Engineering Highly Vascularized Bone Tissues by 3D Bioprinting of Granular Prevascularized Spheroids.

The convergence of 3D bioprinting with powerful manufacturing capability and cellular self-organization that can reproduce intricate tissue microarchitecture and function is a promising direction toward building functional tissues and has yet to be demonstrated. Here, we develop a granular aggregate-prevascularized (GAP) bioink for engineering highly vascularized bone tissues by capitalizing on the condensate-mimicking, self-organization, and angiogenic properties of prevascularized mesenchymal spheroids. The GAP bioink utilizes prevascularized aggregates as building blocks, which are embedded densely in extracellular matrices conducive to spontaneous self-organization. We printed various complex structures with high cell density (∼1.5 × 108 cells/cm3), viability (∼80%), and shape fidelity using GAP bioink. After printing, the prevascularized mesenchymal spheroids developed an interconnected vascular network through angiogenic sprouting. We printed highly vascularized bone tissues using GAP bioink and found that prevascularized spheroids were more conducive to osteogenesis and angiogenesis. We envision that the design of the GAP bioink could be further integrated with human-induced pluripotent stem cell-derived organoids, which opens new avenues to create patient-specific vascularized tissues for therapeutic applications..

3D printing of vascularized hepatic tissues with a high cell density and heterogeneous microenvironment.

Three-dimensional bioprinting has emerged as an appealing approach for creating functional tissues; however, a lack of suitable bioinks with high cell density and printability has greatly limited our ability to print functional tissues. We address this limitation by developing a granular cell aggregate-based biphasic (GCAB) bioink based on densely packed cell aggregates. The GCAB bioink exhibited the desired shear-thinning and shear-recovery properties for extrusion bioprinting and hyperelastic behaviors postprinting for modeling the mechanical characteristics of soft biological tissues. The GCAB bioink displayed a high cell density (∼1.7 × 108cells cm-3) without compromising viability (∼83%). We printed dense hepatic tissue constructs with enhanced vascularization and metabolic functions by preorganization of GCAB bioink with a defined heterogeneous microenvironment. By simultaneously printing the GCAB bioink and an endothelial cell-laden gelatin bioink, we successfully produced functional hepatic tissues with a high cell density and a perfusable vascular network. The design of the generalizable GCAB bioink opens new avenues to create functional tissues for therapeutic applications.

PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene.

Food wastes can be hydrolyzed into soluble microbial substrates, contributing to sustainability. Halomonas spp.-based Next Generation Industrial Biotechnology (NGIB) allows open, unsterile fermentation, eliminating the need for sterilization to avoid the Maillard reaction that negatively affects cell growth. This is especially important for food waste hydrolysates, which have a high nutrient content but are unstable due to batch, sources, or storage conditions. These make them unsuitable for polyhydroxyalkanoate (PHA) production, which usually requires limitation on either nitrogen, phosphorous, or sulfur. In this study, H. bluephagenesis was constructed by overexpressing the PHA synthesis operon phaCABCn (cloned from Cupriavidus necator) controlled by the essential gene ompW (encoding outer membrane protein W) promoter and the constitutive porin promoter that are continuously expressed at high levels throughout the cell growth process, allowing poly(3-hydroxybutyrate) (PHB) production to proceed in nutrient-rich (also nitrogen-rich) food waste hydrolysates of various sources. The recombinant H. bluephagenesis termed WZY278 generated 22 g L-1 cell dry weight (CDW) containing 80 wt% PHB when cultured in food waste hydrolysates in shake flasks, and it was grown to 70 g L-1 CDW containing 80 wt% PHB in a 7-L bioreactor via fed-batch cultivation. Thus, unsterilizable food waste hydrolysates can become nutrient-rich substrates for PHB production by H. bluephagenesis able to be grown contamination-free under open conditions.

Expanding Embedded 3D Bioprinting Capability for Engineering Complex Organs with Freeform Vascular Networks.

Creating functional tissues and organs in vitro on demand is a major goal in biofabrication, but the ability to replicate the external geometry of specific organs and their internal structures such as blood vessels simultaneously remains one of the greatest impediments. Here, this limitation is addressed by developing a generalizable bioprinting strategy of sequential printing in a reversible ink template (SPIRIT). It is demonstrated that this microgel-based biphasic (MB) bioink can be used as both an excellent bioink and a suspension medium that supports embedded 3D printing due to its shear-thinning and self-healing behavior. When encapsulating human-induced pluripotent stem cells, the MB bioink is 3D printed to generate cardiac tissues and organoids by extensive stem cell proliferation and cardiac differentiation. By incorporating MB bioink, the SPIRIT strategy enables the effective printing of a ventricle model with a perfusable vascular network, which is not possible to fabricate using extant 3D printing strategies. This SPIRIT technique offers an unparalleled bioprinting capability to replicate the complex organ geometry and internal structure in a faster manner, which will accelerate the biofabrication and therapeutic applications of tissue and organ constructs.

Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors.

Bacterial outer membrane (OM) is a self-protective and permeable barrier, while having many non-negligible negative effects in industrial biotechnology. Our previous studies revealed enhanced properties of Halomonas bluephagenesis based on positive cellular properties by OM defects. This study further expands the OM defect on membrane compactness by completely deleting two secondary acyltransferases for lipid A modification in H. bluephagenesis, LpxL and LpxM, and found more significant advantages than that of the previous lpxL mutant. Deletions on LpxL and LpxM accelerated poly(3-hydroxybutyrate) (PHB) production by H. bluephagenesis WZY229, leading to a 37% increase in PHB accumulation and 84-folds reduced endotoxin production. Enhanced membrane permeability accelerates the diffusion of γ-butyrolactone, allowing H. bluephagenesis WZY254 derived from H. bluephagenesis WZY229 to produce 82wt% poly(3-hydroxybutyrate-co-23mol%4-hydroxybutyrate) (P(3HB-co-23mol%4HB)) in shake flasks, showing increases of 102% and 307% in P(3HB-co-4HB) production and 4HB accumulation, respectively. The 4HB molar fraction in copolymer can be elevated to 32 mol% in the presence of more γ-butyrolactone. In a 7-l bioreactor fed-batch fermentation, H. bluephagenesis WZY254 supported a 84 g l-1 dry cell mass with 81wt% P(3HB-co-26mol%4HB), increasing 136% in 4HB molar fraction. This study further demonstrated that OM defects generate a hyperproduction strain for high 4HB containing copolymers.