Silk fibroin increases the elasticity of alginate-gelatin hydrogels and regulates cardiac cell contractile function in cardiac bioinks.

Silk fibroin (SF) is a natural protein extracted from Bombyx mori silkworm thread. From its common use in the textile industry, it emerged as a biomaterial with promising biochemical and mechanical properties for applications in the field of tissue engineering and regenerative medicine. In this study, we evaluate for the first time the effects of SF on cardiac bioink formulations containing cardiac spheroids. First, we evaluate if the SF addition plays a role in the structural and elastic properties of hydrogels containing alginate (Alg) and gelatin (Gel). Then, we test the printability and durability of bioprinted SF-containing hydrogels. Finally, we evaluate whether the addition of SF controls cell viability and function of cardiac spheroids in Alg-Gel hydrogels. Our findings show that the addition of 1% (w/v) SF to Alg-Gel hydrogels makes them more elastic without affecting cell viability. However, fractional shortening (FS%) of cardiac spheroids in SF-Alg-Gel hydrogels increases without affecting their contraction frequency, suggesting an improvement in contractile function in the 3D cultures. Altogether, our findings support a promising pathway to bioengineer bioinks containing SF for cardiac applications, with the ability to control mechanical and cellular features in cardiac bioinks.

Endoscopic ultrasound-guided gallbladder drainage as a first approach for jaundice palliation in unresectable malignant distal biliary obstruction: Prospective study.

OBJECTIVES: Endoscopic retrograde cholangiopancreatography (ERCP) represents the gold standard for jaundice palliation in patients with distal malignant biliary obstruction (DMBO). Biliary drainage using electrocautery lumen apposing metal stent (EC-LAMS) is currently a well-established procedure when ERCP fails. In a palliative setting the endoscopic ultrasound-guided gallbladder drainage (EUS-GBD) could represent an easy and valid option. We performed a prospective study with a new EC-LAMS with the primary aim to assess the clinical success rate of EUS-GBD as a first-line approach to the palliation of DMBO. METHODS: In all, 37 consecutive patients undergoing EUS-GBD with a new EC-LAMS were prospectively enrolled. Clinical success was defined as bilirubin level decrease >15% within 24 h and >50% within 14 days after EC-LAMS placement. RESULTS: The mean age was 73.5 ± 10.8 years; there were 17 male patients (45.9%). EC-LAMS placement was technically feasible in all patients (100%) and the clinical success rate was 100%. Four patients (10.8%) experienced adverse events, one bleeding, one food impaction, and two cystic duct obstructions because of disease progression. No stent-related deaths were observed. The mean hospitalization was 7.7 ± 3.4 days. Median overall survival was 4 months (95% confidence interval 1-8). CONCLUSION: Endoscopic ultrasound-guided gallbladder drainage with the new EC-LAMS is a valid option in palliative endoscopic biliary drainage as a first-step approach in low survival patients with malignant jaundice unfit for surgery. A smaller diameter EC-LAMS should be preferred, particularly if the drainage is performed through the stomach, to avoid potential food impaction, which could result in stent dysfunction.

The Use of a New Dedicated Electrocautery Lumen-Apposing Metal Stent for Gallbladder Drainage in Patients with Acute Cholecystitis.

Aims: Lumen-apposing metal stents (LAMSs) in ultrasonography-guided gallbladder drainage (EUS-GBD) have become increasingly important for high-risk surgical patients. Our study aims to evaluate the technical and clinical success, safety, and feasibility of endoscopic ultrasonography-guided gallbladder drainage using a new dedicated LAMS. Methods: This is a retrospective multicenter study that included all consecutive patients not suitable for surgery who were referred to a tertiary center for EUS-GBD using a new dedicated electrocautery LAMS for acute cholecystitis at eight different centers. Results: Our study included 54 patients with a mean age of 76.48 years (standard deviation: 12.6 years). Out of the 54 endoscopic gallbladder drainages performed, 24 (44.4%) were cholecysto-gastrostomy, and 30 (55.4%) were cholecysto-duodenostomy. The technical success of LAMS placement was 100%, and clinical success was achieved in 23 out of 30 patients (76.67%). Adverse events were observed in two patients (5.6%). Patients were discharged after a median of 5 days post-stenting. Conclusions: EUS-GBD represents a valuable option for high-surgical-risk patients with acute cholecystitis. This new dedicated LAMS has demonstrated a high rate of technical and clinical success, along with a high level of safety.

Physically-based simulation of elastic-plastic fusion of 3D bioprinted spheroids.

Spheroids are microtissues containing cells organized in a spherical shape whose diameter is usually less than a millimetre. Depending on the properties of the environment they are placed in, some nearby spheroids spontaneously fuse and generate a tissue. Given their potential to mimic features typical of body parts and their ability to assemble by fusing in permissive hydrogels, they have been used as building blocks to 3D bioprint human tissue parts. Parameters controlling the shape and size of a bioprinted tissue using fusing spheroid cultures include cell composition, hydrogel properties, and their relative initial position. Hence, simulating, anticipating, and then controlling the spheroid fusion process is essential to control the shape and size of the bioprinted tissue. This study presents the first physically-based framework to simulate the fusion process of bioprinted spheroids. The simulation is based on elastic-plastic solid and fluid continuum mechanics models. Both models use the 'smoothed particle hydrodynamics' method, which is based on discretizing the continuous medium into a finite number of particles and solving the differential equations related to the physical properties (e.g. Navier-Stokes equation) using a smoothing kernel function. To further investigate the effects of such parameters on spheroid shape and geometry, we performed sensitivity and morphological analysis to validate our simulations within-vitrospheroids. Through ourin-silicosimulations by changing the aforementioned parameters, we show that the proposed models appropriately simulate the range of the elastic-plastic behaviours ofin-vitrofusing spheroids to generate tissues of desired shapes and sizes. Altogether, this study presented a physically-based simulation that can provide a framework for monitoring and controlling the geometrical shape of spheroids, directly impacting future research using spheroids for tissue bioprinting.

New Perspectives in Endoscopic Treatment of Gastroesophageal Reflux Disease.

Gastroesophageal reflux disease has a high incidence and prevalence in the general population. Clinical manifestations are heterogenous, and so is the response to medical treatment. Proton pump inhibitors are still the most common agents used to control reflux symptoms and for healing esophagitis, but they are not a one-size-fits-all solution for the disease. Patients with persistent troublesome symptoms despite medical therapy, those experiencing some adverse drug reaction, or those unwilling to take lifelong medications deserve valid alternatives. Anti-reflux Nissen fundoplication is an effective option, but the risk of adverse events has limited its spread. In recent years, advancements in therapeutic endoscopy have been made, and three major endoluminal alternatives are now available, including (1) the delivery of radiofrequency energy to the esophago-gastric junction, (2) transoral incisionless fundoplication (TIF), and (3) anti-reflux mucosal interventions (ARMI) based on mucosal resection (ARMS) and mucosal ablation (ARMA) techniques to remodel the cardia. Endoscopic techniques have shown interesting results, but their diffusion is still limited to expert endoscopists in tertiary centers. This review discusses the state of the art in the endoscopic approach to gastroesophageal reflux disease.

Long-term endoscopic surveillance in HBV compensated cirrhotic patients treated with Tenofovir or Entecavir for 11 years.

BACKGROUND: Long-term administration of TDF/ETV in patients with HBV-related compensated cirrhosis reduces HCC and decompensation events but the effect of this regimen on development/regression of oesophageal varices (EV) is currently unknown. AIM: To assess the risk of EV development/progression in this population. METHODS: A total of 186 Caucasian HBV-monoinfected compensated cirrhotics were enrolled in a long-term cohort study from TDF/ETV introduction. Upper GI endoscopies were performed according to Baveno recommendations. Primary endpoint was development/progression of oesophageal/gastric varices over time. RESULTS: At TDF/ETV start, median age was 61 years, 80% males, 60% HBV-DNA undetectable, 63% NUCs previously exposed, 73% normal ALT, 40% platelets <150,000/mmc and 25 (13%) with low-risk varices (LRV). During 11 years of antiviral therapy and 666 endoscopies performed, 9 patients either developed or had a progression of oesophageal or gastric varices with an 11-year cumulative probability of 5.1% (95% CI 3-10%); no patient bled. Out of 161 patients without EV at baseline, the 11-year probably was 4.5% with all varices developing within the first six years of treatment. In 25 patients with LRV at baseline, the 11-year probability of progression or regression was 9.3% and 58%, respectively. Only baseline platelet count (HR 0.96, p = 0.028) was associated with LRV development at multivariate analysis: platelet ≤90,000/mmc (AUROC 0.70) had 98.1% specificity, 42.9% sensitivity, 50% PPV for LRV onset. CONCLUSIONS: In compensated cirrhotic patients under long-term effective TDF/ETV treatment, the 11-year risk of developing/progressing EV is negligible, thus challenging the current endoscopic surveillance recommendations in patients without EV at baseline.

Photosynthetic microorganisms for the oxygenation of advanced 3D bioprinted tissues.

3D bioprinting technology has emerged as a tool that promises to revolutionize the biomedical field, including tissue engineering and regeneration. Despite major technological advancements, several challenges remain to be solved before 3D bioprinted tissues could be fully translated from the bench to the bedside. As oxygen plays a key role in aerobic metabolism, which allows energy production in the mitochondria; as a consequence, the lack of tissue oxygenation is one of the main limitations of current bioprinted tissues and organs. In order to improve tissue oxygenation, recent approaches have been established for a broad range of clinical applications, with some already applied using 3D bioprinting technologies. Among them, the incorporation of photosynthetic microorganisms, such as microalgae and cyanobacteria, is a promising approach that has been recently explored to generate chimerical plant-animal tissues where, upon light exposure, oxygen can be produced and released in a localized and controlled manner. This review will briefly summarize the state-of-the-art approaches to improve tissue oxygenation, as well as studies describing the use of photosynthetic microorganisms in 3D bioprinting technologies. STATEMENT OF SIGNIFICANCE: 3D bioprinting technology has emerged as a tool for the generation of viable and functional tissues for direct in vitro and in vivo applications, including disease modeling, drug discovery and regenerative medicine. Despite the latest advancements in this field, suboptimal oxygen delivery to cells before, during and after the bioprinting process limits their viability within 3D bioprinted tissues. This review article first highlights state-of-the-art approaches used to improve oxygen delivery in bioengineered tissues to overcome this challenge. Then, it focuses on the emerging roles played by photosynthetic organisms as novel biomaterials for bioink generation. Finally, it provides considerations around current challenges and novel potential opportunities for their use in bioinks, by comparing latest published studies using algae for 3D bioprinting.

In vitro modeling of myocardial ischemia/reperfusion injury with murine or human 3D cardiac spheroids.

Myocardial infarction (MI) is the primary cause of death worldwide, but there are no clinically relevant models to study MI. Here, we describe an ischemia/reperfusion (I/R) injury model typical of MI using mouse or human 3D in vitro cardiac spheroids (CSs). First, we demonstrated the culture and maintenance of CSs. Then, we detailed how to expose CSs to pathophysiological oxygen concentrations to induce I/R injury. The protocol can be used in combination with viability, contractility, and mRNA expression level measurements. For complete details on the use and execution of this protocol, please refer to Sharma et al. (2022).

Fibulin-3 Deficiency Protects Against Myocardial Injury Following Ischaemia/ Reperfusion in in vitro Cardiac Spheroids.

Myocardial infarction (MI, or heart attack) is a leading cause of death worldwide. Myocardial ischaemia reperfusion (I/R) injury typical of MI events is also associated with the development of cardiac fibrosis and heart failure in patients. Fibulin-3 is an extracellular matrix component that plays a role in regulating MI response in the heart. In this study, we generated and compared in vitro cardiac spheroids (CSs) from wild type (WT) and fibulin-3 knockout (Fib-3 KO) mice. These were then exposed to pathophysiological changes in oxygen (O2) concentrations to mimic an MI event. We finally measured changes in contractile function, cell death, and mRNA expression levels of cardiovascular disease genes between WT and Fib-3 KO CSs. Our results demonstrated that there are significant differences in growth kinetics and endothelial network formation between WT and Fib-3 KO CSs, however, they respond similarly to changes in O2 concentrations. Fib-3 deficiency resulted in an increase in viability of cells and improvement in contraction frequency and fractional shortening compared to WT I/R CSs. Gene expression analyses demonstrated that Fib-3 deficiency inhibits I/R injury and cardiac fibrosis and promotes angiogenesis in CSs. Altogether, our findings suggest that Fib-3 deficiency makes CSs resistant to I/R injury and associated cardiac fibrosis and helps to improve the vascular network in CSs.

Taking It Personally: 3D Bioprinting a Patient-Specific Cardiac Patch for the Treatment of Heart Failure.

Despite a massive global preventative effort, heart failure remains the major cause of death globally. The number of patients requiring a heart transplant, the eventual last treatment option, far outnumbers the available donor hearts, leaving many to deteriorate or die on the transplant waiting list. Treating heart failure by transplanting a 3D bioprinted patient-specific cardiac patch to the infarcted region on the myocardium has been investigated as a potential future treatment. To date, several studies have created cardiac patches using 3D bioprinting; however, testing the concept is still at a pre-clinical stage. A handful of clinical studies have been conducted. However, moving from animal studies to human trials will require an increase in research in this area. This review covers key elements to the design of a patient-specific cardiac patch, divided into general areas of biological design and 3D modelling. It will make recommendations on incorporating anatomical considerations and high-definition motion data into the process of 3D-bioprinting a patient-specific cardiac patch.

Biofabrication of advancedin vitro3D models to study ischaemic and doxorubicin-induced myocardial damage.

Current preclinicalin vitroandin vivomodels of cardiac injury typical of myocardial infarction (MI, or heart attack) and drug induced cardiotoxicity mimic only a few aspects of these complex scenarios. This leads to a poor translation of findings from the bench to the bedside. In this study, we biofabricated for the first time advancedin vitromodels of MI and doxorubicin (DOX) induced injury by exposing cardiac spheroids (CSs) to pathophysiological changes in oxygen (O2) levels or DOX treatment. Then, contractile function and cell death was analyzed in CSs in control verses I/R and DOX CSs. For a deeper dig into cell death analysis, 3D rendering analyses and mRNA level changes of cardiac damage-related genes were compared in control verses I/R and DOX CSs. Overall,in vitroCSs recapitulated major features typical of thein vivoMI and drug induced cardiac damages, such as adapting intracellular alterations to O2concentration changes and incubation with cardiotoxic drug, mimicking the contraction frequency and fractional shortening and changes in mRNA expression levels for genes regulating sarcomere structure, calcium transport, cell cycle, cardiac remodelling and signal transduction. Taken together, our study supports the use of I/R and DOX CSs as advancedin vitromodels to study MI and DOX-induced cardiac damge by recapitulating their complexin vivoscenario.

Increased complications of COVID-19 in people with cardiovascular disease: Role of the renin-angiotensin-aldosterone system (RAAS) dysregulation.

The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19), has had a dramatic negative impact on public health and economies worldwide. Recent studies on COVID-19 complications and mortality rates suggest that there is a higher prevalence in cardiovascular diseases (CVD) patients. Past investigations on the associations between pre-existing CVDs and susceptibility to coronavirus infections including SARS-CoV and the Middle East Respiratory Syndrome coronavirus (MERS-CoV), have demonstrated similar results. However, the underlying mechanisms are poorly understood. This has impeded adequate risk stratification and treatment strategies for CVD patients with SARS-CoV-2 infections. Generally, dysregulation of the expression of angiotensin-converting enzyme (ACE) and the counter regulator, angiotensin-converting enzyme 2 (ACE2) is a hallmark of cardiovascular risk and CVD. ACE2 is the main host receptor for SARS-CoV-2. Although further studies are required, dysfunction of ACE2 after virus binding and dysregulation of the renin-angiotensin-aldosterone system (RAAS) signaling may worsen the outcomes of people affected by COVID-19 and with preexisting CVD. Here, we review the current knowledge and outline the gaps related to the relationship between CVD and COVID-19 with a focus on the RAAS. Improved understanding of the mechanisms regulating viral entry and the role of RAAS may direct future research with the potential to improve the prevention and management of COVID-19.

Microgravity × Radiation: A Space Mechanobiology Approach Toward Cardiovascular Function and Disease.

In recent years, there has been an increasing interest in space exploration, supported by the accelerated technological advancements in the field. This has led to a new potential environment that humans could be exposed to in the very near future, and therefore an increasing request to evaluate the impact this may have on our body, including health risks associated with this endeavor. A critical component in regulating the human pathophysiology is represented by the cardiovascular system, which may be heavily affected in these extreme environments of microgravity and radiation. This mini review aims to identify the impact of microgravity and radiation on the cardiovascular system. Being able to understand the effect that comes with deep space explorations, including that of microgravity and space radiation, may also allow us to get a deeper understanding of the heart and ultimately our own basic physiological processes. This information may unlock new factors to consider with space exploration whilst simultaneously increasing our knowledge of the cardiovascular system and potentially associated diseases.

A World-First Surgical Instrument for Minimally Invasive Robotically-Enabled Transplantation of Heart Patches for Myocardial Regeneration: A Brief Research Report.

Background: Patch-based approaches to regenerating damaged myocardium include epicardial surgical transplantation of heart patches. By the time this therapy is ready for widespread clinical use, it may be important that patches can be delivered via minimally invasive and robotic surgical approaches. This brief research report describes a world-first minimally invasive patch transplantation surgical device design enabled for human operation, master-slave, and fully automated robotic control. Method: Over a 12-month period (2019-20) in our multidisciplinary team we designed a surgical instrument to transplant heart patches to the epicardial surface. The device was designed for use via uni-portal or multi-portal Video-Assisted Thorascopic Surgery (VATS). For preliminary feasibility and sizing, we used a 3D printer to produce parts of a flexible resin model from a computer-aided design (CAD) software platform in preparation for more robust high-resolution metal manufacturing. Results: The instrument was designed as a sheath containing foldable arms, <2 cm in diameter when infolded to fit minimally invasive thoracic ports. The total length was 35 cm. When the arms were projected from the sheath, three moveable mechanical arms at the distal end were designed to hold a patch. Features included: a rotational head allowing for the arms to be angled in real time, a surface with micro-attachment points for patches and a releasing mechanism to release the patch. Conclusion: This brief research report represents a first step on a potential pathway towards minimally invasive robotic epicardial patch transplantation. For full feasibility testing, future proof-of-concept studies, and efficacy trials will be needed.

Towards engineering heart tissues from bioprinted cardiac spheroids.

Currentin vivoandin vitromodels fail to accurately recapitulate the human heart microenvironment for biomedical applications. This study explores the use of cardiac spheroids (CSs) to biofabricate advancedin vitromodels of the human heart. CSs were created from human cardiac myocytes, fibroblasts and endothelial cells (ECs), mixed within optimal alginate/gelatin hydrogels and then bioprinted on a microelectrode plate for drug testing. Bioprinted CSs maintained their structure and viability for at least 30 d after printing. Vascular endothelial growth factor (VEGF) promoted EC branching from CSs within hydrogels. Alginate/gelatin-based hydrogels enabled spheroids fusion, which was further facilitated by addition of VEGF. Bioprinted CSs contracted spontaneously and under stimulation, allowing to record contractile and electrical signals on the microelectrode plates for industrial applications. Taken together, our findings indicate that bioprinted CSs can be used to biofabricate human heart tissues for long termin vitrotesting. This has the potential to be used to study biochemical, physiological and pharmacological features of human heart tissue.

Proinflammatory Interleukin-33 Induces Dichotomic Effects on Cell Proliferation in Normal Gastric Epithelium and Gastric Cancer.

Interleukin (IL)-33 is a member of the interleukin (IL)-1 family of cytokines linked to the development of inflammatory conditions and cancer in the gastrointestinal tract. This study is designed to investigate whether IL-33 has a direct effect on human gastric epithelial cells (GES-1), the human gastric adenocarcinoma cell line (AGS), and the gastric carcinoma cell line (NCI-N87) by assessing its role in the regulation of cell proliferation, migration, cell cycle, and apoptosis. Cell cycle regulation was also determined in ex vivo gastric cancer samples obtained during endoscopy and surgical procedures. Cell lines and tissue samples underwent stimulation with rhIL-33. Proliferation was assessed by XTT and CFSE assays, migration by wound healing assay, and apoptosis by caspase 3/7 activity assay and annexin V assay. Cell cycle was analyzed by means of propidium iodine assay, and gene expression regulation was assessed by RT-PCR profiling. We found that IL-33 has an antiproliferative and proapoptotic effect on cancer cell lines, and it can stimulate proliferation and reduce apoptosis in normal epithelial cell lines. These effects were also confirmed by the analysis of cell cycle gene expression, which showed a reduced expression of pro-proliferative genes in cancer cells, particularly in genes involved in G0/G1 and G2/M checkpoints. These results were confirmed by gene expression analysis on bioptic and surgical specimens. The aforementioned results indicate that IL-33 may be involved in cell proliferation in an environment- and cell-type-dependent manner.

FXYD1 Is Protective Against Vascular Dysfunction.

[Figure: see text].