MMP modified - Biodegradable hydrogel

Hyperactivation and release of extracellular proteases has been shown to underly many disease pathologies, including neurodegeneration, inflammation, cardiovascular, and cancer1. Matrix metalloproteases (MMPs) are the main group of enzymes involved in extracellular matrix (ECM) degradation and remodeling2. MMPs, which are either soluble or membrane-bound, are critical for the development of multiple different tissues3. MMPs are also upregulated during repair and ECM remodeling3. More recent evidence has additionally implicated the importance of MMPs during neurodevelopment, as it has been suggested MMPs play a critical role in remolding the ECM to allow spinal motor neurons to exit through the basal lamina in order to innervate peripheral targets4. However, MMPs are more commonly studied in disease pathologies. Overexpression of MMPs can induce, and result from, disease states. For example, highly metastatic cancer cells release MMPs to remodel the extracellular matrix and create an invasion phenotype5. This aberrant ECM remodeling is also observed in cardiomyopathy6, osteoarthritis7, and fibrosis8. There are several MMPs that play critical roles in development, healthy tissue function, repair, and disease pathology, including MMP1, MMP2, MMP3, MMP9 and MMP132. These MMPs can target cleavage sites on different ECM proteins. For example, MMP1 can cleave collagen (I, II, III, VII, and X), gelatins, aggrecan, entactin, and tenascin9. Other MMPs also target collagens (MMP2/3/9/13), laminin (MMP3), and fibronectin (MMP3/13)3. Mutations in MMPs can induce a variety of developmental and repair issues, such as the following: defects in tracheal development (MMP110), reduced neovascularization and lung development (MMP211,12), altered development of the neuromuscular junction (MMP33), defects in bone development, neuronal remyelination, vascular remodeling, and angiogenesis (MMP91416), and defects in bone remodeling and increased collagen accumulation in atherosclerotic plaques (MMP1317,18). Therefore, understanding normal and aberrant MMP function, and subsequent changes in ECM structure, in different cell types can aid in furthering our understanding of many diseases.

In order to study MMP activity and ECM degradation, experimenters have developed protease-sensitive hydrogels19. In their seminal work, West and colleagues incorporated MMP1 reactive peptides into polyethylene glycol (PEG) hydrogels and found targeted MMP1-mediated cleavage19. As a result, the incorporation of degradable substrates into 3D matrices has been duplicated in many other studies with PEG, as well as other hydrogels, such as hyaluronic acid20, Pluronic triblock copolymer21, silk and elastin22, collagen23, alginate24, and heparin25.

The use of hydrogels with incorporated MMP substrates has been employed to study cancer cell migration and invasion. Tumor growth and metastasis are often promoted by cancer-associated fibroblasts, which modify the surrounding ECM to create fibrillar structures that may act as tracks for migrating cancer cells26. In order to remodel the ECM and promote migration and invasion, fibroblasts and cancer cells release ECM proteins, such as collagen27, and fibronectin28, as well as MMPs3, 26. By incorporating MMP cleavable substrates, experimenters can measure cell spreading, migration, and invasion in a number of cancer cell models. For example, covalent anchoring of a general MMP cleavable substrate into a PEG hydrogel was shown to support the growth, spreading, and migration of U87 cells, a glioblastoma cell line29. Invasion is often analogous to cancer metastasis, and the application of hydrogels with MMP cleavable substrates allows researchers to measure the level of invasion through ECM barriers. Increased invasion in these modified hydrogels has been observed with multiple cancer cell lines, including melanoma (WM239A,30) and breast (MDA-MB-231,31). As stated above, in addition to cancer cells, fibroblasts release MMPs, which promotes an environment for tumor growth and metastasis. Therefore, researchers have also measured fibroblast migration and invasion in MMP modified hydrogels. Similar to cancer cells, the presence of MMP cleavable substrates promotes migration and invasion of fibroblasts32,33. These data illustrate how MMP substrate modified hydrogels may be employed to study cancer growth and metastasis and prove critical to developing pharmacotherapies for a variety of cancers.

Besides their critical role in tumor growth and metastasis, MMPs are expressed in a number of other cell types during development, injury repair, and tissue function. MMPs regulate morphogenic development of the nervous system4,13, mammary gland34, lung12, and bone14. The development of MMP cleavable hydrogels has allowed for the study of these developmental programs in a controlled 3D environment. Endothelial cell proliferation, adhesion, and migration is a critical step in vascularization35. Endothelial cell survival, adhesion, and migration are enhanced in 3D hydrogel cultures with MMP substrates36. Furthermore, Turturro et. al. (2013) created a 3D hydrogel with gradients of MMP degradable peptides, substrata stiffness, and RGD, an adhesive ligand37. As a model to study vascular sprout formation, the authors used human umbilical vein endothelial and umbilical artery smooth muscle cells and measured aggregate cell invasion. They observed increased aggregate cell invasion toward gradients of higher levels of MMP substrate peptides, further illustrating the role of MMPs in cell migration and invasion. Several other studies have also tested cell migration and invasion in MMP substrate modified hydrogels3840. These studies show the utility of MMP substrate modified hydrogels in studying tissue development.

With the recent explosion of stem cell research scientists have put effort into creating the most accurate 3D cell culture systems to mimic in vivo environments41. This has led scientists to explore different hydrogel types and modifications to promote stem cell survival, proliferation, and differentiation. As a result, studies have tested the role of ECM remodeling, via MMP protease activity, in stem cell proliferation and fate. Accordingly, MMP degradable hydrogels have been shown to promote the expression of differentiation markers in human mesenchymal stem cells42. Moreover, a critical role for MMPs is to degrade the local ECM allowing for the necessary cell growth and spreading needed to induce proliferation and differentiation. Thus, MMP modifiable hydrogels have been shown to enhance cell spreading, traction forces, differentiation, migration, and invasion of both human and mouse mesenchymal stem cells38,43. Finally, increased neuronal differentiation and neurite outgrowth has also been shown in stem cells cultured in MMP degradable hydrogels, likely due to ECM remodeling and greater migratory area44. Overall, these data further demonstrate the variety of cell types and cellular behaviors that may be studied with MMP substrate modifiable hydrogels.

Cell Type Behavior Reference Table for VitroGel MMP

Cell Behavior List for VitroGel MMP (EXPAND)
Tissue/Organ TypeCell TypePeptide SequenceBehavior
BreastMammary gland MCF10AKCGPQG↑IWGQCKIncreased MMP activity in response to TGF-ß1
Cancer/tumorBreast MDA-MB-231Not specifiedEnhanced cell invasion
Glioma U87CGPQGIWGQCSupport cell growth, spreading, and migration
Melanoma WM239AKKCGGPQG↓IWGQGCKKPromoted cell invasion
Connective tissueHuman foreskin fibroblastsGCRD-GPQGIWGQ-DRCGIncreased radial cell migration
Human foreskin fibroblastsGCRD-GPQG↓IWGQ-DRCGIncreased cell invasion and substrata degradation
Stem cellsHuman mesenchymal stem cellsNot specifiedIncreased cell differentiation marker expression
Human mesenchymal stem cellsGCRDGPQGIWGQDRCGPromoted neuronal differentiation and neurite outgrowth
Human mesenchymal stem cellsGCRDVPMS↓MRGGDRCGIncreased cell spreading, traction forces, and differentiation
Mouse mesenchymal stem cellsGCREG-PQGIWGQ-ERCGIncreased cell spreading and migration
VascularHuman umbilical vein endothelial cellGCRDGPQGIWGQDRCGPromoted cell adhesion, migration, and survival
Human umbilical vein endothelial and umbilical artery smooth muscle cellsGGVPMS↓MRGGKIncreased aggregate cell invasion toward gradient of peptide
Human umbilical vein endothelial cellsGDGIPVS↓LRSGGK (MMP2) and GDIPES↓LRAGGKIncreased cell invasion



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