Organization Of Fibers Research Articles

An Image-Analysis-Based Method for the Prediction of Recombinant Protein Fiber Tensile Strength.

Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties. The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers. Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features. However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process. Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers. In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength. Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers.

Rapid Prototypable Biomimetic Peristalsis Bioreactor Capable of Concurrent Shear and Multi-Axial Strain

Peristalsis is a nuanced mechanical stimulus comprised of multi-axial strain (radial and axial strain) and shear stress. Forces associated with peristalsis regulate diverse biological functions including digestion, reproductive function, and urine dynamics. Given the central role peristalsis plays in physiology and pathophysiology, we were motivated to design a bioreactor capable of holistically mimicking peristalsis. We engineered a novel rotating screw-drive based design combined with a peristaltic pump, in order to deliver multi-axial strain and concurrent shear stress to a biocompatible polydimethylsiloxane (PDMS) membrane “wall.” Radial indentation and rotation of the screw drive against the wall demonstrated multi-axial strain evaluated via finite element modeling. Experimental measurements of strain using piezoelectric strain resistors were in close alignment with model-predicted values (15.9 ± 4.2% vs. 15.2% predicted). Modeling of shear stress on the “wall” indicated a uniform velocity profile and a moderate shear stress of 0.4 Pa. Human mesenchymal stem cells (hMSCs) seeded on the PDMS “wall” and stimulated with peristalsis demonstrated dramatic changes in actin filament alignment, proliferation, and nuclear morphology compared to static controls, perfusion, or strain, indicating that hMSCs sensed and responded to peristalsis uniquely. Lastly, significant differences were observed in gene expression patterns of calponin, caldesmon, smooth muscle actin, and transgelin, corroborating the propensity of hMSCs toward myogenic differentiation in response to peristalsis. Collectively, our data suggest that the peristalsis bioreactor is capable of generating concurrent multi-axial strain and shear stress on a “wall.” hMSCs experience peristalsis differently than perfusion or strain, resulting in changes in proliferation, actin fiber organization, smooth muscle actin expression, and genetic markers of differentiation. The peristalsis bioreactor device has broad utility in the study of development and disease in several organ systems.

Effect of electrophysical resources on healing of neurotendinous injury in an experimental model of type I diabetes and kidney disease.

ABSTRACTPurpose:To evaluate and describe the effect of electrophysical resources laser therapy (LLLT), intravascular laser blood irradiation (ILIB), and cryotherapy on the healing process of neurotendinous injury, as well as possible systemic changes, in the experimental model of type 1 diabetes associated with kidney injury.Methods:The animals were randomized into four groups: G1) healthy control with untreated injury; G2) healthy control with injury and treatment; G3) disease control with untreated lesion; G4) disease with injury and treatment. Furthermore, the treated groups were divided into three, according to the type of treatment. All animals were induced to neurotendinous injury and treated according to the therapeutic protocols. Healing and inflammation were analyzed by semiquantitative histopathological study.Results:It was observed in sick animals treated with cryotherapy and ILIB reduction of inflammatory exudate, presence of fibroblasts and organization of collagen, when compared to the effects of LLLT. Moreover, there was reduction in glycemic levels in the group treated with ILIB.Conclusions:Cryotherapy promoted reduction in inflammatory exudate and organization of collagen fibers, in addition to the absence of signs of tissue necrosis, in the groups treated with and without the disease. ILIB therapy showed the same findings associated with significant reduction in glycemic levels in the group of diseased animals. The application of LLLT showed increased inflammatory exudate, low organization of collagen fibers and low sign of tissue degeneration and necrosis. This study in a model of associated diseases (diabetes and kidney disease) whose effects of electrophysical resources studied after neurotendinous injury allows us to verify histopathological variables suggestive of patients with the same comorbidities.

Universal Convolutional Neural Network for Histology-Independent Analysis of Collagen Fiber Organization in Scar Tissue

Histological examination of collagen fiber organization is essential for pathologists to observe the wound healing process. A convolutional neural network (CNN) can be utilized to visually analyze collagen fibers during tissue remodeling in histology images. In this study, a universal CNN (UCNN) independent of the histological staining process is proposed to classify the histology images of burn-induced scar tissues and characterize collagen fiber organization. Normal and scar tissues obtained from an in vivo rodent model are stained using Masson’s Trichrome (MT) and Hematoxylin & Eosin (H&E). The proposed universal model is trained using both MT- and H&E-stained histological image datasets over multiple scales with color augmentation, and classification accuracies of up to 98% and 97% are achieved for the MT- and H&E-stained image datasets, respectively. Regardless of the histological staining process used, the collagen characteristics are visualized by determining the density and directional variance of the normal and scar tissues by using the features extracted with the proposed universal model. Statistical analysis results demonstrated clear differences between scar and normal tissues in terms of collagen fiber organization. The proposed UCNN model can contribute to the development of an intelligent and efficient method that pathologists can use to rapidly evaluate wound healing and tissue remodeling.

Is essential tremor a disorder of GABA dysfunction? No.

Although essential tremor is common, its underlying pathophysiology remains uncertain, and several hypotheses seek to explain the tremor mechanism. The GABA hypothesis states that disinhibition of deep cerebellar neurons due to reduced GABAergic input from Purkinje cells results in increased pacemaker activity, leading to rhythmic output to the thalamo-cortical circuit and resulting in tremor. However, some neuroimaging, spectroscopy, and pathology studies have not shown a clear or consistent GABA deficiency in essential tremor, and animal models have indicated that large reductions of Purkinje cell inhibition may improve tremor. Instead, tremor is increasingly attributable to dysfunction in oscillating networks, where altered (but not necessarily reduced) inhibitory signaling can result in tremor. Hypersynchrony of Purkinje cell activity may account for excessive oscillatory cerebellar output, with potential contributions along multiple sites of the olivocerebellar loop. Although older animal tremor models, such as harmaline tremor, have explored contributions from the inferior olivary body, increasing evidence has pointed to the role of aberrant climbing fiber synaptic organization in oscillatory cerebellar activity and tremor generation. New animal models such as hotfoot17j mice, which exhibit abnormal climbing fiber organization due to mutations in Grid2, have recapitulated many features of ET. Similar abnormal climbing fiber architecture and excessive cerebellar oscillations as measured by EEG have been found in humans with essential tremor. Further understanding of hypersynchrony and excessive oscillatory activity in ET phenotypes may lead to more targeted and effective treatment options.

Morphological signatures of actin organization in single cells accurately classify genetic perturbations using CNNs with transfer learning.

The actin cytoskeleton plays essential roles in countless cell processes, from cell division to migration to signaling. In cancer cells, cytoskeletal dynamics, cytoskeletal filament organization, and overall cell morphology are known to be altered substantially. We hypothesize that actin fiber organization and cell shape may carry specific signatures of genetic or signaling perturbations. We used convolutional neural networks (CNNs) on a small fluorescence microscopy image dataset of retinal pigment epithelial (RPE) cells and triple-negative breast cancer (TNBC) cells for identifying morphological signatures in cancer cells. Using a transfer learning approach, CNNs could be trained to accurately distinguish between normal and oncogenically transformed RPE cells with an accuracy of about 95% or better at the single cell level. Furthermore, CNNs could distinguish transformed cell lines differing by an oncogenic mutation from each other and could also detect knockdown of cofilin in TNBC cells, indicating that each single oncogenic mutation or cytoskeletal perturbation produces a unique signature in actin morphology. Application of the Local Interpretable Model-Agnostic Explanations (LIME) method for visually interpreting the CNN results revealed features of the global actin structure relevant for some cells and classification tasks. Interestingly, many of these features were supported by previous biological observation. Actin fiber organization is thus a sensitive marker for cell identity, and identification of its perturbations could be very useful for assaying cell phenotypes, including disease states.

Multiscale Computational Model Predicts Mouse Skin Kinematics Under Tensile Loading.

Skin is a complex tissue whose biomechanical properties are generally understood in terms of an incompressible material whose microstructure undergoes affine deformations. A growing number of experiments, however, have demonstrated that skin has a high Poisson's ratio, substantially decreases in volume during uniaxial tensile loading, and demonstrates collagen fiber kinematics that are not affine with local deformation. In order to better understand the mechanical basis for these properties, we constructed multiscale mechanical models (MSM) of mouse skin based on microstructural multiphoton microscopy imaging of the dermal microstructure acquired during mechanical testing. Three models that spanned the cases of highly aligned, moderately aligned, and nearly random fiber networks were examined and compared to the data acquired from uniaxially stretched skin. Our results demonstrate that MSMs consisting of networks of matched fiber organization can predict the biomechanical behavior of mouse skin, including the large decrease in tissue volume and nonaffine fiber kinematics observed under uniaxial tension.

Pigmentation Affects Elastic Fiber Patterning and Biomechanical Behavior of the Murine Aortic Valve.

The aortic valve (AoV) maintains unidirectional blood distribution from the left ventricle of the heart to the aorta for systemic circulation. The AoV leaflets rely on a precise extracellular matrix microarchitecture of collagen, elastin, and proteoglycans for appropriate biomechanical performance. We have previously demonstrated a relationship between the presence of pigment in the mouse AoV with elastic fiber patterning using multiphoton imaging. Here, we extended those findings using wholemount confocal microscopy revealing that elastic fibers were diminished in the AoV of hypopigmented mice (KitWv and albino) and were disorganized in the AoV of K5-Edn3 transgenic hyperpigmented mice when compared to wild type C57BL/6J mice. We further used atomic force microscopy to measure stiffness differences in the wholemount AoV leaflets of mice with different levels of pigmentation. We show that AoV leaflets of K5-Edn3 had overall higher stiffness (4.42 ± 0.35 kPa) when compared to those from KitWv (2.22 ± 0.21 kPa), albino (2.45 ± 0.16 kPa), and C57BL/6J (3.0 ± 0.16 kPa) mice. Despite the striking elastic fiber phenotype and noted stiffness differences, adult mutant mice were found to have no overt cardiac differences as measured by echocardiography. Our results indicate that pigmentation, but not melanocytes, is required for proper elastic fiber organization in the mouse AoV and dictates its biomechanical properties.

Nanoimprinting for high-throughput replication of geometrically precise pillars in fused silica to regulate cell behavior

Developing high-throughput nanopatterning techniques that also allow for precise control over the dimensions of the fabricated features is essential for the study of cell-nanopattern interactions. Here, we developed a process that fulfills both of these criteria. Firstly, we used electron-beam lithography (EBL) to fabricate precisely controlled arrays of submicron pillars with varying values of interspacing on a large area of fused silica. Two types of etching procedures with two different systems were developed to etch the fused silica and create the final desired height. We then studied the interactions of preosteoblasts (MC3T3-E1) with these pillars. Varying interspacing was observed to significantly affect the morphological characteristics of the cell, the organization of actin fibers, and the formation of focal adhesions. The expression of osteopontin (OPN) significantly increased on the patterns, indicating the potential of the pillars for inducing osteogenic differentiation. The EBL pillars were thereafter used as master molds in two subsequent processing steps, namely soft lithography and thermal nanoimprint lithography for high-fidelity replication of the pillars on the substrates of interest. The molding parameters were optimized to maximize the fidelity of the generated patterns and minimize the wear and tear of the master mold. Comparing the replicated feature with those present on the original mold confirmed that the geometry and dimensions of the replicated pillars closely resemble those of the original ones. The method proposed in this study, therefore, enables the precise fabrication of submicron- and nanopatterns on a wide variety of materials that are relevant for systematic cell studies. Statement of significanceSubmicron pillars with specific dimensions on the bone implants have been proven to be effective in controlling cell behaviors. Nowadays, numerous methods have been proposed to produce bio-instructive submicron-topographies. However, most of these techniques are suffering from being low-throughput, low-precision, and expensive. Here, we developed a high-throughput nanopatterning technique that allows for control over the dimensions of the features for the study of cell-nanotopography interactions. Assessing the adaptation of preosteoblast cells showed the potential of the pillars for inducing osteogenic differentiation. Afterward, the pillars were used for high-fidelity replication of the bio-instructive features on the substrates of interest. The results show the advantages of nanoimprint lithography as a unique technique for the patterning of large areas of bio-instructive surfaces.

A collagen I derived matricryptin increases aorta vascular wall remodeling after induced thrombosis in mouse

Matricryptins are collagen fragments proteolytically released from the extracellular matrix (ECM) with biological activity that can regulate several processes involved in ECM remodeling. Vessel wall matrix reorganization after lesion is important to the recovery of vascular function. This study aimed to analyze the effect of the peptide p1158/59 (Lindsey, 2015) on thrombosis, neointimal formation, and vascular remodeling of C57BL6 mice abdominal aorta. We used a FeCl3 induced vascular injury mice model and analyzed thrombus size, neointima formation, gelatinase activities in situ, re-endothelization, and collagen fibers organization on the arterial wall using polarization microscopy. As result, we observed that 2 days after injury the treatment with p1158/59 increased thrombus size and gelatinase activity, vascular lesion and it did not recover the endothelium loss induced by the chemical injury. We also observed that the peptide increased neointima growth and collagen birefringence, indicating collagen fibers reorganization. It also promoted increased re-endothelization and decreased activity of gelatinases 14 days after injury. Thus, we conclude that the peptide p1158/59 impaired the initial thrombosis recovery 2 days after injury but was able to induce vascular ECM remodeling after 14 days, improving vessel re-endothelization, collagen fibers deposition, and organization.

Defect of LSS Disrupts Lens Development in Cataractogenesis.

Congenital cataract is one of the leading causes of blindness in children worldwide. About one-third of congenital cataracts are caused by genetic defects. LSS, which encodes lanosterol synthase, is a causal gene for congenital cataracts. LSS is critical in preventing abnormal protein aggregation of various cataract-causing mutant crystallins; however, its roles in lens development remain largely unknown. In our study, we generated a mouse model harboring Lss G589S mutation, which is homologous to cataract-causing G588S mutation in human LSS. LssG589S/G589S mice exhibited neonatal lethality at postal day 0 (P0), whereas these mice showed severe opacity in eye lens. Also, we found that cataract was formed at E17.5 after we examined the opacity of embryonic lens from E13.5 to E18.5. Moreover, disrupted lens differentiation occurred at E14.5 prior to formation of the opacity of eye lens, shown as delayed differentiation of lens secondary fiber and disordered lens fiber organization. In addition, RNA-seq analysis indicated that cholesterol synthesis signaling pathways were significantly downregulated. Overall, our findings provide clear evidence that a mouse model harboring a homozygous Lss G589S mutation can recapitulate human congenital cataract. Our study points out that LSS functions as a critical determinant of lens development, which will contribute to better understanding LSS defects in cataractogenesis and developing therapies for cataracts.

Novel regulatory roles of small leucine-rich proteoglycans in remodeling of the uterine cervix in pregnancy

The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix.

A model of tension-induced fiber growth predicts white matter organization during brain folding

The past decade has experienced renewed interest in the physical processes that fold the developing cerebral cortex. Biomechanical models and experiments suggest that growth of the cortex, outpacing growth of underlying subcortical tissue (prospective white matter), is sufficient to induce folding. However, current models do not explain the well-established links between white matter organization and fold morphology, nor do they consider subcortical remodeling that occurs during the period of folding. Here we propose a framework by which cortical folding may induce subcortical fiber growth and organization. Simulations incorporating stress-induced fiber elongation indicate that subcortical stresses resulting from folding are sufficient to induce stereotyped fiber organization beneath gyri and sulci. Model predictions are supported by high-resolution ex vivo diffusion tensor imaging of the developing rhesus macaque brain. Together, results provide support for the theory of cortical growth-induced folding and indicate that mechanical feedback plays a significant role in brain connectivity.

Beyond muscles: role of intramuscular connective tissue elasticity and passive stiffness in octopus arm muscle function.

The octopus arm is a 'one of a kind' muscular hydrostat, as demonstrated by its high maneuverability and complexity of motions. It is composed of a complex array of muscles and intramuscular connective tissue, allowing force and shape production. In this study, we investigated the organization of the intramuscular elastic fibers in two main muscles composing the arm bulk: the longitudinal (L) and the transverse (T) muscles. We assessed their contribution to the muscles' passive elasticity and stiffness and inferred their possible roles in limb deformation. First, we performed confocal imaging of whole-arm samples and provided evidence of a muscle-specific organization of elastic fibers (more chaotic and less coiled in T than in L). We next showed that in an arm at rest, L muscles are maintained under 20% compression and T muscles under 30% stretching. Hence, tensional stresses are inherently present in the arm and affect the strain of elastic fibers. Because connective tissue in muscles is used to transmit stress and store elastic energy, we investigated the contribution of elastic fibers to passive forces using step-stretch and sinusoidal length-change protocols. We observed a higher viscoelasticity of L and a higher stiffness of T muscles, in line with their elastic fiber configurations. This suggests that L might be involved in energy storage and damping, whereas T is involved in posture maintenance and resistance to deformation. The elastic fiber configuration thus supports the specific role of muscles during movement and may contribute to the mechanics, energetics and control of arm motion.

Immunohistochemical features of the uterus structure in case of fetuses with a gestational term of 29-36 weeks from mothers with the chronic infection of the lower genital tracts

In the following article we present results of the research on the uterus’ wall structure in case of fetuses from mothers, whose pregnancy was complicated by the chronic infection of the lower genital tracts (HILGT), comparing to one in case of fetuses from healthy mothers. The main aim of our research was to disclose immunohistochemical features of the uterus’ wall sructure in case of fetuses with a gestational term of 29-36 weeks. The research methods: macroscopic, organometric, histological, immunohistochemical, morphometric, statistical. All fetuses have died intranatally as a result of an acute violation of utero-placental or placenta-fetal circulation.
 The organometric research has revealed a fact of decrease of the following indexes: namely, the ones of weight, length and thickness of the uterus’ wall in case of fetuses from mothers with a complicated pregnancy. Moreover, the decrease of the thickness’ index is primarily connected with a fact of thinning of the endometrium as a strucutral component of the wall. Staining by picrofuxin according to Van-Gieson has revealed an increase growth of the connective tissue both in endometrium and in myometrium of the uterus wall in case of fetuses from the main group. Moreover, in the muscular component, as a result of the aforementioned phenomenon, we can notice a violation or even in some places a complete lack of the structural organization of muscular fibers. The immunohistochemical research has revealed an increase of an apoptotic index in the endometrium of fetal organs of mothers with a compicated pregnancy comparing to indexes in case of fetuses from healthy mothers, which also contributes to the thinning of the layer as well as atrophic changes in it. In addition, in the strucutre of the connective tissue of the uterus of fetuses from the group of comparison the collagen of the I type prevails, while in case of fetuses from the main group the collagen of the III group prevails. Alongside with aforementioned features in case of fetuses from mothers with HILGT we can emphasize an increased glow of the endotheline-1 in vessels both of arterial and venous types.
 All changes in the connective tissue structure in the uterus of fetuses from mothers with a complicated pregnancy, that were described above, could contribute to the violation of the organ’s maturing as well as stabilization of its functional activity in the future life. The increased endotheline producing leads to the violaton of the blood supply, as well as could enhance collagenogenesis. The prevalence of the collagen of the III type in a structure of the connective tissue is a manifestation of the chronic hipoxia, under the condition of which the organogenesis of fetuses takes place in case of this pathology as well as leads to the violation of the spatial organization of the uterus’ main structural components. All features of the uterus wall structure in case of fetuses from mothers with HILGT, that have been established above, are prescribed by the chronic hipoxia and replication of the viral agents in the embryonic tissues as well as it could lead to make an implantation and pregnancy more difficult, likewise it could be a reason of development of pregnancy weakness in the following ontogenesis.

Atorvastatin Modulates the Efficacy of Electroporation and Calcium Electrochemotherapy

Electroporation is influenced by the features of the targeted cell membranes, e.g., the cholesterol content and the surface tension of the membrane. The latter is eventually affected by the organization of actin fibers. Atorvastatin is a statin known to influence both the cholesterol content and the organization of actin. This work analyzes the effects of the latter on the efficacy of electroporation of cancer cells. In addition, herein, electroporation was combined with calcium chloride (CaEP) to assess as well the effects of the statin on the efficacy of electrochemotherapy. Cholesterol-rich cell lines MDA-MB231, DU 145, and A375 underwent (1) 48 h preincubation or (2) direct treatment with 50 nM atorvastatin. We studied the impact of the statin on cholesterol and actin fiber organization and analyzed the cells’ membrane permeability. The viability of cells subjected to PEF (pulsed electric field) treatments and CaEP with 5 mM CaCl2 was examined. Finally, to assess the safety of the therapy, we analyzed the N-and E-cadherin localization using confocal laser microscopy. The results of our investigation revealed that depending on the cell line, atorvastatin preincubation decreases the total cholesterol in the steroidogenic cells and induces reorganization of actin nearby the cell membrane. Under low voltage PEFs, actin reorganization is responsible for the increase in the electroporation threshold. However, when subject to high voltage PEF, the lipid composition of the cell membrane becomes the regulatory factor. Namely, preincubation with atorvastatin reduces the cytotoxic effect of low voltage pulses and enhances the cytotoxicity and cellular changes induced by high voltage pulses. The study confirms that the surface tension regulates of membrane permeability under low voltage PEF treatment. Accordingly, to reduce the unfavorable effects of preincubation with atorvastatin, electroporation of steroidogenic cells should be performed at high voltage and combined with a calcium supply.

Novel perfusion ex-vivo culture system enables physiological and pathological studies on porcine arteries

Abstract Introduction Routine cardiovascular interventions such as cardiac bypass, balloon angioplasty and stenting provoke vascular activation and remodelling often leading to rehospitalization and further interventions. Conventional in vitro models fail to account for the complex vascular environment essential for vascular tissue fitness. Purpose Our research aims to culture whole porcine arteries in using a novel cost efficient and versatile perfusion system (EasyFlow) and identify the contribution of adventitial progenitors to post-injury remodelling. Methods EasyFlow insert was 3D printed to convert a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine arteries were excised and cultured under constant pulsatile flow for up to 7 days. Injury was performed by balloon catheter at day 0. Tissues were evaluated by doppler ultrasound, immunofluorescence and confocal imaging and PCR, at different timepoints. Results The EasyFlow adaptor takes advantage of 50 ml centrifuge tubes isolating reaction space from the environment and reducing the culture volume. The adaptability of the design facilitates the incubation of vessels of different size and origin while the self-contained perfusion allows parallel cultures and minimal media consumption. EasyFlow perfusion culture of porcine arteries preserves the endothelial coverage and the smooth muscle cell organisation, as compared to static culture. Gene expression analysis and immunofluorescence indicated an increased expression of platelet-derived growth factor receptor beta (PDGFRb) and a decrease in smooth muscle actin (SMA) in the static culture, compared to the perfusion. Furthermore, static culture showed an increased cellular activation in adventitia (proliferating cell nuclear antigen expression), as compared to perfused tissues. Balloon injury followed by flow culture recapitulated the early hallmarks of vascular remodelling, including intimal denudation indicated by loss of platelet endothelial cell adhesion molecule signal, smooth muscle cell disarray shown by loss of actin stress fibre organisation and activation in the media, as supported by confocal microscope and gene expression analysis, accompanied by significant morphological changes based on ultrasound imaging. Conclusion This work lay the basis for future investigations into the pathological remodelling of blood vessels, by providing a robust and controlled culture system for the maintenance of porcine blood vessels in culture. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Surrey En-Face Staining of Arterial Lumen

Investigation of Murine Vaginal Creep Response to Altered Mechanical Loads.

The vagina is a viscoelastic fibromuscular organ that provides support to the pelvic organs. The viscoelastic properties of the vagina are understudied but may be critical for pelvic stability. Most studies evaluate vaginal viscoelasticity under a single uniaxial load; however, the vagina is subjected to dynamic multiaxial loading in the body. It is unknown how varied multiaxial loading conditions affect vaginal viscoelastic behavior and which microstructural processes dictate the viscoelastic response. Therefore, the objective was to develop methods using extension-inflation protocols to quantify vaginal viscoelastic creep under various circumferential and axial loads. Then, the protocol was applied to quantify vaginal creep and collagen microstructure in the fibulin-5 wildtype and haploinsufficient vaginas. To evaluate pressure-dependent creep, the fibulin-5 wildtype and haploinsufficient vaginas (n = 7/genotype) were subjected to various constant pressures at the physiologic length for 100 s. For axial length-dependent creep, the vaginas (n = 7/genotype) were extended to various fixed axial lengths then subjected to the mean in vivo pressure for 100 s. Second-harmonic generation imaging was performed to quantify collagen fiber organization and undulation (n = 3/genotype). Increased pressure significantly increased creep strain in the wildtype, but not the haploinsufficient vagina. The axial length did not significantly affect the creep rate or strain in both genotypes. Collagen undulation varied through the depth of the subepithelium but not between genotypes. These findings suggest that the creep response to loading may vary with biological processes and pathologies, therefore, evaluating vaginal creep under various circumferential loads may be important to understand vaginal function.

Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon.

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin-eosin and immunostaining.

Siglec-15 promotes progression of clear renal cell carcinoma.

Siglec-15 promotes progression of clear renal cell carcinoma.