In vitro fabrication of extracellular matrix-rich tissue equivalents
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1
National University of Ireland Galway, Regenerative, Modular and Developmental Engineering Laboratory (REMODEL), Ireland
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2
National University of Ireland Galway, Centre for Research in Medical Devices (CURAM), Ireland
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3
Covidien, Sofradim Production, France
Introduction: Tendon tissue engineering is becoming increasingly important, as current surgical treatment modalities based on tissue grafts and non-degradable polymers have numerous drawbacks, including disease transmission, foreign body response and scar tissue formation[1]. Injectable cell based therapies are characterised by poor cell localisation at the site of injection[2]. Herein, we hypothesise that provision of a more relevant microenvironment with anisotropic topography and macromolecular crowding will maintain tenogenic phenotype in vitro[3],[4] and facilitate the fabrication of an extracellular matrix-rich tendon equivalent, promoting functional repair and regeneration in vivo.
Materials and Methods: Glycomer™ 631, co-polymer of glycolide (60%), trimethylene carbonate (26%) and dioxanone (14%) was dissolved in hexafluoro-isopropanol (HFIP) at 100mg/ml concentration. Using a rotating collector (3000 RPM) anisotropic electro-spun fibres were produced. Human patellar tendon tenocytes were expanded up to passage 3 in DMEM media, supplemented with 10% foetal bovine serum and 1% penicillin / streptomycin. 50,000 cells / cm2 were subsequently cultured for up to 28 days under the influence of Carragennan 75 μg/ml (crowder). Fibre orientation and fibre diameter distribution were assessed with Scanning Electron Microscopy (SEM). The influence of electro-spun fibres on cell viability and metabolic activity was assessed using Live/Dead® and alamarBlue® assays respectively. Cell morphometric analysis was carried out using DAPI and rhodamine conjugated phalloidin and subsequent image analysis (ImageJ). Tendon specific extracellular matrix (ECM) deposition was assessed with immunocytochemistry.
Results: The scaffold obtained by electrospinning Glycomer™ 631 at 3000RPM, is characterised by high degree of alignment, a fibre diameter distributed at the submicron level and adequate mechanical and thermal properties (Figure 1). Macromolecular crowding enhances tendon-specific matrix deposition from cells. This allows the fabrication of an ECM-rich tendon equivalent in vitro, where tenocytes obtain and maintain their physiological bidirectional morphology (Figure 2).
Conclusion: These in vitro data indicate that anisotropic electro-spun Glycomer™ 631 scaffolds along with macromolecular crowding provide a suitable microenvironment for tenocyte growth, facilitating the in vitro fabrication of a matrix-rich tendon equivalent. Further mechanical analysis, protein and gene assays and in vivo studies are under way.
Irish Research Council
References:
[1] Abbah S.A. et al, Stem Cell Research and Therapy, 5: 1-9, 2014.
[2] Becerra P. et al, Journal of Orthopaedic Research 31:1096-1102, 2013
[3] Spanoudes K. et al Trends in Biotechnology, 32: 472-482, 2014.
[4] Gaspar D. et al, Advanced Drug Delivery Reviews, 84: 240-256, 2015.
Keywords:
Extracellular Matrix,
Tissue Engineering,
Implant
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
New Frontier Oral
Topic:
Biomaterials in constructing tissue substitutes
Citation:
Spanoudes
K,
Bayon
Y,
Pandit
A and
Zeugolis
DI
(2016). In vitro fabrication of extracellular matrix-rich tissue equivalents.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.01640
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.
*
Correspondence:
Dr. Kyriakos Spanoudes, National University of Ireland Galway, Regenerative, Modular and Developmental Engineering Laboratory (REMODEL), Galway, Ireland, Email1
Dr. Yves Bayon, Covidien, Sofradim Production, Trevoux, France, yves.bayon@covidien.com
Dr. Abhay Pandit, National University of Ireland Galway, Centre for Research in Medical Devices (CURAM), Galway, Ireland, abhay.pandit@nuigalway.ie
Dr. Dimitrios I Zeugolis, National University of Ireland Galway, Regenerative, Modular and Developmental Engineering Laboratory (REMODEL), Galway, Ireland, dimitrios.zevgolis@ucd.ie