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The complex fibrillar architecture of native meniscus is essential for proper function and difficult to recapitulate in vitro. In the native meniscus, proteoglycan content is low during the development of collagen fibers and progressively increases with aging. In vitro, fibrochondrocytes produce glycosaminoglycans (GAGs) early in culture, in contrast to native tissue, where they are deposited after collagen fibers have formed. This difference in the timing of GAG production hinders the formation of a mature fiber network in such in vitro models. In this study, we removed GAGs from collagen gel-based tissue engineered constructs using chondroitinase ABC (cABC) and evaluated the effect on the formation and alignment of collagen fibers and the subsequent effect on tensile and compressive mechanical properties. Removal of GAGs during maturation of in vitro constructs improved collagen fiber alignment in tissue engineered meniscus constructs. Additionally, removal of GAGs during maturation improved fiber alignment without compromising compressive strength, and this removal improved not only fiber alignment and formation but also tensile properties. The increased fiber organization in cABC-treated groups also appeared to influence the size, shape, and location of defects in these constructs, suggesting that treatment may prevent the propagation of large defects under loading. This data gives another method of modulating the ECM for improved collagen fiber formation and mechanical properties in tissue engineered constructs.

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Over the past two decades there has been a steady trend towards the development of realistic models of cardiac conduction with increasing levels of detail. However, making models more realistic complicates their personalization and use in clinical practice due to limited availability of tissue and cellular scale data. One such limitation is obtaining information about myocardial fiber organization in the clinical setting. In this study, we investigated a chimeric model of the left atrium utilizing clinically derived patient-specific atrial geometry and a realistic, yet foreign for a given patient fiber organization. We discovered that even significant variability of fiber organization had a relatively small effect on the spatio-temporal activation pattern during regular pacing. For a given pacing site, the activation maps were very similar across all fiber organizations tested.

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OBJECTIVE: The aim of the study is to compare structure tensor imaging (STI) with diffusion tensor imaging (DTI) of the sheep heart (approximately the same size as the human heart). MATERIALS AND METHODS: MRI acquisition on three sheep ex vivo hearts was performed at 9.4 T/30 cm with a seven-element RF coil. 3D FLASH with an isotropic resolution of 150 µm and 3D spin-echo DTI at 600 µm were performed. Tensor analysis, angles extraction and segments divisions were performed on both volumes. RESULTS: A 3D FLASH allows for visualization of the detailed structure of the left and right ventricles. The helix angle determined using DTI and STI exhibited a smooth transmural change from the endocardium to the epicardium. Both the helix and transverse angles were similar between techniques. Sheetlet organization exhibited the same pattern in both acquisitions, but local angle differences were seen and identified in 17 segments representation. DISCUSSION: This study demonstrated the feasibility of high-resolution MRI for studying the myocyte and myolaminar architecture of sheep hearts. We presented the results of STI on three whole sheep ex vivo hearts and demonstrated a good correspondence between DTI and STI.

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Recapitulating the collagen fiber structure of native menisci is one of the major challenges in the development of tissue-engineered menisci. Native collagen fibers are developed by the complex interplay of biochemical and biomechanical signals. In this study, we optimized glucose and transforming growth factor-ß1 (TGF-ß1) concentrations in combination with mechanical anchoring to balance contributions of proteoglycan synthesis and contractile behavior in collagen fiber assembly. Glucose had a profound effect on the final dimensions of collagen-based constructs. TGF-ß1 influenced construct contraction rate and glycosaminoglycan (GAG) production with two half-maximal effective concentration (EC50) ranges, which are 0.23 to 0.28 and 0.53 to 1.71 ng/mL, respectively. At concentrations less than the EC50, for the GAG production and contraction rate, TGF-ß1 treatment resulted in less organized collagen fibers. At concentrations greater than the EC50, TGF-ß1 led to dense, disorganized collagen fibers. Between the two EC50 values, collagen fiber diameter and length increased. The effects of TGF-ß1 on fiber development were enhanced by mechanical anchoring, leading to peaks in fiber diameter, length, and alignment index. Fiber diameter and length increased from 7.9 ± 1.4 and 148.7 ± 16.4 to 17.5 ± 2.1 and 262.0 ± 13.0 µm, respectively. The alignment index reached 1.31, comparable to that of native tissue, 1.40. These enhancements in fiber architecture resulted in significant increases in tensile modulus and ultimate tensile stress (UTS) by 1.6- and 1.4-fold. Correlation analysis showed that tensile modulus and UTS strongly correlated with collagen fiber length, diameter, and alignment, while compressive modulus correlated with GAG content. These outcomes highlight the need for optimization of both biochemical and biomechanical cues in the culture environment for enhancing fiber development within tissue-engineered constructs.

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OBJECTIVES: To explore the pathophysiology of proliferative verrucous leukoplakia, a rare oral disorder that exhibits high rates of recurrence and malignant transformation, through a RNAseq case-control study. MATERIAL AND METHODS: We obtained oral biopsies from 10 patients with verrucous leukoplakia lesions and from the mucosa of 5 healthy individuals for sequencing using RNAseq technology. Using bioinformatic methods, we investigated gene expression and enrichment differences between patients both with and without the disorder. We applied network biology methods to investigate functional relations among those genes that were differentially deregulated. RESULTS: We detected 140 differentially expressed genes with distinct roles in immune surveillance, tissue and organ morphogenesis, development, and organization. Of these 140 genes, 111 have been previously described as cancer expression biomarkers, being oral squamous cell carcinoma the most represented type of cancer among them. Of these 140 genes, 26 were prioritized for further investigation as biomarkers using larger sample sizes. CONCLUSIONS: The gene expression patterns of healthy and unhealthy patients differed in 140 genes whose deregulation has a functional impact on normal functioning of the immune system. This immune expression profile provides a plausible hypothesis to explain the transformation to oral squamous cell carcinoma observed in 6 of the 10 assayed cases. CLINICAL RELEVANCE: By determining the molecular bases of the proliferative verrucous leukoplakia disorder and identifying early biomarkers of malignancy, this can allow us to develop new treatment strategies.

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New methods are described for converting 2D electrospun nanofiber membranes to 3D hierarchical assemblies with structural and compositional gradients. Pore-size gradients are generated by tuning the expansion of 2D membranes in different layers with incorporation of various amounts of a surfactant during the gas-foaming process. The gradient in fiber organizations is formed by expanding 2D nanofiber membranes composed of multiple regions collected by varying rotating speeds of mandrel. A compositional gradient on 3D assemblies consisting of radially aligned nanofibers is prepared by dripping, diffusion, and crosslinking. Bone mesenchymal stem cells (BMSCs) on the 3D nanofiber assemblies with smaller pore size show significantly higher expression of hypoxia-related markers and enhanced chondrogenic differentiation compared to BMSCs cultured on the assemblies with larger pore size. The basic fibroblast growth factor gradient can accelerate fibroblast migration from the surrounding area to the center in an in vitro wound healing model. Taken together, 3D nanofiber assemblies with gradients in pore sizes, fiber organizations, and contents of signaling molecules can be used to engineer tissue constructs for tissue repair and build biomimetic disease models for studying disease biology and screening drugs, in particular, for interface tissue engineering and modeling.

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Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets.

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OBJECTIVE: A focused dissection of the brainstem was performed to study the various fiber bundles and gray matter nuclei of the brainstem and to decipher the intricate anatomy. METHODS: Ten formalin-fixed cadaveric human brains were procured. The fiber dissection techniques described by Klingler were then adopted to study the anatomy. The primary dissection tools used were thin handmade wooden spatulas and fine jeweler's forceps. The fibers were dissected layer by layer with the use of magnification. RESULTS: The brainstem dissection was performed from the dorsal, ventral, and lateral aspects to provide a 3-dimensional perspective of the internal architecture. We have presented a novel classification of the brainstem white matter in accordance with the internal organization. The fiber tracts of the brainstem can be divided into short projection fibers, long projection fibers, traversing fibers, and association fibers. From our dissection findings, we divided these white fibers of the brainstem into 3 zones from laterally to medially. The first or outermost zone consists of short projection fibers connecting the brainstem to the cerebellum and adjacent thalamic nuclei. The second zone or the middle zone lies medial to the first zone and consists of traversing and long projection fibers. These consist of both ascending and descending fibers. The third zone or the innermost zone consists of the brainstem association fibers. CONCLUSIONS: This division of the fiber bundles into zones will help neurosurgeons in understanding the course and anatomy of the fibers, which can be cumbersome to remember when only studying the sectional anatomy of the brainstem.

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Reduced fractional anisotropy (FA) is a well-established correlate of schizophrenia, but it remains unclear whether these tensor-based differences are the result of axon damage and/or organizational changes and whether the changes are progressive in the adult course of illness. Diffusion MRI data were collected in 81 schizophrenia patients (54 first episode and 27 chronic) and 64 controls. Analysis of FA was combined with "fixel-based" analysis, the latter of which leverages connectivity and crossing-fiber information to assess both fiber bundle density and organizational complexity (i.e., presence and magnitude of off-axis diffusion signal). Compared with controls, patients with schizophrenia displayed clusters of significantly lower FA in the bilateral frontal lobes, right dorsal centrum semiovale, and the left anterior limb of the internal capsule. All FA-based group differences overlapped substantially with regions containing complex fiber architecture. FA within these clusters was positively correlated with principal axis fiber density, but inversely correlated with both secondary/tertiary axis fiber density and voxel-wise fiber complexity. Crossing fiber complexity had the strongest (inverse) association with FA (r = -0.82). When crossing fiber structure was modeled in the MRtrix fixel-based analysis pipeline, patients exhibited significantly lower fiber density compared to controls in the dorsal and posterior corpus callosum (central, postcentral, and forceps major). Findings of lower FA in patients with schizophrenia likely reflect two inversely related signals: reduced density of principal axis fiber tracts and increased off-axis diffusion sources. Whereas the former confirms at least some regions where myelin and or/axon count are lower in schizophrenia, the latter indicates that the FA signal from principal axis fiber coherence is broadly contaminated by macrostructural complexity, and therefore does not necessarily reflect microstructural group differences. These results underline the need to move beyond tensor-based models in favor of acquisition and analysis techniques that can help disambiguate different sources of white matter disruptions associated with schizophrenia.

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The fiber arrangement in the pars tensa of the rat tympanic membrane (TM) was observed using a high resolution scanning electron microscope. The entire pars tensa is composed of fibrils with diameter of approximately 25 nm. These fibrils can be grouped into radial, circular, parabolic, and oblique fibers as reported in other mammals. The radial fibrils interweave into a planar form rather than into discrete cylindrical fibers. Before attaching to the manubrium and tympanic ring, the radial fibrils bend and cross neighboring fibrils to form a random fibril network, and change their direction from perpendicular to somewhat parallel to the manubrium and tympanic ring. The circular fibrils form cylindrical fibers near the peripheral part of the TM while closer to the manubrium, they form planar bundles. The observed fiber morphology and arrangement may provide helpful information in improving numerical models for the TM's acoustical response and designing a fibrous graft for the repair of TM perforations. Anat Rec, 299:1531-1539, 2016. © 2016 Wiley Periodicals, Inc.

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Injuries and damage to tendons plague both human and equine athletes. At the site of injuries, various cells congregate to repair and re-structure the collagen. Treatments for collagen injury range from simple procedures such as icing and pharmaceutical treatments to more complex surgeries and the implantation of stem cells. Regardless of the treatment, the level of mechanical stimulation incurred by the recovering tendon is crucial. However, for a given tendon injury, it is not known precisely how much of a load should be applied for an effective recovery. Both too much and too little loading of the tendon could be detrimental during recovery. A mapping of the complex local environment imparted to any cell present at the site of a tendon injury may however, convey fundamental insights related to their decision making as a function of applied load. Therefore, fundamentally knowing how cells translate mechanical cues from their external environment into signals regulating their functions during repair is crucial to more effectively treat these types of injuries. In this paper, we studied systems of tendons with a variety of 2-photon-based imaging techniques to examine the local mechanical environment of cells in both normal and injured tendons. These tendons were chemically treated to instigate various extents of injury and in some cases, were injected with stem cells. The results related by each imaging technique distinguish with high contrast and resolution multiple morphologies of the cells' nuclei and the alignment of the collagen during injury. The incorporation of 2-photon FLIM into this study probed new features in the local environment of the nuclei that were not apparent with steady-state imaging. Overall, this paper focuses on horse tendon injury pattern and analysis with different 2-photon confocal modalities useful for wide variety of application in damaged tissues.

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Although olfaction in birds is known to be involved in a variety of behaviors, there is comparatively little detailed information on the olfactory brain. In the pigeon brain, the olfactory bulb (OB) is known to project to the prepiriform cortex (CPP), piriform cortex (CPi), and dorsolateral corticoid area (CDL), which together are called the olfactory pallium, but centrifugal pathways to the OB have not been fully explored. Fiber connections of CPi and CDL have been reported, but those of other olfactory pallial nuclei remain unknown. The present study examines the fiber connections of OB and CPP in pigeons to provide a more detailed picture of their connections using tract-tracing methods. When anterograde and retrograde tracers were injected in OB, projections to a more extensive olfactory pallium were revealed, including the anterior olfactory nucleus, CPP, densocellular part of the hyperpallium, tenia tecta, hippocampal continuation, CPi, and CDL. OB projected commissural fibers to the contralateral OB but did not receive afferents from the contralateral olfactory pallium. When tracers were injected in CPP, reciprocal ipsilateral connections with OB and nuclei of the olfactory pallium were observed, and CPP projected to the caudolateral nidopallium and the limbic system, including the hippocampal formation, septum, lateral hypothalamic nucleus, and lateral mammillary nucleus. These results show that the connections of OB have a wider distribution throughout the olfactory pallium than previously thought and that CPP provides a centrifugal projection to the OB and acts as a relay station to the limbic system.

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PURPOSE: Recent investigations of human corneal structure and biomechanics have shown that stromal collagen fibers (lamellae) are organized into a complex, highly intertwined three-dimensional meshwork of transverse oriented fibers that increases stromal stiffness and controls corneal shape. The purpose of this study was to characterize the three-dimensional distribution of transverse collagen fibers along the major meridians of the cornea using an automated method to rapidly quantify the collagen fibers' angular orientation. METHODS: Three eyes from three donors were perfusion-fixed under pressure, excised, and cut into four quadrants. Quadrants were physically sectioned using a vibratome and scanned using nonlinear optical high-resolution macroscopy. Planes were analyzed numerically using software to identify collagen fiber angles relative to the corneal surface, stromal depth, and radial position within the anterior 250 µm of the stroma. RESULTS: The range of fiber angles and the fiber percentage having an angular displacement greater than ±3.5° relative to the corneal surface ("transverse fibers") was highest in the anterior stroma and decreased with depth. Numerical analysis showed no significant differences in fiber angles and transverse fibers between quadrants, meridians, and radial position. CONCLUSIONS: These results match our previous observation of a depth-dependent gradient in stromal collagen interconnectivity in the central cornea, and show that this gradient extends from the central cornea to the limbus. The lack of a preferred distribution of angled fibers with regard to corneal quadrant or radial position likely serves to evenly distribute loads and to avoid the formation of areas of stress concentration.

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