Biomimetic modification of dual porosity poly(2-hydroxyethyl methacrylate) hydrogel scaffolds—porosity and stem cell growth evaluation

Abstract

The macroporous synthetic poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels as 3D cellular scaffolds with specific internal morphology, so called dual pore size, were designed and studied. The morphological microstructure of hydrogels was characterized in the gel swollen state and the susceptibility of gels for stem cells was evaluated. The effect of specific chemical groups covalently bound in the hydrogel network by copolymerization on cell adhesion and growth, followed by effect of laminin coating were investigated. The evaluated gels contained either carboxyl groups of the methacrylic acid or quaternary ammonium groups brought by polymerizable ammonium salt or their combinations. The morphology of swollen gel was visualized using the laser scanning confocal microscopy. All hydrogels had very similar porous structures – their matrices contained large pores (up to 102 μm) surrounded with gel walls with small pores (100 μm). The total pore volume in hydrogels swollen in buffer solution ranged between 69 and 86 vol%. Prior to the seeding of the mouse embryonal stem cells, the gels were coated with laminin. The hydrogel with quaternary ammonium groups (with or without laminin) stimulated the cell growth the most. The laminin coating lead to a significant and quaternary ammonium groups. The gel chemical modification influenced also the topology of cell coverage that ranged from individual cell clusters to well dispersed multi cellular structures. Findings in this study point out the laser scanning confocal microscopy as an irreplaceable method for a precise and quick assessment of the hydrogel morphology. In addition, these findings help to optimize the chemical composition of the hydrogel scaffold through the combination of chemical and biological factors leading to intensive cell attachment and proliferation.

Revealing the True Morphological Structure of Macroporous Soft Hydrogels for Tissue Engineering

“…After three or five days of cell cultivation on pHPMA hydrogels, the cells were washed three times with PBS and the cell growth was observed via LSCM. The cell nuclei were stained with Hoechst (5 µg•mL −1 ) for 10 min before imaging, and the number of viable cells growing in yjr hydrogels was evaluated using the Alamar Blue cell viability assay (ThermoFischer Scientific, Brno, Czech Republic), as described previously [32]. Briefly, a section of hydrogel was added to an empty well in 300 µL of fresh media, then 30 µL of AlamarBlue cell viability reagent containing resazurin, a compound which is reduced to fluorescently active resorufin in metabolically active cells, was added to the media with hydrogels.…”

“…The direct measurements of the pore sizes in all samples in Figure 1 (SEM and LSCM, 400 × 400 µm) were quantified using the ImageJ software, as described previously [32]. For each gel type, three different microphotographs were evaluated (see Figure S1).…”

“…LSCM allows the direct observation of samples in their hydrated state without the necessity of freezing, collecting z-plane images (z-stacks) with subsequent gel volume reconstruction to provide information about the size and distribution of pores in hydrogels [30]. Moreover, LSCM enables the observation of native hydrogels, as well as hydrogels with growing cells in situ [31,32].…”

Naturally Formed Chitinous Skeleton Isolated from the Marine Demosponge Aplysina fistularis as a 3D Scaffold for Tissue Engineering

“…Recently, two key routes of scaffold fabrication have been developed. The first involves the production of matrices with previously designed three-dimensional architecture, e.g., by 3D printing [ 16 , 17 , 18 , 19 ], macro-porous hydrogels synthesis [ 20 , 21 , 22 ], or electrospinning [ 23 , 24 ]. The second uses renewable decellularized natural sources of prefabricated scaffolds in their original shape, e.g., autogenic or allogenic ECM [ 25 , 26 , 27 ], skeletons with proper architecture [ 28 , 29 ] or plant-based structure [ 30 ].…”

Combining Biocompatible and Biodegradable Scaffolds and Cold Atmospheric Plasma for Chronic Wound Regeneration

“…Macroporous hydrogels based on cross-linked poly(2-hydroxyethyl methacrylate) (pHEMA) have been assessed in recent years for their swelling and mechanical properties as well as their ability to foster cell adhesion and proliferation with a special focus on stem cells. PHEMA hydrogel properties were assessed with light and electron microscopy, laser scanning confocal microscopy as well as micro-CT analysis and oscillatory shear measurement [79][80][81].…”