Molecularly designed alginate hydrogels susceptible to local proteolysis as three-dimensional cellular microenvironments.

The development of sophisticated three-dimensional (3-D) cell culture microenvironments that recreate some of the complexity of the natural extracellular matrix (ECM) remains a challenging task. Here, the modification of alginate through partial crosslinking with a matrix metalloproteinase (MMP) cleavable peptide (proline-valine-glycine-leucine-isoleucine-glycine, PVGLIG) is described, and its use in the preparation of injectable, in situ crosslinkable hydrogel-like matrices is proposed. PVGLIG-grafted alginates were synthesized by carbodiimide chemistry and characterized. Their biological performance was evaluated by comparing the response of 3-D cultured mesenchymal stem cells (MSCs) to alginate hydrogels containing only cell-adhesion peptides (RGD-alginate) or both peptides (PVGLIG/RGD-alginate). After 1 week, cells remained essentially round within RGD-alginate, while they exhibited an elongated morphology within PVGLIG/RGD-alginate hydrogels, forming cellular networks. This suggests that cells were able to structurally reorganize the matrix, through enzymatic hydrolysis of PVGLIG residues, overcoming biophysical hydrogel resistance. As shown by gelatine-zymography, MSC presented higher activity of MMP-2 when cultured within alginate functionalized with MMP-sensitive peptide, suggesting that the cell's proteolytic phenotype was modulated by the matrix composition. Additionally, PVGLIG/RGD-alginate hydrogels were clearly degraded in cell culture. Taken together, the results demonstrate that the co-incorporation of MMP-labile peptides in cell-adhesive RGD-alginate hydrogels improved their performance as ECM analogues, providing a more dynamic and physiological 3-D cellular microenvironment.

[1]  M. Stack,et al.  Microenvironmental Regulation of Membrane Type 1 Matrix Metalloproteinase Activity in Ovarian Carcinoma Cells via Collagen-induced EGR1 Expression* , 2007, Journal of Biological Chemistry.

[2]  Robert Langer,et al.  Incorporation of a matrix metalloproteinase-sensitive substrate into self-assembling peptides - a model for biofunctional scaffolds. , 2008, Biomaterials.

[3]  D. Mooney,et al.  Designing alginate hydrogels to maintain viability of immobilized cells. , 2003, Biomaterials.

[4]  Motoharu Seiki,et al.  The cell surface: the stage for matrix metalloproteinase regulation of migration. , 2002, Current opinion in cell biology.

[5]  Ivan Stamenkovic,et al.  Functional structure and composition of the extracellular matrix , 2003, The Journal of pathology.

[6]  R. Langer,et al.  Synthesis and characterization of dextran-peptide-methotrexate conjugates for tumor targeting via mediation by matrix metalloproteinase II and matrix metalloproteinase IX. , 2004, Bioconjugate chemistry.

[7]  Andrew J. Ewald,et al.  Matrix metalloproteinases and the regulation of tissue remodelling , 2007, Nature Reviews Molecular Cell Biology.

[8]  David J Mooney,et al.  Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix. , 2007, Biomaterials.

[9]  R. Soares,et al.  Immobilization of human mesenchymal stem cells within RGD-grafted alginate microspheres and assessment of their angiogenic potential. , 2010, Biomacromolecules.

[10]  Jeffrey A. Hubbell,et al.  Polymeric biomaterials with degradation sites for proteases involved in cell migration , 1999 .

[11]  Z. Werb,et al.  ECM signalling: orchestrating cell behaviour and misbehaviour. , 1998, Trends in cell biology.

[12]  David J Mooney,et al.  Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. , 2005, Biomaterials.

[13]  G. Butler,et al.  The Soluble Catalytic Domain of Membrane Type 1 Matrix Metalloproteinase Cleaves the Propeptide of Progelatinase A and Initiates Autoproteolytic Activation , 1996, The Journal of Biological Chemistry.

[14]  M. Barbosa,et al.  Injectability of a bone filler system based on hydroxyapatite microspheres and a vehicle with in situ gel-forming ability. , 2008, Journal of biomedical materials research. Part B, Applied biomaterials.

[15]  Matthias P. Lutolf,et al.  Designing materials to direct stem-cell fate , 2009, Nature.

[16]  S. Kennedy,et al.  A fluorescence-based protein assay for use with a microplate reader. , 1993, Analytical biochemistry.

[17]  Jon A. Rowley,et al.  Controlling Mechanical and Swelling Properties of Alginate Hydrogels Independently by Cross-Linker Type and Cross-Linking Density , 2000 .

[18]  P. Friedl,et al.  The biology of cell locomotion within three-dimensional extracellular matrix , 2000, Cellular and Molecular Life Sciences CMLS.

[19]  David J Mooney,et al.  Alginate hydrogels as biomaterials. , 2006, Macromolecular bioscience.

[20]  Motoharu Seiki,et al.  MT1‐MMP: A potent modifier of pericellular microenvironment , 2006, Journal of cellular physiology.

[21]  P. Neth,et al.  Scientific category: Stem cells in hematology MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines , 2006 .

[22]  Mark W. Tibbitt,et al.  Hydrogels as extracellular matrix mimics for 3D cell culture. , 2009, Biotechnology and bioengineering.

[23]  Z. Werb.  ECM and Cell Surface Proteolysis: Regulating Cellular Ecology , 1997, Cell.

[24]  L. Cantley,et al.  Determination of protease cleavage site motifs using mixture-based oriented peptide libraries , 2001, Nature Biotechnology.

[25]  D J Mooney,et al.  Alginate hydrogels as synthetic extracellular matrix materials. , 1999, Biomaterials.

[26]  N. Peppas,et al.  Synthesis and characterization of insulin-transferrin conjugates. , 2006, Bioconjugate chemistry.

[27]  P. Ma,et al.  Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties. , 2001, Biomaterials.

[28]  D. Mooney,et al.  Cellular cross-linking of peptide modified hydrogels. , 2005, Journal of biomechanical engineering.

[29]  M. Aslam,et al.  Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences , 1998 .

[30]  H. Nagase,et al.  Progress in matrix metalloproteinase research. , 2008, Molecular aspects of medicine.

[31]  R. Bareille,et al.  The effect of the co-immobilization of human osteoprogenitors and endothelial cells within alginate microspheres on mineralization in a bone defect. , 2009, Biomaterials.

[32]  J. Hubbell,et al.  Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering , 2005, Nature Biotechnology.