Three-month outcomes of aortic valve reconstruction using collagenous membranes (biosheets) produced by in-body tissue architecture in a goat model: a preliminary study.

BACKGROUND: Autologous pericardium is widely used as a plastic material in intracardiac structures, in the pulmonary artery, and in aortic valve leaflets. For aortic valve reconstruction (AVRec) using the Ozaki procedure, it has produced excellent clinical results over a 10-year period. In-body tissue architecture (iBTA), which is based on the phenomenon of tissue encapsulation of foreign materials, can be used to prepare autologous prosthetic tissues. In this preliminary study, we examined whether biosheets can be used as valve leaflet material for glutaraldehyde-free AVRec by subchronic implantation experiments in goats and evaluated its performance compared with glutaraldehyde-treated autologous pericardium for AVRec. METHODS: Biosheets were prepared by embedding molds for two months into the dorsal subcutaneous spaces of goats. Autogenic biosheets (n = 4) cut into the shape of the valve were then implanted to the aortic valve annulus of four goats for three months without glutaraldehyde treatment. Autologous pericardium (n = 4) was used in four goats as a control. Valve function was observed using echocardiography. RESULTS: All goats survived the three-month study period. With biosheets, the leaflet surfaces were very smooth and, on histology, partially covered with a thin neointima (including endothelial cells). Biosheets were more thoroughly assimilated into the aortic root compared with autologous pericardium. CONCLUSIONS: For the first time, biosheets were used for large animal AVRec. Biosheets could function as leaflets in the aortic position and may have the ability to assimilate into native valves.

Gorget-like Cuddling Suture: An Anastomosis Reinforcement Technique to Reduce Suture Bleeding for Aortic Surgery.

We developed a new reinforcement technique, the Gorget-like Cuddling (GOCU) suture, to prevent suture line bleeding during aortic surgery. After continuous aortic anastomosis with thick outer felt, an additional 2-0 Ticron (Medtronic, Minneapolis, MN) suture is placed distal from the first suture line. This GOCU suture directly holds the needle holes. Wall tension on the anastomosis can also be reduced to prevent longitudinal dilatation of the aorta. This technique can contribute to hemostasis for a fragile aortic wall in cases like acute aortic dissection.

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.

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.

Bioprosthetic Valve Deterioration: Accumulation of Circulating Proteins and Macrophages in the Valve Interstitium.

Histologic evaluations revealed excessive accumulations of macrophages and absence of fibroblastic interstitial cells in explanted bioprosthetic valves. Comprehensive gene and protein expression analysis and histology unveiled an accumulation of fibrinogen and plasminogen, an activator of infiltrated macrophages, from degenerated valve surfaces in the interstitial spaces. These pathologies were completely reproduced in a goat model replaced with an autologous pericardium-derived aortic valve. Further preclinical animal experiments using goats demonstrated that preventing infiltration of macrophages and circulating proteins by increasing collagen density and leaflet strength is an effective treatment option.

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.

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.

Femoral artery anatomy is a risk factor for limb ischemia in minimally invasive cardiac surgery.

OBJECTIVE: In minimally invasive cardiac surgery (MICS), femoral artery cannulation during cardiopulmonary bypass (CPB) can cause limb ischemia. This study evaluated the association between femoral artery anatomy and the risk of limb ischemia in MICS. METHODS: Eighty-one patients who underwent MICS with CPB using single femoral artery cannulation between 2010 and 2018 were included. The patients were stratified by their femoral artery diameter and anatomy of ectopic side branch, i.e., medial or lateral femoral circumflex arteries: Type A, deep femoral artery (DFA) ≥ superficial femoral artery (SFA); type B, DFA < SFA with an ectopic side branch of the common femoral artery (CFA); type C, DFA < SFA with an ectopic side branch at the CFA bifurcation; and type D, DFA < SFA without an ectopic side branch. The ratio of the postoperative creatine kinase concentration and the cross-sectional area of the femoral muscles (CK/MA) was used as a surrogate marker of limb ischemia. Predictors of high CK/MA were evaluated. RESULTS: No critical limb ischemia was observed in this study. The median postoperative creatine kinase and CK/MA were 1954 (1305-2872) IU/l and 15.2 (9.2-19.8) IU/l/cm2. Multivariable logistic regression found that anatomical type D (odds ratio 4.19, 95% confidence interval: (1.26-14.0); p = 0.020) and prolonged CPB time (OR 1.01, 95% CI (1.00-1.02); p = 0.045) were independent risk factors of high CK/MA. CONCLUSION: Anatomical type D and prolonged CPB time were associated with risk of limb ischemia in MICS.

Aortic valve neocuspidization with in-body tissue-engineered autologous membranes: preliminary results in a long-term goat model.

OBJECTIVES: Aortic valve neocuspidization has shown satisfactory clinical outcomes; however, autologous pericardium durability is a concern for young patients. This study applied an autologous collagenous membrane (Biosheet®), produced by in-body tissue architecture, to aortic valve neocuspidization and investigated its long-term outcome in a goat model. METHODS: Moulds were embedded subcutaneously in 6 goats. After 2 months, Biosheets formed in the moulds. We performed aortic valve neocuspidization using a portion of the sheets with a thickness of 0.20-0.35 mm, measured by optical coherence tomography. Animals were subjected to echocardiography and histological evaluation at 6 months (n = 3) and 12 months (n = 3). As a control, the glutaraldehyde-treated autologous pericardium was used in 4 goats that were similarly evaluated at 12 months. RESULTS: All animals survived the scheduled period. At 6 months, Biosheets maintained valve function and showed a regeneration response: fusion to the annulus, cell infiltration to the leaflets and appearance of elastic fibres at the ventricular side. After 12 months, the regenerative structure had changed little without regression, and there was negligible calcification in the 1/9 leaflets. However, all cases had one leaflet tear, resulting in moderate-to-severe aortic regurgitation. In the pericardium group, three-fourths of the animals experienced moderate-to-severe aortic regurgitation with a high rate of calcification (9/12 leaflets). CONCLUSIONS: Biosheets may have regeneration potential and anti-calcification properties in contrast to autologous pericardium. However, in order to obtain reliable outcome, further improvements are required to strictly control and optimize its thickness, density and homogeneity.

Pseudoaneurysm of the Left Common Carotid Artery With Neurofibromatosis Type 1.

We describe a 54-year-old man with neurofibromatosis type 1 who presented with a left-sided neck mass. Computed tomography demonstrated a left common carotid artery aneurysm (51 × 33 mm). Surgery was performed because of the risk of rupture. The left common carotid artery was found to be a huge aneurysmal dilatation, and the arterial wall partially collapsed and extensively adherent to the surrounding tissues. Left common carotid artery to internal carotid artery bypass grafting was performed with a reversed saphenous vein graft. Histopathological examination revealed vascular fragility of the left carotid arterial wall.

Mechanical characterization of an in-body tissue-engineered autologous collagenous sheet for application as an aortic valve reconstruction material.

The reconstruction of the aortic valve using glutaraldehyde-treated autologous pericardium is known as "aortic valve neo-cuspidization" (AVNeo). In-body tissue architecture (iBTA), a cell-free, in vivo tissue-engineering technology that can form autologous implantable tissues of the desired shape by subcutaneous embedding specially designed molds, was used to prepare sheet-like collagenous tissues called "Biosheets". Cylindrical molds with several line slits arranged in an alternating (n = 30) or parallel (n = 36) pattern were subcutaneously embedded in goats (n = 12) for 2 or 3 months. The tubular tissues formed in the molds were dried and then cut in the longitudinal direction, thus obtaining Biosheets (5 × 7 cm). The success rate was 97.6% when using the alternating-pattern molds and 97.2% for the parallel molds. Thickness mapping of the Biosheets showed that their entire surface, except for the line-projection portions, was smooth without any defects. The average wall thickness could be controlled over a range of ca. 0.2-0.5 mm by changing the size of the gap (0.75-1.5 mm) in the molds. The alternating slit-patterned Biosheets were found to be almost isotropic in their mechanical properties (ultimate tensile strength, fracture strain, and Young's modulus). Although the composition of the Biosheet wall was heterogeneous in terms of its density (which varied with the thickness), the breaking strength of all the alternating-patterned Biosheets increased almost linearly with the thickness within the range of the thickness of clinically used glutaraldehyde-treated pericardium as a control, and was larger than that of human aortic valve leaflets. Therefore, the alternating-patterned Biosheets have potential for use in an alternative aortic leaflet material in AVNeo.

In vitro evaluation of the hemostatic effect of method involving the combined use of Hydrofit® and Spongel®.

OBJECTIVE: We developed an effective hemostatic method using Hydrofit® and a hemostatic gelatin sponge (Spongel®). We evaluated the hemostatic effect in comparison to the conventional silicone sheet method. METHODS: A simulated circuit was created using the pump of a Nipro ventricular assist system and a prosthetic graft. A hole was made in the graft by a needle and three hemostatic methods were applied: the silicone sheet method (SS) using Hydrofit® and a silicone sheet, the bread and butter method (BB) using Hydrofit® and a gelatin sponge instead of a silicone sheet, and French toast method (FT) using Hydrofit® and a gelatin sponge over which water was poured before compression. The amount of leakage before and after the application each of the methods was measured according to the compression time. RESULTS: In the 60 s compression, the amount of leakage after SS, BB, and FT was 0.4 ± 0.8, 0.2 ± 0.6, and 0 ± 0.0 ml, respectively, and FT showed no leakage. In the 30 s compression, the amount of leakage after SS, BB, and FT was 14.2 ± 27.9, 1.0 ± 3.2, and 7.8 ± 22.6 ml, respectively, and did not differ to a statistically significant extent. CONCLUSIONS: The method of combining Hydrofit® and Spongel® could obtain reliable hemostasis in 60 s.