Decellularized Tissue Engineered Constructs Using Cell Sheet Technology Author Farag, Amro Ahmed Mahmoud Published 2016 Thesis Type Thesis (PhD Doctorate) School School of Dentistry and Oral Health DOI https://doi.org/10.25904/1912/2310 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/367720 Griffith Research Online https://research-repository.griffith.edu.au Decellularized Tissue engineered constructs using cell sheet technology Amro Ahmed Mahmoud Farag BDSc, M.Sc. (Periodontics) School of Dentistry Griffith Health Griffith University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy 3/30/2016 I Statement of Originality I, Amro Farag declare that this work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. (Signed)_____________________________ Amro Farag II Contents Statement of Originality ......................................................................................... II Contents ............................................................................................................... III List of Tables ..................................................................................................... VIII List of Figures...................................................................................................... IX Abstract .............................................................................................................. XI Acknowledgements ............................................................................................ XIII Publications and successful grants arising from this thesis .................................. XIV Chapter 1 ............................................................................................................. 1 Introduction and Literature Review .................................................................... 1 Introduction: ............................................................................................................... 1 Literature Review ....................................................................................................... 3 Structure of the periodontium: ................................................................................ 3 Effect of periodontal disease on periodontal tissues: ............................................. 6 Periodontal regeneration: ........................................................................................ 7 Periodontal regenerative therapies: ........................................................................ 8 Guided tissue regeneration (GTR): ......................................................................... 9 Bone Grafting: ...................................................................................................... 12 Bioactive materials & signaling molecules: ......................................................... 15 Tissue engineering of periodontal ligament: ........................................................ 19 Cell sheet technology: .......................................................................................... 21 Tissue engineered decellularized matrices and periodontal regeneration: ........... 22 Research hypothesis: ................................................................................................ 26 Aims: ........................................................................................................................ 26 Potential Significance and Innovation...................................................................... 27 Chapter 2 ........................................................................................................... 28 III Decellularized periodontal ligament cell sheets with recellularization potential ............................................................................................................. 28 Abstract .................................................................................................................... 29 Introduction .............................................................................................................. 30 Materials and methods ............................................................................................. 31 Primary human periodontal ligament cells isolation and culture ......................... 31 Decellularization, immunostaining and confocal imagining of cultured cell monolayers............................................................................................................ 31 Melt Electrospun carrier membrane fabrication ................................................... 32 Cell sheet harvesting ............................................................................................. 33 Decellularization of cell sheets ............................................................................. 33 Confocal imaging of cell sheets ............................................................................ 34 DNA content ......................................................................................................... 34 Scanning electron microscopy (SEM) of cell sheets ............................................ 34 Growth factor ELISA assay .................................................................................. 35 Collagen quantification: ....................................................................................... 35 Recellularization of decellularized sheets ............................................................ 36 Statistical analysis................................................................................................. 36 Results ...................................................................................................................... 37 Scanning electron microscopy .............................................................................. 37 Extracellular matrix characterization .................................................................... 38 DNA quantification .............................................................................................. 39 Growth factors ELISA and collagen quantification ............................................. 39 Cell growth on decellularized sheets .................................................................... 40 Discussion ................................................................................................................ 43 Chapter 3 ........................................................................................................... 47 Assessment of static and perfusion methods for decellularization of PCL membrane supported fibrous cell sheet constructs ...................................... 47 IV Abstract .................................................................................................................... 48 Introduction .............................................................................................................. 49 Materials and methods ............................................................................................. 51 Membrane fabrication via melt electrospinning writing ...................................... 51 Primary human periodontal ligament cells (h-pdl cells) isolation and proliferation .............................................................................................................................. 51 Cell sheet harvesting ............................................................................................. 51 Decellularization protocols ................................................................................... 52 Confocal imaging of cell sheets ............................................................................ 56 Scanning electron microscopy of cell sheets ........................................................ 56 Quantification of DNA ......................................................................................... 57 Collagen quantification: ....................................................................................... 57 Growth factor Bioplex assay ................................................................................ 58 Statistical analysis................................................................................................. 59 Results ...................................................................................................................... 59 DNA removal and DNase effect on decellularization .......................................... 59 Structural preservation of decellularized sheets ................................................... 61 Collagen content preservation .............................................................................. 64 Growth factors retention ....................................................................................... 65 Discussion ................................................................................................................ 65 Chapter 4 ........................................................................................................... 69 The effect of decellularized periodontal ligament constructs on cell differentiation in vitro and periodontal regeneration in vivo. ......................