The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Engineering synthetic hydrogels on a molecular basis to introduce natural features that are important in instructing cell behavior is becoming increasingly crucial in biomaterial-based approaches for regenerative medicine and in cell biology to study cell-matrix interactions in three-dimensions (3D). Here, we used collagen gels and exploited the design flexibility of the biological, biochemical and physical characteristics offered by a PEG-based hydrogel system to systematically study the effect of specific extracellular microenvironments on the behavior of primary human fibroblasts in 3D. We firstly found that the proliferation profiles of fibroblasts from different patients cultured within collagen gels (3D) differed significantly from their behavior observed on tissue culture plastic (2D). Furthermore, using the biomimetic PEG-based matrix we showed that cell proliferation in 3D could be selectively manipulated via alteration of the gel characteristics. In particular, this study revealed that, in spite of matrix sensitivity to proteases (e.g. MMP) and the presence of cell-integrin binding sites, at high stiffness (elastic modulus, G' >1200 Pa) the matrix acts as a barrier for cells cultured in 3D. Finally, a comparison between the biomimetic PEG-based and collagen gels indicated that differences in their viscoelastic behaviours, determined by the nature of network structures and cross-links, may influence the mechanism(s) cells employ to remodel their 3D extracellular microenvironment. In conclusion, these studies highlight that for proliferation in 3D, compared to 2D, cells require strategies to overcome the physical impediment posed by the matrix. We also demonstrate that by exploiting the design flexibility of the characteristics offered by these biomimetic hydrogels, it is possible to separately investigate complex aspects characterizing the cell-matrix interactions in 3D; this has the potential to have great impact in regenerative medicine, as well as in cell biology and cancer research. Abstract Engineering synthetic hydrogels on a molecular basis to introduce natural features that are important in instructing cell behavior is becoming increasingly crucial in biomaterial-based approaches for regenerative medicine and in cell biology to study cell-matrix interactions in three-dimensions (3D). Here, we used collagen gels and exploited the design flexibility of the biological, biochemical and physical characteristics offered by a PEG-based hydrogel system to systematically study the effect of specific extracellular microenvironments on the behavior of primary human fibroblasts in 3D. We firstly found that the proliferation profiles of fibroblasts from different patients cultured within collagen gels (3D) differed significantly from their behavior observed on tissue culture plastic (2D). Furthermore, using the biomimetic PEG-based matrix we showed that cell proliferation in 3D could be selectively manipulated via alteration of the gel characteristics. In particular, this study revealed that, in spite of mat...

We present polymeric hydrogel biomaterials that are biomimetic both in their synthesis and degradation. The design of oligopeptide building blocks with dual enzymatic responsiveness allows us to create polymer networks that are formed and functionalized via enzymatic reactions and are degradable via other enzymatic reactions, both occurring under physiological conditions. The activated transglutaminase enzyme factor XIIIa was utilized for site-specific coupling of prototypical cell adhesion ligands and for simultaneous cross-linking of hydrogel networks from factor XIIIa substrate-modified multiarm poly(ethylene glycol) macromers. Ligand incorporation is nearly quantitative and thus controllable, and does not alter the network's macroscopic properties over a concentration range that elicits specific cell adhesion. Living mammalian cells can be encapsulated in the gels without any noticeable decrease in viability. The degradation of gels can be engineered to occur, for example, via cell-secreted matrix metalloproteinases, thus rendering these gels interesting for biomedical applications such as drug delivery systems or smart implants for in situ tissue engineering.

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

To address the need for bioactive materials toward clinical applications in wound healing and tissue regeneration, an artificial protein was created by recombinant DNA methods and modified by grafting of poly(ethylene glycol) diacrylate. Subsequent photopolymerization of the acrylate-containing precursors yielded protein-graft-poly(ethylene glycol) hydrogels. The artificial protein contained repeating amino acid sequences based on fibrinogen and anti-thrombin III, comprising an RGD integrin-binding motif, two plasmin degradation sites, and a heparin-binding site. Two-dimensional adhesion studies showed that the artificial protein had specific integrin-binding capability based on the RGD motif contained in its fibrinogen-based sequence. Furthermore, heparin bound strongly to the protein's anti-thrombin III-based region. Protein-graft-poly(ethylene glycol) hydrogels were plasmin degradable, had Young's moduli up to 3.5 kPa, and supported three-dimensional outgrowth of human fibroblasts. Cell attachment in three dimensions resulted from specific cell-surface integrin binding to the material's RGD sequence. Hydrogel penetration by cells involved serine-protease mediated matrix degradation in temporal and spatial synchrony with cellular outgrowth. Protein-graft-poly(ethylene glycol) hydrogels represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration.

No abstract

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.