Organization Of Fibers Research Articles

Multiscale modelling of the human lumbar facet capsular ligament: analysing spinal motion from the joint to the neurons.

Due to its high level of innervation, the lumbar facet capsular ligament (FCL) is suspected to play a role in low back pain (LBP). The nociceptors in the lumbar FCL may experience excessive deformation and generate pain signals. As such, understanding the mechanical behaviour of the FCL, as well as that of its underlying nerves, is critical if one hopes to understand its role in LBP. In this work, we constructed a multiscale structure-based finite-element (FE) model of a lumbar FCL on a spinal motion segment undergoing physiological motions of flexion, extension, ipsilateral and contralateral bending, and ipsilateral axial rotation. Our FE model was created for a generic FCL geometry by morphing a previously imaged FCL anatomy onto an existing generic motion segment model. The fibre organization of the FCL in our models was subject-specific based on previous analysis of six dissected specimens. The fibre structures from those specimens were mapped onto the FCL geometry on the motion segment. A motion segment model was used to determine vertebral kinematics under specified spinal loading conditions, providing boundary conditions for the FCL-only multiscale FE model. The solution of the FE model then provided detailed stress and strain fields within the tissue. Lastly, we used this computed strain field and our previous studies of deformation of nerves embedded in fibrous networks during simple deformations (e.g. uniaxial stretch, shear) to estimate the nerve deformation based on the local tissue strain and fibre alignment. Our results show that extension and ipsilateral bending result in largest strains of the lumbar FCL, while contralateral bending and flexion experience lowest strain values. Similar to strain trends, we calculated that the stretch of the microtubules of the nerves, as well as the forces exerted on the nerves' membrane are maximal for extension and ipsilateral bending, but the location within the FCL of peak microtubule stretch differed from that of peak membrane force.

Levator Ani avulsion: The histological composition of this site. A cadaveric study.

The sonographic appearance of a levator muscle "avulsion" representing the literal detachment of the pubovisceral muscle (PVM) enthesis has been contested. The nature of the levator ani "avulsion" is still not fully understood. It is known, that the tensile strength of a tendon is dependent on collagen with increased synthesis of collagen occurring in tissue with increased mechanical load levels. This study aims to perform a quantitative histological evaluation of the PVM enthesis with or without the imaging finding of levator ani "avulsion" to determine if there is a difference in the proportion of muscle and collagen. Three-dimensional translabial ultrasound for PVM "avulsion" was performed on cadavers using a GE Voluson I with a 5-9 MHz electronic probe. Cadavers were meticulously dissected to identify the presence or absence of an anatomical avulsion. The PVM enthesis was excised for further histopathological processing and treated with three different colorations. Quantitative analysis using ImageJ software was conducted to compare tissue composition in samples with or without sonographic "avulsion." All stages were performed by two separate investigators blinded to each other's results. The results were analyzed using SPSS v24, IBM. Twenty-three PVM enthesis with histological staining were procured. Ultrasonographic "avulsions" were seen in 5/23 PVM enthesis. No anatomical avulsions were seen. There was no difference in the overall muscle or collagen content (Kruskal-Wallis, P = 0.864). The mean organized skeletal muscle content was 23% in the sonographic "avulsion" group versus 62% in the no "avulsion" group (Kruskall-Walis, P = 0.02). "Avulsions" were associated with a disorganized appearance at histology. The tissue composition relating to the proportion of muscle and collagen was not significantly different in specimens with or without sonographic "avulsions." However, morphological differences were observed in the organization of the muscle fibres, which requires further evaluation.

Engineered tendon-fibrocartilage-bone composite and bone marrow-derived mesenchymal stem cell sheet augmentation promotes rotator cuff healing in a non-weight-bearing canine model

Reducing rotator cuff failure after repair remains a challenge due to suboptimal tendon-to-bone healing. In this study we report a novel biomaterial with engineered tendon-fibrocartilage-bone composite (TFBC) and bone marrow-derived mesenchymal stem cell sheet (BMSCS); this construct was tested for augmentation of rotator cuff repair using a canine non-weight-bearing (NWB) model. A total of 42 mixed-breed dogs were randomly allocated to 3 groups (n = 14 each). Unilateral infraspinatus tendon underwent suture repair only (control); augmentation with engineered TFBC alone (TFBC), or augmentation with engineered TFBC and BMSCS (TFBC + BMSCS). Histomorphometric analysis and biomechanical testing were performed at 6 weeks after surgery. The TFBC + BMSCS augmented repairs demonstrated superior histological scores, greater new fibrocartilage formation and collagen fiber organization at the tendon-bone interface compared with the controls. The ultimate failure load and ultimate stress were 286.80 ± 45.02 N and 4.50 ± 1.11 MPa for TFBC + BMSCS group, 163.20 ± 61.21 N and 2.60 ± 0.97 MPa for control group (TFBC + BMSCS vs control, P = 1.12E-04 and 0.003, respectively), 206.10 ± 60.99 N and 3.20 ± 1.31 MPa for TFBC group (TFBC + BMSCS vs TFBC, P = 0.009 and 0.045, respectively). In conclusion, application of an engineered TFBC and BMSCS can enhance rotator cuff healing in terms of anatomic structure, collagen organization and biomechanical strength in a canine NWB model. Combined TFBC and BMSCS augmentation is a promising strategy for rotator cuff tears and has a high potential impact on clinical practice.

Centronuclear myopathies under attack: A plethora of therapeutic targets.

Centronuclear myopathies are a group of congenital myopathies characterized by severe muscle weakness, genetic heterogeneity, and defects in the structural organization of muscle fibers. Their names are derived from the central position of nuclei on biopsies, while they are at the fiber periphery under normal conditions. No specific therapy exists yet for these debilitating diseases. Mutations in the myotubularin phosphoinositides phosphatase, the GTPase dynamin 2, or amphiphysin 2 have been identified to cause respectively X-linked centronuclear myopathies (also called myotubular myopathy) or autosomal dominant and recessive forms. Mutations in additional genes, as RYR1, TTN, SPEG or CACNA1S, were linked to phenotypes that can overlap with centronuclear myopathies. Numerous animal models of centronuclear myopathies have been studied over the last 15 years, ranging from invertebrate to large mammalian models. Their characterization led to a partial understanding of the pathomechanisms of these diseases and allowed the recent validation of therapeutic proof-of-concepts. Here, we review the different therapeutic strategies that have been tested so far for centronuclear myopathies, some of which may be translated to patients.

Proteome and transcriptome profiling of equine myofibrillar myopathy identifies diminished peroxiredoxin 6 and altered cysteine metabolic pathways.

Equine myofibrillar myopathy (MFM) causes exertional muscle pain and is characterized by myofibrillar disarray and ectopic desmin aggregates of unknown origin. To investigate the pathophysiology of MFM, we compared resting and 3 h postexercise transcriptomes of gluteal muscle and the resting skeletal muscle proteome of MFM and control Arabian horses with RNA sequencing and isobaric tags for relative and absolute quantitation analyses. Three hours after exercise, 191 genes were identified as differentially expressed (DE) in MFM vs. control muscle with >1 log2 fold change (FC) in genes involved in sulfur compound/cysteine metabolism such as cystathionine-beta-synthase (CBS, ↓4.51), a cysteine and neutral amino acid membrane transporter (SLC7A10, ↓1.80 MFM), and a cationic transporter (SLC24A1, ↓1.11 MFM). In MFM vs. control at rest, 284 genes were DE with >1 log2 FC in pathways for structure morphogenesis, fiber organization, tissue development, and cell differentiation including > 1 log2 FC in cardiac alpha actin (ACTC1 ↑2.5 MFM), cytoskeletal desmoplakin (DSP ↑2.4 MFM), and basement membrane usherin (USH2A ↓2.9 MFM). Proteome analysis revealed significantly lower antioxidant peroxiredoxin 6 content (PRDX6, ↓4.14 log2 FC MFM), higher fatty acid transport enzyme carnitine palmitoyl transferase (CPT1B, ↑3.49 MFM), and lower sarcomere protein tropomyosin (TPM2, ↓3.24 MFM) in MFM vs. control muscle at rest. We propose that in MFM horses, altered cysteine metabolism and a deficiency of cysteine-containing antioxidants combined with a high capacity to oxidize fatty acids and generate ROS during aerobic exercise causes chronic oxidation and aggregation of key proteins such as desmin.

P3.03-19 The Lysyl Oxidase like-1 Promotes NSCLC Tumorigenecity Through Increased Matrix Reorganization and Stiffness

The importance of the stroma in tumorigenicity has been increasingly recognized in the past few years and targeting tumor stroma has become a new strategy for the development of new therapies. The lysyl oxidase like-1 (LOXL1) is a matrix cross-linking enzyme, essentially secreted by cancer-associated fibroblasts (CAFs) in the stroma. We previously showed that LOXL1 expression strongly correlated with integrin α11 expression, a collagen receptor that promotes NSCLC tumorigenecity, suggesting that LOXL1 may also contribute to lung tumor growth and metastasis by modifying the stromal collagen matrix. Primary CAFs (CAF094) isolated from a NSCLC resection specimen and immortalized with hTERT were infected with full length LOXL1 (LOXL1 overexpression), LOXL1 shRNA (LOXL1 knockdown) or with their respective controls, using a lentiviral system. To investigate the impact of LOXL1 on collagen matrix reorganization, we allowed CAFs expressing different levels of LOXL1 to contract collagen matrices. Contracted collagen matrices have then been submitted to second harmonic generation to analyze collagen fibers. We established the role of LOXL1 in lung carcinoma invasion and growth using an in vitro organotypic model and an in vivo xenograft model, respectively. LOXL1-overexpressing CAFs embedded in collagen lattices displayed greater ability to reorganize the collagen matrix than those with lower LOXL1 expression. Furthermore, analysis of the collagen matrix demonstrated that LOXL1 contributed to more linear and dense collagen fibers organization. As a consequence of collagen matrix reorganization, invasion of the lung carcinoma H661 cell line significantly increased in an organotypic model in presence of LOXL1-overexpressing CAFs. Moreover, we showed that loss of Loxl1 in mice inhibited subcutaneous growth of the lung adenocarcinoma H4006 cell line compared to the mouse wild-type counterparts. We demonstrated that overexpression of LOXL1 in NSCLC tumor stroma results in an increased of tumor growth and invasion, due to a change in the matrix reorganization. Thus, LOXL1 appears as an interesting target to improve the outcome of lung cancer.

Inverting Thermal Degradation ( iTD) of Paper Using Chemi- and Physi-Sorbed Modifiers for Templated Material Synthesis.

Fibrous cellulosic materials have been used as templates for material synthesis or organization via thermal degradation of the cellulose. Most of these methods, however, fail to exploit fiber organization, in part due to loss of structure with processing. Herein, we demonstrate that chemi- and physi-sorbed modifiers of cellulose alters the thermal degradation mechanism allowing for controlled deposition of oxide and carbon (incomplete combustion) along the original paper fiber network. We demonstrate that the degradation of the cellulose fibers depends on the amount of physisorbed material due, in part, to effect on the propagation of the ignition event. From the distribution of the residual elements and shape of the deposits, we can infer that the thermal degradation process depends on the nature, and concentration, of filler(s) or occluded.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ± 36 mg) were about 12 and 630 μg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 μg/L and a no-observed-effect concentration of 2.1 μg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 μg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Quantitative MR neurography of brachial plexus lesions based on diffusivity measurements

ObjectivesNew magnetic resonance imaging (MRI) based on 3D fat-suppressed techniques provide high accuracy for evaluation of brachial plexus and peripheral nerve lesions from a qualitative point of view. However, this qualitative evaluation often demonstrates overlapping features among different pathologies (Pullman et al., 2013). Diffusion weighted imaging (DWI) and diffusion tensor imaging (DTI) are functional MRI techniques that have been successfully applied in the imaging of central nervous system lesions, and now are being imported, with promising results for peripheral nerve and plexus evaluation. Several derived parameters from DWI and DTI studies such as apparent diffusion coefficient (ADC) or fractional anisotropy (FA), can be used as potential biomarkers of neural damage by providing information about axonal flow, fiber organization or myelin integrity (Andreou et al., 2015). In this study we assess the role of DW and DT based quantitative measures in characterization of traumatic and non traumatic lesions of brachial plexus and peripheral nerve and its influence on therapeutic management and outcome. Methods34 patients with features ofbrachialplexopathy and peripheral neuropathy were included in the study. MR neurography examinations included conventional MR sequences and unidirectional DW-MRN with background suppression (DWIBS) and DTI. The images were independently reviewed by two radiologists and the findings were recorded at different levels of thebrachial plexus: ganglia, roots, trunks, divisions and cords. Mean ADC and FA values were determined within each lesion and within contra lateral homologous unaffected segment of the plexus. The Mann-Whitney Test was used to calculate the differences in ADC&FA values between diseased and normal sides and the One way ANOVA test between diseased segments of different pathologies with p-values <0.05 being considered statistically significant. Resultspatients were classified according to etiologic causes into four categories: traumatic, compressive degenerative disk, radiation induced plexitis and neoplastic nerve compression or invasion. T2 high signal intensity and swelling of the affected nerve, muscular denervation and complication such as pseudo meningeocele and neuroma were the major findings in qualitatitve sequences. A statistically significant difference in ADC and FA values were found between diseased and normal sides as well as across the traumatic and non traumatic groups and between benign compressive and malignant infiltrative etiologies. ConclusionThe resulted values of newly designed quantitative protocols give us functional and numerical marker by which the diagnosis and differentiation of different pathologies of peripheral nerves and brachial plexus will be more suitable.

Diabetes can change the viscoelastic properties of lymphocytes

Mechanical properties of the cells are among the most highlighted area of interests among researchers for decades. Not only many of the cells’ crucial functional characteristics such as adherence to the cellular substrate, migration abilities and morphological factors are directly influenced by their mechanical properties but also changes in these traits could have importance in diagnosis and even treatments of some serious diseases. The general mechanical properties of the cells are associated with some intercellular characteristics such as arrangement and organization of the actin fibers and cytoskeleton architecture. Any changes due to pathological conditions in the molecular and cellular processes related to these elements can alter the cells’ mechanical characteristics. In this paper, the viscoelastic properties of diabetic and normal lymphocytes were analyzed and compared by application of the iron nanoparticles attached to the cellular membrane and putting the cells in a magnetic field with certain frequency and intensity. Step force was applied to the normal and diabetic lymphocytes and their membrane displacement was tracked by special software and plotted with respect to time. Fitting the experimental data on theoretical formulation of standard linear viscoelastic model, it was demonstrated that diabetic lymphocytes have significantly different viscoelastic characteristics. The results of this paper can be of importance in assessments of diabetic lymphocytes’ abilities to fulfill their immune surveillance tasks.

Endothelial cell activation on 3D-matrices derived from PDGF-BB-stimulated fibroblasts is mediated by Snail1

Carcinomas, such as colon cancer, initiate their invasion by rescuing the innate plasticity of both epithelial cells and stromal cells. Although Snail is a transcriptional factor involved in the Epithelial-Mesenchymal Transition, in recent years, many studies have also identified the major role of Snail in the activation of Cancer-Associated Fibroblast (CAF) cells and the remodeling of the extracellular matrix. In CAFs, Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant. High expression of both SNAI1 and PDGF receptors is associated with poor prognosis in cancer patients, but the mechanism(s) that underlie these connections are not understood. In this study, we demonstrate that PDGF-activated fibroblasts stimulate extracellular matrix (ECM) fiber remodeling and deposition. Furthermore, we describe how SNAI1, through the FAK pathway, is a necessary factor for ECM fiber organization. The parallel-oriented fibers are used by endothelial cells as “tracks”, facilitating their activation and the creation of tubular structures mimicking in vivo capillary formation. Accordingly, Snail1 expression in fibroblasts was required for the co-adjuvant effect of these cells on matrix remodeling and neoangiogenesis when co-xenografted in nude mice. Finally, in tumor samples from colorectal cancer patients a direct association between stromal SNAI1 expression and the endothelial marker CD34 was observed. In summary, our results advance the understanding of PDGF/SNAI1-activated CAFs in matrix remodeling and angiogenesis stimulation.

The actin filament bundling protein α-actinin-4 actually suppresses actin stress fibers by permitting actin turnover

Cells organize actin filaments into contractile bundles known as stress fibers that resist mechanical stress, increase cell adhesion, remodel the extracellular matrix, and maintain tissue integrity. α-actinin is an actin filament bundling protein that is thought to be essential for stress fiber formation and stability. However, previous studies have also suggested that α-actinin might disrupt fibers, making the true function of this biomolecule unclear. Here we use fluorescence imaging to show that kidney epithelial cells depleted of α-actinin-4 via shRNA or CRISPR/Cas9, or expressing a disruptive mutant make more massive stress fibers that are less dynamic than those in WT cells, leading to defects in cell motility and wound healing. The increase in stress fiber mass and stability can be explained, in part, by increased loading of the filament component tropomyosin onto stress fibers in the absence of α-actinin, as monitored via immunofluorescence. We show using imaging and cosedimentation that α-actinin and tropomyosin compete for binding to F-actin and that tropomyosin shields actin filaments from cofilin-mediated disassembly in vitro and in cells. Perturbing tropomyosin in cells lacking α-actinin-4 results in a complete loss of stress fibers. Our results with α-actinin-4 on stress fiber organization are the opposite of what might have been predicted from previous in vitro biochemistry and further highlight how the complex interactions of multiple proteins competing for filament binding lead to unexpected functions for actin-binding proteins in cells.

Human Tendon-Derived Hydrogel Improves Healing of Chronic Tendon–Bone Interface Injuries

Grant support received from: 2016 AFSH Basic Science Grant There is no financial information to disclose. Previously our laboratory reported the positive impact of thermoresponsive, type 1 collagen–rich, decellularized human tendon-derived hydrogel (tHG) on tendon healing using a rat Achilles tendon acute injury model. We hypothesized tHG would facilitate healing of chronic injuries at tendon–bone interface (TBI) in a rotator cuff model. Rotator cuff tears are important causes of pain and disability of upper extremity and involve injury at TBI. In this study a rat chronic rotator cuff injury model was used to evaluate TBI healing after repair. Twenty Sprague–Dawley rats underwent bilateral detachment of supraspinatus (SSp) tendons. Eight weeks later, both SSp tendons were anatomically reattached to humerus. One shoulder underwent a suture to bone repair using bone tunnels (control group, n = 20) and the other underwent the same repair plus tHG injection at repair site (tHG group, n = 20). Eight weeks after repair, SSp tendon–muscle units with humeral bone were harvested after animal death. Biomechanical tests were performed on 15 samples in each group. Five additional samples in each group were evaluated via micro CT, followed by histological examinations. Paired t tests were performed for statistical analyses. Biomechanical testing revealed significantly larger load to failure (27.5 ± 11.8N vs 19.7 ± 5.4N, P = .03) and higher stiffness (15.7 ± 6.3 N/mm vs 11.5 ± 3.5 N/mm, P = .02) in the tHG group versus control. Safranin O staining showed the significantly larger area of new cartilage formation in the tHG group (0.48 ± 0.1 mm2 vs 0.32 ± 0.06 mm2, P = .03). Based on birefringence under polarized microscopy in Picosirius red–stained slides collagen fiber organization was greater in tHG group compared with the control (40.4 ± 9.8 gray scale vs 31.1 ± 4.2 gray scale, P = .07), although it did not significantly differ. Micro CT demonstrated higher bone mineral density (296.1 ± 21.9 mg/cm3 vs 274.6 ± 17.2 mg/cm3, P = .05) and larger trabecular number (1.7 ± 0.1/mm vs 1.5 ± 0.2, P = .07) at the supraspinatus insertion area of the humerus in tHG group than those in the control, although the difference did not reach statistical significance. •Injection of collagen-rich tHG at the rotator cuff repair site statistically significantly enhanced biomechanical properties and increased fibrocartilage formation at the TBI in a chronic injury model.•An improvement collagen fiber organization, higher bone mineral density, and larger trabecular number were also seen in the tHG group versus control.•This human-derived collagen gel is a promising scaffold to augment healing of chronic TBI injuries during surgical intervention. This research was supported by a Basic Science Grant from the American Foundation for Surgery of the Hand.

Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5.

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats’ gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.

Role of Thrombospondin-1 in Mechanotransduction and Development of Thoracic Aortic Aneurysm in Mouse and Humans.

Abnormal mechanosensing of smooth muscle cells (SMCs) resulting from the defective elastin-contractile units has been suggested to drive the formation of thoracic aortic aneurysms; however, the precise molecular mechanism has not been elucidated. The aim of this study was to identify the crucial mediator(s) involved in abnormal mechanosensing and propagation of biochemical signals during the aneurysm formation and to establish a basis for a novel therapeutic strategy. We used a mouse model of postnatal ascending aortic aneurysms ( Fbln4SMKO; termed SMKO [SMC-specific knockout]), in which deletion of Fbln4 (fibulin-4) leads to disruption of the elastin-contractile units caused by a loss of elastic lamina-SMC connections. In this mouse, upregulation of Egr1 (early growth response 1) and angiotensin-converting enzyme leads to activation of Ang II (angiotensin II) signaling. Here, we showed that the matricellular protein, Thbs1 (thrombospondin-1), was highly upregulated in SMKO ascending aortas and in human thoracic aortic aneurysms. Thbs1 was induced by mechanical stretch and Ang II in SMCs, for which Egr1 was required, and reduction of Fbln4 sensitized the cells to these stimuli and led to higher expression of Egr1 and Thbs1. Deletion of Thbs1 in SMKO mice prevented the aneurysm formation in ≈80% of DKO (SMKO;Thbs1 knockout) animals and suppressed Ssh1 (slingshot-1) and cofilin dephosphorylation, leading to the formation of normal actin filaments. Furthermore, elastic lamina-SMC connections were restored in DKO aortas, and mechanical testing showed that structural and material properties of DKO aortas were markedly improved. Thbs1 is a critical component of mechanotransduction, as well as a modulator of elastic fiber organization. Maladaptive upregulation of Thbs1 results in disruption of elastin-contractile units and dysregulation of actin cytoskeletal remodeling, contributing to the development of ascending aortic aneurysms in vivo. Thbs1 may serve as a potential therapeutic target for treating thoracic aortic aneurysms.

Cell regulation of collagen fibril macrostructure during corneal morphogenesis

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Statement of significanceThis work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

Effect of sanguinarine on biomechanics of rat airway smooth muscle cells

This study aimed to evaluate the effect of sanguinarine on biomechanical properties of rat airway smooth muscle cells (rASMCs) including stiffness, traction force and cytoskeletal stress fiber organization. To do so, rASMCs cultured in vitro were treated with sanguinarine solution at different concentrations (0.005~5 μmol/L) for 12 h, 24 h, 36 h, and 48 h, respectively. Subsequently, the cells were tested for their viability, stiffness, traction force, migration and microfilament distribution by using methylthiazolyldiphenyl-tetrazolium bromide assay, optical magnetic twisting cytometry, Fourier transform traction microscopy, scratch wound healing method, and immunofluorescence microscopy, respectively. The results showed that at concentration below 0.5 μmol/L sanguinarine had no effect on cell viability, but caused dose and time dependent effect on cell biomechanics. Specifically, rASMCs treated with sanguinarine at 0.05 μmol/L and 0.5 μmol/L for 12 and 24 h exhibited significant reduction in stiffness, traction force and migration speed, together with disorganization of the cytoskeletal stress fibers. Considering the essential role of airway smooth muscle cells (ASMCs) biomechanics in the airway hyperresponsiveness (AHR) of asthma, these findings suggest that sanguinarine may ameliorate AHR via alteration of ASMCs biomechanical properties, thus providing a novel approach for asthma drug development.

Effect of 3 Preservation Methods (Freezing, Cryopreservation, and Freezing + Irradiation) on Human Menisci Ultrastructure: An Ex Vivo Comparative Study With Fresh Tissue as a Gold Standard

Background: Three main meniscus preservation methods have been advocated: freezing (–80°C), freezing with gamma irradiation (–80°C + 25 kGy), and cryopreservation (–140°C). Hypothesis: All preservation methods will result in structural and architectural properties similar to those of fresh meniscus, defined as the gold standard. Study Design: Controlled laboratory study. Methods: Five human intact menisci were collected from 5 patients undergoing total knee arthroplasty. The inclusion criteria were patients <70 years old with primary unilateral (medial) femorotibial knee osteoarthritis and without surgical or traumatic history on the operated knee. Four cubes (9 mm3) were cut inside of the white, or avascular, area of each specimen’s middle horn and divided into 4 groups: “fresh” control, frozen (–80°C), cryopreserved (–140°C), and frozen + irradiated (–80°C + 25 kGy). Specimens of the control group were evaluated at day 1, and specimens from the frozen, cryopreserved, and frozen + irradiated groups were evaluated after 1 month of storage. Evaluation was performed with electron microscopy according a validated protocol to analyze (1) mean diameters of the collagen fibers in longitudinal and transverse sections in 5 points per section and (2) validated architectural scores. Results: No significant difference was found between the control and cryopreserved groups regarding mean transverse and longitudinal diameters (transverse: 95.39 ± 15.87 nm vs 99.62 ± 19.23 nm, P = .1; longitudinal: 96.31 ± 13.96 nm vs 94.57 ± 16.42 nm, P = .1). Significant differences were found between the control and frozen groups (transverse: 95.39 ± 15.87 nm vs 70.20 ± 13.94 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 71.28 ± 10.64 nm, P < .001) and the control and frozen + irradiated groups (transverse: 95.39 ± 15.87 nm vs 63.1 ± 15.57 nm, P < .001; longitudinal: 96.31 ± 13.96 nm vs 60.9 ± 14.8 nm, P < .001). Regarding architectural score calculation, there were significant differences between the control and frozen groups (4.5 ± 1.3 vs 2.3 ± 1.4, P = .02) and the control and frozen + irradiated groups (4.5 ± 1.3 vs 1.4 ± 0.9, P = .02). Conclusion: Cryopreservation is the only method that preserves fresh meniscus architectural specificities. Freezing and freezing + irradiation methods modify histologic properties of meniscal allograft. Irradiation deeply alters diameters and the organization of collagen fibers, and this method should be used with caution to preserve and sterilize meniscus tissue. Clinical Relevance: The results of our study exhibited detrimental effects of simple freezing and freezing + irradiation on the collagen network of sample meniscus. If those effects occur in menisci prepared for allograft procedures, important differences could appear on the basis of the preservation procedure in terms of the graft’s mechanical properties and, thus, the patient’s outcomes.

Anisotropy of thePassive and Active Rat Vagina Under Biaxial Loading.

Pelvic organ prolapse, the descent of the pelvic organs from their normal anatomical position, is a common condition among women that is associated with mechanical alterations of the vaginal wall. In order to characterize the complex mechanical behavior of the vagina, we performed planar biaxial tests of vaginal specimens in both the passive (relaxed) and active (contracted) states. Specimens were isolated from virgin, female Long-Evans rats (n = 16) and simultaneously stretched along the longitudinal direction (LD) and circumferential direction (CD) of the vagina. Tissue contraction was induced by electric field stimulation (EFS) at incrementally increasing values of stretch and, subsequently, by KCl. On average, the vagina was stiffer in the CD than in the LD (p < 0.001). The mean maximum EFS-induced active stress was significantly higher in the CD than in the LD (p < 0.01). On the contrary, the mean KCl-induced active stress was lower in the CD than in the LD (p < 0.01). When comparing the mean maximum EFS-induced active stress to the mean KCl-induced active stress, no differences were found in the CD (p = 0.366) but, in the LD, the mean active stress was much higher in response to the KCl stimulation (p < 0.001). Collectively, these results suggest that the anisotropic behavior of the vaginal tissue is determined not only by collagen and smooth muscle fiber organization but also by the innervation.

Vitamin D Attenuates Endothelial Dysfunction in Uremic Rats and Maintains Human Endothelial Stability.

BackgroundDysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease (CKD). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD, not only by direct effects on the endothelium but also by an increment of α‐Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia‐induced endothelial damage and the extent to which this was dependent on increased α‐Klotho concentrations.Methods and ResultsIn a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial‐gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α‐Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real‐time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro‐permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial‐cadherin–based cell‐cell junctions and diminished F‐actin stress fiber organization, preventing the formation of endothelial intracellular gaps.ConclusionsOur results demonstrate that paricalcitol attenuates the CKD‐induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell‐cell interactions.