Rizhi Wang

Osteoporosis is a metabolic bone disease that is characterized by low bone mass and a high risk of fracture. It is caused by the increased activity of osteoclasts and the reduced activity of osteoblasts. Therapeutic approaches for osteoporosis include antiresorptive and anabolic drugs. The advanced-stage cancer induced-osteolytic lesions are also caused by abnormally high activity of osteoclasts. Since osteoclastic activity plays an important role in these areas, it has been the focus of many in vitro studies. Currently used assays for assessing osteoclastic activities include bone/dentin slices and synthetic calcium phosphate coatings. However, these assays have various limitations, from poor mechanical adhesion to low feasibility of drug delivery. The purpose of this dissertation was to develop a reliable calcium phosphate nanostructure for the in vitro study of osteoclastic resorption.In the first study, an ammonia-induced mineralization method (AiM) was developed to create a robust nano-structured calcium phosphate coating (CaP) with mineral pins on the track-etched porous membrane (AiM-CaP insert). The mineral pins enhance coating integration and provide diffusion channels. The uniform coating could be used for the test of osteoclastic resorption. The second study demonstrated that the AiM-CaP insert was a stable in vitro platform for evaluating osteoclastic resorption. In the third study, the feasibility of drug delivery was demonstrated with alendronate. Alendronate was delivered either directly onto the AiM-CaP inserts or diffused through the lower chamber of the cell culture system to inhibit osteoclastic resorption. Quantitative results also provided direct evidence about the influence of alendronate on osteoclasts’ actin rings. This study proved that AiM-CaP insert could be used for testing anti-osteoporosis drugs.The fourth study demonstrated that the prostate cancer cell-conditioned medium enhanced osteoclastic resorption. The results suggested that AiM-CaP inserts have a high potential for investigating cell interactions of osteoblasts/osteoclasts lineages with other bone homing cancer cells.In summary, the AiM-CaP coating reported in this thesis is a unique and reliable platform to study osteoclasts. This platform also provides wide opportunities for coating modification, material-bone cell interactions, new anti-osteoporosis drug tests, and bone metastasis studies in the future.

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Over one million hip replacements are performed worldwide to restore hip mobility every year. CoCrMo alloys are widely used in hip implants because of their excellent corrosion resistance and mechanical properties. However, some complications related to corrosion of CoCrMo alloys emerged, leading to unexpected clinical failures such as fracture and adverse local tissue reactions.The purpose of this thesis is thus to investigate the in vivo corrosion mechanisms of retrieved CoCrMo alloys used in total hip implants from two specific clinical failures- mechanical fracture and adverse local tissue reactions.In the mechanical failure study, systematic analyses on the clinically failed CoCrMo based implants revealed a multi-step fracture process. Multiple micro cracks were developed under the combined action of pitting corrosion and dynamic tensile stress on the lateral side of the CoCrMo connection taper, leading to the final catastrophic failure. Such a crack initiation process has not been previously reported on retrieved CoCrMo components. Our findings provide valuable information on the clinical performance of such implants, as well as the material selection and structural designs for future modular stems.In the biological failure study, increasing cases of adverse tissue reactions related to CoCrMo metal release have been observed in hip implants of metal-on-polyethylene articulation (MoP), the most commonly used hip system in recent years. Comprehensive studies were conducted on the origins of CoCrMo metal release in MoP hip system– fretting corrosion at head-neck junction and tribocorrosion at articulating surface. In fretting corrosion, two types of corrosion particles were observed at head-neck junction. Further laboratory tests revealed that the formation of these two types of corrosion particulates was associated with different species,especially the phosphate in electrolytes. This study indicated different electrochemical environments between the inside and opening site of head-neck junction during in vivo fretting corrosion. Meanwhile, retrieval studies revealed evidence of tribocorrosion associated CoCrMo metal release in patients with MoP hip implants, which was rarely reported to our knowledge. It raised the awareness about the risk of tribocorrosion on metal release and the consequent adverse tissue reactions in patients with a MoP hip system.

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About two million people receive hip implants annually, to relieve the pain generated by osteoarthritis. Hip implants are composed of a titanium femoral stem and a Co-Cr-Mo head that articulates with a titanium-alloy acetabular cup, covered by a polyethylene liner (MoP). Other designs directly use a metal (Co-Cr-Mo) acetabular articulating surface (MoM). Concerns have arisen due to the elevated numbers of adverse local tissue reactions (ALTRs) to hip implants that generate pain and soft-tissue destruction. Due to the high rates of ALTRs in MoM implants, the corrosion products or wear particles from the metal surfaces are thought to trigger the immunological reactions. This thesis aimed to understand the mechanisms of ALTRs development through three studies: 1) histological analyses and comparison of ALTRs in MoM and MoP implants. 2) analysis of corrosion products in synovial fluid and tissues. 3) mechanistic study based on gene expression analysis of ALTRs, and cell culture of primary synovial fibroblasts that were exposed to Cr and Co.
 The histological description of ALTRs showed structural similarities between MoM and MoP, with common elements such as tissue necrosis and the presence of perivascular lymphocyte aggregates. Significantly higher levels of metal ions were found in the synovial fluid of ALTR patients, but no differences in the particles present in tissues were found in ALTRs compared to non-ALTRs. The gene expression analysis of lymphocytes aggregates found an identical and non-specific Th1/Th2 reaction in ALTRs in both MoM and MoP, with no evidence of Th17 reaction. Finally, primary synovial fibroblasts responded to concentrations of metal ions observed in the synovial fluid of patients, by releasing pro-inflammatory cytokines. After 24 hours of exposure the secreted cytokines were demonstrated to be chemotactic for human monocytes which is a key processes during inflammation.These results support the hypothesis that metal ions from the hip implants trigger cytokine secretion by synovial fibroblasts, which initiate the immune reaction to hip replacements, providing evidence to reject the hypothesis of hypersensitivity as an etiologic factor of ALTRs.

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Hip fracture has serious repercussions at both the societal and personal levels. For better fracture prevention, it is essential to understand the material changes of femoral cortical bone that contribute to hip fragility, and the deformation and fracture process during hip fractures. Therefore, the aim of this dissertation was to study the mechanisms of hip fracture from both structural and mechanical perspectives. Using quantitative backscattered electron (qBSE) imaging and polarized Raman microspectroscopy, periosteal hypermineralization in aged human proximal femur was found with significantly higher mineral content/mineral-to-matrix ratio than lamellar bone. Accompanying the increased mineralization was the “brittle” cracking behavior upon microindentation in the hypermineralized tissue. Small- and wide-angle X-ray scattering (SAXS/WAXS) measurement showed substantially thinner, shorter and more irregularly distributed mineral platelets in the hypermineralized region, indicating the material changes at the ultrastructural level. Combined second harmonic generation (SHG) and two photon excitation fluorescence (TPEF) techniques were used to study shear microcracking and its association with the organization of collagen fibrils in the femoral cortical bone. Unique arc-shaped shear microcracks, differing from either tensile or compressive microcracks, were identified at the peripheral zone of the osteons. These microcracks were further located within the “bright” lamellae where collagen fibrils are primarily oriented at the circumferential direction to the osteons’ long axes. Microcracking analysis on clinically retrieved femoral neck components identified shear, compressive and tensile microcracks associated with major fractures. The results pointed to the central role of the superior cortex in resisting a hip fracture, whereby higher density of microcracks and buckling failure were found in the superior cortical bone. BSE imaging at the fracture sites found the direct involvement of hypermineralization, which lacked crack deviation and had fewer microcracks than the tough lamellar bone. This dissertation answered fundamental questions regarding the role of femoral cortical bone in clinical hip fractures, and elucidated the underlying failure mechanisms due to microstructural changes and the complex stress states under external loading. The findings thus provided new insights into better identifying at-risk population of hip fracture.

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Titanium (Ti) is a key biomedical material extensively used in orthopaedic implants. Prevention of implant-associated infections has been one of the main challenges in orthopaedic surgery. This challenge is further complicated by the concern over the development of antibiotic resistance as a result of using traditional antibiotics for infection prophylaxis. One of the promising alternatives is the family of antimicrobial peptides (AMPs). The present dissertation develops progressive approaches that enable the loading and local delivery of a unique group of cationic antimicrobial peptides through titanium implant surfaces. In the first technique, a thin layer of micro-porous calcium phosphate (CaP) coating was processed by electrolytic deposition onto the surface of titanium as the drug carrier. The AMP-loaded CaP coating was not cytotoxic for MG-63 osteoblast-like cells, and the implants showed high antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria with 10⁶-fold reductions of both bacterial strains within 30 min and ∼92% and ∼77% inhibition of luminescence at 4 h and 24 h, respectively. Second study investigated the in vitro AMP release, antimicrobial performance, and cytotoxicity of a modified Tet213 (HHC36), as well as the in vivo bone growth of AMP loaded into calcium phosphate coated Ti implants in a rabbit model. Burst release during the first few hours followed by a slow and steady release for 7 days was observed. In vivo bone growth study showed that loading of AMP did not impair bone growth onto the implants. In the last study multilayer thin films of titania nanotubes (NT) and CaP coatings were formulated with AMP and were topped with a thin phospholipid film similar to cell membrane. The films were shown to be non-cytotoxic, hydrophilic, with the potential of tuning loading and release kinetics of AMP. The best model describing the AMP release was first-order model.The first two approaches demonstrated a promising method for an early stage peri-implant infection treatment. The last study proposed a technique to improve the kinetics of AMP release and total loaded AMP quantity, and to increase the Ti interfacial strength while maintain the osteconductivity by applying CaP coating.

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Human bone is a complex biological material with up to seven levels of hierarchical structure. Due to this complexity, it is still not fully understood how the various structures contribute to the macroscopic mechanical response. Such understanding is important to assess the mechanical contributions of the bone material to whole bone fractures. It is well known that microcracking is associated with bone’s inelastic deformation and contributes to its resistance to fracture. Multiple microcracks suggest control over their development. Yet, the structure – microcracking interactions in cortical bone, particularly at the lamellar and Haversian systems levels, are still unclear. Following a qualitative, structure – mechanical function relations approach, the present dissertation provides further insight into how bone resists fracture by distributed microcracking. This was achieved through a detailed study of bone’s deformation and fracture processes using mechanical testing on human cadaver bones and a combination of microscopy techniques, including laser scanning confocal microscopy, to characterize the structure – microcracking relations. Particular interest was given to compression and bending, two loading modes involved in falls resulting in hip fractures. Haversian bone derived part of its fracture resistance through microcracking largely controlled by the concentric lamellae and underlying fibrillar organisation surrounding each Haversian canal. Multiple microcracks developed stably within the osteonal wall due to different fibrillar orientation in each lamella. Such process happened to most osteons resulting in well-distributed damage, hence providing inelastic deformation to the tissue. Haversian bone’s resistance to fracture would thus depend on its intact lamellar structure. Changes in number and organisation of the lamellae would likely alter bone’s ability to control microcracks and may lead to bone fragility. Based on a tibia study, long bones’ fracture resistance in bending was found to be linked to Haversian bone’s behavior. As a result of post-yield strain redistribution associated with tensile and compressive microcracking, bone’s compressive behavior was also found to play an important role in the bending response. Directly applying fundamental research to the clinical field, a preliminary analysis of the superior cortex of fractured femoral necks retrieved from patients revealed compressive microcracking. Such evidence emphasizes the importance of bone’s hierarchical structure in hip fracture.

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