Recombinant Protein-co-PEG Networks as Cell-Adhesive and Proteolytically Degradable Hydrogel Matrixes. Part I: Development and Physicochemical Characteristics

University of Zurich, Zürich, Zurich, Switzerland
Biomacromolecules (Impact Factor: 5.75). 05/2005; 6(3):1226-38. DOI: 10.1021/bm049614c
Source: PubMed


Toward the development of synthetic bioactive materials to support tissue repair, we present here the design, production, and characterization of genetically engineered protein polymers carrying specific key features of the natural extracellular matrix, as well as cross-linking with functionalized poly(ethylene glycol) (PEG) to form hybrid hydrogel networks. The repeating units of target recombinant protein polymers contain a cell-binding site for ligation of cell-surface integrin receptors and substrates for plasmin and matrix metalloproteinases (MMPs), proteases implicated in wound healing and tissue regeneration. Hydrogels were formed under physiological conditions via Michael-type conjugate addition of vinyl sulfone groups of end-functionalized PEG with thiols of cysteine residues, representing designed chemical cross-linking sites within recombinant proteins. Cross-linking kinetics was shown to increase with the pH of precursor solutions. The elastic moduli (G') and swelling ratios (Q(m)) of the resulting hydrogels could be varied as a function of the stoichiometry of the reacting groups and precursor concentration. Optima of G' and Q(m), maximum and minimum, respectively, were obtained at stoichiometry ratios r slightly in excess of 1 (r = cysteine/vinyl sulfone). The pool of technologies utilized here represents a promising approach for the development of artificial matrixes tailored for specific medical applications.

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    • "To understand the crucial mechanism underlying the interaction between EOC cells and their extracellular microenvironment, we have employed a bioengineered hydrogel platform [120] (Fig. 3c). This 3D platform comprises synthetic protease-sensitive hydrogels that are formed from peptide-functionalized polyethylene glycol macromolecules via a factor XIII-catalysed reaction, similar to the fibrinogen cross-linking that occurs during natural fibrin coagulation [121, 122]. Interestingly, over-expression of KLK4–7 did not alter the proliferation rate of OV-MZ-6 EOC cells when cultured as conventional 2D monolayers [118]. "
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    ABSTRACT: Ovarian cancer, in particular epithelial ovarian cancer (EOC), is commonly diagnosed when the tumor has metastasized into the abdominal cavity with an accumulation of ascites fluid. Combining histopathology and genetic variations, EOC can be sub-grouped into Type-I and Type-II tumors, of which the latter are more aggressive and metastatic. Metastasis and chemoresistance are the key events associated with the tumor microenvironment that lead to a poor patient outcome. Kallikrein-related peptidases (KLKs) are aberrantly expressed in EOC, in particular, in the more metastatic Type-II tumors. KLKs are a family of 15 serine proteases that are expressed in diverse human tissues and involved in various patho-physiological processes. As extracellular enzymes, KLKs function in the hydrolysis of growth factors, proteases, cell membrane bound receptors, adhesion proteins, and cytokines initiating intracellular signaling pathways and their downstream events. High KLK levels are differentially associated with the prognosis of ovarian cancer patients, suggesting that they not only have application as biomarkers but also function in disease progression, and therefore are potential therapeutic targets. Recent studies have demonstrated the function of these proteases in promoting and/or suppressing the invasive behavior of ovarian cancer cells in metastasis in vitro and in vivo. Both conventional cell culture methods and three-dimensional platforms have been applied to mimic the ovarian cancer microenvironment of patients, such as the solid stromal matrix and ascites fluid. Here we summarize published studies to provide an overview of our understanding of the role of KLKs in EOC, and to lay the foundation for future research directions.
    Clinical and Experimental Metastasis 09/2013; 31(1). DOI:10.1007/s10585-013-9615-4 · 3.49 Impact Factor
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    • "A different material was therefore required for this study. Another synthetic material, poly (ethylene glycol) (PEG) has been applied as an inert 3D system with tunable elastic properties to culture different cell types (Rizzi and Hubbell, 2005; Nuttelman et al., 2006; Boontheekul et al., 2007; Bryant et al., 2008; Dikovsky et al., 2008; Peyton et al., 2008). Respective studies with chondrocytes , however, did not show facilitation of active stiffness sensing since no adhesion sites were incorporated (Bryant and Anseth, 2002; Park et al., 2004). "
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    ABSTRACT: Cells actively probe the stiffness of their surrounding and respond to it. The authors recently found that maintenance of the chondrogenic phenotype was directly influenced by this property in 2D. Since studies about this process in 3D are still largely absent, this study aimed to transfer this knowledge into a 3D environment. Agarose was modified with RGD to allow active stiffness sensing or RGE as a control. Hydrogels with different mechanical properties were produced by using different concentrations of agarose. Primary chondrocytes were incorporated into the gel, cultured for up to two weeks, and then constructs were analyzed. Cells were surrounded by their own ECM from an early stage and maintained their chondrogenic phenotype, independent of substrate composition, as indicated by a high collagen type II and a lack of collagen type I production. However, softer gels showed higher DNA and GAG content and larger cell clusters than stiff gels in both RGD- and RGE-modified agarose. The authors hypothesize that matrix elasticity in the tested range does not influence the maintenance of the chondrogenic phenotype in 3D but rather the size of the formed cell ECM clusters. The deviation of these findings from previous results in 2D stresses the importance of moving towards 3D systems that more closely mimic in vivo conditions. Copyright © 2011 John Wiley & Sons, Ltd.
    Journal of Tissue Engineering and Regenerative Medicine 11/2012; 6(10). DOI:10.1002/term.501 · 5.20 Impact Factor
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    • "A storage modulus (G′) of 0.5±0.1 kPa (trilysine 1 mg/ml) (Figure 1) was selected as it mimicked the mechanical properties of commonly injected polymers [42], [43]. As expected the G′ is independent of frequency and G″ (loss modulus) is weakly dependent on frequency [33], [44]. "
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    PLoS ONE 06/2011; 6(6):e21571. DOI:10.1371/journal.pone.0021571 · 3.23 Impact Factor
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