In nature, tissues are anisotropic, but most biomaterials used in regenerative medicine applications are not. Patterned biomaterials offer the opportunity to mimic spatially segregating biophysical and biochemical properties found in nature. Engineering such properties allows to study cell-matrix interactions in anisotropic matrices in great detail. The tunable artificial niche this research used is an alginate-based hydrogel with dual crosslinking: Diels-Alder covalent crosslinking (norbornene-tetrazine, non-degradable) and UV-mediated peptide crosslinking (matrix metalloprotease sensitive peptide, enzymatically degradable). This material platform allowed us to generate biomaterials with patterns in diverse multiple biophysical properties to stablish the potential application of this materials for guiding cell behavior in 3D matrices. First, we developed a hydrogel with spatial patterns in degradation and stiffness, featuring two distinct sections: a soft, non-degradable phase (Soft-NoDeg) and a stiff, degradable phase (Stiff-Deg). Rheology was used to characterize single-phase materials, while surface micro-indentation assessed the patterned hydrogels. Over time, the materials exhibited emerging stiffness and degradability patterns. Encapsulated 3D mouse embryonic fibroblasts (MEFs) were analyzed for anisotropic cell-matrix interactions using a novel image-based quantification tool. Live/dead staining revealed no differences in viability but distinct proliferation patterns, with higher cell numbers in Stiff-Deg materials by day 14. Cell morphology analysis showed larger projected cell areas in Soft-NoDeg materials at day 1, due to the difference in stiffness. By day 14, the enzymatic degradation of the Stiff-Deg materials allowed an increase in cell area comparable to Soft-NoDeg, alongside a decrease in circularity and an increase in filopodia number and length significantly higher compared to Soft-noDeg. This demonstrated that stiffness and degradability patterns can control anisotropic cell responses in 3D hydrogels, effectively quantified through image-based approaches. Building on these findings, the next phase compared anisotropic hydrogels with degradation patterns to single-phase materials (Deg and NoDeg), using primary human mesenchymal stem cells (hMSCs). The Deg areas of anisotropic hydrogels showed enhanced cell spreading, collective cell alignment, mechanotransduction, and osteogenic differentiation. Anisotropic hydrogels lead also to spatially patterned osteogenic differentiation, with mid-stage markers (Osteocalcin) expressed by day 14, unlike single-phase Deg materials that showed only early markers (Osterix). Mechanosensing analysis revealed enhanced YAP nuclear translocation and expression in Deg regions of patterned materials, an effect absent in MMP-scrambled peptide or non-RGD-modified hydrogels. These results underscore the potential of degradation patterns in guiding hMSC behavior and boosting osteogenic differentiation by an enhanced mechanotransduction due to the material’s anisotropy. Finally, we focused on interphases for endothelial cell guidance by patterned viscoelasticity and collagen network. To achieve the patterning of these two biophysical properties, we developed a covalently crosslinked alginate and collagen interpenetrating polymer network (IPN). The degree of alginate crosslinking influenced collagen network formation and matrix viscoelasticity. Norbornene (N)- or tetrazine (T)-modified alginate enabled crosslinking via UV (degradable) or non-UV (non-degradable) methods. Confocal reflectance and rheology confirmed that reduced alginate crosslinking improved collagen network formation and viscoelastic properties, while highly crosslinked matrices exhibited elastic behavior and lacked collagen fibers. Photopatterning enabled the creation of high- and low-crosslinked areas within a single matrix, confirmed through confocal reflectance and scanning electron microscopy. These patterns influenced collagen architecture and viscoelasticity, facilitating guided endothelial cell migration and collective invasion. Our findings highlight the importance of a degradable, viscoelastic, collagen-rich matrix in promoting endothelial cell invasion. In summary, this research demonstrates the versatility and potential of patterned biomaterials in guiding cell behaviors by mimicking the anisotropic properties of native tissues. By employing hydrogels with spatially defined stiffness, degradation, viscoelasticity, and collagen organization, we provided evidence that these properties can independently and collectively regulate key cellular processes such as mechanotransduction, alignment, differentiation, and invasion. These findings not only deepen our understanding of cell-matrix interactions in anisotropic environments but also establish a foundation for the design of advanced biomaterials for regenerative medicine applications. Future work will focus on refining these material platforms and exploring their applicability in more complex biological models and therapeutic settings.
