly and proliferate on all ceramic surfaces, exhibiting maximal proliferation and minimal migration on 3YTZP but significantly faster migration on doped-3YTZP. The TZP ceramics were therefore considered to support normal cellular processes and thus were suitable for further study in vivo. A technique based on pressure casting ceramic slurry into a polymer preform of the desire pore structure, followed by polymer burnout and then sintering, was developed for fabricating porous bioceramics containing highly-controlled three-dimensional pore geometries. The ability of a selected pore structure to support bone ingrowth was tested using hydroxyapatite by implanting samples into femoral cortical bone of adult sheep for 4 and 12 weeks. Bone was able to rapidly colonise the porous structure and remodel such that, by 12 weeks implantation time, the majority of the porosity was filled with mature lamellar bone. Porous scaffolds of pure 3YTZP and 3YTZP doped with 1 mol% Mg, 1 mol% Mn, 1 mol% Ca, or 5 mol% Ca were fabricated and tested in the sheep model. Bone ingrowth into the doped compositions was significantly greater than that into pure 3YTZP, and was similar to that into the porous hydroxyapatite, indicating that the dopants significantly promoted osteogenesis within the bioinert scaffolds. This finding has application in clinical applications in that the initial bone ingrowth and, potentially, the long-term maintenance of bone within the porous structure may be improved by the incorporation of small amounts of biologically significant elements.

Additive manufactured porous titanium scaffolds have been extensively investigated for orthopaedic applications. The quantification of tissue response to the biomaterial implants is primarily achieved by analysing a two-dimensional (2D) stained histological section. More recently, three-dimensional X-ray micro-computed tomography (μCT) has become increasingly applied. Although histology is the gold standard, μCT allows non- destructive quantification of 3D tissue structures with minimal sample preparation and high contrast. A methodology to correlate information from both histology and μCT of a single sample might provide greater insights than either examining the results separately. However, this task is challenging because histology and μCT provide different types of information (stained tissue morphology vs. greyscale dependent on the X-ray absorption of material) and dimensionality (2D vs 3D). A semi-automated methodology was developed to directly quantify tissue formation and efficacy within an additive manufactured titanium implant using histology and μCT. This methodology was then extended to correlatively integrate nano-scale elemental information from nano- secondary ion mass spectroscopy (NanoSIMS). The correlative information was applied to investigate the impact of silver release on bone formation within a nano-silver coated additive manufactured implant. The correlative imaging methodology allowed for the quantification of the significant volumetric shrinkage (~15%) that occurs on histology slice preparation. It also demonstrated the importance of the location of the histological sectioning of the tissue and implant, revealing that up to 30% differences in bone ingrowth can be found along the entire length of the porous implant due to preferential bone ingrowth from the periphery to the centre. The quality and quantity of newly formed bone were found to be comparable between the uncoated and nano-silver coated Ti-implants, suggesting that the layer of silver nanoparticles on the Ti-implant does not negatively impact bone formation. Further, the newly formed bone at 2 weeks had a trabecula morphology with bone at the interface of Ti-implant as well as at a distant. This indicates that both contact (bone apposition on implant) and distance (bone ingrowth from host bone) osteogenesis were present in both types of implants. Finally, nanoscale elemental mapping showed silver was present primarily in the osseous tissue and was co-localised to sulphur suggesting that silver sulphide may have formed.

This thesis summarizes the results concerning the manufacture of medical implants for bone replacement using electron beam melting (EBM) which is an additive manufacturing (AM) technology, and aims to satisfy the engineering needs for the medical functionality of manufacturing technology. This thesis has focused on some microscopic properties for surfaces and bone integration. The process parameters of EBM manufacturing were studied to ascertain whether they have impacts on surface appearance, as surface properties have impacts on bone integration and implant performance. EBM manufacturing uses an electron beam to melt metal powder onto each layer in a manner akin to welding. The electron beam is controlled by process parameters that may be altered to a certain extent by the operator. There are individual process parameters for every material, and new parameters are set when developing new materials. In this thesis, process parameters in default settings were altered to ascertain whether it was possible to specify process parameters for implant manufacturing. The blood chamber model was used for thromboinflammation validation, using human whole blood. The model is used to identify early reactions of coagulation and immunoreactions. The material used in this study was Ti6Al4V-ELI, which is corrosion resistant and has the same surface oxide layers as titanium, and CoCr-F75, which has high stiffness, is wear-resistant and is commonly used in articulating joints. The study shows that among the process parameters researched, a combination of speed and current have the most impact on surface roughness and an interaction of parameters were found using design of experiment (DOE). As-built EBM surfaces show thrombogenicity, which in previous studies has been associated with bone ingrowth. Surface structure of as-build EBM manufactured surfaces are similar to implants surfaces described by Pilliar (2005), but with superior material properties than those of implants with sintered metals beads. By altering the process parameters controlling the electron beam, surface roughness of as-build parts may be affected, and the rougher EBM manufactured surfaces tend to be more thrombogen than the finer EBM manufactured surfaces. As-build EBM manufactured surfaces in general show more thrombogenicity than conventional machined implants surfaces. / Denna avhandling behandlar tillverkning av medicinska implantat för integration i ben. I fokus är den additiva tillverkningstekniken ”elektronstrålesmältning” ( Electron Beam Melting –EBM), en av flera tekniker som populärt beskrivs med termen 3D-skrivare. Avhandlingen fokuserar på mikroskopiska ytegenskaper och dess inverkan på benintegration. Processparametrarna för EBM-tillverkning studerades för att fastställa hur de påverkar ytans utseende, efter som ytegenskaper har effekt på implantatens funktion. EBM-tillverkning använder en elektronstråle som likt svetsning smälter ihop metallpulver. Elektronstrålen styrs av processparametrar som till viss mån kan justeras av maskinoperatören. Det finns individuella processparametrar för varje material och nya parametrar utvecklas till varje ny legering. I denna avhandling har ”grundinställningarnas processparametrar” studerats för att ta reda på om det är möjligt att ställa in specifika parametrar till implantattillverkning. Med hjälp av blodkammarmetoden, som använder humant blod, har thromboinflammatoriska egenskaper undersökts. Metoden identifierar tidiga koagulations- och immunologiska reaktioner. Legeringarna som undersökts i denna studie var Ti6Al4V-ELI, som är korrosionsbeständigt med samma uppsättning oxider på ytan som titan har, och CoCr-F75, en legering som har hög styvhet, är slitstarkt och är vanligt förekommande i implantat för leder. Bland de undersökta processparametrarna visar en kombination av hastighet och ström ha mest inverkan på ytjämnhet och en interaktion mellan parametrar identifierades med hjälp av försöksplanering. EBM-tillverkade ytor visade på thrombogena egenskaper som i tidigare studier kan relateras till god integration i benvävnad. Ytstrukturen hos EBM-tillverkade ytor liknar de implantatytor som Pilliar (2005) beskriver, men materialegenskaperna är bättre än de materialegenskaper som implantat, med sintrad yta, har. Genom att ändra processparametrarna som styr elektronstrålen kan ytstrukturen påverkas. Grövre EBM-tillverkade ytor tenderar att vara mer thrombogena än de finare EBM-tillverkade ytorna är. Obehandlade EBM-tillverkade ytor i allmänhet är mer thrombogena än vad konventionellt framställda implantatytor är.

Subscription articles: Theses and dissertations which contain embedded PJAs as part of the formal submission can be posted publicly by the awarding institution with DOI links back to the formal publications on ScienceDirect.doi:10.1016/j.msec.2015.10.069 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19578号 / 医博第4085号 / 新制||医||1013(附属図書館) / 32614 / 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 開 祐司, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A comprehensive understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is essential in the design of advanced biomaterials that better promote bone growth and osseointegration of implants. Dental implants with roughened surfaces and high surface energy are well known to promote osteoblast differentiation in vitro and promote increased bone-to-implant contact in vivo. In addition, increased surface roughness increases osteoblasts response to the vitamin D metabolite 1α,25(OH)2D3. However, the exact mechanisms mediating cell response to surface properties and 1α,25(OH)2D3 are still being elucidated. The central aim of the thesis is to investigate whether integrin signaling in response to rough surface microtopography enhances osteoblast differentiation and responsiveness to 1α,25(OH)2D3. The hypothesis is that the integrin α5β1 plays a role in osteoblast response to surface microtopography and that 1α,25(OH)2D3 acts through VDR-independent pathways involving caveolae to synergistically enhance osteoblast response to surface roughness and 1α,25(OH)2D3. To test this hypothesis the objectives of the studies performed in this thesis were: 1) to determine if α5β1 signaling is required for osteoblast response to surface microstructure; 2) to determine if increased responsiveness to 1α,25(OH)2D3 requires the vitamin D receptor, 3) to determine if rough titanium surfaces functionalized with the peptides targeting integrins (RGD) and transmembrane proteoglycans (KRSR) will enhance both osteoblast proliferation and differentiation, and 4) to determine whether caveolae, which are associated with integrin and 1α,25(OH)2D3 signaling, are required for enhance osteogenic response to surface microstructure and 1α,25(OH)2D3. The results demonstrate that integrins, VDR, and caveolae play important roles in mediating osteoblast response to surface properties and 1α,25(OH)2D3. Silencing of the β1 integrin in osteoblast-like MG63 cells significantly reduced osteogenic response to surface topography and 1α,25(OH)2D3. Silencing of the α5 subunit did not alter the response of MG63 cells to changing surface roughness or chemistry, although future work must confirm these results given similar cell surface α5 integrin expression observed in control and α5-silenced cells. Multifunctional RGD, KRSR, and KSSR coated surfaces show that RGD increased osteoblast proliferation and reduced differentiation, KRSR had no affect on osteoblast phenotype, and KSSR increased osteoblast differentiation. These results suggest that titanium surfaces can be modified to manipulate proliferation and differentiation and that RGD/KSSR functionalized surfaces could be further investigated for use as osteointegrative surfaces. The results using VDR deficient osteoblasts demonstrate that 1α,25(OH)2D3 acts via VDR-dependent mechanisms in cells cultured on titanium surfaces that support terminal differentiation. In caveolae deficient osteoblasts, 1α,25(OH)2D3 affected cell number, alkaline phosphatase activity, and TGF-β1 levels, although levels of osteocalcin and PGE2 were not affected. These results are consistent with the hypothesis that VDR is required for the actions of 1α,25(OH)2D3, but that caveolae-dependent membrane 1α,25(OH)2D3 signaling modulates traditional VDR signaling. The exact mechanisms for this interaction remain to be shown. Overall, these results are important in better understanding the role of β1 integrin partners in mediating osteoblast response to implant surfaces and in understanding how integrin signaling can alter osteoblast differentiation and responsiveness to 1α,25(OH)2D3 via genomic and non-genomic pathways.