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Tissue transglutaminase (TG-2) modified amniotic membrane: a novel scaffold for biomedical applications.

Abstract
The amniotic membrane (AM) is considered as a natural cell culture substrate and has occasionally been exploited in regenerative medicine especially for ocular surface reconstruction and dermal wound healing applications. However, its use is limited by its relatively weak mechanical strength, difficulty during manual handling and susceptibility to proteolytic degradation in vivo. Therefore, in this study we aimed to enhance the mechanical and biological characteristics of the AM by enzymatically cross-linking it using tissue transglutaminase (TG)-a calcium-dependent enzyme capable of forming stable ε(γ-glutamyl)lysine cross-linkages. Using a biological catalyst such as TG does not only prevent denaturation during sample preparation but also minimizes the potential of residual chemical cross-linking agents compared to alternative methodologies. Human AM, sourced from elective caesarean sectioning, were treated with TG, bovine serum albumin and/or a no-treatment control. Samples were then compared in terms of their physical and (scanning electron microscopy (SEM), transparency, mechanical strength, susceptibility to proteolytic degradation) biological characteristics (in vitro cell culture, activation of dendritic cells (DC)) and their in vivo biocompatibility/angiogenic capacity (chick chorioallantoic membrane assay). TG-treated AM exhibited enhanced mechanical strength and greater resistance to proteolytic/collagenase degradation compared to the control(s). SEM imaging of the TG-treated membrane summarized a significantly closer association and greater interconnectivity of individual collagen fibres yet it had no effect on the overall transparency of the AM. In vitro cell culture demonstrated no detrimental effect of TG-treatment on the AM in terms of cell attachment, spreading, proliferation and differentiation. Moreover, an 'immune response' was not elicited based on extended in vitro culture with human-monocyte-derived DC. Interestingly, the TG-treated AM still allowed angiogenesis to occur and in some instances, demonstrated an enhancement compared to the control (n = 5). We hereby demonstrate that treating the AM with the cross-linking enzyme, TG, results in a novel biomaterial with enhanced mechanical and biological characteristics. Above all, this modified membrane demonstrates greater strength, maintains in vitro cell growth, retains optical transparency and allows angiogenesis to occur without inducing an immune response. Altogether, this study demonstrates the feasibility of TG as an alternate cross-linking treatment for the production of novel biomaterials and suggests that TG-treated AM may now be more commonly exploited as a therapeutic dressing for ocular or wound applications.
AuthorsDavid Y S Chau, Sheridan V Brown, Melissa L Mather, Victoria Hutter, Naing L Tint, Harminder S Dua, Felicity R A J Rose, Amir M Ghaemmaghami
JournalBiomedical materials (Bristol, England) (Biomed Mater) Vol. 7 Issue 4 Pg. 045011 (Aug 2012) ISSN: 1748-605X [Electronic] England
PMID22652528 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Biocompatible Materials
  • Cross-Linking Reagents
  • Protein Glutamine gamma Glutamyltransferase 2
  • Transglutaminases
  • Collagenases
  • GTP-Binding Proteins
Topics
  • Amnion (cytology)
  • Animals
  • Biocompatible Materials (chemistry)
  • Cell Culture Techniques
  • Cell Differentiation
  • Cell Membrane (metabolism)
  • Cell Survival
  • Cells, Cultured (cytology)
  • Chick Embryo
  • Chorioallantoic Membrane (metabolism)
  • Collagenases (chemistry)
  • Cross-Linking Reagents (chemistry)
  • Equipment Design
  • Flow Cytometry (methods)
  • GTP-Binding Proteins (metabolism)
  • Humans
  • Materials Testing
  • Microscopy, Electron, Scanning (methods)
  • Models, Statistical
  • Protein Glutamine gamma Glutamyltransferase 2
  • Regeneration
  • Stress, Mechanical
  • Tissue Scaffolds (chemistry)
  • Transglutaminases (metabolism)
  • Wound Healing

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