Glutathione-Triggered Formation of a Fmoc-Protected Short Peptide-Based Supramolecular Hydrogel

A biocompatible method of glutathione (GSH) catalyzed disulfide bond reduction was used to form Fmoc-short peptide-based supramolecular hydrogels. The hydrogels could form in both buffer solution and cell culture medium containing 10% of Fetal Bovine Serum (FBS) within minutes. The hydrogel was characterized by rheology, transmission electron microscopy, and fluorescence emission spectra. Their potential in three dimensional (3D) cell culture was evaluated and the results indicated that the gel with a low concentration of the peptide (0.1 wt%) was suitable for 3D cell culture of 3T3 cells. This study provides an alternative candidate of supramolecular hydrogel for 3D cell culture and cell delivery.     

Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.     

Rheological characterisation of a novel thermosensitive chitosan/poly(vinyl alcohol) blend hydrogel

Thermosensitive hydrogels that are triggered by changes in environmental temperature thus resulting in in situ hydrogel formation have recently attracted the attention of many investigators for biomedical applications. In the current work, the thermosensitive hydrogel was prepared through the mixture of chitosan (CS), poly(vinyl alcohol) (PVA) and sodium bicarbonate. The mixture was liquid aqueous solutions at low temperature (about 4 °C), but a gel under physiological conditions. The hydrogel was characterized by FTIR, swelling and rheological analysis. The effect of hydrogel composition and temperature on both the gel process and the gel strength was investigated from which possible hydrogel formation mechanisms were inferred. In addition, the hydrogel interior morphology as well as porosity of structure was evaluated by scanning electron microscopy (SEM). The potential of the hydrogels as vehicles for delivering bovine serum albumin (BSA) were also examined. In this study, the physically crosslinked chitosan/PVA gel was prepared under mild conditions without organic solvent, high temperature or harsh pH. The viscoelastic properties, as investigated rheologically, indicate that the gel had good mechanical strength. The gel formed implants in situ in response to temperature change, from low temperature (about 4 °C) to body temperature, which was very suitable for local and sustained delivery of proteins, cell encapsulation and tissue engineering.     

Antibacterial activity and biocompatibility of a chitosan-γ-poly(glutamic acid) polyelectrolyte complex hydrogel

In this study, we prepared a polyelectrolyte complex (PEC) hydrogel comprising chitosan as the cationic polyelectrolyte and γ-poly(glutamic acid) (γ-PGA) as the anionic polyelectrolyte. Fourier transform infrared spectroscopy revealed that ionic complex interactions existed in the chitosan-γ-PGA PEC hydrogels. The compressive modulus increased upon increasing the degree of complex formation in the chitosan-γ-PGA PEC hydrogel; the water uptake decreased upon increasing the degree of complex formation. At the same degree of complex formation, the compressive modulus was larger for the chitosan-dominated PEC hydrogels; the water uptake was larger for the γ-PGA-dominated ones. Scanning electron microscopy images revealed the existence of interconnected porous structures (pore size: 30-100 μm) in all of the chitosan-γ-PGA PEC hydrogels. The chitosan-γ-PGA PEC hydrogels also exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, in vitro cell culturing of 3T3 fibroblasts revealed that all the chitosan-γ-PGA PEC hydrogels were effective in promoting cell proliferation, especially the positively charged ones (chitosan-dominated). Therefore, the chitosan-γ-PGA polyelectrolyte hydrogel appears to have potential as a new material for biomedical applications.     

A Genetically Modified Protein-Based Hydrogel for 3D Culture of AD293 Cells

Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence ‘GRGDSP’ to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery.     

Collective Cell Behavior in Mechanosensing of Substrate Thickness

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation—its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 μm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 μm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 μm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.     

Preparation and characterization of chitosan-polyvinyl alcohol blend hydrogels for the controlled release of nano-insulin

Chitosan (CS)-polyvinyl alcohol (PVA) blend hydrogels were prepared using glutaraldehyde as the cross-linking agent. The obtained hydrogels, which have the advantages of both PVA and CS, can be used as a material for the transdermal drug delivery (TDD) of insulin. The nano-insulin-loaded hydrogels were prepared under the following conditions: 1.2 g of polyethylene glycol, 1.5 g of CS, 1.2 g of PVA, 1.2 mL of 1% glutaraldehyde solution, 16 mL of water, and 40 mg of nano-insulin with 12 min of mixing time and 3 min of cross-linking time. The nano-insulin-loaded hydrogels were characterized using scanning electron microscopy, energy dispersive spectrometry, Fourier-transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and its mechanical properties were analyzed. The results show that all molecules in the hydrogel have good compatibility and they formed a honeycomb-like structure. The hydrogel also showed good mechanical and thermal properties. The in vitro drug release of the hydrogel showed that the nano-insulin accorded with Fick's first law of diffusion and it has a high permeation rate (4.421 μg/(cm² h)). These results suggest that the nano-insulin-loaded hydrogels are a promising non-invasive TDD system for diabetes chemotherapy.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.     

Cell proliferation on hydrogels

Summary The adhesion and proliferation of mammalian fibroblasts (Flow 7000) on the surface of hydrophilic (copolymer ofN-vinyl-2-pyrrolidone and methyl methacrylate) and hydrophobic [polymethylmethacrylate (PMMA) stereocomplex] hydrogels with a wide range in water content were studied morphologically and quantitatively. It was demonstrated that cell proliferation on hydrogels by a static culture method decreased as the water content of the gels increased. However, it is remarkable that the cell proliferation on PMMA hydrogels with a high water content is equivalent to that on glass Petri dishes. The results obtained in the proliferation of cells on the surface of these hydrogels closely correspond to the state of cell adhesion. When fresh medium or air was perfused from the popposite side of the PMMA hydrogel membrane on which the cells were proliferating (perfusion method), the cells continued to grow into a higher density than with the conventional static culture method. In the case of fresh medium perfusion, the amount of proliferated cell was dependent on both the permeability of the membrane and the density of the membrane “scaffolding”. Virus multiplication in the cultured cells increased in proportion to the cell density, whereas the cell function was similar in both culture methods.     

3D Differentiation of Neural Stem Cells in Macroporous Photopolymerizable Hydrogel Scaffolds

Neural stem/progenitor cells (NSPCs) are the stem cell of the adult central nervous system (CNS). These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes), thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC) as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm², 10 wt%, 6330±1160 µm², 20 wt%, 7600±1550 µm²) compared with controls (0 wt%, 3150±220 µm²). Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.     

QHREDGS Enhances Tube Formation,Metabolism and Survival of Endothelial Cells in Collagen-Chitosan Hydrogels

Cell survival in complex, vascularized tissues, has been implicated as a major bottleneck in advancement of therapies based on cardiac tissue engineering. This limitation motivates the search for small, inexpensive molecules that would simultaneously be cardio-protective and vasculogenic. Here, we present peptide sequence QHREDGS, based upon the fibrinogen-like domain of angiopoietin-1, as a prime candidate molecule. We demonstrated previously that QHREDGS improved cardiomyocyte metabolism and mitigated serum starved apoptosis. In this paper we further demonstrate the potency of QHREDGS in its ability to enhance endothelial cell survival, metabolism and tube formation. When endothelial cells were exposed to the soluble form of QHREDGS, improvements in endothelial cell barrier functionality, nitric oxide production and cell metabolism (ATP levels) in serum starved conditions were found. The functionality of the peptide was then examined when conjugated to collagen-chitosan hydrogel, a potential carrier for in vivo application. The presence of the peptide in the hydrogel mitigated paclitaxel induced apoptosis of endothelial cells in a dose dependent manner. Furthermore, the peptide modified hydrogels stimulated tube-like structure formation of encapsulated endothelial cells. When integrin αvβ3 or α5β1were antibody blocked during cell encapsulation in peptide modified hydrogels, tube formation was abolished. Therefore, the dual protective nature of the novel peptide QHREDGS may position this peptide as an appealing augmentation for collagen-chitosan hydrogels that could be used for biomaterial delivered cell therapies in the settings of myocardial infarction.     

The effect of tachykinin neuropeptides on amyloid β aggregation

A hallmark of Alzheimer’s disease is production of amyloid β peptides resulting from aberrant cleavage of the amyloid precursor protein. Amyloid β assembles into fibrils under physiological conditions, through formation of neurotoxic intermediate oligomers. Tachykinin peptides are known to affect amyloid β neurotoxicity in cells. To understand the mechanism of this effect, we studied how tachykinins affect Aβ(1–40) aggregation in vitro. Fibrils grown in the presence of tachykinins exhibited reduced thioflavin T (ThT) fluorescence, while their morphology, observed in transmission electron microscopy (TEM), did not alter. Cross linking studies revealed that the distribution of low molecular weight species was not affected by tachykinins. Our results suggest that there may be a specific interaction between tachykinins and Aβ(1–40) that allows them to co-assemble. This effect may explain the reduction of Aβ(1–40) neurotoxicity in cells treated with tachykinins.     

Electrosensitive polyacrylic acid/fibrin hydrogel facilitates cell seeding and alignment

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure.     

Biodegradable and biocompatible synthetic saccharide-Peptide hydrogels for three-dimensional stem cell culture

Saccharide-peptide hydrogels have been developed in our laboratory as new synthetic extracellular matrices for regenerative medicine applications. In this work, we have expanded on our previously reported system and applied copolymerization of cysteine (Cys) and vinyl sulfone (VS)-functionalized saccharide-peptide polymers via Michael-type addition for encapsulation and 3D culture of cells. Specifically, our aims were to (1) develop a novel hydrogel platform, which could be applied for encapsulating and culturing mesenchymal stem cells (MSCs) in a 3D environment, (2) characterize the tunable properties of the hydrogel, specifically, degradation, mechanical, and gel network properties, and (3) determine the biocompatibility of the saccharide-peptide hydrogel material with MSCs. Hydrogel mechanical properties were tunable by varying the VS:Cys ratio (= 0.5, 1, or 2) as well as the pH (6, 7, or 8) of the cross-linking components. Stiffer gels were formed at VS:Cys = 1 and pH 6 or 7. Gels formed at pH 8 or with excess Cys (VS:Cys = 0.5) or VS (VS:Cys = 2) were significantly softer. Cross-linking pH and VS:Cys ratio also had an effect on the degradation behavior of the VS:Cys gels, with higher cross-linking pH resulting in an accelerated loss of mass. On the basis of environmental scanning electron microscopy (ESEM) analysis and fluorescence microscopy, all hydrogels appeared to exhibit porous gel networks. MSCs cultured in monolayer and exposed to soluble Cys or VS copolymers (0.1-5 mg/mL) did not exhibit measurable cytotoxicity. In addition, MSCs were cultured in 3D for up to 14 days in vitro without deleterious effects on cell viability. In summary, we have established and characterized a tunable 3D saccharide-peptide hybrid copolymer hydrogel platform for culturing MSCs. Future studies will focus on utilizing the hydrogel system for controlling the differentiation of MSCs.     

Self‐assembly of pH and calcium dual‐responsive peptide‐amphiphilic hydrogel

Peptide‐based hydrogels have gained much interest for biomedical applications as a result of their biocompatibility. Herein, we reported a synthetic pH‐sensitive and calcium‐responsive peptide‐amphiphilic hydrogel. The sequences of the peptide amphiphiles were derived from the repeat‐in‐toxin (RTX) motif. At a certain peptide‐amphiphile concentration, self‐assembly was accompanied by the formation of a rigid, viscoelastic hydrogel at low pH or the presence of calcium ions. Circular dichroism spectra showed that the peptide amphiphiles adopted beta‐sheet structure. Meanwhile, as revealed by transmission electron microscopy, the peptide‐amphiphile self‐assembly was accompanied by the formation of long interconnected nanofibrillar superstructure. Material properties of the resulting peptide‐amphiphile hydrogel were characterized using oscillatory sheer rheology, and the storage modulus (G′) was found to be one order of magnitude higher than the loss modulus (G″), indicating a moderately rigid viscoelastic material. Furthermore, with systematical residue substitution, it was found that the aspartic acid within the repeat‐in‐toxin sequence of peptide amphiphiles was responsible for the pH and calcium selectivity. The environmental responsiveness, secondary structure, morphology, and mechanical nature of the peptide‐amphiphile hydrogel make it a possible material candidate for biomedical and engineering application. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation of pH- and ionic-strength responsive biodegradable fumaric acid crosslinked carboxymethyl cellulose

A novel biodegradable sodium carboxymethyl cellulose (NaCMC)-based hydrogel was synthesized by using fumaric acid (FA) as a crosslinking agent at various ratios. Hydrogels (CMCF) were characterized using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Swelling behaviors of hydrogels were investigated in distilled water, various salt, and pH solutions. The FTIR results indicated the crosslinking between carboxyl groups of FA with hydroxyl group of NaCMC through ester formation. AFM analyses showed that roughness of hydrogel surface decreased with increasing crosslinker concentration. The swelling capacity decreased with an increase in charge of the metal cation (Al(3+)相似文献   

Inherent interfacial mechanical gradients in 3D hydrogels influence tumor cell behaviors

Cells sense and respond to the rigidity of their microenvironment by altering their morphology and migration behavior. To examine this response, hydrogels with a range of moduli or mechanical gradients have been developed. Here, we show that edge effects inherent in hydrogels supported on rigid substrates also influence cell behavior. A Matrigel hydrogel was supported on a rigid glass substrate, an interface which computational techniques revealed to yield relative stiffening close to the rigid substrate support. To explore the influence of these gradients in 3D, hydrogels of varying Matrigel content were synthesized and the morphology, spreading, actin organization, and migration of glioblastoma multiforme (GBM) tumor cells were examined at the lowest (<50 μm) and highest (>500 μm) gel positions. GBMs adopted bipolar morphologies, displayed actin stress fiber formation, and evidenced fast, mesenchymal migration close to the substrate, whereas away from the interface, they adopted more rounded or ellipsoid morphologies, displayed poor actin architecture, and evidenced slow migration with some amoeboid characteristics. Mechanical gradients produced via edge effects could be observed with other hydrogels and substrates and permit observation of responses to multiple mechanical environments in a single hydrogel. Thus, hydrogel-support edge effects could be used to explore mechanosensitivity in a single 3D hydrogel system and should be considered in 3D hydrogel cell culture systems.     

Modulate the phase transition temperature of hydrogels with both thermosensitivity and biodegradability

The synthesis and characterization of thermoresponsive hydrogels on the basis of N-isoproplyarylamide (NIPAAm) and acrylamide (AAm) copolymers crosslinked with a novel biodegradable crosslinker (PEG-co-PLA) were carried out in this study. Swelling measurement results demonstrated that four gels of PNAM5, PNAM10, PNAM12 and PNAM15 are thermoresponsive. The equilibrium swelling ratio and degradation of the hydrogels strongly depend on hydrogels composition. The morphology of the hydrogels was observed by scanning electron microscopy (SEM), and their thermal property was characterized by differential scanning calorimetry (DSC). The results show that the proportion of AAm in the copolymer has notable effect on the low critical solution temperature (LCST) of the hydrogel. When the molar ratio of AAm to NIPAAm was increased from 1:10 to 3:10 the LCST of the copolymer increased from 39.7 to 64.2 °C. The compression modulus of PNAM15 is of the highest among other hydrogels, because PNAM15 hydrogel has a more compact structure.     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

MMP-Sensitive PEG Diacrylate Hydrogels with Spatial Variations in Matrix Properties Stimulate Directional Vascular Sprout Formation

The spatial presentation of immobilized extracellular matrix (ECM) cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP)-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP). Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10–12 hours in the more crosslinked regions to 4–6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.