Hao Liu

Creating large, functional tissue requires the engineering of a blood capillary network, which is challenging as it necessitates the recapitulation of capillary morphology and spatial distribution. Current approaches employ local mechanical and chemical gradients to modulate capillary morphology or the network's density but they often fail to regulate both parameters to fulfil the needs of tissue engineering. Here, we present a light-mediated biomaterial platform based on the FLight biofabrication technique to regulate blood capillary alignment and density. By modulating local light doses, a highly aligned hydrogel microstructure with spatially tunable matrix stiffness was achieved within a single 3D hydrogel construct. The anisotropic microstructures guided blood capillary elongation and alignment, achieving over 90% alignment in matrices (3.4-5.4 kPa), while spatial variations in stiffness regulated vascularization. Blood capillary density was maximized in regions with intermediate stiffness (4.6-5.0 kPa) and markedly lower in softer (~2.0 kPa) and stiffer (~5.4 kPa) matrices. This study establishes spatial light dosage as an effective engineering parameter for programming matrix mechanics and microvascular organization within a single hydrogel construct, providing a scalable strategy for fabricating anisotropic and spatially heterogeneous capillary networks for vascularized tissue engineering.

The prospects of fabricating human tissue grafts or models using cell‐laden bioresins in space have garnered significant interest in recent years. While there is tremendous progress in extrusion or light‐based bioprinting in microgravity conditions, printing of aligned tissues (e.g., muscle, tendon, cardiac, etc.) remains a challenge. Furthermore, current photoresin formulations do not allow long‐term cell encapsulation and are difficult to handle in microgravity conditions. In this study, a new gravity‐independent filamented light (G‐FLight) biofabrication system, which can create viable muscle constructs within seconds, is demonstrated. New photoresin formulations based on gelatin methacrylate (GelMA) for encapsulation of primary cells (murine myoblasts) and storage in printing cuvettes for at least a week at 4 °C or ‐80 °C are also demonstrated. The tissues printed in microgravity based on the new formulations exhibit higher cell viability, number of proliferating cells, and higher numbers of myotubes and fusion index compared to control formulations (i.e., GelMA dissolved in phosphate‐buffered saline). Importantly, the microgravity‐printed tissues also featured similar myotube density and fusion index to those printed using the same resins on‐ground. The G‐Flight printing concept, together with the new resins enabling refrigeration or cryopreservation with encapsulated cells, offers a promising solution for biofabrication in space.

The plasticity and regenerative capacity of skeletal muscle arise from quiescent stem cells activated upon overload, injury, or disease state. Developing in vitro muscle models to study these properties can advance muscle disease modeling and pre‐clinical evaluation. Here, Filamented Light (FLight) bioprinting is leveraged as a high‐throughput approach for producing mini‐muscle tissues. Using paired box protein 7 (Pax7)‐nGFP primary myoblasts, mini‐muscles are bioprinted from pristine collagen‐fibrinogen (ColFib). The FLight hydrogel consist of aligned microstructures which guide the formation of aligned myotubes. Mini‐muscles demonstrates in vivo‐like tissue organization, including multinucleated myotubes and a Pax7 ⁺ cell pool embedded in newly deposited laminin. Both spontaneous and electrically stimulated contractions are observed. ColFib matrix is promising for maintenance of the Pax7 ⁺ cell pool. Damage from cardiotoxin‐induced injury of the mini‐muscles led to a massive proliferation of Pax7 ⁺ cells and restoration of the contractile properties of myotubes. Notably, small molecules such as Repsox can enhance regeneration. FLight printed mini‐muscles have potential for applications in muscle biology, exercise/atrophy, disease models, and drug screening.

Deep vat printing (DVP) techniques, including tomographic and filamented light (Flight) printing have transformed the tissue engineering landscape by enabling layer‐free bioprinting at high resolution and speeds. Importantly, collagen is one of the most widely used materials in tissue biofabrication, but DVP using collagen‐based photoresins has been under‐explored. In this work, photoclickable collagen‐based resins are demonstrated which are compatible with DVP, enabling prints with up to 50 µm print resolution and speeds less than 20 s per cm ³ . These resins contain iodixanol, which acts as a refractive index matching agent to reduce optical inhomogeneities by added cells and improves the stability of the collagen‐based resins under neutralized conditions. As a potential application, multi‐material tomographic and FLight printing is demonstrated to create intricate tissue constructs featuring muscle and connective tissue interfaces. While tomographic printing allows greater complexity over the overall shape of the constructs, FLight constructs demonstrate enhanced biomimicry through generation of aligned multinucleated myotubes, transition zones between the two cell types, and regularly spaced sarcomere structure (≈2.6 µm average sarcomere length) which facilitates contractility in the muscle regions. The demonstration of photoclickable, isotonic and neutral collagen‐based bioresins offers a new solution for DVP of biomimetic complex tissues and grafts.

The prospects of fabricating human tissue grafts or models using cell-laden bioresins in space has garnered significant interest in recent years. While there has been tremendous progress in extrusion or light-based bioprinting in microgravity conditions, printing of aligned tissues, such as those featuring anisotropic organization of cells and extracellular matrices (e.g., muscle, tendon, cardiac, etc.), remains a challenge. Furthermore, current photoresin formulations do not allow long-term cell encapsulation and are difficult to perform in microgravity. In this study, we demonstrate a new gravity-independent filamented light (G-FLight) biofabrication system with in-built refrigeration and heating units, which can create viable muscle constructs within seconds. We developed new photoresin formulations based on gelatin methacrylate (GelMA) for encapsulation of primary cells (murine myoblasts) and storage in printing cuvettes for at least a week at 4°C or -80°C. The tissues printed in microgravity based on the new formulations exhibited higher cell viability, number of proliferating cells and after maturation higher numbers of myotubes and fusion index compared to control formulations (i.e., GelMA dissolved in phosphate buffered saline). The microgravity-printed tissues also featured similar myotube density and fusion index to those printed using the same resins on-ground. The G-Flight printing concept, together with the new resins enabling refrigeration or cryopreservation with encapsulated cells, offers a promising solution for biofabrication in space.

Light‐based biofabrication techniques have revolutionized the field of tissue engineering and regenerative medicine. Specifically, the projection of structured light, where the spatial distribution of light is controlled at both macro and microscale, has enabled precise fabrication of complex three dimensional structures with high resolution and speed. However, despite tremendous progress, biofabrication processes are mostly limited to benchtop devices which limit the flexibility in terms of where the fabrication can occur. Here, a Fiber‐assisted Structured Light (FaSt‐Light) projection apparatus for rapid in situ crosslinking of photoresins is demonstrated. This approach uses image‐guide fiber bundles which can project bespoke images at multiple wavelengths, enabling flexibility and spatial control of different photoinitiation systems and crosslinking chemistries and also the location of fabrication. Coupling of different sizes of fibers and different lenses attached to the fibers to project small (several mm) or large (several cm) images for material crosslinking is demonstrated. FaSt‐Light allows control over the cross‐section of the crosslinked resins and enables the introduction of microfilaments which can further guide cellular infiltration, differentiation, and anisotropic matrix production. The proposed approach can lead to a new range of in situ biofabrication techniques which improve the translational potential of photofabricated tissues and grafts.

One hallmark of healthy tendon tissue is the high confinement of tenocytes between tightly packed, highly aligned collagen fibers. During tendinopathy, this organization becomes dysregulated, leading to cells with round-shaped morphology and collagen fibers which exhibit crimping and misalignment. The elongated nuclei in healthy tendons are linked to matrix homeostasis through distinct mechanotransduction pathways, and it is believed that the loss of nuclear confinement could upregulate genes associated with abnormal matrix remodeling. Replicating the cell and nuclear morphology of healthy and diseased states of tendon, however, remains a significant challenge for engineered in vitro tendon models. Here we report on a high throughput biofabrication of mini-tendons that mimick the tendon core compartment based on the filamented light (FLight) approach. Each mini-tendon, with a length of 4 mm, was composed of parallel hydrogel microfilaments (2–5 µm diameter) and microchannels (2–10 µm diameter) that confined the cells. We generated four distinct matrices with varying stiffness (7–40 kPa) and microchannel dimensions. After 14 d of culture, 29% of tenocytes in the softest matrix with the largest microchannel diameter were aligned, exhibiting an average nuclear aspect ratio (nAR) of 2.1. In contrast, 84% of tenocytes in the stiffest matrix with the smallest microchannel diameter were highly aligned, with a mean nAR of 3.4. When tenocytes were cultured on the FLight hydrogels (2D) as opposed to within the hydrogels three-dimensional (3D), the mean nAR was less than 1.9, indicating that nuclear morphology is significantly more confined in 3D environments. By tuning the stiffness and microarchitecture of the FLight matrix, we demonstrated that mechanical confinement can be modulated to exert control over the extent of nuclear confinement. This high-throughput, tunable platform offers a promising approach for studying the mechanobiology of healthy and diseased tendons and for eventual testing of drug compounds against tendinopathy.

The plasticity and regenerative capacity of skeletal muscle arise from quiescent stem cells activated upon overload, injury, or inflammation. Developing in vitro muscle models to study these properties could advance muscle disease modeling and pre-clinical evaluation. Here, we leverage Filamented Light (FLight) bioprinting as a high-throughput approach for producing mini-muscle tissues. Using Pax7-nGFP myoblasts, we bioprinted mini-muscles from pristine collagen-fibrinogen. The FLight hydrogel consisted of aligned microstructures which guided the formation of aligned myotubes. Mini-muscles demonstrated in vivo -like tissue organization, including highly aligned myotubes and a Pax7 ⁺ cell pool embedded in newly deposited laminin. Both spontaneous and electrically stimulated contractions were observed. Collagen-fibrinogen matrix was promising for maintenance of the Pax7 ⁺ cell pool. Damage from cardiotoxin-induced injury of the mini-muscles led to a massive proliferation of Pax7 ⁺ cells and restoration of the contractile properties. Notably, small molecules such as Repsox could enhance regeneration. FLight printed mini-muscles have potential for applications in muscle biology, exercise/atrophy, disease models, and drug screening.

One hallmark of healthy tendon tissue is the high confinement of tenocytes between tightly packed, highly aligned collagen fibers. During tendinopathy, this organization becomes dysregulated, leading to cells with round-shaped morphology and collagen fibers which exhibit crimping and misalignment. The elongated nuclei in healthy tendons are linked to matrix homeostasis through distinct mechanotransduction pathways, and it is believed that the loss of nuclear confinement could upregulate genes associated with abnormal matrix remodeling. Replicating the cell and nuclear morphology of healthy and diseased states of tendon, however, remains a significant challenge for engineered in vitro tendon models. Here we report on a high throughput biofabrication of mini-tendons that mimick the tendon core compartment based on the Filamented Light (FLight) approach. Each mini-tendon, with a length of 4 mm, was composed of parallel hydrogel microfilaments (2-5 µm diameter) and microchannels (2-10 µm diameter) that confined the cells. We generated four distinct matrices with varying stiffness (7-40 kPa) and microchannel dimensions. After 14 days of culture, 29% of tenocytes in the softest matrix with the largest microchannel diameter were aligned, exhibiting an average nuclear aspect ratio (nAR) of 2.1. In contrast, 84% of tenocytes in the stiffest matrix with the smallest microchannel diameter were highly aligned, with a mean nAR of 3.4. When tenocytes were cultured on the FLight hydrogels (2D) as opposed to within the hydrogels (3D), the mean nAR was less than 1.9, indicating that nuclear morphology is significantly more confined in 3D environments. By tuning the stiffness and microarchitecture of the FLight matrix, we demonstrated that mechanical confinement can be modulated to exert control over the extent of nuclear confinement. This high-throughput, tunable platform offers a promising approach for studying the mechanobiology of healthy and diseased tendons and for eventual testing of drug compounds against tendinopathy.

Light-based biofabrication techniques have revolutionized the field of tissue engineering and regenerative medicine. Specifically, the projection of structured light, where the spatial distribution of light is controlled at both macro- and micro-scale, has enabled precise fabrication of complex three-dimensional structures with high resolution and speed. However, despite almost two decades of progress, biofabrication processes have been mostly limited to benchtop devices which limit the flexibility in terms of where the fabrication can occur. Here, we demonstrate a Fiber-assisted Structured Light (FaSt-Light) projection apparatus for rapid in situ crosslinking of photoresins. This approach uses image-guide fiber bundles which can project bespoke images at multiple wavelengths, enabling flexibility and spatial control of different photoinitiation systems and crosslinking chemistries and also the location of fabrication. We demonstrate coupling of different sizes of fibers and different lenses attached to the fibers to be able to project small (several mm) or large (several cm) images for material crosslinking. FaSt-Light allows control over the cross-section of the crosslinked resins and enables the introduction of microfilaments which can further guide cellular infiltration, differentiation and anisotropic matrix production. The proposed approach could lead to a new range of in situ biofabrication techniques which improve the translational potential of photo-fabricated tissues and grafts.