- Email: [email protected]
A three dimensional matrix for guided mesenchymal stem cell and urothelial cell growth and differentiation
$578
Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
valve leaflets indicate a difference in mechanical properties between the belly and the commissures after four weeks of culturing. References [1] Mol et al. Ann. Biomed. Eng. 2005; 33(12): 1778-1788. [2] Cox et al. J. Biomech. Eng. 2006; in press. 7012 We-Th, no. 11 (P63) Acellular vascular scaffolds for tissue engineered blood vessels J.L. Berry 1, S.K. Yazdani 1, G. Amiel 2, J.J. Yoo 2, A.A. Atala 2, S. Soker 2. 1Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Wake Forest University, Winston-Salem, NC, USA, 2Wake Forest Institute of Regenerative Medicine, Medical Center Blvd., Winston-Salem, NC, USA Tissue engineered blood vessels (TEBV) represent a potential improvement over autologous or synthetic bypass grafts. Two key requirements of TEBV are (1) normal vascular cell function and (2) matching of mechanical properties of the scaffold with native arterial tissue. The second requirement can be satisfied through the use of decellularized porcine arterial segments. The purpose of this study was to fulfill these design requirements for TEBV. Methods: Carotid arterial segments were obtained from large pigs. Vessels were incubated in a decellularization solution for 5 days, freeze-dried, and sterilized with ethylene oxide. The stress-strain behavior, compliance, and burst pressure were tested followed by histological analysis. Bovine endothelial cells (EC) were seeded onto the inner wall of the decellularized vessel. Results: The porcine arterial segments maintained their size and shape after decellularization. Samples of decellularized vessel were processed for H&E and Movat staining. No porcine cellular components could be detected and only layers of collagen and laminin were observed. Scanning electron microscope images illustrated the structure and the porosity remained intact. Compliance results showed the pressure-diameter curve for the decellularized vessels was similar to the native porcine carotid artery. The diameter change was approximately 5% for native and 10% for decellularized scaffolds over physiologic pulse pressure. The stress-strain behavior of the decellularized vessels was comparable to the native artery. The burst pressure for the decellularized construct was measured at 1340 mmHg (10 times the systolic pressure) and similar native porcine arteries. An EC monolayer was achieved in the lumen after 1 week of preconditioning in a bioreactor that simulated physiologic flow conditions. Conclusion: The novelty in this work is that we can use these scaffolds to mimic native vessel mechanical properties. We have also seeded vascular endothelial cells onto this scaffold material and demonstrated functionality. 5617 We-Th, no. 12 (P63) A three dimensional matrix for guided mesenchymal stem cell and urothelial cell growth and differentiation C. Adel6w, ]7 Segura, J.A. Hubbell, P. Frey. Laboratory for Regenerative Medicine & Pharmacobiology, Ecole Polytechnique F6d#ral de Lausanne, Switzerland Tissue engineering aims to provide a temporary scaffold at the site of injury or disease that is able to support cell attachment and growth while synthesis of matrix proteins and reorganisation take place. Although relatively successful, bladder tissue engineering suffers from the formation of scar tissue partly due to the phenotypic switch of smooth muscle cells (SMC) from a quiescent contractile phenotype to a synthetic proliferative phenotype, known as myofibroblast. In the developing bladder SMCs originate from mesenchyme. However, the signalling mechanisms behind the differentiation of mesenchymal stem cells (MSC) to SMCs are largely unknown. We hypothesise that culturing human MSCs in enzymatically degradable PEG hydrogels modified with integrin binding peptides, and in co-culture with human urothelial cells (UCs) will offer some insight as to the required environment for their subsequent differentiation into quiescent SMCs. We have established protocols for human UC isolation, and characterization, and investigated co-culture conditions for MSCs and UCs. The optimal gel properties in terms of amounts of cell adhesion peptide, PEG and crosslinker content for the growth of cells embedded and/or seeded on top of the hydrogels has been examined using light and fluorescence microscopy, and cell organisation demonstrated by histology. MSCs and UCs were shown to attach, spread and grow for up to 4 weeks in culture within and on top of gels with optimised gel properties, corresponding to rather loose, elastic gels containing higher amounts of cell adhesion peptide. Moreover both cell types demonstrated degradation of the gel implying that both cell types excrete the MMPs required for gel degradation. The development of a simplified extracellular matrix scaffold, which can support the co-culture of MSCs and UC in three dimensions and can incorporate biochemical signals in a controlled manner, can contribute to the understanding of the underlying basic mechanisms of MSCs differentiation to SMCs and has implication to reduced scar tissue formation during bladder reconstruction.
Poster Presentations 7191 We-Th, no. 13 (P63) A strain-gradient model for capturing size-effects in biodegradable nanofibers J. Wang 1, R.ES. Han 2, B. Yuan 3. 1Department of Material Science, Fudan University, Shanghai, China, 2AAME Department, Peking University, Beijing, China and MIE Department, The University of Iowa, Iowa City, USA, 3Department of Engineering Science & Mechanics, Fudan University, Shanghai, China Nanofibers made from biodegradable synthetic polymers are often used to fabricate functional artificial tissues to serve a variety of usage, including as scaffolds to support seeded live cells. Thus, it is crucial that the mechanical performance of these fibers be accurately and adequately characterized. The recent experiments [1-3] from single-fiber bending tests revealed that the Young's moduli for different diameter fibers are size-dependent even though the fibers were fabricated via the same process. The variation in the Young's modulus can be attributed to the adoption of the classical elastic theory for interpreting the experimental data. Furthermore, the isotropic assumption popularly invoked in such a model may be questionable for biodegradable synthetic fibers. Due to the ultrafine-scale size and the intrinsic length scale parameters of the biomaterial, strain gradient effects are expected to play an important role in their deformation energies [4]. To handle and incorporate size-effects in biodegradable nanofibers, a strain gradient model is developed in this paper. Our model is formulated by taking into consideration strain gradient effects under anisotropic condition in the framework of thermodynamics. Parameters related to the biomaterial length scales are identified in the model and they get reduced to 3 when the centrosymmetric and isotropic assumptions are applied. As a demonstration, the model is employed to study mechanical torsion and pure bending behaviors of a single fiber. References [1] Tan E.RS., Ng S.Y., Lim C.T. Tensile testing of a single ultrafine polymeric fiber. Biomaterials 2005; 26: 1453-1456. [2] Tan E.ES., Lim C.T. Physical properties of a single polymeric nanofiber. Applied Physics Letters 2004; 84(9): 1603-1605. [3] Gu S.Y., Wu Q.L., Ren J. and Vancso G.J. Mechanical properties of a single electrospun fiber and its structures. Macromolecular Rapid Communications 2005; 26(9): 716-720. [4] Lam D.C.C., Yang F., Chong A.C.M., Wang J., Tong E Experiments and theory in strain gradient elasticity. J. Mech. Phys. Solids 2003; 51: 1477-1508. 5666 We-Th, no. 14 (P63) CFD model of mass transport with the microarchitecture of engineered cartilage during perfusion culture M. Cioffi 1, J. KiJffer2, S. Str6bel 3, G. Dubini 1, I. Martin 3, D. Wendt 3. 1Laboratory of Biological Structure Mechanics, Politecnico di Milano, Italy, 2Institute for Product and Production-Engineering, University of Applied Sciences Northwestern Switzerland, Switzerland, 3Institute for Surgical Research, University Hospital Basel, Switzerland Introduction: A key step to optimize bioreactor operating conditions for the engineering of functional articular cartilage is to quantify the hydrodynamic and mass transfer phenomena within the 3D construct. The aim of this study was to integrate experimental data with computational models, to determine oxygen concentrations within engineered cartilage during perfusion culture, to help optimize bioreactor flow parameters. A novel CFD model was developed, based on a microcomputed tomography (~tCT) reconstruction of a porous scaffold, to predict local oxygen levels within the scaffold's actual pore microarchitecture. Methodology: A ~tCT-based model of the scaffold (Polyactive foam; 1.2 mm cube) was meshed into 314,000 tetrahedrons. Flow fields and oxygen concentrations were determined with FLUENT. The model simulated fluid flow of culture medium through chondrocyte-seeded foams (8 mm diameter; 5 mm thick) at flow rates of 0.3 and 0.03ml/min, with inlet oxygen tensions of 5%. A first order oxygen consumption rate was used, calculated from oxygen measurements obtained during the culture of engineered cartilage within the perfusion bioreactor. Results and conclusions: CFD simulations were in agreement with simple analytical mass balances and experimental measurements, indicating average oxygen tensions at the scaffold outlet of 4.1% and 0.5% at 0.3 and 0.03 ml/min, respectively (difference <0.15% oxygen tension). However, CFD simulations could reveal more inhomogeneous oxygen profiles within the porous network, particularly at the lower flowrate. For instance, at 0.03ml/min, simulations predicted that oxygen concentrations would drop to anoxic levels (<1%) in particular regions at depths of only 1.9 mm from the scaffold surface, whereas the analytical solution predicted an oxygen tension greater than 2.0%. In conclusion, ~tCT-based models can be used to predict local species concentrations within the tortuous microstructure of engineered constructs, providing a sensitive tool to rationalize and predict experimental data and to aid in efficient bioreactor optimization.
Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
valve leaflets indicate a difference in mechanical properties between the belly and the commissures after four weeks of culturing. References [1] Mol et al. Ann. Biomed. Eng. 2005; 33(12): 1778-1788. [2] Cox et al. J. Biomech. Eng. 2006; in press. 7012 We-Th, no. 11 (P63) Acellular vascular scaffolds for tissue engineered blood vessels J.L. Berry 1, S.K. Yazdani 1, G. Amiel 2, J.J. Yoo 2, A.A. Atala 2, S. Soker 2. 1Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Wake Forest University, Winston-Salem, NC, USA, 2Wake Forest Institute of Regenerative Medicine, Medical Center Blvd., Winston-Salem, NC, USA Tissue engineered blood vessels (TEBV) represent a potential improvement over autologous or synthetic bypass grafts. Two key requirements of TEBV are (1) normal vascular cell function and (2) matching of mechanical properties of the scaffold with native arterial tissue. The second requirement can be satisfied through the use of decellularized porcine arterial segments. The purpose of this study was to fulfill these design requirements for TEBV. Methods: Carotid arterial segments were obtained from large pigs. Vessels were incubated in a decellularization solution for 5 days, freeze-dried, and sterilized with ethylene oxide. The stress-strain behavior, compliance, and burst pressure were tested followed by histological analysis. Bovine endothelial cells (EC) were seeded onto the inner wall of the decellularized vessel. Results: The porcine arterial segments maintained their size and shape after decellularization. Samples of decellularized vessel were processed for H&E and Movat staining. No porcine cellular components could be detected and only layers of collagen and laminin were observed. Scanning electron microscope images illustrated the structure and the porosity remained intact. Compliance results showed the pressure-diameter curve for the decellularized vessels was similar to the native porcine carotid artery. The diameter change was approximately 5% for native and 10% for decellularized scaffolds over physiologic pulse pressure. The stress-strain behavior of the decellularized vessels was comparable to the native artery. The burst pressure for the decellularized construct was measured at 1340 mmHg (10 times the systolic pressure) and similar native porcine arteries. An EC monolayer was achieved in the lumen after 1 week of preconditioning in a bioreactor that simulated physiologic flow conditions. Conclusion: The novelty in this work is that we can use these scaffolds to mimic native vessel mechanical properties. We have also seeded vascular endothelial cells onto this scaffold material and demonstrated functionality. 5617 We-Th, no. 12 (P63) A three dimensional matrix for guided mesenchymal stem cell and urothelial cell growth and differentiation C. Adel6w, ]7 Segura, J.A. Hubbell, P. Frey. Laboratory for Regenerative Medicine & Pharmacobiology, Ecole Polytechnique F6d#ral de Lausanne, Switzerland Tissue engineering aims to provide a temporary scaffold at the site of injury or disease that is able to support cell attachment and growth while synthesis of matrix proteins and reorganisation take place. Although relatively successful, bladder tissue engineering suffers from the formation of scar tissue partly due to the phenotypic switch of smooth muscle cells (SMC) from a quiescent contractile phenotype to a synthetic proliferative phenotype, known as myofibroblast. In the developing bladder SMCs originate from mesenchyme. However, the signalling mechanisms behind the differentiation of mesenchymal stem cells (MSC) to SMCs are largely unknown. We hypothesise that culturing human MSCs in enzymatically degradable PEG hydrogels modified with integrin binding peptides, and in co-culture with human urothelial cells (UCs) will offer some insight as to the required environment for their subsequent differentiation into quiescent SMCs. We have established protocols for human UC isolation, and characterization, and investigated co-culture conditions for MSCs and UCs. The optimal gel properties in terms of amounts of cell adhesion peptide, PEG and crosslinker content for the growth of cells embedded and/or seeded on top of the hydrogels has been examined using light and fluorescence microscopy, and cell organisation demonstrated by histology. MSCs and UCs were shown to attach, spread and grow for up to 4 weeks in culture within and on top of gels with optimised gel properties, corresponding to rather loose, elastic gels containing higher amounts of cell adhesion peptide. Moreover both cell types demonstrated degradation of the gel implying that both cell types excrete the MMPs required for gel degradation. The development of a simplified extracellular matrix scaffold, which can support the co-culture of MSCs and UC in three dimensions and can incorporate biochemical signals in a controlled manner, can contribute to the understanding of the underlying basic mechanisms of MSCs differentiation to SMCs and has implication to reduced scar tissue formation during bladder reconstruction.
Poster Presentations 7191 We-Th, no. 13 (P63) A strain-gradient model for capturing size-effects in biodegradable nanofibers J. Wang 1, R.ES. Han 2, B. Yuan 3. 1Department of Material Science, Fudan University, Shanghai, China, 2AAME Department, Peking University, Beijing, China and MIE Department, The University of Iowa, Iowa City, USA, 3Department of Engineering Science & Mechanics, Fudan University, Shanghai, China Nanofibers made from biodegradable synthetic polymers are often used to fabricate functional artificial tissues to serve a variety of usage, including as scaffolds to support seeded live cells. Thus, it is crucial that the mechanical performance of these fibers be accurately and adequately characterized. The recent experiments [1-3] from single-fiber bending tests revealed that the Young's moduli for different diameter fibers are size-dependent even though the fibers were fabricated via the same process. The variation in the Young's modulus can be attributed to the adoption of the classical elastic theory for interpreting the experimental data. Furthermore, the isotropic assumption popularly invoked in such a model may be questionable for biodegradable synthetic fibers. Due to the ultrafine-scale size and the intrinsic length scale parameters of the biomaterial, strain gradient effects are expected to play an important role in their deformation energies [4]. To handle and incorporate size-effects in biodegradable nanofibers, a strain gradient model is developed in this paper. Our model is formulated by taking into consideration strain gradient effects under anisotropic condition in the framework of thermodynamics. Parameters related to the biomaterial length scales are identified in the model and they get reduced to 3 when the centrosymmetric and isotropic assumptions are applied. As a demonstration, the model is employed to study mechanical torsion and pure bending behaviors of a single fiber. References [1] Tan E.RS., Ng S.Y., Lim C.T. Tensile testing of a single ultrafine polymeric fiber. Biomaterials 2005; 26: 1453-1456. [2] Tan E.ES., Lim C.T. Physical properties of a single polymeric nanofiber. Applied Physics Letters 2004; 84(9): 1603-1605. [3] Gu S.Y., Wu Q.L., Ren J. and Vancso G.J. Mechanical properties of a single electrospun fiber and its structures. Macromolecular Rapid Communications 2005; 26(9): 716-720. [4] Lam D.C.C., Yang F., Chong A.C.M., Wang J., Tong E Experiments and theory in strain gradient elasticity. J. Mech. Phys. Solids 2003; 51: 1477-1508. 5666 We-Th, no. 14 (P63) CFD model of mass transport with the microarchitecture of engineered cartilage during perfusion culture M. Cioffi 1, J. KiJffer2, S. Str6bel 3, G. Dubini 1, I. Martin 3, D. Wendt 3. 1Laboratory of Biological Structure Mechanics, Politecnico di Milano, Italy, 2Institute for Product and Production-Engineering, University of Applied Sciences Northwestern Switzerland, Switzerland, 3Institute for Surgical Research, University Hospital Basel, Switzerland Introduction: A key step to optimize bioreactor operating conditions for the engineering of functional articular cartilage is to quantify the hydrodynamic and mass transfer phenomena within the 3D construct. The aim of this study was to integrate experimental data with computational models, to determine oxygen concentrations within engineered cartilage during perfusion culture, to help optimize bioreactor flow parameters. A novel CFD model was developed, based on a microcomputed tomography (~tCT) reconstruction of a porous scaffold, to predict local oxygen levels within the scaffold's actual pore microarchitecture. Methodology: A ~tCT-based model of the scaffold (Polyactive foam; 1.2 mm cube) was meshed into 314,000 tetrahedrons. Flow fields and oxygen concentrations were determined with FLUENT. The model simulated fluid flow of culture medium through chondrocyte-seeded foams (8 mm diameter; 5 mm thick) at flow rates of 0.3 and 0.03ml/min, with inlet oxygen tensions of 5%. A first order oxygen consumption rate was used, calculated from oxygen measurements obtained during the culture of engineered cartilage within the perfusion bioreactor. Results and conclusions: CFD simulations were in agreement with simple analytical mass balances and experimental measurements, indicating average oxygen tensions at the scaffold outlet of 4.1% and 0.5% at 0.3 and 0.03 ml/min, respectively (difference <0.15% oxygen tension). However, CFD simulations could reveal more inhomogeneous oxygen profiles within the porous network, particularly at the lower flowrate. For instance, at 0.03ml/min, simulations predicted that oxygen concentrations would drop to anoxic levels (<1%) in particular regions at depths of only 1.9 mm from the scaffold surface, whereas the analytical solution predicted an oxygen tension greater than 2.0%. In conclusion, ~tCT-based models can be used to predict local species concentrations within the tortuous microstructure of engineered constructs, providing a sensitive tool to rationalize and predict experimental data and to aid in efficient bioreactor optimization.