<p>Doktorska disertacija razmatra primenu savremenih aditivnih proizvodnih tehnologija u postupku preciznog livenja ortopedskih implantata i njihov uticaj na skraćenje vremena i smanjenje tro&scaron;kova izrade topljivih modela. Konvencionalni postupak preciznog livenja ortopedskih implantata zahteva značajno vreme i tro&scaron;kove za izradu kalupa za topljive modele. U disertaciji je razvijena metoda za &bdquo;brzo precizno livenje&ldquo; kojom se elimini&scaron;e potreba za izradom kalupa za topljive modele ortopedskih implantata. Potrebno je utvrditi da li se pomoću predložene metode &bdquo;brzog preciznog livenja&ldquo; koja predstavlja integraciju aditivnih proizvodnih tehnologija i reverznog inženjerstva sa konvencionalnim preciznim livenjem, može dati značajniji doprinos daljem razvoju u oblasti izrade prilagođenih ortopedskih implantata.</p> / <p>The doctoral thesis discusses the application of modern additive manufacturing technologies in investment casting of orthopaedic implants and their impact on time and cost savings in meltable wax models development. The conventional procedure of investment casting of orthopaedic implants demands considerable time and costs when developing moulds for meltable wax models. The thesis shows a method of &ldquo;rapid investment casting&rdquo; developed to avoid the making the moulds for meltable wax models of orthopaedic implants. It is necessary to establish whether the proposed method of &ldquo;rapid investment casting&rdquo;, which integrates additive manufacturing technologies and reverse engineering with conventional investment casting, may give a significant contribution to further development of manufacturing of customized orthopaedic implants</p>

The aim of this thesis was to investigate some methods for quantitative analysis of bone structure, particularly techniques which might ultimately be applied post-operatively following orthopaedic reconstruction operations. Initially it was decided to explore the efficacy of MRI in quantifying the bone structure at high resolution by comparing high resolution MRI against 'gold standards' such as Scanning Electron Microscopy (SEM) and optical histology. This basic study provided a measure of the distortions in the morphological bone parameters derived from MR images due to susceptibility artefacts and partial volume effects. The study of bone architecture was then extended to a model of advanced renal osteodystrophy in a growing rat. For this study, high-resolution micro computed tomography (microCT) was used and as a result of the high resolution images obtained, three new bone morphological parameters were introduced to characterise the bone structure. The desire to study bone architecture post-operatively in hip replacements led to a preliminary study on ex-vivo sheep acetabulae following total hip replacement, to determine the extent that the bone architecture could be investigated around the acetabulum. The motivation for studying the acetabulum was based on the high occurrence of debonding at the bone / prosthesis interface. This study demonstrated the superior nature of 3D MRI over conventional x-ray radiographs in early quantitation of fibrous membranes located between the host bone and the non-metallic implant and/or the bone cement. The presence of such fibrous membranes is strongly indicative of failure of the prosthesis. When using clinical MRI to image post-operative hip replacement, the image quality is severely affected by the presence of the metallic implant. The head of the prosthesis is shaped like a metal sphere and is located in the acetabular cup. This problem was investigated by performing simulations of MR images in the presence of the field perturbation induced by the presence of a metal sphere, with the effects of slice excitation and frequency encoding incorporated into the simulations. The simulations were compared with experimental data obtained by imaging a phantom comprising a stainless steel ball bearing immersed in agarose gel. The simulations were used to predict the effects of changing imaging parameters that influence artefact size and also to show how current metal artefact reduction techniques such as view angle tilting (VAT) work and to identify their limitations. It was shown that 2D SE and VAT imaging techniques should not be used when metallic prosthesis are present due to extreme slice distortion, whereas 3D MRI provided a method that has no slice distortion, although the effects of using a frequency encoding gradient still remain.

Dans ce travail de thèse, la texturation de surface d‟alliages de titane a été étudiée en utilisant un procédé d'écriture directe par laser femtoseconde dans le but d'améliorer la mouillabilité d‟implants dentaires et orthopédiques par les fluides biologiques et la minéralisation de la matrice (formation osseuse) tout en réduisant l'adhésion bactérienne et la formation de biofilmes. Des surfaces de titane (Ti-6Al-4Vet cp Ti) ont été micro-, nano-texturées par laser femtoseconde et une biofonctionnalisation de ces surfaces a été ajoutée ou non par greffage de peptides d'adhésion cellulaire (peptides RGD) en surface de ces différents matériaux. Les textures de surface peuvent être classées comme suit: (a) structures périodiques de surface induites par laser (LIPSS); (b) étalage de nanopiliers (NP); (c) étalage de micro colonnes recouvertes de LIPSS (MC-LIPSS) formant une distribution bimodale de rugosité. Nous avons montré que la texturation de surface par laser améliore la mouillabilité des surfaces avec de l'eau ainsi qu‟une solution saline tamponnée Hank's (HBSS) et amène une anisotropie de mouillage. Une minéralisation cellulaire est observée pour toutes les surfaces des deux alliages de titane lorsque des Cellules Souches Mésenchymateuses humaines (hMSC) sont cultivées dans un milieu ostéogénique. La minéralisation de la matrice et la formation de nodules osseux sont considérablement améliorées sur les surfaces texturées LIPSS et NP. Parallèlement,l'adhésion de Staphylococcus aureus et la formation de biofilmes sont considérablement réduites pour les surfaces texturées LIPSS et NP. La biofonctionnalisation des différentes surfaces texturées (cp Ti) par laser a été réalisée et caractérisée par spectroscopie de photoélectrons (XPS) et par microscopie à fluorescence en utilisant des peptides fluorescents. L‟ensemble des résultats obtenus suggèrent que la texturation de surface d'alliages de titane (Ti-6Al-4V et cp Ti) en utilisant une technique d‟écriture directe par laser femtoseconde est un procédé prometteur pour l'amélioration de la mouillabilité de la surface d'implants dentaires et orthopédiques par les fluides biologiques et leur ostéointégration (différenciation ostéoblastique et minéralisation de la matrice), tout en réduisant l‟adhésion de Staphylococcus aureus et la formation de biofilmes. Enfin, la combinaison de la texturation par laser et du greffage covalent d‟un principe actif (ici un peptide d‟adhésion cellulaire comme le peptide RGD) amènera indéniablement une bioactivité utile pour favoriser l'adhésion des hMSC et faciliter laformation osseuse. / In the present thesis the surface texturing of Ti alloys using femtosecond laser direct writing method is explored as a potential technique to enhance the wettability of dental and orthopaedic implants by biological fluids and matrix mineralisation (bone formation), while reducing bacteria adhesion and biofilmformation. The surface texture was combined with biofunctionalisation by covalent grafting of a RGD peptide sequence as well. The surface textures can be classified as follows: (a) Laser-Induced Periodic Surface Structures-LIPSS; (b) nanopillars arrays(NP); (c) arrays of microcolumns covered with LIPSS (MC-LIPSS), forming a bimodal roughness distribution. Laser texturing enhances surface wettability by water andHank‟s balanced salt solution (HBSS) and introduces wetting anisotropy, crucial incontrolling the wetting behaviour. Matrix mineralisation is observed for all surfaces of both Ti alloys when human mesenchymal stem cells (hMSCs) are cultured in osteogenic medium. Matrix mineralisation and formation of bone-like nodules are significantly enhanced on LIPSS and NP textured surfaces. On the contrary, Staphylococcus aureusadhesion and biofilm formation are significantly reduced for LIPSS and NP textured surfaces. The biofunctionalisation of the laser textured surfaces of cp Ti is sucessfully achieved. In general, these results suggest that surface texturing of Ti alloys using femtosecond laser direct writing is a promising method for enhancing surface wettability of dental and orthopaedic implants by biological fluids and their osseointegration (osteoblastic differentiation and matrix mineralisation), while reducing Staphylococcus aureus adhesion and biofilm formation. Finally, the combination of laser texturing and covalent grafting of a RGD peptide sequence may be potentially useful for increasing cell adhesion and facilitating bone formation.

The issue of prosthetic infection leading to implant failure due to the formation of bacterial biofilms on biomaterial surfaces has been widely recognized as a major issue, often leading to revision surgery. The growing number of patients requiring synthetic biomaterials as implants is on the rise and so is the risk of infection arising from pre/peri-/post-operative surgical procedures. Traditional antibiotic treatment has led to the emergence of bacterial drug resistance. Therefore, the development of novel bactericidal methods to combat drug resistant microbial pathogens is the need of the hour. The first part of the thesis is an attempt to address prosthetic infection by the development of novel ultrasmall gold nanoparticles (AuNPs) which are cytocompatible and present a therapeutic dosage window for eliciting antimicrobial property. Towards this end, ultrasmall AuNPs with 0.8 nm and 1.4 nm gold core sizes, stabilized by monosulphonated triphenylphosphine ligand shells were synthesized. Such intricately designed AuNPs with ultrasmall gold cores and phosphine-based ligand chemistry were demonstrated to be highly potent bactericidal agents against staphylococci, the most common human pathogen causing biomaterial associated infection. The antibacterial efficacy of these AuNPs was significant even in mature staphylococcal biofilms. In another study, the application of high strength pulse magnetic fields (1-4 Tesla) was examined for bacterial growth inactivation in vitro. A magnetic field strength dependent decrease in bacterial viability with a concomitant increase in the production of reactive oxygen species (ROS) and longer doubling times were recorded. The mechanism of action was explained through an analytical model which involves ion-transport interference of essential ions like Ca2+ and Mg2+ and disruption of FeS clusters leading to inactivation of bacterial redox enzymes. On the contrary, such high magnetic fields did not pose any detrimental effects to eukaryotic cells under similar exposure. Additionally, the potency of low intensity direct current electric field (DC EF: 1V/cm) against biofilm formation by methicillin resistant Staphylococcus aureus (MRSA) was explored on antimicrobial surfaces of hydroxyapatite and Zinc oxide (HA-xZnO; x = 0, 5, 7.5 and 10 wt%). An EF exposure time dependent decline in the viability and stability of MRSA biofilms were noted. Further, EF treatment resulted in bacterial membrane depolarization and reduced biofilm formation on HA-ZnO composites, independent of the substrate composition. In summary, the above three studies were cases of the developmental methods to address prothetic infection. The second part of the thesis is focused on the development of magneto-responsive biomaterials as implants for orthopaedic applications. Under this category, the sintering/ hot pressing of hydroxyapatite-magnetite (HA-xFe3O4; x = 0, 5, 10, 20 and 40 wt%) powders in oxidizing and inert atmospheres was carried out and the resulting phases and microstructure were characterized. A detailed analysis of the phase assemblage by Rietveld refinement of the X-ray diffraction (XRD) data and Mössbauer spectroscopy revealed the major retention of Fe3O4 along with wustite (FeO) formation under reducing conditions while hematite (α-Fe2O3) was the oxidized product of conventional sintering in ambient atmosphere. A good correlation between the unit cell volume increases in HA observed from Rietveld refinements and Fe incorporation into the apatite lattice from Mössbauer spectral parameters was evident. Further, the Mössbauer data analysis indicated a preferential occupancy of Fe at the Ca(1) site under oxidizing conditions and Ca(2) site in inert atmosphere. The above phase analyses were further confirmed by X-ray photoelectron spectroscopy (XPS), Infrared spectroscopy (FT-IR) and CHN analysis. The microstructure of the hot-pressed samples observed under transmission electron microscope (TEM) divulged similar phases as deduced from XRD as well as the formation of translational Moire fringe patterns due to inference of overlapping crystal planes of HA and Fe3O4 in the HA-40 wt% composite. Such translational Moire fringes suggest a preferred arrangement and orientation of the crystallites resulting from hot-pressing, which correlated well with the room temperature magnetic measurements made with the help of a vibrating sample magnetometer (VSM). The compositional similarity of Fe doping in HA to that of the tooth enamel and bone presents these HA-Fe3O4 composites as potent dental/ orthopaedic implant materials. In the conclusive study, the hot-pressed HA-xFe3O4 composites were tested for their efficacy in supporting the osteogenesis of human mesenchymal stem cells (hMSCs) assisted by intermittent static magnetic field exposure. The magneto-responsive substrates were applied as platforms for the culture of hMSCs and the effect of static magnetic field (SMF) exposure on the viability, proliferation and differentiation of hMSCs were elucidated. With a mild compromise in viability, SMF triggered the osteogenic differentiation of hMSCs mediated by proliferative arrest in the G0/G1 phase and elevated intracellular calcium levels. The early bone marker genes - Runx2, Col IA and ALP were significantly up regulated upon SMF exposure on pure HA and HA-Fe3O4 composites. Further, the late osteogenic markers – OCN and OPN were detected exclusively in the HA-xFe3O4 (x = 10 and 40 wt%) composites. Matrix mineralization was enhanced and CaP nodules were detected on similar SMF treated HA-Fe3O4 composites. A substrate magnetization and time dependent modulation of gene expression was recorded which corroborated well with the temporal trending of osteogenic genes during bone development. In conclusion, substrate magnetization can be applied as a tool to modulate the behavior of stem cells and direct them towards osteogenic lineage. Such a pertinent combination of substrate magnetization and external magnetic field stimulation can be applied synergistically for stem cell based bone tissue engineering applications.

Dans cette thèse, nous présentons une méthode de segmentation d’images radiographiques des membres inférieurs en régions d’intérêt (ROIs), une méthode de recalage rigide tridimensionnel (3D) / bidimensionnel (2D) des prothèses du genou sur les deux images biplanaires radiographiques calibrées et une méthode de reconstruction 3D des membres inférieurs à partir de deux images biplanaires radiographiques calibrées. Le premier article présente une méthode de segmentation de rotule, astragale et bassin des images radiographiques en régions d’intérêt basée sur la fusion de multi-atlas et superpixels. Cette méthode utilise l’apprentissage d’une base de données d’images radiographiques de ces os segmentées manuellement et recalées entre elles pour estimer un ensemble de superpixels permettant de tenir compte de toute la variabilité locale et non linéaire existante dans la base, puis la propagation d’étiquettes basée sur le concept d’entropie pour raffiner la carte de segmentations en régions internes afin d’obtenir le résultat final. Le deuxième article présente une méthode de recalage rigide 3D / 2D des composants tibiaux et fémoraux de prothèse du genou sur deux images biplanaires radiographiques calibrées. Cette méthode utilise une mesure de similarité hybride basée sur les notions de contours et régions puis un algorithme d’optimisation stochastique pour estimer la position des composants. La similarité basée sur les régions est stable et robuste contre les bruits. Cependant, cette mesure n’est pas précise car le nombre de pixels aux contours est inférieur au celui à l’intérieur de la région. Au contraire, la similarité basée sur les contours est précise mais plus sensible au bruit ou à d’autres artefacts existant dans les images. C’est pourquoi la combinaison de ces deux similarités fournit une méthode de recalage robuste et précise. Le troisième article représente une méthode statistique biplanaire de reconstruction 3D de rotule, astragale et bassin. Cette méthode utilise un algorithme de réduction de dimensionnalité pour définir un modèle déformable paramétrique qui contient toutes les déformations statistiques admissibles apprises à partir d’une base de données des structures osseuses. Puis un algorithme d’optimisation stochastique est utilisé pour minimiser la différence entre la projection des contours / régions des modèles surfaciques osseux avec ceux segmentés sur les deux images radiographiques. / In this thesis, we present a segmentation method of lower limbs of X-ray images into regions of interest (ROIs), a three-dimensional (3D) / two-dimensional (2D) rigid registration method of knee implant components to biplanar X-ray images, and a 3D reconstruction method of the lower limbs using biplanar X-ray images. The first paper presents a superpixel and multi-atlas-based segmentation method of the patella, talus, and pelvis into regions of interest. This method uses a training dataset of pre-segmented and co-registered X-ray images of these bones to estimate a collection of superpixels allowing to take into account all the nonlinear and local variability existing in the dataset, then a propagation of label based on the entropy concept for refining the segmentation map into internal regions to the final result. The second paper presents a 3D / 2D rigid registration method of tibial and femoral components of knee implants to calibrated biplanar X-ray images. This method uses a hybrid edge- and region-based similarity measure then a stochastic optimization algorithm to estimate the component position. The region-based similarity is stable and robust to noise. However, this measure is not precise because the number of pixels in the border is fewer than the number of pixels inside the region. On the contrary, the edge-based similarity is accurate but more sensitive to noise or other artifacts existing in the images. That’s why the combination of these two similarity types provides a robust and accurate registration method. The third paper presents a statistical biplanar 3D reconstruction method of the patella, talus, and pelvis. This method uses a dimensionality reduction algorithm to define a deformable parametric model which contains all admissible statistical deformations learned from the bone structure dataset. Then a stochastic optimization algorithm is used to minimize the difference between the contour / region projection of bone models and the contours / regions in two segmented X-ray images.