DISC-3D: dual-hydrogel system enhances optical imaging and enables correlative mass spectrometry imaging of invading multicellular tumor spheroids.

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging.

Manifestations of static and dynamic heterogeneity in single molecule translational measurements in glassy systems.

Rotational-translational decoupling in systems near Tg, in which translational diffusion is apparently enhanced relative to rotation, has been observed in ensemble and single molecule experiments and has been linked to dynamic heterogeneity. Here, simulations of single molecules experiencing homogeneous diffusion and static and dynamic heterogeneous diffusion are performed to clarify the contributions of heterogeneity to such enhanced translational diffusion. Results show that time-limited trajectories broaden the distribution of diffusion coefficients in the presence of homogeneous diffusion but not when physically reasonable degrees of static heterogeneity are present. When dynamic heterogeneity is introduced, measured diffusion coefficients uniformly increase relative to input diffusion coefficients, and the widths of output distributions decrease, providing support for the idea that dynamic heterogeneity can drive apparent translational enhancement. Among simulations with dynamic heterogeneity, when the frequency of dynamic exchange is correlated with the initial diffusion coefficient, the measured diffusion coefficient behavior as a function of observation time matches that seen experimentally, the only set of simulations explored in which this occurs. Taken together with experimental results, this suggests that enhanced translational diffusion in glassy systems occurs through dynamic exchange consistent with wide underlying distributions of diffusion coefficients and exchange coupled to local spatiotemporal dynamics.

Engineering collagenous analogs of connective tissue extracellular matrix.

Connective tissue extracellular matrix (ECM) consists of an interwoven network of contiguous collagen fibers that regulate cell activity, direct biological function, and guide tissue homeostasis throughout life. Recently, ECM analogs have emerged as a unique ex vivo culture platform for studying healthy and diseased tissues and in the latter, enabling the screening for and development of therapeutic regimen. Since these tissue models can mitigate the concern that observations from animal models do not always translate clinically, the design and production of a collagenous ECM analogue with relevant chemistry and nano- to micro-scale architecture remains a frontier challenge in the field. Therefore, the objectives of this study are two-fold- first, to apply green electrospinning approaches to the fabrication of an ECM analog with nanoscale mimicry and second, to systematically optimize collagen crosslinking in order to produce a stable, collagen-like substrate with continuous fibrous architecture that supports human cell culture and phenotypic expression. Specifically, the "green" electrospinning solvent acetic acid was evaluated for biofabrication of gelatin-based meshes, followed by the optimization of glutaraldehyde (GTA) crosslinking under controlled ambient conditions. These efforts led to the production of a collagen-like mesh with nano- and micro-scale cues, fibrous continuity with little batch-to-batch variability, and proven stability in both dry and wet conditions. Moreover, the as-fabricated mesh architecture and native chemistry were preserved with augmented mechanical properties. These meshes supported the in vitro expansion of stem cells and the production of a mineralized matrix by human osteoblast-like cells. Collectively these findings demonstrate the potential of green fabrication in the production of a collagen-like ECM analog with physiological relevance. Future studies will explore the potential of this high-fidelity platform for elucidating cell-matrix interactions and their relevance in connective tissue healing.

Single molecule demonstration of Debye-Stokes-Einstein breakdown in polystyrene near the glass transition temperature.

Rotational-translational decoupling, in which translational motion is apparently enhanced over rotational motion in violation of Stokes-Einstein (SE) and Debye-Stokes-Einstein (DSE) predictions, has been observed in materials near their glass transition temperatures (Tg). This has been posited to result from ensemble averaging in the context of dynamic heterogeneity. In this work, ensemble and single molecule experiments are performed in parallel on a fluorescent probe in high molecular weight polystyrene near its Tg. Ensemble results show decoupling onset at approximately 1.15Tg, increasing to over three orders of magnitude at Tg. Single molecule measurements also show a high degree of decoupling, with typical molecules at Tg showing translational diffusion coefficients nearly 400 times higher than expected from SE/DSE predictions. At the single molecule level, higher degree of breakdown is associated with particularly mobile molecules and anisotropic trajectories, providing support for anomalous diffusion as a critical driver of rotational-translational decoupling and SE/DSE breakdown.

Alkaline activation of endogenous latent TGFß1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration.

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFß1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFß1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFß1 from tooth dentin for robust bone marrow stem cell migration. More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

Exploiting differential effects of actomyosin contractility to control cell sorting among breast cancer cells.

In order to gain a greater understanding of the factors that drive spatial organization in multicellular aggregates of cancer cells, we investigate the segregation patterns of 6 breast cell lines of varying degree of mesenchymal character during formation of mixed aggregates. Cell sorting is considered in the context of available adhesion proteins and cellular contractility. It is found that the primary compaction mediator (cadherins or integrins) for a given cell type in isolation plays an important role in compaction speed, which in turn is the major factor dictating preference for interior or exterior position within mixed aggregates. In particular, cadherin-deficient, invasion-competent cells tend to position towards the outside of aggregates, facilitating access to extracellular matrix. Reducing actomyosin contractility is found to have a differential effect on spheroid formation depending on compaction mechanism. Inhibition of contractility has a significant stabilizing effect on cell-cell adhesions in integrin-driven aggregation and a mildly destabilizing effect in cadherin-based aggregation. This differential response is exploited to statically control aggregate organization and dynamically rearrange cells in pre-formed aggregates. Sequestration of invasive cells in the interior of spheroids provides a physical barrier that reduces invasion in three-dimensional culture, revealing a potential strategy for containment of invasive cell types.

A Multifaceted Approach to Improve Physician Communication Scores.

Improving patient satisfaction scores has become a key focus of health-care organizations nationwide but can be a struggle for community hospitals with constrained resources, and particularly challenging for hospitalist programs due to provider variance and turnover. Using the framework of appreciative inquiry, we implemented a multipronged intervention including a rounding model whereby hospitalist leaders rounded on patients and relayed commentary back to their hospitalist providers. We communicated positive feedback preferentially over negative feedback to the entire hospitalist group through regular communication. Providers were encouraged to employ best practices including sitting with the patient, reviewing recommendations using teach back, and providing business cards. Scores improved in the physician communication category by approximately 1% annually from fiscal year 2015 through 2018, with our percentile rank improving 35 percentile points during that time. These findings indicate that a multifaceted approach including best practices is associated with improved patient experience regarding communication with physicians.

Assessment of knowledge and perception about silver diamine fluoride (SDF) for treating older adults among graduating dental students.

PURPOSE/OBJECTIVES: Silver diamine fluoride (SDF) is a promising therapy for arresting and preventing caries in difficult to treat, high-risk populations, including institutionalized older adults. This study investigates the knowledge and perceptions about SDF of graduating dental students in multiple U.S. dental schools, as well as their willingness to use SDF in their practices. METHODS: A survey was designed consisting of 21 total questions: 6 questions regarding students' demographic information and their SDF content exposure contextualization, 8 questions tailored to investigate 2 domains regarding students' knowledge about SDF (properties and indication), and 7 questions aiming to investigate 3 domains regarding students' perceptions about SDF (SDF usefulness, appropriateness of using SDF, and willingness to use SDF when in private practice). The survey was then distributed to graduating dental students at 7 U.S. dental schools. RESULTS: A total of 386 surveys (response rate of 55%) was collected from 7 schools in the Spring of 2019. The median score resulting from the SDF content exposure questions was 3 (SD = 1.43) from a range of 0-5. The median score from knowledge about SDF properties was 4 (SD = 1.18) from a range of 0 to 6. In the multivariate analysis, a linear model found that the covariates "SDF Usefulness", "SDF Appropriateness" and "SDF Patient Willingness to Use" were significantly associated with higher student willingness to use SDF (R2  = 0.395). CONCLUSION: The results indicated that the graduating students have a positive perception of SDF regarding its usefulness and appropriateness. Graduating students appear inclined to utilize SDF upon entering private practice.

Delineating the role of membrane blebs in a hybrid mode of cancer cell invasion in three-dimensional environments.

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional ß1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

Correlating fragility and heterogeneous dynamics in polystyrene through single molecule studies.

Many macroscopic properties of polymers depend on their molecular weight, with one notable example being glass transition temperature: polymers with higher molecular weights typically have higher glass transition temperatures than their lower molecular weight polymeric and oligomeric counterparts. Polymeric systems close to their glass transition temperatures also exhibit interesting properties, showing both high (and molecular weight dependent) fragility and strong evidence of dynamic heterogeneity. While studies have detailed the correlations between molecular weight and fragility, studies clearly detailing correlations between molecular weight and degree of heterogeneous dynamics are lacking. In this study, we use single molecule rotational measurements to investigate the impact of molecular weight on polystyrene's degree of heterogeneity near its glass transition temperature. To this end, two types of fluorescent probes are embedded in films composed of polystyrene ranging from 0.6 to 1364.0 kg mol-1. We find correlation between polystyrene molecular weight, fragility, and degree of dynamic heterogeneity as reported by single molecule stretching exponents but do not find clear correlation between these quantities and time scales associated with dynamic exchange.

Translation and validation of the Greek version of an ageism scale for dental students (ASDS_Gr).

OBJECTIVES: To describe the validation of a new 27-item ageism scale for dental students in Greece. BACKGROUND: A new ageism scale for dental students has been developed by American and European Gerodontology educators and was preliminary validated in the United States. METHODS: The scale was translated into Greek and administered to 8th- and 10th-semester dental students in Athens. Principal components analysis was used to explore the internal structure of the measure; internal consistency reliability was assessed using Cronbach's α coefficient; corrected item-total correlations were calculated to decide which low contributing items should be removed from the scale; and discriminant validity was tested investigating variation in relation to demographic and educational factors. RESULTS: A total of 152 students responded to the questionnaire. The Principal component analysis offered a 15-item scale distributed into four factors that accounted for 56.4%, of the total variance, produced stronger factor loadings, a comparable amount of overall component variance and logical sets of components. The four factors produced were values/ethics about older people (four items, α = 0.71), patient compliance (four items, α = 0.72), barriers to dental care (four items, α = 0.57) and dentist-older patient interaction (three items, α = 0.64). Discriminant validity revealed statistically significant differences in factors and items related to semester of studies, gender and family's permanent residence. CONCLUSION: The preliminary validation of the Greek version of the ageing scale for dental students revealed a 15-item questionnaire that demonstrated acceptable validity and reliability and could be further tested in larger samples.

Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy.

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

Dual institution validation of an ageism scale for dental students.

PURPOSE/AIM: Ageism negatively affects health care. This paper presents an extended validation of a novel scale assessing ageism among dental students. METHOD AND MATERIALS: A previously pilot-tested 27-question scale applied to a larger sample (n = 315) from two U.S. dental schools with Principal Component Analysis used to assess internal structure of the measure. Questions whose deletion increased the overall α loading on >1 factor or those unexpectedly grouped in another factor were thoroughly examined. RESULTS: The scale resulted in five statements (grouped in two factors), which explained 63% of the overall variance with a substantially higher reliability value than other solutions (0.76). Two factors highly correlated were grouped together in a single scale. The five statements are "Elderly people do not take good care of their teeth" (0.62), "Elderly patients do not usually comply with dental advice" (0.93), "The Elderly patient does not live long enough to make it worthwhile to invest time and effort in complex dental treatment" (0.81), "The elderly patient does not live long enough to make it worthwhile to invest money in expensive dental treatment" (0.95), and "Dental treatment of elderly patients is too time-consuming" (0.57). CONCLUSIONS: Five items achieved high reliability toward the validity of this scale.

Which probes can report intrinsic dynamic heterogeneity of a glass forming liquid?

Using extrinsic probes to study a host system relies on the probes' ability to accurately report the host properties under study. Probes have long been used to characterize dynamic heterogeneity, the phenomenon in which a liquid near its glass transition exhibits distinct dynamics as a function of time and position, with molecules within nanometers of each other exhibiting dynamics that may vary by orders of magnitude. The spatial and temporal characteristics of dynamic heterogeneity demand the selection of probes using stringent criteria on their size and dynamics. In this report, we study the dynamic heterogeneity of the prototypical molecular glass former o-terphenyl by investigating single molecule rotation of two perylene dicarboximide probe molecules that differ in size and comparing this to results obtained previously with the probe BODIPY268. It is found that a probe's ability to accurately report dynamic heterogeneity in o-terphenyl depends on whether the reported distribution of dynamics overlaps with the intrinsic dynamics of the host, which is naturally related to the width of the intrinsic dynamics and the magnitude of dynamical shift in probe dynamics relative to the host. We show that a probe that rotates ≈15 times more slowly than the intrinsic dynamics of the host o-terphenyl senses the slowest ≈5% of the full dynamic heterogeneity whereas one that rotates ≈65 times more slowly than the host fails to report dynamic heterogeneity of the host.

Single molecule studies reveal temperature independence of lifetime of dynamic heterogeneity in polystyrene.

Polymeric systems close to their glass transition temperature are known to exhibit heterogeneous dynamics that evolve both over time and space, comparable to the dynamics of small molecule glass formers. It remains unclear how temperature influences the degree of heterogeneous dynamics in such systems. In the following report, a fluorescent perylene dicarboximide probe molecule that reflects the full breadth of heterogeneity of the host was used to examine the temperature dependence of the dynamic heterogeneity lifetime in polystyrene at several temperatures ranging from the glass transition to 10 K above this temperature via single molecule microscopy. Contrary to prior reports, no apparent temperature dependence of time scales associated with dynamic heterogeneity was detected; indeed, the probe molecules report characteristic dynamic heterogeneity lifetimes 100-300 times the average alpha-relaxation time (τα) of the polystyrene host at all temperatures studied.

Predictors of Intravenous Immunoglobulin Nonresponse and Racial Disparities in Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD) is the most common cause of acquired heart disease in American children. Intravenous immunoglobulin (IVIG) nonresponse is a known risk factor for cardiac sequelae. Previously reported risk factors for nonresponse include age, male sex and laboratory abnormalities. We set out to identify additional risk factors for IVIG nonresponse in a racially diverse KD population. METHODS: We conducted a retrospective chart review at a referral center in the Southeastern United States of children meeting ICD-9 (International Statistical Classification of Disease and Related Health Problems) criteria for KD and being treated with IVIG. RESULTS: Four-hundred and fifty-nine children met inclusion criteria, 67 were excluded for subsequent rheumatologic diagnosis, unknown race, or failure to meet the American Heart Association guideline criteria. Our final cohort consisted of 392 subjects, with median age of 2.7 years, 65.1% male, 66.1% White, 24.2% Black, 4.9% Asian and 82.9% responded to a single dose of IVIG. Coronary ectasia or aneurysm developed in 27%; 7.4% developed aneurysms and 2.3% giant coronary aneurysms. Nonresponders were more likely to be Black, have higher white blood cell, erythrocyte sedimentation rate and C-reactive protein, lower hemoglobin, develop ectasia or aneurysm and require critical care and hospital readmission. Responders achieved echocardiographic normalization more often compared with nonresponders (81.3% vs. 60.9%, P = 0.002) and coronary artery pseudonormalization (87.2% vs. 69.7%, P = 0.03) at 1 year. Black nonresponders had the slowest normalization at 1 year (52.9%, P = 0.02). CONCLUSIONS: Nonresponders have higher rates and greater severity of coronary involvement than responders. Our study uniquely demonstrates Black race as a risk factor for nonresponse and for delayed normalization of cardiac involvement at 1-year follow-up.

Development and preliminary validation of an ageism scale for dental students.

PURPOSE/AIM: This work presents the preliminary validation of a novel scale assessing ageism attitudes among dental students. METHOD AND MATERIALS: A 27-question scale was created based on existing ageism scales. The new ageism scale was applied to 144 dental students. Content validity was achieved by experts' consensus. Questions whose deletion increased the overall α, loading < 0.40, loading on more than one factors, or those unexpectedly grouped in another factor were thoroughly examined. Principal Component Analysis assessed internal structure of the measure. RESULTS: The final ageism scale included four items in a single factor that explained 58.5% of the overall variance with substantially higher reliability than other factors. The Cronbach's α for this single four-items factor was 0.75. CONCLUSIONS: This preliminary analysis of a novel ageism scale for dental professionals pointed to four items achieving high reliability, providing guidance for a future definitive validation study with a larger sample.

In†Situ Optical Imaging of the Growth of Conjugated Polymer Aggregates.

Understanding the mechanisms that contribute to conjugated polymer aggregate formation and growth may yield enhanced control of aggregate morphology and functional properties on the mesoscopic scale. In situ optical imaging of the growth of MEH-PPV aggregates in real time in controlled swollen films shows that growth occurs through multiple mechanisms and is more complex than previously described. Direct evidence is provided for both Ostwald ripening and aggregate coalescence as operative modes of aggregate growth in solvent swollen films. These growth mechanisms have a distinct and strong impact on the evolution of morphological order of growing aggregates: while Ostwald ripening allows preservation of highly ordered morphology, aggregate coalescence occurs with no preferential orientation, leading to attenuation in degree of ordering.

Confocal Rheology Probes the Structure and Mechanics of Collagen through the Sol-Gel Transition.

Fibrillar type I collagen-based hydrogels are commonly used in tissue engineering and as matrices for biophysical studies. Mechanical and structural properties of these gels are known to be governed by the conditions under which fibrillogenesis occurs, exhibiting variation as a function of protein concentration, temperature, pH, and ionic strength. Deeper understanding of how macroscopic structure affects viscoelastic properties of collagen gels over the course of fibrillogenesis provides fundamental insight into biopolymer gel properties and promises enhanced control over the properties of such gels. Here, we investigate type I collagen fibrillogenesis using confocal rheology-simultaneous confocal reflectance microscopy, confocal fluorescence microscopy, and rheology. The multimodal approach allows direct comparison of how viscoelastic properties track the structural evolution of the gel on fiber and network length scales. Quantitative assessment and comparison of each imaging modality and the simultaneously collected rheological measurements show that the presence of a system-spanning structure occurs at a time similar to rheological determinants of gelation. Although this and some rheological measures are consistent with critical gelation through percolation, additional rheological and structural properties of the gel are found to be inconsistent with this theory. This study clarifies how structure sets viscoelasticity during collagen fibrillogenesis and more broadly highlights the utility of multimodal measurements as critical test-beds for theoretical descriptions of complex systems.

A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction.

Using a gelatin microbial transglutaminase (gelatin-mTG) cell culture platform tuned to exhibit stiffness spanning that of healthy and diseased glomeruli, we demonstrate that kidney podocytes show marked stiffness sensitivity. Podocyte-specific markers that are critical in the formation of the renal filtration barrier are found to be regulated in association with stiffness-mediated cellular behaviors. While podocytes typically de-differentiate in culture and show diminished physiological function in nephropathies characterized by altered tissue stiffness, we show that gelatin-mTG substrates with Young's modulus near that of healthy glomeruli elicit a pro-differentiation and maturation response in podocytes better than substrates either softer or stiffer. The pro-differentiation phenotype is characterized by upregulation of gene and protein expression associated with podocyte function, which is observed for podocytes cultured on gelatin-mTG gels of physiological stiffness independent of extracellular matrix coating type and density. Signaling pathways involved in stiffness-mediated podocyte behaviors are identified, revealing the interdependence of podocyte mechanotransduction and maintenance of their physiological function. This study also highlights the utility of the gelatin-mTG platform as an in vitro system with tunable stiffness over a range relevant for recapitulating mechanical properties of soft tissues, suggesting its potential impact on a wide range of research in cellular biophysics.