title: Molecularly designed alginate hydrogels susceptible to local proteolysis as three-dimensional cellular microenvironments.
abstract: 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.
authors.affiliation: INEB, Instituto de Engenharia Biomédica, Divisão de Biomateriais, Universidade do Porto, Porto, Portugal.
authors.forename: Keila B
authors.initials: KB
authors.lastname: Fonseca
authors.forename: Sílvia J
authors.initials: SJ
authors.lastname: Bidarra
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Oliveira
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Granja
authors.forename: Cristina C
authors.initials: CC
authors.lastname: Barrias
mesh_terms: D000464:Alginates / Q000737:chemistry / Q000494:pharmacology*; D000595:Amino Acid Sequence; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D020723:Glucuronic Acid / Q000737:chemistry / Q000494:pharmacology; D006603:Hexuronic Acids / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D020100:Hydrogels / Q000737:chemistry / Q000494:pharmacology*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D020100:Hydrogels; D010455:Peptides; D020723:Glucuronic Acid; D020782:Matrix Metalloproteinases