Stretch and stress distributions in the human artery based on two-layer model considering residual stresses.

The objective is to know the stress distributions in the arterial walls under residual stresses based on two-layer model. Human common carotid arteries were analysed to show stress distributions at physiological and supraphysiological intraluminal pressures. The analyses for the loaded states were performed with stretch ratios with reference to a Riemannian stress-free configuration which is a 3D non-Euclidean manifold due to the nonzero Riemann curvature tensor. The experimental data obtained by other literature were used for the common carotid arteries to analyse the stretch and stress distributions in the arterial wall although kinematics is different from the literature. The stretches and stresses were calculated for the unloaded state, i.e. the residual stretches and stresses. And those at the axial stretch ratio 1.1 with reference to the unloaded state were calculated at the intraluminal pressures 16, 50, and 100 kPa. The stresses increased from the inner surface to the outer surface at all pressures analysed. These results suggest that in the human arteries the mechanical loads are mainly supported with the adventitia even though the media and intima play an important role to control of physiological functions.

Three-Dimensional Contractile Mechanics of Artery Accounting for Curl of Axial Strip Sectioned from Vessel Wall.

PURPOSE: It is well known that a sliced ring of arterial wall opens by a radial cut. An axial strip sectioned from arterial wall also curls into an arc. These phenomena imply that there exist residual strains in the circumferential and axial directions. How much do the axial residual strains affect the stress distributions of arterial wall? The aim of the present study is to know stress distributions of arterial wall with the residual strains under the passive and constricted conditions. METHODS: We analyzed the stress distributions under passive and constricted conditions with considering a Riemannian stress-free configuration. In the analysis, we used strain energy functions to describe the passive and active mechanical properties of artery. RESULTS: The present study provided distributions of stretch ratio with reference to the stress-free state (Riemannian stress-free configuration) and stress with and without the curl of axial strip of a homogenous cylindrical arterial model under the passive and constricted smooth muscle conditions. The circumferential and axial stresses with activated smooth muscle (noradrenaline 10-5 M) at the intraluminal pressure 16 kPa and the axial stretch ratio 1.5 with reference to the unloaded vessel decreased by 3.5 and 13.8% at the inner surface with considering the axial residual strain, respectively. CONCLUSIONS: We have shown that the Riemannian stress-free configuration is appropriate tool to analyze stress distributions of arterial wall under passive and activated conditions with the residual stresses.

Biaxial contractile mechanics of common carotid arteries of rabbit.

Few multiaxial constitutive laws under the vasoactive condition have been proposed as compared with those under the passive condition. The biaxial isometric properties of vasoactive rabbit arteries were studied, although the constitutive law was not proposed. The purpose of the present study is also to describe the multiaxial active mechanical properties of arteries. A novel strain energy function for the active stress has been proposed. This function is simple and may describe the multiaxial characteristics of constricted vessels. Although this study used mean stress and mean stretch ratio to determine the mechanical properties of vessels, a triaxial constitutive law of constricted vessels may be developed. There remains the subject of residual strains under active condition. If this problem will be solved, the accurate stress analysis under vasoactive conditions is possible.

Non-Euclidean stress-free configuration of arteries accounting for curl of axial strips sectioned from vessels.

It is well known that arteries are subject to residual stress. In earlier studies, the residual stress in the arterial ring relieved by a radial cut was considered in stress analysis. However, it has been found that axial strips sectioned from arteries also curled into arcs, showing that the axial residual stresses were relieved from the arterial walls. The combined relief of circumferential and axial residual stresses must be considered to accurately analyze stress and strain distributions under physiological loading conditions. In the present study, a mathematical model of a stress-free configuration of artery was proposed using Riemannian geometry. Stress analysis for arterial walls under unloaded and physiologically loaded conditions was performed using exponential strain energy functions for porcine and human common carotid arteries. In the porcine artery, the circumferential stress distribution under physiological loading became uniform compared with that without axial residual strain, whereas a gradient of axial stress distribution increased through the wall thickness. This behavior showed almost the same pattern that was observed in a recent study in which approximate analysis accounting for circumferential and axial residual strains was performed, whereas the circumferential and axial stresses increased from the inner surface to the outer surface under a physiological condition in the human common carotid artery of a two-layer model based on data of other recent studies. In both analyses, Riemannian geometry was appropriate to define the stress-free configurations of the arterial walls with both circumferential and axial residual strains.

Surface density mapping of natural tissue by a scanning haptic microscope (SHM).

To expand the performance capacity of the scanning haptic microscope (SHM) beyond surface mapping microscopy of elastic modulus or topography, surface density mapping of a natural tissue was performed by applying a measurement theory of SHM, in which a frequency change occurs upon contact of the sample surface with the SHM sensor - a microtactile sensor (MTS) that vibrates at a pre-determined constant oscillation frequency. This change was mainly stiffness-dependent at a low oscillation frequency and density-dependent at a high oscillation frequency. Two paragon examples with extremely different densities but similar macroscopic elastic moduli in the range of natural soft tissues were selected: one was agar hydrogels and the other silicon organogels with extremely low (less than 25 mg/cm(3)) and high densities (ca. 1300 mg/cm(3)), respectively. Measurements were performed in saline solution near the second-order resonance frequency, which led to the elastic modulus, and near the third-order resonance frequency. There was little difference in the frequency changes between the two resonance frequencies in agar gels. In contrast, in silicone gels, a large frequency change by MTS contact was observed near the third-order resonance frequency, indicating that the frequency change near the third-order resonance frequency reflected changes in both density and elastic modulus. Therefore, a density image of the canine aortic wall was subsequently obtained by subtracting the image observed near the second-order resonance frequency from that near the third-order resonance frequency. The elastin-rich region had a higher density than the collagen-rich region.

Observation of local elastic distribution in aortic tissues under static strain condition by use of a scanning haptic microscope.

The purpose of this study was to observe variation in the local elastic distribution in aortic tissue walls under different static strain conditions, including physiological strain, by use of a scanning haptic microscope (SHM). Strain was applied by stretching aortic tissues in the circumferential direction by the simple tensile method or by the rod-insertion method to mimic in vivo internal pressure loading. SHM measurements in a saline solution at room temperature were performed on canine thoracic aorta using a glass needle probe with a diameter of ca 5 µm and a scanning area and point pitch of 160 × 80 µm and 2 µm, respectively. Under strain of 0-0.23, corresponding to internal pressure of 0-150 mmHg, wavy-shaped elastin fibers stretched until they were almost straightened, and the average elastic modulus increased almost linearly. Although there was little difference between the images obtained for the two different stretching methods, under high strain (>0.36; 250 mmHg) significant circumferential orientation of the collagen fibrils occurred with an increase in the average elastic modulus. It was concluded that the pressure resistance of the aorta under physiological strain was mainly afforded by elastin fibers; collagen fibrils contributed little except under much higher pressures.

Water-soluble argatroban for antithrombogenic surface coating of tissue-engineered cardiovascular tissues.

Argatroban is a powerful synthetic anticoagulant, but due to its water-insoluble nature, it is unsuitable for use as a coating material to reduce the thrombogenic potential of natural or tissue-engineered blood-contacting cardiovascular tissues. On the other hand, anionic compounds could adsorb firmly onto connective tissues. Therefore, in this study, an anionic form of argatroban was prepared by neutralization from its alkaline solution, dialysis, and freeze-drying. The subsequently obtained argatroban derivative could be easily dissolved in water. Analysis of the surface chemical composition showed that the water-soluble argatroban (WSA) could be adsorbed on the entire surface of tissue-engineered connective tissue sheets composed mainly of collagen. Adsorption was achieved on immersion of the tissue-engineered connective tissue sheet in a saline/WSA solution for only 30 s without any change in the mechanical properties of the tissue-engineered sheets. Complete surface adsorption (ca., 1 mg/cm(2) ) was obtained at WSA concentrations of over 5 mg/mL. WSA adsorption was maintained for at least 7 days with rinsing. Blood coagulation was significantly prevented on the WSA-adsorbed surfaces in acute in vitro experiments. The coating was applied to in vivo tissue-engineered vascular grafts (biotubes) or tri-leaflet tissues (biovalves) under development, ensuring a high likelihood of nonthrombogenicity of their blood-contacting surfaces with high patency, at least in the subchronic phase. It appears that WSA satisfies the initial requirements for a biocompatible aqueous coating material for use in natural or tissue-engineered tissues.

Variations in local elastic modulus along the length of the aorta as observed by use of a scanning haptic microscope (SHM).

Variations in microscopic elastic structures along the entire length of canine aorta were evaluated by use of a scanning haptic microscope (SHM). The total aorta from the aortic arch to the abdominal aorta was divided into 6 approximately equal segments. After embedding the aorta in agar, it was cut into horizontal circumferential segments to obtain disk-like agar portions containing ring-like samples of aorta with flat surfaces (thickness, approximately 1 mm). The elastic modulus and topography of the samples under no-load conditions were simultaneously measured along the entire thickness of the wall by SHM by using a probe with a diameter of 5 µm and a spatial resolution of 2 µm at a rate of 0.3 s/point. The elastic modulus of the wall was the highest on the side of the luminal surface and decreased gradually toward the adventitial side. This tendency was similar to that of the change in the elastin fiber content. During the evaluation of the mid-portion of each tunica media segment, the highest elastic modulus (40.8 ± 3.5 kPa) was identified at the thoracic section of the aorta that had the highest density of elastic fibers. Under no-load conditions, portions of the aorta with high elastin density have a high elastic modulus.

Preparation of well-defined poly(ether-ester) macromers: photogelation and biodegradability.

Two series of poly(ether-ester)-based bis-functional macromers terminated with acrylate groups and a well-defined number of ester bonds were synthesized. One series had a chain of 1, 3 or 5 ester bonds at both ends of the central poly(ethylene glycol) block (molecular weight, about 1000), while the other had an alternating structure of oligo(ethylene glycol) each of them linked to two ester bonds, in which 6 or 10 ester bonds were incorporated equally in the macromer molecules and the total molecular weight was adjusted by about 1000. Irradiation of all poly(ether-ester) macromers mixed with camphorquinone resulted in the formation of gels. Gel yield increased and hydrophilic properties of the gels produced decreased with irradiation time. The elastic modulus of the gels decreased with the number of ester bonds. Upon incubation in a PBS solution (pH 8.04), all gels were gradually degraded with time. At 3 weeks of incubation, the degradation ratio increased linearly with the number of ester bonds per unit of molecular weight of the macromers. The order of in vivo degradation rates determined from weight loss was similar to that of the in vitro study. Thus, these poly(ether-ester) macromers may be useful for biodegradable biomaterials or tissue engineering scaffolds.

Surface elasticity imaging of vascular tissues in a liquid environment by a scanning haptic microscope.

The objective of this study was to make an elasticity distribution image of natural arteries in a liquid environment at high resolution at the micrometer level and at a wide area at the sub-square millimeter level by improving the scanning haptic microscope (SHM), developed previously for characterization of the stiffness of natural tissues. The circumferential sections (thickness, 1.0 mm) of small-caliber porcine arteries (approximately 3-mm diameter) were used as a sample. Measurement was performed by soaking a probe (diameter, 5 microm; spatial resolution, less than 2 microm) in saline solution at an appropriate depth. The vascular tissues were segregated by multi-layering a high elasticity region with mainly elastin (50.8 +/- 13.8 kPa) and a low one with mainly collagen and smooth muscle cells (17.0 +/- 9.0 kPa), as observed previously in high humidity conditions. The elasticity was measured repeatedly with little change for over 4 h in a liquid environment, which enabled observation with maintenance of high precision of a large area of at least 1,200 x 100 microm, whereas the elasticity was increased with time by the dehydration of samples with shrinkage in the air, in which an averaged elasticity in the overall area was approximately doubled within 2 h. This simple, inexpensive system allows observation of the distribution of the surface elasticity at the extracellular matrix level of vascular tissues in a liquid environment close to the natural one.

In vivo evaluation of hydroxyapatite nanocoating on polyester artificial vascular grafts and possibility as soft-tissue compatible material.

The efficacy of hydroxyapatite (HAp) nanocoating on polyester vascular grafts was investigated in animal experiments. The HAp nanocrystals were covalently bonded separately between hydroxyl groups on a nanocrystal and alkoxysilyl groups in gamma-methacryloxypropyl triethoxysilane graft polymerized on a polyester substrate. Twelve HAp-coated polyester grafts and 10 control grafts of 20, 30, or 50 mm in length were implanted in canine common carotid arteries. Serious complications or occlusions were not observed in any of the dogs after implantation. A histologic evaluation was conducted by staining with hematoxylin and eosin (HE), the von Willebrand factor (vWf), and alpha-smooth muscle actin (alpha-SMA) around the inner lumen of the grafts. The number of inflammation cells and giant cells in the HAp-coated group was significantly lower than that in the group receiving noncoated grafts (p < 0.05).

In vitro maturation of "biotube" vascular grafts induced by a 2-day pulsatile flow loading.

Autologous vascular tissues with a small diameter, "biotubes," were developed in vivo using a novel concept in regenerative medicine, "in-body tissue architecture technology." The effect of pulsatile flow in vitro was investigated on the structural and functional properties of the biotubes. Silicone rods (diameter, 3.0 mm; length, 35.0 mm), used as molds, were embedded into dorsal subcutaneous spaces of Wister rats. After 4 weeks, the autologous tubular tissues formed around the rods were harvested. Some tissues were incubated for 2 days under pulsatile flow simulating conditions in the human arteries with small caliber (wall shear stress (WSS), 15.5-77.3 dyn/cm(2); circumferential stress (CS), 0.6-4.5 x 10(5) dyn/cm(2)). Upon flow loading, the sparse, randomly oriented collagen fibers in the biotubes became dense and oriented in the regular circumferential direction. Compliances (beta values) of the control (ca. 30) and flow-loaded (ca. 20) biotubes were equivalent to that of the human coronary arteries and femoral arteries, respectively. Further, upon flow loading, the burst pressure significantly increased from ca. 1000 mmHg to ca. 1800 mmHg, along with the alpha-SMA-positive cell ratio. Pulsatile flow loading in vitro for 2 days could induce biotube maturation in terms of collagen structures and mechanical properties.

Faster and stronger vascular "Biotube" graft fabrication in vivo using a novel nicotine-containing mold.

To accelerate the fabrication of in vivo-tissue engineered autologous vascular prosthetic tissues, the "Biotube," a novel drug-coating mold was designed. The mold was prepared by impregnating nicotine as a model drug into a gelatinous matrix coated on acrylate rods (diameter, 2 mm; length, 20 mm). Upon embedding the molds into dorsal subcutaneous pouches of rats, completely encapsulated Biotubes with significant tissue migration accompanied by rich angiogenesis and having 3.8 times as many neovessels as the uncoated controls, were formed at only 2 weeks. The wall thickness and burst strength of the Biotubes were 399.9 +/- 135.2 microm and 2682.6 +/- 722.6 mmHg, respectively. These values were, respectively, more than 9.6 and 3.2 times greater than the corresponding controls. Therefore, it is confidently expected that the mechanical properties of Biotubes obtained by nicotine coating make them suitable for application as vascular grafts.

Development of salmon collagen vascular graft: mechanical and biological properties and preliminary implantation study.

Elastic salmon collagen (SC) vascular grafts were prepared by incubating a mixture of acidic SC solution and a fibrillogenesis-inducing buffer containing a crosslinking agent [water-soluble carbodiimide (WSC)] in a tubular mold at 4 degrees C for 24 h and then at 60 degrees C for 5 min. Subsequently, re-crosslinking in ethanol solution containing WSC was performed. The dimension of the SC grafts was easily controlled by changing the size of the mold used. The compliance (stiffness parameter: beta) and burst strength of the SC grafts (internal diameter, 2 mm; length, 20 mm; and wall thickness, 0.75 mm) that were prepared for implantation were 18.2 and 1434 mmHg, respectively; both these values were comparable with those of native vessels. Upon placement in rat subcutaneous pouches, the SC grafts were gradually biodegraded with little inflammatory reaction. The SC grafts were preliminarily implanted in rat abdominal aortas by using specially designed vascular connecting system. This system was used because the graft exhibited easy tearing and thus inadequate suturability. There was neither aneurysm formation nor graft rupture, but mild thrombus formation was seen within the 4-week observation period. These grafts may be ideal for use in regenerative medicine because we believe that SC would be completely replaced with native vascular tissues after implantation, although further improvement in the mechanical properties of the graft is needed for anastomosis.

Development of the wing-attached rod for acceleration of "Biotube" vascular grafts fabrication in vivo.

To accelerate the fabrication of in vivo tissue-engineered autologous vascular prosthetic tissues, the "Biotube," a novel wing-attached rod mold was designed for a tissue rolling technique based on a two-step in body tissue incubation (IBTI) process. The new mold consisted of a silicone rod (3-mm diameter, 23-mm length) partly connected to a poly(ethylene terephthalate) film (a wing, 23 x 19 x 0.1 mm). While the molds were embedded into the dorsal subcutaneous pouches of rabbits for 2 weeks (primary IBTI), they were encapsulated fully with thin connective tissues. After removal of the wing materials, the remaining saccular membranous tissues were rolled up on the core tubular tissues that had formed around the silicone rods. Following another 2-week embedding of the assembled tissues (secondary IBTI), the layered tissues fused to each other to form compliant and stiff tubular tissues, "Rolled Biotubes." The wall thickness of the Rolled Biotubes was about 800 microm and the burst strength was about 4000 mmHg, both of which were significantly higher than those of Biotubes prepared by one-step, 4-week IBTI or two-step, 2-week IBTI (p < 0.05). A Rolled Biotube could be applied as middle or large caliber arterial prostheses.

Mechanical responses of a compliant electrospun poly(L-lactide-co-epsilon-caprolactone) small-diameter vascular graft.

To design a "mechano-active" small-diameter artificial vascular graft, a tubular scaffold made of elastomeric poly(L-lactide-co-epsilon-caprolactone) fabrics at different wall thicknesses was fabricated using an electrospinning (ELSP) technique. The wall thickness of the fabricated tube (inner diameter; approximately 2.3-2.5 mm and wall thickness; 50-340 microm) increased proportionally with ELSP time. The wall thickness dependence of mechanical responses including intraluminal pressure-induced inflation was determined under static and dynamic flow conditions. From the compliance-related parameters (stiffness parameter and diameter compliance) measured under static condition, the smaller the wall thickness, the more compliant the tube. Under dynamic flow condition (1 Hz, maximal/minimal pressure of 90 mmHg/45 mmHg) produced by a custom-designed arterial circulatory system, strain, defined as the relative increase in diameter per pulse, increased with the decrease in wall thickness, which approached that of a native artery. Thus, a mechano-active scaffold that pulsates synchronously by responding to pulsatile flow was prepared using elastomeric PLCL as a base material and an ELSP technique.

Mechano-active scaffold design of small-diameter artificial graft made of electrospun segmented polyurethane fabrics.

To fabricate a "mechano-active" tubular scaffold of nonwoven mesh-type small-diameter artificial graft made of the synthetic durable elastomer, segmented polyurethane, the fabrication technique of electrospinning on a mandrel under a high rotation speed and transverse movement was used. Emphasis was placed on how the rotation speed of the mandrel and the fusion or welding states of fibers at contact points affect the compliance (ease of intraluminal pressure-dependent circumferential inflation) and Young's modulus determined by uniaxial stretching in the longitudinal and circumferential directions. The results showed that a high rotation speed is attributed to exhibit isotropic mechanical properties in the entire range of applied strain but reduces the compliance, and a high fusion state, which is produced using a mixed solvent with a high content of high-boiling-point solvent, reduces the compliance but is expected to exhibit high durability in a continuously loaded pulsatile stress field in an arterial circulatory system.

In vivo tissue-engineered small-caliber arterial graft prosthesis consisting of autologous tissue (biotube).

In this study, vascular-like tubular tissues called biotubes, consisting of autologous tissues, were prepared using in vivo tissue engineering. Their mechanical properties were evaluated for application as a small-caliber artificial vascular prosthesis. The biotubes were prepared by embedding six kinds of polymeric rods [poly(ethylene) (PE), poly(fluoroacetate) (PFA), poly(methyl methacrylate) (PMMA), segmented poly(urethane) (PU), poly(vinyl chloride) (PVC), and silicone (Si)] as a mold in six subcutaneous pouches in the dorsal skin of New Zealand White rabbits. For rods apart from PFA, biotubes were constructed after 1 month of implantation by encapsulation around the polymeric implants. The wall thickness of the biotubes ranged from about 50 to 200 microm depending on the implant material and were in the order PFA < PVC < PMMA < PU < PE. As for PE, PMMA, and PVC, the thickness increased after 3 months of implantation and ranged from 1.5-to 2-fold. None of the biotubes were ruptured when a hydrostatic pressure was gradually applied to their lumen up to 200 mmHg. The relationship between the intraluminal pressure and the external diameter, which was highly reproducible, showed a "J"-shaped curve similar to the native artery. The tissue mostly consisted of collagen-rich extracellular matrices and fibroblasts. Generally, the tissue was relatively firm and inelastic for Si and soft for PMMA. For PMMA, PE, and PVC the stiffness parameter (beta value; one of the indexes for compliance) of the biotubes obtained was similar to those of the human coronary, femoral, and carotid arteries, respectively. Biotubes, which possess the ability for wide adjustments in their matrices, mechanics, shape, and luminal surface design, can be applied for use as small-caliber blood vessels and are an ideal implant because they avoid immunological rejection.

Coaxial double-tubular compliant arterial graft prosthesis: time-dependent morphogenesis and compliance changes after implantation.

In order to reduce the compliance mismatch between the native artery and the artificial graft, we have developed a coaxial double-tubular compliant graft, using multiply micropored segmented polyurethane (SPU) thin films, which mimics the relationship between the intraluminal pressure and vessel internal diameter (P-D) of the native artery (termed "J" curve). The graft was coaxially assembled by inserting a high-compliance inner tube with a heparin-immobilized photocured gelatin coating layer into a low-compliance outer tube with a photocured hydrophilic polymer coating layer. Twenty-eight coaxial double-tubular compliant grafts were implanted into the canine common carotid arteries in an end-to-end fashion for up to 12 months. The overall patency rate was 86% (24/28), and neither rupture nor aneurysmal formation was observed. A neoarterial wall was formed via transanastomotic and transmural tissue ingrowth, resulting in neoarterial tissue formation on the luminal surface and into the intertubular space of the double-tubular graft, accompanied by mainly myofibroblasts and inflammatory cells in the early stage and endothelialization and collagen-rich extracellular matrices in the late stage of implantation. Surrounding-tissue adhesion with the outer tube was prevented by the hydrophilic polymer coating. Although the J curve of the implanted prototype model was preserved 1 month after implantation, the impaired J curves were observed because of tissue ingrowth and tissue adhesion between the outer surface of the inner tube and the surrounding tissues 3 and 6 months after implantation. At 12 months after implantation, however, the implanted coaxial double-tubular graft exhibited high compliance due to biodegradation of the SPU films.

Pull-out mechanical measurement of tissue-substrate adhesive strength: endothelial cell monolayer sheet formed on a thermoresponsive gelatin layer.

Although adhesive strength of a single cell on substrates has been reported, the adhesive strength at the tissue-substrate interface has not been reported. However, the tissue-substrate adhesive strength must provide important criteria for performance of implant devices. This article deals with the tissue-substrate adhesive strength for fully endothelialized tissue, which was formed on commercial tissue culture dishes with or without a coating layer of thermoresponsive gelatin (poly(N-isopropylacrylamide)-grafted gelatin, which dissolves in water at room temperature but is precipitated at 37 degrees C). To determine tissue-substrate adhesive strength, a pull-out technique using a glue-coated cover glass was used. The adhesive strength of monolayered tissue on a noncoated dish was approximately 560 Pa or 230 nN/cell at 37 degrees C. For dishes coated with thermoresponsive gelatin, the adhesive strengths were 1050 Pa or 584 nN/cell at 37 degrees C, and 26 Pa or 14 nN/cell at room temperature. For noncoated dishes, delamination occurred mostly at the interface between the extracellular matrix (ECM) secreted by the cells and the dish surface; and for coated dishes, it took place fully at the interface between ECM and the dish surface. This technique enables determination of the adhesive strength between a full monolayered tissue and a substrate.