In Situ UNIversal Orthogonal Network (UNION) Bioink Deposition for Direct Delivery of Corneal Stromal Stem Cells to Corneal Wounds.

The scarcity of human donor corneal graft tissue worldwide available for corneal transplantation necessitates the development of alternative therapeutic strategies for treating patients with corneal blindness. Corneal stromal stem cells (CSSCs) have the potential to address this global shortage by allowing a single donor cornea to treat multiple patients. To directly deliver CSSCs to corneal defects within an engineered biomatrix, we developed a UNIversal Orthogonal Network (UNION) collagen bioink that crosslinks in situ with a bioorthogonal, covalent chemistry. This cell-gel therapy is optically transparent, stable against contraction forces exerted by CSSCs, and permissive to the efficient growth of corneal epithelial cells. Furthermore, CSSCs remain viable within the UNION collagen gel precursor solution under standard storage and transportation conditions. This approach promoted corneal transparency and re-epithelialization in a rabbit anterior lamellar keratoplasty model, indicating that the UNION collagen bioink serves effectively as an in situ -forming, suture-free therapy for delivering CSSCs to corneal wounds. TEASER. Corneal stem cells are delivered within chemically crosslinked collagen as a transparent, regenerative biomaterial therapy.

Embedded 3D Bioprinting of Collagen Inks into Microgel Baths to Control Hydrogel Microstructure and Cell Spreading.

Microextrusion-based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. It is hypothesized that interactions between inks and support baths, which are often composed of granular microgels, can be modulated to control the microscopic structure within these macroscopic-printed constructs. Using printed collagen bioinks crosslinked either through physical self-assembly or bioorthogonal covalent chemistry, it is demonstrated that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero-shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity is modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15-50 µm) results in large pores (≈40 µm) that allow human corneal mesenchymal stromal cells (MSCs) to readily spread, while control samples of cast collagen or collagen printed in non-granular support baths do not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths.

3D printing microporous scaffolds from modular bioinks containing sacrificial, cell-encapsulating microgels.

Microgel-based biomaterials have inherent porosity and are often extrudable, making them well-suited for 3D bioprinting applications. Cells are commonly introduced into these granular inks post-printing using cell infiltration. However, due to slow cell migration speeds, this strategy struggles to achieve depth-independent cell distributions within thick 3D printed geometries. To address this, we leverage granular ink modularity by combining two microgels with distinct functions: (1) structural, UV-crosslinkable microgels made from gelatin methacryloyl (GelMA) and (2) sacrificial, cell-laden microgels made from oxidized alginate (AlgOx). We hypothesize that encapsulating cells within sacrificial AlgOx microgels would enable the simultaneous introduction of void space and release of cells at depths unachievable through cell infiltration alone. Blending the microgels in different ratios produces a family of highly printable GelMA : AlgOx microgel inks with void fractions ranging from 0.03 to 0.35. As expected, void fraction influences the morphology of human umbilical vein endothelial cells (HUVEC) within GelMA : AlgOx inks. Crucially, void fraction does not alter the ideal HUVEC distribution seen throughout the depth of 3D printed samples. This work presents a strategy for fabricating constructs with tunable porosity and depth-independent cell distribution, highlighting the promise of microgel-based inks for 3D bioprinting.

3D bioprinting of dynamic hydrogel bioinks enabled by small molecule modulators.

Three-dimensional bioprinting has emerged as a promising tool for spatially patterning cells to fabricate models of human tissue. Here, we present an engineered bioink material designed to have viscoelastic mechanical behavior, similar to that of living tissue. This viscoelastic bioink is cross-linked through dynamic covalent bonds, a reversible bond type that allows for cellular remodeling over time. Viscoelastic materials are challenging to use as inks, as one must tune the kinetics of the dynamic cross-links to allow for both extrudability and long-term stability. We overcome this challenge through the use of small molecule catalysts and competitors that temporarily modulate the cross-linking kinetics and degree of network formation. These inks were then used to print a model of breast cancer cell invasion, where the inclusion of dynamic cross-links was found to be required for the formation of invasive protrusions. Together, we demonstrate the power of engineered, dynamic bioinks to recapitulate the native cellular microenvironment for disease modeling.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Engineered assistive materials for 3D bioprinting: support baths and sacrificial inks.

Three-dimensional (3D) bioprinting is a promising technique for spatially patterning cells and materials into constructs that mimic native tissues and organs. However, a trade-off exists between printability and biological function, where weak materials are typically more suited for 3D cell culture but exhibit poor shape fidelity when printed in air. Recently, a new class of assistive materials has emerged to overcome this limitation and enable fabrication of more complex, biologically relevant geometries, even when using soft materials as bioinks. These materials include support baths, which bioinks are printed into, and sacrificial inks, which are printed themselves and then later removed. Support baths are commonly yield-stress materials that provide physical confinement during the printing process to improve resolution and shape fidelity. Sacrificial inks have primarily been used to create void spaces and pattern perfusable networks, but they can also be combined directly with the bioink to change its mechanical properties for improved printability or increased porosity. Here, we outline the advantages of using such assistive materials in 3D bioprinting, define their material property requirements, and offer case study examples of how these materials are used in practice. Finally, we discuss the remaining challenges and future opportunities in the development of assistive materials that will propel the bioprinting field forward toward creating full-scale, biomimetic tissues and organs.

3D Bioprinting of Cell-Laden Hydrogels for Improved Biological Functionality.

The encapsulation of cells within gel-phase materials to form bioinks offers distinct advantages for next-generation 3D bioprinting. 3D bioprinting has emerged as a promising tool for patterning cells, but the technology remains limited in its ability to produce biofunctional, tissue-like constructs due to a dearth of materials suitable for bioinks. While early demonstrations commonly used viscous polymers optimized for printability, these materials often lacked cell compatibility and biological functionality. In response, advanced materials that exist in the gel phase during the entire printing process are being developed, since hydrogels are uniquely positioned to both protect cells during extrusion and provide biological signals to embedded cells as the construct matures during culture. Here, an overview of the design considerations for gel-phase materials as bioinks is presented, with a focus on their mechanical, biochemical, and dynamic gel properties. Current challenges and opportunities that arise due to the fact that bioprinted constructs are active, living hydrogels composed of both acellular and cellular components are also evaluated. Engineering hydrogels with consideration of cells as an intrinsic component of the printed bioink will enable control over the evolution of the living construct after printing to achieve greater biofunctionality.

3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks.

Three-dimensional (3D) bioprinting is a promising technology to produce tissue-like structures, but a lack of diversity in bioinks is a major limitation. Ideally each cell type would be printed in its own customizable bioink. To fulfill this need for a universally applicable bioink strategy, we developed a versatile, bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers. We term this family of materials UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin-like protein (ELP), and polyethylene glycol (PEG) were each used as backbone polymers to create inks with storage moduli spanning 200 to 10,000 Pa. Because UNION bioinks are crosslinked by a common chemistry, multiple materials can be printed together to form a unified, cohesive structure. This approach is compatible with any support bath that enables diffusion of UNION crosslinkers. Both matrix-adherent human corneal mesenchymal stromal cells and non-matrix-adherent human induced pluripotent stem cell-derived neural progenitor spheroids were printed with UNION bioinks. The cells retained high viability and expressed characteristic phenotypic markers after printing. Thus, UNION bioinks are a versatile strategy to expand the toolkit of customizable materials available for 3D bioprinting.

Bio-Orthogonally Crosslinked, In Situ Forming Corneal Stromal Tissue Substitute.

In this study, an in situ forming corneal stromal substitute based on collagen type I crosslinked by bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) is presented. The crosslinked collagen gel has greater transparency compared to non-crosslinked collagen gels. The mechanical properties of the gels are controlled by changing functional group ratios and conjugated collagen concentrations. Higher concentrations of conjugated collagen yield enhances mechanical properties, where the storage modulus increases from 42.39 ± 8.95 to 112.03 ± 3.94 Pa after SPAAC crosslinking. Encapsulated corneal keratocytes grow within the SPAAC-crosslinked gels and corneal keratinocytes are supported on top of the gel surfaces. SPAAC-crosslinked gels support more favorable and stable keratinocyte morphology on their surface compared to non-crosslinked gels likely as a result of more optimal substrate stiffness, gel integrity, and resistance to degradation. SPAAC-crosslinked collagen gels with and without encapsulated keratocytes applied to rabbit corneas in an organ culture model after keratectomy exhibit surface epithelialization with multilayered morphology. The novel in situ forming gel is a promising candidate for lamellar and defect reconstruction of corneal stromal tissue.