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Tracheas have a tubular structure consisting of cartilage rings continuously joined by a connective tissue membrane comprising a capillary network for tissue survival. Several tissue engineering efforts have been devoted to the design of scaffolds to produce complex structures. In this study, we successfully fabricated an artificial materials-free autologous tracheal analogue with engraftment ability by combining in vitro cell self-aggregation technique and in-body tissue architecture. The cartilage rings prepared by aggregating chondrocytes on designated culture grooves that induce cell self-aggregation were alternately connected to the connective tissues to form tubular tracheal analogues by subcutaneous embedding as in-body tissue architecture. The tracheal analogues allogeneically implanted into the rat trachea matured into native-like tracheal tissue by covering of luminal surfaces by the ciliated epithelium with mucus-producing goblet cells within eight months after implantation, while maintaining their structural integrity. Such autologous tracheal analogues would provide a foundation for further clinical research on the application of tissue-engineered tracheas to ensure their long-term functionality.

Biotubes, i.e., in vivo tissue-engineered connective tubular tissues, are known to be effective as vascular replacement grafts with a diameter greater than several millimeters. However, the performance of biotubes with smaller diameters is less clear. In this study, MicroBiotubes with diameters 1 mm were prepared, and their patency was evaluated noninvasively by optical coherence tomography (OCT) and magnetic resonance angiography (MRA). MicroBiotube molds, containing seven stainless wires (diameter 0.5 mm) covered with silicone tubes (outer diameter 0.6 mm) per mold, were embedded into the dorsal subcutaneous pouches of rats. After 2 months, the molds were harvested with the surrounding capsular tissues to obtain seven MicroBiotubes (internal diameter 0.59 ± 0.015 mm, burst pressure 4190 ± 1117 mmHg). Ten-mm-long MicroBiotubes were allogenically implanted into the femoral arteries of rats by end-to-end anastomosis. Cross-sectional OCT imaging demonstrated the patency of the MicroBiotubes immediately after implantation. In a 1-month follow-up MRA, high patency (83.3 %, n = 6) was observed without stenosis, aneurysmal dilation, or elongation. Native-like vascular structure was reconstructed with completely endothelialized luminal surfaces, mesh-like elastin fiber networks, regular circumferential orientation of collagen fibers, and α-SMA-positive cells. Although the long-term patency of MicroBiotubes still needs to be confirmed, they may be useful as an alternative ultra-small-caliber vascular substitute.

The treatment of large or wide-necked cerebral aneurysms is extremely difficult, and carries a high risk of rupture, even when surgical or endovascular methods are available. We are developing novel honeycomb microporous covered stents for treating such aneurysms. In this study, 3 experimental animal models were designed and evaluated quantitatively before preclinical study. The stents were prepared using specially designed balloon-expandable stents (diameter 3.5-5.0 mm, length 16-28 mm) by dip-coating to completely cover their struts with polyurethane film (thickness 20 µm) and microprocessing to form the honeycomb pattern after expansion. (1) In an internal carotid artery canine model (n = 4), all stents mounted on the delivery catheter passed smoothly through the tortuous vessel with minimal arterial damage. (2) In an the large, wide-necked, outer-sidewall aneurysm canine model, almost all parts of the aneurysms had embolized immediately after stenting (n = 4), and histological examination at 2 months revealed neointimal formation with complete endothelialization at all stented segments and entirely organized aneurysms. (3) In a perforating artery rabbit model, all lumbar arteries remained patent (n = 3), with minimal change in the vascular flow pattern for over 1 year, even after placement of a second, overlapping stent (n = 3). At 2 months after stenting, the luminal surface was covered with complete thin neointimal formation. Excellent embolization performance of the honeycomb microporous covered stents without disturbing branching flow was confirmed at the aneurysms in this proof-of-concept study.

Small-diameter biotube vascular grafts developed by in-body tissue architecture had high patency at implantation into rabbit carotid arteries or rat abdominal aortas. However, the thin walls (34 ± 14 μm) of the original biotubes made their implantation difficult into areas with low blood flow volumes or low blood pressure due to insufficient mechanical strength to maintain luminal shape. In this study, caged molds with several windows were designed to prepare more robust biotubes. The molds were assembled with silicone tubes (external diameter 2 mm) and cylindrical covers (outer diameter 7 mm) with 12 linear windows (1 × 9 mm). After the molds were embedded into beagle dorsal subcutaneous pouches for 4 weeks, type C (cage) biotubes were obtained by completely extracting the surrounding connective tissues from the molds and removing the molds. The biotube walls (778 ± 31 μm) were formed at the aperture (width 1 mm) between the silicone rods and the covers by connective cell migration through the windows of the covers. Excellent mechanical properties (external pressure resistance, approximately 4 times higher than beagle native femoral arteries; burst strength, approximately 2 times higher than original biotubes) were obtained. In the acute phase of implantation of the biotubes into beagle femoral arteries, perfect patency was obtained with little stenosis and no aneurysmal dilation. The type C biotubes may be useful for implantation into peripheral arteries or veins in addition to aortas.

In autologous valved conduits (biovalves) using in-body tissue architecture, the limited area available for leaflet formation is a concern. In this study, we designed a novel biovalve mold with slits to enhance in vivo cell migration, regardless of size. As a control, the original mold without slits was used. When both types of molds were embedded into subcutaneous pouches in beagle dogs for 8 weeks, the outer surfaces of all molds were completely covered with connective tissue to form conduit tissue. In the molds without slits, the leaflet size was limited to half of the design. In contrast, in the mold with slits, the complete leaflet area was formed. Upon trimming excess peripheral tissues, removing the mold, and cutting the connective tissue formed at the slits, completely autologous connective tissue biovalves with the designed leaflet area were obtained as type S (diameter, 6-28 mm) biovalves. The slit structure customized to the mold was effective for allowing cells to enter, thereby facilitating cell migration and contributing to the successful preparation of reliable biovalves of various physiological sizes suitable for all clinical uses.

We developed an autologous, trileaflet tissue valve ("biovalve") using in-body tissue architecture technology to overcome the disadvantages of current bioprosthetic valves. We designed a novel biovalve with a balloon-expandable stent: the stent biovalve (SBV). This study evaluated the technical feasibility of sutureless aortic valve replacement using the SBV in an orthotopic position, as well as the functionality of the SBV under systemic circulation, in an acute experimental goat model. Three adult goats (54.5-56.1 kg) underwent sutureless AVR under cardiopulmonary bypass (CPB). The technical feasibility and functionality of the SBVs were assessed using angiography, pressure catheterization, and two-dimensional echocardiography. The sutureless AVR was successful in all goats, and all animals could be weaned off CPB. The mean aortic cross-clamp time was 45 min. Angiogram, after weaning the animals off CPB, showed less than mild paravalvular leakage and central leakage was not detected in any of the goats. The mean peak-to-peak pressure gradient was 6.3 ± 5.0 mmHg. Epicardial two-dimensional echocardiograms showed smooth leaflet movement, including adequate closed positions with good coaptation; the open position demonstrated a large orifice area (average aortic valve area 2.4 ± 0.1 cm2). Sutureless AVR, using SBVs, was feasible in a goat model. The early valvular functionalities of the SBV were sufficient; future long-term experiments are needed to evaluate its durability and histological regeneration potential.

The use of stent grafts for endovascular aortic repair has become an important treatment option for aortic aneurysms requiring surgery. This treatment has achieved excellent outcomes; however, problems like type 1 endoleaks and stent graft migration remain. Bio stent grafts (BSGs), which are self-expanding stents covered with connective tissue, were previously developed using "in-body tissue architecture" technology. We assessed their early adaptation to the aorta after transcatheter implantation in a beagle model. BSGs were prepared by subcutaneous embedding of acryl rods mounted with self-expanding nitinol stents in three beagles for 4 weeks (n = 3/dog). The BSGs were implanted as allografts into infrarenal abdominal aortas via the femoral artery of three other beagles. After 1 month of implantation, aortography revealed no stenosis or aneurysmal changes. The luminal surface of the BSGs was completely covered with neointimal tissue, including endothelialization, without any thrombus formation. The cover tissue could fuse the luminal surface of the native aorta with tight conjunctions even at both ends of the stents, resulting in complete impregnation of the strut into the reconstructed vascular wall, which is expected to prevent endoleaks and migration in clinical applications.

In this study, we aimed to describe the development of tissue-engineered self-expandable aortic stent grafts (Bio stent graft) using in-body tissue architecture technology in beagles and to determine its mechanical and histological properties. The preparation mold was assembled by insertion of an acryl rod (outer diameter, 8.6 mm; length, 40 mm) into a self-expanding nitinol stent (internal diameter, 9.0 mm; length, 35 mm). The molds (n = 6) were embedded into the subcutaneous pouches of three beagles for 4 weeks. After harvesting and removing each rod, the excessive fragile tissue connected around the molds was trimmed, and thus tubular autologous connective tissues with the stent were obtained for use as Bio stent grafts (outer diameter, approximately 9.3 mm in all molds). The stent strut was completely surrounded by the dense collagenous membrane (thickness, ∼150 µm). The Bio stent graft luminal surface was extremely flat and smooth. The graft wall of the Bio stent graft possessed an elastic modulus that was almost two times higher than that of the native beagle abdominal aorta. This Bio stent graft is expected to exhibit excellent biocompatibility after being implanted in the aorta, which may reduce the risk of type 1 endoleaks or migration.

We designed a novel method for constructing an autologous heart valve with a stent, called a stent-biovalve. In constructing completely autologous heart valves, named biovalves, which used in-body tissue architecture technology, tissues for leaflets were formed via ingrowths into narrow apertures in the preparation molds, frequently leading to delayed or incomplete biovalve preparation. In this technique, self-expandable nitinol stents after everting were mounted on an acrylic column-shaped part and partially covered with an acrylic cylinder-shaped part with three slits. This assembled mold was placed into subcutaneous abdominal pouches in beagles or goats for 4 weeks. Upon removing the acrylic parts after harvesting and trimming of capsulated tissues, a tubular hollow structure with three pocket-flaps of membranous tissue rigidly fixed to the stent's outer surface was obtained. Then, the stent was turned inside out to the original form, thus moving the pocket-flaps from outside to the inside. Stent-biovalves with a sufficient coaptation area were thus obtained with little tissue damage in all cases. The valve opened smoothly, and high aperture ratio was noted. This novel technique was thus highly effective in constructing a robust, completely autologous stent-biovalve with adequate valve function.

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.

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.

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.

Fructose-U-C 14 and glucose-U-C 14 have been incubated with hemidiaphragm and intact rat diaphragm from normal and diabetic rats in the absence and presence of insulin. In hemidiaphragms from normal and diabetic rats, glucose and fructose metabolic patterns were similar. Insulin increased the incorporation of C 14 into glycogen, but not oxidation to CO 2 . In the intact diabetic diaphragm, glucose oxidation was decreased below normal and insulin increased both oxidation to CO 2 and incorporation into glycogen. Fructose was metabolized to a lesser extent than glucose as measured by these parameters, but hexose uptake was as great in the diabetic as in the normal rat diaphragm. Glucose depressed fructose metabolism, but the presence of fructose had no effect on glucose metabolism. The results are in accord with the occurrence of two separate actions of insulin in the diaphragm: one on cell wall permeability and a second on glycogen synthesis.