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An Adipose Extracellular Matrix-Derived Biomaterial for Soft Tissue Reconstruction

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An Adipose Extracellular Matrix-Derived Biomaterial for Soft Tissue Reconstruction AN ADIPOSE EXTRACELLULAR MATRIX-DERIVED BIOMATERIAL FOR SOFT TISSUE RECONSTRUCTION by Iwen Wu A Dissertation Submitted to Johns Hopkins University in Conformity with the Requirements for the Degree of Doctor of Philosophy Johns Hopkins University Baltimore, Maryland May, 2013 CONTENTS Title ...................................................................................................................................... i Contents .............................................................................................................................. ii List of Tables ....................................................................................................................... v List of Figures ..................................................................................................................... vi Acknowledgments .............................................................................................................. vii Abstract .............................................................................................................................. ix 1. Introduction ................................................................................................................... 1 2. Adipose Tissue Engineering ............................................................................................ 3 2.1 Introduction .......................................................................................................... 3 2.1.1 Current clinical strategies .......................................................................... 4 2.2 Biomaterials ......................................................................................................... 5 2.2.1 Synthetic polymers ................................................................................... 5 2.2.2 Naturally derived scaffolds ........................................................................ 7 2.2.3 Extracellular matrix- based materials .......................................................... 7 2.3 Cell sources ........................................................................................................ 11 2.3.1 Cells of adipose tissue ............................................................................. 11 2.3.2 Adult stem cells ...................................................................................... 11 2.3.3 Embryonic stem cells .............................................................................. 12 2.3.4 Induced pluripotent stem cells .................................................................. 13 3. Development of an Adipose Matrix – Based Biomaterial ................................................. 14 3.1 Introduction ........................................................................................................ 14 3.2 Materials and Methods ........................................................................................ 16 3.2.1 Adipose extracellular matrix preparation .................................................. 16 3.2.2 Material characterization ......................................................................... 16 3.2.3 Crosslinking ........................................................................................... 19 ii 3.3 Results ............................................................................................................... 20 3.3.1 Adipose extracellular matrix .................................................................... 20 3.3.2 Material characterization ......................................................................... 21 3.3.3 Crosslinking ........................................................................................... 27 3.4 Discussion .......................................................................................................... 30 4. In Vitro Bioactivity of the Adipose Extracellular Matrix ................................................. 33 4.1 Introduction ........................................................................................................ 33 4.2 Materials and Methods ........................................................................................ 34 4.2.1 Adipose extracellular matrix preparation .................................................. 34 4.2.2 Adipose-derived stem cell isolation .......................................................... 35 4.2.3 In vitro cell studies .................................................................................. 35 4.3 Results ............................................................................................................... 38 4.3.1 Cell viability on cross-linked matrices ...................................................... 38 4.3.2 Migration assay ...................................................................................... 38 4.3.3 Adipogenic differentiation of cells in 2D culture on the adipose matrices .... 39 4.3.4 Adipogenic differentiation of cells in 3D constructs comparing the adipose and dermis ECM ........................................................................................... 39 4.3.5 HUVEC tube formation assay .................................................................. 41 4.4 Discussion .......................................................................................................... 43 5. The Adipose Extracellular Matrix as a Scaffold for In Vivo Adipose Tissue Regeneration . 46 5.1 Introduction ........................................................................................................ 46 5.2 Materials and Methods ........................................................................................ 47 5.2.1 Adipose extracellular matrix preparation .................................................. 47 5.2.2 In vivo studies ........................................................................................ 48 5.2.3 Histology ............................................................................................... 50 5.2.4 Immunostaining ...................................................................................... 50 5.3 Results ............................................................................................................... 50 5.3.1 In vivo biocompatibility of crosslinked adipose ECM ................................ 50 iii 5.3.2 Long term biocompatibility of adipose ECM ............................................ 51 5.3.3 In vivo volume retention compared to fat grafting ..................................... 53 5.3.4 In vivo adipogenesis of adipose ECM with ASCs ..................................... 54 5.4 Discussion .......................................................................................................... 57 6. Discussion and future work ........................................................................................... 60 BIBLIOGRAPHY ......................................................................................................................... 63 CURRICULUM VITAE ............................................................................................................... 72 iv LIST OF TABLES Table 3.1Denaturation peak temperature .......................................................................... 26 Table 4.1 RT-PCR Primer sequences ............................................................................... 37 v LIST OF FIGURES Figure 3.1 Processed adipose tissue matrix ...................................................................... 20 Figure 3.2 Scanning electron microscopy images of adipose ECM ................................. 21 Figure 3.3 1D SDS-PAGE gels. ........................................................................................ 23 Figure 3.4 Proteomic characterization of the adipose and dermis ECM .......................... 24 Figure 3.5 DSC thermogram of the adipose ECM through the processing stages ........... 26 Figure 3.6 Mechanical properties of the adipose ECM and commercial fillers ............... 28 Figure 3.7 Crosslinked matrices show increased resistance to enzymatic degradation. ... 29 Figure 4.1 LIVE/DEAD staining of cells on crosslinked matrices ................................... 38 Figure 4.2 In vitro adipogenesis of ASCs seeded on adipose matrix in 2D culture ......... 39 Figure 4.3 In vitro chemoattractive properties and adipo-inductive capacity .................. 40 Figure 4.4 HUVEC tube formation assay with adipose ECM. ......................................... 42 Figure 5.1 In vivo biocompatibility of crosslinked matrices ............................................ 51 Figure 5.2 In vivo biocompatibility in a rat model ........................................................... 52 Figure 5.3 De novo adipogenesis at implant periphery .................................................... 52 Figure 5.4 Comparison of in vivo fat grafting and adipose ECM .................................... 53 Figure 5.5 In vivo adipogenesis for adipose ECM with ASC delivery ............................ 56 vi ACKNOWLEDGMENTS There are many individuals whose help and contributions made this work possible. The members of the Elisseeff lab who trained me in the numerous techniques and provided intellectual support were critical to the development of this work, as well as my personal development as a researcher. Dr. Zayna Nahas pioneered much of the early work that would later become the foundation for this thesis. Dr. Alexandra Condé-Green has also been instrumental in the development
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