Speaker
Description
Structurally patterned materials offer various options for guided cell behavior. Cell behaviour can be guided by patterning spatially discretized biochemical, topological or mechanical properties or combinations thereof. Patterned materials allow to integrate multiple characteristics in single materials, resembling an anisotropy found in endogenous tissue regeneration. Previous studies using orthogonal Diels-Alder and thiol-ene crosslinking in alginate hydrogels showed that patterns in 2D affect cell attachment and differentiation. However, to employ the full potential of pattern principles a 3D cell encapsulation in such materials appears mandatory. Therefore, this study aims to evaluate cell responses in 3D alginate hydrogels with spatial patterns using biophysical and biochemical patterning characteristics, which aim to have applications in regenerative medicine.
Single-phase materials were formed using norbornene (N) and tetrazine (T) modified alginate (Diels-Alder reaction, spontaneous non-UV) together with matrix metalloproteinase (MMP) sensitive peptides as a degradable crosslinkers (thiol-ene reaction, UV). The variations in the N-T ratio and the concentration of MMP sensitive peptide crosslinker can tune the mechanical properties and degradability of the material. The patterns on the material were formed using a photomask with UV irradiation which allowed the MMPsens peptides bonds form in the UV regions and the N-T bonds form in the covered stripes. The materials were mechanically characterized using rheology and microindentation. Mouse embryonic fibroblasts (MEFs) were encapsulated in 3D and cell viability (live/dead staining), cell morphology (DAPI/phalloidin) and proliferative state (Ki67) were evaluated at days 1, 7 and 14.
The rheology of single-phase materials revealed similar elastic modulus of ~3kPa in the degradable (UV) and ~2kPa in the non-degradable (non-UV) materials. These results were comparable to the microindentation of patterned hydrogels, showing stripes with soft (~1.8kPa) and stiff regions (~3kPa). 3D encapsulation of mouse embryonic fibroblasts (MEFs) in single-phase and patterned hydrogels showed high viability (>85%) over the 14 days and a significant increase in cell number in degradable materials compared to non degradable. Cell morphology showed a significant increase in cell area and decrease in circularity indicating cell spreading in degradable hydrogels, whereas the cells in non-degradable materials remained round. The main differences in cell morphology were observed in filopodia formation, with significantly higher cell filopodia number and length on softer areas while they were reduced in stiffer ones. The proliferation marker Ki67 was highly expressed in softer materials compared to stiff ones.
The hydrogels showed spatially tunable mechanical and degradation characteristics in 3D that were determined by the crosslinking type. Such differences influenced the morphology and proliferation of MEFs. Further research will look at 3D encapsulation of human mesenchymal stroma cells in patterned materials with the aim to guide cell differentiation.
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