Three-in-one customized bioink for islet organoid: GelMA/ECM/PRP orchestrate pro-angiogenic and immunoregulatory function.

Islet organoids open up new strategies for diabetes treatment and pancreatic tissue engineering. Digital light processing (DLP) bioprinting has been extensively applied to the construction of organoids due to its ability to provide precisely patterned scaffolds with fast printing time while specific tailored bioink is indispensable for islet organoid. Customized bioinks that meet different needs were created and frequently applied based on gelatin methacryloyl (GelMA) mixed with other ingredients. Decellularized extracellular matrix (ECM) retains many organic specific structural and functional components and is widespread utilized to reconstruct the native niche like environment. However, considerable cytokines and growth factors were inevitably lost during the decellularized process, while platelet rich plasma (PRP) contains a string of growth factors which often exerted pro-angiogenic role. Therefore, a customized specific bioink for constructing islet organoid based on GelMA, pancreatic ECM and PRP was prepared in our research. In vitro, tube formation assay, CD31 immunofluorescence and relative mRNA expression of vascular genes indicated that the bioink with distinctively promote angiogenesis potential. Macrophages polarization was also conducted, which exhibited superior expression of CD206 (M2 marker) and inferior expression of iNOS (M1 marker). 3D printed organoids maintain the activity of mouse islet ß-cells (MIN6) with enhanced glucose sensitivity. In vivo, the results of CD31, CD206 and iNOS immunofluorescence were consistent with that in vitro. In summary, we prepared and characterized specific custom-made bioink with orchestrating immune-regulation response indicated by abundant M2-polarized macrophages, attenuated inflammation, and promoted angiogenesis, which provides an underlying bioink for the fabrication of 3D printed islet organoid.

Matrix stiffness triggers chemoresistance through elevated autophagy in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a signature of extremely high matrix stiffness caused by a special desmoplastic reaction, which dynamically stiffens along with the pathological process. The poor prognosis and low five-year survival rate of PDAC are partly owing to chemoresistance triggered by substrate stiffness. Understanding the potential mechanisms of matrix stiffness causing PDAC chemoresistance is of great significance. In this study, methacrylated gelatin hydrogel was used as platform for PANC-1 and MIA-PaCa2 cell culture. The results indicated that compared to soft substrate, stiff substrate distinctively reduced the gemcitabine sensitivity of pancreatic cancer. Intriguingly, transmission electron microscopy, immunofluorescence staining, western blot and qRT-PCR assay showcased that the number of autophagosomes and the expression of LC3 were elevated. The observations indicate that matrix stiffness may regulate the autophagy level, which plays a vital role during chemoresistance. In brief, soft substrate exhibited low autophagy level, while the counterpart displayed elevated autophagy level. In order to elucidate the underlying interaction between matrix stiffness-mediated cell autophagy and chemoresistance, rescue experiments with rapamycin and chloroquine were conducted. We found that inhibiting cell autophagy dramatically increased the sensitivity of pancreatic cancer cells to gemcitabine in the stiff group, while promoting autophagy-driven chemoresistance in the soft group, demonstrating that matrix stiffness modulated chemoresistance via autophagy. Furthermore, RNA-seq results showed that miR-1972 may regulate autophagy level in response to matrix stiffness. Overall, our research shed light on the synergistic therapy of PDAC combined with gemcitabine and chloroquine, which is conducive to promoting a therapeutic effect.

Biomimetic Microenvironmental Stiffness Boosts Stemness of Pancreatic Ductal Adenocarcinoma via Augmented Autophagy.

Pancreatic ductal adenocarcinoma (PDAC) features high recurrence rates and intensified lethality, accompanied by stiffening of the extracellular matrix (ECM) microenvironment, which is mainly due to the deposition, remodeling, and cross-linking of collagen. Boosted stemness plays an essential role during occurrence and progression, which indicates a poor prognosis. Therefore, it is of great importance to understand the effect of the underlying interaction of matrix stiffness and stemness on PDAC. For this purpose, a methacrylated gelatin (GelMA) hydrogel with tunable stiffness was applied for incubating MIA PaCa-2 and PANC-1 cells. The results demonstrated that compared to the soft group (5% GelMA, w/v), the expression of stemness-related genes (SOX2, OCT4, and NANOG) in the stiff group (10% GelMA, w/v) displayed pronounced elevation as well as sphere formation. Intriguingly, we also observed that matrix stiffness regulated autophagy of PDAC, which played a momentous role in stemness promotion. In order to clarify the underlying relationship between matrix stiffness-mediated cell autophagy and stemness, rescue experiments with rapamycin and chloroquine were conducted with transmission electron microscopy, immunofluorescence staining, sphere formation, and qRT-PCR assays to evaluate the level of stemness and autophagy. For exploring the molecular mechanism in depth, RNA-seq and differential expression of miRNAs were carried out, which may sensor and respond to matrix stiffness during the regulation of stemness and autophagy. In conclusion, we validated that blocking autophagy repressed the stemness induced by matrix stiffness in PDAC and provided a potential therapeutic strategy for this aggressive cancer.

Hyaluronic acid methacrylate/pancreatic extracellular matrix as a potential 3D printing bioink for constructing islet organoids.

Islet transplantation has poor long-term efficacy because of the lack of extracellular matrix support and neovascularization; this limits its wide application in diabetes research. In this study, we develop a 3D-printed islet organoid by combining a pancreatic extracellular matrix (pECM) and hyaluronic acid methacrylate (HAMA) as specific bioinks. The HAMA/pECM hydrogel was validated in vitro to maintain islet cell adhesion and morphology through the Rac1/ROCK/MLCK signaling pathway, which helps improve islet function and activity. Further, in vivo experiments confirmed that the 3D-printed islet-encapsulated HAMA/pECM hydrogel increases insulin levels in diabetic mice, maintains blood glucose levels within a normal range for 90 days, and rapidly secretes insulin in response to blood glucose stimulation. In addition, the HAMA/pECM hydrogel can facilitate the attachment and growth of new blood vessels and increase the density of new vessels. Meanwhile, the designed 3D-printed structure was conducive to the formation of vascular networks and it promoted the construction of 3D-printed islet organoids. In conclusion, our experiments optimized the HAMA/pECM bioink composition and 3D-printed structure of islet organoids with promising therapeutic effects compared with the HAMA hydrogel group that can be potentially used in clinical applications to improve the effectiveness and safety of islet transplantation in vivo. STATEMENT OF SIGNIFICANCE: The extraction process of pancreatic islets can easily cause damage to the extracellular matrix and vascular system, resulting in poor islet transplantation efficiency. We developed a new tissue-specific bioink by combining pancreatic extracellular matrix (pECM) and hyaluronic acid methacrylate (HAMA). The islet organoids constructed by 3D printing can mimic the microenvironment of the pancreas and maintain islet cell adhesion and morphology through the Rac1/ROCK/MLCK signaling pathway, thereby improving islet function and activity. In addition, the 3D-printed structures we designed are favorable for the formation of new blood vessel networks, bringing hope for the long-term efficacy of islet transplantation.

[Islet biomimetic microenvironment constructed by chitosan oligosaccharide protects islets from hypoxia-induced damage by reducing intracellular reactive oxygen species].

Objective: Gelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia. Methods: Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively. Islets were cultured under hypoxia (1%O 2) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability. Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O 2) and hypoxia (1%O 2) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions, respectively. Results: GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group ( P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C ( P<0.05). Conclusion: GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.

Particle-specific toxicity and bioavailability of cerium oxide (CeO2) nanoparticles to Arabidopsis thaliana.

The use of manufactured cerium oxide nanoparticles (CeO2-NPs) in consumer products has increased markedly over the past decade, and their release into natural ecosystems is unavoidable. This study investigated the phytotoxicity and uptake of CeO2-NPs in Arabidopsis thaliana grown in an agar medium. Although low concentrations of CeO2-NPs had stimulatory effects on plant growth, at higher concentrations, CeO2-NPs reduced growth and had adverse effects on the antioxidant systems and photosystem. Importantly, the toxicity resulted from the nanoparticles per se, rather than from the dissolved Ce ions. CeO2-NPs were taken up and subsequently translocated to shoot tissues, and transmission electron microscopy (TEM) showed the presence of a large number of needle-like particle aggregations in the intercellular regions and the cytoplasm of leaf cells. The up-translocation factor to shoots was independent of the concentrations of Ce in the roots and the supplied forms of Ce (i.e. CeO2-NPs, CeO2-bulk, and ionic Ce), suggesting that endocytosis is likely to be a general mechanism responsible for the translocation of these Ce compounds. These findings provide important information regarding the toxicity and uptake of CeO2-NPs in plants, which needs to be considered in environmental risk assessment for the safe use and disposal of CeO2-NPs.