3D Bioprinting: from Benches to Translational Applications.

Over the last decades, the fabrication of 3D tissues has become commonplace in tissue engineering and regenerative medicine. However, conventional 3D biofabrication techniques such as scaffolding, microengineering, and fiber and cell sheet engineering are limited in their capacity to fabricate complex tissue constructs with the required precision and controllability that is needed to replicate biologically relevant tissues. To this end, 3D bioprinting offers great versatility to fabricate biomimetic, volumetric tissues that are structurally and functionally relevant. It enables precise control of the composition, spatial distribution, and architecture of resulting constructs facilitating the recapitulation of the delicate shapes and structures of targeted organs and tissues. This Review systematically covers the history of bioprinting and the most recent advances in instrumentation and methods. It then focuses on the requirements for bioinks and cells to achieve optimal fabrication of biomimetic constructs. Next, emerging evolutions and future directions of bioprinting are discussed, such as freeform, high-resolution, multimaterial, and 4D bioprinting. Finally, the translational potential of bioprinting and bioprinted tissues of various categories are presented and the Review is concluded by exemplifying commercially available bioprinting platforms.

Indoor nanoscale particulate matter-induced coagulation abnormality based on a human 3D microvascular model on a microfluidic chip.

BACKGROUND: A growing body of evidence shows that indoor concentrations of airborne particles are often higher than is typically encountered outdoors. Since exposure to indoor PM2.5 is thought to be associated with cardiovascular disease, the health impacts of indoor air pollution need to be explored. Based on animal models, ambient particulate matter has been proved to promote coagulation which is very likely involved in the pathogenic development of cardiovascular disease. However, animal models are insufficient to predict what will happen with any certainty in humans. For this reason, the precise pathogenic mechanisms behind the development of cardiovascular disease in humans have not yet been determined. RESULTS: We generated a 3D functional human microvascular network in a microfluidic device. This model enables human vascular endothelial cells to form tissue-like microvessels that behave very similarly to human blood vessels. The perfusable microvasculature allows the delivery of particles introduced into these generated human-like microvessels to follow the fluid flow. This exposure path effectively simulates the dynamic movement of airborne nanoscale particles (ANPs) within human vessels. In this study, we first identified the existence of ANPs in indoor air pollution. We then showed that ANPs could activate endothelial cells via ROS induced inflammation, and further resulted in abnormal expression of the coagulation factors (TF, TM and t-PA) involved in coagulation cascades. In addition, we found that a protein could cover ANPs, and this biointeraction could interfere with heparan sulfate (HS). Human organotypic 3D microvessel models provide a bridge for how research outcomes can translate to humans. CONCLUSIONS: The 3D human microvessel model was used to determine the physiological responses of human vessels to ANP stimulation. Based on the obtained data, we concluded that ANPs not only disrupts normal coagulation functions, but also act directly on anticoagulant factors in human vessels. These experimental observations provide a potential biological explanation for the epidemiologically established link between ANPs and coagulation abnormality. This organ-on-chip model may provide a bridge from in vitro results to human responses.

Fra-2 is a novel candidate drug target expressed in the podocytes of lupus nephritis.

Lupus nephritis (LN) is a common and devastating complication caused by systemic lupus erythematosus. In this study, we evaluated the expression and mechanism of Fos-related antigen 2 (Fra-2) in LN. The results showed that Fra-2 was significantly increased in kidney biopsies of LN patients compared with healthy controls and other kidney disease in glomerular podocytes. The MRL/lpr mouse strain is a murine model of lupus, and it was used to study the mechanisms of Fra-2 in LN. The results showed that Fra-2 was expressed in the glomerular podocytes. We investigated the effects of inflammatory stimuli on Fra-2 protein expression in the glomerular podocytes, and found that interferon gamma was most effective at increasing Fra-2 protein expression. Knockdown of Fra-2 using siRNA enhanced the protein expression of nephrin. Therefore, Fra-2 may be a specific drug target for podocyte injury in LN.

Digitally Tunable Microfluidic Bioprinting of Multilayered Cannular Tissues.

Despite advances in the bioprinting technology, biofabrication of circumferentially multilayered tubular tissues or organs with cellular heterogeneity, such as blood vessels, trachea, intestine, colon, ureter, and urethra, remains a challenge. Herein, a promising multichannel coaxial extrusion system (MCCES) for microfluidic bioprinting of circumferentially multilayered tubular tissues in a single step, using customized bioinks constituting gelatin methacryloyl, alginate, and eight-arm poly(ethylene glycol) acrylate with a tripentaerythritol core, is presented. These perfusable cannular constructs can be continuously tuned up from monolayer to triple layers at regular intervals across the length of a bioprinted tube. Using customized bioink and MCCES, bioprinting of several tubular tissue constructs using relevant cell types with adequate biofunctionality including cell viability, proliferation, and differentiation is demonstrated. Specifically, cannular urothelial tissue constructs are bioprinted, using human urothelial cells and human bladder smooth muscle cells, as well as vascular tissue constructs, using human umbilical vein endothelial cells and human smooth muscle cells. These bioprinted cannular tissues can be actively perfused with fluids and nutrients to promote growth and proliferation of the embedded cell types. The fabrication of such tunable and perfusable circumferentially multilayered tissues represents a fundamental step toward creating human cannular tissues.

A smart multi-functional coating based on anti-pathogen micelles tethered with copper nanoparticles via a biosynthesis method using l-vitamin C.

A multi-functional anti-pathogen coating with "release-killing", "contact-killing" and "anti-adhesion" properties was prepared from biocompatible polymer encapsulated chlorine dioxide (ClO2) which protected the active ingredient from the outside environment. A slow sustained-release of ClO2 from micelles over fifteen days was detected for long-term release-killing. Micelles only release ClO2 on demand in minimum inhibitory concentrations. We prepared nanoparticles which were covalently clustered on micelle surfaces to improve contact-killing as well as to improve the stability of the micelle. Copper nanoparticles were generated using the biosynthesis method including l-vitamin C, which avoids the toxicity and allows for the preparation of copper nanoparticles in a green environment. Synergistic anti-pathogen activity could be generated by a combination of micelle released ClO2 and ascorbic acid. In addition to release-killing and contact-killing, a pluronic polymer coated surface also provides an additional "anti-adhesion" property through its protein-repelling ability. In this research, the designed coating demonstrated a broad-spectrum of activity to kill drug-resistant bacteria, viruses and spores in short period of time. Based on scanning electron microscopy (SEM), transmission electron microscopy (TEM) and anti-oxidase assays, we found that the designed coatings killed the pathogens via bio-oxidation. We also carried out acute respiratory toxicity tests in this research. Analysis of blood samples, lung function and histopathological slices indicated that the synthesized micelles allowed a controlled and sustained release of ClO2 to kill pathogens while maintaining an overall ClO2 concentration in the air within a safe range.

Correction: Functional human 3D microvascular networks on a chip to study the procoagulant effects of ambient fine particulate matter.

[This corrects the article DOI: 10.1039/C7RA11357A.].

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

Coimplanted endothelial cells improve adipose tissue grafts' survival by increasing vascularization.

BACKGROUND: With goal of improving fat graft survival, many studies have focused on supplementing cells in the graft fat. In these studies, enhanced vascularization is considered the most important mechanism for the improved graft survival. Endothelial cells (ECs) are essential in vessel formation of the vascularization. Therefore, in this study, we coimplanted ECs with adipose tissue to investigate whether the ECs can enhance graft survival in a cell concentration-dependent manner. METHODS: Endothelial cells were isolated from stromal vascular fraction derived from human liposuction aspirates, and the EC characteristics were confirmed by CD31 immunofluorescence staining, measuring acetylated low-density lipoprotein uptake, and observing the formation of capillary-like tubular structures in Matrigel. During the animal experiment, the isolated ECs were labeled, then added to 0.5-mL fat grafts at different numbers (0.5 × 10(6), 1 × 10(6), 2 × 10(6), and 4 × 10(6) cells) before subcutaneous implantation in nude mice. Grafts were harvested at 1 week, 1 month, and 2 months after -transplantation, and graft survival and vascularization were evaluated based on weight measurements, histological assessment, and vascular gene expression. RESULTS: Stromal vascular fraction-derived vascular cells exhibited typical EC characteristics. The observed differences in explanted graft weight, vessel density, vascular gene expression, and cell tracking result indicated that coimplantation with ECs accelerated vascularization that increased graft survival in a concentration-dependent manner. Over the experimental period, fat grafts implanted with 4 × 10(6) ECs showed no weight loss and the greatest increases in measures of vascularization. CONCLUSIONS: Endothelial cells can effectively enhance vascularization in fat grafts, and higher EC concentrations (eg, 4 × 10(6) ECs/0.5 mL adipose tissue) may best support graft survival.