- Email: [email protected]
3D-printed scaffolds for the cultivation of mesenchymal stem cells
6th IFAC Conference on Management and Control of Production and Logistics The International Federation of Automatic Control September 11-13, 2013. Fortaleza, Brazil
3D-printed scaffolds for the cultivation of mesenchymal stem cells Daniela Steffens*, Rodrigo A. Rezende**, Bruna Santi***, Frederico D. A. de S. Pereira**, Paulo Inforçatti Neto, Jorge V. L. da Silva**, Patricia Pranke**** * Post-graduation program of Biological Science – Phisiology; Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brazil (Tel:+5551 3308-5257; e-mail: [email protected]) ** Division of 3D Technologies, Center for Information Technology Renato Archer (CTI), Campinas, SP, Brazil (Tel:+5519 3746-6203; e-mail: [email protected]) *** Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brazil (Tel:+5551 3308-5257; e-mail: [email protected]) **** Post-graduation program of Biological Science – Phisiology; Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul, UFRGS; Stem Cell Research Institute, Porto Alegre, RS, Brazil (Tel: +5551 3308-5275; e-mail: [email protected])
Abstract: Tissue engineering is a tool which is currently under a great deal of investigation for the development and/or restoration of tissue and organs; it combines cell therapy and biomaterials for this aim. Rapid prototyping is a versatile technology which constructs polymer based-scaffolds with strict control of its architecture. The aim of this work, therefore, has been to investigate the interaction between mesenchymal stem cells with the biomaterials obtained by rapid prototyping. Scanning electron microscopy, confocal microscopy and biological assays were performed to analyze this interaction. As results, the number of viable cells attached to the scaffolds was lower when compared to the control group; however, it was possible to observe cells in the scaffolds since day 1 of analysis, with regions of confluence after 21 days of seeding. It could be concluded, therefore, that these biomaterials are interesting for use as a medical device, principally in tissue with prolonged regeneration time and which require good mechanical properties. serve as a structural guide until tissue regeneration is completed and could be desirable for regeneration of large defects (Park et al., 2012).
1. INTRODUCTION The loss or failure of an organ or tissue represents frequent and devastating problems in health care. Thus, the need for substitutes to replace or repair tissue or organ problems is overwhelming. Tissue engineering, therefore, can provide a way of regenerating damaged tissue with the association of stem cells (SCs) with biomaterials. Several techniques for manufacturing polymer-based scaffolds are available today; however, rapid prototyping (recently redefined as additive manufacturing) offers the possibility to strictly control complex details as pore geometry, size, and interconnectivity, as well as the spatial distribution of pores within the structure, providing a 3D scaffold for cell growth in order to generate a matrix (Park et al., 2012, Gloria et al., 2012). The American Society for Testing and Materials (ASTM) defined additive manufacturing by the "process of joining materials to make objects from 3D model data, usually layer upon layer, as opposed to subtractive manufacturing methodologies, such as traditional machining" (ASTM F2792-10, 2010).
2. MATERIALS AND METHODS With the aim of evaluating cell response for 3D-printing scaffolds, PCL molds were fabricated by an extrusion process by additive manufacturing through a 3D printer named Fab@CTI (Figure 1) (Pereira et al., 2009, Lixandrão Filho et al., 2009). It is an open-source design AM machine, based on the Fab@Home (Malone and Lipson, 2007), and it was built and adapted at Center for Information Technology Renato Archer (CTI) with the purpose of collaborating in research initiatives in the bioengineering and biomaterials fields, having partners in many research institutions. Thereby, being an open-source project, Fab@CTI enables applications that request wide flexibility of settings and capability of reproducing complex geometries which are basic requirements in biofabrication. The scaffolds were printed with 5 slices of polymer (Figure 2) with approximately 0.6 mm diameter in a 3D printer. They were produced with different air gaps, as follows: Group 1, 0.15 mm; Group 2, 0.4 mm; Group 3, 0.2 mm; Group 4, 0.3 mm, and; Group 5, 01 mm. Their morphology was evaluated by Scanning Electron Microscopy (SEM). For biological assays, the
The most popular polymers used to construct material are polylactic acid (PLA), polyglycolic acid (PGA), and poly (Ɛcaprolactone) (PCL) (Senedese et al., 2011). PCL takes 2 to 3 years to degrade, owing to its hydrophobic nature but it displays relatively higher ductility (Park et al., 2012). It could 978-3-902823-50-2/2013 © IFAC
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IFAC MCPL 2013 September 11-13, 2013. Fortaleza, Brazil
scaffolds were distributed in cultures plates (96 wells) and 5,000 cells were seeded in each one. The biological analysis consisted of a viability test, performed with WST-8 reagent (Sigma-Aldrich®). To perform this test all the matrices were first change of place, i.e, they were put in another well and the WST-8 reagent was added at this moment. The morphology of the cells was also evaluated by SEM. For this analysis, the scaffolds with the cells were first fixed with glutaraldehyde 3% (w/v) and then dehydrated with increased concentrations of ethanol. The matrices with the cells were then metalized and the analysis was performed. Confocal microscopy was used to evaluate the morphology of the cells. For this test, cells were fixed with parafolmadehyde 4% (w/v) and stained with DAPI to observe the nuclei of the cells and with phalloidin to stain the cytoplasm. All the biological assays were performed after 1, 4, 7, 15 and 21 days after the seeding process. A control group, which consisted of cells directly cultivated on the well, was also evaluated by WST-8 assay and with a confocal microscope.
3.RESULTS The analysis of the morphology of the scaffolds by SEM showed well formed fibers. It is possible to see several layers of fibers in different arrangements. It is also possible to observe that there are differences in terms of fiber diameter and space between them (Fig. 3A-E). Group 5 showed the smallest space between the fibers. A
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Figure 1: The Fab@CTI 3D printer. C
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Figure 2: PCL 3D-scaffolds made by prototyping.
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Figure 5: Viability test of mesenchymal stem cells on the scaffolds groups and control group. E
The number of viable cells on the scaffolds was smaller when compared to the control group (Figure 5). This was expected because the control group was seeded onto the culture plates, which were the gold standard for the cultivation of the cells. The control group had statistically more viable cells (p<0.001 for all comparisons) than the other groups. As the groups in which the cells were seeded on the scaffolds showed lower E absorbance than the control group, a decision was made to investigate where these cells were attached. The absorbance of the well, where the cells were seeded on the scaffolds, was measured in the absence of the scaffold; high absorbance was observed, which was almost comparable to the control group (Figure 6). This analysis was only made with groups 1, 2 and 3 on days 4 and 7 but it confirmed the suspicion that the cells passed through the pores of the matrices.
Figure 3: Morphology of the scaffolds analyzed by SEM. A) Group 1, B) Group 2, C) Group 3, D) Group 4 and, E) Group 5. The viability assay showed that all the scaffolds demonstrated the same behavior, with a little increase in the number of the viable cells, represented by the increase in absorbance (Figure 4). There is no statistical difference between these groups (p>0.550 for all comparisons).
Figure 6: Viability test of mesenchymal stem cells for groups 1, 2 and 3 on days 4 and 7 after seedind the cells on the scaffolds. This analysis evaluated the absorbance of the cells attached on the wells in which the scaffolds were removed before analysis. In relation to time, there was statistical (p<0.001) difference between the number of viable cells over time, i.e, the cells in all the groups were proliferating and increasing in number.
Figure 4: Viability test of mesenchymal stem cells in the scaffolds groups.
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Although the absorbance of the WST-8 assay on the scaffolds was very low in comparison to the cells seeded on the scaffolds, it was possible to observe cells distributed on the fibers that form the matrices in all the groups since day 1 (Figures 7A-D1).
with scaffolds, the cells passed through the pores and adhered in more specific places. In relation to the results obtained by SEM, it was difficult to see cells on the first days of analysis. From day 14 it was possible to see some cells attached to different fibers (Figure 9A-C) in groups 4 and 5. On day 21 it was already possible to note the formation of some colonies of cells attached between the fibers, forming a kind of extracellular matrix in group 5 (Figure 10A-B).
Figure 8A-F shows confocal images of day 1 after seeding for all the groups. These analyses were performed in the wells where the cells were seeded in the scaffolds, but after the remove of the matrices. A control group was also evaluated. From the photographs, it is notable that the cells that adhered to the wells where the scaffolds were present are more grouped, while the cells in the control group are more dispersed. This is probably due to the fact that in the wells
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Figures 7: Cells stained with DAPI and phalloidin. Day 1 - A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5; and, F: Group 6. Day 4 - G: Group 1; H: Group 2; I: Group 3; J: Group 4; K: Group 5; and, L: Group 6. Day 7 - M: Group 1; N: Group 2; O: Group 3; P: Group 4; Q: Group 5; and, R: Group 6. Day 15 - S: Group 1; T: Group 2; U: Group 3; V: Group 4; X: Group 5; and, W: Group 6. Day 21 - Y: Group 1; Z: Group 2; A1: Group 3; B1: Group 4; C1: Group 5; and, D1: Group 6.
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Figure 8: Cells attached on the wells of the culture plates. A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5; and, F: Control.
Figure 9: A-B: cells attached to scaffolds of group 4. C: cells attached to scaffolds of group 5.
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scaffolds. 6th Brazilian Conference On Manufacturing Engineering. Caixias do Sul, RS Brasil.
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Figure 10: A-B: cells attached to scaffolds of group 5.
6. CONCLUSIONS Although the viability test showed a low number of viable cells in the scaffolds, it was possible to visualize cells interacting with the biomaterial and with each other. In addition, the biomaterial worked principally as a 2D environment, since the cells attached more to the surface of the fiber and only a little with the 3D architecture of the matrices. Therefore, the biomaterial developed by rapid prototyping with PCL was an interesting tool to be used in tissue engineering to interact with cells and replace/restore tissues with long periods of regeneration.
REFERENCES Gloria, A., Causa, F., Russo, T., Battista, E., Della Moglie, R., Zeppetelli, S., De Santis, R., Netti, P. A. & Ambrosio, L. (2012). Biomacromolecules, 13, 351021. Lixandrão Filho, A. L. et al. (2009). Construction and Adaptation of an Open Source Rapid Prototyping Machine for Biomedical Research Purposes - a Multinational Collaborative Development. In: Bártolo, P. Innovative Developments in Design and Manufacturing. Leiria: CRC Press, 2009. p. 469473. Malone E, Lipson H (2007). Fab@Home: the personal desktop fabricator kit. Rapid Prototyping Journal. 13:245-55. Park, S. H., Park, D. S., Shin, J. W., Kang, Y. G., Kim, H. K. & Yoon, T. R. 2012. J Mater Sci Mater Med, 23, 2671-8. Pereira, F.D.A.S.,, Inforçatti Neto, P., Lixandrão Filho, A.L., Silveira, Z. C., Da Silva, J.V.L. (2011). Thermoplastic filament extruder head for desktop additive manufacturing machines. In: The International Conference on Advanced Research in Virtual and Rapid Prototyping (VRAP). Senedese ALC, Lixandrão Filho AL, da Silva JVL, Inforçatti Neto P, Pereira FDAS, Maciel Filho R. (2011). Additive manufacturing to build polycaprolactone
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3D-printed scaffolds for the cultivation of mesenchymal stem cells Daniela Steffens*, Rodrigo A. Rezende**, Bruna Santi***, Frederico D. A. de S. Pereira**, Paulo Inforçatti Neto, Jorge V. L. da Silva**, Patricia Pranke**** * Post-graduation program of Biological Science – Phisiology; Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brazil (Tel:+5551 3308-5257; e-mail: [email protected]) ** Division of 3D Technologies, Center for Information Technology Renato Archer (CTI), Campinas, SP, Brazil (Tel:+5519 3746-6203; e-mail: [email protected]) *** Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brazil (Tel:+5551 3308-5257; e-mail: [email protected]) **** Post-graduation program of Biological Science – Phisiology; Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul, UFRGS; Stem Cell Research Institute, Porto Alegre, RS, Brazil (Tel: +5551 3308-5275; e-mail: [email protected])
Abstract: Tissue engineering is a tool which is currently under a great deal of investigation for the development and/or restoration of tissue and organs; it combines cell therapy and biomaterials for this aim. Rapid prototyping is a versatile technology which constructs polymer based-scaffolds with strict control of its architecture. The aim of this work, therefore, has been to investigate the interaction between mesenchymal stem cells with the biomaterials obtained by rapid prototyping. Scanning electron microscopy, confocal microscopy and biological assays were performed to analyze this interaction. As results, the number of viable cells attached to the scaffolds was lower when compared to the control group; however, it was possible to observe cells in the scaffolds since day 1 of analysis, with regions of confluence after 21 days of seeding. It could be concluded, therefore, that these biomaterials are interesting for use as a medical device, principally in tissue with prolonged regeneration time and which require good mechanical properties. serve as a structural guide until tissue regeneration is completed and could be desirable for regeneration of large defects (Park et al., 2012).
1. INTRODUCTION The loss or failure of an organ or tissue represents frequent and devastating problems in health care. Thus, the need for substitutes to replace or repair tissue or organ problems is overwhelming. Tissue engineering, therefore, can provide a way of regenerating damaged tissue with the association of stem cells (SCs) with biomaterials. Several techniques for manufacturing polymer-based scaffolds are available today; however, rapid prototyping (recently redefined as additive manufacturing) offers the possibility to strictly control complex details as pore geometry, size, and interconnectivity, as well as the spatial distribution of pores within the structure, providing a 3D scaffold for cell growth in order to generate a matrix (Park et al., 2012, Gloria et al., 2012). The American Society for Testing and Materials (ASTM) defined additive manufacturing by the "process of joining materials to make objects from 3D model data, usually layer upon layer, as opposed to subtractive manufacturing methodologies, such as traditional machining" (ASTM F2792-10, 2010).
2. MATERIALS AND METHODS With the aim of evaluating cell response for 3D-printing scaffolds, PCL molds were fabricated by an extrusion process by additive manufacturing through a 3D printer named Fab@CTI (Figure 1) (Pereira et al., 2009, Lixandrão Filho et al., 2009). It is an open-source design AM machine, based on the Fab@Home (Malone and Lipson, 2007), and it was built and adapted at Center for Information Technology Renato Archer (CTI) with the purpose of collaborating in research initiatives in the bioengineering and biomaterials fields, having partners in many research institutions. Thereby, being an open-source project, Fab@CTI enables applications that request wide flexibility of settings and capability of reproducing complex geometries which are basic requirements in biofabrication. The scaffolds were printed with 5 slices of polymer (Figure 2) with approximately 0.6 mm diameter in a 3D printer. They were produced with different air gaps, as follows: Group 1, 0.15 mm; Group 2, 0.4 mm; Group 3, 0.2 mm; Group 4, 0.3 mm, and; Group 5, 01 mm. Their morphology was evaluated by Scanning Electron Microscopy (SEM). For biological assays, the
The most popular polymers used to construct material are polylactic acid (PLA), polyglycolic acid (PGA), and poly (Ɛcaprolactone) (PCL) (Senedese et al., 2011). PCL takes 2 to 3 years to degrade, owing to its hydrophobic nature but it displays relatively higher ductility (Park et al., 2012). It could 978-3-902823-50-2/2013 © IFAC
361
10.3182/20130911-3-BR-3021.00117
IFAC MCPL 2013 September 11-13, 2013. Fortaleza, Brazil
scaffolds were distributed in cultures plates (96 wells) and 5,000 cells were seeded in each one. The biological analysis consisted of a viability test, performed with WST-8 reagent (Sigma-Aldrich®). To perform this test all the matrices were first change of place, i.e, they were put in another well and the WST-8 reagent was added at this moment. The morphology of the cells was also evaluated by SEM. For this analysis, the scaffolds with the cells were first fixed with glutaraldehyde 3% (w/v) and then dehydrated with increased concentrations of ethanol. The matrices with the cells were then metalized and the analysis was performed. Confocal microscopy was used to evaluate the morphology of the cells. For this test, cells were fixed with parafolmadehyde 4% (w/v) and stained with DAPI to observe the nuclei of the cells and with phalloidin to stain the cytoplasm. All the biological assays were performed after 1, 4, 7, 15 and 21 days after the seeding process. A control group, which consisted of cells directly cultivated on the well, was also evaluated by WST-8 assay and with a confocal microscope.
3.RESULTS The analysis of the morphology of the scaffolds by SEM showed well formed fibers. It is possible to see several layers of fibers in different arrangements. It is also possible to observe that there are differences in terms of fiber diameter and space between them (Fig. 3A-E). Group 5 showed the smallest space between the fibers. A
A
B
Figure 1: The Fab@CTI 3D printer. C
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Figure 2: PCL 3D-scaffolds made by prototyping.
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IFAC MCPL 2013 September 11-13, 2013. Fortaleza, Brazil
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Figure 5: Viability test of mesenchymal stem cells on the scaffolds groups and control group. E
The number of viable cells on the scaffolds was smaller when compared to the control group (Figure 5). This was expected because the control group was seeded onto the culture plates, which were the gold standard for the cultivation of the cells. The control group had statistically more viable cells (p<0.001 for all comparisons) than the other groups. As the groups in which the cells were seeded on the scaffolds showed lower E absorbance than the control group, a decision was made to investigate where these cells were attached. The absorbance of the well, where the cells were seeded on the scaffolds, was measured in the absence of the scaffold; high absorbance was observed, which was almost comparable to the control group (Figure 6). This analysis was only made with groups 1, 2 and 3 on days 4 and 7 but it confirmed the suspicion that the cells passed through the pores of the matrices.
Figure 3: Morphology of the scaffolds analyzed by SEM. A) Group 1, B) Group 2, C) Group 3, D) Group 4 and, E) Group 5. The viability assay showed that all the scaffolds demonstrated the same behavior, with a little increase in the number of the viable cells, represented by the increase in absorbance (Figure 4). There is no statistical difference between these groups (p>0.550 for all comparisons).
Figure 6: Viability test of mesenchymal stem cells for groups 1, 2 and 3 on days 4 and 7 after seedind the cells on the scaffolds. This analysis evaluated the absorbance of the cells attached on the wells in which the scaffolds were removed before analysis. In relation to time, there was statistical (p<0.001) difference between the number of viable cells over time, i.e, the cells in all the groups were proliferating and increasing in number.
Figure 4: Viability test of mesenchymal stem cells in the scaffolds groups.
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IFAC MCPL 2013 September 11-13, 2013. Fortaleza, Brazil
Although the absorbance of the WST-8 assay on the scaffolds was very low in comparison to the cells seeded on the scaffolds, it was possible to observe cells distributed on the fibers that form the matrices in all the groups since day 1 (Figures 7A-D1).
with scaffolds, the cells passed through the pores and adhered in more specific places. In relation to the results obtained by SEM, it was difficult to see cells on the first days of analysis. From day 14 it was possible to see some cells attached to different fibers (Figure 9A-C) in groups 4 and 5. On day 21 it was already possible to note the formation of some colonies of cells attached between the fibers, forming a kind of extracellular matrix in group 5 (Figure 10A-B).
Figure 8A-F shows confocal images of day 1 after seeding for all the groups. These analyses were performed in the wells where the cells were seeded in the scaffolds, but after the remove of the matrices. A control group was also evaluated. From the photographs, it is notable that the cells that adhered to the wells where the scaffolds were present are more grouped, while the cells in the control group are more dispersed. This is probably due to the fact that in the wells
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Figures 7: Cells stained with DAPI and phalloidin. Day 1 - A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5; and, F: Group 6. Day 4 - G: Group 1; H: Group 2; I: Group 3; J: Group 4; K: Group 5; and, L: Group 6. Day 7 - M: Group 1; N: Group 2; O: Group 3; P: Group 4; Q: Group 5; and, R: Group 6. Day 15 - S: Group 1; T: Group 2; U: Group 3; V: Group 4; X: Group 5; and, W: Group 6. Day 21 - Y: Group 1; Z: Group 2; A1: Group 3; B1: Group 4; C1: Group 5; and, D1: Group 6.
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Figure 8: Cells attached on the wells of the culture plates. A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5; and, F: Control.
Figure 9: A-B: cells attached to scaffolds of group 4. C: cells attached to scaffolds of group 5.
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scaffolds. 6th Brazilian Conference On Manufacturing Engineering. Caixias do Sul, RS Brasil.
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Figure 10: A-B: cells attached to scaffolds of group 5.
6. CONCLUSIONS Although the viability test showed a low number of viable cells in the scaffolds, it was possible to visualize cells interacting with the biomaterial and with each other. In addition, the biomaterial worked principally as a 2D environment, since the cells attached more to the surface of the fiber and only a little with the 3D architecture of the matrices. Therefore, the biomaterial developed by rapid prototyping with PCL was an interesting tool to be used in tissue engineering to interact with cells and replace/restore tissues with long periods of regeneration.
REFERENCES Gloria, A., Causa, F., Russo, T., Battista, E., Della Moglie, R., Zeppetelli, S., De Santis, R., Netti, P. A. & Ambrosio, L. (2012). Biomacromolecules, 13, 351021. Lixandrão Filho, A. L. et al. (2009). Construction and Adaptation of an Open Source Rapid Prototyping Machine for Biomedical Research Purposes - a Multinational Collaborative Development. In: Bártolo, P. Innovative Developments in Design and Manufacturing. Leiria: CRC Press, 2009. p. 469473. Malone E, Lipson H (2007). Fab@Home: the personal desktop fabricator kit. Rapid Prototyping Journal. 13:245-55. Park, S. H., Park, D. S., Shin, J. W., Kang, Y. G., Kim, H. K. & Yoon, T. R. 2012. J Mater Sci Mater Med, 23, 2671-8. Pereira, F.D.A.S.,, Inforçatti Neto, P., Lixandrão Filho, A.L., Silveira, Z. C., Da Silva, J.V.L. (2011). Thermoplastic filament extruder head for desktop additive manufacturing machines. In: The International Conference on Advanced Research in Virtual and Rapid Prototyping (VRAP). Senedese ALC, Lixandrão Filho AL, da Silva JVL, Inforçatti Neto P, Pereira FDAS, Maciel Filho R. (2011). Additive manufacturing to build polycaprolactone
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