Vapor-induced phase-separation-enabled versatile direct ink writing.

Versatile printing of polymers, metals, and composites always calls for simple, economic approaches. Here we present an approach to three-dimensional (3D) printing of polymeric, metallic, and composite materials at room conditions, based on the polymeric vapor-induced phase separation (VIPS) process. During VIPS 3D printing (VIPS-3DP), a dissolved polymer-based ink is deposited in an environment where nebulized non-solvent is present, inducing the low-volatility solvent to be extracted from the filament in a controllable manner due to its higher chemical affinity with the non-solvent used. The polymeric phase is hardened in situ as a result of the induced phase separation process. The low volatility of the solvent enables its reclamation after the printing process, significantly reducing its environmental footprint. We first demonstrate the use of VIPS-3DP for polymer printing, showcasing its potential in printing intricate structures. We further extend VIPS-3DP to the deposition of polymer-based metallic inks or composite powder-laden polymeric inks, which become metallic parts or composites after a thermal cycle is applied. Furthermore, spatially tunable porous structures and functionally graded parts are printed by using the printing path to set the inter-filament porosity as well as an inorganic space-holder as an intra-filament porogen.

[Ozone Sensitivity Analysis in Urban Beijing Based on Random Forest].

The basis and key step to developing ozone (O3) prevention and control measures is determining the non-linear relationship between O3 and its precursors. Based on online observations of O3, volatile organic compounds (VOCs), nitrogen oxides (NOx), and meteorological elements from April to September 2020 at an urban site in Beijing, we analyzed the pollution characteristics of O3 and its precursors, explored key factors affecting O3 using the random forest (RF) model combined with SHAP values, and explored the O3-VOCs-NOx sensitivity through a multi-scenarios analysis. The results of correlation analysis showed that the hourly concentration of O3 was significantly positively correlated with temperature (T) and negatively correlated with TVOCs and NOx. However, in terms of the daily values, O3 was significantly positively correlated with T, TVOCs, and NOx. The simulated O3 values by the RF model agreed with the measured values. The SHAP values of each characteristic variable were further calculated. The results suggested that T and NOx showed the two highest effects on O3, with positive and negative values, respectively. Based on the average NOx and VOCs on O3 pollution days during the observation period (the base scenario), multi-scenarios with different NOx and VOCs were set up. The RF model was used to calculate O3 under different scenarios and obtain the O3 isopleth (EKMA curve). The results showed that the O3-VOCs-NOx sensitivity in urban areas of Beijing was in the VOCs-limited regime, which was consistent with the results obtained from the observation-based box model(OBM). This indicated that the RF model could be used as a complementary method for O3-VOCs-NOx sensitivity analysis.

Lack of racial and ethnic diversity in lung cancer cell lines contributes to lung cancer health disparities.

Lung cancer is the leading cause of cancer death in the United States and worldwide, and a major source of cancer health disparities. Lung cancer cell lines provide key in vitro models for molecular studies of lung cancer development and progression, and for pre-clinical drug testing. To ensure health equity, it is imperative that cell lines representing different lung cancer histological types, carrying different cancer driver genes, and representing different genders, races, and ethnicities should be available. This is particularly relevant for cell lines from Black men, who experience the highest lung cancer mortality in the United States. Here, we undertook a review of the available lung cancer cell lines and their racial and ethnic origin. We noted a marked imbalance in the availability of cell lines from different races and ethnicities. Cell lines from Black patients were strongly underrepresented, and we identified no cell lines from Hispanic/Latin(x) (H/L), American Indian/American Native (AI/AN), or Native Hawaiian or other Pacific Islander (NHOPI) patients. The majority of cell lines were derived from White and Asian patients. Also missing are cell lines representing the cells-of-origin of the major lung cancer histological types, which can be used to model lung cancer development and to study the effects of environmental exposures on lung tissues. To our knowledge, the few available immortalized alveolar epithelial cell lines are all derived from White subjects, and the race and ethnicity of a handful of cell lines derived from bronchial epithelial cells are unknown. The lack of an appropriately diverse collection of lung cancer cell lines and lung cancer cell-of-origin lines severely limits racially and ethnically inclusive lung cancer research. It impedes the ability to develop inclusive models, screen comprehensively for effective compounds, pre-clinically test new drugs, and optimize precision medicine. It thereby hinders the development of therapies that can increase the survival of minority and underserved patients. The noted lack of cell lines from underrepresented groups should constitute a call to action to establish additional cell lines and ensure adequate representation of all population groups in this critical pre-clinical research resource.

Pollution characteristics, sources, and photochemical roles of ambient carbonyl compounds in summer of Beijing, China.

Ambient carbonyls are important precursors of radicals and ground-level ozone (O3). In this study, sources, precursors, and impacts on radicals and O3 of carbonyls were investigated based on online observations of volatile organic compounds (VOCs) at an urban site in Beijing during June 2021. Carbonyls accounted for 36% and 42% of mixing ratios and OH reactivity for total measured VOCs, respectively. Formaldehyde was the most abundant carbonyl, with the mean level of 4.13 ± 2.28 ppb. Source apportionment results based on the multi linear regression (MLR) method suggested that secondary production contributed 41%, 25%, 36%, and 30% of formaldehyde, acetaldehyde, propanal, and acetone, respectively. Key precursors of carbonyls were then identified based on the calculation of their production rates. It was found that alkenes contributed 59%-80% of aldehydes production. Impacts of carbonyls on HOx radicals (OH and HO2) and O3 production were explored using a box model based on observations (OBM). Photolysis of HONO, formaldehyde, and O3 were the dominant primary sources of HOx radicals during daytime of O3 pollution days, with average relative contributions of 52%, 28%, and 19% to the total primary production rate of HOx, respectively. Aldehydes accounted for 32% (20% from formaldehyde) of average HOx removal rates. The relative incremental reactivity (RIR) values of NOx determined by the OBM were negative, suggesting that the O3-VOCs-NOx sensitivity was in the VOCs-limited regime. Using the observed concentrations of carbonyls as constraints of OBM, the absolute values of RIR for NOx tended to increase but those for anthropogenic VOCs tended to decrease. Formaldehyde showed the largest RIR value for anthropogenic VOCs during O3 pollution days. These findings indicated the important impacts of carbonyls on O3 production and O3-VOCs-NOx sensitivity.

Evaluation of Low-dose Radiation-induced DNA Damage and Repair in 3D Printed Human Cellular Constructs.

ABSTRACT: DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) are considered to be the most critical lesion that when unrepaired or misrepaired leads to genomic instability or cell death depending on the radiation exposure dose. The potential health risks associated with exposures of low-dose radiation are of concern since they are being increasingly used in diverse medical and non-medical applications. Here, we have used a novel human tissue-like 3-dimensional bioprint to evaluate low-dose radiation-induced DNA damage response. For the generation of 3-dimensional tissue-like constructs, human hTERT immortalized foreskin fibroblast BJ1 cells were extrusion printed and further enzymatically gelled in a gellan microgel-based support bath. Low-dose radiation-induced DSBs and repair were analyzed in the tissue-like bioprints by indirect immunofluorescence using a well-known DSB surrogate marker, 53BP1, at different post-irradiation times (0.5 h, 6 h, and 24 h) after treatment with various doses of γ rays (50 mGy, 100 mGy, and 200 mGy). The 53BP1 foci showed a dose dependent induction in the tissue bioprints after 30 min of radiation exposure and subsequently declined at 6 h and 24 h in a dose-dependent manner. The residual 53BP1 foci number observed at 24 h post-irradiation time for the γ-ray doses of 50 mGy, 100 mGy, and 200 mGy was not statistically different from mock treated bioprints illustrative of an efficient DNA repair response at these low-dose exposures. Similar results were obtained for yet another DSB surrogate marker, γ-H2AX (phosphorylated form of histone H2A variant) in the human tissue-like constructs. Although we have primarily used foreskin fibroblasts, our bioprinting approach-mimicking a human tissue-like microenvironment-can be extended to different organ-specific cell types for evaluating the radio-response at low-dose and dose-rates of IR.

Characteristics, sources of volatile organic compounds, and their contributions to secondary air pollution during different periods in Beijing, China.

Continuous measurements of volatile organic compounds (VOCs), ozone (O3), fine particulate matter (PM2.5), and related parameters were conducted between April 2020 and March 2021 in Beijing, China, to characterize potential sources of VOCs and their impacts on secondary organic aerosols (SOAs) and O3 levels. The annual average mixing ratio of VOCs was 17.4 ± 10.1 ppbv, with monthly averages ranging from 11.6 to 25.2 ppbv. According to the empirical kinetic modeling approach (EKMA), O3 formation during O3 season was "VOCs-limited", while it was in a "transition" regime during O3 pollution episodes. In the O3 season, higher ozone formation potential (OFP) of m/p-xylene, o-xylene, toluene, isopentane, and n-butane were evident during O3 pollution episodes, in line with the increasing contributions of solvent usage and coating, as well as gasoline evaporation to OFP obtained through a matrix factorization model (PMF). Aromatics contributed the most to the secondary organic aerosol formation potential (SOAFP). In the non-O3 season, the contribution of vehicle exhaust to SOAFP elevated on hazy days, thereby revealing the importance of traffic-derived VOCs for PM2.5 pollution. Our results indicate that the prior control of different VOC sources should vary by season, thereby facilitating the synergistic control of O3 and PM2.5 in Beijing.

Design and Realization of Lung Organoid Cultures for COVID-19 Applications.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading globally and threatening public health. Advanced in vitro models that recapitulate the architecture and functioning of specific tissues and organs are in high demand for COVID-19-related pathology studies and drug screening. Three-dimensional (3D) in vitro cultures such as self-assembled and engineered organoid cultures surpass conventional two-dimensional (2D) cultures and animal models with respect to the increased cellular complexity, better human-relevant environment, and reduced cost, thus presenting as promising platforms for understanding viral pathogenesis and developing new therapeutics. This review highlights the recent advances in self-assembled and engineered organoid technologies that are used for COVID-19 studies. The challenges and future perspectives are also discussed.

Study of sacrificial ink-assisted embedded printing for 3D perfusable channel creation for biomedical applications.

For an engineered thick tissue construct to be alive and sustainable, it should be perfusable with respect to nutrients and oxygen. Embedded printing and then removing sacrificial inks in a cross-linkable yield-stress hydrogel matrix bath can serve as a valuable tool for fabricating perfusable tissue constructs. The objective of this study is to investigate the printability of sacrificial inks and the creation of perfusable channels in a cross-linkable yield-stress hydrogel matrix during embedded printing. Pluronic F-127, methylcellulose, and polyvinyl alcohol are selected as three representative sacrificial inks for their different physical and rheological properties. Their printability and removability performances have been evaluated during embedded printing in a gelatin microgel-based gelatin composite matrix bath, which is a cross-linkable yield-stress bath. The ink printability during embedded printing is different from that during printing in air due to the constraining effect of the matrix bath. Sacrificial inks with a shear-thinning property are capable of printing channels with a broad range of filaments by simply tuning the extrusion pressure. Bi-directional diffusion may happen between the sacrificial ink and matrix bath, which affects the sacrificial ink removal process and final channel diameter. As such, sacrificial inks with a low diffusion coefficient for gelatin precursor are desirable to minimize the diffusion from the gelatin precursor solution to minimize the post-printing channel diameter variation. For feasibility demonstration, a multi-channel perfusable alveolar mimic has been successfully designed, printed, and evaluated. The study results in the knowledge of the channel diameter controllability and sacrificial ink removability during embedded printing.

Distribution and transport characteristics of fine particulate matter in beijing with mobile lidar measurements from 2015 to 2018.

Accurately quantifying the concentration and transport flux of atmospheric fine particulate matter (PM2.5) is vital when attempting to thoroughly identify the pollution formation mechanism. In this study, the mobile lidar measurements in Beijing on heavily polluted days in December from 2015 to 2018 are presented. The lidar was mounted on a vehicle, which could perform measurements along designated routes. On the basis of mobile lidar measurements along closed circuits of the 6th Ring Road around Beijing, the spatial distribution and transport flux of PM2.5 in Beijing were determined with information of wind field. In the spatial distribution, both the concentration and transport of PM2.5 were revealed to be more significant in the southern section of Beijing. The regional transport layer at heights < 1.3 km plays an important role in pollution formation. The maximum transport flux reached 1600 µg/(m2*sec) on 11 December 2016. With the aerosol boundary layer height determined from the image edge detection (IED) method, the inter-annual variations of the aerosol boundary layer height (ABLH) were also analysed. The ABLH decreased from 0.73 to 0.46 km during the same heavy pollution period from 2015 to 2018. Increasingly adverse aerosol boundary layer (ABL) meteorological factors, including lower ABLH, light winds, temperature inversions, and accumulated moisture, have become necessary for pollution formation in Beijing.

Effects of transglutaminase cross-linking process on printability of gelatin microgel-gelatin solution composite bioink.

Three-dimensional (3D) bioprinting has emerged as a powerful engineering approach for various tissue engineering applications, particularly for the development of 3D cellular structures with unique mechanical and/or biological properties. For the jammed gelatin microgel-gelatin solution composite bioink, comprising a discrete phase of microgels (enzymatically gelled gelatin microgels) and a cross-linkable continuous gelatin precursor solution-based phase containing transglutaminase (TG), its rheological properties and printability change gradually due to the TG enzyme-induced cross-linking process. The objective of this study is to establish a direct mapping between the printability of the gelatin microgel-gelatin solution based cross-linkable composite bioink and the TG concentration and cross-linking time, respectively. Due to the inclusion of TG in the composite bioink, the bioink starts cross-linking once prepared and is usually prepared right before a printing process. Herein, the bioink printability is evaluated based on the three metrics: injectability, feature formability, and process-induced cell injury. In this study, the rheological properties such as the storage modulus and viscosity have been first systematically investigated and predicted at different TG concentrations and times during the cross-linking process using the first-order cross-linking kinetics model. The storage modulus and viscosity have been satisfactorily modeled as exponential functions of the TG concentration and time with an experimentally calibrated cross-linking kinetic rate constant. Furthermore, the injectability, feature formability, and process-induced cell injury have been successfully correlated to the TG concentration and cross-linking time via the storage modulus, viscosity, and/or process-induced shear stress. By combing the good injectability, good feature formability, and satisfactory cell viability zones, a good printability zone (1.65, 0.61, and 0.31 h for the composite bioinks with 1.00, 2.00, and 4.00% w/v TG, respectively) has been established during the printing of mouse fibroblast-based 2% gelatin B microgel-3% gelatin B solution composite bioink. This printability zone approach can be extended to the use of other cross-linkable bioinks for bioprinting applications.

[Heavy Pollution Characteristics and Assessment of PM2.5 Predicted Model Results in Beijing-Tianjin-Hebei Region and Surrounding Areas During November 23 to December 4, 2018].

This study discusses the concentration characteristics of PM2.5 and PM10, as well as pollution meteorology in large-scale and long-term heavy pollution in the Beijing-Tianjin-Hebei region and its surrounding areas from November 23 to December 4, 2018, where the primary pollutants are comprised of PM2.5 and PM10. The monitoring results obtained from ground-based and vehicle-mounted lidars, as well as the HYSPLIT-4 backward trajectory combined with meteorological factors analysis are discussed. The accuracy and uncertainty of the air quality forecast model of NAQPMS, CMAQ, and CAMx during heavy air pollution were analyzed retrospectively. The results show that PM2.5 and sand dust in most cities in the south-central region contribute to severe pollution levels. The hourly peak concentrations of PM10 in Zhangjiakou, Beijing, Shijiazhuang, Handan, and Zhengzhou were 1589, 864, 794, 738, and 766 µg·m-3, respectively. The respective hourly peak concentrations of PM2.5 were 239, 319, 387, 321, and 380 µg·m-3. Ground static pressure field, high humidity, inversion, and other static and stable conditions, as well as sand dust transmitted from the northwest, were important pollution meteorological and weather factors. The monitoring data of ground-based lidar and vehicle-mounted lidar combined with the HYSPLIT-4 backward trajectory analysis showed that the air pollutant transmitted from the Southwest and Southeast during the heavy pollution period was primarily PM2.5. The air pollutant transmitted from the Northwest during the two sand dust processes. Moreover, the model of NAQPMS, CMAQ, and CAMx performed well in forecasting the heavy pollution process in the Beijing-Tianjin-Hebei region and its surrounding areas. However there are slight deviations for some individual cities, related to uncertainty in the meteorological model prediction, atmospheric chemical reaction mechanism, and pollution source list. Furthermore, the reduction in pollution source emissions caused by pollution emergency measures was also one of the main reasons for the overestimation.

Injectable Gelatin Microgel-Based Composite Ink for 3D Bioprinting in Air.

Injectable hydrogels have attracted much attention in tissue engineering and regenerative medicine for their capability to replace implantation surgeries with a minimally invasive injection procedure and ability to fill irregular defects. The proposed composite ink is a gelatin microgel-based yield-stress and shear-thinning composite material that is injectable and solidifies quickly after injection at room temperature, which can be utilized for the creation of three-dimensional parts in air directly. The gelatin composite ink consists of a microgel solid phase (gelled gelatin microgels) and a cross-linkable solution phase (gelatin solution-based acellular or cellular suspension). The gelatin composite ink can be injected or printed directly in air and solidifies as physical cross-linking to hold printed structures at room temperature. The fabricated part further undergoes a chemical cross-linking process when immersed in a transglutaminase solution to enzymatically gel the gelatin solution, making a physiologically stable construct as needed. Lattice, tube-shaped, cup-shaped, and human anatomical (ear and nose) structures are printed to demonstrate the feasibility of the proposed composite ink for printing applications. The morphology and metabolic activity of cells cultured in the gelatin composite ink are further analyzed to confirm the suitability of the proposed composite ink to provide a beneficial physiological environment for bioprinting needs.

Comprehensive study of regional haze in the North China Plain with synergistic measurement from multiple mobile vehicle-based lidars and a lidar network.

Recently, haze pollution has emerged as a regional characteristic that needs to be monitored and mitigated sensibly in China, particularly in the North China Plain (NCP). Clarifying the distribution and source characteristics of haze is necessary to better understand its formation mechanism on a regional scale. In this study, a comprehensive study of regional haze using synergistic measurement from multiple mobile vehicle-based lidars, a ground-based lidar network, and in suit instruments is presented. To investigate the distribution and source characteristics of regional haze in the NCP during the winter of 2017, simultaneous measurements of aerosol under different wind conditions are conducted. The regional distribution characteristics of the aerosol were observed using three sets of mobile vehicle-based lidars, and the source characteristics were achieved using an analysis of transport flux (with the ground-based lidar network and the WRF-Chem model). High aerosol extinction was observed on the southwest pathway under a southern wind. Backward trajectories also indicated that the air masses at 500 m were primarily from the southwest. The transport flux at the boundary of Beijing (BJ) and Baoding (BD) on the southwest pathway was calculated. Below 500 m, the transport flux from BD to BJ was positive under a southern wind and negative under a northern wind. In addition to the transport layer below 500 m, an upper transport layer was observed both on November 6, 2017 and January 15, 2018. The upper transport layer from 500 m to 1500 m on November 6, 2017 was obviously noticeable, which decreased dramatically with a maximum transport flux of 539.53 µg m2 s. The significant transport layer at 1250 m with a maximum flux of 614.93 µg m2 s was observed on January 15, 2018, while it had no impact on the ground because it had not yet fallen.

Cross-Linkable Microgel Composite Matrix Bath for Embedded Bioprinting of Perfusable Tissue Constructs and Sculpting of Solid Objects.

Tissue engineering is a rapidly growing field, which requires advanced fabrication technologies to generate cell-laden tissue analogues with a wide range of internal and external physical features including perfusable channels, cavities, custom shapes, and spatially varying material and/or cell compositions. A versatile embedded printing methodology is proposed in this work for creating custom biomedical acellular and cell-laden hydrogel constructs by utilizing a biocompatible microgel composite matrix bath. A sacrificial material is patterned within a biocompatible hydrogel precursor matrix bath using extrusion printing to create three-dimensional features; after printing, the matrix bath is cross-linked, and the sacrificial material is flushed away to create perfusable channels within the bulk composite hydrogel matrix. The composite matrix bath material consists of jammed cross-linked hydrogel microparticles (microgels) to control rheology during fabrication along with a fluid hydrogel precursor, which is cross-linked after fabrication to form the continuous phase of the composite hydrogel. For demonstration, gellan or enzymatically cross-linked gelatin microgels are utilized with a continuous gelatin hydrogel precursor solution to make the composite matrix bath herein; the composite hydrogel matrix is formed by cross-linking the continuous gelatin phase enzymatically after printing. A variety of features including discrete channels, junctions, networks, and external contours are fabricated in the proposed composite matrix bath using embedded printing. Cell-laden constructs with printed features are also evaluated; the microgel composite hydrogel matrices support cell activity, and printed channels enhance proliferation compared to solid constructs even in static culture. The proposed method can be expanded as a solid object sculpting method to sculpt external contours by printing a shell of sacrificial ink and further discarding excess composite hydrogel matrix after printing and cross-linking. While aqueous alginate solution is used as a sacrificial ink, more advanced sacrificial materials can be utilized for better printing resolution.

Laser printing-enabled direct creation of cellular heterogeneity in lab-on-a-chip devices.

Lab-on-a-chip devices, capable of culturing living cells in continuously perfused, micrometer-sized channels, have been intensively investigated to model physiological microenvironments for cell-related testing and evaluation applications. Various chemical, physical, and/or biological culture cues are usually expected in a designed chip to mimic the in vivo environment with defined spatial heterogeneity of cells and biomaterials. To create such heterogeneity within a given chip, typical methods rely heavily on sophisticated fabrication and cell seeding processes, and chips fabricated with these methods are difficult to readily adapt for other applications. In this study, laser-induced forward transfer (LIFT)-based printing has been implemented to create heterogeneous cellular patterns in a lab-on-a-chip device to achieve the efficiency in creating heterogeneous cellular patterns as well as the flexibility in adapting different evaluation configurations in lab-on-a-chip devices. Two applications, parallel evaluation of cellular behavior and targeted drug delivery to cancer cells, have been implemented as proof-of-concept demonstrations of the proposed fabrication method. For the first application, the morphology of cells in different extracellular matrix (ECM) materials cultured under varying conditions has been investigated. It is found that less stiff ECM and dynamic culturing are preferred for spreading of fibroblasts. For the second application, different drug carriers have been utilized for targeted delivery of anticancer drugs to breast cancer cells. It is found that targeted drug delivery is important to realize effective chemotherapy and drug release rate from drug carriers affects the chemotherapy effect. Consequently, the proposed laser printing-based method enables direct creation of heterogeneous cellular patterns within lab-on-a-chip devices which improves the efficiency and versatility of cell-related sensing and evaluation using lab-on-a-chip devices.

Fabrication of Stand-Alone Cell-Laden Collagen Vascular Network Scaffolds Using Fugitive Pattern-Based Printing-Then-Casting Approach.

Vascular networks are of great significance in tissue engineering and viewed as the first step to fabricate human tissues. Although various techniques have been investigated to create vascular and vascular-like networks, the fabrication of stand-alone pure collagen-based vascular constructs is still a challenge because of the poor extrudability, weak mechanical property, and long cross-linking time of pure collagen solutions. In this study, a fugitive pattern-based printing-then-casting approach is investigated. The proposed alginate-based fugitive ink has excellent mechanical strength (by adding Laponite nanoclay), printability (by adding Laponite nanoclay), and controllable gelation rate (by adding disodium hydrogen phosphate). Using this fugitive ink, complex vascular-like structures can be easily printed and cross-linked in Laponite EP bath as fugitive vascular tree patterns. Each fugitive vascular tree pattern is then embedded in a gelatin bath to make a gelatin mold with the tree patterns. With the help of sodium citrate, the fugitive vascular tree pattern is liquefied and removed to create the gelatin mold with vascular channels. Finally, a stand-alone collagen vascular network scaffold embedded with fibroblasts can be fabricated by casting the cell-laden collagen suspension into the gelatin mold and releasing it from the mold at 37 °C. The cell-related investigations indicate that the cells grow and spread well in the pure collagen vascular network scaffold. The proposed hybrid printing-then-casting approach also provides a feasible technology to fabricate with materials having low viscosity, long gelation time, and poor mechanical property.

Printability study of hydrogel solution extrusion in nanoclay yield-stress bath during printing-then-gelation biofabrication.

Yield-stress support bath-enabled extrusion printing is emerging as a promising filament-based direct-write strategy for different applications in tissue engineering and regenerative medicine. Central to the printing quality of complex three-dimensional structures fabricated by the support bath-enabled fabrication approach is the formation of a continuous filament with well-defined geometry. The objective of this research is to study the printability of hydrogel precursor solutions in a Laponite nanoclay yield-stress bath during extrusion printing where the printed hydrogel precursor solutions remain liquid. The printability herein is mainly evaluated based on the morphology and dimensions of printed liquid filaments. Seven filament types are observed during extrusion in the nanoclay bath: three types of well-defined filaments (swelling, equivalent diameter, and stretched) and four types of irregular filaments (rough surface, over-deposited, compressed, and discontinuous). When the alginate concentration increases, the diameter of filaments made of alginate-gelatin blends decreases. The nanoclay concentration significantly affects the morphology of deposited filaments: low concentration Laponite bath (such as 0.5% (w/v)) may lead to the formation of irregular filaments such as rough surface and over-deposited filaments while high concentration bath (such as 8.0% (w/v)) may result in the formation of compressed filaments. Operating conditions affect the filament diameter and morphology similar to those as observed during conventional extrusion printing. The printability knowledge enables the successful fabrication of cellular vascular constructs in the Laponite nanoclay bath.

Study of gelatin as an effective energy absorbing layer for laser bioprinting.

Laser-induced forward transfer printing, also commonly known as laser printing, has been widely implemented for three-dimensional bioprinting due to its unique orifice-free nature during printing. However, the printing quality has the potential to be further improved for various laser bioprinting applications. The objectives of this study are to investigate the feasibility of using gelatin as an energy absorbing layer (EAL) material for laser bioprinting and its effects on the quality of printed constructs, bioink printability, and post-printing cell viability and process-induced DNA damage. The gelatin EAL is applied between the quartz support and the coating of build material, which is to be printed. Printing quality can be improved by EAL-assisted laser printing when using various alginate solutions (1%, 2%, and 4%) and cell-laden bioinks (2% alginate and 5 × 106 cells ml-1 in cell culture medium). The required laser fluence is also reduced due to a higher absorption coefficient of gelatin gel, in particular when to achieve the best printing type/quality. The post-printing cell viability is improved by ∼10% and DNA double-strand breaks are reduced by ∼50%. For all the build materials investigated, the gelatin EAL helps reduce the droplet size and average jet velocity.

Printing-induced cell injury evaluation during laser printing of 3T3 mouse fibroblasts.

Three-dimensional bioprinting has emerged as a promising solution for the freeform fabrication of living cellular constructs, which can be used for tissue/organ transplantation and tissue models. During bioprinting, some living cells are unavoidably injured and may become necrotic or apoptotic cells. This study aims to investigate the printing-induced cell injury and evaluates injury types of post-printing cells using the annexin V/7-aminoactinomycin D and FAM-DEVD-FMK/propidium iodide assays during laser printing of NIH 3T3 mouse fibroblasts. As observed, the percentage of post-printing early apoptotic mouse fibroblasts increases with the incubation time, indicating that post-printing apoptotic mouse fibroblasts have different initiation lag times of apoptosis due to different levels of mechanical stress exerted during laser printing. Post-printing necrotic mouse fibroblasts can be detected immediately after printing, while post-printing early apoptotic mouse fibroblasts need time to develop into a late apoptotic stage. The minimum time needed for post-printing early apoptotic mouse fibroblasts to complete their apoptosis pathway and transition into late apoptotic mouse fibroblasts is from 4 h to 5 h post-printing. The resulting knowledge of the evolution of different apoptotic post-printing mouse fibroblasts will help better design future experiments to quantitatively determine, model, and mitigate the post-printing cell injury based on molecular signal pathway modeling.

Self-Supporting Nanoclay as Internal Scaffold Material for Direct Printing of Soft Hydrogel Composite Structures in Air.

Three dimensional (3D) bioprinting technology enables the freeform fabrication of complex constructs from various hydrogels and is receiving increasing attention in tissue engineering. The objective of this study is to develop a novel self-supporting direct hydrogel printing approach to extrude complex 3D hydrogel composite structures in air without the help of a support bath. Laponite, a member of the smectite mineral family, is investigated to serve as an internal scaffold material for the direct printing of hydrogel composite structures in air. In the proposed printing approach, due to its yield-stress property, Laponite nanoclay can be easily extruded through a nozzle as a liquid and self-supported after extrusion as a solid. Its unique crystal structure with positive and negative charges enables it to be mixed with many chemically and physically cross-linked hydrogels, which makes it an ideal internal scaffold material for the fabrication of various hydrogel structures. By mixing Laponite nanoclay with various hydrogel precursors, the hydrogel composites retain their self-supporting capacity and can be printed into 3D structures directly in air and retain their shapes before cross-linking. Then, the whole structures are solidified in situ by applying suitable cross-linking stimuli. The addition of Laponite nanoclay can effectively improve the mechanical and biological properties of hydrogel composites. Specifically, the addition of Laponite nanoclay results in a significant increase in the Young's modulus of each hydrogel-Laponite composite: 1.9-fold increase for the poly(ethylene glycol) diacrylate (PEGDA)-Laponite composite, 7.4-fold increase for the alginate-Laponite composite, and 3.3-fold increase for the gelatin-Laponite composite.