Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture is developed. Hyaluronic acid-based hydrogels are utilized, amorphously packed with acellular cartilage tissue particulated to ≈125-250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300 kPa. Remarkably, primary chondrocytes recellularize particles within 48 h, a process driven by chemotaxis, exhibit distributed cellularity in large engineered composites, and express genes consistent with native cartilage repair. In addition, broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in an in vivo mouse model is demonstrated. The findings suggest optimal material architectures to balance concurrent demands for large-scale mechanical properties while also supporting recellularization and integration of dense musculoskeletal and connective tissues.

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies require two-dimensional (2D) cell expansion processes to achieve large cell populations critical for the repair of damaged (e.g. connective and musculoskeletal) tissues. However, the influence of mechanical memory on cell fate following expansion is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we show that the organization of histone H3 trimethylated at lysine 9 (H3K9me3) and expression of tissue-identifying genes in primary cartilage cells (chondrocytes) transferred to three-dimensional (3D) hydrogels depends on the number of previous population doublings on tissue culture plastic during 2D cell expansion. Decreased levels of H3K9me3 occupying promoters of dedifferentiation genes after the 2D culture were also retained in 3D culture. Suppression of H3K9me3 during expansion of cells isolated from a murine model similarly resulted in the loss of the chondrocyte phenotype and global remodeling of nuclear architecture. In contrast, increasing levels of H3K9me3 through inhibiting H3K9 demethylases partially rescued the chondrogenic nuclear architecture and gene expression, which has important implications for tissue repair therapies, where expansion of large numbers of phenotypically-suitable cells is required. Overall, our findings indicate mechanical memory in primary cells is encoded in the chromatin architecture, which impacts cell fate and the phenotype of expanded cells.SIGNIFICANCE STATEMENTTissue regeneration procedures, such as cartilage defect repair (e.g. Matrix-induced Autologous Chondrocyte Implantation) often require cell expansion processes to achieve sufficient cells to transplant into an in vivo environment. However, the chondrocyte cell expansion on 2D stiff substrates induces epigenetic changes that persist even when the chondrocytes are transferred to a different (e.g. 3D) or in vivo environment. Treatments to alter epigenetic gene regulation may be a viable strategy to improve existing cartilage defect repair procedures and other tissue engineering procedures that involve cell expansion.

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Articular cartilage is a layered tissue with a complex, heterogenous structure and lubricated surface which is challenging to reproduce using traditional tissue engineering methods. 3D printing techniques have enabled engineering of complex scaffolds for cartilage regeneration, but constructs fail to replicate the unique zonal layers, and limited cytocompatible crosslinkers exist. To address the need for mechanically robust, layered scaffolds, we developed an extracellular matrix particle-based biomaterial ink (pECM biomaterial ink) which can be extruded, polymerizes via disulfide bonding, and restores surface lubrication. Our cartilage pECM biomaterial ink utilizes functionalized hyaluronan, a naturally occurring glycosaminoglycan, crosslinked directly to decellularized tissue particles (ø 40-100 µm). We experimentally determined that hyaluronan functionalized with thiol groups (t-HA) forms disulfide bonds with the ECM particles to form a 3D network. We show that two inks can be co-printed to create a layered cartilage scaffold with bulk compressive and surface (friction coefficient, adhesion, and roughness) mechanics approaching values measured on native cartilage. We demonstrate that our printing process enables the addition of macropores throughout the construct, increasing the viability of introduced cells by 10%. The delivery of these 3D printed scaffolds to a defect is straightforward, customizable to any shape, and adheres to surrounding tissue.

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