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.

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.

[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.

Repairing whole facial nerve defects with xenogeneic acellular nerve grafts in rhesus monkeys.

Acellular nerve allografts conducted via chemical extraction have achieved satisfactory results in bridging whole facial nerve defects clinically, both in terms of branching a single trunk and in connecting multiple branches of an extratemporal segment. However, in the clinical treatment of facial nerve defects, allogeneic donors are limited. In this experiment, we exposed the left trunk and multiple branches of the extratemporal segment in six rhesus monkeys and dissected a gap of 25 mm to construct a monkey model of a whole left nerve defect. Six monkeys were randomly assigned to an autograft group or a xenogeneic acellular nerve graft group. In the autograft group, the 25-mm whole facial nerve defect was immediately bridged using an autogenous ipsilateral great auricular nerve, and in the xenogeneic acellular nerve graft group, this was done using a xenogeneic acellular nerve graft with trunk-branches. Examinations of facial symmetry, nerve-muscle electrophysiology, retrograde transport of labeled neuronal tracers, and morphology of the regenerated nerve and target muscle at 8 months postoperatively showed that the faces of the monkey appeared to be symmetrical in the static state and slightly asymmetrical during facial movement, and that they could actively close their eyelids completely. The degree of recovery from facial paralysis reached House-Brackmann grade II in both groups. Compound muscle action potentials were recorded and orbicularis oris muscles responded to electro-stimuli on the surgical side in each monkey. FluoroGold-labeled neurons could be detected in the facial nuclei on the injured side. Immunohistochemical staining showed abundant neurofilament-200-positive axons and soluble protein-100-positive Schwann cells in the regenerated nerves. A large number of mid-graft myelinated axons were observed via methylene blue staining and a transmission electron microscope. Taken together, our data indicate that xenogeneic acellular nerve grafts from minipigs are safe and effective for repairing whole facial nerve defects in rhesus monkeys, with an effect similar to that of autologous nerve transplantation. Thus, a xenogeneic acellular nerve graft may be a suitable choice for bridging a whole facial nerve defect if no other method is available. The study was approved by the Laboratory Animal Management Committee and the Ethics Review Committee of the Affiliated Wuxi No. 2 People's Hospital of Nanjing Medical University, China (approval No. 2018-D-1) on March 15, 2018.

Pancreatic Extracellular Matrix/Alginate Hydrogels Provide a Supportive Microenvironment for Insulin-Producing Cells.

Type 1 diabetes mellitus (T1DM), as an autoimmune deficiency disease, is associated with an absolute deficiency of insulin subject to islet ß-cell destruction. Insulin-producing cells (IPCs) differentiated from induced pluripotent stem cells are an ideal replacement origin of ß-cells, which can be applied for cell transplantation therapies in T1DM. At present, more strategies focus on inducing and differentiating to obtain IPCs; however, the unsatisfactory differentiation efficiency and the lack of ideal carriers for in vivo transplantation limited their application. It is necessary to consider the cell microenvironment by constructing a biomimetic niche to improve the differentiation and transplantation efficiency. The main components of the extracellular matrix derived from pancreatic (the niche of ß-cells) decellularization were retained, which could provide the ideal extracellular microenvironment for IPCs. In this research, a hydrogel prepared with alginate (Alg) and the pancreatic extracellular matrix (pECM) was assessed for the beneficial outcomes on encapsulated IPCs. The results showed that pECM/Alg improved the differentiation efficiency and promoted insulin secretion and the expression of insulin-related genes as well. Besides, pECM/Alg-encapsulated IPCs exhibited obvious biocompatibility in vivo, which can prolong the transplantation effect and hypoglycemic function by reducing the inflammatory reaction. RNA-seq indicated that the PI3K/Akt pathway may be related to the improvement of the differentiation efficiency and function of IPCs. In general, the pECM/Alg hydrogel provides an ideal biomimetic microenvironment for IPCs and is suitable for in vivo transplantation.

Comparative pharmacokinetics of florfenicol in healthy and Pasteurella multocida-infected Gaoyou ducks.

Pasteurella multocida is the causative agent of fowl cholera, and florfenicol (FF) has potent antibacterial activity against P. multocida and is widely used in the poultry industry. In this study, we established a P. multocida infection model in ducks and studied the pharmacokinetics of FF in serum and lung tissues after oral administration of 30 mg/kg bodyweight. The maximum concentrations reached (Cmax) were lower in infected ducks (13.88 ± 2.70 µg/ml) vs. healthy control animals (17.86 ± 1.57 µg/ml). In contrast, the mean residence time (MRT: 2.35 ± 0.13 vs. 2.27 ± 0.18 hr) and elimination half-life (T½ß : 1.63 ± 0.08 vs. 1.57 ± 0.12 hr) were similar for healthy and diseased animals, respectively. As a result, the area under the concentration curve for 0-12 hr (AUC0-12 hr ) for FF in healthy ducks was significantly greater than that in infected ducks (49.47 ± 5.31 vs. 34.52 ± 8.29 µg hr/ml). The pharmacokinetic differences of FF in lung tissues between the two groups correlated with the serum pharmacokinetic differences. The Cmax and AUC0-12 hr values of lung tissue in healthy ducks were higher than those in diseased ducks. The concentration of FF in lung tissues was approximately 1.2-fold higher than that in serum both in infected and healthy ducks indicating that FF is effective in treating respiratory tract infections in ducks.

Dynamic tracking of pathogenic receptor expression of live cells using pyrenyl glycoanthraquinone-decorated graphene electrodes.

Expression of specific transmembrane receptors by cells frequently represents an important signature of diseases, but this dynamic event can hardly be monitored directly with live cells due to the limitation of current biochemical techniques. Here we develop a pyrenyl glycoanthraquinone construct that can be firmly immobilized on a graphene-spotted screen printed electrode via strong π-interactions. The inherent current signal produced by the surface-confined glycoquinone can be used to detect selective sugar-protein recognitions with simple electrochemical techniques and portable facilities. Importantly, we demonstrate that the level of pathogenic receptors expressed by different types of live cells can be tracked with the electrode system in a label-free manner, providing a useful tool for the on-demand disease diagnosis as well as basic biochemical studies.

Anthraquinonyl glycoside facilitates the standardization of graphene electrodes for the impedance detection of lectins.

BACKGROUND: Construction of electrochemical impedance sensors by the self-assembly technique has become a promising strategy for the 'label-free' detection of protein-ligand interactions. However, previous impedance sensors are devoid of an inherent electrochemical signal, which limits the standardization of the sensors for protein recognition in a reproducible manner. RESULTS: We designed and synthesized an anthraquinonyl glycoside (AG) where the anthraquinone (AQ) moiety can bind to the surface of a graphene-based working electrode while the glycoside serving as a ligand for lectin. By measuring the inherent voltammetric signal of AQ, the glycosides decorated on the working electrode could be simply quantified to obtain electrodes with a unified signal window. Subsequently, impedance analysis showed that the 'standardized' electrodes gave a reproducible electrochemical response to a selective lectin with no signal variation in the presence of unselective proteins. CONCLUSION: Anthraquinone-modified ligands could be used to facilitate the standardization of electrochemical impedance sensors for the reproducible, selective analysis of ligand-protein interactions.

Evaluation of an augmented virtual reality and haptic control interface for psychomotor training.

This study investigated the design of a virtual reality (VR) simulation integrating a haptic control interface for motor skill training. Twenty-four healthy participants were tested and trained in standardized psychomotor control tasks using native and VR forms with their nondominant hands in order to identify VR design features that might serve to accelerate motor learning. The study was also intended to make preliminary observations on the degree of specific motor skill development that can be achieved with a VR-based haptic simulation. Results revealed significant improvements in test performance following training for the VR with augmented haptic features with insignificant findings for the native task and VR with basic haptic features. Although performance during training was consistently better with the native task, a correspondence between the VR training and test task interfaces led to greater improvement in test performance as reported by a difference between baseline and post-test scores. These findings support use of VR-based haptic simulations of standardized psychomotor tests for motor skill training, including visual and haptic enhancements for effective pattern recognition and discrete movement of objects. The results may serve as an applicable guide for design of future haptic VR features.

Comparison of Infant Car Seat grip orientations and lift strategies.

The rear-facing Infant Car Seat (ICS) is designed to meet federal requirements for transporting children less than 1 year old. Typical use includes transfer in and out of a vehicle, which is shown to be a difficult lift. Despite the frequency of this lift, manufacturers provide little guidance for users. Review of relevant literature suggested an ICS featuring an angled handle, promoting a neutral wrist posture, would increase grip stability and decrease lifting effort. Popular press suggested a foot-in-car stance for the ICS lift would do the same. An experiment was conducted in which wrist deviations from neutral posture were recorded along with lifting muscle activation levels (multiple flexor muscles and biceps brachii) and overall perceived exertion for straight versus a new bent handle design and conventional stance versus foot-in-car. Foot position was examined to test the recommendations in the popular press. Surprisingly, wrist deviation was not significantly affected by the new bent handle design (due to compensatory behavior with the straight handle) but was related to foot placement (p=0.04). Results revealed the bent handle to significantly reduce flexor activation compared with the straight handle (p=0.0003); however, the level of biceps activation increased. Biceps activation also significantly increased for foot-in-car stance (p=0.035) but not flexor activation. In general, the bent handle enabled the user to lift the ICS with a steadier grip and less effort.