Abstract
Decellularized tissue-engineered constructs have the potential to promote regeneration by providing a biomimetic extracellular matrix that directs tissue-specific regeneration when implanted in situ. Recently, the use of cell sheets has shown promising results in promoting periodontal regeneration. Here, we describe the fabrication of decellularized periodontal cell sheets with intact extracellular matrix structural and biological properties. Melt electro spun polycaprolactone (PCL) scaffolds are used as a carrier for the inherently fragile cell sheets, to provide support during the processes of decellularization. An optimized decellularization method is outlined using perfusion with a combination of NH4OH and Triton X-100 together with a DNase treatment step for DNA removal. The maintenance of extracellular matrix structural and biological integrity is important, and here we describe the assessment of these properties using immunostaining for extracellular matrix proteins and ELISA for growth factor quantification.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ishikawa I, Iwata T, Washio K, Okano T, Nagasawa T, Iwasaki K, Ando T (2009) Cell sheet engineering and other novel cell-based approaches to periodontal regeneration. Periodontol 2000 51:220–238
Flores MG, Yashiro R, Washio K, Yamato M, Okano T, Ishikawa I (2008) Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats. J Clin Periodontol 35:1066–1072
Akizuki T, Oda S, Komaki M, Tsuchioka H, Kawakatsu N, Kikuchi A, Yamato M, Okano T, Ishikawa I (2005) Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs. J Periodontal Res 40:245–251
Vaquette C, Fan W, Xiao Y, Hamlet S, Hutmacher DW, Ivanovski S (2012) A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex. Biomaterials 33:5560–5573
Dan H, Vaquette C, Fisher AG, Hamlet SM, Xiao Y, Hutmacher DW, Ivanovski S (2014) The influence of cellular source on periodontal regeneration using calcium phosphate coated polycaprolactone scaffold supported cell sheets. Biomaterials 35:113–122
Gawlitta D, Benders KE, Visser J, van der Sar AS, Kempen DH, Theyse LF, Malda J, Dhert WJ (2015) Decellularized cartilage-derived matrix as substrate for endochondral bone regeneration. Tissue Eng Part A 21:694–703
Sadr N, Pippenger BE, Scherberich A, Wendt D, Mantero S, Martin I, Papadimitropoulos A (2012) Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix. Biomaterials 33:5085–5093
Brown TD, Dalton PD, Hutmacher DW (2011) Direct writing by way of melt electrospinning. Adv Mater 23:5651–5657
Acknowledgments
This work was supported by Australia’s National Health and Medical Research Council project grant 1086181.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Farag, A., Vaquette, C., Hutmacher, D.W., Bartold, P.M., Ivanovski, S. (2017). Fabrication and Characterization of Decellularized Periodontal Ligament Cell Sheet Constructs. In: Seymour, G., Cullinan, M., Heng, N. (eds) Oral Biology. Methods in Molecular Biology, vol 1537. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6685-1_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6685-1_23
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6683-7
Online ISBN: 978-1-4939-6685-1
eBook Packages: Springer Protocols