Functional Analysis of the Cortical Transcriptome and Proteome Reveal Neurogenesis, Inflammation, and Cell Death after Repeated Traumatic Brain Injury In vivo.

The pathological effects of repeated traumatic brain injuries (TBIs) are largely unknown. To gain a detailed understanding of the cortical tissue acute biological response after one or two TBIs, we utilized RNA-sequencing and protein mass spectrometry techniques. Using our previously validated C57Bl/6 weight-drop model, we administered one or two TBIs of a mild or moderate severity. Double injury conditions were spaced 7 days apart, and cortical tissue was isolated 24 h after final injury. Analysis was carried out through functional gene annotation, utilizing Gene Ontology, for both the proteome and transcriptome. Major themes across the four different conditions include: neurogenesis; inflammation and immune response; cell death; angiogenesis; protein modification; and cell communication. Proteins associated with neurogenesis were found to be upregulated after single injuries. Transcripts associated with angiogenesis were upregulated in the moderate single, mild double, and moderate double TBI conditions. Genes associated with inflammation and immune response were upregulated in every condition, with the moderate single condition reporting the most functional groups. Proteins or genes involved in cell death, or apoptosis, were upregulated in every condition. Our results emphasize the significant differences found in proteomic and transcriptomic changes in single versus double injuries. Further, cortical omics analysis offers important insights for future studies aiming to deepen current knowledge on the development of secondary injuries and neurobehavioral impairments after brain trauma.

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.

Blood-Brain Barrier Breakdown and Astrocyte Reactivity Evident in the Absence of Behavioral Changes after Repeated Traumatic Brain Injury.

Repeated traumatic brain injuries (TBIs) cause debilitating effects. Without understanding the acute effects of repeated TBIs, treatment options to halt further degeneration and damage cannot be developed. This study sought to examine the acute effects of blood-brain barrier (BBB) dysfunction, edema, inflammation and behavioral changes after either a single or double TBI using a C57BL/6 mouse model. We examined the effects of one or two TBIs, of either a mild or moderate severity. Double injuries were spaced 7 days apart, and all analysis was performed 24 h post-injury. To examine edema and inflammation, protein levels of glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B, interleukin-6, and matrix metallopeptidase 9 (MMP9) were analyzed. Aquaporin-4 (AQP4) and zonula occludens-1 (ZO-1) were analyzed to observe BBB dysfunction. Ionized calcium-binding adapter molecule 1 (IBA1) was analyzed to observe microglial activation. Rotarod, beam walking, and grip strength tests were used to measure changes in physical behavior post-injury. A sample size of ≥5 was used for all analysis. Double injuries led to an increase in BBB breakdown, as indicated by altered MMP-9, AQP4, and ZO-1 protein expression. Single injuries showed an increase in microglial activation, astrocyte activation, and BBB breakdown. Behavioral tasks showed no significant differences between injured and control groups. Based on our findings, we suggest that behavioral studies should not be used as the sole clinical indicator on brain tissue recovery. Analysis of markers such as IBA1, GFAP, MMP-9, AQP4, and ZO-1 provide valuable insight on pathophysiological response to injury.

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.

Anisotropic Fiber-Reinforced Glycosaminoglycan Hydrogels for Heart Valve Tissue Engineering.

This study investigates polymer fiber-reinforced protein-polysaccharide-based hydrogels for heart valve tissue engineering applications. Polycaprolactone and gelatin (3:1) blends were jet-spun to fabricate aligned fibers that possessed fiber diameters in the range found in the native heart valve. These fibers were embedded in methacrylated hydrogels made from gelatin, sodium hyaluronate, and chondroitin sulfate to create fiber-reinforced hydrogel composites (HCs). The fiber-reinforced gelatin glycosaminoglycan (GAG)-based HC possessed interconnected porous structures and porosity higher than fiber-only conditions. These fiber-reinforced HCs exhibited compressive modulus and biaxial mechanical behavior comparable to that of native porcine aortic valves. The fiber-reinforced HCs were able to swell higher and degraded less than the hydrogels. Elution studies revealed that less than 20% of incorporated gelatin methacrylate and GAGs were released over 2 weeks, with a steady-state release after the first day. When cultured with porcine valve interstitial cells (VICs), the fiber-reinforced composites were able to maintain higher cell viability compared with fiber-only samples. Quiescent VICs expressed alpha smooth muscle actin and calponin showing an activated phenotype, along with a few cells expressing the proliferation marker Ki67 and negative expression for RUNX2, an osteogenic marker. Our study demonstrated that compared with the hydrogels and fibers alone, combining both components can yield durable, reinforced composites that mimic heart valve mechanical behavior, while maintaining high cell viability and expressing positive activation as well as proliferation markers.

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.

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.

Repeated In Vitro Impact Conditioning of Astrocytes Decreases the Expression and Accumulation of Extracellular Matrix.

Pathological changes to the physical and chemical properties of brain extracellular matrix (ECM) occur following injury. It is generally assumed that astrocytes play an important role in these changes. What remain unclear are the triggers that lead to changes in the regulation of ECM by astrocytes following injury. We hypothesize that mechanical stimulation triggers genotypic and phenotypic changes to astrocytes that could ultimately reshape the ECM composition of the central nervous system following injury. To explore astrocyte mechanobiology, an in vitro drop test bioreactor was employed to condition primary rat astrocytes using short duration (10 ms), high deceleration (150G) and strain (20%) impact stimuli. Experiments were designed to explore the effect of single and repeated impact (single vs. double) on mechano-sensitive behavior including cell viability; ECM gene (collagens I and IV, fibronectin, neurocan, versican) and reactivity gene [glial fibrillary acidic protein (GFAP), S100B, vimentin] expression; matrix regulatory cytokine secretion [matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 1 (TIMP1), transforming growth factor beta 1 (TGFß1)]; and matrix accumulation [collagen and glycosaminoglycan (GAG)]. Experiments revealed that both ECM and reactive gliosis gene expression was significantly decreased in response to impact conditioning. The decreases for several genes, including collagen, versican, and GFAP were sensitive to impact number, suggesting mechano-sensitivity to repeated impact conditioning. The measured decreases in ECM gene expression were supported by longer-term in vitro experiments that demonstrated significant decreases in chondroitin sulfate proteoglycan (CSPG) and collagen accumulation within impact conditioned 3-D scaffolds accompanied by a 25% decrease in elastic modulus. Overall, the general trend across all samples was towards altered ECM and reactive gliosis gene expression in response to impact. These results suggest that the regulation of ECM production by astrocytes is sensitive to mechanical stimuli, and that repeated impact conditioning may increase this sensitivity.

Characterization of uniaxial high-speed stretch as an in vitro model of mild traumatic brain injury on the blood-brain barrier.

Traumatic brain injury (TBI) occurs when external mechanical forces induce brain damage as result of impact, penetration or rapid acceleration/deceleration that causes deformation of brain tissue. Depending on its severity, TBI can be classified as mild, moderate or severe and can lead to blood-brain barrier (BBB) dysfunction. In the present study, we evaluated the effects of uniaxial high-speed stretch (HSS) at 0, 5, 10 and 15% on a pure culture of primary rat brain endothelial cells as an in vitro model of TBI to the BBB. LDH release, viability and apoptosis analysis, expression of tight junction proteins and endothelial permeability were evaluated 24 h after a single stretch episode. HSS slightly increased cell death and apoptosis at 10 and 15%, while LDH release was increased only at 15% stretch. Occludin expression was increased at 10% stretch, while claudin-5 expression was increased at 5% stretch, which also decreased the endothelial permeability. In summary, 15% HSS induced low levels of cell death, consistent with mild TBI and very low percentages of HSS (5%) enhanced the BBB properties, promoting the formation of a stronger barrier. These data support the use of 15% HSS as valuable tool in the study of mild TBI to the BBB in vitro.

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.

Characterization of Biaxial Stretch as an In Vitro Model of Traumatic Brain Injury to the Blood-Brain Barrier.

Traumatic brain injury (TBI) is one of the major causes of disability in the USA. It occurs when external mechanical forces induce brain damage that causes deformation of brain tissue. TBI is also associated with alterations of the blood-brain barrier (BBB). Using primary rat brain microvascular endothelial cells as an in vitro BBB model, the effects of biaxial stretch were characterized at 5, 10, 15, 25, and 50% deformation using a commercially available system. The results were compared to the effects of mild and moderate TBI in vivo, induced by the weight-drop method in mice. In vitro, live/dead cells, lactate dehydrogenase (LDH) release, caspase 3/7 staining, and tight junction (TJ) protein expression were evaluated 24 h after a single stretch episode. In vivo, Evans blue extravasation, serum levels of S100ß, and TJ protein expression were evaluated. Stretch induced a deformation-dependent increase in LDH release, cell death, and activation of caspase 3/7, suggesting the induction of apoptosis. Interestingly, low magnitudes of deformation increased the expression of TJ proteins, likely in an attempt to compensate for stretch damage. High magnitudes of deformation decreased the expression of TJ proteins, suggesting that the damage was too severe to counteract. In vivo, mild TBI did not affect BBB permeability or the expression of TJ proteins. However, moderate TBI significantly increased BBB permeability and decreased the expression of these proteins, similar to the results obtained with a high magnitude deformation. These data support the use biaxial stretch as valuable tool in the study of TBI in vitro.

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

Fabrication of a matrigel-collagen semi-interpenetrating scaffold for use in dynamic valve interstitial cell culture.

The study of heart valve homeostatic and disease mechanisms are often limited by the challenges in simulating the in vivo milieu, where valve cells are surrounded by the extracellular matrix in a three-dimensional (3D) environment and experience multiple dynamic mechanical forces. Type I collagen is typically the most common 3D matrix used to culture valve cells in vitro. Unfortunately, this material has poor mechanical behavior due to an inherent propensity to compact significantly, unlike native valve leaflets. We hypothesized that incorporation of matrigel, which contains other heart valve-relevant matrix components such as type IV collagen and sulfated proteoglycans, to type I collagen would provide an appropriate physiological milieu for in vitro valve interstitial cell culture. Different semi-interpenetrating mixtures of collagen type I and matrigel were prepared and a thorough characterization of their physical, mechanical and biocompatibility properties was performed. We observed that the matrigel-collagen hydrogel was porous and degradable with tunable swelling behavior. Incorporation of matrigel not only enhanced the mechanical behavior of the composite hydrogel but also presented the cultured valve interstitial cells with a more enriched extracellular matrix network for in vitro culture. We showed that cells cultured in the composite hydrogel had comparable viability, proliferation and cell phenotype as compared with those in a collagen only gel. Importantly, the composite hydrogel was also amenable to in vitro cyclic stretching culture for 48 h. Overall, we report here the potential use of the matrigel-collagen hydrogel as a three dimensional scaffold for the dynamic mechanical culture of valve interstitial cells in vitro.

Engineering anisotropic biphasic Janus-type polymer nanofiber scaffold networks via centrifugal jet spinning.

Biphasic materials, comprised of an ordered arrangement of two different material phases within a material, have the potential for a wide variety of applications including filtration, protective clothing and tissue engineering. This study reports for the first time, a process for engineering biphasic Janus-type polymeric nanofiber (BJPNF) networks via the centrifugal jet spinning technique. BJPNF alignment and fiber diameter was dependent on fabrication rotational speed as well as solution composition. The biphasic character of these BJPNFs, which was controlled via the rotational speed of fabrication, was confirmed at the individual nanofiber scale using energy dispersive X-ray spectroscopy, and at the bulk, macro-scale using attenuated total reflectance-Fourier transform infrared spectroscopy. Biphasic character was also demonstrated at the functional level via differing affinities on either side of the BJPNF for cell attachment. Our work thus presents a method for fabricating BJPNF scaffold networks where there might be a need for different properties on either side of a material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2455-2464, 2017.

Valve interstitial cell contractile strength and metabolic state are dependent on its shape.

The role of valvular interstitial cell (VIC) architecture in regulating cardiac valve function and pathology is not well understood. VICs are known to be more elongated in a hypertensive environment compared to those in a normotensive environment. We have previously reported that valve tissues cultured under hypertensive conditions are prone to acute pathological alterations in cell phenotype and contractility. We therefore aimed to rigorously study the relationship between VIC shape, contractile output and other functional indicators of VIC pathology. We developed an in vitro model to engineer VICs to take on the same shapes as those seen in normal and hypertensive conditions. VICs with longer cellular and nuclear shapes, as seen in hypertensive conditions, had greater contractile response to endothelin-1 that correlated with increased anisotropy of the actin architecture. These elongated VICs also demonstrated altered cell metabolism through a decreased optical redox ratio, which coincided with increased cellular proliferation. In the presence of actin polymerization inhibitor, however, these functional responses were significantly reduced, suggesting the important role of cytoskeletal actin organization in regulating cellular responses to abnormal shape. Overall, these results demonstrate the relationship between cell shape, cytoskeletal and nuclear organization, with functional output including contractility, metabolism, and proliferation.

Acetazolamide Mitigates Astrocyte Cellular Edema Following Mild Traumatic Brain Injury.

Non-penetrating or mild traumatic brain injury (mTBI) is commonly experienced in accidents, the battlefield and in full-contact sports. Astrocyte cellular edema is one of the major factors that leads to high morbidity post-mTBI. Various studies have reported an upregulation of aquaporin-4 (AQP4), a water channel protein, following brain injury. AZA is an antiepileptic drug that has been shown to inhibit AQP4 expression and in this study we investigate the drug as a therapeutic to mitigate the extent of mTBI induced cellular edema. We hypothesized that mTBI-mediated astrocyte dysfunction, initiated by increased intracellular volume, could be reduced when treated with AZA. We tested our hypothesis in a three-dimensional in vitro astrocyte model of mTBI. Samples were subject to no stretch (control) or one high-speed stretch (mTBI) injury. AQP4 expression was significantly increased 24 hours after mTBI. mTBI resulted in a significant increase in the cell swelling within 30 min of mTBI, which was significantly reduced in the presence of AZA. Cell death and expression of S100B was significantly reduced when AZA was added shortly before mTBI stretch. Overall, our data point to occurrence of astrocyte swelling immediately following mTBI, and AZA as a promising treatment to mitigate downstream cellular mortality.

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.