Orientation Of Smooth Muscle Cells Research Articles

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study.

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns-10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15-20% higher LD50, p < 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30-40% lower LD50 (p < 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation.

AORTOCORONARY SAPHENOUS VEIN GRAFT ANEURYSM: A SILENT COMPLICATION

AORTOCORONARY SAPHENOUS VEIN GRAFT ANEURYSM: A SILENT COMPLICATION

Stage-dependent sequential organization of nascent smooth muscle cells and its implications for the gut coiling morphogenesis in Xenopus larva

In vertebrates, gut coiling proceeds left-right asymmetrically during expansion of the gastrointestinal tract with highly organized muscular structures facilitating peristalsis. In this report, we explored the mechanisms of larval gut coiling morphogenesis relevant to its nascent smooth muscle cells using highly transparent Xenopus early larvae. First, to visualize the dynamics of intestinal smooth muscle cells, whole-mount specimens were immunostained with anti-smooth muscle-specific actin (SM-actin) antibody. We found that the nascent gut of Xenopus early larvae gradually expands the SM-actin-positive region in a stage-dependent manner. Transverse orientation of smooth muscle cells was first established, and next, the cellular longitudinal orientation along the gut axis was followed to make a meshwork of the contractile cells. Finally, anisotropic torsion by the smooth muscle cells was generated in the center of gut coiling, suggesting that twisting force might be involved in the late phase of coiling morphogenesis of the gut. Administration of S-(–)-Blebbistatin to attenuate the actomyosin contraction in vivo resulted in cancellation of coiling of the gut. Development of decapitation embryos, trunk ‘torso’ explants, and gut-only explants revealed that initial coiling of the gut proceeds without interactions with the other parts of the body including the central nervous system. We newly developed an in vitro model to assess the gut coiling morphogenesis, indicating that coiling pattern of the nascent Xenopus gut is partially gut-autonomous. Using this gut explant culture technique, inhibition of actomyosin contraction was performed by administrating either actin polymerization inhibitor, myosin light chain kinase inhibitor, or calmodulin antagonist. All of these reagents decreased the extent of gut coiling morphogenesis in vitro. Taken together, these results suggest that the contraction force generated by actomyosin-rich intestinal smooth muscle cells during larval stages is essential for the normal coiling morphogenesis of this muscular tubular organ.

Biomimetic dual-oriented/bilayered electrospun scaffold for vascular tissue engineering

Natural blood vessels have a multi-layered, cell-specific oriented spatial structure, mimicking of this structure is a promising way for blood vessel regeneration. In this study, a newly developed dual-oriented/bilayered small-diameter tubular scaffold was electrospun using a mixture of poly (ε-caprolactone) (PCL), poly (D, L-lactide-co-glycolide) (PLGA) and gelatin. The nanofiber orientations of the bilayers were spatially perpendicular to each other, aiming at guiding cell-specific orientation of smooth muscle cells (SMCs) and endothelial cells (ECs) in vitro respectively. The results showed that the hydrophilicity of scaffold was greatly improved by gelatin, and the mechanical property of this scaffold was the best among all. The in vitro degradation demonstrated that by mixing of three biodegradable polymers, a relatively fast degradation rate was achieved. After SMCs and ECs were seeded on scaffolds, cell viability, cellular morphology, and cytoskeleton behavior were investigated. The results revealed that as-electrospun scaffolds could promote both SMCs and ECs proliferation. Moreover, topographic cues offered by oriented nanofibers could guide the growth and orientation of SMCs and ECs. Therefore, the dual-oriented/bilayered electrospun scaffold is a superior structural and functional analogue to natural blood vessel and a potential candidate for vascular tissue engineering.

The organisation of vascular smooth muscle cells; a quantitative Fast Fourier Transform (FFT) based assessment

BackgroundThe existence of a helical arrangement of vascular smooth muscle cells in the wall of relatively large blood vessels has been known of for at least 80 years. In that time various workers have proposed mathematical models of vascular function that have incorporated one or more helix. However, most of this work has focussed on large conduit vessels such as aorta and carotid arteries. The smaller resistance arteries are more difficult to study and do not have such an apparent helical arrangement, although there are suggestions of its existence in the patterning of smooth muscle cells in the tunica media. MethodWe have employed confocal laser scanning microscopy (CLSM) and a relatively simple Fast Fourier Transform (FFT) based image analysis method to examine a collection of extended focus (z-projection) images of the tunica media of mouse superior mesenteric artery. A fluorescent nuclear stain (Syto 61) was used to identify smooth muscle cell orientation and arrangement. Thirty-six CLSM datasets representing the tunica media of mesenteric arteries taken from 12 C57/black mice were collected for analysis. ResultsMeasurements of Z-projection (extended focus) images, of the tunica media, were compared with measurements made on inverse FFT images where the high frequency information was removed. The results, of both measurement types, indicated the existence of an underlying basic helical arrangement of cells that has a pitch angle of 49°. 3D electron microscopy-based models of individual smooth muscle cells (from the tail artery) were generated to highlight their heterogeneity and orientational position within the tunica media. ConclusionInverse FFT images provide a more objective means of measuring cell organisation and therefore could be used in automated processes. Whilst we propose the existence of an underlying helical arrangement of smooth muscle cells, we do not discount the existence of other helices each with different angles, but these may be more difficult to detect and may extend radially (deep within the tunica media) as well as longitudinally.

Complete Myogenic Differentiation of Adipogenic Stem Cells Requires Both Biochemical and Mechanical Stimulation.

Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by treatment with transforming growth factor-β, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (αSMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased αSMA and calponin expression by either pulsatile (12.48-fold; p < 0.01 and 38.15-fold; p = 0.07) or static incubation plus TSB pre-treatment (8.91-fold; p < 0.05 and 37.69-fold; p < 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (p < 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.

Multifibrillar bundles of a self-assembling hyaluronic acid derivative obtained through a microfluidic technique for aortic smooth muscle cell orientation and differentiation.

A hyaluronic acid derivative that is able to physically crosslink in a saline aqueous environment was employed for the production of fibers with a mean diameter of 50 μm using a microfluidic technique. The microfibers were collected in a tailored rotating collector and assembled to form multifibrillar bundles. The orientation of the microfibers on the collected bundles was evaluated by microCT analysis. The bundles were biofunctionalized by physical addition of fibronectin or chemical tethering of a cyRGDC peptide to achieve control of Aortic Smooth Muscle Cell (AoSMC) attachment, elongation and alignment. The mechanical performances of these bundles were evaluated by elongation tests, related to the kind of biological functionalization and compared to non-functionalized samples. The alignment and differentiation of AoSMCs on single fibers and on the bundles were evaluated by microscopy and histochemical analyses.

Mechanical induction of bi-directional orientation of primary porcine bladder smooth muscle cells in tubular fibrin-poly(vinylidene fluoride) scaffolds for ureteral and urethral repair using cyclic and focal balloon catheter stimulation.

To restore damaged organ function or to investigate organ mechanisms, it is necessary to prepare replicates that follow the biological role model as faithfully as possible. The interdisciplinary field of tissue engineering has great potential in regenerative medicine and might overcome negative side effects in the replacement of damaged organs. In particular, tubular organ structures of the genitourinary tract, such as the ureter and urethra, are challenging because of their complexity and special milieu that gives rise to incrustation, inflammation and stricture formation. Tubular biohybrids were prepared from primary porcine smooth muscle cells embedded in a fibrin gel with a stabilising poly(vinylidene fluoride) mesh. A mechanotransduction was performed automatically with a balloon kyphoplasty catheter. Diffusion of urea and creatinine, as well as the bursting pressure, were measured. Light and electron microscopy were used to visualise cellular distribution and orientation. Histological evaluation revealed a uniform cellular distribution in the fibrin gel. Mechanical stimulation with a stretch of 20% leads to a circumferential orientation of smooth muscle cells inside the matrix and a longitudinal alignment on the outer surface of the tubular structure. Urea and creatinine permeability and bursting pressure showed a non-statistically significant trend towards stimulated tissue constructs. In this proof of concept study, an innovative technique of intraluminal pressure for mechanical stimulation of tubular biohybrids prepared from autologous cells and a composite material induce bi-directional orientation of smooth muscle cells by locally and cyclically applied mechanical tension. Such geometrically driven patterns of cell growth within a scaffold may represent a key stage in the future tissue engineering of implantable ureter replacements that will allow the active transportation of urine from the renal pelvis into the bladder.

Development of Dynamic Liquid and Conjugated Electrospun Poly(L-lactide-co-caprolactone)/Collagen Nanoyarns for Regulating Vascular Smooth Muscle Cells Growth.

Simulating the modeling of smooth muscle layer in the vascular structure makes a great difference for vascular tissue regeneration. A functional tissue engineered vascular media shall promote the aligned organization and three-dimensional penetration of smooth muscle cells (SMCs) into the scaffold. To this aim, dynamic liquid and conjugated nanoyarns based on poly(L-lactide-co-caprolactone) (P(LLA-CL)) and collagen (COL) with a weight ratio at 3:1 were fabricated by electrospinning methods, with random and aligned nanofibers as control groups. The Fourier transform infrared spectroscopy and X-ray diffraction analyses confirmed the preservation of P(LLA-CL)/COL components and structure. Scanning electron microscope (SEM) results indicated a significant increase of yarn diameters at 19.27 ± 6.16 μm (dynamic liquid) and 10.24 ± 3.09 μm (conjugated), and both of the nanoyarns had improved mechanical tensile properties than the random nanofibers. Compared with random and aligned nanofibers, the nanoyarns presented significant higher porosity and larger pore diameter, leading to a decrease of water contact angle and a promotion of SMCs proliferation and migration. Better SMCs orientation was observed on the conjugated nanoyarns, while superior SMCs penetration was achieved on the dynamic liquid nanoyarns, owing to the differences in yarns microstructure. Herein, this study demonstrated that the aligned and porous P(LLA-CL)/COL nanoyarns fabricated by dynamic liquid and conjugated electrospinning were beneficial to regulating vascular SMCs outgrowth, which had important implications for functional reconstruction of vascular media.

A Dynamically Tunable, Bioinspired Micropatterned Surface Regulates Vascular Endothelial and Smooth Muscle Cells Growth at Vascularization.

Regulation of the growth of vascular endothelial cells (ECs) and smooth muscle cells (SMCs) with artificial vascular grafts at vascularization is well-known to regenerate functional blood vessels for treating cardiovascular disease; however, little research has been published on this subject. Here, a novel polymer vascular graft is presented, whose inner surface contains an assembled circular microgroove pattern decorated with a combination of concentric circular microgrooves and radial, straight microgrooves inspired by the orientation of SMCs and ECs in natural tissues. The surface micropatterns can produce dynamically tunable variations via the thermally switched shape memory. The results from the in vitro EC/SMC co-cultures reveal that the surface micropatterns have a great capacity to regulate the specific distribution of ECs/SMCs because the ECs grow along the radial, straight microgrooves and the SMCs grow along concentric circular microgrooves. The in vivo vascularization is further analyzed by implanting the vascular graft in the rabbit carotid artery. Both histological analysis and immunofluorescence staining demonstrate that it is capable of highly effectively capturing ECs and SMCs in the blood and subsequent regeneration of new blood vessels. Therefore, this study opens a new possibility for regenerating neovessels to replace and repair damaged vessels for cardiovascular diseases treatment.

Aortic aneurysm in Erdheim's idiopathic cystic medial necrosis in autopsy and forensic medical practice

To date, the modern Russian literature has not covered morphological studies of aortic aneurysm in Erdheim's idiopathic cystic medial necrosis on autopsy and surgical materials, by using immunohistochemical studies, and it has not estimated the magnitude of pathohistochemical changes in the aortic media either. to conduct a morphological examination of aortic aneurysm in Erdheim's idiopathic cystic medial necrosis, by using the material of pathology and forensic medical departments. 41 surgical samples from patients with idiopathic cystic medial necrosis of the ascending aorta and autopsy samples from 17 sudden deaths from aortic rupture were examined. The aortic wall was histologically and histochemically studied. Immunohistochemical examination was done using antibodies to smooth muscle actin, collagens types I and III, elastin, CD3 (T lymphocytes), CD20 (B lymphocytes), CD45 (leukocyte common antigen), CD68 (macrophages), apoptotic marker p53, tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1). Morphological examination showed lamellar unit destruction, fibrosis, medial necrosis, and elastic fiber fragmentation. These signs were assessed by their degree. The immunohistochemical examination of collagens types I and III determined fascicles of randomly positioned fibers. The multidirectional orientation of smooth muscle cells was confirmed by the expression of smooth muscle actin. There was an obvious expression of the apoptotic marker p53 in the smooth muscle cells of the aortic media and in the aortic adventitia. The expression of TNF-α was revealed in the cells of an inflammatory infiltrate in the aneurysm wall and that of ICAM-1 was found in the aortic endothelium, vasa vasorum, and in the cells of an inflammatory infiltrate in the media and adventitia. The paper proposes a design of postmortem and forensic medical diagnosis, which reflects the components of pathogenesis and tanatogenesis in Erdheim's idiopathic cystic medial necrosis and which allows ICD-10 diagnosis coding. The main histopathological signs of aortic aneurysm in Erdheim's idiopathic cystic medial necrosis were presented; structural changes in the primary components of the aortic wall were characterized; the cells of an inflammatory infiltrate were investigated; and the formulation of autopsy and forensic medical diagnoses was proposed.

A method for three-dimensional quantification of vascular smooth muscle orientation: application in viable murine carotid arteries.

When studying in vivo arterial mechanical behaviour using constitutive models, smooth muscle cells (SMCs) should be considered, while they play an important role in regulating arterial vessel tone. Current constitutive models assume a strictly circumferential SMC orientation, without any dispersion. We hypothesised that SMC orientation would show considerable dispersion in three dimensions and that helical dispersion would be greater than transversal dispersion. To test these hypotheses, we developed a method to quantify the 3D orientation of arterial SMCs. Fluorescently labelled SMC nuclei of left and right carotid arteries of ten mice were imaged using two-photon laser scanning microscopy. Arteries were imaged at a range of luminal pressures. 3D image processing was used to identify individual nuclei and their orientations. SMCs showed to be arranged in two distinct layers. Orientations were quantified by fitting a Bingham distribution to the observed orientations. As hypothesised, orientation dispersion was much larger helically than transversally. With increasing luminal pressure, transversal dispersion decreased significantly, whereas helical dispersion remained unaltered. Additionally, SMC orientations showed a statistically significant (p < 0.05) mean right-handed helix angle in both left and right arteries and in both layers, which is a relevant finding from a developmental biology perspective. In conclusion, vascular SMC orientation (1) can be quantified in 3D; (2) shows considerable dispersion, predominantly in the helical direction; and (3) has a distinct right-handed helical component in both left and right carotid arteries. The obtained quantitative distribution data are instrumental for constitutive modelling of the artery wall and illustrate the merit of our method.

Preliminary Evidence for Aortopathy and an X-Linked Parent-of-Origin Effect on Aortic Valve Malformation in a Mouse Model of Turner Syndrome.

Turner syndrome (TS), most frequently caused by X-monosomy (45,X), is characterized in part by cardiovascular abnormalities, including aortopathy and bicuspid aortic valve (BAV). There is a need for animal models that recapitulate the cardiovascular manifestations of TS. Extracellular matrix (ECM) organization and morphometrics of the aortic valve and proximal aorta were examined in adult 39,XO mice (where the parental origin of the single X was paternal (39,XPO) or maternal (39,XMO)) and 40,XX controls. Aortic valve morphology was normal (tricuspid) in all of the 39,XPO and 40,XX mice studied, but abnormal (bicuspid or quadricuspid) in 15% of 39,XMO mice. Smooth muscle cell orientation in the ascending aorta was abnormal in all 39,XPO and 39,XMO mice examined, but smooth muscle actin was decreased in 39,XMO mice only. Aortic dilation was present with reduced penetrance in 39,XO mice. The 39,XO mouse demonstrates aortopathy and an X-linked parent-of-origin effect on aortic valve malformation, and the candidate gene FAM9B is polymorphically expressed in control and diseased human aortic valves. The 39,XO mouse model may be valuable for examining the mechanisms underlying the cardiovascular findings in TS, and suggest there are important genetic modifiers on the X chromosome that modulate risk for nonsyndromic BAV and aortopathy.

Biomechanistic Study of Smooth Muscle Cell Sheet during Circumferential Alignment in Circular Micropatterns.

Engineered functional blood vessels play an important role in tissue regeneration and wound healing, but they remain far from clinical practices because of the complex three-layer capillary histology. In general, the reconstruction of tunica media from vascular smooth muscle cells (SMCs) that mediate vasoconstriction and vasodilation of circulatory system is critical for engineering functional blood vessels. To date, the mechanotransduction during circumferential alignment of SMC layer in tunica media remains to be elucidated. In this study, concentric microwalls fabricated on a polydimethylsiloxane (PDMS) substrate were developed for cell traction force microscopy (CTFM) of SMC layer. First, the cell shape, density, and alignment orientation of SMCs during circumferential cell sheet formation were measured during 7-day cell culture. Second, a collective CTFM assay was carried out to measure the traction forces exerted by the confluent cell layer at various stages of circumferential alignment. It was found that the mechanotransduction of SMC layer grown between the circular microwalls was significantly altered compared to the SMC layer grown on a flat substrate. The results indicated the presence of an optimal region for formation of circumferentially aligned SMC layer verified by both quantitative analysis and biomechanical study. Moreover, the distributions of traction forces and von Mises stresses during cell alignment were affected by the cellular mechanotransduction from the extracellular physical constraints and cell-cell interactions. The immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR) analyses of actin filaments further revealed the relation between the monolayer-generated traction forces and the direction of aligned actin filaments.

Engineering blood vessels through micropatterned co-culture of vascular endothelial and smooth muscle cells on bilayered electrospun fibrous mats with pDNA inoculation

Although engineered blood vessels have seen important advances during recent years, proper mechanical strength and vasoactivity remain unsolved problems. In the current study, micropatterned fibrous mats were created to load smooth muscle cells (SMC), and a co-culture with endothelial cells (EC) was established through overlaying on an EC-loaded flat fibrous mat to mimic the layered structure of a blood vessel. A preferential distribution of SMC was determined in the patterned regions throughout the fibrous scaffolds, and aligned fibers in the patterned regions provided topological cues to guide the orientation of SMC with intense actin filaments and extracellular matrix (ECM) production in a circumferential direction. Plasmid DNA encoding basic fibroblast growth factors and vascular endothelial growth factor were integrated into electrospun fibers as biological cues to promote SMC infiltration into fibrous mats, and the viability and ECM production of both EC and SMC. The layered fibrous mats with loaded EC and SMC were wrapped into a cylinder, and engineered vessels were obtained with compact EC and SMC layers after co-culture for 3months. Randomly oriented ECM productions of EC formed a continuous endothelium covering the entire lumenal surface, and a high alignment of ECM was shown in the circumferential direction of SMC layers. The tensile strength, strain at failure and suture retention strength were higher than those of the human femoral artery, and the burst pressure and radial compliance were in the same range as the human saphenous vein, indicating potential as blood vessel substitutes for transplantation in vivo. Thus, the establishment of topographical cues and biochemical signals in fibrous scaffolds demonstrates advantages in modulating cellular behavior and organization found in complex multicellular tissues.

Three-dimensional vascular smooth muscle orientation as quantitatively assessed by multiphoton microscopy: mouse carotid arteries do show a helix.

Smooth muscle cells (SMCs) play a pivotal role in regulating vascular tone in arteries, and are therefore an essential part of constitutive models of the artery wall. In the present study, we developed a method to quantify in 3D the orientation of SMCs in the intact artery wall. We stained cell nuclei in excised mouse carotid arteries mounted between micropipettes and imaged these in 3D using two-photon laser scanning microscopy. A clustering method was used to identify individual nuclei. Orientations of these nuclei (as a representative of the SMC orientations) were found by calculating the inertia matrix eigenvectors. Subsequently, SMC locations and orientations were converted to cylindrical and spherical coordinate systems, respectively. We found SMCs to be arranged in two distinct layers. For each of these layers, SMC orientations were described by a Bingham distribution. Distributions showed a statistically significant helical and transversal angular component in both inner and outer layers. In conclusion, this study demonstrates that SMC orientation can be quantified in 3D, and shows a distinct helical as well as transversal orientation. The acquired distribution data are essential to improve current constitutive models of the artery wall, by describing physiological SMC orientation and dispersion.

Segmental differences in the orientation of smooth muscle cells in the tunica media of porcine aortae.

The orientation of vascular smooth muscle cells of porcine aortae was assessed to test the widely accepted assumption that these smooth muscle cells are arranged in two helices. We used tangential histological sections of 82 samples of five anatomical segments of thoracic and abdominal porcine aortae and three age groups in animals ranging in age from 5 to 210 days. The distribution of the orientation of smooth muscle cell nuclei in five proximodistal segments of the porcine aortae was determined using an algorithm that fitted a mixture of one to five von Mises probability distributions of the data retrieved from histological micrographs. Automated tracking of the nuclei was confirmed by and consistent with manual histological analysis. The orientation of the vascular smooth muscle cells was successfully fitted using two von Mises distributions in most of the samples with different ages, wall thicknesses, and anatomical positions, which corresponds to two populations of vascular smooth muscle cells. A minor fraction of samples also required a tertiary von Mises distribution to describe the orientation of the smooth muscle cell nuclei. The distribution of vascular smooth muscle cells in five aortic segments ranging from the thoracic ascending aorta to the abdominal intrarenal aorta exhibited similar main directions but different shapes. These results are consistent with the widely used model of two muscular helices intermingling in the arterial wall. Furthermore, we calculated the central angles of symmetry and the mean value of angles between the two assumed smooth muscle directions. We also successfully approximated the orientation of the smooth muscle cells using a mixture of von Mises distributions and our open-source software named dist_mixtures. This method is openly available to researchers who are interested in mathematically assessing the orientation of cell nuclei in various tissues.

Collective cell traction force analysis on aligned smooth muscle cell sheet between three-dimensional microwalls.

During the past two decades, novel biomaterial scaffold for cell attachment and culture has been developed for applications in tissue engineering, biosensing and regeneration medicine. Tissue engineering of blood vessels remains a challenge owing to the complex three-layer histology involved. In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of vascular smooth muscle cells (SMCs) inside the tunica media, which is known to be critical for vasoconstriction and vasodilation of the circulatory system. Until now, there has been a lack of understanding on the mechanotransduction of the SMC layer during the transformation from viable synthetic to quiescent contractile phenotypes. In this study, microfabricated arrays of discontinuous microwalls coated with fluorescence microbeads were developed to probe the mechanotransduction of the SMC layer. First, the system was exploited for stimulating the formation of a highly aligned orientation of SMCs in native tunica medium. Second, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of a polyacrylamide gel layer coated on the discontinuous microwall arrays. Third, the conventional traction force assay for single cell measurement was extended for applications in three-dimensional cell aggregates. Then, the biophysical effects of discontinuous microwalls on the mechanotransduction of the SMC layer undergoing cell alignment were probed. Generally, the cooperative multiple cell-cell and cell-microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite-element modelling. The results show that the traction forces of highly aligned cells lying in the middle region between two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of Von Mises stress during the cell alignment process were dependent on the collective cell layer orientation. Immunostaining of the SMC sheet further demonstrated that the collective mechanotransduction induced by three-dimensional topographic cues was correlated with the reduction of actin and vinculin expression. In addition, the online two-dimensional LC-MS/MS analysis verified the modulation of focal adhesion formation under the influence of microwalls through the regulation in the expression of three key cytoskeletal proteins.

Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers

Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration.

Impact of Cyclic Stretch on Induced Elastogenesis Within Collagenous Conduits

In vitro tissue engineering of vascular conduits requires a synergy between several external factors, including biochemical supplementation and mechanotranductive stimulation. The goal of this study was to improve adult human vascular smooth muscle cell orientation and elastic matrix synthesis within 3D tubular collagen gel constructs. We used a combination of elastogenic factors (EFs) previously tested in our lab, along with cyclic circumferential strains at low amplitude (2.5%) delivered at a range of frequencies (0.5, 1.5, and 3 Hz). After 21 days of culture, the constructs were analyzed for elastic matrix outcomes, activity of matrix metalloproteinases (MMPs)-2 and -9, cell densities and phenotype, and mechanical properties of constructs. While cell densities remained unaffected by the addition of stretch, contractile phenotypic markers were elevated in all stretched constructs relative to control. Constructs cultured with EFs stretched at 1.5 Hz exhibited the maximum elastin mRNA expression and total matrix elastin (over sixfold vs. the static EFs control). MMP-2 content was comparable in all treatment conditions, but MMP-9 levels were elevated at the higher frequencies (1.5 and 3 Hz). Minimal circumferential orientation was achieved and the mechanical properties remained comparable among the treatment conditions. Overall, constructs treated with EFs and stretched at 1.5 Hz exhibited the most elastogenic outcomes.