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Increasingly, titanium is being used as an implantable material to make best use of its ability to integrate with bone. The replacement of missing teeth is likely the most widespread use of this technology but there is an increasing number of uses in orthopaedics and audiology. One of the challenges with this technique is being able to assess whether the bone-titanium interface is intact or is being subject to breakdown. Currently there is not a reliable and sensitive instrument able to monitor changes in bone-titanium interface. This study sought to develop a reliable and simple-to-use test for monitoring osseointegration using dental implants as a model system with an approach that would allow early detection of compromise to the bone to implant interface. A series of systematic investigations were conducted to examine the reliability of the acoustic emission technique (AE) for measuring changes in the osseointegration of dental implants using an in vitro model system. The model system involved dental implants installed into bovine rib bones with models for; primary stability, partial and full osseointegration, and degraded osseointegrated interfaces. The AE (a high-frequency ultrasonic wave) was produced by a simple source and was injected into the abutment of the implant. The transmitted energy was measured on the surface of the rib bone using a proprietary sensor. Some energy is lost at the implant-bone interface, but also in transmission along and through the bone. The effect of bone micro- and macro-structure on acoustic transmission through and along bone has been measured in the primary stability model and a quantitative relationship developed to allow this patient-specific aspect to be taken into account in a clinical situation. The primary stability model simply involved installing the implant using the normal surgical procedure. For secondary stability, glass ionomer cement was used as a model interfacial material giving partial and full coverage. It has been found that the transmitted energy could distinguish between primary stability and partial and full integration. Finally, to gauge how effective the acoustic emission technique could be in detecting early changes in the marginal bone around osseointegrated implants, simulated circumferential and vertical peri-implant bone defects of various vertical and circumferential extent were tested. It was found that the acoustic energy could effectively detect small changes in marginal bone level around osseointegrated implants. Changes in transmission of the AE signal were able to show both circumferential and narrow vertical bone defects including the most coronal 1 mm of the marginal bone. These findings suggest a role for AE in monitoring the development of osseointegration in the weeks following implant placement and could be coupled with an assessment of bone density on an individual patient basis. The technique could also have a potential application in the early diagnosis of the peri-implantitis in the oral environment or other forms of loss of integration when used elsewhere in the body. These findings are promising, although a number of practical issues need to be resolved before the technique can be validated in the clinical setting. Whereas the dental application is a useful model system, a clinical validation could lead to more general application in cases of monitoring bone-implant integration.

Titanium (Ti) and Ti alloys are widely used as medical implant materials due to their remarkable biocompatibility and corrosion resistance properties. Thanks to their great durability and high strength to weight ratio, Ti-based devices are often chosen as a skeletal replacement therapy to improve quality of life of patients suffering from bone fracture and osteolysis. Researchers around the globe have tried multiple approaches to increase the unmet needs of cost-effectiveness, to improve restorative time and the osseointegration of Ti-based implants. This thesis aims to investigate the optimisation of Ti-based materials through surface coating, nanostructured modifications, and electrical stimulation for use in bone and dental applications. Surface coating using osteoinductive coating materials such as hydroxyapatite (HA), brushite and other orthophosphates can enhance bone growth and osseointegration. In collaboration with the University of Bath and Plasma Coatings Ltd., the alloyed Ti (Ti-6Al-4V) substrates were modified to investigate the biocompatibility of the commercially viable thin layer calcium orthophosphates coatings on metal substrates using different coating methodologies: thermal spraying HA, grit-blasting, HA via brushite conversion, and electro-precipitating brushite. A more uniform coating layer with sophisticated morphology was achieved using electrodeposition techniques, which presented approximately 6 times thinner coating thickness in comparison to thermally coated samples. However, the subsequent biocompatibility test using MC3T3-E1 osteoblastic cells revealed the possible dissociation of undesirable ions and structural instability of thinly coated brushite and converted HA samples, as opposed to more biocompatible and morphologically stable thermal coated HA and grit-blasted samples. Surface nanostructuring using electrochemical anodisation techniques has gained increasing interest due to its ability to create uniform titania (TiO2) nanotube arrays that provide a nanoengineered biomimetic environment to promote osteogenesis and bone cell mineralisation. This section provides a methodological optimisation of the anodisation device to gain enough specimens to be able to meet statistically informed decisions on the suitability of this surface modification technique in biomedical applications. An average of approximately 7.5-fold increase in cost and time efficiency was attained with regards to the reproducible and consistent generation of 20, 50 and 100 nm pore diameter nanotube via anodising pure Ti and Ti-6Al-4V. The optimised device allows the manufacturing of a high number of anodised specimens with customisable specimen shape and tunable pore diameters for use in in-vitro assessments. The biocompatibility and functional assays closed the gaps present in the literature regarding the effect of different nanotube pore diameter on different human cell lineage, revealing that human mesenchymal progenitors exhibited high differentiation rate on 20 nm pore diameter TiO2 nanotubes; while osteoblast lineage differentiates better on TiO2 nanotube specimens with 50 nm pore diameter. Electrical stimulation (ES) can control cell growth, orientation and bone remodelling appreciated by the electrical signals as important stimuli. Combining pure Ti and ES, this section involves software modelling of the electric field (EF) and current density in different bioreactor designs. The novel ES bioreactor with capacitive stimulation delivery system features long-term stimulation with homogeneous EF, biocompatible, sterilisable and cost-effective in the manufacturing process. A continuous capacitive stimulation regime on Ti with 200 mV/mm simulated EF could influence stem cell orientation and promote nuclei elongation, proliferation, differentiation and matrix production as compared to non-stimulated controls. The three approaches investigated in this thesis provide novel engineering approaches to influence cell activities. In addition to filling the gaps present in the literature, the interaction of the cells and materials was also explored to elucidate the underlying mechanisms affecting the cellular behaviours. As a whole, the novel methodologies introduced in this research provide a thoughtful fundamental basis for future studies in the area of biomaterials and regenerative medicine.

Introduction: The survival of dental implants depends on osseointegration. Modifications to the implant surface are attractive for promoting the success of the implant. Abrading the surface of the implant with bioactive glass is an attractive option for improving the speed of osseointegration. Aims: To develop a bioactive glass that has the ability to enhance the surface roughness of the implant by embedding particles into the surface and to investigate the effect of grit blast parameters on the removal of the titanium from the surface. Methods: Three glasses based on SiO2-CaO-Na2O-P2O5-CaF2 were synthesized by a melt quench technique. The glasses were characterised and investigated for their bioactivity. Titanium discs were abraded with coarse glass particles by using a grit blast technique with different parameters, such as, distance, air pressure and speed. The depth, width of the abraded line and surface roughness (Ra) were measured by light profilometer. The distribution of the glasses on the titanium surface was measured by SEM-EDX. The biocompatibility of the abraded discs was tested in vitro using MC3T3-E1 cell line. Results: All glasses exhibited an amorphous structure with varied bioactivity. Changing the abrasion parameters influenced the amount of titanium removed and the surface coverage. The harder the glass the lower the amount of titanium removed and the wider the width of the abraded area. The Ra was significantly increased from 0.1μm to 1.6μm. The SEM-EDX analysis confirmed that the glasses were widely distributed and a higher coverage was seen with the harder glass. The abraded discs showed good biocompatibility in vitro. Conclusions: The designed compositions were successfully modifying the surface of the titanium. They are abrasive enough to significantly embed into the titanium surface by using the grit blast technique and increase their surface roughness. TheIntroduction: The survival of dental implants depends on osseointegration. Modifications to the implant surface are attractive for promoting the success of the implant. Abrading the surface of the implant with bioactive glass is an attractive option for improving the speed of osseointegration. Aims: To develop a bioactive glass that has the ability to enhance the surface roughness of the implant by embedding particles into the surface and to investigate the effect of grit blast parameters on the removal of the titanium from the surface. Methods: Three glasses based on SiO2-CaO-Na2O-P2O5-CaF2 were synthesized by a melt quench technique. The glasses were characterised and investigated for their bioactivity. Titanium discs were abraded with coarse glass particles by using a grit blast technique with different parameters, such as, distance, air pressure and speed. The depth, width of the abraded line and surface roughness (Ra) were measured by light profilometer. The distribution of the glasses on the titanium surface was measured by SEM-EDX. The biocompatibility of the abraded discs was tested in vitro using MC3T3-E1 cell line. Results: All glasses exhibited an amorphous structure with varied bioactivity. Changing the abrasion parameters influenced the amount of titanium removed and the surface coverage. The harder the glass the lower the amount of titanium removed and the wider the width of the abraded area. The Ra was significantly increased from 0.1μm to 1.6μm. The SEM-EDX analysis confirmed that the glasses were widely distributed and a higher coverage was seen with the harder glass. The abraded discs showed good biocompatibility in vitro. Conclusions: The designed compositions were successfully modifying the surface of the titanium. They are abrasive enough to significantly embed into the titanium surface by using the grit blast technique and increase their surface roughness. The glass abraded discs show both good bioactivity and biocompatibility in vitro.

Due to an aging population and younger patients presenting with musculoskeletal disorders, there is a need for orthopaedic implants with improved healing rates and longer implant life. Numerous research has developed implant surfaces with micro-topography and biomolecules to imitate the native extra cellular matrix (also known as biomimetic surfaces). This research has utilised such a biomimetic approach by immobilising the cell adhesive peptide, RGD (Arginine-Glycine-Aspartic Acid), to a titanium alloy Ti6Al4V surface. This research polymerised Hyperbranched Polyglycerol (HBPG) from the titanium surface using Ring Opening Multi-Branching Polymerisation (ROMBP). HBPG is a biologically compatible and non-toxic synthetic biopolymer, able to reduce non-specific protein adsorption, increase the titanium surface wetting (hydrophilicity), thereby limiting foreign body reactions. Extensive hydroxyl groups at the periphery of HBPG provides conjugation sites for biomolecule attachment. In this work the RGD peptide was conjugated to the polymer via a siloxane layer. This research developed a novel passivation solution for the preparation of the titanium alloy surface, using a mixture of hydrogen peroxide and nitric acid (a passivation mixture not used in the literature). This novel mixture was shown to etch the titanium surface, producing micro and nano surface features, both of which have been shown to improve cellular function in the literature. The hydrogen peroxide/nitric acid solution showed extensive oxidising ability on titanium, leading to the formation of reactable hydroxyl groups. Contact-angle measurements showed that the novel passivating solution produces a hydrophilic surface similar to that of peroxidation for 12-hours, but achieved in only 2-hours. In conjunction with the etching and oxidising abilities of hydrogen peroxide, the nitric acid reacts with the titanium surface, leading to the formation of a protective titanium oxide layer, enhancing corrosion resistance and improving biocompatibility. Biological investigations with the pre-osteoblast cell line MC3T3-E1 showed greater osteoblast cell attachment and adhesion strength, as well as improved bone matrix mineralisation on the passivated titanium surface functionalised with HBPG and the RGD peptide, compared to the raw and passivated titanium surfaces. Antibacterial testing of HBPG revealed substantially reduced bacterial cell colonies on the passivated/polymerised titanium surface, possibly arising from electrostatic and hydrophobic repulsion. This research has successfully developed a new titanium passivation solution (hydrogen peroxide/nitric acid) that can yield a contact-angle of around 35° in just 2-hours, rivalling the Piranha solution. The successful immobilisation of a cyclic RGD (cyclic-RGDfc) to a titanium surface functionalised with HBPG, has been shown in this research to drastically improve mineralised bone matrix production from the MC3T3-E1 cell line. This indicates earlier osseointegration of the implant may be possible, thereby improving patient healing times.

The primary events occurring in vivo after implant placement are very important in achieving osseointegration. The objective of this research study was to investigate these primary processes and its variations with different surface treatments of titanium. This may further aid in finding surface characteristics which have a positive effect on osseointegration. Firstly, ion adsorption from a physiological solution and ion and protein adsorption from a simulated in vivo environment to polished, glass bead blasted and alkali etched + heat-treated titanium surfaces were tested with the aid of x-ray photoelectron spectroscopy. Secondly, the adhesion strength of bone cells to the 3 different titanium surfaces in two force directions (normal and tangential to the surface) was assessed with the aid of centrifugal accelerations. The theory here was that ions are adsorbed first to the surfaces, proteins then bind to the ions, and then cells can bind to the RGD sequence in certain proteins. Calcium (Ca) and phosphorus (P) were found to be adsorbed to all titanium surfaces from the physiological solution to varying degrees, however, only trace levels of Ca and P could be detected on all surfaces after exposure to a simulated in vivo environment, instead large amounts of protein were found. Bone cell adhesion strength was found to vary (between < 2 x 10-8 N and 8 x 10-8 N) statistically significant (p < 0.001 - p < 0.05) with surface treatment and force direction. Conclusion: although the surfaces exhibited different responses for individual factors of ion adsorption, protein adsorption or cell adhesion, on balance for all three factors the performance of glass bead blasted titanium surfaces showed the most positive results.

Abstract: Osseointegrated implants rely on the direct structural and functional attachment of an implant to the surrounding bone tissues. These implants are used in various fields including dentistry, bone conduction hearing and lower-limb amputations. Determining the quality of the bond between the implant and the surrounding bone (implant stability) is important in evaluating the surgical outcomes, early failure-detection and optimising rehabilitation. Vibration analysis can non-invasively quantify the bone-implant-interface (BII) properties. The Advanced System for Implant Stability Testing (ASIST) is a vibration-based stability measurement system that has been used with Bone Anchored Hearing Aids (BAHA). It evaluates the BII stiffness by matching the measured acceleration to the prediction of a mathematical model. This work focuses on the mathematical and experimental analysis of the dynamic behavior of the implant for Transfemoral lower-limb Amputations (TFA) and BAHA systems. The analysis extends the ASIST approach to TFA systems and builds on the existing knowledge of BAHA systems using novel modelling approaches. The TFA bone-implant system was first analysed using 3D Finite Element (FE) modal analysis and benchtop experiments under different BII, loading, boundary and implant-adapter conditions. The analysis indicated that a measurement system based on the axial modes is: 1) more sensitive to the BII condition, 2) more immune to the femoral boundary condition and 3) more characterizable for different implant-adapter configurations compared to the transverse modes. Therefore, a 1D FE model of the TFA system was developed using axial bar elements. The model was validated by: 1) testing the 1D FE formulation using a uniform cylinder, 2) comparing the 1D FE modal behavior with a 3D FE model for the actual implant geometry and 3) using the 1D FE model to analyse signals generated from 3D FE implicit and benchtop models. The 1D FE formulation converged to the analytical and 3D FE solutions for the uniform cylinder. The 1D model was representative of the TFA system and predicted similar axial modes (maximum frequency difference of 2.07%) compared to the 3D FE model. The 1D FE model was then incorporated into a custom-built MATLAB® application that extracts the BII stiffness by matching the input signal to the model’s prediction using an optimization routine. The approach was highly sensitive to the BII condition and predicted interface stiffness values of 4.75×106, 1.43×108 and 2.21×109 N/m for the experimental LOW, INTERMEDIATE and HIGH conditions respectively. The model also predicted similar damping ratios compared to the 3D FE simulations. The optimization routine successfully matched the signals, with frequency domain similarity scores of 94-100%. 1D and 3D FE models of the BAHA systems were developed. Although the previous work involved modelling BAHA implants using an analytical 4 Degree of Freedom (DOF) and benchtop setups, the developed FE models tested the implant system under a wider range of conditions. The 3D FE signals were analysed with the ASIST utility, the ASIST Stability Coefficient (ASC; directly computed from the BII stiffness) was more sensitive to the BII condition and less sensitive to the abutment geometry compared to the natural frequencies. The 3D models were also validated with benchtop and clinical signals that represented extreme BII cases and were then used to study the nature of the BII and its influencing parameters. Finally, a 1D FE model was developed for BAHA systems using Timoshenko beam elements. The 1D FE model behaved similarly to the previously developed 4 DOF model, where they both predicted similar natural frequencies with differences of -0.77 to 4.40% and -1.07 to -2.76% for the 1st and 2nd modes respectively. The consistently lower 2nd mode frequencies of the 1D FE model (due the larger number of DOF) resulted in lower ASC scores but the trends between both models were preserved. The mathematical analysis performed in this work offers a comprehensive understanding of the dynamic behavior of both implant systems. This is the first attempt to model the Osseointegrated Prosthetic Limb (OPL) TFA system and the first one to propose a vibration-based implant stability measurement method for it. The 1D FE models for both bone-implant systems (TFA and BAHA) show high promise in modelling them and evaluating the BII stiffness. This approach paves the way for the non-invasive evaluation of the BII properties in clinical settings.

The development of bioactive materials that can mimic bone tissue has been an area of active research to try and improve the process of implant integration. Wool-derived keratin, a readily available natural polymer, has been shown in vivo to enhance bone healing and as such may be a novel material for use in the development of biomimetic devices. Coating titanium with keratin, therefore, may be a promising approach to achieve this, but effective bonding between keratin and titanium and the molecular response of osteoblasts to keratin has not been fully delineated. Using aminosilane (APTES) and glutaraldehyde (GA) to prepare the titanium surface, two coating strategies were employed in the present study i.e., solvent casting and molecular grafting, to try and improve the coating's chemical and mechanical properties and improve the interaction between keratin and adherent osteoblastic cells. This thesis had three main goals. First, to characterize the physiochemical and mechanical properties of the keratin coating using various analytical techniques. Second, to evaluate the cytocompatibility of the keratin coating with osteoblastic MG-63 cells in vitro. Finally, to investigate the bone healing response to keratin-coated titanium in vivo using a rat calvarial defect model. [...]

The development of patient-specific implants for reconstructive applications involving unique deformities or injuries has demonstrated the promise of additive manufacturing (AM) in the medical setting. Biomimetic lattice structures can facilitate internal bone growth due to their porous nature, which can reduce stress shielding and improve the lifespan of an implant. In addition, pulsed electromagnetic field therapy can be implemented to accelerate the osseointegration process internally in the lattice structures by improving cellular proliferation and attachment. In this research, cubic and body-centered cubic (BCC) unit cell geometries were 3D printed using selective laser melting and biocompatible Ti-6Al-4V feedstock powder. The lattices were designed with four different pore sizes (400, 500, 600, and 900 µm, representing 40-90% porosity in a 10 mm cube). Compression and tensile testing were performed to evaluate the mechanical properties of the lattice structures, with results compared to human cancellous bone. SEM microstructural characterization of the manufactured lattices exhibited deviations in AM pore sizes and strut diameters when compared to original CAD designs in nTopology. It was found that the elastic modulus of human cancellous bone (10 - 900 MPa) could be matched for both tensile (92.7 - 129.6 MPa) and compressive (185.2 - 996.1 MPa) elastic modulus of cubic and BCC lattices. BCC lattices exhibited higher compressive properties over cubic, whereas cubic lattices exhibited superior tensile properties over BCC. To determine the effects of porosity and unit cell geometry on the osteogenesis of human MG-63 osteoblastic cell lines in vitro, glucose consumption, alkaline phosphate (ALP), and end-of-culture cell count activity was analyzed as markers for osteogenic growth. The results indicated that lattices with a 900 µm pore size exhibited the highest glucose consumption and the greatest change in alkaline phosphate activity when compared to other pore sizes, for both strut geometries. On average, the cubic unit cell geometry exhibited higher glucose consumption, and a larger cell count when compared to their counterparts with body-centered cubic strut geometries. Irrespective of a particular pore size or unit cell geometry, it was found that all lattices can promote osteogenic growth. A pulsed electromagnetic field (PEMF) was then applied (15 Hz, 300 µs pulse width, 20 VPP, 0.5 mT field intensity) to cell cultures in vitro (2 hours/day for 14 days) and compared to non-PEMF stimulated controls. The results indicated that PEMF had a greater impact on glucose concentration than ALP activity for all lattices. This may indicate that at lower field intensities, PEMF had an increased effect on the early formation (surface attachment) stage of bone cell differentiation.

Limb loss poses a significant challenge to mobility and the performance of daily activities. Currently, nearly half of the amputees using powered prosthetics abandon their device due to inadequate limb coordination and movement control abilities. In the healthy system, passive and neural dynamics facilitate the synchronized activation of muscles necessary for coordinated movement. However, in the amputee, the system is disrupted. The goal of this research is to use neural and mechanical dynamics to inform the design of prosthetic devices in order to restore intuitive control and coordinated limb movement. The first aim of this research is to investigate neural dynamics for prosthetic control, and establish patterns of muscle activity during symmetric and asymmetric precise stepping and apply two common dimensionality reduction techniques to identify independent neuromuscular control signals in an animal model. Future work will be done to incorporate these control signals with musculoskeletal models in order to predict intended movement in the prosthetic limb. We find that there does exist a stereotypical pattern, or template, of muscle activity during the precise stepping task, and a meaningful set of dynamic control signals can be extracted using dimensionality reduction techniques. To investigate mechanical dynamics in the amputee, we studied changes in limb coordination after replacing the socket prosthetic with osseointegrated components. Using an analysis of spatiotemporal gait characteristics, muscle activity and kinematics, we show significant improvements in locomotor coordination in three participants. The results from this work will guide the development of advanced prosthetic devices that will incorporate dynamic templates of muscle activity into control algorithms and improve mechanical dynamics through osseointegration. This proposed design incorporates neural and mechanical dynamics to improve limb coordination and intuitive control, thereby reducing the rate of prosthetic device abandonment and improving the quality of life for amputees.

Zirconia has been established as a promising material for dental implants. Various surface treatment methods have been utilized to promote better osseointegration and improve the success rate of dental implants. However, a better understanding of the influences of topographic characteristics on cell attachment, proliferation, and differentiation is needed. Different surface topographic zirconia specimens, As sintered, Mild rough, Moderate rough, and Rough zirconia groups were fabricated with sandblasting method in various distances and stages. The surface texture, microstructure, and wettability were inspected with the optical profiler, SEM, and contact angle measurement respectfully. Human primary osteoblast cells were cultured on the four groups of zirconia with different surface modifications in 24 well plates and on plates without test material as control. Crystal violet and triton X-100 solution were used to evaluate cell attachment efficiency at 9 hours and proliferation rate at 7, 14, and 21 days after seeding. ALP activity was measured with fluorometric assay. The expression of osteocalcin was measured with an ELISA kit. Alizarin red staining was conducted to evaluate the mineralization. The cell morphologies were inspected under SEM after cell fixation and critical point drying process. The data were analyzed with one-way ANOVA for experiment on each time interval and two-way ANOVA for all time points. Tukey post hoc test was used for pairwise comparison. P value < 0.05 was considered statistically significant. Topographic parameters and contact angle measured in As sintered, Mild rough, Moderate rough, and Rough surface groups were as follow: Sa = 0.23, 0.50, 2.13, 5 µm, Sal = 49.88, 21.20, 30.42, 49.87 µm, Sdq = 64.64, 248.60, 511, 734.66 µm/mm, Sk = 0.7, 1.54, 4.19,16 µm, Spk = 0.31, 0.64, 1.47, 5.13 µm, Svk = 0.35, 0.71, 5.96, 6.18 µm respectively, and contact angle = 64.6°, 55.2°, 43.5°, 38.6° respectively. The result showed that Rough zirconia group induced the highest cell attachment efficiency at 9 hours (p

AIM: To measure marginal bone loss around endosseous dental implants at 2nd stage abutment surgery and retrospectively evaluate the association of pre-surgical variables with bone findings. MATERIALS AND METHODS: Two implant systems were included in the study design. Straumann Bone Level Tapered SLActive and Nobel Biocare Replace Conical Connection. Implants were placed by the same resident from the Periodontics department. Clinical measurements of bone level were recorded at the time of placement and then again at 2nd stage surgery at four different points (mesial, lingual, buccal, distal). RESULTS: Five implants were evaluated in the study. Bone loss averages ranged from 0-1.0mm. Nobel implants had an average loss of .75mm and Straumann implants had an average loss of .67mm. CONCLUSION: Many variables contribute to marginal bone loss around endosseous dental implants at 2nd stage abutment surgery. The current research was designed to understand the relationship between these variables and early marginal bone loss. All implant samples included here were defined as control because of ≤1.0mm bone loss per IRB protocol.

New understanding of immune system biology has led a paradigm shift in the development of biomaterials away from classically ‘inert’, to ‘immuno-modulatory’ biomaterials that have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling responses as opposed to persistent inflammation and scar tissue formation. This project aims to examine macrophage behavior and in particular integrin expression at the cell–biomaterial interface in vitro, in order to delineate the underlying molecular events occurring during biomaterial-mediated osseous healing. After reviewing the current literature to identify research gaps that this thesis should focus on, the activation of macrophages with controlled timing, and modulation of their interactions with other cell types involved in wound healing, emerged as key strategies to improve biomaterial efficacy. Careful design of biomaterial structure and controlled release of immunomodulators can be employed to manipulate macrophage phenotype for the maximization of the wound healing response with enhanced tissue integration and repair, however, to elicit predictable immune responses there is a need for a thorough understanding of how the biomaterial properties can be tuned to harness a designed immunological outcome. Our systematic review of in vitro studies suggested that the initial immune response of macrophages to titanium may be modulated by its surface characteristics both topographically and physiochemically. We therefore assessed the potential effects of three novel titanium surfaces; Machined, Blasted and Fluoride-treated, on macrophage phenotype. We chose a monocytic cell-line as our macrophage source rather than using primary macrophages, as the inflammatory status of primary cells could not be readily defined nor reproduced between experiments. This was crucial to the overall study if we were to clearly assess any changes in macrophage phenotype as a result of their II interaction with the titanium surfaces. We subsequently analysed in some detail the experimental processes involved in the optimization of macrophage cell culture, macrophage differentiation and phenotype characterization. We used microscopy, profilometry and elemental analysis to understand how the roughness of the titanium surfaces affected macrophage viability and proliferation in both non-activated ‘M0’ and activated inflammatory ‘M1’ macrophages. Subsequent analysis of one of the key mediators of cell attachment to biomaterials showed for the first time that αM, β1, β2 and α2 integrins play an important role in the adhesion of non-polarised M0 macrophages onto both smooth and rough titanium surfaces. Moreover, the distinct temporal expression profile of αM integrin expression correlated broadly with the subsequent secretion profiles of inflammatory cytokines IL-1β and TNF-α in these cells. Specific integrin expression also played an important role in the adhesion of polarised inflammatory M1 macrophages onto surface-modified titanium surfaces. In these cells, early integrin αM and β1 in particular, along with integrin α2 was associated with modulation of the subsequent cytokine response in these cells. Furthermore, the surface induced differential expression of integrins on the different titanium surfaces showed the Fluoride-modified surface in general induced greater cytokine and integrin modulation than either the Machined or Blasted surfaces. While now biologically plausible, further studies to fully determine how integrin-derived modulation of the macrophage-associated inflammatory response following macrophage attachment as suggested by these studies, can enhance wound healing and osteogenesis are required. Despite this, ‘harnessing the immune response’ is clearly a potential goal of engineered titanium implant surfaces in order ‘to enhance osseointegration’.

Stability of implant-tissue interface is vital for implant integration with adjacent tissue and it influences directly short- and long-term surgical outcome. The literature provides comprehensive research on implants engaged in soft as well as in hard tissues. Various factors have been found to significantly influence the interface stability. This comprehensive work incorporated several research projects with main focus on implant-tissue interface stability, which was evaluated using biomechanical assessment tools such as pullout, four-point bending as well as reverse torque test. Implants were evaluated under ex vivo conditions, immediately after being placed within tissue as well as under in vivo conditions, 2- and 6-months following the implantation surgery in goat and equine animal model. Several viewpoints of implant - tissue interface have been addressed in the following studies. Stability of the interface has been found to be primarily influenced by amount of load experienced by the implants, stress applied to adjacent tissue as well as bioactive coatings. The following studies offer novel technologies which improved implant - tissue interface stability and significantly enhanced tissue regeneration. These technologies include novel design laryngeal clamps and novel technology polyurethane scaffolds coated with bioactive nanophase Hydroxyapatite. Projects included in this dissertation concern stresses applied by the suture to adjacent laryngeal cartilage or collateral ligaments, enthesis restoration using commercially available suture anchors, as well as stability of suture anchor and stainless-steel screw within bone tissue under different loading conditions. The final chapters concern integration of tissue scaffold coated bioactive agents.Research incorporated in this work found that increased tissue and implant contact area resulted in increased interface stability and better osseointegration. Further, osseointegration process varied between each screw used to fix dynamic compression plate to bone varied. Bioactive coatings such as nanophase Hydroxyapatite were found to significantly enhance osseointegration of bone scaffolds and resulted in significantly enhanced bone regeneration.

Regenerative therapies, or bone substitutes, for long bone fractures are on the verge of becoming standard practice. Development of a wide variety of synthetic materials has been undertaken in effort to improve healing of debilitating fractures. An ideal bone substitutes would mimic natural bone physiology. The form and function of long bones must first be understood in order to create the ideal regenerative material. From there, basic fracture healing provides key insights as to where and how fractures may advance to stages of non-healing. The ideal bone substitute would be both osteo-inductive and osteo-conductive. An effective material to promote bone healing in large defects has yet to be developed, and large animal preclinical models are lacking. Few large animal studies looking at bone regeneration exceed ninety days making long-term osseointegration of the bone substitute difficult. One of the most studied platforms for synthetic bone substitutes are nanohydroxyapatite and polyurethane composites due to their biocompatibility and bioresorbability. The studies detailed here focus on the biological assessment of a bone substitute that contains polyurethane, nanohydroxyapatite, and decellularized bone particles. A multitude of in vivo assessments were carried out to assess the impact of the bone substitute on a novel preclinical large animal model of long term bone healing. Baseline gait assessment characteristics were able to be determined for goat models relating to apparently healthy goats prior to the start of the bone healing model. Positive results were associated with long term integration of the bone substitute when the material was impregnated with the growth factor bone morphogenetic protein-2. The most catastrophic complication of any bone substitute used for long bone fractures, infection was encountered. Phenotypic and whole genomic characterization of the Staphylococcal associated infections, and subsequent osteomyelitis, were performed. It was recognized that there was an initial bone proliferation associated with Staphylococcus aureus associated osteomyelitis cases. The successful large animal preclinical model may provide an alternative to study bone substitutes. Conventional fixation methodologies may be removed, after sufficient healing time, to allow for further investigation into the integration and rehabilitation of the bone substitute with the native bone.

BACKGROUND: Implant therapy has been the gold standard in the past decade when it comes to replacing partially or complete edentulous oral cavities. Patients favor this line of treatment since it does mimic their natural teeth in esthetic, function and phonetics. Unfortunately, some initially integrated implants end up diagnosed with peri-implantitis, which threatens the longevity of those implants in their respective alveolar bone. Several methods have been discussed aiming to either salvage the diseased implant or prolong the life of it in patients’ oral cavities. In this protocol we studied the efficacy of one of the suggested protocols that has been used frequently in periodontal practices aiming to decontaminate the surface of previously diseased implants. MATERIALS AND METHODS: This study looked at the efficacy through two analyses, a descriptive and a quantitative. In the descriptive, several peri-implantitis diagnosed implants were collected and distributed over 4 groups: Test, Negative Control, Positive control and compared to pristine implants. Osteoprogenitor cells were prepared in-vitro and seeded over these implants after applying the protocol on Test group only. The quantitative analysis used the EDX analysis to study the percentages of Titanium and Oxygen on contaminated implants before and after applying the protocol on. Deposits removal was tested as well to ensure efficacy of decontamination protocol. RESULTS: Descriptive analysis showed that osteoprogenitor cells had higher attachment and proliferation on implants that followed the decontamination protocol vs. other groups. Quantitative analysis showed statistically significant higher titanium percentages after decontamination. Oxygen levels were higher as well but not statistically significant. Deposits were statistically significant in removal after decontamination protocol. CONCLUSION: Decontamination of previously diseased implants following the mentioned protocol has efficiently increased the chances of re-establishment of osseointegration in previously contaminated implants.

Despite advances in technology and surgical procedures, periprosthetic infection of orthopaedic implants remains a common and costly cause of implant failure and revision. Novel materials and coatings have been developed in an effort to improve implant resistance to the onset of infection, including those utilising the antimicrobial properties of silver ions. The aims of this study were to evaluate the osseointegration of a novel silver-doped hydroxyapatite coating and a second acid-etching process, applied to a titanium implant substrate implanted using a well-established ovine model. The subsequent results from the silver-doped coating could be compared to conventional hydroxyapatite coatings, to evaluate the effect of silver ions upon potential bone ongrowth. In this study, the silver HA coated implants demonstrated significantly higher shear stresses of mechanical pushout when compared to the acid etched implants after 4 weeks and 12 weeks in situ. Similarly, the bone-implant contact percentage was significantly higher for the silver HA coated implants indicating greater osseointegration at both timepoints. When compared to conventional HA coated implants in an identical ovine model, there was no significant difference observed in mechanical pushout or bone-implant contact of the silver HA coated implants. These results indicate that the novel silver-doped HA coating does not adversely affect the osseointegration of titanium implants. Therefore, this represents a viable solution for an antimicrobial implant coating which continues to facilitate effective osseointegration following implantation.

The goal of this study was to investigate initial bone response to the presence of the endoprosthesis and investigate the effects of low intensity vibration therapy on this bone response. We hypothesized that a press fit between the endoprosthesis and bone would provide stable fixation and that low intensity vibration would improve osseointegration without jeopardizing mechanical stability. To test this hypotheses a press fit, percutaneous endoprosthesis was successfully implemented in an ovine model to allow immediate weight bearing after the attachment of an exprosthesis. Animals were split into SHAM and LIV groups. The LIV group received vibration treatment by standing on a platform that produced 0.3 g of acceleration at a frequency of 45 Hz for 10 minutes a day, five days a week. The SHAM group stood on a platform that did not produce any vibrations. Nine animals completed a 12 week study with implants that were well fixed to the bone. Gait data were recorded before surgery (week 0) and at weeks 4, 8, and 12. Computed tomography (CT) scans were obtained at weeks 0 and 12 and dexamorphometry (DXA) was performed at weeks 4, 8 and 12. Fluorochrome labels were administered at the beginning and end of weeks 4 and 8. After animals were sacrificed, the implanted bone was sectioned for histomorphometry and mechanical push-out testing. Fluorescent microscopy was performed to image the fluorochrome labeling after which the slides were stained and imaged again. Mechanical push-out testing was performed to determine the bone-endoprosthesis interface strength. MicroCT scans were performed on the push-out sections before and after testing to evaluate three dimensional morphometry and measure the amount of osseointegration.The animals with osseointegrated endoprostheses at the end of the 12 week study displayed strong bone-endoprosthesis interface strength as determined by mechanical push-out testing. Animals with stable endoprosthesis fixation at 12 weeks did not display statistically significant lameness as determined by gait asymmetry. Asymmetrical gait was shown to be an indication of osseointegration failure. Finite element modeling predicted that bone-endoprosthesis press fits greater than 50 µm would cause bone damage at endoprosthesis implantation and the amount of damage would increase over the subsequent 24 hours after surgery. The finite element model predicted areas of damage seen in the animal model, giving the computation model and hoop strain based failure criterion experimental validity.LIV was shown to increase osseointegration between a press-fit, percutaneous, skeletally anchored endoprosthesis for prosthetic attachment by 18% by stimulating new bone growth in the intracortical region, and no adverse effects to this treatment were found. An interaction effect between the experimental group (SHAM or LIV) and the amount of osseointegration was found to influence the bone-implant interface mechanics. DXA data was normalized by the depth of the bone to obtain bone mineral apparent density (BMAD) data. BMAD in the AP view for the LIV group remained constant between weeks 0 and 8 and then increased between weeks 8 and 12. Newly formed bone tissue in the intracortical compartment was shown to have less mineralization than the surrounding cortical bone, indicating that the length of the study was insufficient to determine if LIV treatment will result in increased bone mineral density in the implanted bone. LIV was not found to increase bone remodeling rates at weeks 4 or 8. Due to the positive results found in this study and lack of adverse effects to LIV treatment, a longer study with more subjects should be performed to evaluate a new endoprosthesis design and to determine the long term effects of LIV on endoprosthesis stability and bone quality in the residual limb.

Titanium dental implants are currently one of the best treatment alternatives for replacing missing teeth. The direct bond that forms between the jawbone and titanium in a process defined as osseointegration has made titanium the material of choice for such devices. Whilst the underlying biological mechanisms responsible for the osseointegration process have not been fully elucidated, high demand by both patients and clinicians has driven the clinical application of titanium implants. Modification of the surface properties of titanium has been proven to be effective in promoting osseointegration, and thus understanding the fundamental cellular and molecular mechanisms responsible for the clinically observed outcomes as a result of titanium surface modification is the focus of considerable research. Previous research in this field has, as expected, focused on osteoblasts as they are the bone forming cells. This however, ignores the possible fundamental role of early mediators of the healing process, such as platelets and macrophages, which may interact with the implant surface well ahead of osteoblasts. As platelets and macrophages have well documented roles in modulating the function of other cells including osteoblasts, it is reasonable to postulate that the events that occur during the very early stages of the healing process may modulate repair in later stages, and ultimately influence the final osseointegration outcome.

The most important factor contributing to short-term and long-term success of cementless total joint arthroplasties is osseointegration. Osseointegration is the process by which a direct structural and functional connection between living bone and the surface of an implant is made. Surface contaminants may remain on orthopaedic implants after sterilization procedures and impair osseointegration. For example, specific lots of hip replacement Sulzer Inter-OPTM acetabular shells that were associated with impaired osseointegration and early failure rates were found to be contaminated with both bacterial debris and machine oil residues. However, few osseointegration studies have focused on surface contaminants and their effect on implant integration is unknown. Therefore, we developed a novel murine model that provides quantitative and reproducible measurements of osseointegration to study the effect of surface contaminants. We found that a rigorous cleaning procedure significantly enhances osseointegration compared to implants that were autoclaved. The most likely interpretation of these results is that surface contaminants on the autoclaved implants inhibit osseointegration. Using our murine model, we also found that Gram-negative bacterial derived LPS or machine oil residues on implants inhibit osseointegration. Ultrapure LPS adherent to titanium alloy discs had no detectable effect on early stages of MC3T3-E1 osteogenesis in vitro such as attachment, spreading or growth but inhibited later stages of osteogenic differentiation and mineralization. In contrast, machine oil on titanium alloy discs inhibited osteoblast attachment, spreading, growth, differentiation and mineralization in vitro. These results demonstrate that both adherent LPS and machine oil can significantly impair osseointegration through direct effects on osteoblasts; however, they do so by inhibiting distinct steps in the process. The presence of both LPS and machine oil could lead to synergistic impairment of osseointegration, which may account for the impaired osseointegration in the failed lots of Sulzer Inter-OPTM implants. These results highlight bacterial debris and machine oil as types of surface contaminants that can impair the osseointegration of orthopaedic implants. In addition, these results justify the need for the development of better detection and removal techniques for contaminants on orthopaedic implants.

Ever since Dr. Brånemark discovered that titanium was biocompatible with bone, extensive research has been done to improve the osseointegration of dental implants. As advances in medicine continue, the average life span of the population is ever increasing. Today, people of all ages are investing more money and time into dental treatment than ever before. Patients are becoming more educated on dental treatment options and expect the best treatment possible. As a result, the replacement of missing teeth with implants is becoming more and more commonplace in dental practices.The purpose of the present study is to evaluate bone-implant interaction of functionally graded, thin film hydroxyapatite (HA) coatings in an animal model. The rationale for the graded coatings is such that they elicit different biological responses from different layers within the thin film (less than two microns). As such in this study, the graded coatings consist of an initial layering of crystalline HA coatings followed by the layering of an amorphous coating on the crystalline HA surface. Controls for this study are (a) plasma-sprayed HA, (b) amorphous HA, (c) crystalline HA, and (d) non-coated Titanium (Ti). The long-term goal is to improve the bone-implant interface leading to improved design and construction of implants and improved long term success. With this development it is expected that dental implants will be restored following shorter post-operative healing periods and patients will more quickly regain masticatory function resulting in the overall improvement of physical and mental health.This study focused on mechanical and histological analysis of HA-coated and non-coated Titanium implants placed into the left femur of 40 male Sprague-Dawley rats. The rat was selected for this experiment because the specimen is affordable and historically proven to be a very good model for initial evaluation of the bone healing response to metal and metal coated implants. In vivo experimentation is preferred for these studies because bone wound healing is a complex process requiring the interaction of many cell types and factors. These conditions can not presently be predictably duplicated with in vitro experimentation.In this study, two implants were placed into the left femur of each rat. At two time intervals (three weeks and nine weeks after implantation) 20 rats were euthanized and the femur containing the implants excised. A total of 40 implants were yielded at each time point. Since there were five groups (titanium, plasma sprayed HA, amorphous HA, crystalline HA and graded HA), there were eight implants per group per time point (40/5 = 8). Of these eight implants per time point per group, six were used for mechanical testing and two for histological evaluation. To evaluate the interfacial strength of the implants at the bone-implant interface, push-out test were performed. The evaluation of the bone response to implant was done using histological analysis.At three weeks following implantation, there was no significant difference in the interfacial strength between the different implants. The interfacial strength had increased in all groups by nine weeks. The plasma sprayed HA implants had the greater interfacial strength followed by the graded HA, crystalline HA, amorphous HA and titanium implants. At three weeks post implantation, the presence of connective tissue at the tissue-implant interface was noted for all implant groups tested. By nine weeks post implantation, all HA coated implants exhibited more bone formation at the bone-implant interface when compared to the non-coated Ti implants. The greatest response was seen in the plasma sprayed implants. A similar response was seen between the graded and crystalline as well as the amorphous and pure titanium. This study suggested that graded HA is a viable option for implant coatings.

Bone is a connective tissue with nanosized particles of carbonated apatitic calcium phosphate dispersed in a hydrated collagen matrix. With the ageing of the baby boomer population, an increasing number of people sustain bone fractures and defects. Hence, efforts are underway to develop materials to hasten the healing and repairing of such defects. These materials are termed as artificial bone substitutes. This study represents innovative techniques for development of bone implant materials and improving the existing substitute materials. Emphasis was on three different kinds of materials: Metals (titanium and alloys), Ceramics (calcium phosphates), and Polymers (collagen). The bioactivity of titanium and alloys, resorptivity of calcium phosphates and biocompatibility of collagen were the major issues with these materials. These issues are appropriately addressed in this dissertation. For titanium and alloys, biomimetic coating methodology was developed for uniformly and evenly coating 3-D titanium structures. Cracks were observed in these coatings and a protocol was developed to form crack-free biomimetic coatings. In calcium phosphates, increasing the resorption rate of HA (hydroxyapatite) and decreasing the resorption rate of beta-TCP (beta-tricalcium phosphate) were studied. HA-based ceramics were synthesized with Na+ and CO32- ions dopings, and development of biphasic mixtures of HA-TCP and HA-Rhenanite was performed. Similarly, beta-TCP ceramics were synthesized with Zn2+ ion doping and development of TCP-HA biphasic mixtures was performed. In case of collagen, a biomimetic coating process was developed that decreased the time to coat the collagen substrates and also increased biocompatibility, as determined by the response of mouse osteoblasts.

Purpose: The objective of this study was to evaluate the osseointegration between plasma sprayed (PS) hydroxyapatite (HA) coated implants and electrophoresis deposited (EPD) nano hydroxyapatite coated implants in an animal model. The quantity of osseointegration was inferred by interfacial strength and percent tissue-contact length.Materials and Methods: Thirty-six cylindrical implants (18 PS and 18 EPD) were placed into dog mandibles. Nine implants from each group were evaluated with pull out testing and histological studies at 12 weeks after implantation. The remaining implants were occlusally loaded for 9 months and then evaluated by pull out testing and histological studies.Results: Twelve weeks after implantation, no statistical difference in pull out strength was observed between PS HA (70.43 ± 16.65 lbf) and EPD nano HA (86.35 ± 28.07 lbf) coated implants . After loading for 9 months, it was observed that the interfacial strength of implants coated by EPD nano HA (199.9 ± 35.1 lbf) was statistically higher (P

Bone tissue engineering represents a strategy for the repair or regeneration of damaged bone in the body. The science underlying this clinical therapy bridges the traditional fields of cell biology, materials science and mechanical engineering with the aim to identify how cells behave on physiologically relevant materials with natural mechanical stimuli. The objectives of this research were to develop and characterize calcium phosphate ceramic scaffolds matched to the local architecture of natural trabecular bone and to apply tissue engineering strategies for the study of cell behavior in both in vitro and in vivo models.The specific role of environment on cell stress pathways was evaluated on three dimensional (3-D) calcium phosphate scaffolds resembling vertebral trabecular bone. A scaffold foam dipping technique was employed in the fabrication of fully sintered hydroxyapatite and tricalcium phosphate scaffolds. Study of the early cell behavior on two dimension (2-D) controls and scaffolds was performed using human embryonic palatal mesenchyme cells (HEPM), an osteoblast precursor cell line. Cell stress signaling was identified in response to the 3-D architecture using; members of the mitogen activated protein kinase cascade, cell survival signals and adhesion dependant proteins. The application of low intensity pulsed ultrasound (LIPUS) or fluid perfusion further stimulated cell-scaffold hybrids for short and long term in vitro study. Additionally, an animal model was characterized using the scaffolds for the repair of a segmental defect in the canine mandible.Study of the cell stress signaling mechanisms identified high activation of stress pathways on 3-D materials compared to controls with a corresponding increase in anti- apoptosis signaling. Similar trends were found with LIPUS stimulation demonstrating that changes in adhesion proteins during attachment may account for the alteration in stress pathways activated by bone precursors. The absence of cell death and the activation of an anti-apoptosis signal suggest that cells are able to manage these stress levels which may be required for proper function. Supporting this theory, long term in vitro perfusion studies demonstrated that the process of cell transition into a mature bone phenotype was improved with the fluid shear forces of perfusion. Finally, the scaffolds were applied for repair of a segmental defect in the canine mandible and demonstrated extensive bone in-growth and partially-organized, lamellar collagen fiber assembly characteristic of organized bone. The open architecture of the scaffold design also allowed for substantial blood vessel infiltration.This research demonstrated the importance of architecture on bone cell response for in vitro cell study and for clinical application. The scaffold design provides a bridge between laboratory based signaling mechanisms and the development of clinical therapies in regenerative orthopedics.

The clinical success of dental implants is determined mostly by surface properties of implants and interactions with surrounding tissues. Furthermore, clinical success of dental implants is directed by implant surfaces and bone cell responses that promote osseointegration and long-term stability. Hydroxyapatite (HA)-coated Titanium (Ti) implants have been widely used due to its effect on bone response. However, microbial infection is common. One material known to have anti-microbial properties is silver (Ag). However, the effects Ag has on osteoblastic activity as well as decrease bacterial load has yet to be determined. In this study, the effect of Ag-doped HA coatings on initial bacterial adhesion and osteoblast cell proliferation, differentiation, and mineralization was investigated. Using a sol-gel process, HA coatings doped with 2 wt % AgNO3 were prepared. Coated surfaces were characterized using Xray diffraction (XRD). The osteoblast cell attachment, differentiation which is measured by alkaline phosphatase activity, and mineralization which is measured by osteocalcin activity were evaluated using human embryonic palatal mesenchyme cells (HEPM), an osteoblast precursor cell line. Initial bacteria adhesion was evaluated using an RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus. A significant difference in osteoblastic cell attachment over time was observed with 2% Ag-doped HA having the least cell attachment. The use of HEPM cells indicated no significant difference in alkaline phosphatase specific activity or osteocalcin activity among the 2% Ag-doped HA, HA, and Ti surfaces. Furthermore, the in vitro bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on Ag-doped HA surfaces when compared to HA and Ti surfaces. Overall, it was concluded that 2% Ag-doped HA surfaces have similar osteoconductive activity when compared to HA and Ti surfaces. It was also concluded that the doping of HA with Ag minimized the adhesion of bacteria on its surface. Further studies on Ag-doped HA surfaces should involve long and short-term animal studies to evaluate its biocompatibility and ability to induce bone formation for implant stability, as well as bacterial adhesion properties. Furthermore, a decreased concentration of Ag may aid in an increasing osteogenic activity as well as have an antimicrobial effect.