Use of a 3D inkjet‐printed model to access dust particle toxicology in the human alveolar barrier

Fine dust particles in the air travel into our body via the airway tract and cause severe respiratory diseases. Thus, the analysis of the effects of dust particles on the respiratory system has been receiving significant research interest. However, most studies on the toxicity of dust particles involve two‐dimensional (2D) cell cultures, animal models, and epidemiology. Here, we inkjet‐printed a three‐dimensional (3D) alveolar barrier model to study how dust particles cause respiratory diseases. The three‐layered in vitro model was exposed to A2 fine test dust with varying concentrations and exposure durations. The results highlighted the destruction of the tissue architecture along with apoptosis in the bioprinted alveolar barrier. The damage at the cellular level induced an increase in the amount of pro‐inflammatory cytokines secreted, followed by triggering of the signal transduction pathway and activation of transcription factors. As a consequence of the release of cytokines, the extracellular matrix was degraded, which led to the collapse of the cell structure, loss of cell polarity, and a decrease in barrier tightness. Further, the pulmonary surfactant protein‐related genes in the dust‐treated alveolar tissue were investigated to evaluate the possible role of dust particles in pulmonary surfactant dysfunction. This study demonstrated the use of 3D‐printed tissue model to evaluate the physiological impact of fine dust particles on cytotoxicity, alveolar barrier rigidity, and surfactant secretion of an alveolar barrier.

The incidence of these diseases has been mainly identified in epidemiological clinical analysis and animal studies. Epidemiologically, the concentration of fine dust particles in confined areas has been measured over the years, and its association with the incidence of lung cancer in the local residents of the confined areas has been investigated (Consonni et al., 2018). After controlling most of the potential variables in the enclosed areas, suggestive evidence of a positive correlation between particulate matter exposure and lung cancer was established. Sogl et al. (2012) investigated the exposureresponse relationship between silica and lung cancer risk using data from the German uranium miner cohort study. The cohort includes 58,677 workers between 1946 and 2003, with individual information on occupational exposure to silica and the potential confounders such as radon and arsenic. Consequently, the study confirmed a positive relationship between silica and lung cancer, particularly for high exposures.
Meanwhile, an increase in lymphocytes, macrophages, and inflammatory cytokines was confirmed in mice exposed to these particles (Chan et al., 2019). Inflammatory cytokine activation has been observed in patients with asthma, chronic obstructive pulmonary disease, and pulmonary inflammation, suggesting that continuous exposure to dust particles can increase susceptibility to these conditions. He et al. (2017) identified the allergic inflammatory response mechanism of urban PM 2.5 , or fine inhalable particles with diameters that are generally 2.5 µm or smaller, which is known to exacerbate asthma. This study demonstrated that urban PM 2.5 may exacerbate allergic inflammation in the murine lung via a TLR2/TLR4/ MyD88-signaling pathway. The results suggested that inhalation of urban PM 2.5 imposes a significant risk for inflammatory and allergic lung diseases. Furthermore, over the decades, the risk of dust particles has been examined by cellular biological analysis, which has been improved to assess the detrimental effects of particles from microscopic and systematic perspectives. Representatively, particulate matter has been demonstrated to aggravate the factors that induce asthma through the growth factor-related signaling pathways of oxidative stress and expression in cultured human bronchial epithelial cells exposed to air (Hyseni et al., 2012). Thus, treatment with growth-factor-receptor antagonist drugs has been proposed to suppress the secretion of asthma-causing substances. Leclercq et al. (2018) studied oxidative, inflammatory, and apoptotic in human lung epithelial cells exposed to air pollution-derived PM 2.5 and concluded that PM 2.5 can cause cytoplasmic reactive oxygen species (ROS) overproduction, causing oxidative damage and activating oxygensensitive NF-kB signaling pathways, as well as continuous mitochondrial dysfunction that lowers cell energy supply.
As stated previously, there are numerous studies on the toxicity of dust particles on epidemiological investigation, 2D cell culture, and animal models; however, based on available information, no relevant study has been conducted involving 3D alveolar tissue models containing multiple layers. The 3D in vitro tissue model based on cells originating from humans can provide more in-depth information on the cell-cell and cell-ECM interactions in the in vivo environment.
Since almost all cells in the in vivo environment are surrounded threedimensionally by other cells and ECMs, 3D cell cultures are more appropriate considering the natural microenvironment of cells (Edmondson et al., 2014). Therefore, 3D artificial tissue possesses significant potential for application in translational studies and is expected to bridge the gap between cell cultures and animal models (Jensen & Teng, 2020). Meanwhile, bioprinting is a rapidly emerging technology used for creating these 3D culture models. With bioprinting, the simultaneous positioning of biomaterials and living cells in a prescribed layer-by-layer stacking arrangement can be achieved to afford engineered tissues and organs (Moroni et al., 2018;Sun et al., 2020). Our recent study demonstrated that the threelayered alveolar barrier model created by inkjet-bioprinting exhibited the physiological functions of the natural human alveolar barrier (Kang et al., 2021). Since inkjet bioprinting can precisely position cells with a high resolution Park et al., 2017;Yoon et al., 2018), it is suitable for mimicking extremely thin and fine structures, such as the alveoli. The tissue produced by the automated integrated inkjet bioprinting system is acceptable for use as a test model for toxicological testing of substances with the capability of quality controlled tissue production.
Here, the detrimental effect of fine dust particles was investigated using an alveolar barrier model fabricated by inkjet printing following the process developed in a previous study. After exposing the fine dust particles to the 3D alveolar barrier, various detailed evaluations associated with respiratory function were performed including tissue metabolism and apoptosis assays, tissue structure changes and barrier integrity verification, and gene analysis.
Consequently, physiological and pathological structural changes and gene-level regulation were observed in the tissue exposed to the particles. The results of these studies suggest the correlations between fine dust particles and respiratory diseases.

| Engineering and 3D culture of the alveolar barrier
The alveolar barrier construct was fabricated by printing the four different cell types in a layer-by-layer manner using the drop-ondemand inkjet bioprinting system (Jetlab II; MicroFab). The cells were directly printed on the porous surface of transwells. The total numbers of printed NCI-H1703, NCI-H441, MRC5, and HULEC-5a cells were 8 × 10 4 , 1.6 × 10 5 , 1.8 × 10 5 , and 3 × 10 5 , respectively, which was consistent with the numbers of cells in the natural human alveoli (Pinkerton et al., 2015). The all-inkjet-printed alveolar barrier was cultured in a 1:1:1 mixture of the complementary cell-culture medium (RPMI-1640, MEM α, and MCDB 131) with aprotinin (100 Kallikrein inhibitor unit; A1153, Sigma-Aldrich). The growth medium (500 µl) was added to the basolateral side of the 12-mm cell culture insert, whereas the apical side was exposed to air during the incubation. The culture medium was replaced once a day. The cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 for 7 days before the dust-particle assessment. The information on detailed fabrication process can be described elsewhere (Kang et al., 2021).

| Dust preparation and tissue exposure
As model fine dust particles, Arizona A2 fine test dust (ISO 12103-1, Powder Tech. Inc.) was used, which consists of 69%-77% quartz. The standardized particles were employed to increase the reproducibility of experimental outcomes. The particles were stirred in Dulbecco's phosphate-buffered saline (DPBS, HyClone) at concentrations of 67.2, 224, and 672 μg/ml, after which they were vortexed for a few seconds before use as solution. After 7 days of air exposure, the alveolar barrier constructs were treated with dust particles at concentrations of 30, 100, and 300 μg/cm 2 in DPBS solution. To determine the concentration of dust particles per unit area, 500 μL of the solution was administered to the tissue epithelial layer over an area of 1.12 cm 2 . The epithelial layer of the control group was immersed in a 500 μl DPBS solution without dust. Dust particles were introduced at a single point in time, and the apical side was not washed during the exposure period. The dust-particle-exposure durations were 1, 3, 5, and 7 days. Over time, the dust particles settled in the epithelial layer.

| Tissue proliferation assay
Tissue proliferation and metabolic activity were assessed in the dusttreated alveolar barrier constructs using Cell Counting Kit 8 (CCK 8; Dojindo Laboratories). The assay reagent was mixed with the cell growth medium in the ratio of 1:9. Thereafter, 0.5 ml of the mixture was added to the basolateral side of the 12-mm cell-culture insert and incubated for 1.5 h. The ultraviolet A (UVA) absorbance of the mixture was measured at 450 nm at each time point using a microplate reader (Spark; Tecan).

| Histological analysis
For the histological evaluation of the dust-treated alveolar barrier models, the samples were fixed in 1 ml of a 4% paraformaldehyde solution that was added to the tissue basolateral chamber overnight.
The samples containing a porous membrane were cut out from the cell culture insert. The samples were embedded in optimal cutting temperature compounds (Leica Biosystems) and frozen at −80°C.
Serial 10-µm-thick sections were obtained using a cryostat (CM1860; Leica Biosystems). The tissue sections were stained with hematoxylin (Mayer's; Dako) and eosin Y (0.5% alcohol; Merck) to visualize and compare the structures of the tissues treated with dust particles. The sections were stained with Alcian blue (pH: 2.5; Sigma-Aldrich) for observation of secreted surfactant, and nuclear fast red (Sigma-Aldrich) stained the cell nuclei.
To determine the effect of the dust-particle treatment on the apoptosis in the alveolar barrier construct, the frozen sections were examined for the presence of apoptotic cells with fragmented DNA by the TdT-mediated dUTP nick-end labeling (TUNEL) assay using a commercially available kit (G3250, Promega), as detailed in the manufacturer's protocol. The cell nuclei were stained with propidium iodide (Sigma-Aldrich).
For the histological analysis, all the bright field and fluorescence images were captured by a microscope (Ti-S; Nikon).

| RNA extraction and gene analysis
The tissues were washed with PBS and centrifuged. The total RNA was extracted from pellets using the RNeasy Mini Kit (Qiagen).
Afterward, it was measured quantitatively using an ultraviolet-visible (UV-Vis) spectrophotometer (NanoDrop One, Thermo Fisher Scientific). The extracted RNA was reverse-transcribed using the oligo dT and high-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's protocols. Each cDNA was detected using a real-time polymerase chain reaction (PCR) system (StepOne Plus, Applied Biosystems) with the SYBR Green Master Mix (Applied Biosystems). The sequences of the forward and reverse primer pairs are shown in Supporting Information: Table S1. The reference gene, glyceraldehyde 3-phosphate dehydrogenase, was used to normalize the raw C T (cycle threshold) values.

| Statistical analysis
Quantitative data are expressed as means, with error bars representing ± standard error of the mean. One-way analysis of variance (ANOVA) with Fisher test was carried out to determine the statistical significance of the differences between experimental groups using the OriginPro 2016 software (OriginLab). The sample size (n) is 3, and only the control (0 μg/cm 2 ) comparison p values are indicated and the representation of statistical significance is denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

| RESULTS
3.1 | Exposure of the alveolar barrier construct to dust The hazard of atmospheric fine dust particles was evaluated using allinkjet-printed alveolar barrier construct. On a permeable membrane, endothelial cells, type-I collagen, fibroblast-containing collagen, and types I and II alveolar epithelial cells were sequentially deposited by a high-resolution drop-on-demand inkjet printer that could generate picolitre cell-laden droplets at a high frequency (Figure 1a). The printed models had a three-layered structure with a thickness of approximately 10 μm and consisted of an endothelium, a basement membrane, and an epithelium (Figure 1b). They were cultured for 7 days with the epithelium exposed to air for tissue maturation. After maturation, the epithelium of the tissues was exposed to various concentrations of fine dust particles (0, 30, 100, or 300 μg/cm 2 ), and was further cultured for 1, 3, 5, or 7 days for analysis (Figure 1c).

| Changes in the metabolic activity and barrier functions induced by dust particles
Most studies reported that particulate matter causes the generation of ROS and mitochondrial dysfunction, which is involved in cellular respiration, reduces metabolic activity, and consequently induces apoptosis of cells (Peixoto et al., 2017). To verify the harmful influence of dust particles on tissue proliferation and metabolic activity, a CCK-8 assay was performed at each time point (Figure 2a).
The results showed that the tissues and control group treated with 30 μg/cm 2 of dust particles demonstrated almost identical F I G U R E 1 Schematic diagram of the research process. (a) The alveolar barrier constructs fabricated by the sequentially stacking of alveolar microvascular endothelial cells, collagen and lung fibroblasts, and types Ⅰ and Ⅱ alveolar epithelial cells using inkjet bioprinting technology. (b) The printed alveolar barrier construct cultured for 7 days with the epithelium exposed to air for tissue maturation. (c) Alveolar barrier constructs treated with different concentrations of dust (0-300 μg/cm 2 ) for different durations (from 1 to 7 days). A cross-sectional image of the alveolar barrier model stained with hematoxylin & eosin Y. The black arrows indicate the dust particles on the epithelium. Scale bar = 50 μm.
proliferations from Days 1 to 5. The proliferation rate of the tissues exposed to 100 and 300 μg/cm 2 of dust particles gradually decreased from Day 3, and a value approximately 50% lower than that in the control group was observed on Day 7. This result does not indicate the absolute number of dead cells in the tissues; however, it reflects that the dust particles had an adverse effect on the metabolic activity of the tissues.
The changes in alveolar barrier functions were investigated by measuring the electrical resistance of an alveolar barrier (Figure 2b). At all time points, the tissues exposed to 30 μg/cm 2 of dust particles did not show any significant change in the TEER, similar to the control group result. The 5-day exposure to particles for 100 and 300 μg/cm 2 groups resulted in a significant reduction in the barrier resistance.
Tissue permeability tests were also conducted by measuring the FITC-dextran fluorescence from the basolateral chamber. Similar to the case in the TEER measurements, the significantly increased permeability to 4 kDa FITC-dextran was observed for 100 or 300 μg/cm 2 groups from Day 5 (Figure 2c).
To observe the apoptosis in the dust-treated alveolar barrier constructs, a TUNEL assay was conducted (Figure 3). The TUNEL staining results showed that the exposure of a dust concentration of 100 μg/cm 2 or more led to more apoptotic cells as the dust-exposure duration increased. With the apoptosis, changes in tissue structure were observed. While the three-layer structure of the tissue was F I G U R E 2 Effects of the dust concentration and exposure times on the viability and properties of the alveolar barrier constructs. Analysis of the alveolar barrier constructs treated with different concentrations of dust (0-300 μg/cm 2 ) for various exposure durations (1-7 days). (a) CCK-8 assay was implemented to detect tissue proliferation. Absorbance was normalized using the control group on Day 1 (data normalization = 1). (b) TEER was measured to identify the tissue barrier integrity post exposure to dust. (c) Barrier permeability was measured according to the amount of FITC-dextran passing through the tissue barrier. Fluorescence intensity was normalized using the blank as the permeable membrane without cells (data normalization = 100).
F I G U R E 3 Observation of the barrier collapse and DNA fragmentation in the alveolar barrier constructs after dust exposure. The threelayered structure collapse because of the concentration-and time-dependent effects of the dust particles. The number of TUNEL-stained (green) apoptotic cells was significantly increased after treatment with high concentrations of dust. The arrows indicate apoptotic cells. The nuclei were counterstained with PI (red). Scale bar = 50 μm. maintained until Day 7 for the control and 30 μg/cm 2 groups, the basement membranes of 100 and 300 μg/cm 2 groups were decomposed and their boundary between the epithelium and endothelium was not distinguished.

| Basement-membrane degradation and surfactant dysfunctions in the dust-treated tissues
Immunohistochemically analysis was performed to demonstrate the structural composition of the basement membrane in the three-layer tissue barriers at 7 days after exposure to fine dust. We investigated the distribution of laminin in the basement membrane whose degradation was confirmed by the TUNEL assay (Figure 4a). Laminin is a major component of the basement membrane and is involved in cell adhesion, differentiation, and control of cell functions in ECM.
Generally, the deposition of laminins indicates the endothelial and epithelial cells aligned at the basement membrane with polarity. In the tissues exposed to dust, laminin accumulation and typical alignment along the epithelium and endothelium were not observed.
The laminins synthesized by some cells attached to the degraded ECM, which remained unstable, were detected; however, the polarity of the cells in the tissue was not maintained. Furthermore, to determine the other components of the basement membrane ECM, immunostaining of collagen IV was performed (Figure 4b). Resultantly, a homogeneous distribution of collagen IV in the basement membrane was not observed in the tissues exposed to dust. Collagen IV was unevenly distributed in the tissues treated with dust particles of 100 μg/cm 2 or more, although the overall fluorescence intensity did not decrease. Finally, the surfactant-secretion function of the ATII cells in the epithelium was investigated by Alcian blue staining ( Figure 4c). This distinguishable epithelium revealed that the secreted pulmonary surfactant stained in light blue was aligned along with the epithelial cells. For the tissue exposed to 300 μg/cm 2 of dust, only the dust particles administered on the epithelium were stained blue, and there was no part observed in the similar morphology of the stained surfactant.

| Effect of dust on the expression of mRNAs in the alveolar barrier constructs
To investigate the effect of fine dust particles, quantitative PCR (qPCR) was conducted to determine the mRNA expression levels associated with extracellular matrix degradation and lung dysfunction. We chose genes that encode pro-inflammatory cytokines The result showed a remarkable increase in the MMP-1 mRNA levels F I G U R E 4 Functional characterization of the alveolar barrier constructs after dust exposure for 7 days. (a, b) Immunofluorescence microscopic images using specific antibodies (green) and the nuclei staining reagent, Hoechst33342 (blue); the basement membrane constituent (a) laminin and (b) collagen IV. (c) Microscopic cross-sectional image after staining with Alcian blue and nuclear fast red. Nuclei in cells and the secreted surfactant stained in purple and light blue, respectively. The black arrows indicate the dust particles on the epithelium, while the blue arrows indicate the stained surfactant. Scale bar = 50 μm.
after the tissues were exposed to 300 μg/cm 2 of dust particles for 7 days (Figure 5c). In addition, the expression level of MMP-9, which digests collagen IV substrates, was investigated. The expression level of MMP-9 increased significantly concentration-dependently from Day 3 onwards. For the tissue exposed to the highest concentration of dust for the longest time, a remarkable increase in the MMP-9 expression level was confirmed (Figure 5d). It was demonstrated that dust particles contribute to the high expression level of the enzyme that causes ECM turnover and structural changes.
In contrast, the mRNA expression levels of integrins and surfactant proteins decreased as the fine dust concentration and exposure time increased. ITGA1 and ITGB1, which encode integrin subunits α1 and β1, are transmembrane receptors that promote cell-cell or cell-ECM adhesion and activate signaling pathways that mediate various cellular signals. The expression level of ITGA1 showed a significant downregulation from Day 3 in the tissues exposed to the highest concentration of dust particles (Figure 5e). In particular, in the tissue exposed to fine dust for 7 days, the expression level of ITGA1 significantly reduced, regardless of the dust concentration. Further, the expression level of IGTB1 on Day 7 decreased as the concentration of dust increased, although the decrease was insignificant (Figure 5f). Finally, the expression levels of hydrophilic SP-A and hydrophobic SP-B were determined, which represent the pulmonary surfactants in the alveoli. Marked differences were noted among the control and experimental groups, with the lowest gene expression levels observed in the dust-exposed tissues. The SP-A expression level in the tissue exposed to 300 μg/ cm 2 of dust was significantly decreased from Day 1 compared to the control group. A significant decrease was observed in the control group and in the tissues exposed to 30 μg/cm 2 or more of dust particles on Day 7 (Figure 5g). The expression level of SP-B was significantly lower in most of the tissues exposed to dust from Days 1 to 7 (Figure 5h). Thus, dust particles inhibit the secretion of respiratory surfactants in alveolar tissues and have the potential to cause surfactant-related physiological dysfunction.

| DISCUSSION
The use of 3D in vitro tissue constructs in toxicology has been increasing over the last decade as a new preclinical testing platform and as an alternative to animal testing. In this study, we introduced a 3D in vitro alveolar barrier model to evaluate the harmful effects of fine dust particles. Our findings demonstrated that the all-inkjetprinted alveolar barrier model closely mimics the morphological, functional, and micro-environmental features of the human alveolar tissue and could be a successful testing platform to evaluate the disease-inducing effects of inhalable particles potential for toxicology.
Lung epithelial cells are the first site of entry for atmospheric particles and infectious organisms from the external environment and provide early signals for the inflammatory response via the release of pro-inflammatory cytokines and inflammatory mediators. proinflammatory cytokines, which are critical to the initiation of an inflammatory response, trigger the signal transduction pathway, leading to the activation of transcription factors. This affects a variety of genes involved in numerous biological processes, such as cell survival, apoptosis, and diseases (Kataoka, 2009). The expression level of pro-inflammatory cytokines was increased in dust exposed tissues (Figure 5a,b). Generally, the release of pro-inflammatory cytokines leads to tight junction degradation and loss of barrier integrity (Wang et al., 2012;Zhang et al., 2020). Tight junctions are F I G U R E 5 Expression of mRNAs in the alveolar barrier constructs. Relative mRNA expression was measured via qPCR using specific markers involved in pro-inflammatory cytokines (a, b), metalloproteases (c, d), integrin subunits (e, f), and pulmonary surfactant protein (g, h). The expression level of each gene was normalized using the expression level in the control group on Day 1 (data normalization = 1). known to play a role in trapping airborne particulate matter and removing it from the respiratory tract, and particulate matter-induced damage is a major cause of epithelial barrier disruption in lung inflammation (Wittekindt, 2017). As high concentrations of dust particles induced upregulation of pro-inflammatory cytokine expression, barrier resistance decreased and permeability increased, indicating a loss of epithelial junctional function (Figure 2b,c).
Additionally, we identified the expression levels of laminin and collagen IV, which are major components of the basement membrane ECM, through immunohistochemical analysis and proved that the collapse of the structure is related to the decomposition of the basement membrane (Figure 4a,b).
MMPs are the major enzymes involved in ECM degradation and the cleavage of ECM components with broad substrate specificity.
Normally, the activity of MMPs is low; however, the activity increases during repair or remodeling and in diseased tissues, inflamed tissue, and tissues exposed to air pollutants (Bonnans et al., 2014;Dagouassat et al., 2012). MMP-induced ECM breakdown contributes significantly to the progression of several lung pathologies, including cancer and epithelial-mesenchymal transition (Balestrini & Niklason, 2015;Borthwick et al., 2009). The upregulation of MMP along with the secretion of pro-inflammatory cytokines was observed ( Figure 5c,d), and it is known that the upregulation of MMPs is mediated by the secretion of pro-inflammatory cytokines, such as IL-1β and TNF-α (Manicone & Mcguire, 2008). Additionally, the integrins function as receptors for cell-cell and cell-ECM adhesion, and they affect the orientation of the intracellular actin cytoskeleton, which induced by the integrin interactions is essential for the cell polarity signals that define the position of the basal domain (Giancotti, 1999;Manninen, 2015). Cells require polarity to establish their barrier function, and cell polarity is essential for cell migration in the correct direction, creating distinct leading and trailing structures (Campanale et al., 2017). Loss of cell polarity and tissue disorganization are the hallmarks of cancer and tumor initiation (Lee et al., 2014). The upregulation of MMPs and loss of the barrier integrity, induced by the pro-inflammatory cytokines, have been employed as markers of respiratory diseases (Ostridge et al., 2016;Parks & Shapiro, 2001;Tatsuta et al., 2019). In this study, the expression level of integrin receptors decreased with an increase in MMP and degradation of the ECM (Figure 5e,f). Thus, exposure to particulate matter may be responsible for the initiation of lung inflammation and the development of lung cancer.
Finally, the effects of dust particles on the secretion of the pulmonary surfactant were investigated. This surfactant is essential for breathing as it lines the alveoli to reduce surface tension, thereby preventing atelectasis. In the tissues exposed to dust particles, the reduction of pulmonary surfactant expression was observed (Figure 5g,h).
Malformation in surfactant production or function is associated with several pulmonary diseases, simultaneously, and pulmonary inflammation changes the surfactant metabolism. Representatively, the deficiency of surfactants has been linked to a variety of diseases, such as respiratory distress syndrome, idiopathic pulmonary fibrosis, and asthma (Han & Mallampalli, 2015). The all-inkjet-printed alveolar barrier exhibits the risk potential of fine dust particles to induce severe respiratory diseases, such as fibrosis and asthma.
The first limitation of this study is the exclusion of alveolar macrophages in the all-inkjet-printed alveolar barrier constructs.
Generally, alveolar macrophages account for 9.4% of cells in the human alveoli (Pinkerton et al., 2015). Inhaled particles can modify immune function by directly affecting antigen-presenting macrophages, thus upregulating the normal lymphocytic response to antigens in the lung (Hamilton et al., 2004). To simulate and implement the immune system and disease induction that occurs in the human body, further investigations are required using artificial alveoli that include immune cells. Second, there are no mechanical stresses in our alveolar barrier construct that can simulate breathing motions. Replicating mechanical strain can be the next step in fabricating advanced alveolar barrier constructs with high biomimetic.
Application of an appropriate mechanical strain that simulates breathing motion may increase the transmittance of particulates, which may lead to more serious consequences (Huh et al., 2010). If these limitations are overcome in future works and the alveolar barrier contains immune cells and mechanical strain, this method has the potential to pave the way for investigations of the toxicity of inhaled particles and the associated respiratory diseases.

| CONCLUSION
In summary, the analysis of all-inkjet-printed alveolar barrier constructs afforded three major new insights regarding the harmful effects of inhaled dust particles. The phenomena in the alveolar tissue exposed to high concentrations of fine dust particles include the following: (i) an increased expression of pro-inflammatory cytokines, (ii) destruction and disorganization of tissue architecture, and (iii) up-or downregulation of genes associated with respiratory diseases. The findings of this study suggest that the in vitro artificial tissue structure created using bioprinting technology can be used as an innovative testing platform to evaluate the disease-inducing effects of inhalable particles. Products fabricated using bioprinting can be customized by introducing complexity or patientspecificity into the system, as required, to meet the requirements of users. Therefore, it is expected to be used in various fields. In addition, since some of the phenomena occurring in the body are mimicked, the fundamental understanding of the infection process will be improved, and tests for toxic substances and antigens will be accelerated. The all-inkjetprinted alveolar barrier model is capable of simulating the physiological phenomena in the body with a high degree of accuracy. This suggests that it can be used as a preclinical model in toxicology and facilitate the translation of new findings into effective treatment strategies.