Heart valve replacement is a common treatment for severe valvular heart disease. Mechanical valves or bioprosthetic valves are usually used for the substitutes. However, both of them have major disadvantages; mechanical valves need for anticoagulants1 and bioprosthetic valves cause time-related structural deterioration early.2 , 3 There are some attempts to develop tissue-engineered heart valves (TEHVs) for substitutes.4 , 5 Tissue-engineered heart valves are expected to be promising valve substitutes which overcome these disadvantages. They have better hemodynamic characteristics, do not require long-term anticoagulation, and (if they are autologous TEHVs) do not require chemical treatment to reduce immunogenicity, which could accelerate their degeneration.
Tissue-engineered heart valves are currently categorized in two ways, either by treating the TEHV with a decellularization process4 or by implanting a scaffold graft directly into the patient for in situ conversion into a living tissue by the host’s cells (in situ TEHV).6
Tissue-engineered heart valves are also expected additional advantage that they may settle down on the recipient as viable grafts and even have growth potential. Someone use stem cells awaiting for differentiation4 and others expect to undifferentiated host’s cells migrating into scaffolds.6 However, it is not well known how TEHVs take time-course change in the recipient after implantation.
As another recent topic in heart valve replacement, transcatheter heart valve implantations (TAVI) have been introduced into the clinical routine expanding the treatment options for elderly inoperable patients. Although TAVI is expected to expand to younger patient populations in the near future, the available heart valve prostheses for these techniques are bioprostheses and associated with progressive degeneration which is a serious problem for younger patient. Tissue-engineered heart valve is expected to be a promising substitute for TAVI.4 , 5
We are developing a novel autologous TEHV (biovalve) with other unique method named in-body tissue engineering which is based on a tissue encapsulation phenomenon in the living body.7 , 8 We are also developing new type of biovalves for transcatheter implantation named stent biovalve.
In this study, we investigated feasibility of stent biovalves and time-course histological transition in a large animal model.
Materials and Methods
Twenty-seven adult goats (body weight, 41.3 ± 14.3 kg) were used in this study. All animals received care according to the Principles of Laboratory Animal Care (formulated by the National Institutes of Health, Publication No. 56-23, received 1985), and all protocols were reviewed and approved by the Animal Subjects Committee of the National Cerebral and Cardiovascular Center (No. 17013).
Mold and Stent Assembling
Three kinds of self-expandable stents by Piolax Medical Devices (Yokohama, Japan) or Goodman Co. (Nagoya, Japan) were used for this study. These were prepared according to our previous report.9–12 Figure 1A shows the photograph of finished stent biovalves. The mold for the stent biovalve was obtained by assembling the stent and specially designed acrylic parts (Figure 1B). All acrylic parts were prepared using a 3D printer (CONNEX 260 Objet, Stratasys [Rehovot, Israel]).
Preparation of Stent Biovalves
The preparation method of the typical stent biovalve (type 2 in Figure 1) is shown in Figure 2. The specially designed molds with stent were placed into the dorsal subcutaneous pouches of the goats under anesthesia that was induced with 10 mg/kg of ketamine and maintained with 1–3% isoflurane (Figure 2A). After about 2 months, we harvested the implants (molds with stent and biovalve) en bloc (Figure 2B), which were completely encapsulated with robust connective tissue.10 , 11 After treatment with 70% ethanol for 10 min, the fragile, irregular, and redundant tissues around the developed tubular tissue were gently cut, the acrylic mold was removed, and a tubular hollow structure of membranous connective tissue rigidly fixed to the stent’s outer surface was obtained (Figure 2C). Then the half of the tubular hollow structure with the three pillar parts was completely folded in half inwards (Figure 2D, E). The folded pillar parts were sutured to the outside pillar parts as commissures to form three leaflets (Figure 2F), and the stent biovalve with tubular leaflets (20 or 25 mm) was obtained (Figure 2G). Other two stent biovalves (types 1 and 3 in Figure 1) only need to be carefully removed the acrylic mold (no need process D, E, and F). All stent biovalves were stored at room temperature in 70% ethanol.
Eight goats were used for the insertion of the molds into the subcutaneous pouches.
Transcatheter Implantation of Stent Biovalve
To evaluate the stent biovalve in vivo as the aortic valve (AV) and the pulmonary valve (PV), we used transcatheter valve implantation via the left ventricle (LV) or the right ventricle (RV) of goats. Anesthesia was induced with 10 mg/kg of ketamine and maintained with 1–3% isoflurane. The heart was exposed through a thoracotomy at the fifth subcostal region. After purse-string suture at the LV or RV apex, a custom-made sheath (diameter: 10 mm) was inserted into the ventricular cavity. After injection of heparin sodium (100 U/kg), a stent biovalve in a catheter introducer was inserted into the ventricular cavity via the sheath and finally reached to the AV or PV position under fluoroscopic guidance. After the angiography, the catheter introducer was gradually removed and the stent biovalve was deployed at the AV or PV position. Pressure catheters were inserted into the RV and the pulmonary artery, and both the pressures were continuously monitored. An angiography was performed after implantation. Postoperatively, no systemic anticoagulation was performed.
Total 19 goats received implantation of stent biovalves (10 goats received AV implantation and nine goats received PV implantation).
Qualitative tissue analyses: (immuno-)histology.
The stent biovalves were extracted at several durations after implantation (every month until 6 months and as long as possible) to observe the transition of implanted tissues. The extracted specimens were fixed with 10% formalin, embedded in paraffin, sliced into longitudinal sections, and finally stained with hematoxylin–eosin (H&E), Masson’s Trichrome (collagen stain), Elastica-van Gieson (elastin stain), and von Kossa (calcium stain). In addition, a few sections of biovalve were also stained for α-smooth muscle actin (α-SMA) by immunohistochemical techniques; the protein was detected using monoclonal antibodies (Abcam, Cambridge, UK).
Quantitative tissue analyses: achievement of endothelialization and cell migration.
The achievement of endothelialization and cell migration in the leaflets was analyzed using observation of cells in the longitudinal sections of HE-stained tissues. In each stent biovalve, each leaflet was divided four sections at even intervals from base to tip and it was judged whether endothelialized cells and migrated cells have reached to the border of the sections or not and was determined the progression rate among 0%, 25%, 50%, 75%, and 100% in response to degree of achievement.
Data are represented as mean ± standard deviation. To identify differences in endothelialization or cell migration between the preimplanted valve leaflets and the explanted leaflets at each duration, or between 2 explanted leaflets at different duration, a one-way analysis of variance was performed with Bonferroni’s post hoc testing. Differences were considered significant when p < 0.05. Statistics were performed using Microsoft Excel for Mac software (version 15.30, Redmond, WA).
Summary of the Transcatheter Implantation
Total 12 goats (five for AV implantation and seven for PV implantation) were successfully implanted and survived more than 1 month. All of the cause of failed implantation were translocation of the stents because, in animal experiment, healthy goats which have neither calcified nor atherosclerotic annuli were used and the stent valves had easily translocated by changing the annular diameter in a cardiac cycle. The summary of successfully implanted animal cases is shown in Table 1. In succeeded cases, the angiogram showed smooth movement of the leaflets with a little regurgitation under the aortic or pulmonary circulation. For each animal, the biovalve was extracted at one of these durations (1, 2, 3, 4, 5, 6 months or as long as possible) to have a progressive time-course transition at 1 month step (from 1 to 6 months) and also at the maximum duration, which have become19 months as a result.
Time-Course Histological Change of Implanted Stent Biovalves
The maximum duration reached to 19 months. In macroscopic and endoscopic view at in situ position after the extraction en bloc, the leaflets of the biovalve kept their shape and elasticity even after 19 months (Figure 3). Neither calcification nor thrombi were observed in any cases and duration.
The tissue of preimplanted stent biovalve is presented in Figure 4. Almost only the collagen was main component, and there was no endothelialization on the surface.
The tissues extracted at 1 and 3 and 19 months are presented in Figures 5–7. The time-course achievement of endothelialization and cell migration in the leaflets is shown in Figure 8A, B, respectively. Histological examination showed the cell migration into the biovalve body and laminar endothelialization on the surface of the valve leaflets even after 1 month (Figures 5 and 8A). Their histogenesis had gradually advanced in 3 months without any hyperplasia (Figures 6 and 8B), but was interrupted when the biovalve tissues were not attached enough on the native vascular wall surface. The recipients’ cells have also spread to the tip of leaflets gradually in 19 months without any hyperplasia and finally constructed characteristic three layered tissues like native leaflets (Figure 7A, B). The distal aspect of the leaflet is well stained in Collagen stain (Figure 7A2) that means it is composed primarily of collagen fibers, and the proximal aspect of the leaflet is well stained in Elastica-van Gieson stain (Figure 7A3) that means it contains elastin fiber well. α-SMA–positive cells were partly seen at central aspect of the leaflet layer and α-SMA–negative cells were mainly seen at both side aspects of it (Figure 9A). In histology, the biovalve was almost free from calcification even after 19 months (Figure 9B).
The most notable result in this study is implanted TEHV leaflets have adapted their histological structure like native ones. The mature semilunar valve structure (of both AV and PV) is composed of the extracellular matrix (ECM), valvular interstitial cells (VICs), and overlying endothelial cells (Figure 7B).13 The ECM is composed of three highly organized overlapping layers with distinct mechanical properties arranged in orientation to blood flow in the semilunar valves.14–16 The primary components of these layers are collagens, proteoglycans, and elastin: In AVs, the fibrosa, or arterial aspect of the cusp, is composed primarily of collagen fibers; the spongiosa, or central aspect, consists predominantly of loosely arranged proteoglycans; and the ventricularis, or ventricular aspect, contains elastin fiber. The preimplanted biovalves harvested from the subcutaneous portion mainly consist of the fibroblasts and their product, the collagen, and do not have three laminar structure that is a characteristic of the native valve leaflets. The harvested biovalves were soaked in 70% of ethanol and preserved before implant, and when the implantation, the fibroblasts perish and almost only the collagen was main component (Figure 4). Thinking of the histology in 1, 3, and 19 months, it is supposed that host cells in the tissues attached to implanted biovalve have immigrated into the collagen body of the biovalve, gradually advanced to the tip of the leaflets, and finally constructed the characteristic histological structures at the valve-leaflet region. And also supposed that the beautiful three layered structures have been completed in between 3 and 19 months. It is reported that stem-cell TEHVs underwent extensive remodeling in vivo to resemble native heart valves.17 , 18 However, thinking of our result, stem cells (or other undifferentiated cells) do not necessarily need as VICs to create TEHVs. Even adult recipient cells can migrate into the TEHV leaflets and compose similar ECM under the environment in which stress is loaded because of 100,000 times of opening and closing each day to maintain unidirectional blood flow through the heart. These migrated cells were mainly α-SMA positive in the tissue of 1 month, but in 19 months, α-SMA–positive cells were partly seen at central aspect of the leaflet layer and α-SMA–negative cells were mainly seen at both side aspects of it. This may mean that migrated fibroblasts have transformed into VIC-like cells and produced collagen and elastin fibers in each side aspect to adapt the environment, respectively. Anyhow, it is reported that host cells can migrate into TEHV,19 , 20 but this is the first report that the cells can compose appropriate histological structure like native valve leaflets. This histological adaptation gives us the expectation that some implanted TEHVs can sufficiently exert the functions as a native valve and purposively increase the flexibility and durability for a long while.
Compared with immigration of the cells into the collagen body, covering the surface of the leaflets and walls advances faster and almost completes within 1 month. However, it took longer if the contact of the graft with the wall of the host was insufficient. This suggests that host cells crawled along the contact region between the host and the graft and spread to the entire graft. This is supported that such endothelialization seldom occurred like scattered islands on the surface. It is reported that fibroblasts transform into endothelial cells (or endothelial-like cells).21 , 22 This contributed to the stent biovalves being free from thrombi on the surface without anticoagulation usage through the experiment.
It is reported that TEHVs and decellularized heart valves sometimes cause calcification,23 , 24 but all the biovalves in this study were almost free from calcification through the experiment. This would also be a potent advantage for durable valve substitute.
In terms of the type of stents, we are improving them in durability and avoiding translocation and used three types in this study. Although stent valves easily translocate in healthy-animal experiments as mentioned in the results, these three types have not reached the level for clinical usage, yet. A little more improvement is necessary for the design of the stent.
Implanted biovalves can adapt their histological structure to the environment. They have a potential to be used for viable bioprosthesis and to keep better function and biocompatibility longer than current valve substitutes.
1. Hammermeister K, Sethi GK, Henderson WG, Grover FL, Oprian C, Rahimtoola SHOutcomes 15 years after valve replacement with a mechanical versus a bioprosthetic valve: Final report of the Veterans Affairs randomized trial. J Am Coll Cardiol 2000.36: 1152–1158,
2. Cosgrove DM, Lytle BW, Taylor PC, et alThe Carpentier-Edwards pericardial aortic valve. Ten-year results. J Thorac Cardiovasc Surg 1995.110: 651–662,
3. Arsalan M, Walther TDurability of prostheses for transcatheter aortic valve implantation. Nat Rev Cardiol 2016.13: 360–367,
4. Driessen-Mol A, Emmert MY, Dijkman PE, et alTranscatheter implantation of homologous “off-the-shelf” tissue-engineered heart valves
with self-repair capacity: Long-term functionality and rapid in vivo
remodeling in sheep. J Am Coll Cardiol 2014.63: 1320–1329,
5. Schlegel F, Salameh A, Oelmann K, et alInjectable tissue engineered pulmonary heart valve implantation into the pig model: A feasibility study. Med Sci Monit Basic Res 2015.21: 135–140,
6. Kluin J, Talacua H, Smits AI, et alIn situ heart valve tissue engineering
using a bioresorbable elastomeric implant - From material design to 12 months follow-up in sheep. Biomaterials 2017.125: 101–117,
7. Takewa Y, Yamanami M, Kishimoto Y, et alIn vivo
evaluation of an in-body, tissue-engineered, completely autologous valved conduit (biovalve type VI) as an aortic valve in a goat model. J Artif Organs 2013.16: 176–184,
8. Nakayama Y, Takewa Y, Sumikura H, et alIn-body tissue-engineered aortic valve (Biovalve type VII) architecture based on 3D printer molding. J Biomed Mater Res B Appl Biomater 2015.103: 1–11,
9. Sumikura H, Nakayama Y, Ohnuma K, Kishimoto S, Takewa Y, Tatsumi EIn vitro
hydrodynamic evaluation of a biovalve with stent (tubular leaflet type) for transcatheter pulmonary valve implantation. J Artif Organs 2015.18: 307–314,
10. Sumikura H, Nakayama Y, Ohnuma K, Takewa Y, Tatsumi EDevelopment of a stent-biovalve with round-shaped leaflets: In vitro
hydrodynamic evaluation for transcatheter pulmonary valve implantation (TPVI). J Artif Organs 2016.19: 357–363,
11. Funayama M, Sumikura H, Takewa Y, Tatsumi E, Nakayama YDevelopment of self-expanding valved stents with autologous tubular leaflet tissues for transcatheter valve implantation. J Artif Organs 2015.18:228–235,
12. Mizuno T, Takewa Y, Sumikura H, et alPreparation of an autologous heart valve with a stent (stent-biovalve) using the stent eversion method. J Biomed Mater Res B Appl Biomater 2014.102: 1038–1045,
13. Tao G, Kotick JD, Lincoln JHeart valve development, maintenance, and disease: The role of endothelial cells. Curr Top Dev Biol 2012.100: 203–232,
14. Hinton RB Jr, Lincoln J, Deutsch GH, et alExtracellular matrix remodeling and organization in developing and diseased aortic valves. Circ Res 2006.98: 1431–1438,
15. Eisenberg LM, Markwald RRMolecular regulation of atrioventricular valvuloseptal morphogenesis. Circ Res 1995.77: 1–6,
16. Person AD, Klewer SE, Runyan RBCell biology of cardiac cushion development. Int Rev Cytol 2005.243: 287–335,
17. Sutherland FW, Perry TE, Yu Y, et alFrom stem cells to viable autologous semilunar heart valve. Circulation 2005.111: 2783–2791,
18. Lincoln J, Garg VEtiology of valvular heart disease-genetic and developmental origins. Circ J 2014.78: 1801–1807,
19. Lichtenberg A, Tudorache I, Cebotari S, et alPreclinical testing of tissue-engineered heart valves
re-endothelialized under simulated physiological conditions. Circulation 2006.114(suppl 1): I559–I565,
20. Rieder E, Seebacher G, Kasimir MT, et alTissue engineering of heart valves
: Decellularized porcine and human valve scaffolds differ importantly in residual potential to attract monocytic cells. Circulation 2005.111: 2792–2797,
21. Kon K, Fujiwara TTransformation of fibroblasts into endothelial cells during angiogenesis. Cell Tissue Res 1994.278: 625–628,
22. Shibuya T, Kambayashi J, Okahara K, et alSubendothelial layer of pseudointima of polytetrafluoroethylene graft is formed by transformation of fibroblasts migrated from extravascular space. Eur J Vasc Surg 1994.8: 276–285,
23. Tepeköylü C, Lobenwein D, Blunder S, et alAlteration of inflammatory response by shock wave therapy leads to reduced calcification of decellularized aortic xenografts in mice†. Eur J Cardiothorac Surg 2015.47: e80–e90,
24. Brizard CP, Brink J, Horton SB, Edwards GA, Galati JC, Neethling WMNew engineering treatment of bovine pericardium confers outstanding resistance to calcification in mitral and pulmonary implantations in a juvenile sheep model. J Thorac Cardiovasc Surg 2014.148: 3194–3201,
Keywords:Copyright © 2018 by the American Society for Artificial Internal Organs
tissue engineering; heart valves; tissue adaptation; transcatheter implantation; bioprosthesis; viable graft