- Email: [email protected]
Influence of cell density on dedifferentiated chondrocytes in alginate bead culture under mechanical stimulation
$220
Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
(Martin et al., 1998), thus occluding pores and reducing nutrient diffusion to the inner tissue phase. It is essential that a scaffold possess suitable interconnectivity and pore size in order to maintain diffusion during the initial stages of cell proliferation and osteoid formation prior to vascularisation. To address these issues we have developed a hydroxyapatite scaffold with a tri-modal pore distribution in order to provide non-competing domains for (1) cell seeding and diffusion, (2) cell proliferation and mineral formation, (3) vascularisation. A ceramic suspension is subjected to a controlled freeze-drying cycle, which results in a micro(<5 ~tm) and mesoporous (90-100 ~tm) interconnected pore structure. Unidirectional macrochannels (500 ~tm) with a spacing of 500 ~tm are introduced into the porous structure through CNC-machining. Scaffold discs of 5 mm diameter and 5 mm height are then sintered to 1500°C. Total porosity of these saffolds was calculated to be 84%. The large unidirectional macropores provide three functions. Firstly to aid in uniform cell seeding; secondly to provide a direct diffusion route for oxygen and nutrients to the scaffold interior to promote matrix formation and thirdly to permit vascularisation once implanted in vive. The mesopores provide a second diffusion route as well as a providing a domain for cell proliferation and migration. Finally the microporosity provides a third albeit limited diffusion route and the volume for pre-saturating the scaffold with biochemical agents. Finite element models are being employed to determine the optimal pore configuration. References
Ishaug-Riley et al. (1998). Biomaterials 19: 1405-1412. Martin et al. (1998). J Orthop Res. 16: 181-189. 7401 We, 12:15-12:30 (P31) Modeling o f large bone implant culture in a perfusion bioreactor J. Pierre 1, B. David 2, K. Oudina 2, H. Petite 2, C. Ribreau 1, C. Oddou 1. 1B2OA, Facult~ des Sciences, Universit6 Paris 12, Cr6teil, France, 2B20A, Universit~ Paris 7, UMR CNRS 7052, Paris, France Successful bone cells culture in large implants remains a challenge for biologists and requires a strict control of the physico-chemical and mechanical environment. The goal of this study was to describe the limits of a perfusion bioreactor for bone cells culture with fibrous and porous large implants (2.5 cm in length, volume around some cubic centimetres, fibers diameter of 250 ~tm, porosity around 60%). A two dimensional mathematical model, based upon time dependent mass and momentum transport through the implant, during the first week of culture, was proposed and numerically solved. The modeling challenge resides in the consideration of non-linear terms, in boundary conditions of the convection diffusion equation, imposed by both oxygen consumption and cell proliferation. A simple 2D periodic geometry of the implant architecture was assumed. Numerical solutions were obtained with a commercial code (Femlab ® 3.1; Comsol, Stockholm, Sweden). For a perfusion velocity of the order of 10-4 m s -1 , fluid dynamics equations at low Reynolds number were solved and revealed a consequent non uniformity in the shear stresses applied to the cells (in the range of 10-6 Pa to 5.10 -3 Pa). Coupled equations characterizing the convection-diffusion processes of the oxygen and time evolution of the cell density were then solved. Oxygen repartition in space within the implant was then obtained and an oxygen penetration length within the porous medium was defined and evaluated around 10 mm after first 6 days of culture. Furthermore, was then performed a parametric analysis concerning the influence, upon oxygen penetration length, of the experimental parameters such as upstream oxygen tension, perfusion velocity and oxygen consumption rate by individual cells. Such a study has thus shown that numerical simulation turns out to be a powerful complementary tool for the designing of performing bioreactors and evaluation of the associated culture conditions, particularly for both oxygen concentration and shear stresses distribution. Acknowledgements: We are grateful for thesis financial support of J. Pierre by DGA-CNRS and would like to thank Dr. Dietmar Hutmacher for providing PCL scaffolds.
Oral Presentations Chondrocytes were isolated from metacarpal-phalangeal joint of steers using a sequential enzyme digestion process, and seeded in 4% ultra-low gelling temperature agarose (type IX). Resultant cylindrical chondrocyte-agarose constructs, with a diameter of 3mm and a height of 2.5mm, were cultured in sterile culture medium (DMEM + 20% FBS) within a humidified tissue culture incubatorcontrolled at 37°C and 5% CO2.15% compressive strain was applied to constructs cyclically at 1 Hz for 6 hours a day during their culture period. After culture periods of 1, 8, 15 and 22 days, their mechanical properties, tangent modulus and equilibrium modulus, were evaluated by the unconfined compression test. At the same time, the proteoglycan content of constructs and three dimensional morphology of elaborated collagen type II network were examined by the dimethylmethylene blue assay and the confocal laser scanning microscopy (CLSM) observation with fluorescent antibody staining method respectively. As results, the proteoglycan content and the tangent modulus of the cyclically compressed constructs became significantly higher than that of the control after 15 days culture. Although the equilibrium modulus was also increased by the cyclic compression, the difference between the compressed group and the control group was not significant. On the other hand, the apparent effect on the construction of the three dimensional collagen networks was not observed. These results indicated that the cyclic compression could stimulate the proteoglycan synthesis of seeded chondrocytes and increase the tangent modulus of constructs; however it had a little effect on the development of collagen network and could not increase the equilibrium modulus. 5662 Th, 08:30-08:45 (P38) Dynamic c o m p r e s s i o n o f IL-16 stimulated chondrocyte-agarose constructs influences the release of. NO and PGE 2 via MAPK pathways T. Chowdhury, D. Lee, D. Bader. Medical Engineering Division, Dept of Engineering, Queen Mary University of London, London, UK The Interleukin-ll~ (IL-11~) induced release of both nitric oxide (-NO) and prostaglandin E2 (PGE2) in articular chondrocytes can be reversed with dynamic compression [1]. It has also been shown that IL-11~ can activate three members of the mitogen-activated protein kinase (MAPK) family, namely c-Jun N-terminal kinase (JNK), protein 38 (p38) and extracellular-signal regulated kinase (ERK) [2]. Suc complex processes may involve phosphorylation of p38 MAPK, JNK2, 3 and activation of transcription factors, NF6B and AP-I. The current study examines whether inhibition of these pathways can influence. NO and PGE2 release in IL-11~stimulated bovine chondrocytes cultured in agarose constructs and subjected to dynamic compression. Bovine chondrocytes seeded in agarose gel were cultured under free-swelling conditions, for 48 hrs in medium containing 0 or 10 ng.m1-1 IL-11~,supplemented with a variety of inhibitors for p38 MAPK, JNK-1, NFkB activation and AP-I. Separate constructs were then subjected to 15% dynamic compression at 1 Hz for 48 hrs under similar test conditions. Dynamic compression counteracted the IL-11~ induced nitrite release. Compression-induced inhibition of nitrite release was abolished by the inhibitors for both MAPKs, the transcription factor NFkB, but only partially reduced for JNK I1. The application of dynamic compression resulted in the strain-induced inhibition of PGE2 release in IL-11~ stimulated constructs. This effect was abolished with some of the inhibitors. These results suggest the involvement of MAPKs and transcription factors in the IL-11~ induced release of. NO and PGE2. Further work will determine the sequential activation of the signalling cascades associated with mechanical loading and IL-11~. Such an approach can provide important information for the pharmacological and biophysical treatment of degenerative joint disease as well as the biomechnical control of tissue engineered cartilage. This project was supported by the Wellcome Trust UK. References
[1] Chowdhury T.T., et al. Biochem Biophys Res Commun. 2001; 285: 1168-74. [2] Chowdhury T.T., et al. Osteoarthritis Cartilage 2003; 11: 688-96.
9.2. Cartilage - M e n i s c u s T i s s u e E n g i n e e r i n g
7637
5650
Y. Sawae 1, A. Hanaki 2, E. Suzuki 2, T. Murakami 1. 1Faculty of Engineering, Kyushu University, Fukuoka, Japan, 2Graduate School of Engineering, Kyushu University, Fukueka, Japan
bead culture under mechanical stimulation '~ Wang 1,2, N. de Isla 1, C. Gigant-Huselstein 1, S. Muller, B.H. Wang 2, J.E Stoltz 1. 1M6canique et Ing~nierie Cellulaire et Tissulaire, UMR-CNRSINPL 7563 LEMTA et IFR111, Facult6 de M~decine, Vandoeuvre-les-Nancy, France, 2Department of Biochemistry and Molecular Biology, School of Medicine, Wuhan University, Wuhan, China
On approach for cartilage tissue engineering, freshly isolated chondrocytes are cultured in 3D scaffold and exposed to some kinds of mechanical loading to stimulate their biosynthesis. In this study, cyclic compression was applied to the model system of tissue engineered cartilage, chondrocyte-agarose construct, and effects of the mechanical loading on the extra-cellular matrix synthesis and mechanical properties of cultured constructs were examined.
Chondrocytes in three-dimensional culture like alginate hydrogel was a useful model for research of the mechanism of chondrocytes differentiation and rededifferentiation. In this study, we investigated the effect of cell density on cell proliferation, viability and biosynthetic activity of dedifferentiated chondrocytes encapsulated in alginate gel under mechanical stimulation. Rat chondrocytes undergoing dedifferentiation upon serial monolayer culture up to Passage
Th, 08:15-08:30 (P38)
Effects o f cyclic compression on ECM synthesis and mechanical property in cultured chondrocyte-agarose construct
Th, 08:45-09:00 (P38)
Influence o f cell density on dedifferentiated chondrocytes in alginate
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
$221
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.
Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
(Martin et al., 1998), thus occluding pores and reducing nutrient diffusion to the inner tissue phase. It is essential that a scaffold possess suitable interconnectivity and pore size in order to maintain diffusion during the initial stages of cell proliferation and osteoid formation prior to vascularisation. To address these issues we have developed a hydroxyapatite scaffold with a tri-modal pore distribution in order to provide non-competing domains for (1) cell seeding and diffusion, (2) cell proliferation and mineral formation, (3) vascularisation. A ceramic suspension is subjected to a controlled freeze-drying cycle, which results in a micro(<5 ~tm) and mesoporous (90-100 ~tm) interconnected pore structure. Unidirectional macrochannels (500 ~tm) with a spacing of 500 ~tm are introduced into the porous structure through CNC-machining. Scaffold discs of 5 mm diameter and 5 mm height are then sintered to 1500°C. Total porosity of these saffolds was calculated to be 84%. The large unidirectional macropores provide three functions. Firstly to aid in uniform cell seeding; secondly to provide a direct diffusion route for oxygen and nutrients to the scaffold interior to promote matrix formation and thirdly to permit vascularisation once implanted in vive. The mesopores provide a second diffusion route as well as a providing a domain for cell proliferation and migration. Finally the microporosity provides a third albeit limited diffusion route and the volume for pre-saturating the scaffold with biochemical agents. Finite element models are being employed to determine the optimal pore configuration. References
Ishaug-Riley et al. (1998). Biomaterials 19: 1405-1412. Martin et al. (1998). J Orthop Res. 16: 181-189. 7401 We, 12:15-12:30 (P31) Modeling o f large bone implant culture in a perfusion bioreactor J. Pierre 1, B. David 2, K. Oudina 2, H. Petite 2, C. Ribreau 1, C. Oddou 1. 1B2OA, Facult~ des Sciences, Universit6 Paris 12, Cr6teil, France, 2B20A, Universit~ Paris 7, UMR CNRS 7052, Paris, France Successful bone cells culture in large implants remains a challenge for biologists and requires a strict control of the physico-chemical and mechanical environment. The goal of this study was to describe the limits of a perfusion bioreactor for bone cells culture with fibrous and porous large implants (2.5 cm in length, volume around some cubic centimetres, fibers diameter of 250 ~tm, porosity around 60%). A two dimensional mathematical model, based upon time dependent mass and momentum transport through the implant, during the first week of culture, was proposed and numerically solved. The modeling challenge resides in the consideration of non-linear terms, in boundary conditions of the convection diffusion equation, imposed by both oxygen consumption and cell proliferation. A simple 2D periodic geometry of the implant architecture was assumed. Numerical solutions were obtained with a commercial code (Femlab ® 3.1; Comsol, Stockholm, Sweden). For a perfusion velocity of the order of 10-4 m s -1 , fluid dynamics equations at low Reynolds number were solved and revealed a consequent non uniformity in the shear stresses applied to the cells (in the range of 10-6 Pa to 5.10 -3 Pa). Coupled equations characterizing the convection-diffusion processes of the oxygen and time evolution of the cell density were then solved. Oxygen repartition in space within the implant was then obtained and an oxygen penetration length within the porous medium was defined and evaluated around 10 mm after first 6 days of culture. Furthermore, was then performed a parametric analysis concerning the influence, upon oxygen penetration length, of the experimental parameters such as upstream oxygen tension, perfusion velocity and oxygen consumption rate by individual cells. Such a study has thus shown that numerical simulation turns out to be a powerful complementary tool for the designing of performing bioreactors and evaluation of the associated culture conditions, particularly for both oxygen concentration and shear stresses distribution. Acknowledgements: We are grateful for thesis financial support of J. Pierre by DGA-CNRS and would like to thank Dr. Dietmar Hutmacher for providing PCL scaffolds.
Oral Presentations Chondrocytes were isolated from metacarpal-phalangeal joint of steers using a sequential enzyme digestion process, and seeded in 4% ultra-low gelling temperature agarose (type IX). Resultant cylindrical chondrocyte-agarose constructs, with a diameter of 3mm and a height of 2.5mm, were cultured in sterile culture medium (DMEM + 20% FBS) within a humidified tissue culture incubatorcontrolled at 37°C and 5% CO2.15% compressive strain was applied to constructs cyclically at 1 Hz for 6 hours a day during their culture period. After culture periods of 1, 8, 15 and 22 days, their mechanical properties, tangent modulus and equilibrium modulus, were evaluated by the unconfined compression test. At the same time, the proteoglycan content of constructs and three dimensional morphology of elaborated collagen type II network were examined by the dimethylmethylene blue assay and the confocal laser scanning microscopy (CLSM) observation with fluorescent antibody staining method respectively. As results, the proteoglycan content and the tangent modulus of the cyclically compressed constructs became significantly higher than that of the control after 15 days culture. Although the equilibrium modulus was also increased by the cyclic compression, the difference between the compressed group and the control group was not significant. On the other hand, the apparent effect on the construction of the three dimensional collagen networks was not observed. These results indicated that the cyclic compression could stimulate the proteoglycan synthesis of seeded chondrocytes and increase the tangent modulus of constructs; however it had a little effect on the development of collagen network and could not increase the equilibrium modulus. 5662 Th, 08:30-08:45 (P38) Dynamic c o m p r e s s i o n o f IL-16 stimulated chondrocyte-agarose constructs influences the release of. NO and PGE 2 via MAPK pathways T. Chowdhury, D. Lee, D. Bader. Medical Engineering Division, Dept of Engineering, Queen Mary University of London, London, UK The Interleukin-ll~ (IL-11~) induced release of both nitric oxide (-NO) and prostaglandin E2 (PGE2) in articular chondrocytes can be reversed with dynamic compression [1]. It has also been shown that IL-11~ can activate three members of the mitogen-activated protein kinase (MAPK) family, namely c-Jun N-terminal kinase (JNK), protein 38 (p38) and extracellular-signal regulated kinase (ERK) [2]. Suc complex processes may involve phosphorylation of p38 MAPK, JNK2, 3 and activation of transcription factors, NF6B and AP-I. The current study examines whether inhibition of these pathways can influence. NO and PGE2 release in IL-11~stimulated bovine chondrocytes cultured in agarose constructs and subjected to dynamic compression. Bovine chondrocytes seeded in agarose gel were cultured under free-swelling conditions, for 48 hrs in medium containing 0 or 10 ng.m1-1 IL-11~,supplemented with a variety of inhibitors for p38 MAPK, JNK-1, NFkB activation and AP-I. Separate constructs were then subjected to 15% dynamic compression at 1 Hz for 48 hrs under similar test conditions. Dynamic compression counteracted the IL-11~ induced nitrite release. Compression-induced inhibition of nitrite release was abolished by the inhibitors for both MAPKs, the transcription factor NFkB, but only partially reduced for JNK I1. The application of dynamic compression resulted in the strain-induced inhibition of PGE2 release in IL-11~ stimulated constructs. This effect was abolished with some of the inhibitors. These results suggest the involvement of MAPKs and transcription factors in the IL-11~ induced release of. NO and PGE2. Further work will determine the sequential activation of the signalling cascades associated with mechanical loading and IL-11~. Such an approach can provide important information for the pharmacological and biophysical treatment of degenerative joint disease as well as the biomechnical control of tissue engineered cartilage. This project was supported by the Wellcome Trust UK. References
[1] Chowdhury T.T., et al. Biochem Biophys Res Commun. 2001; 285: 1168-74. [2] Chowdhury T.T., et al. Osteoarthritis Cartilage 2003; 11: 688-96.
9.2. Cartilage - M e n i s c u s T i s s u e E n g i n e e r i n g
7637
5650
Y. Sawae 1, A. Hanaki 2, E. Suzuki 2, T. Murakami 1. 1Faculty of Engineering, Kyushu University, Fukuoka, Japan, 2Graduate School of Engineering, Kyushu University, Fukueka, Japan
bead culture under mechanical stimulation '~ Wang 1,2, N. de Isla 1, C. Gigant-Huselstein 1, S. Muller, B.H. Wang 2, J.E Stoltz 1. 1M6canique et Ing~nierie Cellulaire et Tissulaire, UMR-CNRSINPL 7563 LEMTA et IFR111, Facult6 de M~decine, Vandoeuvre-les-Nancy, France, 2Department of Biochemistry and Molecular Biology, School of Medicine, Wuhan University, Wuhan, China
On approach for cartilage tissue engineering, freshly isolated chondrocytes are cultured in 3D scaffold and exposed to some kinds of mechanical loading to stimulate their biosynthesis. In this study, cyclic compression was applied to the model system of tissue engineered cartilage, chondrocyte-agarose construct, and effects of the mechanical loading on the extra-cellular matrix synthesis and mechanical properties of cultured constructs were examined.
Chondrocytes in three-dimensional culture like alginate hydrogel was a useful model for research of the mechanism of chondrocytes differentiation and rededifferentiation. In this study, we investigated the effect of cell density on cell proliferation, viability and biosynthetic activity of dedifferentiated chondrocytes encapsulated in alginate gel under mechanical stimulation. Rat chondrocytes undergoing dedifferentiation upon serial monolayer culture up to Passage
Th, 08:15-08:30 (P38)
Effects o f cyclic compression on ECM synthesis and mechanical property in cultured chondrocyte-agarose construct
Th, 08:45-09:00 (P38)
Influence o f cell density on dedifferentiated chondrocytes in alginate
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
$221
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.