Digital light processing of hydrogel molds to guide cell mechanosensing and the fabrication of meniscal tissue constructs

The organization of cells and extracellular matrix (ECM) informs tissue function. This structure/function relationship is especially evident in musculoskeletal (MSK) tissues in which a specific ECM organization (e.g., anisotropy) guides directional properties under load. Injury disrupts the ECM structure, and new methods are needed to recapitulate the organization of cells and ECM within MSK tissue constructs to improve tissue models as well as to fabricate implants for repair. To address this, we use digital light processing (DLP) to rapidly 3D print custom molds that support large (centimeter-scale) meniscal tissue construct formation from meniscal fibrochondrocytes embedded within collagen gels. Importantly, these hydrogel molds include multiple pillars designed to anchor the tissue constructs and provide biophysical cues to direct tissue organization. Here, the effect of pillar spacing aspect ratio, mold size, and mold curvature on tissue contraction and cellular organization is investigated both experimentally and <i>in silico</i>. Pillar placement results in either disorganized (1:1 pillar spacing) or anisotropic (1:2 or 1:4 pillar spacing) cell spreading, with anisotropy observed in molds ranging from 6 mm to 2.4 cm in length. The introduction of mold curvature does not impact final construct width but does increase cellular anisotropy relative to molds without curvature. Furthermore, culture in the presence of the contractility inhibitor Cytochalasin D reduces construct contraction. These observations of cell behavior and construct compaction based on mold design are supported by coarse-grain simulations. Overall, this work establishes an adaptable DLP-based platform to grow custom MSK constructs for tissue models or repair.