Modular Multiwell Viscoelastic Hydrogel Platform for Two- and Three-Dimensional Cell Culture Applications.

Hydrogels have gained significant popularity as model platforms to study reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within the same 96-well plate for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and is used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for two-dimensional (2D) cell culture applications was demonstrated by measuring both population-level and single-cell-level metrics via microplate reader and high-content imaging. Finally, a 96-well hydrogel array was utilized for three-dimensional (3D) cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adaptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

Hydrogels have gained significant popularity as model platforms to study the reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within 96-well plates for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and was used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for cell culture applications was demonstrated by measuring both population-level and single cell-level metrics via microplate reader and high-content imaging. Finally, the 96-well hydrogel array was utilized for 3D cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adoptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Supramolecular Fibrous Hydrogel Augmentation of Uterosacral Ligament Suspension for Treatment of Pelvic Organ Prolapse.

Uterosacral ligament suspension (USLS) is a common surgical treatment for pelvic organ prolapse (POP). However, the relatively high failure rate of up to 40% underscores a strong clinical need for complementary treatment strategies, such as biomaterial augmentation. Herein, the first hydrogel biomaterial augmentation of USLS in a recently established rat model is described using an injectable fibrous hydrogel composite. Supramolecularly-assembled hyaluronic acid (HA) hydrogel nanofibers encapsulated in a matrix metalloproteinase (MMP)-degradable HA hydrogel create an injectable scaffold showing excellent biocompatibility and hemocompatibility. The hydrogel can be successfully delivered and localized to the suture sites of the USLS procedure, where it gradually degrades over six weeks. In situ mechanical testing 24 weeks post-operative in the multiparous USLS rat model shows the ultimate load (load at failure) to be 1.70 ± 0.36 N for the intact uterosacral ligament (USL), 0.89 ± 0.28 N for the USLS repair, and 1.37 ± 0.31 N for the USLS + hydrogel (USLS+H) repair (n = 8). These results indicate that the hydrogel composite significantly improves load required for tissue failure compared to the standard USLS, even after the hydrogel degrades, and that this hydrogel-based approach can potentially reduce the high failure rate associated with USLS procedures.

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

Genome-wide Profiling Identifies DNA Methylation Signatures of Aging in Rod Photoreceptors Associated with Alterations in Energy Metabolism.

Aging-associated functional decline is accompanied by alterations in the epigenome. To explore DNA modifications that could influence visual function with age, we perform whole-genome bisulfite sequencing of purified mouse rod photoreceptors at four ages and identify 2,054 differentially methylated regions (DMRs). We detect many DMRs during early stages of aging and in rod regulatory regions, and some of these cluster at chromosomal hotspots, especially on chromosome 10, which includes a longevity interactome. Integration of methylome to age-related transcriptome changes, chromatin signatures, and first-order protein-protein interactions uncover an enrichment of DMRs in altered pathways that are associated with rod function, aging, and energy metabolism. In concordance, we detect reduced basal mitochondrial respiration and increased fatty acid dependency with retinal age in ex vivo assays. Our study reveals age-dependent genomic and chromatin features susceptible to DNA methylation changes in rod photoreceptors and identifies a link between DNA methylation and energy metabolism in aging.

Case series, contemporary review and imaging guided diagnostic and management approach of congenital pericardial defects.

Objective: Congenital pericardial defect (CPD) is a rare entity with an estimated frequency of 0.01%-0.04%. The recognition of this anomaly is important since it can be associated with serious complications. The aim of this study and review was to describe clinical and imaging features that help in establishing the diagnosis of this condition. Methods: We retrospectively reviewed all adult patients at the Cleveland Clinic Health System with the diagnosis of CPD between the years 2000 and 2015. Baseline clinical characteristics, clinical manifestations, ECG, transthoracic echocardiography (TTE), cardiac CT and cardiac magnetic resonance (CMR) images were reviewed. Results: Eight patients were included in the study. Sixty-three percent of patients were males with mean age at diagnosis of 48 years, 63% had a partial pericardial defect on the left side and right ventricular (RV) dilation on TTE. Three patients had CMR. Levocardia was present in all CMRs. One patient had greater than 60° clockwise rotation and none of the CMRs showed ballooning of the left ventricular apex. One patient required surgical pericardioplasty. The remaining seven patients had a median follow-up of 17.3 months (5-144.9 months) and all remained asymptomatic. Conclusion: CPDs are more likely to be partial on the left side and patients often have RV dilation on the TTE and levocardia on CMR. Most patients remain stable and do not require surgical intervention. TTE and CMR play an important role in making the diagnosis of this anomaly.

Arrhythmias and Adaptations of the Cardiac Conduction System in Former National Football League Players.

Background Habitual high-intensity endurance exercise is associated with increased atrial fibrillation (AF) risk and impaired cardiac conduction. It is unknown whether these observations extend to prior strength-type sports exposure. The primary aim of this study was to compare AF prevalence in former National Football League (NFL) athletes to population-based controls. The secondary aim was to characterize other conduction system parameters. Methods and Results This cross-sectional study compared former NFL athletes (n=460, age 56±12 years, black 47%) with population-based controls of similar age and racial composition from the cardiovascular cohort Dallas Heart Study-2 (n=925, age 54±9 years, black 53%). AF was present in 28 individuals (n=23 [5%] in the NFL group; n=5 [0.5%] in the control group). After controlling for other cardiovascular risk factors in multivariable regression analysis, former NFL participation remained associated with a 5.7 (95% CI: 2.1-15.9, P<0.001) higher odds ratio of AF. Older age, higher body mass index, and nonblack race were also independently associated with higher odds ratio of AF, while hypertension and diabetes mellitus were not. AF was previously undiagnosed in 15/23 of the former NFL players. Previously undiagnosed NFL players were rate controlled and asymptomatic, but 80% had a CHA2DS2-VASc score ≥1. Former NFL players also had an 8-fold higher prevalence of paced cardiac rhythms (2.0% versus 0.25%, P<0.01), compared with controls. Furthermore, former athletes had lower resting heart rates (62±11 versus 66±11 beats per minute, P<0.001), and a higher prevalence of first-degree atrioventricular block (18% versus 9%, P<0.001). Conclusions Former NFL participation was associated with an increased AF prevalence and slowed cardiac conduction when compared with a population-based control group. Former NFL athletes who screened positive for AF were generally rate controlled and asymptomatic, but 80% should have been considered for anticoagulation based on their stroke risk.

Characteristics and Outcomes in a Contemporary Group of Patients With Suspected Significant Mitral Stenosis Undergoing Treadmill Stress Echocardiography.

BACKGROUND: In contemporary patients with suspected significant mitral stenosis (MS) undergoing rest and treadmill stress echocardiography, we assessed characteristics and factors associated with longer-term survival. METHODS: We studied 515 consecutive patients (asymptomatic/atypical symptoms, mean left ventricular ejection fraction 58±2%; 43% male) with suspected at least moderate MS ([1] native mitral valve [MV]: resting mean MV gradient ≥5 mm Hg or area ≤1.5 cm2 and [2] prosthetic valve: resting mean MV gradient ≥5 mm Hg or effective orifice area ≤2 cm) who underwent rest and treadmill stress echocardiography between 1/2003 and 12/2013. MS was categorized as rheumatic (n=170, 33%), postsurgical (prior mitral repair/replacement, n=245, 48%), and primary nonrheumatic (n=100, 19%). Primary outcome was all-cause mortality. RESULTS: Mean resting MV gradient and right ventricular systolic pressure were 8.5±3 and 39±13 mm Hg. Patients achieved 95±29% age-sex predicted metabolic equivalents; peak-stress MV gradient and right ventricular systolic pressure were 17±7 and 61±14 mm Hg, respectively. At 54 days (median), 224 (44%) underwent invasive mitral procedure. At 6±4 years, 76 (15%) died. On survival analysis, primary nonrheumatic MS (hazard ratio [HR], 4.92), higher Society of Thoracic Surgeons score (HR, 1.92), lower % age-sex predicted metabolic equivalents (HR, 1.22), and higher peak-stress right ventricular systolic pressure (HR, 1.35), was associated with higher mortality, while invasive mitral procedures were associated with improved survival (HR, 0.67; all P<0.01). CONCLUSIONS: In asymptomatic patients (or with atypical symptoms) with significant MS undergoing treadmill stress echocardiography, higher mortality was associated with primary nonrheumatic MS, lower % age-sex predicted metabolic equivalents, and higher peak-stress right ventricular systolic pressure, while invasive MV procedures were associated with survival.

Pericardial diseases in patients with hypothyroidism.

Hypothyroidism is a well-known cause of pericardial effusion (with an incidence of 3%-37%) and can cause cardiac tamponade in severe cases. In this review, we present the current knowledge on the epidemiology of hypothyroid-induced pericardial diseases, the mechanism through which low thyroid hormone levels affect the pericardium, the associated clinical manifestations, diagnostic tests and management options. Hypothyroidism causes pericardial effusion through increased permeability of the epicardial vessels and decreased lymphatic drainage of albumin, resulting in accumulation of fluid in the pericardial space. Interestingly, autoimmunity does not seem to play a major role in the pathophysiology, and a majority of effusions are asymptomatic due to slow fluid accumulation. The diagnosis is generally made when the pericardial disease is associated with an elevated thyroid-stimulating hormone level, and other secondary causes are excluded. Management consists of thyroid replacement therapy, along with pericardial drainage in case of tamponade.In conclusion, hypothyroidism-induced pericardial diseases are underdiagnosed. Initiating treatment early in the disease process and preventing complications relies on early diagnosis through systematic screening per guidelines.

Interobserver Variability in Applying American Society of Echocardiography/European Association of Cardiovascular Imaging 2016 Guidelines for Estimation of Left Ventricular Filling Pressure.

BACKGROUND: Assessment of left ventricular (LV) filling pressure is among the important components of a comprehensive echocardiographic report. Previous studies noted wide limits of agreement using 2009 American Society of Echocardiography/European Association of Echocardiography guidelines, but reproducibility of 2016 guidelines update in estimating LV filling pressure is unknown. METHODS: Echocardiographic and hemodynamic data were obtained from 50 patients undergoing cardiac catheterization for clinical indications. Clinical and echocardiographic findings but not invasive hemodynamics were provided to 4 groups of observers, including experienced echocardiographers and cardiology fellows. Invasively acquired LV filling pressure was the gold standard. RESULTS: In group I of 8 experienced echocardiographers from the guidelines writing committee, sensitivity for elevated LV filling pressure was 92% for all observers, and specificity was 93±6%. Fleiss κ-value for the agreement in group I was 0.80. In group II of 4 fellows in training, sensitivity was 91±2%, and specificity was 95±2%. Fleiss κ-value for the agreement in group II was 0.94. In group III of 9 experienced echocardiographers who had not participated in drafting the guidelines, sensitivity was 88±5%, and specificity was 91±7%. Fleiss κ-value for the agreement in group III was 0.76. In group IV of 7 other fellows, sensitivity was 91±3%, and specificity was 92±5%. Fleiss κ-value for the agreement in group IV was 0.89. CONCLUSIONS: There is a good level of agreement and accuracy in the estimation of LV filling pressure using the American Society of Echocardiography/European Association of Cardiovascular Imaging 2016 recommendations update, irrespective of the experience level of the observer.

Transvenous Extraction of Pacemaker and Defibrillator Leads and the Risk of Tricuspid Valve Regurgitation.

OBJECTIVES: The aims of this study were to detect and quantify acute increases in tricuspid regurgitation (TR) severity following transvenous lead extraction (TLE) and to evaluate the associated risk factors. BACKGROUND: Although established as a safe and effective method for lead removal, TLE is sometimes complicated by TR. METHODS: In 208 consecutive patients undergoing TLE, acute changes in TR severity were assessed by transesophageal echocardiography. A significant acute TR increase (TRI) was defined as a ≥1 grade increase in TR severity and post-extraction TR severity that was moderate or greater. RESULTS: Overall, 266 ventricular leads (mean lead age, 11.8 ± 7.3 years) were extracted from the 208 patients. A significant acute TRI was observed in 24 (11.5%) of these patients. Acute TRI was associated with longer lead implant duration, extraction of pacemaker rather than defibrillator leads, anatomic injury to the tricuspid valve (TV), and longer post-extraction hospital stays. Multivariate analysis yielded only lead implant duration as an independent predictor of TLE-related acute TRI (odds ratio: 1.05; 95% confidence interval: 1.01 to 1.11; p = 0.046). When the patients were divided into 4 subgroups according to quartiles of lead age, there was a graded elevation in the rates of acute TRI (p trend = 0.048) and TV injury (p trend = 0.009) with lead implant duration. CONCLUSIONS: Following TLE, TV damage and acute TRI were commonly detected by transesophageal echocardiography, particularly in patients with advanced lead age. Lead abandonment strategies, which prolong implantation duration of future leads requiring extraction, should consider the potential long-term deleterious impact on TV function.

Application of a High-Resolution Quadrupole/Orbital Trapping Mass Spectrometer Coupled to a Gas Chromatograph for the Determination of Persistent Organic Pollutants in Cow's and Human Milk.

A quadrupole/orbital trapping mass spectrometer or Q-Exactive (QE) interfaced with a gas chromatograph (GC) was optimized for measuring polychlorinated dibenzo- p-dioxins, dibenzofurans (PCDD/Fs), and polychlorinated biphenyls (PCBs) in foods. Figures of merit include (1) an instrument detection limit (IDL) for 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) of 9 femtograms (fg), (2) quantitative mass resolution from PCDD interferences (e.g., PCBs, methoxy-PCBs DDTs, polychlorodibenzylphenyl ethers, polychloroxanthenes, methyl-polychlorodibenzofuran, and polychlorodibenzothiophenes), and (3) mass accuracy <1 ppm at the IDL. The QE measured the concentrations of PCDD/Fs and PCBs in whole cow's milk with no known source of contamination (e.g., TCDD 33 fg/g fat). A National Institute of Standards and Technology (NIST) unfortified human milk standard reference material (SRM) 1953 was measured determining 27 PCDD/F and PCB congeners with an average difference of 7.6% from the certified results. The QE-GC is a benchtop instrument, easy to service, easy to operate, and requires no lock masses, mass preselection, or chemical ionization conditions. The QE-GC demonstrated that it can be an alternative to the double focusing magnetic sector instruments (sector) for the high-resolution measurement of PCDD/Fs and PCBs in dairy products.

Nanoengineered Ionic-Covalent Entanglement (NICE) Bioinks for 3D Bioprinting.

We introduce an enhanced nanoengineered ionic-covalent entanglement (NICE) bioink for the fabrication of mechanically stiff and elastomeric 3D biostructures. NICE bioink formulations combine nanocomposite and ionic-covalent entanglement (ICE) strengthening mechanisms to print customizable cell-laden constructs for tissue engineering with high structural fidelity and mechanical stiffness. Nanocomposite and ICE strengthening mechanisms complement each other through synergistic interactions, improving mechanical strength, elasticity, toughness, and flow properties beyond the sum of the effects of either reinforcement technique alone. Herschel-Bulkley flow behavior shields encapsulated cells from excessive shear stresses during extrusion. The encapsulated cells readily proliferate and maintain high cell viability over 120 days within the 3D-printed structure, which is vital for long-term tissue regeneration. A unique aspect of the NICE bioink is its ability to print much taller structures, with higher aspect ratios, than can be achieved with conventional bioinks without requiring secondary supports. We envision that NICE bioinks can be used to bioprint complex, large-scale, cell-laden constructs for tissue engineering with high structural fidelity and mechanical stiffness for applications in custom bioprinted scaffolds and tissue engineered implants.

Ascending Aortic Dimensions in Former National Football League Athletes.

BACKGROUND: Ascending aortic dimensions are slightly larger in young competitive athletes compared with sedentary controls, but rarely >40 mm. Whether this finding translates to aortic enlargement in older, former athletes is unknown. METHODS AND RESULTS: This cross-sectional study involved a sample of 206 former National Football League (NFL) athletes compared with 759 male subjects from the DHS-2 (Dallas Heart Study-2; mean age of 57.1 and 53.6 years, respectively, P<0.0001; body surface area of 2.4 and 2.1 m2, respectively, P<0.0001). Midascending aortic dimensions were obtained from computed tomographic scans performed as part of a NFL screening protocol or as part of the DHS. Compared with a population-based control group, former NFL athletes had significantly larger ascending aortic diameters (38±5 versus 34±4 mm; P<0.0001). A significantly higher proportion of former NFL athletes had an aorta of >40 mm (29.6% versus 8.6%; P<0.0001). After adjusting for age, race, body surface area, systolic blood pressure, history of hypertension, current smoking, diabetes mellitus, and lipid profile, the former NFL athletes still had significantly larger ascending aortas (P<0.0001). Former NFL athletes were twice as likely to have an aorta >40 mm after adjusting for the same parameters. CONCLUSIONS: Ascending aortic dimensions were significantly larger in a sample of former NFL athletes after adjusting for their size, age, race, and cardiac risk factors. Whether this translates to an increased risk is unknown and requires further evaluation.

Vacancy-Driven Gelation Using Defect-Rich Nanoassemblies of 2D Transition Metal Dichalcogenides and Polymeric Binder for Biomedical Applications.

A new approach of vacancy-driven gelation to obtain chemically crosslinked hydrogels from defect-rich 2D molybdenum disulfide (MoS2 ) nanoassemblies and polymeric binder is reported. This approach utilizes the planar and edge atomic defects available on the surface of the 2D MoS2 nanoassemblies to form mechanically resilient and elastomeric nanocomposite hydrogels. The atomic defects present on the lattice plane of 2D MoS2 nanoassemblies are due to atomic vacancies and can act as an active center for vacancy-driven gelation with a thiol-activated terminal such as four-arm poly(ethylene glycol)-thiol (PEG-SH) via chemisorption. By modulating the number of vacancies on the 2D MoS2 nanoassemblies, the physical and chemical properties of the hydrogel network can be controlled. This vacancy-driven gelation process does not require external stimuli such as UV exposure, chemical initiator, or thermal agitation for crosslinking and thus provides a nontoxic and facile approach to encapsulate cells and proteins. 2D MoS2 nanoassemblies are cytocompatible, and encapsulated cells in the nanocomposite hydrogels show high viability. Overall, the nanoengineered hydrogel obtained from vacancy-driven gelation is mechanically resilient and can be used for a range of biomedical applications including tissue engineering, regenerative medicine, and cell and therapeutic delivery.

Latent Presentation of Decompression Sickness After Altitude Chamber Training in an Active Duty Flier.

BACKGROUND: Decompression sickness (DCS) is a potential danger and risk for both divers and aircrew alike. DCS is also a potential side effect of altitude (hypobaric) chamber training as well and can present long after training occurs. Literature review shows that altitude chamber induced DCS has approximately a 0.25% incidence. CASE REPORT: A 32-yr-old, active duty military member developed symptoms of DCS 3 h after his hypobaric chamber training. Unfortunately, he did not seek treatment for DCS until 48 h after the exposure. His initial treatment included ground level oxygen therapy for 30 min at 12 L of oxygen per minute using a nonrebreathing mask. He achieved complete symptom resolution and was returned to duty. However, 12 d after his initial Flight Medicine evaluation, the patient returned complaining of a right temporal headache, multijoint pains, and fatigue. He was treated in the hyperbaric chamber and had complete resolution of symptoms. He was returned to flying status and 5 mo later denied any return of symptoms. DISCUSSION: Hypobaric chamber familiarity training is a requirement for all military aircrew personnel to allow them assess their ability to identify symptoms of hypoxia. This training method is not only costly to maintain, but it also places aircrew and chamber technicians at risk for potential long-term side effects from failed recompression treatment of DCS. We are presenting a case of recurrent DCS symptoms 12 d after initial ground level oxygen therapy.Gentry J, Rango J, Zhang J, Biedermann S. Latent presentation of decompression sickness after altitude chamber training in an active duty flier. Aerosp Med Hum Perform. 2017; 88(4):427-430.