A tissue-engineered, decellularized, connective tissue membrane for allogeneic arterial patch implantation.

BACKGROUND: We have previously applied in vivo tissue-engineered vascular grafts constructed in patients' subcutaneous spaces. However, since the formation of these vascular grafts depends on host health, their application is challenging in patients with suppressed regenerative ability. Therefore, the allogeneic implantation of grafts from healthy donors needs to be evaluated. This study aimed to fabricate allogeneic cardiovascular grafts in animals. MATERIALS AND METHODS: Silicone rod molds were implanted into subcutaneous pouches in dogs; the implants, along with surrounding connective tissues, were harvested after four weeks. Tubular connective tissues were decellularized and stored before they were cut open, trimmed to elliptical sheets, and implanted into the common carotid arteries of another dog as vascular patches (n = 6); these were resected and histologically evaluated at 1, 2, and 4 weeks after implantation. RESULTS: No aneurysmal changes were observed by echocardiography. Histologically, we observed neointima formation on the luminal graft surface and graft wall cell infiltration. At 2 and 4 weeks after implantation, α-SMA-positive cells were observed in the neointima and graft wall. At 4 weeks after implantation, the endothelial lining was observed at the grafts' luminal surfaces. CONCLUSION: Our data suggest that decellularized connective tissue membranes can be prepared and stored for later use as allogeneic cardiovascular grafts.

Genetically Modified Cell Transplantation Through Macroencapsulated Spheroids with Scaffolds to Treat Fabry Disease.

Cell transplantation is expected to be another strategy to treat lysosomal diseases, having several advantages compared to enzyme replacement therapy, such as continuous enzyme secretion and one-time treatment to cure diseases. However, cell transplantation for lysosomal diseases holds issues to be resolved for the clinical field. In this study, we developed a new ex vivo gene therapy platform using a transplant pack, which consists of a porous membrane made of ethylene-vinyl alcohol in the pack-type and spheroids with scaffolds. These membranes have countless pores of less than 0.1 µm2 capable of secreting proteins, including alpha-galactosidase enzyme, and segregating the contents from the host immune system. When the packs were subcutaneously transplanted into the backs of green fluorescent protein (GFP) mice, no GFP-positive cells migrated to the transplanted pack in either autogenic or allogenic mice. The transplanted cells in the pack survived for 28 days after transplantation. When cells overexpressing alpha-galactosidase were used as donor cells for the packs and implanted into Fabry disease model mice, the accumulation of the alpha-galactosidase enzyme was also observed in the livers. In this study, we reported a new ex vivo therapeutic strategy combining macroencapsulation and cellular spheroids with scaffolds. This pack, macroencapsulated spheroids with scaffolds, can also be applied to other types of lysosomal diseases by modifying genes of interest.

Modifications of the mechanical properties of in vivo tissue-engineered vascular grafts by chemical treatments for a short duration.

In vivo tissue-engineered vascular grafts constructed in the subcutaneous spaces of graft recipients have functioned well clinically. Because the formation of vascular graft tissues depends on several recipient conditions, chemical pretreatments, such as dehydration by ethanol (ET) or crosslinking by glutaraldehyde (GA), have been attempted to improve the initial mechanical durability of the tissues. Here, we compared the effects of short-duration (10 min) chemical treatments on the mechanical properties of tissues. Tubular tissues (internal diameter, 5 mm) constructed in the subcutaneous tissues of beagle dogs (4 weeks, n = 3), were classified into three groups: raw tissue without any treatment (RAW), tissue dehydrated with 70% ET (ET), and tissue crosslinked with 0.6% GA (GA). Five mechanical parameters were measured: burst pressure, suture retention strength, ultimate tensile strength (UTS), ultimate strain (%), and Young's modulus. The tissues were also autologously re-embedded into the subcutaneous spaces of the same dogs for 4 weeks (n = 2) for the evaluation of histological responses. The burst pressure of the RAW group (1275.9 ± 254.0 mm Hg) was significantly lower than those of ET (2115.1 ± 262.2 mm Hg, p = 0.0298) and GA (2570.5 ± 282.6 mm Hg, p = 0.0017) groups. Suture retention strength, UTS or the ultimate strain did not differ significantly among the groups. Young's modulus of the ET group was the highest (RAW: 5.41 ± 1.16 MPa, ET: 12.28 ± 2.55 MPa, GA: 7.65 ± 1.18 MPa, p = 0.0185). No significant inflammatory tissue response or evidence of residual chemical toxicity was observed in samples implanted subcutaneously for four weeks. Therefore, short-duration ET and GA treatment might improve surgical handling and the mechanical properties of in vivo tissue-engineered vascular tissues to produce ideal grafts in terms of mechanical properties without interfering with histological responses.

Effects of Short-Duration Ethanol Dehydration on Mechanical Properties of Porcine Pericardium.

PURPOSE: Autologous pericardium is an ideal material for cardiovascular reconstruction including pulmonary artery plasty. Despite the fact that dehydration by ethanol has been used to improve its surgical handling, the effects of the ethanol on mechanical properties of the pericardium have not been previously investigated. The effects of short-duration ethanol dehydration on the mechanical properties of porcine pericardium were evaluated. METHODS: Porcine pericardia (n = 3) were separated into three groups: the raw group with no treatments (RAW), the group immersed in 70% ethanol for 10 min (ET group), and the group immersed in 0.6% glutaraldehyde for 10 min (GA). We measured five parameters of mechanical properties as specified in ISO 7198. RESULTS: ET treatment improved surgical handling as well as GA treatment. There were no significant differences in burst pressure (P = 0.639), suture retention strength (P = 0.529), ultimate tensile strength (UTS; P = 0.486), or Young's modulus (P = 0.408). Only the ultimate strain of the GA group was significantly higher among the three groups (RAW: 33.34% ± 2.02%, ET: 37.48% ± 1.84%, GA: 44.74% ± 2.87%; P = 0.046). CONCLUSIONS: Short-duration ethanol dehydration did not compromise its mechanical properties while maintaining its surgical handling improvements.

Cell Transplantation Combined with Recombinant Collagen Peptides for the Treatment of Fabry Disease.

Fabry disease is caused by a decrease in or loss of the activity of alpha-galactosidase, which causes its substrates globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) to accumulate in cells throughout the body. This accumulation results in progressive kidney injury due to glomerulosclerosis and in heart failure due to hypertrophy. Enzyme replacement therapy (ERT) has been used as the standard therapy for Fabry disease, but it causes a significant financial burden, and regular administration is inconvenient for patients. Because of the short half-life of alpha-galactosidase in vivo, therapeutic methods that can supplement or replace ERT are expected to involve continuous release of alpha-galactosidase, even at low doses. Cell transplantation therapy is one of these methods; however, its use has been hindered by the short-term survival of transplanted cells. CellSaic technology, which utilizes cell spheroids that form after cells are seeded simultaneously with a recombinant collagen peptide scaffold called a µ-piece, has been used to improve cell survival upon implantation. In this study, syngeneic murine embryonic fibroblasts were used to generate CellSaic that were transplanted into Fabry mice. These spheroids survived for 28 days in the renal subcapsular space with forming blood vessels. These results indicate CellSaic technology could be a platform to promote cellular graft survival and may facilitate the development of cell transplantation methods for lysosomal diseases.

Histology and Mechanics of In Vivo Tissue-Engineered Vascular Graft for Children.

PURPOSE: This study sought to evaluate the histologic and mechanical properties of autologous in vivo tissue-engineered vascular grafts (in vivo TEVGs) used for pediatric heart surgery. DESCRIPTION: Molds of in vivo TEVGs made of silicone drain tubes were embedded into subcutaneous spaces in 2 boys during their first operation and were used as patch materials to treat pulmonary artery stenosis during the second operation. The remaining pieces of the patches were evaluated histologically and mechanically. EVALUATION: In vivo TEVGs had very smooth luminal surfaces, and their walls mainly comprised collagen fibers and small numbers of fibroblasts. Mean wall thickness was 200 µm, mean suture retention strength was 2.26 N, and burst pressure was 3057 mm Hg. CONCLUSIONS: Human in vivo TEVGs mainly comprise collagen fibers, and their mechanical properties prove them safe for pulmonary arterioplasty. Therefore, human in vivo TEVGs may be promising alternatives to autologous pericardium for pediatric cardiovascular surgical procedures that often require multistage operations.

Development of xenogeneic decellularized biotubes for off-the-shelf applications.

In earlier studies, we developed in vivo tissue-engineered, autologous, small-caliber vascular grafts, called "biotubes," which withstand systemic blood pressure and exhibit excellent performance as small-caliber vascular prostheses in animal models. However, biotube preparation takes 4 weeks; therefore, biotubes cannot be applied in emergency situations. Moreover, for responses to various types of surgery, grafts should ideally be readily available in advance. The aim of this study was to develop novel, off-the-shelf, small-caliber vascular grafts by decellularizing in vivo tissue-engineered xenogeneic tubular materials. Silicone rod molds (diameter: 2 mm, length: 70 mm) placed in subcutaneous pouches of a beagle dog for 4 weeks were harvested with their surrounding connective tissues. Tubular connective tissues were obtained after pulling out the impregnated molds. Subsequently, they were decellularized by perfusion with sodium dodecyl sulfate and Triton X-100. They were stored as off-the-shelf grafts at -20°C for 1 week. The decellularized grafts derived from the beagle dog were xenogeneically transplanted to the abdominal aortas of rats (n = 3). No signs of abnormal inflammation or immunological problems due to the xenogeneic material were observed. Echocardiography confirmed the patency of the grafts at 1 month after implantation. Histological evaluation revealed that the grafts formed neointima on the luminal surface, and that the graft walls had cell infiltration. Little accumulation of CD68-positive macrophages in the graft wall was observed. Xenogeneic decellularized tubular tissues functioned as small-caliber vascular grafts, as well as autologous biotubes. This technology enables the easy fabrication of grafts from xenogeneic animals in advance and their storage for at least a week, satisfying the conditions for off-the-shelf grafts.

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.

Implantation study of a tissue-engineered self-expanding aortic stent graft (bio stent graft) in a beagle model.

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-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.

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.

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 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.

Delayed-onset systolic anterior motion of the mitral valve after aortic valve replacement for severe aortic stenosis.

Systolic anterior motion (SAM) of the mitral valve after aortic valve replacement (AVR) for severe aortic stenosis (AS) is one of the causes of perioperative left ventricular outflow tract (LVOT) obstruction in older patients. A 90-year-old woman underwent AVR with a 19-mm bioprosthesis for symptomatic aortic valve stenosis. Preoperative transthoracic echocardiography (TTE) showed left ventricular hypertrophy, with LVOT obstruction and mild mitral regurgitation (MR). Intraoperative transesophageal echocardiography and postoperative TTE showed that the degree of MR was unchanged after surgery. The patient's postoperative course was uneventful. However, she developed shortness of breath 6 months after discharge. A subsequent TTE showed significant LVOT obstruction and SAM, which resulted in moderate to severe MR. Because of the patient's advanced age, cibenzoline was administered to decrease the left ventricular pressure gradient (LVPG) and improve the left ventricular diastolic function. Two months after administration of cibenzoline, a TTE showed decreased LVPG, trivial MR, and the absence of SAM. This case clearly demonstrated that cibenzoline improved the SAM of the mitral valve that arose after AVR for AS in a remote postoperative period.

Implantation study of small-caliber "biotube" vascular grafts in a rat model.

We developed autologous vascular grafts, called "biotubes," by simple and safe in-body tissue architecture technology, which is a practical concept of regenerative medicine, without using special sterile conditions or complicated in vitro cell treatment processes. In this study, biotubes of extremely small caliber were first auto-implanted to rat abdominal aortas. Biotubes were prepared by placing silicone rods (outer diameter 1.5 mm, length 30 mm) used as a mold into dorsal subcutaneous pouches in rats for 4 weeks. After argatroban coating, the obtained biotubes were auto-implanted to abdominal aortas (n = 6) by end-to-end anastomosis using a custom-designed sutureless vascular connecting system under microscopic guidance. Graft status was evaluated by contrast-free time-of-flight magnetic resonance angiography (TOF-MRA). All grafts were harvested at 12 weeks after implantation. The patency rate was 66.7 % (4/6). MRA showed little stenosis and no aneurysmal dilation in all biotubes. The original biotube had wall thickness of about 56.2 ± 26.5 µm at the middle portion and mainly random and sparse collagen fibers and fibroblasts. After implantation, the wall thickness was 235.8 ± 24.8 µm. In addition, native-like vascular structure was regenerated, which included (1) a completely endothelialized luminal surface, (2) a mesh-like elastin fiber network, and (3) regular circumferential orientation of collagen fibers and α-SMA positive cells. Biotubes could be used as small-caliber vascular prostheses that greatly facilitate the healing process and exhibit excellent biocompatibility in vascular regenerative medicine.

In vivo evaluation of an in-body, tissue-engineered, completely autologous valved conduit (biovalve type VI) as an aortic valve in a goat model.

Using simple, safe, and economical in-body tissue engineering, autologous valved conduits (biovalves) with the sinus of Valsalva and without any artificial support materials were developed in animal recipients' bodies. In this study, the feasibility of the biovalve as an aortic valve was evaluated in a goat model. Biovalves were prepared by 2-month embedding of the molds, assembled using two types of specially designed plastic rods, in the dorsal subcutaneous spaces of goats. One rod had three projections, resembling the protrusions of the sinus of Valsalva. Completely autologous connective tissue biovalves (type VI) with three leaflets in the inner side of the conduit with the sinus of Valsalva were obtained after removing the molds from both terminals of the harvested implants with complete encapsulation. The biovalve leaflets had appropriate strength and elastic characteristics similar to those of native aortic valves; thus, a robust conduit was formed. Tight valvular coaptation and a sufficient open orifice area were observed in vitro. Biovalves (n = 3) were implanted in the specially designed apico-aortic bypass for 2 months as a pilot study. Postoperative echocardiography showed smooth movement of the leaflets with little regurgitation under systemic circulation (2.6 ± 1.1 l/min). α-SMA-positive cells appeared significantly with rich angiogenesis in the conduit and expanded toward the leaflet tip. At the sinus portions, marked elastic fibers were formed. The luminal surface was covered with thin pseudointima without thrombus formation. Completely autologous biovalves with robust and elastic characteristics satisfied the higher requirements of the systemic circulation in goats for 2 months with the potential for valvular tissue regeneration.

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.

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.

Long-term animal implantation study of biotube-autologous small-caliber vascular graft fabricated by in-body tissue architecture.

A mold for the preparation of an in-body tissue architecture-induced autologous vascular graft, termed "biotube," was prepared by covering a main silicone rod (outer diameter, 3 mm; length, 30 mm) with two pieces of polyurethane sponge tubes (internal diameter, 3 mm; length, 3 mm) at both ends. The molds were embedded into the dorsal subcutaneous pouch of rabbits (weighing ca. 2 kg) for 2 months. After harvesting the rods with the formed surrounding tissues, the rods were removed to create biotubes impregnated with anastomotic reinforcement cuffs at both ends. The biotubes had homogeneous, thin connective tissue wall (thickness, 76 ± 37 µm) that was primarily composed of collagen and fibroblasts. One biotube was loaded with argatroban and autoimplanted in the carotid artery for 26 months. Neither antiplatelet nor anticoagulant agents were administered, except for an intraoperative heparin injection. Follow-up angiography showed no aneurysm formation, rupturing, or stenosis during implantation. At the end of implantation, the wall thickness of biotube (212 ± 24 µm at the anastomosis portion and 150 ± 14 µm at the midportion) was similar to that of native artery (189 ± 23 µm). The luminal surface was completely covered with endothelial cells on the formed lamina elastica interna-like layer. The regenerated vascular walls comprised multilayered smooth muscle cells and dense collagen fibers with regular circumferential orientation. A remarkable multilayered elastin fiber network was observed near the anastomosis portion. Biotubes could thus be used as small-caliber vascular prostheses that greatly facilitate the healing process and exhibit excellent biocompatibility.

In-body optical stimulation formed connective tissue vascular grafts, "biotubes," with many capillaries and elastic fibers.

The autologous biotube, developed by using in-body tissue architecture technology, is one of the most promising small-diameter vascular grafts in regenerative medicine. The walls of the biotubes obtained by a traditional silicone mold-based method were very thin, and this is still the primary obstacle while handling anastomosis, even though these biotubes have adequate pressure resistance ability. This pilot study showed the effect of optical stimulation of subcutaneous tissue formation in the body during the preparation of the biotubes. A blue light-emitting diode (LED) was embedded into a silicone rod as a mold. The biotube was prepared by placing the luminescent molds into the dorsal subcutaneous pouches of a pair of beagles (each weighing ~10 kg) for 2 weeks under photoirradiation. The wall thickness of the obtained biotubes was 506.9 ± 185.7 µm, which was remarkably more than that of the previous biotubes prepared by 2 months of embedding similarly in beagles' subcutaneous pouches (thickness, 77.2 ± 14.8 µm). Many capillaries with smooth muscle cells were infiltrated into the wall and concentrated in the internal layer. Interestingly, the formation of elastic fibers had already started along with collagen fibers, mostly with a regular circumferential orientation. The short-term in-body optical stimulation resulted in the rapid formation of a biotube. These phenomena will allow easy surgical handling and may induce vascular maturation in histology during the acute phase after implantation.

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. CONCLUSIONS: We developed the first completely autologous BIOVALVE and successfully implanted these BIOVALVEs in a beagle model in a pilot study.