Breaking the Limit of Cardiovascular Regenerative Medicine: Successful 6-Month Goat Implant in World's First Ascending Aortic Replacement Using Biotube Blood Vessels.

This study investigated six-month outcomes of first models of ascending aortic replacement. The molds used to produce the Biotube were implanted subcutaneously in goats. After 2-3 months, the molds were explanted to obtain the Biotubes (inner diameter, 12 mm; wall thickness, 1.5 mm). Next, we performed ascending aortic replacement using the Biotube in five allogenic goats. At 6 months, the animals underwent computed tomography (CT) and histologic evaluation. As a comparison, we performed similar surgeries using glutaraldehyde-fixed autologous pericardial rolls or pig-derived heterogenous Biotubes. At 6 months, CT revealed no aneurysmalization of the Biotube or pseudoaneurysm formation. The histologic evaluation showed development of endothelial cells, smooth muscle cells, and elastic fibers along the Biotube. In the autologous pericardium group, there was no evidence of new cell development, but there was calcification. The histologic changes observed in the heterologous Biotube group were similar to those in the allogenic Biotube group. However, there was inflammatory cell infiltration in some heterologous Biotubes. Based on the above, we could successfully create the world's first Biotube-based ascending aortic replacement models. The present results indicate that the Biotube may serve as a scaffold for aortic tissue regeneration.

Carotid Artery Bypass Surgery of In-Body Tissue Architecture-Induced Small-Diameter Biotube in a Goat Model: A Pilot Study.

Biotubes are autologous tubular tissues developed within a patient's body through in-body tissue architecture, and they demonstrate high potential for early clinical application as a vascular replacement. In this pilot study, we used large animals to perform implantation experiments in preparation for preclinical testing of Biotube. The biological response after Biotube implantation was histologically evaluated. The designed Biotubes (length: 50 cm, internal diameter: 4 mm, and wall thickness: 0.85 mm) were obtained by embedding molds on the backs of six goats for a predetermined period (1-5 months). The same goats underwent bypass surgery on the carotid arteries using Biotubes (average length: 12 cm). After implantation, echocardiography was used to periodically monitor patency and blood flow velocity. The maximum observation period was 6 months, and tissue analysis was conducted after graft removal, including the anastomosis. All molds generated Biotubes that exceeded the tensile strength of normal goat carotid arteries, and eight randomly selected Biotubes were implanted. Thrombotic occlusion occurred immediately postoperatively (1 tube) if anticoagulation was insufficient, and two tubes, with insufficient Biotube strength (<5 N), were ruptured within a week. Five tubes maintained patency for >2 months without aneurysm formation. The spots far from the anastomosis became stenosed within 3 months (3 tubes) when Biotubes had a wide intensity distribution, but the shape of the remaining two tubes remained unchanged for 6 months. The entire length of the bypass region was walled with an αSMA-positive cell layer, and an endothelial cell layer covered most of the lumen at 2 months. Complete endothelial laying of the luminal surface was obtained at 3 months after implantation, and a vascular wall structure similar to that of native blood vessels was formed, which was maintained even at 6 months. The stenosis was indicated to be caused by fibrin adhesion on the luminal surface, migration of repair macrophages, and granulation formation due to the overproliferation of αSMA-positive fibroblasts. We revealed the importance of Biotubes that are homogeneous, demonstrate a tensile strength > 5 N, and are implanted under appropriate antithrombotic conditions to achieve long-term patency of Biotube. Further, we clarified the Biotube regeneration process and the mechanism of stenosis. Finally, we obtained the necessary conditions for a confirmatory implant study planned shortly.

Pre-implantation evaluation of a small-diameter, long vascular graft (Biotube®) for below-knee bypass surgery in goats.

There are no small-diameter, long artificial vascular grafts for below-knee bypass surgery in chronic limb-threatening ischemia. We have developed tissue-engineered vascular grafts called "Biotubes®" using a completely autologous approach called in-body tissue architecture (iBTA). This study aimed at pre-implantation evaluation of Biotube and its in vivo preparation device, Biotube Maker, for use in below-knee bypass surgery. Forty nine makers were subcutaneously embedded into 17 goats for predetermined periods (1, 2, or 3 months). All makers produced Biotubes as designed without inflammation over all periods, with the exception of a few cases with minor defects (success rate: 94%). Small hole formation occurred in only a few cases. All Biotubes obtained had an inner diameter of 4 mm and a length of 51 to 52 cm with a wall thickness of 594 ± 97 µm. All Biotubes did not kink when completely bent under an internal pressure of 100 mmHg and did not leak without any deformation under a water pressure of 200 mmHg. Their burst strength was 2409 ± 473 mmHg, and suture retention strength was 1.75 ± 0.27 N, regardless of the embedding period, whereas tensile strength increased from 7.5 ± 1.3 N at 1 month to 9.7 ± 2.0 N at 3 months with the embedding period. The amount of water leakage from the needle holes prepared in the Biotube wall was approximately 1/7th of that in expanded polytetrafluoroethylene vascular grafts. The Biotubes could be easily connected to each other without cutting or anastomosis leaks. They could be stored for at least 1 year at room temperature. This study confirmed that even Biotubes formed 1 month after embedding of Biotube Makers had properties comparable to arteries.

Acute Phase Pilot Evaluation of Small Diameter Long iBTA Induced Vascular Graft "Biotube" in a Goat Model.

OBJECTIVE: There is a need for small diameter vascular substitutes in the absence of available autologous material. A small diameter, long tissue engineered vascular graft was developed using a completely autologous approach called "in body tissue architecture technology (iBTA)". The aim of this pilot study was to evaluate "Biotubes", iBTA induced autologous collagenous tubes, for their potential use as small diameter vascular bypass conduits. METHODS: Biotubes (internal diameter 4 mm, length 50 cm, wall thickness 0.85 mm) were prepared by subcutaneous embedding of plastic moulds (Biotube Maker) in three goats for approximately two months. Allogenic Biotubes (length 10 cm [n = 2], 15 cm [n = 2], 22 cm [n = 2]) were bypassed to both carotid arteries by end to side anastomosis with their ligation between the anastomoses in another three goats. Residual Biotubes were examined for their mechanical properties. After four weeks, the harvested Biotubes were evaluated histologically. RESULTS: All Biotubes had sufficient pressure resistance, approximately 3000 mmHg. Although wall thickening occurred at two proximal anastomosis sites, all six grafts were patent without luminal thrombus formation, stenosis, or aneurysm deformation throughout the implantation period. Endothelial cells covered both anastomosis sites almost completely, with partial covering in the central portion of the grafts. Furthermore, α smooth muscle actin positive cells infiltrated the middle layer along almost the entire graft length. CONCLUSION: This preliminary study showed that small diameter, long, tissue engineered Biotubes could function properly as arterial bypass conduits in a large animal for one month without any abnormal change in vascular shape. Thus, small diameter, long Biotubes are potentially viable conduits, which are biocompatible and labour non-intensive, and therefore, suitable for clinical practice. Additionally, Biotubes can start the regeneration process in a short period of time.

Mechanism of pancreatic juice reflux in pancreaticobiliary maljunction: A fluid dynamics model experiment.

BACKGROUND: Pancreatic juice reflux to the common bile duct and gallbladder is observed in the pancreaticobiliary maljunction (PBM), and various pathological conditions occur in the biliary tract. However, the mechanism of pancreatic juice reflux has not been discussed yet. This study aimed to investigate the mechanism of this phenomenon from the perspective of the fluid dynamics theory. METHODS: A fluid dynamics model of PBM without biliary dilatation having gallbladder function and of the pressure of sphincter of Oddi was developed. Water (as bile juice and pancreatic juice) was flowed to these models with a flow rate similar to that in humans. Pancreatic and bile juice flow and bile duct pressure were observed in three phases of gallbladder function. Moreover, the same experiment was performed in the PBM without biliary dilatation model without gallbladder. RESULTS: Pancreatic juice reflux could be observed when the gallbladder was passively expanded with the pressure in the bile duct lower than that in the sphincter of Oddi. However, pancreatic juice reflux was not observed in the model without gallbladder. CONCLUSIONS: Gallbladder function may be strongly involved in pancreatic juice reflux in PBM without biliary dilatation. Cholecystectomy may be able to stop the reflux of pancreatic juice.

Construction of 3 animal experimental models in the development of honeycomb microporous covered stents for the treatment of large wide-necked cerebral aneurysms.

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.

Successful implantation of autologous valved conduits with self-expanding stent (stent-biovalve) within the pulmonary artery in beagle dogs.

OBJECTIVES: To evaluate the functionality of an autologous heart valve with stent (Stent-biovalve or SBV) after implantation in the pulmonic valve position in beagle dogs. ANIMALS: Five beagle dogs. METHODS: A mold with an aperture of a tri-leaflet structure was constructed from a pair of concave and convex rods to which a nitinol (NiTi) stent was mounted. This mold was embedded in a dorsal subcutaneous pouch in beagle dogs for 4 weeks. At the time of the removal, the surfaces of the molds were completely covered with connective tissues, tri-leaflet valves were formed and the NiTi stent was tightly connected to the structure. RESULTS: The mean burst strength of the SBV leaflet was 2710 mmHg (range 2280-3116 mmHg), which was approximately equal to that of the native pulmonic valve leaflet. After implantation in the pulmonary position, the SBV showed good functionality as a pulmonic valve. At 84 days after implantation, the SBV was replaced with autologous fibroblasts and collagenous tissues, and showed organization similar to that of native heart valves. CONCLUSION: Stent-Biovalves achieved good valvular function with laminar flow in the pulmonic valve position of beagle dogs.

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.

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.

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.

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.

Preparation of in-vivo tissue-engineered valved conduit with the sinus of Valsalva (type IV biovalve).

A novel autologous valved conduit with the sinus of Valsalva-defined as a type IV biovalve-was created in rabbits by "in-body tissue-architecture" technology with a specially designed mold for the valve leaflets and the sinus of Valsalva and a microporous tubular scaffold for the conduit. The mold included 2 rods composed of silicone substrates. One was concave shaped, with 3 projections resembling the sinus of Valsalva; the other was convex shaped. The connection between the rods was designed to resemble the closed form of a trileaflet valve. The 2 rods were connected with a small aperture of 500-800 microm, which bound membranous connective tissue obtained from the dorsal subcutaneous layer of a rabbit. The rods were placed in a polyurethane scaffold that had many windows in its center. Both ends of the scaffold were tied with thread for fixation, and this assembly was embedded for 1 month in a subcutaneous pouch in the same Japanese white rabbit from which the connective tissue was obtained. After 1 month, all the surfaces of the implant were found to be completely covered with newly developed connective tissue. The substrates were removed from both sides of the harvested cylindrical implant, and homogenous well-balanced trileaflet-shaped membranous tissue was found inside the developed conduit with 3 protrusions resembling the sinus of Valsalva. The trileaflet valve closed and opened rapidly in synchrony with the backward and forward flow of a pulsatile flow circuit in vitro.

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.

"In vivo tissue-engineered" valved conduit with designed molds and laser processed scaffold.

BACKGROUND: "In body tissue architecture" technology is a practical concept of regenerative medicine that uses the living recipient body's reaction to a foreign object as a reactor for autologous tissue organization. A novel autologous valved conduit was produced by creating a specially designed conduit-mold composite and elastomeric scaffold for this unique in vivo tissue engineering. METHODS: Convex and concave plastic molds assembled with a small aperture of 500-800 microm were inserted into a microporous elastomeric conduit scaffold. The assembly was placed in a subcutaneous pocket of Japanese white rabbits for 1 month. The molds were pulled out from the edge of the harvested implant to obtain the valved conduit. RESULTS: Homogenous and well-balanced trileaflet of membranous tissue was developed in the optimized aperture of molds. The valve leaflet closed and opened rapidly in synchronization with the backward and forward flow of the pulsatile flow circuit in vitro. CONCLUSIONS: A tissue-engineered conduit incorporated with a functional autologous trileaflet valve was developed in an in vivo reactor by optimizing the microstructures of conduit scaffolds and newly designing the composite molds. The method holds promise for a safe, biocompatible, and economical heart valve prosthesis.

Architecture of an in vivo-tissue engineered autologous conduit "Biovalve".

As a practical concept of regenerative medicine, we have focused on in vivo tissue engineering utilizing the foreign body reaction. Plastic substrates for valvular leaflet organization, consisting of two pieces assembled with a small aperture were inserted into a microporous polyurethane conduit scaffold. The assembly was placed in the subcutaneous spaces of Japanese white rabbits for 1 month. After the substrates were pulled out from the harvested implant, valve leaflet-shaped membranous tissue was formed inside the tubular scaffold as designed. The valve leaflet was composed of the same collagen-rich tissue, with the absence of any elastic fiber, as that which had ingrown or covered the scaffold. No abnormal collection or infiltration of inflammatory cells in the leaflet and the scaffold could be demonstrated. According to the immunohistochemical staining, the leaflet was comprised of numerous vimentin- or alpha-SMA-positive cells, corresponding to fibroblasts or myofibroblats, but contained no desmin-positive cells. The analysis of the video data of the valve movement showed that, in synchronization with the backward flow in the diastolic phase, the valve closed rapidly and tightly and, in the transition phase of the flow direction, the valve opened smoothly without flapping or hitting the scaffold wall. Using mold designs, consisting of two different plastic substrates and the tubular scaffold, in conjunction with "in body tissue architecture," the complex 3-dimensional autologous conduit-typed Biovalve was developed for the first time.