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SummaryLoss of muscle stem cell (MuSC) self-renewal with aging reflects a combination of influences from the intracellular (e.g., post-transcriptional modifications) and extracellular (e.g., matrix stiffness) environment. Whereas conventional single cell analyses have revealed valuable insights into factors contributing to impaired self-renewal with age, most are limited by static measurements that fail to capture nonlinear dynamics. Using bioengineered matrices mimicking the stiffness of young and old muscle, we showed that while young MuSCs were unaffected by aged matrices, old MuSCs were phenotypically rejuvenated by young matrices. Dynamical modeling of RNA velocity vector fieldsin silicorevealed that soft matrices promoted a self-renewing state in old MuSCs by attenuating RNA decay. Vector field perturbations demonstrated that the effects of matrix stiffness on MuSC self-renewal could be circumvented by fine-tuning the expression of the RNA decay machinery. These results demonstrate that post-transcriptional dynamics dictate the negative effect of aged matrices on MuSC self-renewal.Graphical AbstractGraphical abstract description:The balance of self-renewal and differentiation in young muscle stem cells (MuSCs) is robust to perturbations of the biophysical microenvironment. In contrast, aged MuSCs are highly sensitive to extrinsic perturbations, and exposure to a youthful microenvironment rejuvenates the self-renewing potential of aged MuSCs by modulating post-transcriptional RNA dynamics.

Cardiovascular disease is the leading cause of death worldwide, often associated with coronary artery occlusion. A common intervention for arterial blockage utilizes a vascular graft to bypass the diseased artery and restore downstream blood flow; however, current clinical options exhibit high long-term failure rates. Our goal was to develop an off-the-shelf tissue-engineered vascular graft capable of delivering a biological payload based on the monocyte recruitment factor C-C motif chemokine ligand 2 (CCL2) to induce remodeling. Bi-layered silk scaffolds consisting of an inner porous and outer electrospun layer were fabricated using a custom blend of Antherea Assama and Bombyx Mori silk (lyogel). Lyogel silk scaffolds alone (LG), and lyogel silk scaffolds containing microparticles (LGMP) were tested. The microparticles (MPs) were loaded with either CCL2 (LGMP+) or water (LGMP-). Scaffolds were implanted as abdominal aortic interposition grafts in Lewis rats for 1 and 8 weeks. 1-week implants exhibited patency rates of 50% (7/14), 100% (10/10), and 100% (5/5) in the LGMP-, LGMP+, and LG groups, respectively. The significantly higher patency rate for the LGMP+ group compared to the LGMP- group (p = 0.0188) suggests that CCL2 can prevent acute occlusion. Immunostaining of the explants revealed a significantly higher density of macrophages (CD68+ cells) within the outer vs. inner layer of LGMP- and LGMP+ constructs but not in LG constructs. After 8 weeks, there were no significant differences in patency rates between groups. All patent scaffolds at 8 weeks showed signs of remodeling; however, stenosis was observed within the majority of explants. This study demonstrated the successful fabrication of a custom blended silk scaffold functionalized with cell-mimicking microparticles to facilitate controlled delivery of a biological payload improving their in vivo performance. Statement of significance This study outlines the development of a custom blended silk-based tissue–engineered vascular graft (TEVG) for use in arterial bypass or replacement surgery. A custom mixture of silk was formulated to improve biocompatibility and cellular binding to the tubular scaffold. Many current approaches to TEVGs include cells that encourage graft cellularization and remodeling; however, our technology incorporates a microparticle based delivery platform capable of delivering bioactive molecules that can mimic the function of seeded cells. In this study, we load the TEVGs with microparticles containing a monocyte attractant and demonstrate improved performance in terms of unobstructed blood flow versus blank microparticles. The acellular nature of this technology potentially reduces risk, increases reproducibility, and results in a more cost-effective graft when compared to cell-based options.

Vascular tissue engineering is aimed at developing regenerative vascular grafts to restore tissue function by bypassing or replacing defective arterial segments with tubular biodegradable scaffolds. Scaffolds are often combined with stem or progenitor cells to prevent acute thrombosis and initiate scaffold remodeling. However, there are limitations to cell-based technologies regarding safety and clinical translation. Extracellular vesicles (EVs) are nanosized particles released by most cell types, including stem and progenitor cells, that serve to transmit protein and RNA cargo to target cells throughout the body. EVs have been shown to replicate the therapeutic effect of their parent cells; therefore, EVs derived from stem or progenitor cells may serve as a more translatable, cell-free, therapeutic base for vascular scaffolds. Our study aims to determine if EV incorporation provides a positive effect on graft patency and remodeling

The success of tissue-engineered vascular graft (TEVG) predominantly relies on the selection of a suitable biomaterial and graft design. Natural biopolymer silk has shown great promise for various tissue-engineering applications. This study is the first to investigate Indian endemic non-mulberry silk (Antheraea assama-AA) – which inherits naturally superior mechanical and biological traits (e.g., RGD motifs) compared to Bombyx mori-BM silk, for TEVG applications. We designed bi-layered biomimetic small diameter AA-BM silk TEVGs adopting a new fabrication methodology. The inner layer showed ideally sized (~40 µm) pores with interconnectivity to allow cellular infiltration, and an outer dense electrospun layer that confers mechanical resilience. Biodegradation of silk TEVGs into amino acids as resorbable byproducts corroborates their in vivo remodeling ability. Following our previous reports, we surgically implanted human adipose tissue-derived stromal vascular fraction (SVF) seeded silk TEVGs in Lewis rats as abdominal aortic interposition grafts for 8 weeks. Adequate suture retention strength (0.45 ± 0.1 N) without any blood seepage post-implantation substantiate the grafts’ viability. AA silk-based TEVGs showed superior animal survival and graft patency compared to BM silk TEVGs. Histological analysis revealed neo-tissue formation, host cell infiltration and graft remodeling in terms of extracellular matrix turnover. Altogether, this study demonstrates promising aspects of AA silk TEVGs for vascular tissue engineering applications. Statement of significance Clinical ‘off the shelf’ implementation of tissue-engineered vascular grafts (TEVGs) remains a challenge. Achieving optimal blood vessel regeneration requires the use of bioresorbable materials having suitable degradation rates while producing minimal or no toxic byproducts. Host cell recruitment and preventing acute thrombosis are other pre-requisites for successful graft remodeling. In this study, for the first time we explored the use of naturally derived Indian endemic non-mulberry Antheraea assama silk in combination with Bombyx mori silk for TEVG applications by adopting a new biomimetic approach. Our bi-layered silk TEVGs were optimally porous, mechanically resilient and biodegradable. In vivo implantation in rat aorta showed long-term patency and graft remodeling by host cell infiltration and extracellular matrix deposition corroborating their clinical feasibility.

OBJECTIVE: To assess ascending aortic distensibility and build geometry and distensibility-based patient-specific stress distribution maps in patients sustaining type A aortic dissection (TAAD) using pre-dissection non-invasive imaging. METHODS: Review of charts from patients undergoing surgical repair of TAAD (n=351) led to the selection of a subset population (n=7) with two or more pre-dissection CTAs and echocardiograms at least one year prior to dissection. Ascending aortic wall biomechanical properties (aortic strain, distensibility and stiffness) were compared to age and size matched non-dissected non-aneurysmal controls. Patient-specific aortic strain served as an input in aortic geometry-based simulated three-dimensional reconstructions to generate longitudinal and circumferential wall stress maps. Inspection of peri-operative dissection scans and intraoperative visual examination confirmed primary tear locations. RESULTS: Pre-dissection echocardiography revealed ascending aortas of patients sustaining TAAD to exhibit decreased aortic wall strain (14.50 ± 1.13% vs 8.49 ± 1.08%; p < 0.01), decreased distensibility (4.26 ± 0.44 vs 2.39 ± 0.33 10(−6)cm(2)dyne(−1); p < 0.01), increased stiffness (3.84 ± 0.24 vs 7.48 ± 1.05; p < 0.001), and increased longitudinal wall stress (246 ± 22 vs 172 ± 37 kPa; p < 0.01). There was no significant difference in circumferential wall stress. Pre-dissection CTA models revealed overlap between regions of increased longitudinal wall stress and primary tear sites. CONCLUSIONS: Using pre-dissection imaging, we identified increased stiffness and longitudinal wall stress in ascending aortas of dissection patients. Patient-specific imaging-derived biomechanical property maps like these may be instrumental toward designing better prediction models of aortic dissection potential.

The aims of the present study were to assess the relative proportion of collagen and elastin in the arterial wall and to evaluate the collagen microstructure from the aortic root to the external iliac artery.Arterial wall tissue samples sampled during post-mortem examination from 16 sites in 14 individuals without aneurysm disease were fixed and stained for collagen and elastin. Stained sections were imaged and analysed to calculate collagen and elastin content as a percentage of overall tissue area. Scanning electron microscopy was used to quantify the collagen microstructure at six specific arterial regions.From the aortic root to the level of the suprarenal aorta, the percentages (area fractions) of collagen (ascending, descending, and suprarenal aorta respectively with 95% confidence interval [CI] 37.5%, 31.7 - 43.2; 38.9%, 33.1 - 44.7; 44.8%, 37.4 - 52.1) and elastin (43.0%, 37.3 - 48.8; 40.3%, 34.8 - 46.1; 32.4%, 25.2 - 39.6) in the aortic wall were similar. From the suprarenal aorta to the internal iliac arteries, the percentage of collagen increased (abdominal aorta, common and internal iliac arteries and external iliac artery respectively with 95% CI 50.6%, 42.7 - 58.7; 51.2%, 45.5 - 56.9; 49.2%, 42.0 - 56.4) reaching a double percentage for elastin (23.6%, 15.7 - 31.6; 20.8%, 15.1 - 26.5; 22.2%, 14.9 - 29.5). Mean collagen fibre diameter (MFD) and average segment length (ASL) were significantly larger in the external iliac artery (MFD 6.03, 95% CI 5.95 - 6.11; ASL 22.21, 95% CI 20.80 - 23.61) than in the ascending aorta (MFD 5.81, 5.72 - 5.89; ASL 19.47, 18.07 - 20.88) and the abdominal aorta (MFD 5.92, 5.84 - 6.00; ASL 21.10, 19.69 - 22.50).In subjects lacking aneurysmal disease, the aorta and iliac arteries are not structurally uniform along their length. There is an increase in collagen percentage and decrease in elastin percentage progressing distally along the aorta. Mean collagen fibre diameter and average segment length are larger in the external iliac artery, compared with the ascending and the abdominal aorta.

This study addresses a crucial gap in the literature by characterising the relationship between urethral tissue mechanics, composition and gross structure. We then utilise these data to develop a biomimetic urethral scaffold with physical properties that more accurately mimic the native tissue than existing gold standard scaffolds; small intestinal submucosa (SIS) and urinary bladder matrix (UBM). Nine human urethra samples were mechanically characterised using pressure-diameter and uniaxial extension testing. The composition and gross structure of the tissue was determined using immunohistological staining. A pressure stiffening response is observed during the application of intraluminal pressure. The elastic and viscous tissue responses to extension are free of regional or directional variance. The elastin and collagen content of the tissue correlates significantly with tissue mechanics. Building on these data, a biomimetic urethral scaffold was fabricated from collagen and elastin in a ratio that mimics the composition of the native tissue. The resultant scaffold is comprised of a dense inner layer and a porous outer layer that structurally mimic the submucosa and corpus spongiosum layers of the native tissue, respectively. The porous outer layer facilitated more uniform cell infiltration relative to SIS and UBM when implanted subcutaneously (p The tissue characterisation data presented herein paves the way for the development of biomimetic urethral grafts, and the novel scaffold we develop demonstrates positive findings that warrant furtherin vivoevaluation.

Background The pia arachnoid complex (PAC) is a cerebrospinal fluid-filled tissue conglomerate that surrounds the brain and spinal cord. Pia mater adheres directly to the surface of the brain while the arachnoid mater adheres to the deep surface of the dura mater. Collagen fibers, known as subarachnoid trabeculae (SAT) fibers, and microvascular structure lie intermediately to the pia and arachnoid meninges. Due to its structural role, alterations to the biomechanical properties of the PAC may change surface stress loading in traumatic brain injury (TBI) caused by sub-concussive hits. The aim of this study was to quantify the mechanical and morphological properties of ovine PAC. Methods Ovine brain samples (n = 10) were removed from the skull and tissue was harvested within 30 min post-mortem. To access the PAC, ovine skulls were split medially from the occipital region down the nasal bone on the superior and inferior aspects of the skull. A template was used to remove arachnoid samples from the left and right sides of the frontal and occipital regions of the brain. 10 ex-vivo samples were tested with uniaxial tension at 2 mm s−1, average strain rate of 0.59 s−1, until failure at Results The elastic modulus, ultimate stress, and ultimate strain were found to be 7.7 ± 3.0, 2.7 ± 0.76 MPa, and 0.60 ± 0.13, respectively. No statistical significance was found across brain dissection locations in terms of biomechanical properties. SHG images were post-processed to obtain average SAT fiber intersection density, concentration, porosity, tortuosity, segment length, orientation, radial counts, and diameter as 0.23, 26.14, 73.86%, 1.07 ± 0.28, 17.33 ± 15.25 µm, 84.66 ± 49.18°, 8.15%, 3.46 ± 1.62 µm, respectively. Conclusion For the sizes, strain, and strain rates tested, our results suggest that ovine PAC mechanical behavior is isotropic, and that the Mooney-Rivlin model is an appropriate curve-fitting constitutive equation for obtaining material parameters of PAC tissues.

High lethality of aortic dissection necessitates accurate predictive metrics for dissection risk assessment. The not infrequent incidence of dissection at aortic diameters

Summary Age‐related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age‐related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age‐related increase in muscle stiffness drives YAP/TAZ‐mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate.

Endovascular coil embolization is now widely used to treat cerebral aneurysms (CA) as an alternative to surgical clipping. It involves filling the aneurysmal sac with metallic coils to reduce flow, induce clotting, and promote the formation of a coil/thrombus mass which protects the aneurysm wall from hemodynamic forces and prevents rupture. However, a significant number of aneurysms are incompletely coiled leading to aneurysm regrowth and/or recanalization. Computational models of aneurysm coiling may provide important new insights into the effects of intrasaccular coil and thrombus on aneurysm wall stresses. Porcine blood and platinum coils were used to construct an in vitro coil thrombus mass (CTM) for mechanical testing. A uniaxial compression test was performed with whole blood clots and CTM, with coil packing densities (CPDs) of 10%, 20%, and 30% to obtain compressive stress/strain responses. A fourth-order polynomial mechanical response function was fit to the experimentally obtained stress/strain responses for each CPD in order to represent their mechanical properties for computational simulations. Patient-specific three-dimensional (3D) geometries of three aneurysms with simple geometry and four with complex geometry were reconstructed from digital subtraction angiography (DSA) images. The CPDs were digitally inserted in the aneurysm geometries and finite element modeling was used to determine transmural peak/mean wall stress (MWS) with and without coil packing. Reproducible stress/strain curves were obtained from compression testing of CTM and the polynomial mechanical response function was found to approximate the experimental stress/strain relationship obtained from mechanical testing to a high degree. An exponential increase in the CTM stiffness was observed with increasing CPD. Elevated wall stresses were found throughout the aneurysm dome, neck, and parent artery in simulations of the CAs with no filling. Complete, 100% filling of the aneurysms with whole blood clot and CPDs of 10%, 20%, and 30% significantly reduced MWS in simple and complex geometry aneurysms. Sequential increases in CPD resulted in significantly greater increases in MWS in simple but not complex geometry aneurysms. This study utilizes finite element analysis to demonstrate the reduction of transmural wall stress following coil embolization in patient-specific computational models of CAs. Our results provide a quantitative measure of the degree to which CPD impacts wall stress and suggest that complex aneurysmal geometries may be more resistant to coil embolization treatment. The computational modeling employed in this study serves as a first step in developing a tool to evaluate the patient-specific efficacy of coil embolization in treating CAs.

Macromolecular components of the vascular extracellular matrix (ECM), particularly elastic fibers and collagen fibers, are critical for the proper physiological function of arteries. When the unique biomechanical combination of these fibers is disrupted, or in the ultimate extreme where fibers are completely lost, arterial disease can emerge. Bioengineers in the realms of vascular tissue engineering and regenerative medicine must therefore ideally consider how to create tissue engineered vascular grafts containing the right balance of these fibers and how to develop regenerative treatments for situations such as an aneurysm where fibers have been lost. Previous work has demonstrated that the primary cells responsible for vascular ECM production during development, arterial smooth muscle cells (SMCs), can be induced to make new elastic fibers when exposed to secreted factors from adipose-derived stromal cells. To further dissect how this signal is transmitted, in this study, the factors were partitioned into extracellular vesicle (EV)-rich and EV-depleted fractions as well as unseparated controls. EVs were validated using electron microscopy, dynamic light scattering, and protein quantification before testing for biological effects on SMCs. In 2D culture, EVs promoted SMC proliferation and migration. After 30 days of 3D fibrin construct culture, EVs promoted SMC transcription of the elastic microfibril gene FBN1 as well as SMC deposition of insoluble elastin and collagen. Uniaxial biomechanical properties of strand fibrin constructs were no different after 30 days of EV treatment versus controls. In summary, it is apparent that some of the positive effects of adipose-derived stromal cells on SMC elastogenesis are mediated by EVs, indicating a potential use for these EVs in a regenerative therapy to restore the biomechanical function of vascular ECM in arterial disease.

Incorporation of collagen structural information into the study of biomechanical behavior of ascending thoracic aortic (ATA) wall tissue should provide better insight into the pathophysiology of ATA. Structurally motivated constitutive models that include fiber dispersion and recruitment can successfully capture overall mechanical response of the arterial wall tissue. However, these models cannot examine local microarchitectural features of the collagen network, such as the effect of fiber disruptions and interaction between fibrous and non-fibrous components, which may influence emergent biomechanical properties of the tissue. Motivated by this need, we developed a finite element based three-dimensional structural model of the lamellar units of the ATA media that directly incorporates the collagen fiber microarchitecture. The fiber architecture was computer generated utilizing network features, namely fiber orientation distribution, intersection density and areal concentration, obtained from image analysis of multiphoton microscopy images taken from human aneurysmal ascending thoracic aortic media specimens with bicuspid aortic valve (BAV) phenotype. Our model reproduces the typical J-shaped constitutive response of the aortic wall tissue. We found that the stress state in the non-fibrous matrix was homogeneous until the collagen fibers were recruited, but became highly heterogeneous after that event. The degree of heterogeneity was dependent upon local network architecture with high stresses observed near disrupted fibers. The magnitude of non-fibrous matrix stress at higher stretch levels was negatively correlated with local fiber density. The localized stress concentrations, elucidated by this model, may be a factor in the degenerative changes in aneurysmal ATA tissue.

Type A Aortic Dissection (TAAD) is a life-threatening condition involving delamination of ascending aortic media layers. While current clinical guidelines recommend surgical intervention for aneurysm diameter > 5.5 cm, high incidence of TAAD in patients below this diameter threshold indicates the pressing need for improved evidence-based risk prediction metrics. Construction of such metrics will require the knowledge of the biomechanical failure properties of the aortic wall tissue under biaxial loading conditions. We utilized a fiber-level finite element based structural model of the aortic tissue to quantify the relationship between aortic tissue strength and physiologically relevant biaxial stress state for nonaneurysmal and aneurysmal patient cohorts with tricuspid aortic valve phenotype. We found that the model predicted strength of the aortic tissue under physiologic biaxial loading conditions depends on the stress biaxiality ratio, defined by the ratio of the longitudinal and circumferential components of the tissue stress. We determined that predicted biaxial tissue strength is statistically similar to its uniaxial circumferential strength below biaxiality ratios of 0.68 and 0.69 for nonaneurysmal and aneurysmal cohorts, respectively. Beyond this biaxiality ratio, predicted biaxial strength for both cohorts reduced drastically to a magnitude statistically similar to its longitudinal strength. We identified fiber-level failure mechanisms operative under biaxial stress state governing aforementioned tissue failure behavior. These findings are an important first step towards the development of mechanism-based TAAD risk assessment metrics for early identification of high-risk patients.

Determination of correlations between transmural mechanical and morphological properties of aorta would provide a quantitative baseline for assessment of preventive and therapeutic strategies for aortic injuries and diseases. A multimodal and multidisciplinary approach was adopted to characterize the transmural morphological properties of descending porcine aorta. Histology and multi-photon microscopy were used for describing the media layer micro-architecture in the circumferential-radial plane, and Fourier Transform infrared imaging spectroscopy was utilized for determining structural protein, and total protein content. The distributions of these quantified properties across the media thickness were characterized and their relationship with the mechanical properties from a previous study was determined. Our findings indicate that there is an increasing trend in the instantaneous Young[U+05F3]s modulus (E), elastic lamella density (ELD), structural protein (SPR), total protein (TPR), and elastin and collagen circumferential percentage (ECP and CCP) from the inner towards the outer layers. Two regions with equal thickness (inner and outer halves) were determined with significantly different morphological and material properties. The results of this study represent a substantial step toward anatomical characterization of the aortic wall building blocks and establishment of a foundation for quantifying the role of microstructural components on the functionality of aorta.

Nerve blocks are frequently performed by anesthesiologists to control pain. For sciatic nerve blocks, the optimal placement of the needle tip between its paraneural sheath and epineurial covering is challenging, even under ultrasound guidance, and frequently results in nerve puncture. We performed needle penetration tests on cadaveric isolated paraneural sheath (IPS), isolated nerve (IN), and the nerve with overlying paraneural sheath (NPS), and quantified puncture force requirement and fracture toughness of these specimens to assess their role in determining the clinical risk of nerve puncture. We found that puncture force (123 ± 17 mN) and fracture toughness (45.48 ± 9.72 J m−2) of IPS was significantly lower than those for NPS (1440 ± 161 mN and 1317.46 ± 212.45 Jm−2, respectively), suggesting that it is not possible to push the tip of the block needle through the paraneural sheath only, without pushing it into the nerve directly, when the sheath is lying directly over the nerve. Results of this study provide a physical basis for tangential placement of the needle as the ideal situation for local anesthetic deposition, as it allows for the penetration of the sheath along the edge of the nerve without entering the epineurium.

Background and Purpose: Ureteral injuries such as avulsion are directly related to mechanical damage of the ureter. Understanding the tensile strength of this tissue may assist in prevention of iatrogenic injuries. Few published studies have looked at the mechanical properties of the animal ureter and, of those, none has determined the tensile strength of the human ureter. Therefore, the purpose of this work was to determine the tensile strength of the human ureter. Methods: We harvested 11 human proximal ureters from patients who were undergoing nephrectomy for either kidney tumors or nonfunctioning kidney. The specimens were then cut into multiple circumferentially and longitudinally oriented tissue strips for tensile testing. Strips were uniaxially stretched to failure in a tensile testing machine. The corresponding force and displacement were recorded. Finally, stress at failure was noted as the tensile strength of the sample. Circumferential tensile strength was also compared in the proximal...

Objectives Patients with bicuspid aortic valves (BAV) are predisposed to developing ascending thoracic aortic aneurysms (TAA) at an earlier age than patients who develop degenerative TAAs and have a tricuspid aortic valve (TAV). The hypothesis tested is that BAV-associated aortopathy is mediated by a mechanism of matrix remodeling that is distinct from that seen in TAAs of patients with tricuspid aortic valves. Methods Aortic specimens were collected during ascending aortic replacement, aortic valve replacement, and heart transplants from nonaneurysmal (NA) donors and recipients. Matrix architecture of the aortic media was assessed qualitatively using multiphoton microscopy followed by quantification of collagen and elastin fiber orientation. α-Elastin was determined and matrix maturity was assessed by quantifying immature and mature collagen and lysyl oxidase (Lox) expression and activity in aortic specimens. Matrix metalloproteinase-2/9 activity was quantified in aortic smooth muscle cells. Results Elastin and collagen fibers were more highly aligned in BAV-NA and BAV-TAA cases than in TAV-TAA cases, whereas TAV-TAA cases were more disorganized than TAV-NA cases. α-Elastin content was unchanged. Immature collagen was reduced in BAV-NA and BAV-TAA cases when compared with TAV-NA and TAV-TAA cases. Mature collagen was elevated in TAV-TAA cases compared with TAV-NA and BAV-TAA cases. There was a trend toward elevated Lox gene expression and activity and matrix metalloproteinase-2/9 activity for TAV-TAA, BAV-NA, and BAV-TAA specimens. Conclusions The highly aligned matrix architecture in patients with BAVs indicates that wall remodeling is distinct from TAV-TAA. Altered matrix architecture and reduced collagen maturity suggest that the effector molecules mediating the remodeling of TAAs are different in BAV and TAV cases.

The aorta possesses a micro-architecture that imparts and supports a high degree of compliance and mechanical strength. Alteration of the quantity and/or arrangement of the main load-bearing components of this micro-architecture - the elastin and collagen fibers - leads to mechanical, and hence functional, changes associated with aortic disease and aging. Therefore, in the future, the ability to rigorously characterize the wall fiber micro-architecture could provide insight into the complicated mechanisms of aortic wall remodeling in aging and disease. Elastin and collagen fibers can be observed using state-of-the-art multi-photon microscopy. Image-analysis algorithms have been effective at characterizing fibrous constructs using various microscopy modalities. The objective of this study was to develop a custom MATLAB-language automated image-based analysis tool to describe multiple parameters of elastin and collagen micro-architecture in human soft fibrous tissue samples using multi-photon microscopy images. Human aortic tissue samples were used to develop the code. The tool smooths, cleans and equalizes fiber intensities in the image before segmenting the fibers into a binary image. The binary image is cleaned and thinned to a fiber skeleton representation of the image. The developed software analyzes the fiber skeleton to obtain intersections, fiber orientation, concentration, porosity, diameter distribution, segment length and tortuosity. In the future, the developed custom image-based analysis tool can be used to describe the micro-architecture of aortic wall samples in a variety of conditions. While this work targeted the aorta, the software has the potential to describe the architecture of other fibrous materials, tube-like networks and connective tissues

We recently reported a mechanistic model to link micro-architectural information to the delamination strength (Sd) of human ascending thoracic aorta (ATA). That analysis demonstrated that the number density (N) and failure energy (Uf) of the radially-oriented collagen fibers contribute to the Sd of both aneurysmal (ATAA) and non-aneurysmal (CTRL-ATA) aortic tissue. Among the set of ATAA samples, we studied specimens from patients displaying bicuspid (BAV) and tricuspid aortic valve (TAV) morphologic phenotypes. Results from our prior work were based on the assumption that the Uf was independent of dissection direction. In the current study, we excluded that assumption and hypothesized that Uf correlates with the Sd of ATAA. To test the hypothesis, we used previously-reported experimentally-determined Sd measurements and N of radially-oriented collagen fibers as input in our validated mechanistic model to calculate Uf for BAV-ATAA, TAV-ATAA and CTRL-ATA tissue specimens. The results of our analysis revealed that Uf is significantly lower for both BAV-ATAA and TAV-ATAA compared to CTRL-ATA cases, and does not differ between BAV-ATAA and TAV-ATAA. Furthermore, we found that Uf is consistent between circumferential-radial and longitudinal-radial planes in either of BAV-ATAA, TAV-ATAA or CTRL-ATA specimens. These findings employ a novel mechanistic model to increase our understanding of the putative interrelationship between biomechanical properties, extracellular matrix biology, and failure energy of aortic dissection.

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues.

Crohn’s disease is a challenging inflammatory process with a propensity for focal gastro-intestinal tract inflammation and stricture. Surgically, Crohn’s is often treated with resection. However, a subtype of diffuse disease with multiple strictures is treated by strictureplasty procedures in hope of avoiding short-gut syndrome. Prior work by Pocivavsek et al. defined the geometry of a Heineke–Mikulicz strictureplasty. Here, we bring this analysis one step closer to clinical and biological relevance by calculating the mechanical stresses and strains that the strictureplasty deformation generates on a model intestinal wall. The small bowel is simulated as a linearly elastic isotropic deformable cylindrical shell using finite element modeling. Data show a divergence in elastic response between the anti-mesenteric and mesenteric halves. The anti-mesenteric surface shows a bending dominated elastic response that correlates with the prior purely geometric analysis. However, the mesenteric half is not a neutral bystander during strictureplasty formation, as geometric arguments predict. Strong in-plane stretching strains develop in a rim around the image of the transverse closure, which may impact local perfusion and serve as sites of disease recurrence. Lastly, nearly all the deformation energy is stored in the central vertex stitch, placing this part at highest risk of dehiscence. This study enhances our understanding of mechanical response in complex nonlinear cylindrical geometries like the surgically manipulated intestinal tract. The developed framework serves as a platform for future addition of more complex clinically relevant parameters to our model, including real tissue properties, anisotropy, blood supply modeling, and patient deriver anatomic factors.

Interleukin 33 (IL-33), an inflammatory and mechanically responsive cytokine, is an important component of a TLR4-dependent innate immune process in mucosal epithelium. Although TLR4 also plays a role in sensing biomechanical stretch, a pathway of stretch-induced TLR4-dependent IL-33 biosynthesis has not been revealed. In the current study, we show that short term (6 h) cyclic stretch (CS) of cultured murine respiratory epithelial cells (MLE-12) increased intracellular IL-33 expression in a TLR4 dependent fashion. There was no detectable IL-33 in conditioned media in this interval. CS, however, increased release of the notable alarmin, HMGB1, and a neutralizing antibody (2G7) to HMGB1 completely abolished the CS mediated increase in IL-33. rHMGB1 increased IL-33 synthesis and this was partially abrogated by silencing TLR4 suggesting additional receptors for HMGB1 are involved in its regulation of IL-33. Collectively, these data reveal a HMGB1/TLR4/IL-33 pathway in the response of respiratory epithelium to mechanical stretch.

Current commercial tensile testing systems use spring-loaded or other compression-based grips to clamp materials in place posing a problem for very soft or delicate materials that cannot withstand this mechanical clamping force. In order to perform uniaxial tensile tests on soft tissues or materials, we have created a novel vacuum-assisted anchor (VAA). Fibrin gels were subjected to uniaxial extension, and the testing data was used to determine material mechanical properties. Utilizing the VAA, we achieved successful tensile breaks of soft fibrin gels while finding statistically significant differences between the mechanical properties of gels fabricated at two different fibrinogen concentrations.

Many preclinical evaluations of autologous small-diameter tissue-engineered vascular grafts (TEVGs) utilize cells from healthy humans or animals. However, these models hold minimal relevance for clinical translation, as the main targeted demographic is patients at high cardiovascular risk such as individuals with diabetes mellitus or the elderly. Stem cells such as adipose-derived mesenchymal stem cells (AD-MSCs) represent a clinically ideal cell type for TEVGs, as these can be easily and plentifully harvested and offer regenerative potential. To understand whether AD-MSCs sourced from diabetic and elderly donors are as effective as those from young nondiabetics (i.e., healthy) in the context of TEVG therapy, we implanted TEVGs constructed with human AD-MSCs from each donor type as an aortic interposition graft in a rat model. The key failure mechanism observed was thrombosis, and this was most prevalent in grafts using cells from diabetic patients. The remainder of the TEVGs was able to generate robust vascular-like tissue consisting of smooth muscle cells, endothelial cells, collagen, and elastin. We further investigated a potential mechanism for the thrombotic failure of AD-MSCs from diabetic donors; we found that these cells have a diminished potential to promote fibrinolysis compared to those from healthy donors. Together, this study served as proof of concept for the development of a TEVG based on human AD-MSCs, illustrated the importance of testing cells from realistic patient populations, and highlighted one possible mechanistic explanation as to the observed thrombotic failure of our diabetic AD-MSC-based TEVGs.

Abdominal aortic aneurysm (AAA) intervention and surveillance is currently based on maximum transverse diameter, even though it is recognized that this might not be the best strategy. About 10% of patients with small AAA transverse diameters, for whom intervention is not considered, still rupture; while patients with large AAA transverse diameters, for whom intervention would have been recommended, have stable aneurysms that do not rupture. While maximum transverse diameter is easy to measure and track in clinical practice, one of its main drawbacks is that it does not represent the whole AAA and rupture seldom occurs in the region of maximum transverse diameter. By following maximum transverse diameter alone clinicians are missing information on the shape change dynamics of the AAA, and clues that could lead to better patient care. We propose here a method to register AAA surfaces that were obtained from the same patient at different time points. Our registration method could be used to track the local changes of the patient-specific AAA. To achieve registration, our procedure uses a consistent parameterization of the AAA surfaces followed by strain relaxation. The main assumption of our procedure is that growth of the AAA occurs in such a way that surface strains are smoothly distributed, while regions of small and large surface growth can be differentiated. The proposed methodology has the potential to unravel different patterns of AAA growth that could be used to stratify patient risks.

Abdominal aortic aneurysm (AAA) is a degenerative disease of the aorta characterized by severe disruption of the structural integrity of the aortic wall and its major molecular constituents. From the early stages of disease, elastin in the aorta becomes highly degraded and is replaced by collagen. Questions persist as to the contribution of collagen content, quality and maturity to the potential for rupture. Here, using our recently developed Fourier transform infrared imaging spectroscopy (FT-IRIS) method, we quantified collagen content and maturity in the wall of AAA tissues in pairs of specimens with different wall stresses. CT scans of AAAs from 12 patients were used to create finite element models to estimate stress in different regions of tissue. Each patient underwent elective repair of the AAA, and two segments of the AAA tissues from anatomic regions more proximal or distal with different wall stresses were evaluated by histology and FT-IRIS after excision. For each patient, collagen content was generally greater in the tissue location with lower wall stress, which corresponded to the more distal anatomic regions. The wall stress/collagen ratio was greater in the higher stress region compared to the lower stress region (1.01 ± 1.09 vs. 0.55 ± 0.084, p = 0.02). The higher stress region also corresponded to the location with reduced intraluminal thrombus thickness. Further, collagen maturity tended to decrease with increased collagen content (p = 0.068, R = 0.38). Together, these results suggest that an increase in less mature collagen content in AAA patients does not effectively compensate for the loss of elastin in the aortic wall, and results in a reduced capability to endure wall stresses.

Objectives: The acute dissection of an ascending thoracic aortic aneurysm (ATAA) represents a devastating separation of elastic layers occurring when the hemodynamic loads on the diseased wall exceed the adhesive strength between layers. At present, the mechanics underlying aortic dissection are largely unclear, and the biomechanical delamination properties of the aneurysmal aorta are not defined. Individuals with bicuspid aortic valve (BAV) are particularly predisposed to ascending aortic aneurysm formation, with a marked risk of aortic dissection. The purpose of this study was to evaluate and compare the dissection properties of nonaneurysmal and aneurysmal human ascending thoracic aorta from patients with BAV morphology or normal tricuspid aortic valve (TAV) morphology using biomechanical delamination testing.Methods: The influence on the delamination strength (S-d) of the aorta associated with BAV was compared with that in patients with TAV. After complete delamination of ATAA tissue samples, tensile tests were performed on each delaminated half for comparison of their tensile strengths.Results: The results showed that the aneurysmal aortas with BAV and TAV have lower S-d than nonaneurysmal aortas and that ATAA with BAV has a lower S-d than that with TAV. We have found a significant difference in S-d between longitudinal and circumferential directions of the nondiseased aorta, suggesting anisotropic dissection properties.Conclusions: The tensile testing results suggest that the weaker intimal half of the aortic wall might fail before the outer adventitial half. Scanning electron microscope analyses suggest different failure modalities of dissection between the two morphologies, and the lower S-d in ATAAs appears to be associated with a disorganized microstructure. BAV ATAAs have a lower S-d than TAV ATAAs, suggesting a greater propensity for aortic dissection. (J Thorac Cardiovasc Surg 2012;143:460-7)

Stress urinary incontinence is often seen in postmenopausal women but limited information is available on hormone dependent changes of urethral function. Thus, we examined how ovariectomy and estrogen replacement affect urethral continence mechanisms.In female nulliparous Sprague-Dawley® rats under urethane anesthesia after ovariectomy with or without estrogen replacement we measured urethral response amplitude during sneezing, urethral baseline pressure and sneeze induced leak point pressure. Whole urethras were tested for ex vivo urethral properties.Urethral response amplitude during sneezing was significantly decreased in 3 and 6-week ovariectomized rats. Urethral baseline pressure was significantly decreased only in 6-week ovariectomized rats. After estrogen replacement urethral baseline pressure but not urethral response amplitude during sneezing was significantly increased. Neither 3-week ovariectomized nor sham operated rats leaked during sneezing but fluid leakage was observed in 63% of 6-week ovariectomized rats. Estrogen replacement decreased the stress urinary incontinence incidence to 25%. Ex vivo testing revealed a significant increase in middle urethral compliance and a decrease in β stiffness at the proximal and middle urethras in 6-week ovariectomized rats.Results indicate that ovariectomy significantly impairs urethral function from the early stage (3 weeks) but does not induce stress urinary incontinence until the late stage (6 weeks). Also, estrogen replacement restores only the urethral baseline pressure parameter, leading to partial prevention of stress urinary incontinence. Since urethral baseline pressure and urethral response amplitude during sneezing parameters are related to urethral smooth and striated muscle activity, respectively, based on our previous studies, hormone replacement therapy may be partially effective for stress urinary incontinence by enhancing smooth muscle mediated urethral activity under stress conditions such as sneezing.

Aims The Sprague–Dawley (SD) rat, an out-bred, all-purpose strain, has served well for lower urinary tract research. However, to test new cellular therapies for conditions such as stress urinary incontinence, an in-bred rat strain with immune tolerance, such as the Lewis rat, may be more useful. The objective of this study was to reveal any differences in lower urinary tract continence mechanisms between the Lewis and SD rat. Methods The contribution of (1) the striated and smooth muscle to the mechanical and functional properties of the urethra in vitro, and (2) the striated sphincter to leak point pressure (LPP) and reflex continence mechanisms in vivo were assessed in normal (control) Lewis and SD rats and in a model of stress urinary incontinence produced by bilateral pudendal nerve transection. Results Control, Lewis rats had significantly lower LPP, significantly less fast-twitch skeletal muscle and relied less on the striated sphincter for continence than control, SD rats, as indicated by the failure of neuromuscular blockade with alpha-bungarotoxin to reduce LPP. Nerve transection significantly decreased LPP in the SD rat, but not in the Lewis rat. Although the Lewis urethra contained more smooth muscle than the SD rat, it was less active in vitro as indicated by a low urethral baseline pressure and lack of response to phenylephrine. Conclusions We have observed distinct differences in functional and mechanical properties of the SD and Lewis urethra and have shown that the Lewis rat may not be suitable as a chronic model of SUI via nerve transection. Neurourol. Urodynam. Neurourol. Urodynam. 30: 1652–1658, 2011. © 2011 Wiley Periodicals, Inc.

Objective Tissue-engineered vascular grafts containing adipose-derived mesenchymal stem cells offer an alternative to small-diameter vascular grafts currently used in cardiac and lower-extremity revascularization procedures. Adipose-derived, mesenchymal stem cell–infused, tissue-engineered vascular grafts have been shown to promote remodeling and vascular homeostasis in vivo and offer a possible treatment solution for those with cardiovascular disease. Unfortunately, the time needed to cultivate adipose-derived mesenchymal stem cells remains a large hurdle for tissue-engineered vascular grafts as a treatment option. The purpose of this study was to determine if stromal vascular fraction (known to contain progenitor cells) seeded tissue-engineered vascular grafts would remain patent in vivo and remodel, allowing for a “same-day” process for tissue-engineered vascular graft fabrication and implantation. Methods Stromal vascular fraction, obtained from adult human adipose tissue, was seeded within 4 hours after acquisition from the patient onto poly(ester urethane)urea bilayered scaffolds using a customized rotational vacuum seeding device. Constructs were then surgically implanted as abdominal aortic interposition grafts in Lewis rats. Results Findings revealed patency in 5 of 7 implanted scaffolds at 8 weeks, along with neotissue formation and remodeling occurring in patent tissue-engineered vascular grafts. Patency was documented using angiography and gross inspection, and remodeling and vascular components were detected using immunofluorescent chemistry. Conclusions A “same-day” cell-seeded, tissue-engineered vascular graft can remain patent after implantation in vivo, with neotissue formation and remodeling occurring by 8 weeks.

Birth trauma and pelvic injury have been implicated in the etiology of stress urinary incontinence (SUI). This study aimed to assess changes in the biomechanical properties and adrenergic-evoked contractile responses of the rat urethra after simulated birth trauma induced by vaginal distension (VD). Urethras were isolated 4 days after VD and evaluated in our established ex vivo urethral testing system that utilized a laser micrometer to measure the urethral outer diameter at proximal, middle, and distal positions. Segments were precontracted with phenylephrine (PE) and then exposed to intralumenal static pressures ranging from 0 to 20 mmHg to measure urethral compliance. After active assessment, the urethra was rendered passive with EDTA and assessed. Pressure and diameter measurements were recorded via computer. Urethral thickness was measured histologically to calculate circumferential stress-strain response and functional contraction ratio (FCR), a measure of smooth muscle activity. VD proximal urethras exhibited a significantly increased response to PE compared with that in controls. Conversely, proximal VD urethras had significantly decreased circumferential stress and FCR values in the presence of PE, suggesting that VD reduced the ability of the proximal segment to maintain smooth muscle tone at higher pressures and strains. Circumferential stress values for VD middle urethral segments were significantly higher than control values. Histological analyses using antibodies against general (protein gene product 9.5) and sympathetic (tyrosine hydroxylase) nerve markers showed a significant reduction in nerve density in VD proximal and middle urethral segments. These results strongly suggest that VD damages adrenergic nerves and alters adrenergic responses of proximal and middle urethral smooth muscle. Defects in urethral storage mechanisms, involving changes in adrenergic regulation, may contribute to stress urinary incontinence induced by simulated birth trauma.

peer-reviewed In the event of abdominal aortic aneurysm (AAA) rupture, the outcome is often death. This paper aims to experimentally identify the rupture locations of in vitro AAA models and validate these rupture sites using finite element analysis (FEA). Silicone rubber AAA models were manufactured using two different materials (Sylgard 160 and Sylgard 170, Dow Corning) and imaged using computed tomography (CT). Experimental models were inflated until rupture with high speed photography used to capture the site of rupture. 3D reconstructions from CT scans and subsequent FEA of these models enabled the wall stress and wall thickness to be determined for each of the geometries. Experimental models ruptured at regions of inflection, not at regions of maximum diameter. Rupture pressures (mean ± SD) for the Sylgard 160 and Sylgard 170 models were 650.6 ± 195.1 mmHg and 410.7 ± 159.9 mmHg, respectively. Computational models accurately predicted the locations of rupture. Peak wall stress for the Sylgard 160 and Sylgard 170 models was 2.15 ± 0.26 MPa at an internal pressure of 650 mmHg and 1.69 ± 0.38 MPa at an internal pressure of 410 mmHg, respectively. Mean wall thickness of all models was 2.19 ± 0.40 mm, with a mean wall thickness at the location of rupture of 1.85 ± 0.33 mm and 1.71 ± 0.29 mm for the Sylgard 160 and Sylgard 170 materials, respectively. Rupture occurred at the location of peak stress in 80% (16/20) of cases and at a high stress regions but not peak stress in 10% (2/20) of cases. 10% (2/20) of models had defects in the AAA which moved the rupture location away from regions of elevated stress. The results presented may further contribute to the understanding of AAA biomechanics and ultimately AAA rupture prediction.