Mosaic tissue-engineered porcine pulmonary artery valved conduit: long-term follow-up after implantation in an ovine model RETRACTED

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1 Interactive CardioVascular and Thoracic Surgery (2014) 1 5 doi: /icvts/ivu350 CONGENITAL a b c d Mosaic tissue-engineered porcine pulmonary artery valved conduit: long-term follow-up after implantation in an ovine model Zhiwei Xu a,b,, Yan Tan c,, Juyi Wan a,,haowu b, Da Gong a, Qiuxia Shi a, Zifan Zhou a, Xiufang Xu d and Wenbin Li a, * Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing, China Department of Cardiac Surgery, The Affiliated Hospital of Hangzhou Normal University, Hangzhou, China Department of Intensive Care Unit, The Affiliated Hospital of Hangzhou Normal University, Hangzhou, China Department of Molecular Biology, Institute of Heart, Lung and Blood Vessel, Beijing Anzhen Hospital, Capital Medical University, Beijing, China * Corresponding author. Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing , China. Tel: ; @163.com (W. Li). Received 1 July 2014; received in revised form 12 September 2014; accepted 22 September 2014 Abstract OBJECTIVES: To determine the effects of implanting a novel mosaic tissue-engineered porcine pulmonary artery valved conduit into the right ventricular outflow tract of sheep at a long-term follow-up. METHODS: The designed mosaic tissue-engineered porcine pulmonary artery valved conduits were implanted between the right ventricular outflow tract and distal pulmonary artery in sheep using the off-pump method. The sheep weight, conduit diameter, pulmonary valve annular diameter, left ventricular end-diastolic diameter, calcification and regurgitation of the pulmonary valve were measured preoperatively and at 6 and 12 months postoperatively. Macroscopic observation, ultrastructural analysis, endothelialization and detection of calcium content were performed after sacrificing the sheep at 12 months after surgery. RESULTS: The average sheep weight at 12 months after surgery was significantly higher than that preoperatively (P <0.05), indicating that the sheep continued to grow well. The transplanted conduit showed unobstructed blood flow, soft walls and a smooth inner wall, but no ectasia or stenosis. The valve of the conduit was partially stiff, able to open and close and mild-to-medium regurgitation was present. The conduit diameter, pulmonary valve annular diameter and the left ventricular end-diastolic diameter were each significantly increased (P <0.05). Haematoxylin eosin staining and scanning electron microscopy revealed regularly arranged cells with slight inflammatory cell infiltration and a clear, fibrous texture. Immunohistochemical staining indicated that endothelial cell marker CD31-positive cells had formed a continuous film-like structure on the inner wall of the conduit. Scattered smooth muscle actin-positive cells were found in the middle layer of the conduit. CONCLUSIONS: The mosaic tissue-engineered porcine pulmonary artery valved conduit demonstrated good biocompatibility, did not cause an immune rejection response, contributed to endothelial coverage and has the potential to adapt to the needs of the growth and development of the body. Keywords: Tissue engineering Porcine pulmonary artery Valved conduit Mosaic Ovine model INTRODUCTION During cardiac surgery, the application of pulmonary artery valved conduits to reconstruct the right ventricular outflow tract (the connection point of the right ventricle and pulmonary artery) is a major method for anatomical correction of various complex congenital heart diseases, including pulmonary atresia, double-outlet right ventricle and transposition of the great arteries, and for transplantation of an autologous pulmonary valve (Ross operation). The pulmonary artery valved conduits that are currently used in the clinic include vascular prostheses with biological valves, allogeneic valved conduits and the Contegra conduit. The incidence of reoperation because of long-term stenosis, calcification or decay of Contributed equally to this article. these conduits is high, and their inability to grow obviously precludes their application to paediatric cardiac surgery [1, 2]. The development of a more appropriate pulmonary artery valved conduit would have a large impact on the field of cardiac surgery. The morphology, tissue components and mechanical strength and elasticity of porcine pulmonary artery are similar to human pulmonary artery. Moreover, the porcine pulmonary artery is easily obtained, is in adequate supply and is a very good heterogeneous pulmonary artery scaffold. Recently, a series of basic studies focused on the construction of a tissue-engineered porcine pulmonary artery valved conduit using decellularized porcine pulmonary artery was reported in vitro, but it did not solve the problem of decay or the calcification of the valve and conduit induced by immunological rejection [3]. The Author Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

2 2 Z. Xu et al. / Interactive CardioVascular and Thoracic Surgery The goal of the present study was to verify the low immunogenicity, endothelial cell coverage, lack of calcification and growth potential of a novel mosaic tissue-engineered porcine pulmonary artery valved conduit at a 12-month follow-up after in vivo implantation into the circulatory system of a large animal model (ovine). Such data would demonstrate the utility of this tissueengineered pulmonary artery valved conduit. MATERIALS AND METHODS Animals A total of 12 young pigs of both genders weighing 15.2 ± 1.3 kg ( kg), as well as 12 young sheep of both genders weighing 17.5 ± 2.7 kg ( kg) were used in this study. All protocols involving the animals were performed according to the guidelines set forth by the American Association for Accreditation of Laboratory Animal Care. Preparation of the mosaic porcine pulmonary artery valved conduits Following a previously published mosaic method [4], the pulmonary artery valved conduits of young pigs were aseptically harvested, including 1 cm of the myocardial tissue below the valve. The main pulmonary artery was reserved anterior to the bifurcation of the left and right pulmonary arteries. The fresh porcine pulmonary artery valved conduits were decellularized using the trypsin, Triton X-100 and hyaluronidase digestion method, resulting in pure, porous pulmonary artery valved conduit fibrous scaffolds. The scaffolds were then treated with gelatin to create the mosaic. Next, the gelatin was embedded into the pores of the scaffolds. Then the conduits were fixed by carbodiimide crosslinking to create the mosaic tissue-engineered porcine pulmonary artery valved conduits. Mosaic here means that the gelatin was firmly embedded into the pores of the porous scaffolds. The physical properties of these pulmonary artery valved conduits were assessed, including water content, thickness and tensile strength. The water content of the conduits was 0.83 ± 0.03%, the thickness was 0.79 ± 0.04 mm and the tensile strength of the conduits recovered to be no different from that of a normal porcine pulmonary artery (20.47 ± 1.98 vs ± 1.66 MPa, P >0.05). The mosaic pulmonary artery valved conduit is shown in Fig. 1. Implantation of the pulmonary artery valved conduits Following a previously published method [5], young sheep were anaesthetized with 30 mg/kg ketamine, and general anaesthesia was maintained with 2 mg/kg propofol. Systemic anticoagulation was maintained using a low dose of intravenous heparin (200 IU/ kg). A 4- to 5-cm anterolateral incision was made along the left third rib, and the main pulmonary artery was dissociated. The main pulmonary (proximal to the bifurcation) was partially occluded with a side-biting auricular clamp and incised. The distal end of the conduit was then sutured end-to-side to the main pulmonary artery with 5-0 polypropylene suture. A clamp was applied to the conduit and the PA clamp was released and re-applied in a similar fashion to the proximal PA at the level of the sinuses. A similar anastomosis was created and clamps were released. The main PA was ligated between the two anastomoses. After anastomosis, the newly replaced conduit was implanted as a bridge over the native pulmonary artery, establishing continuity between the sinus of the pulmonary trunk and the distal pulmonary artery (Fig. 2). Protamine (1 mg/kg) was used to neutralize the administered heparin. After complete haemostasis, the chest was sutured closed in layers. An intercostal nerve block with 0.25% bupivacaine was used to reduce postoperative chest pain. There was no need to place a drainage tube in the thoracic cavity. Intravenous penicillin was administered for 3 days after surgery, but no anticoagulant was administered. Experimental measurements Echocardiography and computed tomography (CT) angiography were performed on the young sheep at 6 and 12 months after conduit implantation. The weight of the animals was measured after 12 months and compared with the weights of healthy domestic sheep of the same age. The sheep were sacrificed by Figure 1: Mosaic pulmonary artery valved conduit. Figure 2: The replaced conduit was implanted as a bridge above the native pulmonary artery, establishing continuity between the sinus of the pulmonary trunk and the distal pulmonary artery. The native autochthonous pulmonary artery was ligated between the distal and proximal anastomoses.

3 Z. Xu et al. / Interactive CardioVascular and Thoracic Surgery 3 injecting 20 ml of 15% KCl into an ear vein. The implanted pulmonary artery valved conduit was aseptically harvested and subjected to macroscopic observation, haematoxylin eosin staining, scanning electron microscopy, immunohistochemistry for CD31 and SMA and detection of calcium content. Statistical analysis The data are expressed as mean ± SD and were analysed using the SPSS v17.0 software. Differences between groups were assessed using analysis of variance. A value of P <0.05 was considered statistically significant. RESULTS Postoperative survival rate and weights The mosaic tissue-engineered porcine pulmonary artery valved conduits were successfully implanted in 12 sheep. One sheep died after 93 days because of atelectasis, but the remaining 11 sheep survived until 12 months. The sheep weights were significantly higher than the preoperative weights (44.7 ± 4.2 vs 17.9 ± 1.3 kg, P <0.05). Compared with healthy domestic sheep of the same age, no significant difference was detected (44.7 ± 4.2 vs 46.3 ± 3.7 kg, P >0.05). Valved conduit and ventricular growth At 6 and 12 months postoperatively in the 11 sheep, no blood flow was found in the autologous pulmonary artery by echocardiography and CT angiography (Fig. 3). The transplanted conduit showed unobstructed blood flow with no local ectasia or stenosis. The valve of the conduit was partially stiff, was able to open and close and showed mild to medium regurgitation. The left Figure 3: No blood flow was found in the autologous pulmonary artery, while unobstructed blood flow was seen in the conduit with no local ectasia or stenosis. SC: surgery care unit; RHA: right hand anterior. ventricular end-diastolic diameter was significantly longer at 12 months than at 6 months by echocardiography. The conduit diameter and pulmonary valve annular diameter were also shown to be significantly longer at 12 months than at 6 months by CT angiography (Table 1). Macroscopic observation of the valved conduits At autopsy 12 months after implantation, the conduits were unobstructed and had soft walls and smooth inner walls with no local ectasia or stenosis. Small parts of the valve were calcified and stiff in all 11 sheep. Histology and ultrastructure of the valved conduits Postoperative haematoxylin eosin staining of the conduits showed regularly arranged cells with slight inflammatory cell infiltration and a clear fibrous texture at 12 months (Fig. 4). Scanning electron microscopy revealed that the smooth inner wall of the conduit was covered by a continuous intimal layer. The cell morphology was similar to that of endothelial cells. Collagen and elastic fibres were seldom noted (Fig. 5). Immunohistochemical endothelial and smooth muscle staining Endothelial cell marker CD31-positive cells formed a continuous film-like structure on the inner wall of the conduit (Fig. 6). Scattered smooth muscle actin (SMA)-positive cells were found in the middle layer of the conduit (Fig. 7). Calcification of the implanted conduits The calcium content of the implanted conduits was significantly higher than that of normal autologous pulmonary artery (273.8 ± 33.8 vs ± 18.4 mg/kg, P <0.05). DISCUSSION The use of a valved conduit to connect the right ventricle and pulmonary artery is a key milestone in reconstructing the ventricular outflow tract for the correction of complex congenital heart diseases. An appropriate pulmonary artery valved conduit should adapt to physical growth and development, be biocompatible and resist thrombosis, calcification and decay. Moreover, it should be applicable to diverse cases and convenient to use. However, the conduits currently used in the clinic do not satisfy these requirements. Many studies have constructed tissue-engineered heterogeneous porcine pulmonary artery valved conduits using decellularization methods [2, 6]. However, decellularized heterogeneous pulmonary artery tissue has been reported to be immunogenic, inducing monocyte infiltration that results in an inflammatory reaction [7]. Normal pulmonary artery conduit wall and valve are composed of cells, interstitial matrix and collagen fibres. Collagen fibres are the major factors that maintain the biomechanical properties of the pulmonary artery conduit wall and valve. The cells reside in gaps between fibres surrounded by matrix. The matrix

4 4 Z. Xu et al. / Interactive CardioVascular and Thoracic Surgery Table 1: Geometry of the valved conduit and ventricular development in sheep Diameter Preoperatively (mm) 6 months postoperatively # (mm) 12 months postoperatively* (mm) P-value ( # vs*) Conduit 10.1 ± ± ± Pulmonary valve annulus 11.2 ± ± ± Left ventricular end-diastolic diameter 25.9 ± ± Figure 4: Interstitial cells, a few infiltrated inflammatory cells, and a clear fibrous texture in a postoperative conduit and the adjacent myocardium (haematoxylin eosin staining, 400). mainly consists of proteoglycans and glycoproteins. Several explanations for rejection, calcification, thrombosis and unsatisfactory mechanical properties after implanting decellularized porcine pulmonary artery valved conduits exist, including the following: (i) Conventional decellularization techniques remove the cell components, but the remaining extracellular matrix can also induce immune rejection [8, 9] and can lead to monocyte infiltration and an inflammatory reaction [10, 11]. (ii) The loss of biological signals after decellularization can reduce the adhesion of endothelial cells and endothelialization of the conduit. Exposed heterogeneous fibres likely cause immune rejection after implantation in the blood circulation [12], and simultaneously activate blood platelets and evoke thrombosis. (iii) After decellularization, partial heterogeneous fibres can break, decreasing the mechanical properties of the tissue [13]. To address these problems, we proposed the theory of mosaic tissue reconstitution. Detergents and enzymatic digestion were employed to remove both the cells and as much of the interstitial matrix as possible. Only the collagen and elastic fibres remained in the pulmonary artery wall, forming pure, porous pulmonary artery fibre scaffolds. Gelatin was embedded into the pores of these fibre scaffolds and fixed with carbodiimide cross-linking. Gelatin has a simple structure composed of straight-chain amino acids, has weak immunogenicity, and displays desirable physical properties such as affinity, coverage and toughness. The crosslinking agent carbodiimide has low cytotoxicity, and its crosslinked C-terminus contains abundant antigenic determinants in a polypeptide chain-based biomaterial, resulting in decreased antigenicity and increased biological stability [14]. Thus, the pulmonary artery fibre scaffold was coated with gelatin and cross-linked Figure 5: Smooth surface of the conduit channel was coated by a continuous intima. The cell morphology appeared similar to endothelial cells, and few collagen and elastic fibres were present (electron microscopy, 3000). with carbodiimide. This mosaic pulmonary artery valved conduit has the following advantages: (i) removing the interstitial matrix drastically reduces antigenicity of the heterologous tissue, resulting in a biocompatible scaffold; (ii) the gelatin coating prevents thrombosis induced by direct exposure of heterologous fibres to the blood; (iii) the toughness of gelatin restores the original mechanical strength of the pure fibre scaffold; (iv) the mosaic gelatin promotes the adhesion and proliferation of host endothelial cells after implantation and (v) the mosaic gelatin decreases the degradation rate of the scaffold, ensuring that the heterologous pulmonary artery tissue has enough time to regenerate the pulmonary artery tissue in vivo, forming a pulmonary artery valved conduit that has the potential to adapt to the needs of the growth and development of the body. In a previously published pilot study, we determined the effects of the enzyme digestion protocol on the decellularized matrix [4]. The aim of the present study was to investigate the long-term follow-up results of conduit implantation in young sheep. The weight of the young sheep increased after implantation and no obvious limitations were found, indicating that this conduit can adapt to the body s growth and development. Echocardiography and CT angiography results showed that the left ventricular enddiastolic diameter and pulmonary valve annular diameter were increased with the growth and development of the young sheep. Immunohistochemical results demonstrated that the inner wall of the mosaic tissue-engineered porcine pulmonary artery valved conduit was coated with endothelial cells, and interstitial cells and

5 Z. Xu et al. / Interactive CardioVascular and Thoracic Surgery 5 Figure 6: Many endothelial cell marker CD31-positive cells (blue brown) were found in the inner wall of the conduit, showing a continuous, film-like structure (light microscopy, 400). Figure 7: Smooth muscle actin (SMA)-positive cells (blue brown) scattered in the middle layer of the conduit (light microscopy, 400). smooth muscle cells were visible in the middle layer. These results suggest that endothelialization occurred in the inner wall of the conduit. Endothelial growth can resist thrombosis and eventually form its own pulmonary artery valved conduit. The calcium content in the conduit increased after implantation, suggesting that mild calcification occurred in the mosaic pulmonary artery valved conduit. One potential explanation of this calcification is mild calcification of the valve. Because the thicknesses of the valve and artery wall are different, the same decellularization method may not be appropriate for both tissues. In addition, the decellularization method for valves could be further improved. In summary, the mosaic tissue-engineered porcine pulmonary artery valved conduit has several advantages derived from the tissue engineering methods used to create it, including low immunogenicity, endothelial coverage, biocompatibility and the potential to adapt to the needs of the growth and development of the body. Funding This study was funded by the National Natural Science Foundation of China ( ). Conflict of interest: none declared. REFERENCES [1] Prior N, Alphonso N, Arnold P, Peart I, Thorburn K, Venugopal P et al. Bovine jugular vein valved conduit: up to 10 years follow-up. J Thorac Cardiovasc Surg 2011;141: [2] Dohmen PM, Konertz W. Tissue-engineered heart valve scaffolds. Ann Thorac Cardiovasc Surg 2009;15: [3] Rüffer A, Wittmann J, Potapov S, Purbojo A, Glöckler M, Koch AM et al. Mid-term experience with the Hancock porcine-valved Dacron conduit for right ventricular outflow tract reconstruction. Eur J Cardiothorac Surg 2012;42: [4] Xu XF, Guo HP, Gong D, Ma JH, Xu ZW, Wan JY et al. Decellularized porcine pulmonary arteries cross-linked by carbodiimide. Int J Clin Exp Med 2013;6: [5] Wu H, Xu ZW, Liu XM, Gong DA, Wan JY, Xu XF et al. An in vivo model of in situ implantation using pulmonary valved conduit in large animals under off-pump condition. Chin Med J 2013;126: [6] Dohmen PM, Lembcke A, Holinski S, Kivelitz D, Braun JP, Pruss A et al. Mid-term clinical results using a tissue-engineered pulmonary valve to reconstruct the right ventricular outflow tract during the Ross procedure. Ann Thorac Surg 2007;84: [7] Bayrak A, Tyralla M, Ladhoff J, Schleicher M, Stock UA, Volk HD et al. Human immune responses to porcine xenogeneic matrices and their extrdecellularization matrix constituents in vitro. Biomaterials 2010;31: [8] Simon P, Kasimir MT, Seebacher G, Weigel G, Ullrich R, Salzer-Muhar U et al. Early failure of the tissue engineered porcine heart valve SYNERGRAFT in pediatric patients. Eur J Cardiothorac Surg 2003;23: [9] Kasimir MT, Rieder E, Seebacher G, Nigisch A, Dekan B, Wolner E et al. Decellularization does not eliminate thrombogenicity and inflammatory stimulation in tissue-engineered porcine heart valves. J Heart Valve Dis 2006;15: [10] Li QZ, Xu ZY, Huang SD. Immunogenicity of decellularized porcine heart valve. J Clin Rehabil Tissue Eng Res 2009;13: [11] Mathapati S, Verma RS, Cherian KM, Guhathakurta S. Inflammatory responses of tissue-engineered xenografts in a clinical scenario. Interact CardioVasc Thorac Surg 2011;12: [12] Rieder E, Seebacher G, Kasimir MT, Eichmair E, Winter B, Dekan B et al. Tissue engineering of heart valves: decellularized porcine and human valve scaffolds differ importantly in residual potential to attract monocytic cells. Circulation 2005;111: [13] Zhang J, Wu Y, Chen L. Simplified preparation and relative evaluation of decellularized porcine aortic scaffold. Chin J Reparative Reconstr Surg 2008;3: [14] Yu X, Cheng M, Chen H. Methods for the pre-treatment of biological tissues for vascular scaffold. J Biomed Eng 2004;21: