The cellular response to transglutaminase-cross-linked collagen
Introduction
Collagen is a very popular biomaterial due to its biocompatibility, i.e. the ability to support cell adhesion and proliferation. It is also biodegradable and only weakly antigenic—able to persist in the body without developing a foreign body response that could lead to its premature rejection [1]. The replacement of skin with artificial collagen–GAG matrices has been investigated since the early 1980s and is now in clinical use [2], [3]. The primary reason for the usefulness of collagen in biomedical applications is that collagen can form fibres with extra strength and stability through its self-aggregation and in vivo cross-linking [4]. Unfortunately, collagen, like many natural polymers, once extracted from its original source and then reprocessed, suffers from weak mechanical properties, thermal instability and ease of proteolytic breakdown. To overcome these problems, collagen has been cross-linked by a variety of agents—a subject of much recent research to find methods of preventing rapid absorption by the body [4]. However, these methods suffer from the problem that the residual catalysts, initiators and unreacted or partially reacted cross-linking agents used can be toxic or cause inflammatory responses if not fully removed or, simply, not cost-effective or practical at the large scale [5], [6], [7]. As a consequence, research continues to find alternative methods to stabilise collagen which are natural, milder, efficient and more practical.
Transglutaminases (EC 2.3.2.13) are a group of enzymes that can catalyse several types of post-translational modifications to proteins. The most important of these reactions results in the cross-linking of peptides or proteins to form multimers via a ε(γ-glutamyl)lysine linkage using the side chains of lysine and glutamine residues. Transglutaminases are also able to covalently attach primary amine containing compounds to peptide bound glutamine, facilitating modification of the physical, chemical and biological properties of proteins [8]. For these reasons, transglutaminases have been utilised by the commercial sector in many different processes and have attracted much attention from the research community [9]. Microbial transglutaminase has been used to cross-link gelatin matrices to further increase their strength [10] and, also, to incorporate cell adhesion factors within the gel matrix, resulting in an enhancement of cell proliferation [11].
Interestingly, a novel component of the cell/tissue response to cell damage and stress is tissue transglutaminase (tTG), a Ca2+-dependent mammalian form of the enzyme, which modulates cell–matrix interactions, tissue stability and a variety of other cell functions [12], [13]. The entire tissue repair process is regulated by the interaction of cells with the surrounding extracellular matrix (ECM), ensuring cell adhesion, survival and proliferation [14], [15]. To date, the cross-linking function of tTG in the ECM leading to ECM stabilisation/remodelling has been identified in a number of biological processes important for tissue repair [12]: in addition, at least three of the nine genes so far characterised are thought to be naturally involved in the wound healing response process [see review, 16].
The aim of this study was to investigate the use of the two different transglutaminases; the mammalian (tTG; TG2; TG-2; isolated from guinea pig liver) and the microbial enzyme (mTG; isolated from Streptoverticillium mobaraense) in the modification of collagen type I with the view to investigate potential application as a biocompatible natural polymer for use in soft and hard tissue repair.
Section snippets
Materials and methods
All water used was deionised using an Elgastat System 2 water purifier (ELGA Ltd., UK) and a Milli-Q water purifier (Millipore Waters, UK). All chemicals were purchased from Sigma-Aldrich, Poole, UK, unless otherwise stated. Sterile preparation of stock solutions and chemicals were performed either by filtration through a 0.22 μm Whatmann sterile filter and/or autoclaving at 121 °C at 1 bar for 1 h.
Cross-linking of collagen by microbial and tissue transglutaminases
Native collagen (type I) was treated with both tTG and mTG, separately, in order to catalyse the formation of ε-(γ-glutamyl)lysine cross-linking. The extent of cross-linking for each of the TG treatments is shown in Table 1. Treatment of collagen with increasing concentrations of TG led to a corresponding increase in the amount of ε-(γ-glutamyl)lysine bonds present—with up to 1 mol of cross-link per mole of collagen monomer. Treatment with mTG gave a much greater increase (almost two-fold) in
Discussion
We have confirmed previous work [25], in demonstrating that treatment of collagen type I matrices with transglutaminases results in the incorporation of ε(γ-glutamyl)lysine, with both mTG and tTG introducing similar amounts of cross-link per unit activity. It has been previously demonstrated that collagen type I shows greater resistance to proteolytic degradation by matrix metalloproteinase 1 (MMP-1) in vitro after cross-linking by tTG [26]. Importantly, we have shown that collagen modified
Conclusion
In conclusion, the cellular response of HFDF and HOB cells grown on transglutaminase-cross-linked collagen is altered in such a manner that they show enhanced attachment, spreading and proliferation. Another important finding was that HOB cells differentiated faster on the cross-linked collagen. The modified collagen was also degraded at a much slower rate than native collagen further enhancing its in vivo efficacy as a biomaterial. Transglutaminases, therefore, show considerable potential as
Acknowledgements
The above work has been filed as a GB patent application and supported by grant number GR/521755/01 from the EPSRC and BLC Leather Technology Centre Ltd., Northampton, UK.
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