Immersive virtual reality-based learning as a supplement for biomedical engineering labs: challenges faced and lessons learned.

Introduction: Immersive virtual reality (VR) based laboratory demonstrations have been gaining traction in STEM education as they can provide virtual hands-on experience. VR can also facilitate experiential and visual learning and enhanced retention. However, several optimizations of the implementation, in-depth analyses of advantages and trade-offs of the technology, and assessment of receptivity of modern techniques in STEM education are required to ensure better utilization of VR-based labs. Methods: In this study, we developed VR-based demonstrations for a biomolecular engineering laboratory and assessed their effectiveness using surveys containing free responses and 5-point Likert scale-based questions. Insta360 Pro2 camera and Meta Quest 2 headsets were used in combination with an in-person lab. A cohort of 53 students watched the experimental demonstration on VR headsets in the lab after a brief lab overview in person and then performed the experiments in the lab. Results: Only 28.29% of students reported experiencing some form of discomfort after using the advanced VR equipment as opposed to 63.63% of students from the previous cohort. About 40% of the students reported that VR eliminated or reduced auditory and visual distractions from the environment, the length of the videos was appropriate, and they received enough information to understand the tasks. Discussion: The traditional lab method was found to be more suitable for explaining background information and lab concepts while the VR was found to be suitable for demonstrating lab procedures and tasks. Analyzing open-ended questions revealed several factors and recommendations to overcome the potential challenges and pitfalls of integrating VR with traditional modes of learning. This study provides key insights to help optimize the implementation of immersive VR to effectively supplement in-person learning experiences.

Effect of Cyclic Uniaxial Mechanical Strain on Endothelial Progenitor Cell Differentiation.

PURPOSE: Endothelial progenitor cells (EPCs) have been used as an autologous or allogeneic source in multiple tissue engineering applications. EPCs possess high proliferative and tissue regeneration potential. The effect of shear stress on EPCs has been extensively studied but the role of cyclic mechanical strain on EPCs remains to be understood. In this study, we focused on examining the role of uniaxial cyclic strain on EPCs cultured on three-dimensional (3D) anisotropic composites that mimic healthy and diseased aortic valve tissue matrix compositions. METHODS AND RESULTS: The composites were fabricated by combining centrifugal jet spun fibers with photocrosslinkable gelatin and glycosaminoglycan hydrogels. A custom-designed uniaxial cyclic stretcher was used to provide the necessary cyclic stimulation to the EPC-seeded 3D composites. The samples were cyclically strained at a rate of 1 Hz at 15% strain mimicking the physiological condition experienced by aortic valve, with static conditions serving as controls. Cell viability was high in all conditions. Immunostaining revealed reduced endothelial marker (CD31) expression with increased smooth muscle cell marker, SM22α, expression when subjected to cyclic strain. Functional analysis through Matrigel assay agreed with the immunostaining findings with reduced tubular structure formation in strained conditions compared to EPC controls. Additionally, the cells showed reduced acLDL uptake compared to controls which are in alignment with the EPCs undergoing differentiation. CONCLUSION: Overall, we show that EPCs lose their endothelial progenitor phenotype, and have the potential to be differentiated into mesenchymal-like cells through cyclic mechanical stimulation.

Local Renin-Angiotensin System Signaling Mediates Cellular Function of Aortic Valves.

The renin-angiotensin system (RAS) is activated in aortic valve disease, yet little is understood about how it affects the acute functional response of valve interstitial cells (VICs). Herein, we developed a gelatin-based valve thin film (vTF) platform to investigate whether the contractile response of VICs can be regulated via RAS mediators and inhibitors. First, the impact of culture medium (quiescent, activated, and osteogenic medium) on VIC phenotype and function was assessed. Contractility of VICs was measured upon treatment with angiotensin I (Ang I), angiotensin II (Ang II), angiotensin-converting enzyme (ACE) inhibitor, and Angiotensin II type 1 receptor (AT1R) inhibitor. Anisotropic cell alignment on gelatin vTF was achieved independent of culture conditions. Cells cultured in activated and osteogenic conditions were found to be more elongated than in quiescent medium. Increased α-SMA expression was observed in activated medium and no RUNX2 expression were observed in cells. VIC contractile stress increased with increasing concentrations (from 10-10 to 10-6 M) of Ang I and Ang II. Moreover, cell contraction was significantly reduced in all ACE and AT1R inhibitor-treated groups. Together, these findings suggest that local RAS is active in VICs, and our vTF may provide a powerful platform for valve drug screening and development.

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is the most common valvular heart disease. CAVD results in a considerable socio-economic burden, especially considering the aging population in Europe and North America. The only treatment standard is surgical valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. Novel diagnostic techniques and biomarkers for early detection and monitoring of CAVD progression are thus a pressing need. Additionally, non-destructive tools are required for longitudinal in vitro and in vivo assessment of CAVD initiation and progression that can be translated into clinical practice in the future. Multiphoton microscopy (MPM) facilitates label-free and non-destructive imaging to obtain quantitative, optical biomarkers that have been shown to correlate with key events during CAVD progression. MPM can also be used to obtain spatiotemporal readouts of metabolic changes that occur in the cells. While cellular metabolism has been extensively explored for various cardiovascular disorders like atherosclerosis, hypertension, and heart failure, and has shown potential in elucidating key pathophysiological processes in heart valve diseases, it has yet to gain traction in the study of CAVD. Furthermore, MPM also provides structural, functional, and metabolic readouts that have the potential to correlate with key pathophysiological events in CAVD progression. This review outlines the applicability of MPM and its derived quantitative metrics for the detection and monitoring of early CAVD progression. The review will further focus on the MPM-detectable metabolic biomarkers that correlate with key biological events during valve pathogenesis and their potential role in assessing CAVD pathophysiology.

Aortic valve cell microenvironment: Considerations for developing a valve-on-chip.

Cardiac valves are sophisticated, dynamic structures residing in a complex mechanical and hemodynamic environment. Cardiac valve disease is an active and progressive disease resulting in severe socioeconomic burden, especially in the elderly. Valve disease also leads to a 50% increase in the possibility of associated cardiovascular events. Yet, valve replacement remains the standard of treatment with early detection, mitigation, and alternate therapeutic strategies still lacking. Effective study models are required to further elucidate disease mechanisms and diagnostic and therapeutic strategies. Organ-on-chip models offer a unique and powerful environment that incorporates the ease and reproducibility of in vitro systems along with the complexity and physiological recapitulation of the in vivo system. The key to developing effective valve-on-chip models is maintaining the cell and tissue-level microenvironment relevant to the study application. This review outlines the various components and factors that comprise and/or affect the cell microenvironment that ought to be considered while constructing a valve-on-chip model. This review also dives into the advancements made toward constructing valve-on-chip models with a specific focus on the aortic valve, that is, in vitro studies incorporating three-dimensional co-culture models that incorporate relevant extracellular matrices and mechanical and hemodynamic cues.

Label-free optical biomarkers detect early calcific aortic valve disease in a wild-type mouse model.

BACKGROUND: Calcific aortic valve disease (CAVD) pathophysiology is a complex, multistage process, usually diagnosed at advanced stages after significant anatomical and hemodynamic changes in the valve. Early detection of disease progression is thus pivotal in the development of prevention and mitigation strategies. In this study, we developed a diet-based, non-genetically modified mouse model for early CAVD progression, and explored the utility of two-photon excited fluorescence (TPEF) microscopy for early detection of CAVD progression. TPEF imaging provides label-free, non-invasive, quantitative metrics with the potential to correlate with multiple stages of CAVD pathophysiology including calcium deposition, collagen remodeling and osteogenic differentiation. METHODS: Twenty-week old C57BL/6J mice were fed either a control or pro-calcific diet for 16 weeks and monitored via echocardiography, histology, immunohistochemistry, and quantitative polarized light imaging. Additionally, TPEF imaging was used to quantify tissue autofluorescence (A) at 755 nm, 810 nm and 860 nm excitation, to calculate TPEF 755-860 ratio (A860/525/(A755/460 + A860/525)) and TPEF Collagen-Calcium ratio (A810/525/(A810/460 + A810/525)) in the murine valves. In a separate experiment, animals were fed the above diets till 28 weeks to assess for later-stage calcification. RESULTS: Pro-calcific mice showed evidence of lipid deposition at 4 weeks and calcification at 16 weeks at the valve commissures. The valves of pro-calcific mice also showed positive expression for markers of osteogenic differentiation, myofibroblast activation, proliferation, inflammatory cytokines and collagen remodeling. Pro-calcific mice exhibited lower TPEF autofluorescence ratios, at locations coincident with calcification, that correlated with increased collagen disorganization and positive expression of osteogenic markers. Additionally, locations with lower TPEF autofluorescence ratios at 4 and 16 weeks exhibited increased calcification at later 28-week timepoints. CONCLUSIONS: This study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology.

Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression.

Calcific aortic valve disease (CAVD) is the most common form of valve disease where the only available treatment strategy is surgical valve replacement. Technologies for the early detection of CAVD would benefit the development of prevention, mitigation and alternate therapeutic strategies. Two-photon excited fluorescence (TPEF) microscopy is a label-free, non-destructive imaging technique that has been shown to correlate with multiple markers for cellular differentiation and phenotypic changes in cancer and wound healing. Here we show how specific TPEF markers, namely, the optical redox ratio and mitochondrial fractal dimension, correlate with structural, functional and phenotypic changes occurring in the aortic valve interstitial cells (VICs) during osteogenic differentiation. The optical redox ratio, and fractal dimension of mitochondria were assessed and correlated with gene expression and nuclear morphology of VICs. The optical redox ratio decreased for VICs during early osteogenic differentiation and correlated with biological markers for CAVD progression. Fractal dimension correlated with structural and osteogenic markers as well as measures of nuclear morphology. Our study suggests that TPEF imaging markers, specifically the optical redox ratio and mitochondrial fractal dimension, can be potentially used as a tool for assessing early CAVD progression in vitro.

Cancer Stem Cells Equipped with Powerful Hedgehog Signaling and Better Epigenetic Memory: Avenues to Look for Cancer Therapeutics.

Complex nature of the tumor is depicted at the cellular landscape by showing heterogeneity in the presence of cancer cells, cancer-associated stromal cells, mesenchymal stem cells and cancer stem cells (CSCs). One of the plausible views in cancer formation is suggested as the theory of cancer CSCs that is known as a source of initiation of tumorigenesis. In essence, these powerful CSCs are equipped with high Sonic Hedgehog (SHH) signaling and epigenetic memory power that support various tumor hallmarks. Truly, nature justifies its intent by limiting these stem cells with a potential to turn into CSCs and in turn suppressing the high risk of humans and other organisms. In short, this mini-review addresses the contribution of SHH signaling to allow reprogramming of epigenetic memory within CSCs that support tumor hallmarks. Besides, this paper explores therapeutic approaches to mitigate SHH signaling that may lead to a blockade of the pro-tumor potential of CSCs.

Extrachromosomal circular DNAs: an extra piece of evidence to depict tumor heterogeneity.

The tumor microenvironment (TME) comprises a heterogeneous number and type of cellular and noncellular components that vary in the context of molecular, genomic and epigenomic levels. The genotypic diversity and plasticity within cancer cells are known to be affected by genomic instability and genome alterations. Besides genomic instability within the chromosomal linear DNA, an extra factor appears in the form of extrachromosomal circular DNAs (eccDNAs; 2-20 kbp) and microDNAs (200-400 bp). This extra heterogeneity within cancer cells in the form of an abundance of eccDNAs adds another dimension to the expression of procancer players, such as oncoproteins, acting as a driver for cancer cell survival and proliferation. This article reviews research into eccDNAs centering around cancer plasticity and hallmarks, and discusses these facts in light of therapeutics and biomarker development.

The role of fibroblast growth factor 1 and 2 on the pathological behavior of valve interstitial cells in a three-dimensional mechanically-conditioned model.

BACKGROUND: More than five million Americans suffer from heart valve disease annually, a condition that worsens cardiac function and gradually leads to heart failure if appropriate treatment is not performed on time. Currently no medication can cure heart valve disease, leaving surgical intervention as the only viable option for patients at late stages of cardiac valve disease. Tremendous efforts have been undertaken to elucidate how resident cells in the valves respond to pathological stimulation as well as the underlying mechanisms that regulate these responses, to identify potential therapeutic targets for non-surgical treatment of valvular heart disease. RESULTS: Cardiac valve interstitial cells (VICs) naturally reside in a complex three-dimensional environment under varying hemodynamics, which is difficult to replicate in vitro. As a result, most cell signaling studies in the field have traditionally been conducted on two-dimensional models or in the absence of hemodynamic forces. Previously, we reported the fabrication of a hydrogel scaffold that could be used to culture valve cells under dynamic mechanical stimulation in a valve-mimetic environment. This model, therefore appeared to be suitable for VIC signaling studies as it provided cells a three-dimensional environment with the ability to incorporate mechanical stretching stimulation. Utilizing this model, we investigated the possible role of fibroblast growth factor 1 and 2 (FGF1 and FGF2) via FGFR1 receptor signaling in regulating valve cell activation under physiological (10% stretch) and pathological (20% stretch) mechanical conditions as well as in mediating cell proliferation and metabolism via the Akt/mTOR pathways. We reported that 1) FGF1 and FGF2 treatment was able to maintain the quiescent phenotype of VICs; 2) Cells increased proliferation as determined by optical redox ratios under elevated cyclic stretch via Akt/mTOR pathways; and 3) FGF1 and 2 signaling via the FGFR1 reduced VIC proliferation and activation under elevated cyclic stretch conditions. CONCLUSIONS: Overall, these results suggested that targeting FGFR1 receptor signaling may represent a possible therapeutic strategy for preventing heart valve disease progression.

Macrophage Flipping from Foe to Friend: A Matter of Interest in Breast Carcinoma Heterogeneity Driving Drug Resistance.

Tumor heterogeneity within various cancer types including breast carcinoma is pivotal in the manifestations of tumor hallmarks. Tumor heterogeneity is seen as a common landscape where intra-tumoral components including cellular and non-cellular factors create an interface with outside environment that leads to the unique identity of a specific cancer type. Among various contributors to tumor heterogeneity, cellular heterogeneity immensely plays a role in drug resistance and relapse of cancer. Within cellular heterogeneity of tumor, tumor-associated macrophages (TAMs) are the pro-tumor type of immune cells that promote growth, metastasis and drug resistance in breast carcinoma and other cancer types. Revealing the molecular aspects of TAMs can provide a breakthrough to remove therapeutics blockade to existing drugs and this understanding in future will pave the way for a new class of cancer immunotherapeutic. This review addresses current understanding of the role of TAMs in breast carcinoma hallmarks and clarifies the current scenario of pre-clinical drugs directed to tame pro-cancer TAMs.

Induction of Apoptotic Death and Cell Cycle Arrest in HeLa Cells by Extracellular Factors of Breast Cancer Cells

Background: There are evidences on the role of extracellular factors in cellular communication between cancer cells and non-cancerous cells to support tumor progression and a phenomenon of cancer cachexia. However, evidences are scarce to show the effects of extracellular factors from one carcinoma microenvironment upon growth and survival of another carcinoma. Methodology: To address the above issue, we have selected excised breast carcinoma tissue samples and in vitro grown MCF-7 sources of extracellular factors and tested their effects to evaluate growth and proliferation inhibitory potential against a cervical carcinoma cell line HeLa. Results: Data from the in vitro experiments like Trypan blue dye exclusion, MTT assay, cell cycle assay and annexin V/PI staining lead us to suggest that the extracellular factors collected from the culture medium of in vitro grown MCF-7 and excised breast carcinoma tissue play an apoptosis inducing and cell cycle arrest role in HeLa. In these in vitro experiments, we detected the presence of up to 40-50% apoptotic cell death in HeLa cells and increase in G2-M cell cycle phase from 11%-25% due to treatment with extracellular factors from human breast carcinoma cells. Discussion and Conclusion: These observations are novel and suggest that extracellular factors from breast carcinoma play an apoptosis inducing and growth inhibitory role upon on HeLa cells. This study can also support the concept of cancer cachexia and a possible hypothesis for rare chance of synchronous two or more primary tumor in a single patient.

Mutual concessions and compromises between stromal cells and cancer cells: driving tumor development and drug resistance.

BACKGROUND: Various cancers have been found to be associated with heterogeneous and adaptive tumor microenvironments (TMEs) and to be driven by the local TMEs in which they thrive. Cancer heterogeneity plays an important role in tumor cell survival, progression and drug resistance. The diverse cellular components of the TME may include cancer-associated fibroblasts, adipocytes, pericytes, mesenchymal stem cells, endothelial cells, lymphocytes and other immune cells. These components may support tumor development through the secretion of growth factors, evasion from immune checkpoints, metabolic adaptations, modulations of the extracellular matrix, activation of oncogenes and the acquisition of drug resistance. Here, we will address recent advances in our understanding of the molecular mechanisms underlying stromal-tumor cell interactions, with special emphasis on basic and pre-clinical information that may facilitate the design of novel personalized cancer therapies. CONCLUSIONS: This review presents a holistic view on the translational potential of the interplay between stromal cells and cancer cells. This interplay is currently being employed for the development of promising preclinical and clinical biomarkers, and the design of small molecule inhibitors, antibodies and small RNAs for (combinatorial) cancer treatment options. In addition, nano-carriers, tissue scaffolds and 3-D based matrices are being developed to precisely and safely deliver these compounds.

Elevated Serotonin Interacts with Angiotensin-II to Result in Altered Valve Interstitial Cell Contractility and Remodeling.

While the valvulopathic effects of serotonin (5HT) and angiotensin-II (Ang-II) individually are known, it was not clear how 5HT and Ang-II might interact, specifically in the context of the mechanobiological responses due to altered valve mechanics potentiated by these molecules. In this context, the hypothesis of this study was that increased serotonin levels would result in accelerated progression toward disease in the presence of angiotensin-II-induced hypertension. C57/BL6 J mice were divided into four groups and subcutaneously implanted with osmotic pumps containing: PBS (control), 5HT (2.5 ng/kg/min), Ang-II (400 ng/kg/min), and 5HT + Ang-II (combination). Blood pressure was monitored using the tail cuff method. Echocardiography was performed on the mice before surgery and every week thereafter to assess ejection fraction. After three weeks, the mice were sacrificed and their hearts excised, embedded and sectioned for analysis of the aortic valves via histology and immunohistochemistry. In separate experiments, porcine valve interstitial cells (VICs) were directly stimulated with 5HT (10-7 M), Ang-II (100 nM) or both and assayed for cellular contractility, cytoskeletal organization and collagen remodeling. After three weeks, average systolic blood pressure was significantly increased in the 5HT, Ang-II and combination groups compared to control. Echocardiographic analysis demonstrated significantly reduced ejection fraction in Ang-II and the combination groups. H&E staining demonstrated thicker leaflets in the combination groups, suggesting a more aggressive remodeling process. Picrosirius red staining and image analysis suggested that the Ang-II and combination groups had the largest proportion of thicker collagen fibers. VIC orientation, cellular contractility and collagen gene expression was highest for the 5HT + Ang-II combination treatment compared to all other groups. Overall, our results suggest that 5HT and Ang-II interact to result in significantly detrimental alteration of function and remodeling in the valve.

Valve interstitial cell shape modulates cell contractility independent of cell phenotype.

Valve interstitial cells are dispersed throughout the heart valve and play an important role in maintaining its integrity, function, and phenotype. While prior studies have detailed the role of external mechanical and biological factors in the function of the interstitial cell, the role of cell shape in regulating contractile function, in the context of normal and diseased phenotypes, is not well understood. Thus, the aim of this study was to elucidate the link between cell shape, phenotype, and acute functional contractile output. Valve interstitial cell monolayers with defined cellular shapes were engineered via constraining cells to micropatterned protein lines (10, 20, 40, 60 or 80µm wide). Samples were cultured in either normal or osteogenic medium. Cellular shape and architecture were quantified via fluorescent imaging techniques. Cellular contractility was quantified using a valve thin film assay and phenotype analyzed via western blotting, zymography, and qRT-PCR. In all pattern widths, cells were highly aligned, with maximum cell and nuclear elongation occurring for the 10µm pattern width. Cellular contractility was highest for the most elongated cells, but was also increased in cells on the widest pattern (80µm) that also had increased CX43 expression, suggesting a role for both elongated shape and increased cell-cell contact in regulating contractility. Cells cultured in osteogenic medium had greater expression of smooth muscle markers and correspondingly increased contractile stress responses. Cell phenotype did not significantly correlate with altered cell shape, suggesting that cellular shape plays a significant role in the regulation of valve contractile function independent of phenotype.

Fluorescent silk cocoon creating fluorescent diatom using a "Water glass-fluorophore ferry".

Fluorophores are ubiquitous in nature. Naturally occurring fluorophores are exceptionally stable and have high quantum yield. Several natural systems have acquired fluorescent signature due to the presence of these fluorophores. Systematic attempt to harvest these fluorophores from natural systems could reap rich commercial benefit to bio-imaging industry. Silk cocoon biomaterial is one such example of natural system, which has acquired a fluorescent signature. The objective of this study is to develop simple, rapid, commercially viable technique to isolate silk cocoon membrane fluorophores and exploring the possibility of using them as fluorescent dye in bio-imaging. Here, we report an innovative water glass (Na2SiO3) based strategy to isolate the silk cocoon fluorophores. Isolated fluorophore is majorly quercetin derivatives and exhibited remarkable photo- and heat stability. Fluorescence and mass spectrometric analysis confirmed presence of a quercetin derivative. We further used this fluorophore to successfully label the silicate shell of diatom species Nitzschia palea.