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Articular cartilage functions as a shock absorber and facilitates the free movement of joints. Currently, there are no therapeutic drugs that promote the healing of damaged articular cartilage. Limitations associated with the two clinically relevant cell populations, human articular chondrocytes and mesenchymal stem cells, necessitate finding an alternative cell source for cartilage repair. Human embryonic stem cells (hESCs) provide a readily accessible population of self-renewing, pluripotent cells with perceived immunoprivileged properties for cartilage generation. We have developed a robust method to generate 3D, scaffold-free, hyaline cartilage tissue constructs from hESCs that are composed of numerous chondrocytes in lacunae, embedded in an extracellular matrix containing Type II collagen, sulphated glycosaminoglycans and Aggrecan. The elastic (Young’s) modulus of the hESC-derived cartilage tissue constructs (0.91 ± 0.08 MPa) was comparable to full-thickness human articular cartilage (0.87 ± 0.09 MPa). Moreover, we have successfully scaled up the size of the scaffold-free, 3D hESC-derived cartilage tissue constructs to between 4.5 mm and 6 mm, thus enhancing their suitability for clinical application.

Background Metabolic changes in obese pregnant women, such as changes of plasma lipids beyond physiological levels, may subsequently affect fetal development in utero. These metabolic derangements may remain in the offspring and continue throughout life. The placenta mediates bidirectional exchange of nutrients between mother and fetus. The impact of prepregnancy obesity on placental transfer of lipids is still unknown. Objective We aimed to examine materno-to-fetal free fatty acid (FFA) transfer by a combined experimental and modeling approach. Flux of 13C-labeled FFA was evaluated by ex vivo perfusion of human placentae as a function of prepregnancy obesity. Mathematical modeling complemented ex vivo results by providing FFA kinetic parameters. Results Obesity was strongly associated with elevated materno-to-fetal transfer of applied 13C-FFA. Clearance of polyunsaturated 13C-docosahexaenoic acid (DHA) was most prominently affected. The use of the mathematical model revealed a lower tissue storage capacity for DHA in obese compared with lean placentae. Conclusion Besides direct materno-to-fetal FFA transfer, placental mobilization accounts for the fetal FA supply. Together, with metabolic changes in the mother and an elevated materno-fetal FFA transfer shown in obesity, these changes suggest that they may be transmitted to the fetus, with yet unknown consequences.

The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block‐face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies. Term human placental villi were aldehyde‐fixed and processed for imaging by SBF SEM. Manual segmentation was carried out on a terminal villous capillary and an intermediate villous arteriole and venule. Twenty‐seven SBF SEM stacks from terminal villi were analysed using stereological approaches to determine the volumes of microvascular components and the proportions of pericyte coverage. SBF SEM analysis of capillary endothelial cells revealed the presence of interendothelial protrusions (IEPs) originating from the donor cell at the endothelial junction and forming deep thin projections up to 7 μm into the adjacent endothelial cells. IEP density was estimated to be in the order of 35 million cm–3 placental tissue. Pericytes cover 15% of the fetal capillary surface area in terminal villi. In comparison, the cytotrophoblast covered 24% of the syncytiotrophoblast basal membrane. A trans‐endothelial channel was observed in a region of the vasculo‐syncytial capillary. Pericyte coverage was extensive in both arteriole and venule. Three‐dimensional imaging of the placental microvasculature identified novel ultrastructural features and provided an insight into factors that may influence capillary permeability and placental function. We hypothesise that the IEPs may allow mechanosensing between adjacent endothelial cells to assist in the maintenance of vessel integrity. The numbers of endothelial junctions, the presence of trans‐endothelial channels and the extent of pericyte coverage all provide an insight into the factors determining capillary permeability., Serial block‐face scanning electron microscopy to create a 3D reconstruction of endothelial cells. Interendothelial protrusions were observed originating from a donor endothelial cell and tunnelling inside a recipient endothelial cell. These structures constitute a novel intercellular connection.

Placental function is essential for fetal development and establishing the foundations for lifelong health. The placental villous stroma is a connective tissue layer that supports the fetal capillaries and villous trophoblast. All the nutrients that cross the placenta must also cross the stroma, and yet little is known about this region. This study uses high‐resolution three‐dimensional imaging to explore the structural complexity of this region within the placental villi. Serial block‐face scanning electron microscopy and confocal microscopy were used to image the placental villous stroma in three‐dimensions. Transmission electron microscopy (TEM) was used to generate high resolution two‐dimensional images. Stereological approaches were used to quantify volumes of stromal constituents. Three‐dimensional imaging identified stromal extracellular vesicles, which constituted 3.9% of the villous stromal volume. These stromal extracellular vesicles were ovoid in shape, had a median length of 2750 nm (range 350–7730 nm) and TEM imaging confirmed that they were bounded by a lipid bilayer. Fifty‐nine per cent of extracellular vesicles were in contact with a fibroblast‐like stellate cell and these vesicles were significantly larger than those where no contact was observed. These stellate cells formed local networks with adherent junctions observed at contact points. This study demonstrates that the villous stroma contains extracellular macrovesicles which are considerably larger than any previously described in tissue or plasma. The size and abundance of these macrovesicles in the villous stroma highlight the diversity of extracellular vesicle biology and their roles within connective tissues., Extracellular vesicles are membrane‐bound structures released from cells into the plasma or stroma. Extracellular vesicles are thought to contain chemical messengers and have a signalling function. The two main classes of extracellular vesicle are exosomes and microvesicles, although because of their overlapping size distributions these cannot always be readily distinguished. Stromal extracellular vesicles are typically

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (

Extracellular matrix stiffness has a profound effect on the behaviour of many cell types. Adherent cells apply contractile forces to the material on which they adhere, and sense the resistance of the material to deformation – its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (

Cartilage tissue repair procedures currently under development aim to create a construct in which patient-derived cells are seeded and expanded ex vivo before implantation back into the body. The key challenge is producing physiologically realistic constructs that mimic real tissue structure and function. One option with vast potential is to print strands of material in a 3D structure called a scaffold that imitates the real tissue structure; the strands are composed of gel seeded with cells and so provide a template for cartilaginous tissue growth. The scaffold is placed in the construct and pumped with nutrient-rich culture medium to supply nutrients to the cells and remove waste products, thus promoting tissue growth. In this paper we use asymptotic homogenization to determine the effective flow and transport properties of such a printed scaffold system. These properties are used to predict the distribution of nutrient/waste products through the construct, and to specify design criteria for the scaffold that will optimize the growth of functional tissue. © 2009 Elsevier Ltd. All rights reserved.

In 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.

Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a ‘guiding’ structure to support the body’s self-healing process.

Based 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.

A multilevel finite element approach is applied to predict local cell deformations in engineered tissue constructs. Cell deformations are predicted from detailed nonlinear FE analysis of the microstructure, consisting of an arrangement of cells embedded in matrix material. Effective macroscopic tissue behavior is derived by a computational homogenization procedure. To illustrate this approach, we simulated the compression of a skeletal muscle tissue construct and studied the influence of microstructural heterogeneity on local cell deformations. Results show that heterogeneity has a profound impact on local cell deformations, which highly exceed macroscopic deformations. Moreover, microstructural heterogeneity and the presence of neighboring cells leads to complex cell shapes and causes non-uniform deformations within a cell.

Significant oxygen gradients occur within tissue engineered cartilaginous constructs. Although oxygen tension is an important limiting parameter in the development of new cartilage matrix, its precise role in matrix formation by chondrocytes remains controversial, primarily due to discrepancies in the experimental setup applied in different studies. In this study, the specific effects of oxygen tension on the synthesis of cartilaginous matrix by human articular chondrocytes were studied using a combined experimental-computational approach in a “scaffold-free” 3D pellet culture model. Key parameters including cellular oxygen uptake rate were determined experimentally and used in conjunction with a mathematical model to estimate oxygen tension profiles in 21-day cartilaginous pellets. A threshold oxygen tension (pO2 ≈ 8% atmospheric pressure) for human articular chondrocytes was estimated from these inferred oxygen profiles and histological analysis of pellet sections. Human articular chondrocytes that experienced oxygen tension below this threshold demonstrated enhanced proteoglycan deposition. Conversely, oxygen tension higher than the threshold favored collagen synthesis. This study has demonstrated a close relationship between oxygen tension and matrix synthesis by human articular chondrocytes in a “scaffold-free” 3D pellet culture model, providing valuable insight into the understanding and optimization of cartilage bioengineering approaches. Biotechnol. Bioeng. 2014;111: 1876–1885.

Amino acid transfer to the fetus is dependent on several different factors. While these factors can be understood in isolation, it is still not possible to predict the function of the system as a whole. In order to do this an integrated approach is required which incorporates the interactions between the different determinants of amino acid transfer. Computational modelling of amino acid transfer in the term human placenta provides a mechanism by which this integrated approach can be delivered. Such a model would be invaluable for understanding amino acid transfer in both normal and pathological pregnancies.In order to develop a computational model it is necessary to determine all the biological factors which are important contributors to net amino acid transfer and the ways in which they interact. For instance, how different classes of amino acid transporter must interact to transfer amino acids across the placenta. Mathematically, the kinetics of each type of transporter can be represented by separate equations that describe their transfer rate as a non-linear function of amino acid concentrations. These equations can then be combined in the model to predict the overall system behaviour. Testing these predictions experimentally will demonstrate the strengths and weaknesses of the model, which can then be refined with increasing complexity and retested in an iterative fashion.In this way we hope to develop a functional computational model which will allow exploration of the factors that determine amino acid transfer across the placenta. This model may also allow the development of strategies to optimise placental transfer in pathologies associated with impaired amino acid transfer such as fetal growth restriction

The 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 microCT 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.

Improved biological and mechanical functionality of musculoskeletal tissue-engineered constructs is required for clinical application, which can only be achieved by comprehensive multidisciplinary research. This review focuses on the contribution of computational modelling as a framework for obtaining an integrated understanding of key processes, which include: nutrient transport and utilization, matrix formation, cell population dynamics, cell attachment and migration, and local cell-cell interactions. Such an integrated perspective of these key aspects will be critical to open up new directions in tissue engineering research, as significant progress can be made by combining existing computational and experimental methods. Furthermore, theoretical modelling has enormous potential in applications ranging from the interpretation of experimental results and the identification of the main governing processes, to the optimization of practical tissue engineering protocols with implications therein for an increasing ageing population.

Endochondral 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.

Different 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.