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.

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. © 2012 Sieh et al.

β-glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice

Objective The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17–dependent inflammatory arthritis developed after dectin 1–mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. Methods SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti–IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. Results After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell– and IL-23–dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. Conclusion Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.

The effects of dietary fish oil on inflammation, fibrosis and oxidative stress associated with obstructive renal injury in rats.

Scope: We examined whether dietary supplementation with fish oil modulates inflammation, fibrosis and oxidative stress following obstructive renal injury. Methods and results: Three groups of Sprague-Dawley rats (n = 16 per group) were fed for 4 wk on normal rat chow (oleic acid), chow containing fish oil (33 g eicosapentaenoic acid and 26 g docosahexaenoic acid per kg diet), or chow containing safflower oil (60 g linoleic acid per kg diet). All diets contained 7% fat. After 4 wk, the rats were further subdivided into four smaller groups (n = 4 per group). Unilateral ureteral obstruction was induced in three groups (for 4, 7 and 14 days). The fourth group for each diet did not undergo surgery, and was sacrificed as controls at 14 days. When rats were sacrificed, plasma and portions of the kidneys were removed and frozen; other portions of kidney tissue were fixed and prepared for histology. Compared with normal chow and safflower oil, fish oil attenuated collagen deposition, macrophage infiltration, TGF-beta expression, apoptosis, and tissue levels of arachidonic acid, MIP-1 alpha, IL-1 beta, MCP-1 and leukotriene B(4). Compared with normal chow, fish oil increased the expression of HO-1 protein in kidney tissue. Conclusions: Fish oil intake reduced inflammation, fibrosis and oxidative stress following obstructive renal injury.

Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles

Articular cartilage defects are common after joint injuries. When left untreated, the biomechanical protective function of cartilage is gradually lost, making the joint more susceptible to further damage, causing progressive loss of joint function and eventually osteoarthritis (OA). In the process of translating promising tissue-engineering cartilage repair approaches from bench to bedside, pre-clinical animal models including mice, rabbits, goats, and horses, are widely used. The equine species is becoming an increasingly popular model for the in vivo evaluation of regenerative orthopaedic approaches. As there is also an increasing body of evidence suggesting that successful lasting tissue reconstruction requires an implant that mimics natural tissue organization, it is imperative that depth-dependent characteristics of equine osteochondral tissue are known, to assess to what extent they resemble those in humans. Therefore, osteochondral cores (4-8 mm) were obtained from the medial and lateral femoral condyles of equine and human donors. Cores were processed for histology and for biochemical quantification of DNA, glycosaminoglycan (GAG) and collagen content. Equine and human osteochondral tissues possess similar geometrical (thickness) and organizational (GAG, collagen and DNA distribution with depth) features. These comparable trends further underscore the validity of the equine model for the evaluation of regenerative approaches for articular cartilage.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3alpha mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Feasibility of agent-based modelling of articular cartilage including a conceptual representation of its structure

Articular cartilage is a complex structure with an architecture in which fluid-swollen proteoglycans constrained within a 3D network of collagen fibrils. Because of the complexity of the cartilage structure, the relationship between its mechanical behaviours at the macroscale level and its components at the micro-scale level are not completely understood. The research objective in this thesis is to create a new model of articular cartilage that can be used to simulate and obtain insight into the micro-macro-interaction and mechanisms underlying its mechanical responses during physiological function. The new model of articular cartilage has two characteristics, namely: i) not use fibre-reinforced composite material idealization ii) Provide a framework for that it does probing the micro mechanism of the fluid-solid interaction underlying the deformation of articular cartilage using simple rules of repartition instead of constitutive / physical laws and intuitive curve-fitting. Even though there are various microstructural and mechanical behaviours that can be studied, the scope of this thesis is limited to osmotic pressure formation and distribution and their influence on cartilage fluid diffusion and percolation, which in turn governs the deformation of the compression-loaded tissue. The study can be divided into two stages. In the first stage, the distributions and concentrations of proteoglycans, collagen and water were investigated using histological protocols. Based on this, the structure of cartilage was conceptualised as microscopic osmotic units that consist of these constituents that were distributed according to histological results. These units were repeated three-dimensionally to form the structural model of articular cartilage. In the second stage, cellular automata were incorporated into the resulting matrix (lattice) to simulate the osmotic pressure of the fluid and the movement of water within and out of the matrix; following the osmotic pressure gradient in accordance with the chosen rule of repartition of the pressure. The outcome of this study is the new model of articular cartilage that can be used to simulate and study the micromechanical behaviours of cartilage under different conditions of health and loading. These behaviours are illuminated at the microscale level using the socalled neighbourhood rules developed in the thesis in accordance with the typical requirements of cellular automata modelling. Using these rules and relevant Boundary Conditions to simulate pressure distribution and related fluid motion produced significant results that provided the following insight into the relationships between osmotic pressure gradient and associated fluid micromovement, and the deformation of the matrix. For example, it could be concluded that: 1. It is possible to model articular cartilage with the agent-based model of cellular automata and the Margolus neighbourhood rule. 2. The concept of 3D inter connected osmotic units is a viable structural model for the extracellular matrix of articular cartilage. 3. Different rules of osmotic pressure advection lead to different patterns of deformation in the cartilage matrix, enabling an insight into how this micromechanism influences macromechanical deformation. 4. When features such as transition coefficient were changed, permeability (representing change) is altered due to the change in concentrations of collagen, proteoglycans (i.e. degenerative conditions), the deformation process is impacted. 5. The boundary conditions also influence the relationship between osmotic pressure gradient and fluid movement at the micro-scale level. The outcomes are important to cartilage research since we can use these to study the microscale damage in the cartilage matrix. From this, we are able to monitor related diseases and their progression leading to potential insight into drug-cartilage interaction for treatment. This innovative model is an incremental progress on attempts at creating further computational modelling approaches to cartilage research and other fluid-saturated tissues and material systems.

Decoration of titania nanofibres with anatase nanoparticles as efficient photocatalysts for decomposing pesticides and phenols

Using a series of partial phase transitions, an effective photocatalyst with fibril morphology was prepared. The catalytic activities of these materials were tested against phenol and herbicide in water. Both H-titanate and TiO2-(B) fibres decorated with anatase nanocrystals were studied. It was found that anatase coated TiO2-(B) fibres prepared by a 45 h hydrothermal treatment followed by calcination were not only superior photocatalysts but could also be readily separated from the slurry after photocatalytic reactions due to its fibril morphology.

Sport biomechanics

Biomechanics involves research and analysis of the mechanisms of living organisms. This can be conducted on multiple levels and represents a continuum from the molecular, wherein biomaterials such as collagen and elastin are considered, to the tissue, organ and whole body level. Some simple applications of Newtonian mechanics can supply correct approximations on each level, but precise details demand the use of continuum mechanics. Sport biomechanics uses the scientific methods of mechanics to study the effects of forces on the sports performer and considers aspects of the behaviour of sports implements, equipment, footwear and surfaces. There are two main aims of sport biomechanics, that is, the reduction of injury and the improvement of performance (Bartlett, 1999). Aristotle (384-322 BC) wrote the first book on biomechanics, De Motu Animalium, translated as On the Movement of Animals. He saw animals' bodies as mechanical systems, but also pursued questions that might explain the physiological difference between imagining the performance of an action and actually doing it. Some simple examples of biomechanics research include the investigation of the forces that act on limbs, the aerodynamics of animals in flight, the hydrodynamics of objects moving through water and locomotion in general across all forms of life, from individual cells to whole organisms...

Dermal fibroblast infiltration of poly(ε-caprolactone) scaffolds fabricated by melt electrospinning in a direct writing mode

Melt electrospinning in a direct writing mode is a recent additive manufacturing approach to fabricate porous scaffolds for tissue engineering applications. In this study, we describe porous and cell-invasive poly (ε-caprolactone) scaffolds fabricated by combining melt electrospinning and a programmable x–y stage. Fibers were 7.5 ± 1.6 µm in diameter and separated by interfiber distances ranging from 8 to 133 µm, with an average of 46 ± 22 µm. Micro-computed tomography revealed that the resulting scaffolds had a highly porous (87%), three-dimensional structure. Due to the high porosity and interconnectivity of the scaffolds, a top-seeding method was adequate to achieve fibroblast penetration, with cells present throughout and underneath the scaffold. This was confirmed histologically, whereby a 3D fibroblast-scaffold construct with full cellular penetration was produced after 14 days in vitro. Immunohistochemistry was used to confirm the presence and even distribution of the key dermal extracellular matrix proteins, collagen type I and fibronectin. These results show that melt electrospinning in a direct writing mode can produce cell invasive scaffolds, using simple top-seeding approaches.

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.

The interplay between chondrocyte redifferentiation pellet size and oxygen concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Computer modeling of diffusion in biological tissues

Molecular-level computer simulations of restricted water diffusion can be used to develop models for relating diffusion tensor imaging measurements of anisotropic tissue to microstructural tissue characteristics. The diffusion tensors resulting from these simulations can then be analyzed in terms of their relationship to the structural anisotropy of the model used. As the translational motion of water molecules is essentially random, their dynamics can be effectively simulated using computers. In addition to modeling water dynamics and water-tissue interactions, the simulation software of the present study was developed to automatically generate collagen fiber networks from user-defined parameters. This flexibility provides the opportunity for further investigations of the relationship between the diffusion tensor of water and morphologically different models representing different anisotropic tissues.

The acute response of tendon to loading: implications for rehabilitation

Achilles tendinopathy is a common disorder involving physically active and sedentary individuals alike. Although the processes underlying its development are poorly understood, tendinopathy is widely regarded as an ‘overuse’ injury in which the tendon fails to adapt to prevalent loading conditions. Paradoxically, there is emerging evidence that heavy eccentric loading of the Achilles tendon may be an effective conservative approach for treatment of tendinopathy, with success rates of 60–80% reported. Interestingly, loading exercises involving other forms of muscle action, such as concentric activation, have been shown to be less effective treatment options. However, little is known about the acute response of tendon to exercise at present, and there are few plausible explanatory mechanisms for the observed beneficial effects of eccentric exercise, as opposed to other forms of strain stimuli. This paper presents the findings from a series of experiments undertaken to evaluate the effect of various strain stimuli on the time-dependent response of human Achilles tendon in vivo. It was shown for the first time, that heavy resistive ankle plantarflexion/ dorsiflexion exercises induced an immediate and significant decrease in Achilles tendon thickness (~15%). While thickness returned to pre-exercise levels within 24 hours, the recovery was exponential, with primary recovery occurring in less than 6 hours post-exercise. We proposed that such a diametral strain response with tensile loading reflects collagen realignment, Poison’s effects and radial extrusion of water from the tendon core. With unloading, the recovery of tendon dimensions likely reflects the re-diffusion of water via osmotic and/or inflammatory driven processes. Interestingly, prolonged walking was found to induce a similar diametral strain response. In subsequent studies, we demonstrated that eccentric exercise resulted in a greater reduction (-21%) in Achilles tendon thickness than isolated concentric exercise alone (-5%), despite a similar loading impulse. These novel findings, coupled with observations of a reduced diametral strain response with tendon pathology, highlight the importance of fluid movement to tendon function, nutrition and health. They also provide new insights into potential mechanisms underlying Achilles tendinopathy that impact rehabilitation strategies.

Feasibility of ranking articular cartilage conditions with non-destructive near-infrared spectroscopy with extension to the Mankin grading system

Grading osteoarthritic tissue has, until now, been a laboratory process confined to research activities. This thesis establishes a scientific protocol that extends osteoarthritic tissue ranking to surgical practice. The innovative protocol, which now incorporates the structural degeneration of collagen, enhances the traditional Modified Mankin ranking system, enabling its application to real time decision during surgery. Because it is fast and without time consuming laboratory process, it would potentially enable the cataloguing of tissues in osteoarthritic joints in all compartments of diseased joints during surgery for epistemological study and insight into the manifestation of osteoarthritis across age, gender, occupation, physical activities and race.