Hans Van Oosterwyck

Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor-α

Osteoporosis and muscle frailty are important health problems in elderly men and may be partly related to biological androgen activity. This androgen action can be mediated directly through stimulation of the androgen receptor (AR) or indirectly through stimulation of estrogen receptor‐alpha (ERα) following aromatization of androgens into estrogens. To assess the differential action of AR and ERα pathways on bone and body composition, AR‐ERα double‐knockout mice were gener‐ated and characterized. AR disruption decreased trabec‐ular bone mass, whereas ERα disruption had no additional effect on the AR‐dependent trabecular bone loss. In contrast, combined AR and ERα inactivation additionally reduced cortical bone and muscle mass compared with either AR or ERα disruption alone. ERα inactivation—in the presence or absence of AR—increased fat mass. We demonstrate that AR activation is solely responsible for the development and maintenance of male trabecular bone mass. Both AR and ERα activation, however, are needed to optimize the acquisition of cortical bone and muscle mass. ERα activation alone is sufficient for the regulation of fat mass. Our findings clearly define the relative importance of AR and ERα signaling on trabecu‐lar and cortical bone mass as well as body composition in male mice.—Callewaert, F., Venken, K., Ophoff, J., De Gendt, K., Torcasio, A., van Lenthe, G. H., Van Ooster‐wyck, H., Boonen, S., Bouillon, R., Verhoeven, G., Vanderschueren, D. Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor‐α. FASEB J. 23, 232‐240 (2009)

Modeling fluid flow through irregular scaffolds for perfusion bioreactors

Direct perfusion of 3D tissue engineered constructs is known to enhance osteogenesis, which can be partly attributed to enhanced nutrient and waste transport. In addition flow mediated shear stresses are known to upregulate osteogenic differentiation and mineralization. A quantification of the hydrodynamic environment is therefore crucial to interpret and compare results of in vitro bioreactor experiments. This study aims to deal with the pitfalls of numerical model preparation of highly complex 3D bone scaffold structures and aims to provide more accurate wall shear stress (WSS) estimates. µCT imaging techniques were used to reconstruct the geometry of both a titanium (Ti) and a hydroxyapatite scaffold, starting from 430 images with a resolution of 8 µm. To tackle the tradeoff between model size and mesh resolution we selected two concentric regions of interest (cubes with a volume of 1 and 3.375 mm3, respectively) for both scaffolds. A flow guidance in front of the real inlet surface of the scaffold was designed to mimic realistic inlet conditions. With a flow rate of 0.04 mL/min perfused through a 5 mm diameter scaffold at an inlet velocity of 33.95 µm/s we obtained average WSSs of 1.10 and 1.46 mPa for the 1 mm3 and the 3.375 mm3 model of the hydroxyapatite scaffold compared to 1.40 and 1.95 mPa for the 1 mm3 model and the 3.375 mm3 model of the Ti scaffold, showing the important influence of the scaffold micro‐architecture heterogeneity and the proximity of boundaries. To assess that influence we selected cubic portions, of which the WSS data were analyzed, with the same size and the same location within both 1 and 3.375 mm3 cubic models. Varying the size of the inner portions simultaneously in both model selections gives a quantification of the sensitivity to boundary neighborhood. This methodology allows to get more insight in the complex concept of tissue engineering and will likely help to understand and eventually improve the fluid‐mechanical aspects. Biotechnol. Bioeng. 2009;103: 621–630.

MOSAIC: a multiscale model of osteogenesis and sprouting angiogenesis with lateral inhibition of endothelial cells.

The healing of a fracture depends largely on the development of a new blood vessel network (angiogenesis) in the callus. During angiogenesis tip cells lead the developing sprout in response to extracellular signals, amongst which vascular endothelial growth factor (VEGF) is critical. In order to ensure a correct development of the vasculature, the balance between stalk and tip cell phenotypes must be tightly controlled, which is primarily achieved by the Dll4-Notch1 signaling pathway. This study presents a novel multiscale model of osteogenesis and sprouting angiogenesis, incorporating lateral inhibition of endothelial cells (further denoted MOSAIC model) through Dll4-Notch1 signaling, and applies it to fracture healing. The MOSAIC model correctly predicted the bone regeneration process and recapitulated many experimentally observed aspects of tip cell selection: the salt and pepper pattern seen for cell fates, an increased tip cell density due to the loss of Dll4 and an excessive number of tip cells in high VEGF environments. When VEGF concentration was even further increased, the MOSAIC model predicted the absence of a vascular network and fracture healing, thereby leading to a non-union, which is a direct consequence of the mutual inhibition of neighboring cells through Dll4-Notch1 signaling. This result was not retrieved for a more phenomenological model that only considers extracellular signals for tip cell migration, which illustrates the importance of implementing the actual signaling pathway rather than phenomenological rules. Finally, the MOSAIC model demonstrated the importance of a proper criterion for tip cell selection and the need for experimental data to further explore this. In conclusion, this study demonstrates that the MOSAIC model creates enhanced capabilities for investigating the influence of molecular mechanisms on angiogenesis and its relation to bone formation in a more mechanistic way and across different time and spatial scales.

Micro-CT-based screening of biomechanical and structural properties of bone tissue engineering scaffolds

The development of successful scaffolds for bone tissue engineering requires a concurrent engineering approach that combines different research fields. In order to limit in vivo experiments and reduce trial and error research, a scaffold screening technique has been developed. In this protocol seven structural and three biomechanical properties of potential scaffold materials are quantified and compared to the desired values. The property assessment is done on computer models of the scaffolds, and these models are based on micro-CT images. As a proof of principle, three porous scaffolds were evaluated with this protocol: stainless steel, hydroxyapatite, and titanium. These examples demonstrate that the modelling technique is able to quantify important scaffold properties. Thus, a powerful technique for automated screening of bone tissue engineering scaffolds has been developed that in a later stage may be used to tailor the scaffold properties to specific requirements.

Towards a quantitative understanding of oxygen tension and cell density evolution in fibrin hydrogels

The in vitro culture of hydrogel-based constructs above a critical size is accompanied by problems of unequal cell distribution when diffusion is the primary mode of oxygen transfer. In this study, an experimentally-informed mathematical model was developed to relate cell proliferation and death inside fibrin hydrogels to the local oxygen tension in a quantitative manner. The predictive capacity of the resulting model was tested by comparing its outcomes to the density, distribution and viability of human periosteum derived cells (hPDCs) that were cultured inside fibrin hydrogels in vitro. The model was able to reproduce important experimental findings, such as the formation of a multilayered cell sheet at the hydrogel periphery and the occurrence of a cell density gradient throughout the hydrogel. In addition, the model demonstrated that cell culture in fibrin hydrogels can lead to complete anoxia in the centre of the hydrogel for realistic values of oxygen diffusion and consumption. A sensitivity analysis also identified these two parameters, together with the proliferation parameters of the encapsulated cells, as the governing parameters for the occurrence of anoxia. In conclusion, this study indicates that mathematical models can help to better understand oxygen transport limitations and its influence on cell behaviour during the in vitro culture of cell-seeded hydrogels.

Mathematical modeling of fracture healing in mice: comparison between experimental data and numerical simulation results

The combined use of experimental and mathematical models can lead to a better understanding of fracture healing. In this study, a mathematical model, which was originally established by Bailón-Plaza and van der Meulen (J Theor Biol 212:191–209, 2001), was applied to an experimental model of a semi-stabilized murine tibial fracture. The mathematical model was implemented in a custom finite volumes code, specialized in dealing with the model’s requirements of mass conservation and non-negativity of the variables. A qualitative agreement between the experimentally measured and numerically simulated evolution in the cartilage and bone content was observed. Additionally, an extensive parametric study was conducted to assess the influence of the model parameters on the simulation outcome. Finally, a case of pathological fracture healing and its treatment by administration of growth factors was modeled to demonstrate the potential therapeutic value of this mathematical model.

A hybrid bioregulatory model of angiogenesis during bone fracture healing

Bone fracture healing is a complex process in which angiogenesis or the development of a blood vessel network plays a crucial role. In this paper, a mathematical model is presented that simulates the biological aspects of fracture healing including the formation of individual blood vessels. The model consists of partial differential equations, several of which describe the evolution in density of the most important cell types, growth factors, tissues and nutrients. The other equations determine the growth of blood vessels as a result of the movement of leading endothelial (tip) cells. Branching and anastomoses are accounted for in the model. The model is applied to a normal fracture healing case and subjected to a sensitivity analysis. The spatiotemporal evolution of soft tissues and bone, as well as the development of a blood vessel network are corroborated by comparison with experimental data. Moreover, this study shows that the proposed mathematical framework can be a useful tool in the research of impaired healing and the design of treatment strategies.

Occurrence and Treatment of Bone Atrophic Non-Unions Investigated by an Integrative Approach

Recently developed atrophic non-union models are a good representation of the clinical situation in which many non-unions develop. Based on previous experimental studies with these atrophic non-union models, it was hypothesized that in order to obtain successful fracture healing, blood vessels, growth factors, and (proliferative) precursor cells all need to be present in the callus at the same time. This study uses a combined in vivo-in silico approach to investigate these different aspects (vasculature, growth factors, cell proliferation). The mathematical model, initially developed for the study of normal fracture healing, is able to capture essential aspects of the in vivo atrophic non-union model despite a number of deviations that are mainly due to simplifications in the in silico model. The mathematical model is subsequently used to test possible treatment strategies for atrophic non-unions (i.e. cell transplant at post-osteotomy, week 3). Preliminary in vivo experiments corroborate the numerical predictions. Finally, the mathematical model is applied to explain experimental observations and identify potentially crucial steps in the treatments and can thereby be used to optimize experimental and clinical studies in this area. This study demonstrates the potential of the combined in silico-in vivo approach and its clinical implications for the early treatment of patients with problematic fractures.

Oxygen as a critical determinant of bone fracture healing - a multiscale model

A timely restoration of the ruptured blood vessel network in order to deliver oxygen and nutrients to the fracture zone is crucial for successful bone healing. Indeed, oxygen plays a key role in the aerobic metabolism of cells, in the activity of a myriad of enzymes as well as in the regulation of several (angiogenic) genes. In this paper, a previously developed model of bone fracture healing is further improved with a detailed description of the influence of oxygen on various cellular processes that occur during bone fracture healing. Oxygen ranges of the cell-specific oxygen-dependent processes were established based on the state-of-the art experimental knowledge through a rigorous literature study. The newly developed oxygen model is compared with previously published experimental and in silico results. An extensive sensitivity analysis was also performed on the newly introduced oxygen thresholds, indicating the robustness of the oxygen model. Finally, the oxygen model was applied to the challenging clinical case of a critical sized defect (3mm) where it predicted the formation of a fracture non-union. Further model analyses showed that the harsh hypoxic conditions in the central region of the callus resulted in cell death and disrupted bone healing thereby indicating the importance of a timely vascularization for the successful healing of a large bone defect. In conclusion, this work demonstrates that the oxygen model is a powerful tool to further unravel the complex spatiotemporal interplay of oxygen delivery, diffusion and consumption with the several healing steps, each occurring at distinct, optimal oxygen tensions during the bone repair process.

Analysis of initial cell spreading using mechanistic contact formulations for a deformable cell model.

Adhesion governs to a large extent the mechanical interaction between a cell and its microenvironment. As initial cell spreading is purely adhesion driven, understanding this phenomenon leads to profound insight in both cell adhesion and cell-substrate interaction. It has been found that across a wide variety of cell types, initial spreading behavior universally follows the same power laws. The simplest cell type providing this scaling of the radius of the spreading area with time are modified red blood cells (RBCs), whose elastic responses are well characterized. Using a mechanistic description of the contact interaction between a cell and its substrate in combination with a deformable RBC model, we are now able to investigate in detail the mechanisms behind this universal power law. The presented model suggests that the initial slope of the spreading curve with time results from a purely geometrical effect facilitated mainly by dissipation upon contact. Later on, the spreading rate decreases due to increasing tension and dissipation in the cells cortex as the cell spreads more and more. To reproduce this observed initial spreading, no irreversible deformations are required. Since the model created in this effort is extensible to more complex cell types and can cope with arbitrarily shaped, smooth mechanical microenvironments of the cells, it can be useful for a wide range of investigations where forces at the cell boundary play a decisive role.