Bioprinted Injectable Hierarchically Porous Gelatin Methacryloyl Hydrogel Constructs with Shape-Memory Properties.

Direct injection of cell-laden hydrogels shows high potentials in tissue regeneration for translational therapy. The traditional cell-laden hydrogels are often used as bulk space fillers to tissue defects after injection, likely limiting their structural controllability. On the other hand, patterned cell-laden hydrogel constructs often necessitate invasive surgical procedures. To overcome these problems, herein, we report a unique strategy for encapsulating living human cells in a pore-forming gelatin methacryloyl (GelMA)-based bioink to ultimately produce injectable hierarchically macro-micro-nanoporous cell-laden GelMA hydrogel constructs through three-dimensional (3D) extrusion bioprinting. The hydrogel constructs can be fabricated into various shapes and sizes that are defect-specific. Due to the hierarchically macro-micro-nanoporous structures, the cell-laden hydrogel constructs can readily recover to their original shapes, and sustain high cell viability, proliferation, spreading, and differentiation after compression and injection. Besides, in vivo studies further reveal that the hydrogel constructs can integrate well with the surrounding host tissues. These findings suggest that our unique 3D-bioprinted pore-forming GelMA hydrogel constructs are promising candidates for applications in minimally invasive tissue regeneration and cell therapy.

A Tumor-on-a-Chip System with Bioprinted Blood and Lymphatic Vessel Pair.

Current in vitro anti-tumor drug screening strategies are insufficiently portrayed lacking true perfusion and draining microcirculation systems, which may post significant limitation in reproducing the transport kinetics of cancer therapeutics explicitly. Herein, we report the fabrication of an improved tumor model consisting of bioprinted hollow blood vessel and lymphatic vessel pair, hosted in a three-dimensional (3D) tumor microenvironment-mimetic hydrogel matrix, termed as the tumor-on-a-chip with bioprinted blood and lymphatic vessel pair (TOC-BBL). The bioprinted blood vessel was perfusable channel with opening on both ends while the bioprinted lymphatic vessel was blinded on one end, both of which were embedded in a hydrogel tumor mass, with vessel permeability individually tunable through optimization of the composition of the bioinks. We demonstrated that systems with different combinations of these bioprinted blood/lymphatic vessels exhibited varying levels of diffusion profiles for biomolecules and anti-cancer drugs. Our TOC-BBL platform mimicking the natural pathway of drug-tumor interactions would have the drug introduced through the perfusable blood vessel, cross the vascular wall into the tumor tissue via diffusion, and eventually drained into the lymphatic vessel along with the carrier flow. Our results suggested that this unique in vitro tumor model containing the bioprinted blood/lymphatic vessel pair may have the capacity of simulating the complex transport mechanisms of certain pharmaceutical compounds inside the tumor microenvironment, potentially providing improved accuracy in future cancer drug screening.

A stable, reusable, and highly active photosynthetic bioreactor by bio-interfacing an individual cyanobacterium with a mesoporous bilayer nanoshell.

An individual cyanobacterium cell is interfaced with a nanoporous biohybrid layer within a mesoporous silica layer. The bio-interface acts as an egg membrane for cell protection and growth of outer shell. The resulting bilayer shell provides efficient functions to create a single cell photosynthetic bioreactor with high stability, reusability, and activity.

Near-Infrared-II Nanoparticles for Cancer Imaging of Immune Checkpoint Programmed Death-Ligand 1 and Photodynamic/Immune Therapy.

Development of second near-infrared (NIR-II) nanoparticles (NPs) with high biocompatibility, low toxicity, and high singlet oxygen quantum yield (ΦΔ) to prevent tumor recurrence is highly desirable in molecular imaging and photodynamic/immune combination therapy. Here, theranostic photosensitizer BODIPY (BDP)-I-N-anti-PD-L1 NPs were developed by encapsulating the photosensitizer BDP-I-N with amphipathic poly(styrene-co-chloromethylstyrene)-graft-poly(ethylene glycol) nanocarriers through self-assembly functionalization with programmed cell death-ligand 1 (PD-L1) monoclonal antibody. These NPs exhibit highly intensive luminescence in the NIR-II window (1000-1700 nm) to real-time imaging of immune checkpoint PD-L1, high singlet oxygen quantum yield (ΦΔ = 73%), and an eliminating effect of primary cancers. The NPs also allow for profiling PD-L1 expression as well as accumulating in MC38 tumor and enabling molecular imaging in vivo. Upon an 808 nm laser excitation, the targeted NPs produce an emission wavelength above 1200 nm to image a tumor to a normal tissue signal ratio (T/NT) at an approximate value of 14.1. Moreover, the MC38 tumors in mice are eliminated by combining photodynamic therapy and immunotherapy within 30 days, with no tumor recurrence within a period of 40 days. In addition, the tumors do not grow in the rechallenged mice within 7 days of inoculation. Such a strategy shows a durable immune memory effect against tumor rechallenging without toxic side effects to major organs.

Thiophene donor for NIR-II fluorescence imaging-guided photothermal/photodynamic/chemo combination therapy.

Organic fluorophores/photosensitizers have been widely used in biological imaging and photodynamic and photothermal combination therapy in the first near-infrared (NIR-I) window. However, their applications in the second near-infrared (NIR-II) window are still limited primarily due to low fluorescence quantum yields (QYs). Here, a boron dipyrromethene (BDP) is created as a molecularly engineered thiophene donor unit with high QYs to the redshift. Thiophene insertion initiates substantial redshifts of the absorbance as compared to its counterparts in which iodine is introduced. The fluorescent molecule can be triggered by an NIR laser with a single wavelength, thereby producing emission in the NIR-II windows. Single NIR laser-triggered phototherapeutic nanoparticles (NPs) are developed by encapsulating the BDP and the chemotherapeutic drug docetaxel (DTX) by using a synthetic amphiphilic poly(styrene-co-chloromethyl styrene)-graft-poly(ethylene glycol) functionalized with folic acid (FA). These BDP-T-N-DTX-FA NPs not only show superior solubility and high singlet oxygen QY (ΦΔ=62%) but also demonstrate single NIR laser-triggered multifunctional characteristics. After intravenous administration of the NPs into 4T1 tumor-bearing mice, the accumulation of the NPs in the tumor showed a high signal-to-background ratio (11.8). Furthermore, 4T1 tumors in mice were almost eradicated by DTX released from the BDP-T-N-DTX-FA NPs under single NIR laser excitation and the combination of photodynamic therapy (PDT) and photothermic therapy (PTT). STATEMENT OF SIGNIFICANCE: The application of organic photosensitizers is still limited primarily due to low fluorescence quantum yields (QYs) in the second near-infrared (NIR-II) window. Here, a boron dipyrromethene (BDP) as a molecularly engineered thiophene donor unit with high QYs to the redshift is created. Phototherapeutic nanoparticles (NPs) are developed by encapsulating the BDP and docetaxel (DTX) using a synthetic amphiphilic poly(styrene-co-chloromethyl styrene)-graft-poly(ethylene glycol) functionalized with folic acid (FA). These BDP-T-N-DTX-FA NPs not only show high singlet oxygen QY (ΦΔ=62%) but also demonstrate single NIR laser-triggered multifunctional characteristics and a high signal-to-background ratio (11.8). Furthermore, 4T1 tumors in mice were almost eradicated by DTX released from the BDP-T-N-DTX-FA NPs under single NIR laser excitation and the PDT/PTT combination therapy.

Symbiotic Photosynthetic Oxygenation within 3D-Bioprinted Vascularized Tissues.

In this study, we present the photosynthetic oxygen (O2) supply to mammalian cells within a volumetric extracellular matrix-like construct, whereby a three-dimensional (3D)-bioprinted fugitive pattern encapsulating unicellular green algae, Chlamydomonas reinhardtii (C. reinhardtii), served as a natural photosynthetic O2-generator. The presence of bioprinted C. reinhardtii enhanced the viability and functionality of mammalian cells while reducing the hypoxic conditions within the tissues. We were able to subsequently endothelialize the hollow perfusable microchannels formed after enzymatic removal of the bioprinted C. reinhardtii-laden patterns from the matrices following the initial oxygenation period, to obtain biologically relevant vascularized mammalian tissue constructs. The feasibility of co-culture of C. reinhardtii with human cells, the printability and the enzymatic degradability of the fugitive bioink, as well as the exploration of C. reinhardtii as a natural, eco-friendly, cost-effective, and sustainable source of O2 would likely promote the development of engineered tissues, tissue models, and food for various applications.

A miniaturized optical tomography platform for volumetric imaging of engineered living systems.

Volumetric optical microscopy approaches that enable acquisition of three-dimensional (3D) information from a biological sample are attractive for numerous non-invasive imaging applications. The unprecedented structural details that these techniques provide have helped in our understanding of different aspects of architecture of cells, tissues, and organ systems as they occur in their natural states. Nonetheless, the instrumentation for most of these techniques is sophisticated, bulky, and costly, and is less affordable to most laboratory settings. Several miniature imagers based on webcams or low-cost sensors featuring easy assembly have been reported, for in situ imaging of biological structures at low costs. However, they have not been able to achieve the ability of 3D imaging throughout the entire volumes for spatiotemporal analyses of the structural changes in these specimens. Here we present a miniaturized optical tomography (mini-Opto) platform for low-cost, volumetric characterization of engineered living systems through hardware optimizations as well as applications of an optimized algebraic algorithm for image reconstruction.

Tacrolimus- and Nerve Growth Factor-Treated Allografts for Neural Tissue Regeneration.

Treatment of injured peripheral nerves, especially long-distance nerve defects, remains a significant challenge in regenerative medicine due to complex biological conditions and a lack of biomaterials for effective nerve reconstruction. Without proper treatment, nerve injury leads to motor and sensory dysfunction. Here, we have developed an efficacious nerve allograft treated with a dual drug containing acrolimus and nerve growth factor to bridge the nerve gap and achieve rapid neural tissue recovery without immunological rejection. The recovery of the structure, activity, and function of rats treated with the dual drug-treated allograft was investigated by walking track analysis and electrophysiological measurement. The sciatic functional index was measured to be -3.0 after a 12-week treatment. The nerve conduction velocity, peak latency, and peak amplitude of the nerve action potentials demonstrate the functional recovery of the nerve. To study the synergistic effect of the dual drug on the growth of neurites, a neural cell hypoxia model was created. The dual drug exhibited a high efficiency in promoting the growth of nerve cells under the nerve injury-induced hypoxic condition. The dual drug could protect the cells against antioxidative damage from hypoxia by the expression of heat shock protein, hypoxia-inducible factor, ß-tubulin, and vimentin.

Aqueous Two-Phase Emulsion Bioink-Enabled 3D Bioprinting of Porous Hydrogels.

3D bioprinting technology provides programmable and customizable platforms to engineer cell-laden constructs mimicking human tissues for a wide range of biomedical applications. However, the encapsulated cells are often restricted in spreading and proliferation by dense biomaterial networks from gelation of bioinks. Herein, a cell-benign approach is reported to directly bioprint porous-structured hydrogel constructs by using an aqueous two-phase emulsion bioink. The bioink, which contains two immiscible aqueous phases of cell/gelatin methacryloyl (GelMA) mixture and poly(ethylene oxide) (PEO), is photocrosslinked to fabricate predesigned cell-laden hydrogel constructs by extrusion bioprinting or digital micromirror device-based stereolithographic bioprinting. The porous structure of the 3D-bioprinted hydrogel construct is formed by subsequently removing the PEO phase from the photocrosslinked GelMA hydrogel. Three different cell types (human hepatocellular carcinoma cells, human umbilical vein endothelial cells, and NIH/3T3 mouse embryonic fibroblasts) within the 3D-bioprinted porous hydrogel patterns show enhanced cell viability, spreading, and proliferation compared to the standard (i.e., nonporous) hydrogel constructs. The 3D bioprinting strategy is believed to provide a robust and versatile platform to engineer porous-structured tissue constructs and their models for a variety of applications in tissue engineering, regenerative medicine, drug development, and personalized therapeutics.

A bilayered nanoshell for durable protection of single yeast cells against multiple, simultaneous hostile stimuli.

Single cell surface engineering provides the most efficient, non-genetic strategy to enhance cell stability. However, it remains a huge challenge to improve cell stability in complex artificial environments. Here, a soft biohybrid interfacial layer is fabricated on individual living-cell surfaces by their exposure to a suspension of gold nanoparticles and l-cysteine to form a protecting functional layer to which porous silica layers were bound yielding pores with a diameter of 3.9 nm. The living cells within the bilayered nanoshells maintained high viability (96 ± 2%) as demonstrated by agar plating, even after five cycles of simultaneous exposure to high temperature (40 °C), lyticase and UV light. Moreover, yeast cells encapsulated in bilayered nanoshells were more recyclable than native cells due to nutrient storage in the shell.

"Self-repairing" nanoshell for cell protection.

Self-repair is nature's way of protecting living organisms. However, most single cells are inherently less capable of self-repairing, which greatly limits their wide applications. Here, we present a self-assembly approach to create a nanoshell around the cell surface using nanoporous biohybrid aggregates. The biohybrid shells present self-repairing behaviour, resulting in high activity and extended viability of the encapsulated cells (eukaryotic and prokaryotic cells) in harsh micro-environments, such as under UV radiation, natural toxin invasion, high-light radiation and abrupt pH-value changes. Furthermore, an interaction mechanism is proposed and studied, which is successful to guide design and synthesis of self-repairing biohybrid shells using different bioactive molecules.

Amino acid-based biohybrids for nano-shellization of individual desulfurizing bacteria.

Amino acid-based biohybrids have been developed to self-assemble on the surface of desulfurizing bacteria to form nanothin and nanoporous shells. The shells not only endow the encapsulated cells with reusability, but also offer platforms to incorporate titania and magnetic nanoparticles to improve the desulfurizing activity and the separation efficiency.