70 research outputs found
Experimental–computational evaluation of human bone marrow stromal cell spreading on trabecular bone structures
No full textThe clinical application of macro-porous scaffolds for bone regeneration is significantly affected by the problem of insufficient cell colonization. Given the wide variety of different scaffold structures used for tissue engineering it is essential to derive relationships for cell colonization independent of scaffold architecture. To study cell population spreading on 3D structures decoupled from nutrient limitations, an in vitro culture system was developed consisting of thin slices of human trabecular bone seeded with Human Bone Marrow Stromal Cells, combined with dedicated ?CT imaging and computational modeling of cell population spreading. Only the first phase of in vitro scaffold colonization was addressed, in which cells migrate and proliferate up to the stage when the surface of the bone is covered as a monolayer, a critical prerequisite for further tissue formation. The results confirm the model’s ability to represent experimentally observed cell population spreading. The key advantage of the computational model was that by incorporating complex 3D structure, cell behavior can be characterized quantitatively in terms of intrinsic migration parameters, which could potentially be used for predictions on different macro-porous scaffolds subject to additional experimental validation. This type of modeling will prove useful in predicting cell colonization and improving strategies for skeletal tissue engineering
Computational modeling to predict the temporal regulation of chondrocyte metabolism in response to various dynamic compression regimens
No full textBased on previously published experimental work, computational models were developed to simulate the effect of different dynamic compression regimens on the activity of chondrocytes seeded in agarose constructs. In particular, the balance between proliferation and matrix synthesis can be adjusted by applying different intervals of continuous or intermittent mechanical compression. A phenomenological compartment based-modeling approach was used as first model. A more mechanistic cell cycle model was used as the second model. The compartment-based modeling approach was found to be useful in representing a balance between proliferation and proteoglycan synthesis, when the effect of a certain stimulation protocol is known. In order to predict the response to different intervals of mechanical stimulation, however, a more mechanistic cell cycle-based approach is required. The cell cycle model supports an important role of the onset of loading. In addition, an inhibitory effect of further loading is required, which is more likely to be related to cell cycle progression velocity than to a decreased probability of commitment to the cell cycle. The mechanisms behind this inhibitory effect and the computational implementation, however, require further investigation
Can the growth factors PTHrP, Ihh and VEGF, together regulate the development of a long bone?
No full textEndochondral ossification is the process of differentiation of cartilaginous into osseous tissue. Parathyroid hormone related protein (PTHrP), Indian hedgehog (Ihh) and vascular endothelial growth factor (VEGF), which are synthesized in different zones of the growth plate, were found to have crucial roles in regulating endochondral ossification. The aim of this study was to evaluate whether the three growth factors PTHrP, Ihh and VEGF, together, could regulate longitudinal growth in a normal human, fetal femur. For this purpose, a one-dimensional finite element (FE) model, incorporating growth factor signaling, was developed of the human, distal, femoral growth plate. It included growth factor synthesis in the relevant zones, their transport and degradation and their effects. Simulations ran from initial hypertrophy in the center of the bone until secondary ossification starts at approximately 3.5 months postnatal. For clarity, we emphasize that no mechanical stresses were considered. The FE model showed a stable growth plate in which the bone growth rate was constant and the number of cells per zone oscillated around an equilibrium. Simulations incorporating increased and decreased PTHrP and Ihh synthesis rates resulted, respectively, in more and less cells per zone and in increased and decreased bone growth rates. The FE model correctly reflected the development of a growth plate and the rate of bone growth in the femur. Simulations incorporating increased and decreased PTHrP and Ihh synthesis rates reflected growth plate pathologies and growth plates in PTHrP-/- and Ihh-/- mice. The three growth factors, PTHrP, Ihh and VEGF, could potentially together regulate tissue differentiation
Tissue regeneration in porous structures for bone engineering applications
No full textScaffold design for bone regeneration is currently widely investigated as pore architecture can dramatically impact tissue formation in porous biomaterials used in regenerative medicine. A wide variety of 3D structures is used for this purpose, which has become even more important given the geometric freedom offered by emerging rapid prototyping techniques. Therefore, optimal design of pore architecture to maximize tissue formation and ingrowth is required. Tissue formation is frequently assessed in certain established scaffold structures, produced mainly as the end result of a particular fabrication process and its limitations. However, instead, scaffold architecture design should be based on the knowledge of how tissue actually forms in porous structures in the first place.Tissue formation within porous structures can be dependent on several parameters, such as cell generated forces, cell division, cytoskeleton and extracellular matrix arrangement. Tissue differentiation is also an important aspect as once cells commit to a lineage, the proliferation could decrease. These aspects have been extensively shown to be modulated biochemically. However, the impact of different 3D structures is still largely unclear. Therefore, in this thesis, it is aimed to characterise 3D tissue formation within different structures. For this purpose, an in vitro system with well-defined open pore slots of 1cm length, 1mm thickness and varying width (hundreds of micrometres) was used to characterise tissue growth solely as a function of pore geometry. This system provided a 3D environment for neo-tissue formation while minimizing nutrient limitations associated with full 3D constructs.This thesis is the outcome of three studies. The first was focused on tissue formation kinetics in four different pore widths of 200, 300, 400 and 500 ?m. For this purpose, a unique system made of calcium phosphate cement with open pores was designed and fabricated. Several types of microscopy were used such as optical microscopy, time-lapse microscopy, epi-fluorescence microscopy and confocal microscopy. Results demonstrated that the material was biocompatible with Human Bone Marrow Stromal Cells and that tissue formation was strongly influenced by pore geometry. Both velocity of tissue invasion and area of tissue formed increased as pores became narrower. This was associated with distinct patterns of actin cytoskeleton organisation depending on pore width, indicating the role of active cell generated forces.The second study is a more detailed characterisation of the type of tissue regenerated and its organisation. The neo-tissue was seen to display an osteoid-like collagen matrix. The main elements constituting a tissue i.e. cells, actin cytoskeleton and collagen matrix were imaged and their organisation was quantified with various image analysis methods. Results showed a significantly higher alignment with the longitudinal pore axis in the 200 ?m compared to the 500 ?m pores for all the tissue components analysed. By relating tissue orientation with its expansion rate, the results suggested that increased tissue alignment could be an important factor enhancing tissue formation.In the third study, tissue differentiation was assessed as a function of pore size. Expression of intermediate and late bone markers was assessed, Alkaline Phosphatase and Osteopontin respectively. Results showed that both markers were expressed, indicating that the neo-tissue regenerated reached late state of differentiation and is prepared for mineralization. The expression of these markers was semi-quantitatively evaluated. ALP expression was expressed increasingly as tissue “age” increased. A gradient was observed with increasing staining intensity towards the starting point of tissue formation. Thus, the results revealed presence of distinct zones in which cells are in different states associated with various functions (proliferation or differentiation). Additionally, the expression of Osteopontin assessed semi quantitatively did not show any notable differences between pore widths. However, the results obtained displayed high variability between replicates. Therefore, it was only concluded that neo-tissue formed in both structures was able to express early (second study, chapter 4), intermediate and late osteogenic markers, although no significant differences were found between the different pore widths. This demonstrated that the tissue regenerated had committed to the osteogenic pathway, with the potential for full differentiation into mineralized tissue, which needs to be confirmed in future studies.Overall, the results presented in this thesis provide evidence for the hypothesis that pore geometry affects tissue growth capacity by modulating tissue organisation. Key factors governing tissue formation in vitro were elucidated, highlighting the importance of the interplay between cell division, cell mechanics, cytoskeleton dynamics, tissue spatial organisation, matrix deposition and differentiation in relation to porous structure
Experimental-computational evaluation of human bone marrow stromal cell migration on trabecular bone structures
No full textComputational study of culture conditions and nutrient supply in cartilage tissue engineering
No full textDifferent culture conditions for cartilage tissue engineering were evaluated with respect to the supply of oxygen and glucose and the accumulation of lactate. A computational approach was adopted in which the culture configurations were modeled as a batch process and transport was considered within constructs seeded at high cell concentrations and of clinically relevant dimensions. To assess the extent to which mass transfer can be influenced theoretically, extreme cases were distinguished in which the culture medium surrounding the construct was assumed either completely static or well mixed and fully oxygenated. It can be concluded that severe oxygen depletion and lactate accumulation can occur within constructs for cartilage tissue engineering. However, the results also indicate that transport restrictions are not insurmountable, providing that the medium is well homogenized and oxygenated and the construct's surfaces are sufficiently exposed to the medium. The large variation in uptake rates of chondrocytes indicates that for any specific application the quantification of cellular utilization rates, depending on the cell source and culture conditions, is an essential starting point for optimizing culture protocols
Modelling the effect of intervillous flow on solute transfer based on 3D imaging of the human placental microstructure
No full textIntroduction: A healthy pregnancy depends on placental transfer from mother to fetus. Placental transfer takes place at the micro scale across the placental villi. Solutes from the maternal blood are taken up by placental villi and enter the fetal capillaries. This study investigated the effect of maternal blood flow on solute uptake at the micro scale. Methods: A 3D image based modelling approach of the placental microstructures was undertaken. Solute transport in the intervillous space was modelled explicitly and solute uptake with respect to different maternal blood flow rates was estimated. Fetal capillary flow was not modelled and treated as a perfect sink. Results: For a freely diffusing small solute, the flow of maternal blood through the intervillous space was found to be limiting the transfer. Ignoring the effects of maternal flow resulted in a 2.4 ± 0.4 fold over-prediction of transfer by simple diffusion, in absence of binding. Villous morphology affected the efficiency of solute transfer due to concentration depleted zones. Interestingly, less dense microvilli had lower surface area available for uptake which was compensated by increased flow due to their higher permeability. At super-physiological pressures, maternal flow was not limiting, however the efficiency of uptake decreased. Conclusions: This study suggests that the interplay between maternal flow and villous structure affects the efficiency of placental transfer but predicted that flow rate will be the major determinant of transfer
Mathematical modelling of tissue formation in chondrocyte filter cultures
No full textIn the field of cartilage tissue engineering, filter cultures are a frequently used three-dimensional differentiation model. However, understanding of the governing processes of in vitro growth and development of tissue in these models is limited. Therefore, this study aimed to further characterise these processes by means of an approach combining both experimental and applied mathematical methods. A mathematical model was constructed, consisting of partial differential equations predicting the distribution of cells and glycosaminoglycans (GAGs), as well as the overall thickness of the tissue. Experimental data was collected to allow comparison with the predictions of the simulation and refinement of the initial models. Healthy mature equine chondrocytes were expanded and subsequently seeded on collagen-coated filters and cultured for up to 7 weeks. Resulting samples were characterised biochemically, as well as histologically. The simulations showed a good representation of the experimentally obtained cell and matrix distribution within the cultures. The mathematical results indicate that the experimental GAG and cell distribution is critically dependent on the rate at which the cell differentiation process takes place, which has important implications for interpreting experimental results. This study demonstrates that large regions of the tissue are inactive in terms of proliferation and growth of the layer. In particular, this would imply that higher seeding densities will not significantly affect the growth rate. A simple mathematical model was developed to predict the observed experimental data and enable interpretation of the principal underlying mechanisms controlling growth-related changes in tissue composition
The local matrix distribution and the functional development of tissue engineered cartilage, a finite element study
No full textAssessment of the functionality of tissue engineered cartilage constructs is hampered by the lack of correlation between global measurements of extra cellular matrix constituents and the global mechanical properties. Based on patterns of matrix deposition around individual cells, it has been hypothesized previously, that mechanical functionality arises when contact occurs between zones of matrix associated with individual cells. The objective of this study is to determine whether the local distribution of newly synthesized extracellular matrix components contributes to the evolution of the mechanical properties of tissue engineered cartilage constructs. A computational homogenization approach was adopted, based on the concept of a periodic representative volume element. Local transport and immobilization of newly synthesized matrix components were described. Mechanical properties were taken dependent on the local matrix concentration and subsequently the global aggregate modulus and hydraulic permeability were derived. The transport parameters were varied to assess the effect of the evolving matrix distribution during culture. The results indicate that the overall stiffness and permeability are to a large extent insensitive to differences in local matrix distribution. This emphasizes the need for caution in the visual interpretation of tissue functionality from histology and underlines the importance of complementary measurements of the matrix's intrinsic molecular organization
The Disabled Female Body and the Spirit: A Comprehensive Study of Justitia Sengers and her Commentary on the 69th Psalm
No full textIn 1586, a Lutheran religious commentary was published out of a printing press in Magdeburg, Germany, and was titled Trostbüchlein: Uber den Neun und Sechtzigsten Psalm (Little Book of Comfort over the Sixty-Ninth Psalm). The author signed their name at the end of the foreword as Justitia Sengers. Justitia Sengers described herself in the foreword as a young woman from Braunschweig, Germany, and congenitally blind. Her text was well-received, which resulted in a reprint of the text in 1593 out of a printing press in Hamburg, this time under another title, Des Heiligen Geistes Beschreibung vom Leiden und Sterben (The Holy Spirit's Description of the Suffering and Death of our Lord Jesus Christ), whose subtitle would now explicitly name Justitia Sengers as the young blind woman who authored the text. It would continue to be a popular text and would be circulated and reprinted into the 1610’s, nearly thirty years after its initial print, under this new title. This religious commentary, while popular and widely circulated during its time, is severely understudied. This thesis is therefore a microhistory, with Justitia’s text at the center of the study. The main purpose of this thesis is to better understand the gendered, disabled, and religious aspects of the text, as well as the society and historical world from which it originated. The main avenues through which this is achieved is via interdisciplinary methodologies, literature analysis, and case studies of the known female owners, handlers, circulators, and (re)publishers of the text. This thesis therefore offers important insights into sixteenth and seventeenth-century gender, disability, and popular religion in the Lower Saxony region of Germany, as well as new insights into Justitia Sengers and her “Little Book of Comfort.
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