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Collagen networks within 3D PEG hydrogels support valvular interstitial cell matrix mineralization.

ABSTRACT: Enzymatically degradable hydrogels were designed for the 3D culture of valvular interstitial cells (VICs), and through the incorporation of various functionalities, we aimed to investigate the role of the tissue microenvironment in promoting the osteogenic properties of VICs and matrix mineralization. Specifically, porcine VICs were encapsulated in a poly(ethylene glycol) hydrogel crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinker (KCGPQG↓IWGQCK) and formed via a thiol-ene photoclick reaction in the presence or absence of collagen type I to promote matrix mineralization. VIC-laden hydrogels were treated with osteogenic medium for up to 15 days, and the osteogenic response was characterized by the expression of RUNX2 as an early marker of an osteoblast-like phenotype, osteocalcin (OCN) as a marker of a mature osteoblast-like phenotype, and vimentin (VIM) as a marker of the fibroblast phenotype. In addition, matrix mineralization was characterized histologically with Von Kossa stain for calcium phosphate. Osteogenic response was further characterized biochemically with calcium assays, and physically via optical density measurements. When the osteogenic medium was supplemented with calcium chloride, OCN expression was upregulated and mineralization was discernable at 12 days of culture. Finally, this platform was used to screen various drug therapeutics that were assessed for their efficacy in preventing mineralization using optical density as a higher throughput readout. Collectively, these results suggest that matrix composition has a key role in supporting mineralization deposition within diseased valve tissue.

SUBMITTER: Schroeder ME

PROVIDER: S-EPMC7738375 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Characterization of valvular interstitial cell function in three dimensional matrix metalloproteinase degradable PEG hydrogels.

Project description:Valvular interstitial cells (VICs) maintain functional heart valve structure and display transient fibroblast and myofibroblast properties. Most cell characterization studies have been performed on plastic dishes; while insightful, these systems are limited. Thus, a matrix metalloproteinase (MMP) degradable poly(ethylene glycol) (PEG) hydrogel system is proposed in this communication as a useful tool for characterizing VIC function in 3D. When encapsulated, VICs attained spread morphology, and proliferated and migrated as shown through real-time cell microscopy. Additionally, fibronectin derived pendant RGD was incorporated into the system to promote integrin binding. As RGD concentration increased from 0 to 2000 microM, VIC process extension and integrin alpha(v)beta(3) binding increased within two days. By day 10, integrin binding was equalized between conditions. VIC morphology and rate of process extension were also increased through decreasing the hydrogel matrix density presented to the cells. VIC differentiation in response to exogenously delivered transforming growth factor-beta1 (TGF-beta1) was also examined within the hydrogel networks. TGF-beta1 increased expression of alpha smooth muscle actin (alphaSMA) and collagen-1 at both the mRNA and protein level by day 2 of culture, indicating myofibroblast differentiation, and was sustained over the course of the study (2 weeks). These studies demonstrate the utility, flexibility, and biological activity of this MMP-degradable system for the characterization of VICs, an important cell population for tissue engineering viable valve replacements and understanding valvular pathobiology.

| S-EPMC2772097 | biostudies-literature

Transcriptional profiles of valvular interstitial cells cultured on tissue culture polystyrene, on 2D hydrogels, or within 3D hydrogels.

Project description:Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis (Chen and Simmons, 2011) [1]. To better understand and quantify how microenvironmental cues influence VIC phenotype, we compared expression profiles of VICs cultured on/in poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. Here, we present both the raw and processed microarray data from these culture conditions. Interpretation of this data can be found in a research article entitled "Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype" (Mabry et al., 2015) [2].

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Role of cell-matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels.

Project description:Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but understanding these effects on VIC function better is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands, derived from fibronectin (RGDS), elastin (VGVAPG) and collagen-1 (P15). By day 14, VICs became significantly more elongated in RGDS-containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS-functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α-smooth muscle actin (αSMA) at day 14. The percentage of αSMA+ VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at days 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin and chondroitin sulphate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG-functionalized gels had a significantly higher collagen-X:collagen-1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide-functionalized PEG hydrogels are a useful system for culturing VICs three-dimensionally and, with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. Copyright © 2013 John Wiley & Sons, Ltd.

| S-EPMC4077989 | biostudies-literature

3D Printing Low-Stiffness Silicone Within a Curable Support Matrix.

Project description:Embedded 3D printing processes involve extruding ink within a support matrix that supports the ink throughout printing and curing. In once class of embedded 3D printing, which we refer to as "removable embedded 3D printing," curable inks are printed, cured, then removed from the uncured support matrix. Removable embedded 3D printing is advantageous because low-viscosity inks can be patterned in freeform geometries which may not be feasible to create via casting and other printing processes. When printing solid-infill geometries, however, uncured support matrix becomes trapped within the prints, which may be undesirable. This study builds on previous work by formulating a support matrix for removable embedded 3D printing that cures when mixed with the printed silicone ink to solve the problem of trapped, uncured support matrix within solid-infill prints. Printed specimens are shown to have a nearly isotropic elastic modulus in directions perpendicular and parallel to the printed layers, and a decreased modulus and increased elongation at break compared to specimens cast from the ink. The rheological properties of the support matrix are reported. The capabilities of the printer and support matrix are demonstrated by printing a variety of geometries from four UV and addition-cure silicone inks. Shapes printed with these inks range by nearly two orders of magnitude in stiffness and have failure strains between approximately 50 and 250%, suggesting a wide range of potential applications for this printing process.

| S-EPMC7946128 | biostudies-literature

3D Printed Osteoblast-Alginate/Collagen Hydrogels Promote Survival, Proliferation and Mineralization at Low Doses of Strontium Calcium Polyphosphate.

Project description:The generation of biomaterials via 3D printing is an emerging biotechnology with novel methods that seeks to enhance bone regeneration. Alginate and collagen are two commonly used biomaterials for bone tissue engineering and have demonstrated biocompatibility. Strontium (Sr) and Calcium phosphate (CaP) are vital elements of bone and their incorporation in composite materials has shown promising results for skeletal repair. In this study, we investigated strontium calcium polyphosphate (SCPP) doped 3D printed alginate/collagen hydrogels loaded with MC3T3-E1 osteoblasts. These cell-laden scaffolds were crosslinked with different concentrations of 1% SCPP to evaluate the effect of strontium ions on cell behavior and the biomaterial properties of the scaffolds. Through scanning electron microscopy and Raman spectroscopy, we showed that the scaffolds had a granular surface topography with the banding pattern of alginate around 1100 cm-1 and of collagen around 1430 cm-1. Our results revealed that 2 mg/mL of SCPP induced the greatest scaffold degradation after 7 days and least amount of swelling after 24 h. Exposure of osteoblasts to SCPP induced severe cytotoxic effects after 1 mg/mL. pH analysis demonstrated acidity in the presence of SCPP at a pH between 2 and 4 at 0.1, 0.3, 0.5, and 1 mg/mL, which can be buffered with cell culture medium. However, when the SCPP was added to the scaffolds, the overall pH increased indicating intrinsic activity of the scaffold to buffer the SCPP. Moreover, cell viability was observed for up to 21 days in scaffolds with early mineralization at 0.3, 0.5, and 1 mg/mL of SCPP. Overall, low doses of SCPP proved to be a potential additive in biomaterial approaches for bone tissue engineering; however, the cytotoxic effects due to its pH must be monitored closely.

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2021-03-05 | GSE168303 | GEO

Varying PEG density to control stress relaxation in alginate-PEG hydrogels for 3D cell culture studies.

Project description:Hydrogels are commonly used as artificial extracellular matrices for 3D cell culture and for tissue engineering. Viscoelastic hydrogels with tunable stress relaxation have recently been developed, and stress relaxation in the hydrogels has been found to play a key role in regulating cell behaviors such as differentiation, spreading, and proliferation. Here we report a simple but precise materials approach to tuning stress relaxation of alginate hydrogels with polyethylene glycol (PEG) covalently grafted onto the alginate. Hydrogel relaxation was modulated independent of the initial elastic modulus by varying molecular weight and concentration of PEG along with calcium crosslinking of the alginate. Increased concentration and molecular weight of the PEG resulted in faster stress relaxation, a higher loss modulus, and increased creep. Interestingly, we found that stress relaxation of the hydrogels is determined by the total mass amount of PEG in the hydrogel, and not the molecular weight or concentration of PEG chains alone. We then evaluated the utility of these hydrogels for 3D cell culture. Faster relaxation in RGD-coupled alginate-PEG hydrogels led to increased spreading and proliferation of fibroblasts, and enhanced osteogenic differentiation of mesenchymal stem cells (MSCs). Thus, this work establishes a new materials approach to tuning stress relaxation in alginate hydrogels for 3D cell culture.

| S-EPMC6463514 | biostudies-literature

Ascorbic acid promotes extracellular matrix deposition while preserving valve interstitial cell quiescence within 3D hydrogel scaffolds.

Project description:Current options for aortic valve replacements are non-viable and thus lack the ability to grow and remodel, which can be problematic for paediatric applications. Toward the development of living valve substitutes that can grow and remodel, porcine aortic valve interstitial cells (VICs) were isolated and encapsulated within proteolytically degradable and cell-adhesive poly(ethylene glycol) (PEG) hydrogels, in an effort to study their phenotypes and functions. The results showed that encapsulated VICs maintained high viability and proliferated within the hydrogels. The VICs actively remodelled the hydrogels via secretion of matrix metalloproteinase-2 (MMP-2) and deposition of new extracellular matrix (ECM) components, including collagens I and III. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from an activated myofibroblastic phenotype to a quiescent, unactivated phenotype, evidenced by the loss of α-smooth muscle actin expression upon encapsulation. In an effort to promote VIC-mediated ECM production, ascorbic acid (AA) was supplemented in the medium to investigate its effects on VIC function and phenotype. AA treatment enhanced VIC spreading and proliferation, and inhibited apoptosis. AA treatment also promoted VIC-mediated ECM remodelling by increasing MMP-2 activity and depositing collagens I and III. AA treatment did not significantly influence the expression of α-smooth muscle actin (myofibroblast activation marker) and alkaline phosphatase (osteogenic differentiation marker). No calcification or nodule formation was observed within the cell-laden hydrogels, with or without AA treatment. These results suggest the potential of this system and the beneficial effect of AA in heart valve tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

| S-EPMC4987276 | biostudies-literature

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