Masashi Yamanami

Development of a Completely Autologous Valved Conduit With the Sinus of Valsalva Using In-Body Tissue Architecture Technology A Pilot Study in Pulmonary Valve Replacement in a Beagle Model

Background— We developed autologous prosthetic implants by simple and safe in-body tissue architecture technology. We present the first report on the development of autologous valved conduit with the sinus of Valsalva (BIOVALVE) by using this unique technology and its subsequent implantation in the pulmonary valves in a beagle model. Methods and Results— A mold of BIOVALVE organization was assembled using 2 types of specially designed silicone rods with a small aperture in a trileaflet shape between them. The concave rods had 3 projections that resembled the protrusions of the sinus of Valsalva. The molds were placed in the dorsal subcutaneous spaces of beagle dogs for 4 weeks. The molds were covered with autologous connective tissues. BIOVALVEs with 3 leaflets in the inner side of the conduit with the sinus of Valsalva were obtained after removing the molds. These valves had adequate burst strength, similar to that of native valves. Tight valvular coaptation and sufficient open orifice area were observed in vitro. These BIOVALVEs were implanted to the main pulmonary arteries as allogenic conduit valves (n=3). Postoperative echocardiography demonstrated smooth movement of the leaflets with trivial regurgitation. Histological examination of specimens obtained at 84 days showed that the surface of the leaflet was covered by endothelial cells and neointima, including an elastin fiber network, and was formed at the anastomosis sides on the luminal surface of the conduit. Conclusion— We developed the first completely autologous BIOVALVE and successfully implanted these BIOVALVEs in a beagle model in a pilot study.

In-body tissue-engineered aortic valve (Biovalve type VII) architecture based on 3D printer molding

In-body tissue architecture--a novel and practical regeneration medicine technology--can be used to prepare a completely autologous heart valve, based on the shape of a mold. In this study, a three-dimensional (3D) printer was used to produce the molds. A 3D printer can easily reproduce the 3D-shape and size of native heart valves within several processing hours. For a tri-leaflet, valved conduit with a sinus of Valsalva (Biovalve type VII), the mold was assembled using two conduit parts and three sinus parts produced by the 3D printer. Biovalves were generated from completely autologous connective tissue, containing collagen and fibroblasts, within 2 months following the subcutaneous embedding of the molds (success rate, 27/30). In vitro evaluation, using a pulsatile circulation circuit, showed excellent valvular function with a durability of at least 10 days. Interposed between two expanded polytetrafluoroethylene grafts, the Biovalves (N = 3) were implanted in goats through an apico-aortic bypass procedure. Postoperative echocardiography showed smooth movement of the leaflets with minimal regurgitation under systemic circulation. After 1 month of implantation, smooth white leaflets were observed with minimal thrombus formation. Functional, autologous, 3D-shaped heart valves with clinical application potential were formed following in-body embedding of specially designed molds that were created within several hours by 3D printer.

Preparation of a completely autologous trileaflet valve-shaped construct by in-body tissue architecture technology

The aim of this study was to prepare completely autologous heart-valve-shaped constructs without using any artificial scaffold materials by in-body tissue architecture technology, which is a practical concept of regenerative medicine based on the biological defense mechanism against foreign bodies. Silicone rods were used as molds to achieve the tubular shape of the arteries, which were implanted in the subcutaneous spaces of rabbits. After 2 weeks of primary in-body tissue incubation, the silicone rods were completely encapsulated within a thin membranous connective tissue mainly consisting of collagen and having a thickness of approximately 100 microm. To achieve the trileaflet shape of the valve, the cylindrical tissues obtained were rolled up with polyurethane belts cut in the shape of three semi-ovals. The assembled tissues were reimplanted for 2 weeks for secondary incubation. The resulting tissues were over-encapsulated with the newly developed membranous connective tissue having a thickness of approximately 200-400 microm. The newly formed membranes were completely fused to the previously developed inner membrane. After the removal of the two artificial materials, tubular constructs with trileaflet-shaped internal surface were obtained. By controlling the formation of the encapsulating tissue in the two-step in-body tissue incubation process, we were able to develop completely autologous trileaflet valve-shaped constructs.

A completely autologous valved conduit prepared in the open form of trileaflets (type VI biovalve): Mold design and valve function in vitro†

In-body tissue, architecture technology represents a promising approach for the development of living heart valve replacements and preparation of a series of biovalves. To reduce the degree of regurgitation and increase the orifice ratio, we designed a novel mold for a type VI biovalve. The mold had an outer diameter of 14 mm for implantation in beagles, and it was prepared by assembling two silicone rods with a small aperture (1 mm) between them. One rod had three protrusions of the sinus of Valsalva, whereas the other was almost cylindrical. When the molds were embedded in the subcutaneous pouches of beagles for 1 month, the native connective tissues that subsequently developed covered the entire outer surface of the molds and migrated into the aperture between the rods. The mold from both sides of the harvested cylindrical implant was removed, and homogenous well-balanced trileaflets were found to be separately formed in the open form with a small aperture at the three commissure parts inside the developed conduit, which had a thick homogenous wall even in the sinus of Valsalva. Exposure of the obtained biovalves to physiological aortic valve flow in beagles revealed proper opening motion with a wide orifice area. The closure dynamics were suboptimal, probably due to the reduction in the size of the sinus of Valsalva. The mechanical behavior of this biovalve might allow its use as a living aortic valve replacement.

Preparation of an autologous heart valve with a stent (stent-biovalve) using the stent eversion method

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 stents 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.

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.

3-Tesla magnetic resonance angiographic assessment of a tissue-engineered small-caliber vascular graft implanted in a rat.

In the development of small-caliber vascular grafts (diameter; less than 3 mm), animal implantation studies have been mostly performed by using rat abdominal aortas, and their certain patency must evaluate with sacrificing every observation periods, which is both labor-intensive and time-consuming when performing a large number of experiments. This study is the first to demonstrate the application of 3-Tesla contrast-free time-of-flight magnetic resonance angiography (TOF-MRA) in the continuous assessment of the status of a tissue-engineered vascular graft in rat. As a model graft, a single connective tubular tissue (diameter; 1.5 mm), prepared by embedding the silicone rod (diameter; 1.5 mm) into a subcutaneous pouch of a rat for 2 weeks an in vivo tissue-engineering, was used. The graft was implanted in the abdominal aorta (diameter; 1.3 mm) of the rat by end-to-end anastomosis. Repeated TOF-MRA imaging of the graft obtained over a 3-month follow-up period after implantation made it possible to evaluate the patency of the graft, both simply and noninvasively. It also permitted visualization of the connected abdominal aorta and renal and common iliac arteries having smaller caliber (diameter; less than 1 mm). In addition, the degree of the stenosis or aneurysm could also be detected. 3-Tesla MRA allowed the simplified and noninvasive assessment of the status on the vascular graft, including the formation of a stenosis or aneurysm, in the same rat at different times, which will be contributing to enhance the development of tissue-engineered vascular grafts even with small caliber.

Development of tissue-engineered self-expandable aortic stent grafts (Bio stent grafts) using in-body tissue architecture technology in beagles.

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.

First Successful Clinical Application of the In Vivo Tissue-Engineered Autologous Vascular Graft

PURPOSE The ideal material for pediatric pulmonary artery (PA) augmentation is autologous pericardium. However, its utility for multistaged operations is limited. In this study, we applied an in vivo tissue-engineered autologous Biotube graft to a patient with congenital heart disease for the first time. DESCRIPTION For molds of the Biotubes, two silicone 19F drain tubes were embedded in the subcutaneous spaces of a 2-year-old girl with a diagnosis of pulmonary atresia and ventricular septal defect with major aortopulmonary collateral arteries during palliative surgical procedures. When definitive repair was performed after 8 months, the implants were removed to prepare Biotubes, one of which was cut open and autologously implanted into the PA for patch augmentation. EVALUATION Seven months after implantation, the Biotube patch-augmented PA tolerated balloon angioplasty (BAP) for residual stenosis of the peripheral PA. Computed tomography images after BAP showed the well-preserved shape of the Biotube patch-augmented PA. Neither aneurysm formation nor stenosis was observed. CONCLUSIONS The safety and feasibility of Biotubes for pediatric PA patch augmentation are described. Because Biotubes are completely autologous, they may be ideal material for pediatric PA augmentation.