Matrix Metalloproteinase-sensitive Hydrogels Research Articles

IDG-SW3 Osteocyte Differentiation and Bone Extracellular Matrix Deposition Are Enhanced in a 3D Matrix Metalloproteinase-Sensitive Hydrogel

Osteocytes reside within a heavily mineralized matrix making them difficult to study in vivo and to extract for studies in vitro. IDG-SW3 cells are capable of producing mineralized collagen matrix and transitioning from osteoblasts to mature osteocytes, thus offering an alternative to study osteoblast to late osteocyte differentiation in vitro. The goal for this work was to develop a 3D degradable hydrogel to support IDG-SW3 differentiation and deposition of bone ECM. In 2D, the genes Mmp2 and Mmp13 increased during IDG-SW3 differentiation and were used as targets to create a MMP-sensitive poly(ethylene glycol) hydrogel containing the peptide crosslink GCGPLG-LWARCG and RGD to promote cell attachment. IDG-SW3 differentiation in the MMP-sensitive hydrogels improved over non-degradable hydrogels and standard 2D culture. Alkaline phosphatase activity at day 14 was higher, Dmp1 and Phex were 8.1-fold and 3.8-fold higher, respectively, and DMP1 protein expression was more pronounced in the MMP-sensitive hydrogels compared to non-degradable hydrogels. Cell-encapsulation density (cells/ml precursor) influenced formation of dendrite-like cellular process and mineral and collagen deposition with 80×106 performing better than 2×106 or 20×106, while connexin 43 was not affected by cell density. The cell density effects were more pronounced in the MMP-sensitive hydrogels over non-degradable hydrogels. This study identified that high cell encapsulation density and a hydrogel susceptible to cell-mediated degradation enhanced mineralized collagen matrix and osteocyte differentiation. Overall, a promising hydrogel is presented that supports IDG-SW3 cell maturation from osteoblasts to osteocytes in 3D.

Surface-Mediated Stimuli-Responsive Gene Delivery Based on Breath Figure Film Combined with Matrix Metalloproteinase-Sensitive Hydrogel.

Surface-mediated gene delivery appears to be potential gene delivery modes for various applications. Still, controlled and smart delivery manners are required especially considering the need for gene therapy to deliver gene with selectivity. A surface that can effectively payload DNA, promote cell adhesion, and stimuli response is an important prerequisite. Here, we report a matrix metalloproteinase (MMP)-responsive surface-mediated gene delivery system by combining MMP-degradable hydrogel with a breath figure (BF) porous film. The MMP-degradable hydrogel containing plasmid DNA was loaded into the surface pores of the BF film as DNA reservoirs. The uppersurface without hydrogel on the BF film served as footholds of integrin adhesions. MMP is one of the important endogenous signals in tumor-related pathologic changes, and MMP expressions in cancer cells are significantly higher than those in normal cells. Consequently, our surface-mediated gene delivery locally and rapidly released the payload DNA in response to cancer cells and transfected them. This work highlights the importance of the combination of stimuli-response and surface-mediated gene delivery to functional materials, showing good potential applications in the field of gene therapy.

A three-dimensional ex vivo tri-culture model mimics cell-cell interactions between acute myeloid leukemia and the vascular niche.

Ex vivo studies of human disease, such as acute myeloid leukemia, are generally limited to the analysis of two-dimensional cultures which often misinterpret the effectiveness of chemotherapeutics and other treatments. Here we show that matrix metalloproteinase-sensitive hydrogels prepared from poly(ethylene glycol) and heparin functionalized with adhesion ligands and pro-angiogenic factors can be instrumental to produce robust three-dimensional culture models, allowing for the analysis of acute myeloid leukemia development and response to treatment. We evaluated the growth of four leukemia cell lines, KG1a, MOLM13, MV4-11 and OCI-AML3, as well as samples from patients with acute myeloid leukemia. Furthermore, endothelial cells and mesenchymal stromal cells were co-seeded to mimic the vascular niche for acute myeloid leukemia cells. Greater drug resistance to daunorubicin and cytarabine was demonstrated in three-dimensional cultures and in vascular co-cultures when compared with two-dimensional suspension cultures, opening the way for drug combination studies. Application of the C-X-C chemokine receptor type 4 (CXCR4) inhibitor, AMD3100, induced mobilization of the acute myeloid leukemia cells from the vascular networks. These findings indicate that the three-dimensional tri-culture model provides a specialized platform for the investigation of cell-cell interactions, addressing a key challenge of current testing models. This ex vivo system allows for personalized analysis of the responses of patients’ cells, providing new insights into the development of acute myeloid leukemia and therapies for this disease.

Matrix metalloproteinase-sensitive hydrogel microparticles for pulmonary drug delivery of small molecule drugs or proteins.

Hydrogel microparticles are particularly attractive for pulmonary drug delivery. Their size can be engineered for efficient delivery into the bronchi, where they subsequently swell, avoiding macrophage uptake. In this study, enzyme-responsive peptide functionalized poly(ethylene glycol) (PEG) based hydrogel microparticles were synthesized by an emulsion polymerization. Here, we demonstrate that these microparticles are nontoxic and demonstrated their viability as a drug carrier by studying the encapsulation and release of three types of drugs: a hydrophobic (dexamethasone), a hydrophilic (methylene blue) and a protein (horseradish peroxidase)-based drug. The release of each of these three drugs was studied in the presence of varying concentrations of matrix metalloproteinase (MMP). Each of the three types of drugs were able to be encapsulated in the microparticles, and we further showed that the protein is still functional after release.

Biomimetic Hydrogels with Immobilized EphrinA1 for Therapeutic Angiogenesis

The formation of a microvasculature is regulated in large part by cell-cell interactions. Ephrins and their Eph receptors mediate cell adhesion, repulsion, and migration, all critical processes in angiogenesis. (1) Here we use a covalently immobilized ephrinA1, conjugated to poly(ethylene glycol), to induce vessel formation both in vitro and in vivo in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Human umbilical vein endothelial cell (HUVEC) tubulogenesis in matrix metalloproteinase-sensitive hydrogels was visualized from 6 h to 7 days in response to three different concentrations of PEG-ephrinA1. The deposition of extracellular matrix proteins collagen IV and laminin that stabilize tubule formation were imaged, quantified, and found to be dependent on PEG-ephrinA1 concentration. To confirm the importance of the EphA2-ephrinA1 interaction in tubule formation, soluble EphA2 was used to disrupt the EphA2-ephrinA1 interaction between a coculture of HUVEC and human brain vascular pericyte cells. HUVECs seeded onto PEGDA hydrogels displayed a dose-dependent reduction in tubule formation in response to the soluble EphA2. Finally, hydrogels with releasable platelet-derived growth factor (PDGF), immobilized RGDS, and covalently immobilized PEG-ephrinA1 were implanted into the mouse cornea micropocket. These hydrogels induced a more robust vascular response with an increase in vessel density as compared with hydrogels with releasable PDGF alone. As such, PEG-ephrinA1 may represent a promising molecule to regulate cell adhesion and migration for formation of a microvasculature in tissue-engineered constructs.

In vivo evaluation of MMP sensitive high‐molecular weight HA‐based hydrogels for bone tissue engineering

Hyaluronic acid (170 kDa)-based hydrogel was synthesized using acrylated hyaluronic acid (HA) and matrix metalloproteinase (MMP) sensitive HA-based hydrogels were then prepared by conjugation with two different peptides: cell adhesion peptides containing integrin-binding domains (Arg-Gly-Asp: RGD) and a cross-linker with MMP degradable peptides to mimic the remodeling characteristics of natural extracellular matrices by cell-derived MMPs. Mechanical properties of these hydrogels were evaluated with different weight percentages (2.5 and 3.5 wt %) by measuring elastic modulus, viscous modulus, and swelling ratio. Human mesenchymal stem cells (hMSCs) were then cultured in MMP-sensitive or insensitive HA-based hydrogels and/or immobilized cell adhesive RGD peptides in vitro. Actin staining and image analysis proved that cells cultured in the MMP-sensitive hydrogel with RGD peptides showed extensive cell spreading and sprouting. Gene expression analysis showed that bone specific genes such as alkaline phosphatase, osteocalcin, and osteopontin increased in MMP-sensitive hydrogels as biomolecules such as BMPs and cells were added in the gels. For in vivo calvarial defect regeneration, five different samples (MMP insensitive hydrogel, MMP sensitive hydrogel, MMP sensitive hydrogel with BMP-2, MMP sensitive hydrogel with hMSC, and MMP sensitive hydrogel with BMP-2 and hMSC) were prepared. After 4 weeks of implantation, the Masson-Trichrome staining and micro computed tomography scan results demonstrated that the MMP sensitive hydrogels with BMP-2 and hMSCs have the highest mature bone formation. The MMP sensitive HA-based hydrogel could become useful scaffolds in bone tissue engineering with improvements on tissue remodeling rates and regeneration activity.

Synthesis and characterization of matrix metalloprotease sensitive-low molecular weight hyaluronic acid based hydrogels

Hyaluronic acid is a naturally derived glycosaminoglycan (GAG) involved in biological processes. A low molecular weight hyaluronic acid (50 kDa)-based hydrogel was synthesized using acrylated hyaluronic acid. Matrix metalloproteinase (MMP) sensitive hyaluronic acid-based hydrogels were prepared by conjugation with two different peptides: cell adhesion peptides containing integrin binding domains (Arg-Gly-Asp: RGD) and a cross-linker with MMP degradable peptides to mimic the remodeling characteristics of natural extracellular matrices (ECMs) by cell-derived MMPs. Mechanical properties of these hydrogels were evaluated with different molecular weights of acrylated hyaluronic acid (10 kDa and 50 kDa) cross-linked by MMP sensitive peptides by measuring elastic modulus, viscous modulus, swelling ratio and degradation rate. The MMP sensitive hydrogel based on the 50 kDa hyaluronic acid showed a 31.5-fold shorter gelation time, 4.7-fold higher storage modulus and 0.51-fold smaller swelling ratio than those of the hydrogel based on the 10 kDa. Degradation rate was dependent on MMP sensitivity of the peptide cross-linker. MMP sensitive hyaluronic acid based hydrogels were degraded faster than MMP insensitive-hyaluronic acid-based hydrogels. Human mesenchymal stem cells (MSCs) were cultured in MMP-sensitive or insensitive hyaluronic acid-based hydrogels (50 kDa hyaluronic acid) and/or immobilized cell adhesive RGD peptides. Cells cultured in the MMP-sensitive hydrogel with RGD peptides showed dramatic cell spreading compared with that of the control, which remained round. This MMP-sensitive low molecular weight hyaluronic acid-based hydrogel could be useful in tissue engineering by improving tissue defect regeneration and tissue remodeling.

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

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.