Selectively Cross-Linked Tetra-PEG Hydrogels Provide Control over Mechanical Strength with Minimal Impact on Diffusivity.

Synthetic hydrogels formed from poly(ethylene glycol) (PEG) are widely used to study how cells interact with their extracellular matrix. These in vivo-like 3D environments provide a basis for tissue engineering and cell therapies but also for research into fundamental biological questions and disease modeling. The physical properties of PEG hydrogels can be modulated to provide mechanical cues to encapsulated cells; however, the impact of changing hydrogel stiffness on the diffusivity of solutes to and from encapsulated cells has received only limited attention. This is particularly true in selectively cross-linked "tetra-PEG" hydrogels, whose design limits network inhomogeneities. Here, we used a combination of theoretical calculations, predictive modeling, and experimental measurements of hydrogel swelling, rheological behavior, and diffusion kinetics to characterize tetra-PEG hydrogels' permissiveness to the diffusion of molecules of biologically relevant size as we changed polymer concentration, and thus hydrogel mechanical strength. Our models predict that hydrogel mesh size has little effect on the diffusivity of model molecules and instead predicts that diffusion rates are more highly dependent on solute size. Indeed, our model predicts that changes in hydrogel mesh size only begin to have a non-negligible impact on the concentration of a solute that diffuses out of hydrogels for the smallest mesh sizes and largest diffusing solutes. Experimental measurements characterizing the diffusion of fluorescein isothiocyanate (FITC)-labeled dextran molecules of known size aligned well with modeling predictions and suggest that doubling the polymer concentration from 2.5% (w/v) to 5% produces stiffer gels with faster gelling kinetics without affecting the diffusivity of solutes of biologically relevant size but that 10% hydrogels can slow their diffusion. Our findings provide confidence that the stiffness of tetra-PEG hydrogels can be modulated over a physiological range without significantly impacting the transport rates of solutes to and from encapsulated cells

In vitro and in vivo delivery of a sustained release nanocarrier-based formulation of an MRTF/SRF inhibitor in conjunctival fibrosis

Abstract Background Sustained drug delivery is a large unmet clinical need in glaucoma. Here, we incorporated a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor, CCG-222740, into slow release large unilamellar vesicles derived from the liposomes DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and tested their effects in vitro and in vivo. Results The vesicles were spherical particles of around 130 nm and were strongly cationic. A large amount of inhibitor could be incorporated into the vesicles. We showed that the nanocarrier CCG-222740 formulation gradually released the inhibitor over 14 days using high performance liquid chromatography. Nanocarrier CCG-222740 significantly decreased ACTA2 gene expression and was not cytotoxic in human conjunctival fibroblasts. In vivo, nanocarrier CCG-222740 doubled the bleb survival from 11.0 ± 0.6 days to 22.0 ± 1.3 days (p = 0.001), decreased conjunctival scarring and did not have any local or systemic adverse effects in a rabbit model of glaucoma filtration surgery. Conclusions Our study demonstrates proof-of-concept that a nanocarrier-based formulation efficiently achieves a sustained release of a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor and prevents conjunctival fibrosis in an established rabbit model of glaucoma filtration surgery.https://deepblue.lib.umich.edu/bitstream/2027.42/146540/1/12951_2018_Article_425.pd

Receptor-targeted liposome-peptide-siRNA nanoparticles represent a novel and efficient therapeutic approach to prevent conjunctival fibrosis.

There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye

Neighboring cells override 3D hydrogel matrix cues to drive human MSC quiescence

Physical properties of modifiable hydrogels can be tuned to direct stem cell differentiation in a role akin to that played by the extracellular matrix in native stem cell niches. However, stem cells do not respond to matrix cues in isolation, but rather integrate soluble and non-soluble signals to balance quiescence, self-renewal and differentiation. Here, we encapsulated single cell suspensions of human mesenchymal stem cells (hMSC) in hyaluronic acid-based hydrogels at high and low densities to unravel the contributions of matrix- and non-matrix-mediated cues in directing stem cell response. We show that in high-density (HD) cultures, hMSC do not rely on hydrogel cues to guide their fate. Instead, they take on characteristics of quiescent cells and secrete a glycoprotein-rich pericellular matrix (PCM) in response to signaling from neighboring cells. Preventing quiescence precluded the formation of a glycoprotein-rich PCM and forced HD cultures to differentiate in response to hydrogel composition. Our observations may have important implications for tissue engineering as neighboring cells may act counter to matrix cues provided by scaffolds. Moreover, as stem cells are most regenerative if activated from a quiescent state, our results suggest that ex vivo native-like niches that incorporate signaling from neighboring cells may enable the production of clinically relevant, highly regenerative cells

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation

Matrix metalloproteinase distribution during early corneal wound healing

Aim: To compare matrix metalloproteinase (MMP) localisation in anterior keratectomy (AK) and lamellar keratectomy (LK) wounds. Methods: Wounds were produced in one eye of 24 rabbits. The AK wounds were made to approximately 120 mm in depth and then allowed to re-epithelialise. The LK wounds were of similar depth, but the anterior stroma and epithelium were replaced after a second deeper keratectomy had been performed. Immunohistochemistry was used to localise the MMP-1, -2, -3, and -9 at intervals from 4 h to 14 days following surgery. The contralateral eyes acted as controls. Results: After an AK wound MMP-1 was present at the leading edge of migrating epithelium after 18 h, while MMP-2 and -9 were localised behind the advancing epithelial edge. The presence of these enzymes rapidly fell to low levels after epithelial closure. There was only faint MMP-3 localisation between days 3 and 7. After an LK wound, MMP-1, -3, and -9 were not detected in the stromal interface, but MMP-2 was present at all time points. Conclusions: This study suggests that after an AK wound, MMP-1 is a key mediator of epithelial migration, while MMP-2 and -9, and to a lesser extent MMP-3, may participate in the remodelling of corneal stroma and the reformation of epithelial basement membrane. In contrast, an LK wound results in a much lower stimulus for MMP activation. The action of MMP-2 in stromal repair is thus partly independent of epithelial injury