Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics

Abstract

Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (k(cat) and K(m)) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 microg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.

Figure 1

A Michael-type addition reaction between vinyl sulfone-functionalized multiarm PEGs and mono-cysteine adhesion peptides (step 1, in high stoichiometric deficit) or bis-cysteine MMP substrate peptides (step 2, to come up to stoichiometric equivalence) was used to form gels from aqueous solutions in the presence of cells. These elastic networks were designed to locally respond to local protease activity at the cell surface (step 3).

Figure 2

Kinetic analysis of MMP-1 degradation of two oligopeptides, GCRD-GPQG↓IAGQ-DRCG (●) and GCRD-GPQG↓IWGQ-DRCG (□), bearing MMP substrates flanked by charged amino acids and a Cys on both sides. Degradation was followed fluorometrically by the reaction of the free amine group with fluorescamine. Lineweaver–Burk plots yielded the corresponding kinetic parameters k_cat and K_m, for the enzymatic reaction.

Figure 3

Degradation kinetics of hydrogels respond to the MMP activity of the incorporated substrates: □, MMP(W)_X; ●, MMP(A)_X; ▵, (DF)_X. Kinetics was measured by a quantitative fluorescamine assay yielding a linear increase of the fluorescent signal with time for both sample types (A) and by conducting swelling measurements (i.e., the volume increase of the gel samples) (B).

Figure 4

(A) Fibroblasts radially invaded the adhesive and MMP-sensitive synthetic hydrogel matrix (bar = 250 μm). (B) Migration of spindle-like-shaped fibroblasts occurred in a cohort manner (bar = 150 μm). (C) Cell invasion distances increased approximately linear with culture time. (D) Cell invasion rate depended on the proteolytic activity of the incorporated peptide substrates. (E) Migration rate depended on adhesion ligand density, i.e., the concentration of RGD-containing peptide sites fixed throughout the three-dimensional material, in a biphasic manner. (F) Crosslink density of hydrogels influenced cell invasion dramatically.

Figure 5

Healing of critical size rat calvarial defects 4 wk after implantation. The ability of various matrices to promote healing was assessed by radiography (A–C Left) and histology (A–C Right). The healing outcome of the excised samples is reported as percentage of the total area of the original defect covered by mineralized tissue. Healing in vivo depended on the enzymatic sensitivity of the incorporated substrate: gels that were not susceptible to degradation by MMPs [A, the gel (DF)_X] or moderately sensitive [B, the gel MMP-(A)_X] showed significantly less bone formation than was observed in materials that were highly susceptible to invasion [C, the gel MMP-(W)_X]. This relationship was also observed quantitatively by radiography [D, P < 0.01 for both (DF)_X and MMP-(A)_X relative to MMP-(W)_X].