JBNB  Vol.4 No.4 , October 2013
A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of Breast Cancer Cells
ABSTRACT

In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional (3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining technique in an attempt to create an in vitro model for the culture of breast cancer cells. The morphology, pore structure, and mechanical performance of the obtained patterned macroporous BC (PM-BC) scaffolds were characterized by scanning electron microscopy (SEM), mercury intrusion porosimeter, and mechanical testing. A human breast cancer cell (MDA-MB-231) line was cultured onto the PM-BC scaffolds to investigate the role of macropores in the control of cancer cell behavior. MTT assay, SEM, and hematoxylin and eosin (H&E) staining were employed to determine cell adhesion, growth, proliferation, and infiltration. The PM-BC scaffolds were found to be able to promote cellular adhesion and proliferation on the scaffolds, and further to allow for cell infiltration into the PM-BC scaffolds. The results demonstrated that BC scaffolds with laser-patterned macropores were promising for the in vitro 3D culture of breast cancer cells.


Cite this paper
G. Xiong, H. Luo, F. Gu, J. Zhang, D. Hu and Y. Wan, "A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of Breast Cancer Cells," Journal of Biomaterials and Nanobiotechnology, Vol. 4 No. 4, 2013, pp. 316-326. doi: 10.4236/jbnb.2013.44040.
References
[1]   R. Langer and J. P. Vacanti, “Tissue Engineering,” Science, Vol. 260, No. 5110, 1993, pp. 920-926. http://dx.doi.org/10.1126/science.8493529

[2]   C. M. Ghajar and M. J. Bissell, “Tumor Engineering: The Other Face of Tissue Engineering,” Tissue Engineering Part A, Vol. 16, No. 7, 2010, pp. 2153-2156. http://dx.doi.org/10.1089/ten.tea.2010.0135

[3]   Y. Aizawa, S. C. Owen and M. S. Shoichet, “Polymers Used to Influence Cell Fate in 3D Geometry: New Trends,” Progress in Polymer Science, Vol. 37, No. 5, 2012, pp. 645-658. http://dx.doi.org/10.1016/j.progpolymsci.2011.11.004

[4]   G. Y. Lee, P. A. Kenny, E. H. Lee and M. J. Bissell, “Three-Dimensional Culture Models of Normal and Malignant Breast Epithelial Cells,” Nature Methods, Vol. 4, No. 4, 2007, pp. 359-365. http://dx.doi.org/10.1038/nmeth1015

[5]   C. Fischbach, R. Chen, T. Matsumoto, T. Schmelzle, J. S. Brugge, P. J. Polverini and D. J. Mooney, “Engineering Tumors with 3D Scaffolds,” Nature Methods, Vol. 4, No. 10, 2007, pp. 855-860. http://dx.doi.org/10.1038/nmeth1085

[6]   M. A. Cichon, V. G. Gainullin, Y. Zhang and D. C. Radisky, “Growth of Lung Cancer Cells in Three-Dimensional Microenvironments Reveals Key Features of Tumor Malignancy,” Integrative Biology, Vol. 4, No. 4, 2012, pp. 440-448. http://dx.doi.org/10.1039/c1ib00090j

[7]   J. L. Horning, S. K. Sahoo, S. Vijayaraghavalu, S. Dimitrijevic, J. K. Vasir, T. K. Jain, A. K. Panda and V. Labhasetwar, “3-D Tumor Model for in Vitro Evaluation of Anticancer Drugs,” Molecular Pharmacology, Vol. 5, No. 5, 2008, pp. 849-862. http://dx.doi.org/10.1021/mp800047v

[8]   M. Mitra, C. Mohanty, A. Harilal, U. K. Maheswari, S. K. Sahoo and S. Krishnakumar, “A Novel in Vitro Three-Dimensional Retinoblastoma Model for Evaluating Chemotherapeutic Drugs,” Molecular Vision, Vol. 18, No. 142-145, 2012, pp. 1361-1378.

[9]   L. Chen, Z. Xiao, Y. Meng, Y. Zhao, J. Han, G. Su, B. Chen and J. Dai, “The Enhancement of Cancer Stem Cell Properties of MCF-7 Cells in 3D Collagen Scaffolds for Modeling of Cancer and Anti-Cancer Drugs,” Biomaterials, Vol. 33, No. 5, 2012, pp. 1437-1444. http://dx.doi.org/10.1016/j.biomaterials.2011.10.056

[10]   C. S. Szot, C. F. Buchanan, J. W. Freeman and M. N. Rylander, “3D in Vitro Bioengineered Tumors Based on Collagen I Hydrogels,” Biomaterials, Vol. 32, No. 31, 2011, pp. 7905-7912. http://dx.doi.org/10.1016/j.biomaterials.2011.07.001

[11]   F. M. Kievit, S. J. Florczyk, M. C. Leung, O. Veiseh, J. O. Park, M. L. Disis and M. Zhang, “Chitosan-Alginate 3D Scaffolds as a Mimic of the Glioma Tumor Microenvironment,” Biomaterials, Vol. 31, No. 22, 2010, pp. 5903-5910. http://dx.doi.org/10.1016/j.biomaterials.2010.03.062

[12]   K. A. Beningo, M. Dembo and Y. Wang, “Responses of Fibroblasts to Anchorage of Dorsal Extracellular Matrix Receptors,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 52, 2004, p. 18024. http://dx.doi.org/10.1073/pnas.0405747102

[13]   S. K. Sahoo, A. K. Panda and V. Labhasetwar, “Characterization of Porous PLGA/PLA Microparticles as a Scaffold for Three Dimensional Growth of Breast Cancer Cells,” Biomacromolecules, Vol. 6, No. 2, 2005, pp. 1132-1139. http://dx.doi.org/10.1021/bm0492632

[14]   N. Rhodes, J. Srivastava, R. Smith and C. Longinotti, “Metabolic and Histological Analysis of Mesenchymal Stem Cells Grown in 3-D Hyaluronan-Based Scaffolds,” Journal of Materials Science: Materials in Medicine, Vol. 15, No. 4, 2004, pp. 391-395. http://dx.doi.org/10.1023/B:JMSM.0000021108.74004.7e

[15]   S. Talukdar, M. Mandal, D. W. Hutmacher, P. J. Russell, C. Soekmadji and S. C. Kundu, “Engineered Silk Fibroin Protein 3D Matrices for in Vitro Tumor Model,” Biomaterials, Vol. 32, No. 8, 2010, pp. 2149-2159. http://dx.doi.org/10.1016/j.biomaterials.2010.11.052

[16]   J. Lannutti, D. Reneker, T. Ma, D. Tomasko and D. F. Farson, “Electrospinning for Tissue Engineering Scaffolds,” Materials Science and Engineering C, Vol. 27, No. 3, 2007, pp. 504-509. http://dx.doi.org/10.1016/j.msec.2006.05.019

[17]   S. Agarwal, J. H. Wendorff and A. Greiner, “Progress in the Field of Electrospinning for Tissue Engineering Applications,” Advanced Materials, Vol. 21, No. 32-33, 2009, pp. 3343-3351. http://dx.doi.org/10.1002/adma.200803092

[18]   W. Yang, F. Yang, Y. Wang, S. K. Both and J. A. Jansen, “In Vivo Bone Generation via the Endochondral Pathway on Three-Dimensional Electrospun Fibers,” Acta Biomaterialia, Vol. 9, No. 1, 2013, pp. 4505-4512. http://dx.doi.org/10.1016/j.actbio.2012.10.003

[19]   T. Dvir, B. P. Timko, D. S. Kohane and R. Langer, “Nanotechnological Strategies for Engineering Complex Tissues,” Nature Nanotechnology, Vol. 6, No. 1, 2011, pp. 13-22. http://dx.doi.org/10.1038/nnano.2010.246

[20]   T. G. Kim, H. Shin and D. W. Lim, “Biomimetic Scaffolds for Tissue Engineering,” Advanced Functional Materials, Vol. 22, No. 12, 2012, pp. 2446-2468. http://dx.doi.org/10.1002/adfm.201103083

[21]   C. Vaquette and J. Cooper-White, “A Simple Method for Fabricating 3-D Multilayered Composite Scaffolds,” Acta Biomaterialia, Vol. 9, No. 1, 2013, pp. 4599-4608. http://dx.doi.org/10.1016/j.actbio.2012.08.015

[22]   J. Mao, S. Duan, A. Song, Q. Cai, X. Deng and X. Yang, “Macroporous and Nanofibrous Poly(Lactide-co-Glycolide)(50/50) Scaffolds via Phase Separation Combined with Particle-Leaching,” Materials Science and Engineering C, Vol. 32, No. 6, 2012, pp. 1407-1414. http://dx.doi.org/10.1016/j.msec.2012.04.018

[23]   N. Petersen and P. Gatenholm, “Bacterial Cellulose-Based Materials and Medical Devices: Current State and Perspectives,” Applied Microbiology and Biotechnology, Vol. 91, No. 5, 2011, pp. 1277-1286. http://dx.doi.org/10.1007/s00253-011-3432-y

[24]   D. Klemm, F. Kramer, S. Moritz, T. Lindstrom, M. Ankerfors, D. Gray and A. Dorris, “Nanocelluloses: A New Family of Nature-Based Materials,” Angewandte Chemie International Edition, Vol. 50, No. 24, 2011, pp. 5438-5466. http://dx.doi.org/10.1002/anie.201001273

[25]   S. Tanpichai, F. Quero, M. Nogi, H. Yano, R. J. Young, T. Lindstrom, W. W. Sampson and S. J. Eichhorn, “Effective Young’s Modulus of Bacterial and Microfibrillated Cellulose Fibrils in Fibrous Networks,” Biomacromolecules, Vol. 13, No. 5, 2012, pp. 1340-1349. http://dx.doi.org/10.1021/bm300042t

[26]   R. A. N. Pertile, S. Moreira, R. M. Gil da Costa, A. Correia, L. Guardao, F. Gartner, M. Vilanova and M. Gama, “Bacterial Cellulose: Long-Term Biocompatibility Studies,” Journal of Biomaterials Science Polymer Edition, Vol. 23, No. 10, 2012, pp. 1339-1354.

[27]   F. K. Andrade, N. Alexandre, I. Amorim, F. Gartner, A. C. Maurício, A. L. Luís and M. Gama1, “Studies on the Biocompatibility of Bacterial Cellulose,” Journal of Bioactive and Compatable Polymers, Vol. 28, No. 1, 2013, pp. 97-112.

[28]   P. M. Favi, R. S. Benson, N. R. Neilsen, R. L. Hammonds, C. C. Bates, C. P. Stephens and M. S. Dhar, “Cell Proliferation, Viability, and in Vitro Differentiation of Equine Mesenchymal Stem Cells Seeded on Bacterial Cellulose Hydrogel Scaffolds,” Materials Science and Engineering C, Vol. 33, No. 4, 2013, pp. 1935-1944. http://dx.doi.org/10.1016/j.msec.2012.12.100

[29]   Q. Shi, Y. Li, J. Sun, H. Zhang, L. Chen, B. Chen, H. Yang and Z. Wang, “The Osteogenesis of Bacterial Cellulose Scaffold Loaded with Bone Morphogenetic Protein-2,” Biomaterials, Vol. 33, No. 28, 2012, pp. 6644-6649. http://dx.doi.org/10.1016/j.biomaterials.2012.05.071

[30]   S. Saska, R. M. Scarel-Caminaga, L. N. Teixeira, L. P. Franchi, R. A. Dos Santos, A. M. M. Gaspar, P. T. de Oliveira, A. L. Rosa, C. S. Takahashi, Y. Messaddeq, S. J. L. Ribeiro and R. Marchetto, “Characterization and in Vitro Evaluation of Bacterial Cellulose Membranes Functionalized with Osteogenic Growth Peptide for Bone Tissue Engineering,” Journal of Materials Science Materials in Medicine, Vol. 23, No. 9, 2012, pp. 2253-2266. http://dx.doi.org/10.1007/s10856-012-4676-5

[31]   K. Hirayama, T. Okitsu, H. Teramae, D. Kiriya, H. Onoe and S. Takeuchi, “Cellular Building Unit Integrated with Microstrand-Shaped Bacterial Cellulose,” Biomaterials, Vol. 34, No. 10, 2013, pp. 2421-2427. http://dx.doi.org/10.1016/j.biomaterials.2012.12.013

[32]   C. S. Szot, C. F. Buchanan, P. Gatenholm, M. N. Rylander and J. W. Freeman, “Investigation of Cancer Cell Behavior on Nanofibrous Scaffolds,” Materials Science and Engineering C, Vol. 31, No. 1, 2011, pp. 37-42. http://dx.doi.org/10.1016/j.msec.2009.12.005

[33]   J. M. Taboas, R. D. Maddox, P. H. Krebsbach and S. J. Hollister, “Indirect Solid Free form Fabrication of Local and Global Porous, Biomimetic and Composite 3D Polymer-Ceramic Scaffolds,” Biomaterials, Vol. 24, No. 1, 2003, pp. 181-194. http://dx.doi.org/10.1016/S0142-9612(02)00276-4

[34]   C. Martinez-Ramos, A. Valles-Lluch, J. M. Garcia Verdugo, J. L. Gomez Ribelles, J. Antonio Barcia, A. Baiget Orts, J. M. Soria Lopez and M. Monleon Pradas, “Channeled Scaffolds Implanted in Adult Rat Brain,” Journal of Biomedial Materials Research Part A, Vol. 100A, No. 12, 2012, pp. 3276-3286.

[35]   J. Wang, C. Yang, Y. Wan, H. Luo, F. He, K. Dai and Y. Huang, “Laser Patterning of Bacterial Cellulose Hydrogel and Its Modification with Gelatin and Hydroxyapatite for Bone Tissue Engineering,” Soft Materials, Vol. 11, No. 2, 2013, pp. 173-180. http://dx.doi.org/10.1080/1539445X.2011.611204

[36]   J. C. Le Huec, T. Schaeverbeke, D. Clement, J. Faber and A. Le Rebeller, “Influence of Porosity on the Mechanical Resistance of Hydroxyapatite Ceramics under Compressive Stress,” Biomaterials, Vol. 16, No. 2, 1995, pp. 113-118. http://dx.doi.org/10.1016/0142-9612(95)98272-G

[37]   L. Hong, Y. L. Wang, S. R. Jia, Y. Huang, C. Gao and Y. Z. Wan, “Hydroxyapatite/Bacterial Cellulose Composites Synthesized via a Biomimetic Route,” Materials Letters, Vol. 60, No. 13-14, 2006, pp. 1710-1713. http://dx.doi.org/10.1016/j.matlet.2005.12.004

[38]   Y. Z. Wan, L. Hong, S. R. Jia, Y. Huang, Y. Zhu, Y. L. Wang and H. J. Jiang, “Synthesis and Characterization of Hydroxyapatite-Bacterial Cellulose Nanocomposites,” Composites Science and Technology, Vol. 66, No. 11-12, 2006, pp. 1825-1832. http://dx.doi.org/10.1016/j.compscitech.2005.11.027

[39]   A. I. Itala, H. O. Ylanen, C. Ekholm, K. H. Karlsson and H. T. Aro, “Pore Diameter of More than 100 mu m Is Not Requisite for Bone Ingrowth in Rabbits,” Journal of Biomedial Materials Research, Vol. 58, No. 6, 2001, pp. 679-683. http://dx.doi.org/10.1002/jbm.1069

[40]   H. E. Gotz, M. Muller, A. Emmel, U. Holzwarth, R. G. Erben and R. Stangl, “Effect of Surface Finish on the Osseointegration of Laser-Treated Titanium Alloy Implants,” Biomaterials, Vol. 25, No. 18, 2004, pp. 4057-4064. http://dx.doi.org/10.1016/j.biomaterials.2003.11.002

[41]   V. V. Kancharla and S. C. Chen, “Fabrication of Biodegradable Polymeric Micro-Devices Using Laser Micromachining,” Biomedical Microdevices, Vol. 4, No. 2, 2002, pp. 105-109. http://dx.doi.org/10.1023/A:1014679013888

[42]   S. Chen, V. V. Kancharla and Y. Lu, “Laser-Based Microscale Patterning of Biodegradable Polymers for Biomedical Applications,” International Journal of Materials and Product Technology, Vol. 18, No. 4-6, 2003, pp. 457-468.

[43]   C. A. Aguilar, Y. Lu, S. Mao and S. C. Chen, “Direct Micro-Patterning of Biodegradable Polymers Using Ultraviolet and Femtosecond Lasers,” Biomaterials, Vol. 26, No. 36, 2005, pp. 7642-7649. http://dx.doi.org/10.1016/j.biomaterials.2005.04.053

[44]   C. Gao, Y. Z. Wan, C. X. Yang, K. R. Dai, T. T. Tang, H. L. Luo and J. H. Wang, “Preparation and Characterization of Bacterial Cellulose Sponge with Hierarchical Pore Structure as Tissue Engineering Scaffold,” Journal of Porous Materials, Vol. 18, 2011, pp. 139-145. http://dx.doi.org/10.1007/s10934-010-9364-6

[45]   V. Karageorgiou and D. Kaplan, “Porosity of 3D Biornaterial Scaffolds and Osteogenesis,” Biomaterials, Vol. 26, No. 27, 2005, pp. 5474-5491. http://dx.doi.org/10.1016/j.biomaterials.2005.02.002

[46]   M. Flaibani and N. Elvassore, “Gas Anti-Solvent Precipitation Assisted Salt Leaching for Generation of Microand Nano-Porous Wall in Bio-Polymeric 3D Scaffolds,” Materials Science and Engineering C, Vol. 32, No. 6, 2012, pp. 1632-1639. http://dx.doi.org/10.1016/j.msec.2012.04.054

[47]   N. D. Evans, E. Gentleman and J. M. Polak, “Scaffolds for Stem Cells,” Materials Today, Vol. 9, No. 12, 2006, pp. 26-33. http://dx.doi.org/10.1016/S1369-7021(06)71740-0

[48]   B. J. Papenburg, J. Liu, G. A. Higuera, A. M. C. Barradas, J. de Boer, C. A. van Blitterswijk, M. Wessling and D. Stamatialis, “Development and Analysis of Multi-Layer Scaffolds for Tissue Engineering,” Biomaterials, Vol. 30, No. 31, 2009, pp. 6228-6239. http://dx.doi.org/10.1016/j.biomaterials.2009.07.057

[49]   A. A. Al-Munajjed, M. Hien, R. Kujat, J. P. Gleeson and J. Hammer, “Influence of Pore Size on Tensile Strength, Permeability and Porosity of Hyaluronan-Collagen Scaffolds,” Journal of Materials Science Materials in Medicine, Vol. 19, No. 8, 2008, pp. 2859-2864. http://dx.doi.org/10.1007/s10856-008-3422-5

[50]   E. Tsuruga, H. Takita, H. Itoh, Y. Wakisaka and Y. Kuboki, “Pore Size of Porous Hydroxyapatite as the Cell-Substratum Controls BMP-Induced Osteogenesis,” Journal of Biochemistry, Vol. 121, No. 2, 1997, pp. 317-324. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a021589

 
 
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