Improving in vivo models of fracture fixation associated osteomyelitis

This project’s aim was to create new experimental models in small animals for the investigation of infections related to bone fracture fixation implants. Animal models are essential in orthopaedic trauma research and this study evaluated new implants and surgical techniques designed to improve standardisation in these experiments, and ultimately to minimise the number of animals needed in future work. This study developed and assessed procedures using plates and inter-locked nails to stabilise fractures in rabbit thigh bones. Fracture healing was examined with mechanical testing and histology. The results of this work contribute to improvements in future small animal infection experiments.

Mimicking breast cancer-induced bone metastasis in vivo: Current transplantation models and advanced humanized strategies

Bone metastasis is a complication that occurs in 80 % of women with advanced breast cancer. Despite the prevalence of bone metastatic disease, the avenues for its clinical management are still restricted to palliative treatment options. In fact, the underlying mechanisms of breast cancer osteotropism have not yet been fully elucidated due to a lack of suitable in vivo models that are able to recapitulate the human disease. In this work, we review the current transplantation-based models to investigate breast cancer-induced bone metastasis and delineate the strengths and limitations of the use of different grafting techniques, tissue sources, and hosts. We further show that humanized xenograft models incorporating human cells or tissue grafts at the primary tumor site or the metastatic site mimic more closely the human disease. Tissue-engineered constructs are emerging as a reproducible alternative to recapitulate functional humanized tissues in these murine models. The development of advanced humanized animal models may provide better platforms to investigate the mutual interactions between human cancer cells and their microenvironment and ultimately improve the translation of preclinical drug trials to the clinic.

Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Breast cancer in its advanced stage has a high predilection to the skeleton. Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities. This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet. This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue. Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis. Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour. Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions. Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind. Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone. Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour. Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix. The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold. Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP scaffold, fabricated using a fused deposition modelling technique. After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique was chosen using the same polymer. Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice. Ectopic bone formation with sufficient vascularisation could be observed. After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed. Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment.

Tissue engineered humanized bone supports human hematopoiesis in vivo

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.

In vivo performance of melimine as an antimicrobial coating for contact lenses in models of CLARE and CLPU

Purpose: One strategy to minimize bacteria-associated adverse responses such as microbial keratitis, contact lens–induced acute red eye (CLARE), and contact lens induced peripheral ulcers (CLPUs) that occur with contact lens wear is the development of an antimicrobial or antiadhesive contact lens. Cationic peptides represent a novel approach for the development of antimicrobial lenses.---------- Methods: A novel cationic peptide, melimine, was covalently incorporated into silicone hydrogel lenses. Confirmation tests to determine the presence of peptide and anti-microbial activity were performed. Cationic lenses were then tested for their ability to prevent CLPU in the Staphylococcus aureus rabbit model and CLARE in the Pseudomonas aeruginosa guinea pig model. ---------- Results: In the rabbit model of CLPU, melimine-coated lenses resulted in significant reductions in ocular symptom scores and in the extent of corneal infiltration (P < 0.05). Evaluation of the performance of melimine lenses in the CLARE model showed significant improvement in all ocular response parameters measured, including the percentage of eyes with corneal infiltrates, compared with those observed in the eyes fitted with the control lens (P ≤ 0.05). ---------- Conclusions: Cationic coating of contact lenses with the peptide melimine may represent a novel method of prevention of bacterial growth on contact lenses and consequently result in reduction of the incidence and severity of adverse responses due to Gram-positive and -negative bacteria during lens wear.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated.

In vivo evaluation of an ultra-thin polycaprolactone film as a wound dressing

The use of ultra-thin films as dressings for cutaneous wounds could prove advantageous in terms of better conformity to wound topography and improved vapour transmission. For this purpose, ultra-thin poly(epsilon-caprolactone) (PCL) films of 5-15 microm thickness were fabricated via a biaxial stretching technique. To evaluate their in vivo biocompatibility and feasibility as an external wound dressing, PCL films were applied over full and partial-thickness wounds in rat and pig models. Different groups of PCL films were used: untreated, NaOH-treated, untreated with fibrin, NaOH-treated with perforations, and NaOH-treated with fibrin and S-nitrosoglutathione. Wounds with no external dressings were used as controls. Wound contraction rate, histology and biomechanical analyses were carried out. Wounds re-epithelialized completely at a comparable rate. Formation of a neo-dermal layer and re-epithelialization were observed in all the wounds. A lower level of fibrosis was observed when PCL films were used, compared to the control wounds. Ultimate tensile strength of the regenerated tissue in rats reached 50-60% of that in native rat skin. Results indicated that biaxially-stretched PCL films did not induce inflammatory reactions when used in vivo as a wound dressing and supported the normal wound healing process in full and partial-thickness wounds.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

The active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.

Measuring mechanical properties of tendon in vivo

Introduction: Understanding the mechanical properties of tendon is an important step to guiding the process of improving athletic performance, predicting injury and treating tendinopathies. The speed of sound in a medium is governed by the bulk modulus and density for fluids and isotropic materials. However, for tendon,which is a structural composite of fluid and collagen, there is some anisotropy requiring an adjustment for Poisson’s ratio. In this paper, these relationships are explored and modelled using data collected, in vivo, on human Achilles tendon. Estimates for elastic modulus and hysteresis based on speed of sound data are then compared against published values from in vitro mechanical tests. Methods: Measurements using clinical ultrasound imaging, inverse dynamics and acoustic transmission techniques were used to determine dimensions, loading conditions and longitudinal speed of sound for the Achilles tendon during a series of isometric plantar flexion exercises against body weight. Upper and lower bounds for speed of sound versus tensile stress in the tendon were then modelled and estimates derived for elastic modulus and hysteresis. Results: Axial speed of sound varied between 1850 to 2090 m.s−1 with a non-linear, asymptotic dependency on the level of tensile stress in the tendon 5–35 MPa. Estimates derived for the elastic modulus ranged between 1–2 GPa. Hysteresis derived from models of the stress-strain relationship, ranged from 3–11%. These values agree closely with those previously reported from direct measurements obtained via in vitro mechanical tensile tests on major weight bearing tendons. Discussion: There is sufficiently good agreement between these indirect (speed of sound derived) and direct (mechanical tensile test derived) measures of tendon mechanical properties to validate the use of this non-invasive acoustic transmission technique. This non-invasive method is suitable for monitoring changes in tendon properties as predictors of athletic performance, injury or therapeutic progression.

Myogel, a novel, basement membrane-rich, extracellular matrix derived from skeletal muscle, is highly adipogenic in vivo and in vitro

Background/Aims Biological and synthetic scaffolds play important roles in tissue engineering and are being developed towards human clinical applications. Based on previous work from our laboratory, we propose that extracellular matrices from skeletal muscle could be developed for adipose tissue engineering. Methods Extracellular matrices (Myogels) extracted from skeletal muscle of various species were assessed using biochemical assays including ELISA and Western blotting. Biofunctionality was assessed using an in vitro differentiation assay and a tissue engineering construct model in the rat. Results Myogels were successfully extracted from mice, rats, pigs and humans. Myogels contained significant levels of laminin α4- and α2-subunits and collagen I compared to Matrigel™, which contains laminin 1 (α1β1γ1) and collagen IV. Levels of growth factors such as fibroblast growth factor 2 were significantly higher than Matrigel, vascular endothelial growth factor-A levels were significantly lower and all other growth factors were comparable. Myogels reproducibly stimulated adipogenic differentiation of preadipocytes in vitro and the growth of adipose tissue in the rat. Conclusions We found Myogel induces adipocyte differentiation in vitroand shows strong adipogenic potential in vivo, inducing the growth of well-vascularised adipose tissue. Myogel offers an alternative for current support scaffolds in adipose tissue engineering, allowing the scaling up of animal models towards clinical adipose tissue engineering applications.

A humanized tissue-engineered in vivo model to dissect interactions between human prostate cancer cells and human bone

Currently used xenograft models for prostate cancer bone metastasis lack the adequate tissue composition necessary to study the interactions between human prostate cancer cells and the human bone microenvironment. We introduce a tissue engineering approach to explore the interactions between human tumor cells and a humanized bone microenvironment. Scaffolds, seeded with human primary osteoblasts in conjunction with BMP7, were implanted into immunodeficient mice to form humanized tissue engineered bone constructs (hTEBCs) which consequently resulted in the generation of highly vascularized and viable humanized bone. At 12 weeks, PC3 and LNCaP cells were injected into the hTEBCs. Seven weeks later the mice were euthanized. Micro-CT, histology, TRAP, PTHrP and osteocalcin staining results reflected the different characteristics of the two cell lines regarding their phenotypic growth pattern within bone. Microvessel density, as assessed by vWF staining, showed that tumor vessel density was significantly higher in LNCaP injected hTEBC implants than in those injected with PC3 cells (p\0.001). Interestingly, PC3 cells showed morphological features of epithelial and mesenchymal phenotypes suggesting a cellular plasticity within this microenvironment. Taken together, a highly reproducible humanized model was established which is successful in generating LNCaP and PC3 tumors within a complex humanized bone microenvironment. This model simulates the conditions seen clinically more closely than any other model described in the literature to date and hence represents a powerful experimental platform that can be used in future work to investigate specific biological questions relevant to bone metastasis.

Study of reproducibility of human arterial plaque reconstruction and its effects on stress analysis based on multispectral in vivo magnetic resonance imaging

Purpose: To quantify the uncertainties of carotid plaque morphology reconstruction based on patient-specific multispectral in vivo magnetic resonance imaging (MRI) and their impacts on the plaque stress analysis. Materials and Methods: In this study, three independent investigators were invited to reconstruct the carotid bifurcation with plaque based on MR images from two subjects to study the geometry reconstruction reproducibility. Finite element stress analyses were performed on the carotid bifurcations, as well as the models with artificially modified plaque geometries to mimic the image segmentation uncertainties, to study the impacts of the uncertainties to the stress prediction. Results: Plaque reconstruction reproducibility was generally high in the study. The uncertainties among interobservers are around one or the subpixel level. It also shows that the predicted stress is relatively less sensitive to the arterial wall segmentation uncertainties, and more affected by the accuracy of lipid region definition. For a model with lipid core region artificially increased by adding one pixel on the lipid region boundary, it will significantly increase the maximum Von Mises Stress in fibrous cap (>100%) compared with the baseline model for all subjects. Conclusion: The current in vivo MRI in the carotid plaque could provide useful and reliable information for plaque morphology. The accuracy of stress analysis based on plaque geometry is subject to MRI quality. The improved resolution/quality in plaque imaging with newly developed MRI protocols would generate more realistic stress predictions.