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Published Online: 27 June 2014

Decellularization of Fibroblast Cell Sheets for Natural Extracellular Matrix Scaffold Preparation

Publication: Tissue Engineering Part C: Methods
Volume 21, Issue Number 1

Abstract

The application of cell-derived extracellular matrix (ECM) in tissue engineering has gained increasing interest because it can provide a naturally occurring, complex set of physiologically functional signals for cell growth. The ECM scaffolds produced from decellularized fibroblast cell sheets contain high amounts of ECM substances, such as collagen, elastin, and glycosaminoglycans. They can serve as cell adhesion sites and mechanically strong supports for tissue-engineered constructs. An efficient method that can largely remove cellular materials while maintaining minimal disruption of ECM ultrastructure and content during the decellularization process is critical. In this study, three decellularization methods were investigated: high concentration (0.5 wt%) of sodium dodecyl sulfate (SDS), low concentration (0.05 wt%) of SDS, and freeze-thaw cycling method. They were compared by characterization of ECM preservation, mechanical properties, in vitro immune response, and cell repopulation ability of the resulted ECM scaffolds. The results demonstrated that the high SDS treatment could efficiently remove around 90% of DNA from the cell sheet, but significantly compromised their ECM content and mechanical strength. The elastic and viscous modulus of the ECM decreased around 80% and 62%, respectively, after the high SDS treatment. The freeze-thaw cycling method maintained the ECM structure as well as the mechanical strength, but also preserved a large amount of cellular components in the ECM scaffold. Around 88% of DNA was left in the ECM after the freeze-thaw treatment. In vitro inflammatory tests suggested that the amount of DNA fragments in ECM scaffolds does not cause a significantly different immune response. All three ECM scaffolds showed comparable ability to support in vitro cell repopulation. The ECM scaffolds possess great potential to be selectively used in different tissue engineering applications according to the practical requirement.

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References

1.
Daley W.P., Peters S.B., and Larsen M. Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci 121, 255, 2008.
2.
Mostafavi-Pour Z., Askari J.A., Parkinson S.J., Parker P.J., Ng T.T.C., and Humphries M.J. Integrin-specific signaling pathways controlling focal adhesion formation and cell migration. J Cell Biol 161, 155, 2003.
3.
Bourget J.-M., Gauvin R., Larouche D., Lavoie A., Labbe R., Auger F.A., et al. Human fibroblast-derived ECM as a scaffold for vascular tissue engineering. Biomaterials 33, 9205, 2012.
4.
Lu H.X., Hoshiba T., Kawazoe N., Koda I., Song M.H., and Chen G.P. Cultured cell-derived extracellular matrix scaffolds for tissue engineering. Biomaterials 32, 9658, 2011.
5.
Sadr N., Pippenger B.E., Scherberich A., Wendt D., Mantero S., Martin I., et al. Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix. Biomaterials 33, 5085, 2012.
6.
Pei M., He F., and Kish V.L. Expansion on extracellular matrix deposited by human bone marrow stromal cells facilitates stem cell proliferation and tissue-specific lineage potential. Tissue Eng Part A 17, 3067, 2011.
7.
Scobie L., Padler-Karavani V., Le Bas-Bernardet S., Crossan C., Blaha J., Matouskova M., et al. Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts. J Immunol 191, 2907, 2013.
8.
Grenier G., Remy-Zolghadri M., Larouche D., Gauvin R., Baker K., Bergeron F., et al. Tissue reorganization in response to mechanical load increases functionality. Tissue Eng 11, 90, 2005.
9.
Xing Q., Vogt C., Leong K.W., and Zhao F. Highly aligned nanofibrous scaffold derived from decellularized human fibroblasts. Adv Funct Mater 24, 3027, 2014.
10.
Datta N., Pham Q.P., Sharma U., Sikavitsas V.I., Jansen J.A., and Mikos A.G. In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation. Proc Natl Acad Sci U S A 103, 2488, 2006.
11.
Cukierman E., Pankov R., Stevens D.R., and Yamada K.M. Taking cell-matrix adhesions to the third dimension. Science 294, 1708, 2001.
12.
El Ghalbzouri A., Commandeur S., Rietveld M.H., Mulder A.A., and Willemze R. Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products. Biomaterials 30, 71, 2009.
13.
Grinnell F., Fukamizu H., Pawelek P., and Nakagawa S. Collagen processing, crosslinking and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic-acid. Exp Cell Res 181, 483, 1989.
14.
Ishikawa O., Kondo A., Okada K., Miyachi Y., and Furumura M. Morphological and biochemical analyses on fibroblasts and self-produced collagens in a novel three-dimensional culture. Br J Dermatol 136, 6, 1997.
15.
Ahlfors J.-E.W., and Billiar K.L. Biomechanical and biochemical characteristics of a human fibroblast-produced and remodeled matrix. Biomaterials 28, 2183, 2007.
16.
Calve S., Dennis R.G., Kosnik P.E., Baar K., Grosh K., and Arruda E.M. Engineering of functional tendon. Tissue Eng 10, 755, 2004.
17.
L'Heureux N., Paquet S., Labbe R., Germain L., and Auger F.A. A completely biological tissue-engineered human blood vessel. FASEB J 12, 47, 1998.
18.
L'Heureux N., Dusserre N., Konig G., Victor B., Keire P., Wight T.N., et al. Human tissue-engineered blood vessels for adult arterial revascularization. Nat Med 12, 361, 2006.
19.
Konig G., McAllister T.N., Dusserre N., Garrido S.A., Iyican C., Marini A., et al. Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery. Biomaterials 30, 1542, 2009.
20.
Crapo P.M., Gilbert T.W., and Badylak S.F. An overview of tissue and whole organ decellularization processes. Biomaterials 32, 3233, 2011.
21.
Gilbert T.W. Strategies for tissue and organ decellularization. J Cell Biochem 113, 2217, 2012.
22.
Du L.Q., Wu X.Y., Pang K.P., and Yang Y.M. Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold. Br J Ophthalmol 95, 410, 2011.
23.
Xu C.C., Chan R.W., and Tirunagari N. A biodegradable, acellular xenogeneic scaffold for regeneration of the vocal fold lamina propria. Tissue Eng 13, 551, 2007.
24.
Patel N., Solanki E., Picciani R., Cavett V., Caldwell-Busby J.A., and Bhattacharya S.K. Strategies to recover proteins from ocular tissues for proteomics. Proteomics 8, 1055, 2008.
25.
Elder B.D., Kim D.H., and Athanasiou K.A. Developing an articular cartilage decellularization process toward facet joint cartilage replacement. Neurosurgery 66, 722, 2010.
26.
Boer U., Lohrenz A., Klingenberg M., Pich A., Haverich A., and Wilhelmi M. The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts. Biomaterials 32, 9730, 2011.
27.
Petersen T.H., Calle E.A., Colehour M.B., and Niklason L.E. Matrix composition and mechanics of decellularized lung scaffolds. Cells Tissues Organs 195, 222, 2012.
28.
Zhao F., Veldhuis J.J., Duan Y.J., Yang Y., Christoforou N., Ma T., et al. Low oxygen tension and synthetic nanogratings improve the uniformity and stemness of human mesenchymal stem cell layer. Mol Ther 18, 1010, 2010.
29.
Kim J., and Ma T. Autocrine fibroblast growth factor 2-mediated interactions between human mesenchymal stem cells and the extracellular matrix under varying oxygen tension. J Cell Biochem 114, 716, 2013.
30.
Reing J.E., Brown B.N., Daly K.A., Freund J.M., Gilbert T.W., Hsiong S.X., et al. The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials 31, 8626, 2010.
31.
Gauvin R., Ahsan T., Larouche D., Levesque P., Dube J., Auger F.A., et al. A novel single-step self-assembly approach for the fabrication of tissue-engineered vascular constructs. Tissue Eng Part A 16, 1737, 2010.
32.
Portmann-Lanz C.B., Ochsenbein-Kolble N., Marquardt K., Luthi U., Zisch A., and Zimmermann R. Manufacture of a cell-free amnion matrix scaffold that supports amnion cell outgrowth in vitro. Placenta 28, 6, 2007.
33.
Zheng M.H., Chen J., Kirilak Y., Willers C., Xu J., and Wood D. Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation. J Biomed Mater Res Part B 73B, 61, 2005.
34.
Giusti S., Bogetti M.E., Bonafina A., and de Plazas S.F. An improved method to obtain a soluble nuclear fraction from embryonic brain tissue. Neurochem Res 34, 2022, 2009.
35.
Elder B.D., Eleswarapu S.V., and Athanasiou K.A. Extraction techniques for the decellularization of tissue engineered articular cartilage constructs. Biomaterials 30, 3749, 2009.
36.
Cortiella J., Niles J., Cantu A., Brettler A., Pham A., Vargas G., et al. Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation. Tissue Eng Part A 16, 2565, 2010.
37.
Stamov D.R., and Pompe T. Structure and function of ECM-inspired composite collagen type I scaffolds. Soft Matter 8, 10200, 2012.
38.
Nuttelman C.R., Mortisen D.J., Henry S.M., and Anseth K.S. Attachment of fibronectin to poly(vinyl alcohol) hydrogels promotes NIH3T3 cell adhesion, proliferation, and migration. J Biomed Mater Res 57, 217, 2001.
39.
Cribb A.M., and Scott J.E. Tendon response to tensile-stress—an ultrastructural investigation of collagen-proteoglycan interactions in stressed tendon. J Anat 187, 423, 1995.
40.
Lovekamp J.J., Simionescu D.T., Mercuri J.J., Zubiate B., Sacks M.S., and Vyavahare N.R. Stability and function of glycosaminoglycans in porcine bioprosthetic heart valves. Biomaterials 27, 1507, 2006.
41.
Badylak S.F., Freytes D.O., and Gilbert T.W. Extracellular matrix as a biological scaffold material: structure and function. Acta Biomater 5, 1, 2009.
42.
Keane T.J., Londono R., Turner N.J., and Badylak S.F. Consequences of ineffective decellularization of biologic scaffolds on the host response. Biomaterials 33, 1771, 2012.
43.
Esposito E., and Cuzzocrea S. TNF-alpha as a therapeutic target in inflammatory diseases, ischemia-reperfusion injury and trauma. Curr Med Chem 16, 3152, 2009.
44.
Saraiva M., and O'Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol 10, 170, 2010.
45.
Martinez F.O., Sica A., Mantovani A., and Locati M. Macrophage activation and polarization. Front Biosci (Landmark Ed) 13, 453, 2008.
46.
Youngstrom D.W., Barrett J.G., Jose R.R., and Kaplan D.L. Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications. PLoS One 8, 2013.
47.
Franz S., Rammelt S., Scharnweber D., and Simon J.C. Immune responses to implants—a review of the implications for the design of immunomodulatory biomaterials. Biomaterials 32, 6692, 2011.
48.
Salek-Ardakani S., Arrand J.R., Shaw D., and Mackett M. Heparin and heparan sulfate bind interleukin-10 and modulate its activity. Blood 96, 1879, 2000.
49.
Rammelt S., Schulze E., Bernhardt R., Hanisch U., Scharnweber D., Worch H., et al. Coating of titanium implants with type-I collagen. J Orthop Res 22, 1025, 2004.
50.
de Fougerolles A.R., and Koteliansky V.E. Regulation of monocyte gene expression by the extracellular matrix and its functional implications. Immunol Rev 186, 208, 2002.

Information & Authors

Information

Published In

cover image Tissue Engineering Part C: Methods
Tissue Engineering Part C: Methods
Volume 21Issue Number 1January 2015
Pages: 77 - 87
PubMed: 24866751

History

Published in print: January 2015
Published ahead of print: 30 June 2014
Published online: 27 June 2014
Published ahead of production: 27 May 2014
Accepted: 7 May 2014
Received: 28 October 2013

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Affiliations

Qi Xing, PhD
Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan.
Keegan Yates
Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan.
Mitchell Tahtinen
Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan.
Emily Shearier, BSc
Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan.
Zichen Qian, BSc
Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan.
Feng Zhao, PhD
Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan.

Notes

Address correspondence to:Feng Zhao, PhDDepartment of Biomedical EngineeringMichigan Technological University1400 Townsend DriveHoughton, MI 49931E-mail: [email protected]

Disclosure Statement

The authors declare no competing financial interests.

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