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Low intensity pulsed ultrasound does not stimulate cartilage matrix synthesis in 3d agarose constructs
Track 9. Tissue Engineering 6, were encapsulated in 1.2% alginate bead as low density culture (LDC, 0.5 105/bead) or as high density culture (HDC, 1.5 105/bead). After 28 days culture, these static cultures were exposed to mechanical stimulation with agitation on a gyroscopic rocker for 48 hours. Cell number was determined using the trypan blue dye exclusion method. Cell viability was investigated using vibrant/apoptosis assay Kit. The content of sulfated glycosaminoglycan (sGAG) in the beads was measured by dimethtlmethylene blue dye. The expression of chondrocytes hallmark collagen II was revealed by immunofluorescence. At the end of 28-days culture, chondrocytes in the two cultures maintained round in shape and showed positive expression of collagen II, demonstrating that chondrocytes resulting from these systems regain their phenotype. The cell number in LDC and in HDC increased to 1.05 105/bead and 2.6 105/bead respectively, suggesting that the cells in LDC proliferate more quickly than those in HDC. Cell viability of LDC (91.1%) was higher than that of the HDC (86.3%). Compared with static culture, 48 h mechanical stimulation did not affect cell proliferation or cell viability, but induced noticeable increase in both sGAG content and collagen II expression. However, there were no considerable differences between the LDC and HDC. These results showed that chondrocytes in LDC have the same cell reactions to the mechanical loading as those in HDC, but have higher proliferation rate and viability than those in HDC. Our findings suggested that the LDC model is more appropriate than HDC model for the research purpose and clinical applications. Acknowledgement: This work was supported in part by the fellowship from France embassy and Lorraine region. 6880 Th, 09:00-09:15 (P38) Mechanical properties of synovial cell-seeded 3-D constructs for cartilage regeneration: Effects of cyclic compressive stress D. Katakai 1, H. Fujie 1, Y. Muroi 2, K. Nakata 2. 1Biomechanics Laboratory, Kogakuin University, Tokyo, Japan, 2Department of Orthopaedic Surgery, Osaka University Medical School, Osaka, Japan Introduction: Previous reports indicated that stress application to biological cells had the ability to induce cell proliferation, differentiation, and extracellular production 12) . . The objective of the study was to determine the effect of stress application on the compressive and frictional behaviors of synovial cell-seeded 3-D constructs. Methods: Human synovium-derived cells were seeded into a collagen scaffold to build 3-D constructs. In groups I and II, the constructs were cultured for 5 days (group I) and 10 days (group II) in DMEM in an incubator without mechanical stress application. In group III, the constructs were initially cultured without mechanical stress application for 5 days, and thereafter cultured with cyclic compressive stress for 1 hour a day for 5 days. After the culture, the constructs were subjected to a quasi-static unconfined compression test (4 and 100 ?~trn/s of rate) as well as a cyclic friction test (20 mm/s of speed). Results and Discussion: At 4 ~tm/s of compression rate, the tangent modulus of the constructs at 5% strain were 187kPa in control group, and were decreased to 143, 136 and 28kPa in groups I, II, and III, respectively. A significant decrease of the modulus was observed in group III as compared with groups II, although the difference disappeared at 100 ~tm/s of rate. In the friction test, the coefficient of friction of the constructs against a glass plate was significantly lower in all groups than in control. These results suggested that the stress application decreased the elasticity of the scaffold and provided the constructs with viscoelastic properties. References [1] Mauck R., et al. Annales of Biomedical Engineering 2002. [2] Park J., et al. Biotechnology and Bioengineering 2004. 5638 Th, 09:15-09:30 (P38) Low intensity pulsed ultrasound does not stimulate cartilage matrix synthesis in 3d agarose constructs N. Vaughan, D. Bader, M. Knight. Medical Engineering Division, Dept. ef Engineering, Queen Mary University of London, London, UK Low Intensity Pulsed Ultrasound (LIPUS) has been proposed as a mechanism for stimulating articular cartilage repair, either in vive, or in vitro as part of a conditioning strategy for tissue engineering. Previous studies using chondrocytes in monolayer culture and pellet culture, have suggested that LIPUS may stimulate glycosaminoglycan (GAG) synthesis [1,2]. The present study tested the hypothesis that LIPUS stimulates GAG synthesis via a calcium signalling pathway in chondrocytes seeded in 3D agarose constructs. Bovine articular chondrocytes were isolated by enzyme digestion, seeded in 3% agarose gel and cast to a depth of 3 mm in each well of a 6 well plate. One plate was subjected to LIPUS once a day at 30 mW.cm 2, while a control plate remained unstimulated. At days 1, 2, 5, 9, 12, 16 and 20, six core specimens, 5 mm in diameter, were removed from each plate for analysis of GAG content using the DMB assay. In a separate study, 5 5 5mm chondrocyte-agarose constructs at day 1 of culture were labelled with the calcium indicator, Fluo4 AM
9.3. Ligament and Tendon Tissue Engineering
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and mounted in a test rig on the stage of an inverted confocal microscope. One group of constructs were subjected to LIPUS, between 30 and 200 mW/cm 2 over a 20 minute period, while a control group remained unstimulated. Although chondrocytes elaborated GAG montonically with time, differences between the GAG content for LIPUS-stimulated and controls were not statistically significant (p >0.05 at all time points). Furthermore LIPUS had no significant effect on intracellular calcium signalling, with no increase in percentage number of cells exhibiting Ca 2+ transients. These results suggest that the nature of the model system is critical if LIPUS is to be used to stimulate cartilage matrix as part of a tissue engineering repair strategy. References [1] Parvizi J., et al. J Orthop Res. 1999; 17: 488-494. [2] Mukai et al. J. Ultrasound in Medicine and Biol. 2005; 31: 1713-1721. 6540 Th, 09:30-09:45 (P38) Multiscale modeling of diffusion hindrance in tissue engineered cartilage G.E. Chao, C.W.J. Oomens, C.C. van Donkelaar, F.P.T. Baaijens. Materials Technology Group, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands Macroscopic mechanical properties of tissue engineered constructs depend on their composition, which evolves in time following synthesis, transport, binding and degradation of biomolecules. A thorough understanding of these phenomena is relevant to improve the development of artificial tissues during culturing. From a purely mechanical point of view, diffusion plays a fundamental role in the transport of newly synthesized material across the tissue. Diffusion mechanisms are highly inhomogeneous in developing biological tissues, in which large aggregating matrix molecules such as collagen and GAGs are continually synthesized by individuals cells. In these systems, diffusivity decreases due to an increase in the tortuosity of the extracellular matrix. In this work we address the effects of diffusion hindrance on global mechanical properties of tissue engineered cartilage. We also study the influence of the continuous accumulation of bound material and the developing microgeometry of the tissue on the diffusion of aggregating molecules. The study is based on a continuous model for diffusion, binding and a posteriori degradation of matrix components. Diffusion hindrance is modeled in terms of a random walk approximation. The governing equations are solved using finite element methods at tissue and RVE scales. The numerical results show a significant effect of diffusion hindrance on the concentration distribution of immobilized GAG in tissue engineered cartilage as well as on the mechanical properties of the construct. Diffusion hindrance causes a higher accumulation of GAG around the cells, hampering the diffusion of newly synthesized material. On a macroscopic scale, the aggregate modulus and the permeability are sensitive to the distribution of the extracellular matrix. The enhanced localization of the extracellular matrix contributes to a softening of the construct, which becomes apparent from fifteen days of culture.
9.3. Ligament and Tendon Tissue Engineering 5794 Mo, 15:15-15:30 (P10) Mechanical characterisation of rabbit Achilles tendon for functional tissue engineering C. Kahn, C. Vaquette, S. Slimani, R. Rahouadj, X. Wang. Group ef Cell and Tissue Engineering, LEMTA UMR 7563 CNRS, Vandoeuvre-les-Nancy, Fran ce In functional tissue engineering, the knowledge of the mechanical properties of native tissues is essential for the design of scaffolds and the evaluation of reconstructed tissues. In this study, we characterised the tensile properties of Achilles tendons of New-Zealand rabbit (N=5) with the help of a traction machine: Adamel Lhomargy D'~22 (MTS). Our mechanical tests consisted in series of traction-relaxation of the Achilles tendons which were maintained in a saline fluid at 37°C. Each tendon was tested within 2 hours after euthanasia of the animal. Experimental results show that the stress-strain curves of healthy tendons had a typical behaviour with three zones: a 'toe region' (~<3%), a linear elastic zone (3% <~ <9%) with a relaxed elastic modulus at the order of 25 MPa and a zone of damage (~ > 9%). Moreover, relaxation tests showed that the tendons had a viscoelastic behaviour with a relaxation time of about 20 minutes. Healthy tendons were compared to defected tendons created in the rabbit 14 weeks before the mechanical tests. The operation consisted in a 1-cm-long gap defect on one of the three bundles of the Achilles tendon into which a resorbable scaffold was implanted. Thus the defected tendon was composed of two intact bundles and a reconstructed tissue in place of the defect. The comparison showed that the strength of the defected tendons was lower than the healthy one. However, the normalised stress-strain curves for the healthy tendons were superimposed with those of the defect tendons. Based on these experimental results we are working on a theoretical model to approach the Achilles tendon mechanical properties.
9.3. Ligament and Tendon Tissue Engineering
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and mounted in a test rig on the stage of an inverted confocal microscope. One group of constructs were subjected to LIPUS, between 30 and 200 mW/cm 2 over a 20 minute period, while a control group remained unstimulated. Although chondrocytes elaborated GAG montonically with time, differences between the GAG content for LIPUS-stimulated and controls were not statistically significant (p >0.05 at all time points). Furthermore LIPUS had no significant effect on intracellular calcium signalling, with no increase in percentage number of cells exhibiting Ca 2+ transients. These results suggest that the nature of the model system is critical if LIPUS is to be used to stimulate cartilage matrix as part of a tissue engineering repair strategy. References [1] Parvizi J., et al. J Orthop Res. 1999; 17: 488-494. [2] Mukai et al. J. Ultrasound in Medicine and Biol. 2005; 31: 1713-1721. 6540 Th, 09:30-09:45 (P38) Multiscale modeling of diffusion hindrance in tissue engineered cartilage G.E. Chao, C.W.J. Oomens, C.C. van Donkelaar, F.P.T. Baaijens. Materials Technology Group, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands Macroscopic mechanical properties of tissue engineered constructs depend on their composition, which evolves in time following synthesis, transport, binding and degradation of biomolecules. A thorough understanding of these phenomena is relevant to improve the development of artificial tissues during culturing. From a purely mechanical point of view, diffusion plays a fundamental role in the transport of newly synthesized material across the tissue. Diffusion mechanisms are highly inhomogeneous in developing biological tissues, in which large aggregating matrix molecules such as collagen and GAGs are continually synthesized by individuals cells. In these systems, diffusivity decreases due to an increase in the tortuosity of the extracellular matrix. In this work we address the effects of diffusion hindrance on global mechanical properties of tissue engineered cartilage. We also study the influence of the continuous accumulation of bound material and the developing microgeometry of the tissue on the diffusion of aggregating molecules. The study is based on a continuous model for diffusion, binding and a posteriori degradation of matrix components. Diffusion hindrance is modeled in terms of a random walk approximation. The governing equations are solved using finite element methods at tissue and RVE scales. The numerical results show a significant effect of diffusion hindrance on the concentration distribution of immobilized GAG in tissue engineered cartilage as well as on the mechanical properties of the construct. Diffusion hindrance causes a higher accumulation of GAG around the cells, hampering the diffusion of newly synthesized material. On a macroscopic scale, the aggregate modulus and the permeability are sensitive to the distribution of the extracellular matrix. The enhanced localization of the extracellular matrix contributes to a softening of the construct, which becomes apparent from fifteen days of culture.
9.3. Ligament and Tendon Tissue Engineering 5794 Mo, 15:15-15:30 (P10) Mechanical characterisation of rabbit Achilles tendon for functional tissue engineering C. Kahn, C. Vaquette, S. Slimani, R. Rahouadj, X. Wang. Group ef Cell and Tissue Engineering, LEMTA UMR 7563 CNRS, Vandoeuvre-les-Nancy, Fran ce In functional tissue engineering, the knowledge of the mechanical properties of native tissues is essential for the design of scaffolds and the evaluation of reconstructed tissues. In this study, we characterised the tensile properties of Achilles tendons of New-Zealand rabbit (N=5) with the help of a traction machine: Adamel Lhomargy D'~22 (MTS). Our mechanical tests consisted in series of traction-relaxation of the Achilles tendons which were maintained in a saline fluid at 37°C. Each tendon was tested within 2 hours after euthanasia of the animal. Experimental results show that the stress-strain curves of healthy tendons had a typical behaviour with three zones: a 'toe region' (~<3%), a linear elastic zone (3% <~ <9%) with a relaxed elastic modulus at the order of 25 MPa and a zone of damage (~ > 9%). Moreover, relaxation tests showed that the tendons had a viscoelastic behaviour with a relaxation time of about 20 minutes. Healthy tendons were compared to defected tendons created in the rabbit 14 weeks before the mechanical tests. The operation consisted in a 1-cm-long gap defect on one of the three bundles of the Achilles tendon into which a resorbable scaffold was implanted. Thus the defected tendon was composed of two intact bundles and a reconstructed tissue in place of the defect. The comparison showed that the strength of the defected tendons was lower than the healthy one. However, the normalised stress-strain curves for the healthy tendons were superimposed with those of the defect tendons. Based on these experimental results we are working on a theoretical model to approach the Achilles tendon mechanical properties.