Mesenchymal stem cells derived-exosomes enhanced amniotic membrane extract promotes corneal keratocyte proliferation.

Amniotic membrane extract (AME) and Wharton's jelly mesenchymal stem cells derived-exosomes (WJ-MSC-Exos) are promising therapeutic solutions explored for their potential in tissue engineering and regenerative medicine, particularly in skin and corneal wound healing applications. AME is an extract form of human amniotic membrane and known to contain a plethora of cytokines and growth factors, making it a highly attractive option for topical applications. Similarly, WJ-MSC-Exos have garnered significant interest for their wound healing properties. Although WJ-MSC-Exos and AME have been used separately for wound healing research, their combined synergistic effects have not been studied extensively. In this study, we evaluated the effects of both AME and WJ-MSC-Exos, individually and together, on the proliferation of corneal keratocytes as well as their ability to promote in vitro cell migration, wound healing, and their impact on cellular morphology. Our findings indicated that the presence of both exosomes (3 × 105 Exo/mL) and AME (50 µg/mL) synergistically enhance the proliferation of corneal keratocytes. Combined use of these solutions (3 × 105 Exo/mL + 50 µg/mL) increased cell proliferation compared to only 50 µg/mL AME treatment on day 3 (**** p < 0.0001). This mixture treatment (3 × 105 Exo/mL + 50 µg/mL) increased wound closure rate compared to isolated WJ-MSC-Exo treatment (3 × 105 Exo/mL) (*p < 0.05). Overall, corneal keratocytes treated with AME and WJ-MSC-Exo (3 × 105 Exo/mL + 50 µg/mL) mixture resulted in enhanced proliferation and wound healing tendency. Utilization of combined use of AME and WJ-MSC-Exo can pave the way for a promising foundation for corneal repair research.

Synthesis and characterization of stimuli-responsive hydrogels: evaluation of external stimuli influence on L929 fibroblast viability.

In this study, poly(2-hydroxyethyl methacrylate) [p(HEMA)] based hydrogels responsive to the pH, temperature and magnetic field were synthesized. The surface properties of p(HEMA) were improved by designing the stimuli-responsive hydrogels made of MAGA, NIPAAm and methacrylate-decorated magnetite nanoparticles as a function of pH-, thermo- and magnetic responsive cell culture surfaces. These materials were then modified an abundant extracellular matrix component, type I collagen, which has been considered as a biorecognition element to increase the applicability of hydrogels to cell viability. Based on results from scanning electron microscopy (SEM) and thermal gravimetric analysis (TGA), stimuli-responsive hydrogel demonstrated improved non-porous structures and thermal stability with a high degree of cross-linking. Mechanical analyses of the hydrogels also showed that stimuli-responsive hydrogels are more elastomeric due to the polymeric chains and heterogeneous amorphous segments compared to plain hydrogels. Furthermore, surface modification of hydrogels with collagen provided better biocompatibility, which was confirmed with L929 fibroblast cell adhesion. Produced stimuli-responsive hydrogels modulated cellular viability by changing pH and magnetic field.

Rose petal topography mimicked poly(dimethylsiloxane) substrates for enhanced corneal endothelial cell behavior.

Low proliferation capacity of corneal endothelial cells (CECs) and worldwide limitations in transplantable donor tissues reveal the critical need of a robust approach for in vitro CEC growth. However, preservation of CEC-specific phenotype with increased proliferation has been a great challenge. Here we offer a biomimetic cell substrate design, by optimizing mechanical, topographical and biochemical characteristics of materials with CEC microenvironment. We showed the surprising similarity between topographical features of white rose petals and corneal endothelium due to hexagonal cell shapes and physiologically relevant cell density (≈ 2000 cells/mm2). Polydimethylsiloxane (PDMS) substrates with replica of white rose petal topography and cornea-friendly Young's modulus (211.85 ± 74.9 kPa) were functionalized with two of the important corneal extracellular matrix (ECM) components, collagen IV (COL 4) and hyaluronic acid (HA). White rose petal patterned and COL 4 modified PDMS with optimized stiffness provided enhanced bovine CEC response with higher density monolayers and increased phenotypic marker expression. This biomimetic approach demonstrates a successful platform to improve in vitro cell substrate properties of PDMS for corneal applications, suggesting an alternative environment for CEC-based therapies, drug toxicity investigations, microfluidics and organ-on-chip applications.

Bioinspired hydrogel surfaces to augment corneal endothelial cell monolayer formation.

Corneal endothelial cells (CECs) have limited proliferation ability leading to corneal endothelium (CE) dysfunction and eventually vision loss when cell number decreases below a critical level. Although transplantation is the main treatment method, donor shortage problem is a major bottleneck. The transplantation of in vitro developed endothelial cells with desirable density is a promising idea. Designing cell substrates that mimic the native CE microenvironment is a substantial step to achieve this goal. In the presented study, we prepared polyacrylamide (PA) cell substrates that have a microfabricated topography inspired by the dimensions of CECs. Hydrogel surfaces were prepared via two different designs with small and large patterns. Small patterned hydrogels have physiologically relevant hexagon densities (∼2000 hexagons/mm2 ), whereas large patterned hydrogels have sparsely populated hexagons (∼400 hexagons/mm2 ). These substrates have similar elastic modulus of native Descemet's membrane (DM; ∼50 kPa) and were modified with Collagen IV (Col IV) to have biochemical content similar to native DM. The behavior of bovine corneal endothelial cells on these substrates was investigated and results show that cell proliferation on small patterned substrates was significantly (p = 0.0004) higher than the large patterned substrates. Small patterned substrates enabled a more densely populated cell monolayer compared to other groups (p = 0.001 vs. flat and p < 0.0001 vs. large patterned substrates). These results suggest that generating bioinspired surface topographies augments the formation of CE monolayers with the desired cell density, addressing the in vitro development of CE layers.

Substrate stiffness effects on SH-SY5Y: The dichotomy of morphology and neuronal behavior.

Like many other cell types, neuroblastoma cells are also known to respond to mechanical cues in their microenvironment in vitro. They were shown to have mechanotransduction pathways, which result in enhanced neuronal morphology on stiff substrates. However, in previous studies, the differentiation process was monitored only by morphological parameters. Motivated by the lack of comprehensive studies that investigate the effects of mechanical cues on neuroblastoma differentiation, we used SH-SY5Y cells differentiated on polyacrylamide (PA) gels as a model. Cells differentiated on the surface of PA hydrogels with three different elastic moduli (0.1, 1, and 50 kPa) were morphologically evaluated and their electrophysiological responsiveness was probed using calcium imaging. Immunodetection of neural marker TUJ1 and p-FAK was used for biochemical characterization. Groups with defined stiffness that are matching and nonmatching to neural tissue extracellular matrix were used to distinguish biomimetic results from other effects. Results show that while cells display morphologies that do not resemble neurons on soft substrates, they are in fact electrophysiologically more responsive and abundant in neuronal marker TUJ1. Our findings suggest that while neuronal differentiation occurs more efficiently in microenvironments mechanically mimicking neural tissue, the SH-SY5Y model demonstrates morphologies that conflict with neuronal behavior under these conditions. These results are expected to contribute considerable input to researchers that use SH-SY5Y as a neuron model.