13:30
Soft-tissue models
Chair: Bart Koopman
13:30
15 mins
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IMPROVED CELL SEEDING RESULTS IN IMPROVED MINERALIZATION
Johanna Melke, Keita Ito, Sandra Hofmann
Abstract: Seeding cells onto a 3D scaffold is the first step for tissue engineering (TE). Ideally, cells are seeded efficiently and are distributed uniformly in a reproducible manner before they start depositing extracellular matrix (ECM). Even though dynamic seeding methods have been reported to be superior to static ones due to a more homogeneous cell distribution using kinetic forces [1], many laboratories still apply cells statically by pipetting them on top of the scaffolds as it is an easy to use technique. With this study, we applied a dynamic seeding method using an orbital shaker and examined whether it enhances seeding efficiency and results in an homogeneous distribution of human mesenchymal stem cells (hMSCs) in porous silk fibroin (SF) scaffolds. We also examined whether hMSCs seeded dynamically show bone-like tissue formation to test the feasibility for bone TE.
SF scaffolds, were manufactured as reported previously [2]. Static seeding of hMSCs was performed by pipetting a cell suspension (10^6 cells/20 µL) onto pre-wetted scaffolds and incubating for 90 min before adding cell culture medium. For dynamic seeding, scaffolds were incubated with a cell suspension (10^6 cells/4 mL) in 50-mL tubes on an orbital shaker for either 2 h, 4 h or 6 h. Cell number and distribution was assessed by DNA quantification and DAPI staining, respectively. The constructs were further cultured in osteogenic medium for 3 weeks and visualized with microcomputed tomography imaging.
Cell number was significantly higher using the dynamic seeding approach for 4 h and 6 h. Dynamic seeding resulted in a more homogenous cell distribution throughout the scaffold volume and interestingly, also an increased, uniform ECM mineralization.
The dynamic seeding method using an orbital shaker is not only as simple as static seeding but also superior to the static approach in terms of seeding efficiency and cellular distribution within scaffolds. It is a highly effective seeding method for engineering bone-like tissues but can go beyond bone TE and be used for seeding similar porous scaffolds with hMSCs.
REFERENCES
[1] P. Thevenot et. al, “Method to analyze three-dimensional cell distribution and infiltration in degradable scaffolds”, Tissue Eng Part C Methods, Vol. 14, pp. 319–31. (2008).
[2] S. Hofmann et al. ”Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds”, Biomaterials, Vol. 28, pp 1152–62, (2007).
ACKNOWLEDGEMENTS
This project was supported by the European Union’s Seventh Framework Programme (FP/2007-2013) / grant agreement No. 336043.
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13:45
15 mins
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MODELING THE HUMAN SKIN AS A NON-LINEAR VISCOELASTIC MATERIAL REINFORCED WITH ANISOTROPIC NON-LINEAR ELASTIC FIBERS
Marc van Vijven, Jibbe Soetens, Cees Oomens
Abstract: The skin is composed of multiple layers, namely the stratum corneum, viable epidermis and dermis. Each layer has its own function, composition and mechanical properties. Superposition of the layers results in complex mechanical behaviour that can be described as non-linear viscoelastic, anisotropic and heterogeneous over depth. Measuring, describing and modelling this mechanical behaviour can be relevant for several applications, for example research involving pressure ulcers [1]. A mechanical model in which all components of the behaviour are captured does not exist yet, although during previous research in our group (data under review) the heterogeneous, non-linear viscoelastic part of a newly introduced constitutive model was successfully fitted on shear experiments on ex vivo human skin. The in-plane fibre part of this model, describing non-linear elastic behaviour with the option to include anisotropy, was evaluated in this research.
Biaxial tensile tests were performed on ex vivo human skin, using digital image correlation to determine local deformations. A Gaussian fit describing collagen fibre orientations in a skin sample was created, based on CNA35 staining. The fibre distribution was applied in the constitutive model, and the parameters describing fibre stiffness and non-linearity in this material law were determined using finite element modelling and iterative parameter estimation. The anisotropic material model was evaluated by comparing it to its isotropic version and a two-term Ogden model (the current golden standard in skin modelling [2]), for each of which the same parameter estimation was performed.
Large variations in fibre distribution between and within individuals were observed, especially in the standard deviation and the ratio of isotropic and anisotropic fibres. Despite these variations, anisotropic characteristics in stress-stretch curves correlated with the found fibre distributions. Surface areas between the curves of experimental and simulated reaction forces over time for both variants of the introduced constitutive model were around two times smaller than for the Ogden material law for all samples.
Independent of the assigned fibre distribution, the reported constitutive material model had high predictive value for both biaxial tensile tests and shear experiments with the same set of parameters. It outperformed the established Ogden model, which can only predict skin mechanical behaviour with a separate set of parameters per experiment. This makes the newly introduced constitutive model very promising for application in skin modelling.
REFERENCES
[1] Oomens, C.W.J. et al. (2013). A numerical study to analyse the risk for pressure ulcer development on a spine board. Clinical Biomechanics, 28(7), 736-742.
[2] Flynn, C. et al. (2011). Modeling the mechanical response of in vivo human skin under a rich set of deformations. Annals of Biomedical Engineering, 39(7), 1935-1946.
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14:00
15 mins
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ARTHROSCOPIC AIRBRUSH TECHNOLOGY FOR CELL-BASED TREATMENT OF KNEE CARTILAGE DEFECTS – A PRE-CLINICAL IN VITRO STUDY USING 3D PRINTING TECHNOLOGY
Koen Dijkstra, Tommy de Windt, Lucienne Vonk, Daniël Saris
Abstract: Purpose
Recent research has shown the feasibility of arthroscopic airbrush assisted filling of cartilage defects using fibrin glue [1]. However, currently available fibrin spray devices can be optimized with 3D-printed custom-made nozzles. Current changes include internal to external mixing to prevent fibrin clogging and developing a 45-degree tip for increased arthroscopic maneuverability. Our work aims at (I) optimizing current technology with the design of custom-made spray nozzles and (II) at investigating cell viability after spraying at varying pressures.
Methods and materials
Custom-made spray nozzles were designed with 3D CAD software and produced by high-resolution 3D printing. Two commercially available fibrin spray devices using an internal mixing nozzle (Baxter® DuploSpray, for endoscopic use), and external mixing nozzle (Baxter® EasySpray, for topical use only) were used as a comparison. To study the influence of the spraying pressure on cell viability, human chondrocytes, human MSCs, and immortalized MSCs were sprayed in vitro in culture medium under increasing pressure ranging from 0 to 1.5 bar, from a distance of 1.5 cm. Cell viability was determined after 24 hrs (AlamarBlue) and normalized to the DNA content (PicoGreen).
Results
Spraying under increasing air pressure (0-1.5 bar) with an internal mixing nozzle (DuploSpray) had a highly destructive effect on cell viability (<20% viability at 1.0 bar), while an external mixing nozzle (EasySpray, custom nozzles) had a limited effect on cell viability (>90% viability at 1.0 bar). Using custom-made external mixing nozzles for the endoscopic DuploSpray system, the destructive effect of increasing air pressure on cell viability of hMSCs, iMSCs and chondrocytes was not statistically significant for pressures up to 1.0 bar (>90% viability).
Conclusion
The current commercially available spray nozzles are not applicable for arthroscopic cell spraying due to their design or influence on cell viability. However, modification of this technology with 3D-printed custom-made nozzles enabled cell spraying at a short distance without significantly affecting cell viability. This provides an easy-to-use technology for arthroscopic application of (cell-laden) hydrogels.
REFERENCES:
[1] T. S. de Windt, L. A. Vonk, J. K. Buskermolen, J. Visser, M. Karperien, R. L. A. W. Bleys, W. J. A. Dhert, and D. B. F. Saris, “Arthroscopic airbrush assisted cell implantation for cartilage repair in the knee: a controlled laboratory and human cadaveric study.,” Osteoarthritis Cartilage, vol. 23, no. 1, pp. 143–50, Jan. 2015.
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14:15
15 mins
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PREDICTING CELLULAR ORIENTATION IN ENGINEERED SOFT TISSUES WITH DIFFERENT COLLAGEN CONCENTRATIONS
Tommaso Ristori, Thomas Notermans, Frank Baaijens, Sandra Loerakker
Abstract: Cellular orientation is one of the most important determinants of the mechanical behavior of engineered soft tissue. In fact, along their main direction, cells exert traction forces and produce collagen, the main load-bearing component of most soft tissues. Therefore, to analyze the mechanical behavior of these constructs and ultimately optimize their design with finite element models, predicting and understanding cellular orientation is essential.
Contractile cells usually align along the direction of actin stress fibers, the main contractile component of the cytoskeleton. Cells are generally able to remodel the direction of these fibers in response to mechanical stimuli. However, collagen fibers can influence this process of realignment, a phenomenon which seems to depend on collagen concentration. In particular, by culturing cells in biaxially constrained and uniaxially cyclically strained engineered tissues with different collagen concentrations, it has been observed that high collagen concentrations can hinder stress fiber remodeling [1,2].
Recently, a computational model able to predict stress fiber alignment in response to a range of mechanical stimuli has been developed [3]. However, this model could not capture the impact that collagen concentration has on stress fiber remodeling. To overcome this limitation, we extended the model by considering the two following hypotheses:
A) collagen fibers provide cells with topographical cues, such that stress fibers tend to align along the directions with higher collagen densities;
B) collagen fibers constitute a spatial obstruction for cellular realignment, such that cells can reorient only when the collagen density in their surrounding is below a certain threshold.
The two hypotheses were tested separately and in combination by simulating stress fiber remodeling occurring in biaxially constrained collagen gels that were uniaxially cyclically stretched, varying both the amplitude of the cyclic strain and the concentration of collagen. With both hypotheses, the predicted stress fiber orientations were qualitatively in agreement with the experimental results [1,2]. Therefore, the computational simulations demonstrate that predicting cellular orientation in tissue-engineered soft tissue with different collagen concentrations is possible, although future studies are necessary to identify the exact processes that cause the effects of collagen fibers on stress fiber remodeling.
REFERENCES
[1] J. Foolen, V.S. Deshpande, F.M.W. Kanters and F.P.T. Baaijens, “The influence of matrix integrity on stress-fiber remodeling in 3D”, Biomaterials, Vol. 33, pp. 75087518, (2012).
[2] J. Foolen, M.W.J.T. Janssen-van den Broek, F.P.T. Baaijens, “Synergy between Rho signaling and matrix density in cyclic stretch-induced stress fiber organization”, Acta Biomaterialia, Vol. 10, pp. 18761885, (2014).
[3] C. Obbink-Huizer, C.W. J. Oomens, S. Loerakker, J. Foolen, C.V.C. Bouten, F.P.T. Baaijens, “Computational model predicts cell orientation in response to a range of mechanical stimuli”, Biomech Model Mechanobiol, Vol. 13, pp. 227–236, (2014).
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14:30
15 mins
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NUCLEUS PULPOSUS TONICITY AND INFLAMMATION
Meike Kleuskens, Bart van Dijk, Stefan de Vries, Keita Ito
Abstract: The healthy nucleus pulposus (NP), at the center of the intervertebral disc, is characterized by a high tissue tonicity due to an abundance of negatively charged proteoglycans. Previous studies have demonstrated that free swelling conditions can induce an inflammatory reaction by NP tissue1, and that this may also be true for milder hypotonic conditions.2 Hence, we investigated, in a well controlled environment, whether hypotonicity induces NP inflammation, and whether NP in a hypotonic environment are more sensitive to an inflammatory stimulus.
First, a bioreactor was designed to control the swelling pressure of bovine NP tissue samples. The bioreactor consisted of an inner container that holds the sample, confined at the top and bottom by porous plates to allow medium diffusion. The complete inner container is placed inside an outer container to which culture medium is added. The lid of the outer container presses onto the top plate of the inner container, transducing load applied to the lid to the NP sample.
To validate the controllability of the NP swelling pressure, bovine NP tissue was placed in the bioreactor. Subsequently, static preloads of 0.02 MPa and 0.075 MPa (hypotonic, n=3 each), and 0.2 MPa (native, n=6) were applied for a period of 16 hours after which the inner container maximum volume was fixed, limiting NP swelling. Thereafter, a stress-relaxation test was performed at 2% strain, resulting in equilibrium stresses of 0.03 ± 0.02 MPa, 0.11 ± 0.01 MPa and 0.22 ± 0.02 MPa respectively. Equilibrium stresses were slightly higher than the swelling pressures induced by the pre-loading, however these data suggest that hypotonic and native NP environments were indeed produced in a well controlled manner.
NP culture is ongoing to evaluate whether hypotonicity induces and/or facilitates inflammation. Bovine NP tissue is cultured for 5 days within the bioreactor, under free swelling, 0.02 MPa and 0.2 MPa swelling pressures, with and without an inflammatory stimulus (100 ng/ml TNFα + 10 ng/ml IL-1β, added at day 1 and day 3, n=6 per group) for each tonicity level. The swelling pressure during the culture period will be confirmed by a stress relaxation test at day 1 and 5. Additionally, real-time PCR will be performed at day 5 to evaluate the effect of hypotonicity, and hypotonicity in combination with an inflammatory stimulus on expression levels of inflammatory genes (i.e. IL-1β, IL-6, IL-8, TNFα, COX2).
REFERENCES
[1] B.G.M. van Dijk, E. Potier, M. Langelaan, N. Papen-Botterhuis and K. Ito. ”Reduced tonicity stimulates an inflammatory response in nucleus pulposus tissue that can be limited by a COX-2-specific inhibitor”, J Orthop Res 33(11), 1724-1731, 2015.
[2] I.T.M. Arkesteijn, B.G.M. van Dijk, K. Würtz-Kozak and K. Ito. “Hypotonicity differentially affects the production of inflammatory markers by nucleus pulposus tissue in simulated disc degeneration versus disc herniation”, In preparation.
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14:45
15 mins
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THE EFFECTS OF SHEAR AND PRESSURE ON TISSUE VIABILITY.
Iris Hoogendoorn, Jelle ten Brinke, Jasper Reenalda, Bart Koopman, Hans Rietman
Abstract: Pressure ulcers are a significant problem in health care (prevalence 5-20% in Dutch healthcare institutions), decreasing the quality of life of patients and leading to high healthcare costs. A pressure ulcers develops when tissue is devitalized due to prolonged mechanical loading (pressure and/or shear). Mechanical loading results in deformation and internal stresses and strains [1], causing direct cell death and a change in skin blood flow and perfusion. Shear is thought to cause twisting of the blood vessels and increasing the internal strains in the tissue, and thereby decreasing the tissue viability. The effect of pressure on skin viability has been studied broadly. Although shear is considered as of great importance and perhaps as more harmful compared to pressure alone, the effect of shear is handled more in a descriptive manner, rather than a quantitative manner. Therefore, the aim of this study was to quantify the effect of shear and the effect of pressure on skin viability in-vivo.
To quantify the effect of shear on tissue viability, four magnitudes of shear were applied, each in combination with two magnitudes of horizontal force (pressure). Each condition started with a one-minute baseline (no load), followed by 5 minutes of loading and 5 minutes of recovery after load removal. The conditions were performed in random order on the sacrum of ten healthy young human subjects (IRB was obtained). The mechanical loading was applied with a custom-made device including two force sensors, and a special designed indenter that fit two sensors of the ‘Oxygen to See’ (O2C). The ‘O2C’ combines laser Doppler flowmetry and white light spectroscopy, to measure oxygen saturation (%) and blood flow (AU) continuously and simultaneously at two measurement depths (approximately 1mm and 8mm).
In general the response of oxygen saturation and skin blood flow (SBF) shows a decrease during load application and a rapid increase after load removal called the post-reactive hyperaemic peak (PRH peak), to compensate for the deficit during load application.
Higher pressure magnitudes resulted in a further decrease of oxygenation and SBF during load application, but did not result in a larger PRH peak. The effect of shear seems to be twofold depending on the amount of pressure. With low pressure magnitudes, shear in general shows a negative effect on oxygenation and SBF, however this is not dependent on the amount of shear. With high pressure magnitudes, the effect of shear is more prominent and higher shear magnitudes results in a stronger decrease in oxygenation and SBF during load application. Also the PRH peak shows an increase with higher shear values.
This study showed that shear, especially with high pressure values, results in a stronger decrease in tissue viability and thereby increasing the risk of tissue damage.
REFERENCES
[1] Oomens CWJ, Loerakker S, Bader DL. The importance of internal strain as opposed to interface pressure in the prevention of pressure related deep tissue injury. J Tissue Viability 2010;19:35–42. doi:10.1016/j.jtv.2009.11.002.
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