ORIGINAL ARTICLE – ADULT CARDIAC
Interactive CardioVascular and Thoracic Surgery 18 (2014) 556–561 doi:10.1093/icvts/ivt544 Advance Access publication 27 January 2014
Healing process of a novel zero-porosity vascular graft Tetsuro Morota* and Shinishi Takamoto* Department of Cardiothoracic Surgery, The University of Tokyo, Tokyo, Japan * Corresponding author. Department of Cardiothoracic Surgery, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan. Tel: +81-3-38155411 (T.M.)/ +81-3-38629111 (S.T.); fax: +81-3-58008654 (T.M.)/+81-3-38629233 (S.T.); e-mail: [email protected] (T.M.); [email protected] (S.T.) Received 31 July 2013; received in revised form 25 September 2013; accepted 21 October 2013
Abstract OBJECTIVES: We report on a study of thoracic aortic replacement in a canine model in order to demonstrate experimentally the process of tissue healing facilitated by the uniquely structured Triplex® graft. METHODS: Twelve Triplex® grafts were used in this study with 12 collagen-coated vascular grafts (Hemashield®, Boston Scientific, Natick, MA, USA) as the controls in 24 dogs; the grafts were taken out 4 or 26 weeks after implantation and were analysed histopathologically. RESULTS: The results demonstrate, at 4 weeks after implantation, that the degree of pseudointima formation was comparable between the Triplex® grafts and the control grafts, although significant inflammatory reactivity was observed in the control grafts. After 26 weeks of implantation, significant lymphocytic infiltration was found in one animal treated with a Triplex® graft and significant neutrophil infiltration was found in one animal implanted with a control graft. In the other animals implanted with Triplex® or control grafts, the observed inflammatory reactions were similar. Specifically, in both animals implanted with Triplex® or control grafts, significant numbers of immature mesenchymal cells, fibroblasts and collagen fibres were observed at 26 weeks after implantation, and foreign-body reactions found in animals implanted with the control graft at 4 weeks after implantation were not observed at 26 weeks after implantation. Thus, the xenobiotic property and the degree of encapsulation were comparable between both the animals implanted with the Triplex® graft and those treated with the control graft. CONCLUSIONS: Our study demonstrated that Triplex® vascular grafts, which have a unique structure that conventional grafts do not possess, induced mild inflammation in the acute phase after the implant compared with the control grafts, and contributed to tissue healing comparable with the control graft 26 weeks after implantation. Keywords: Vascular graft • Sealed graft • Zero-porosity • Less leakage • Tissue healing • Animal implantation study
INTRODUCTION In recent years, macroangiopathy including aortic aneurysm and dissection is increasing. For these disorders, a treatment method using vascular grafts has been established, in which polyester grafts are employed in many cases; especially in the thoracic region, polyester vascular grafts coated with some bioabsorbable material are typically employed. The newly developed vascular graft, Triplex® (Terumo corporation), has a unique three-layered structure that comprises a middle layer of an elastomer non-porous matrix without using bioabsorbable materials such as collagen and gelatin. Also, for the inner and outer layers, Triplex® incorporates a porous matrix in order to facilitate cell infiltration and enhances its connectivity with the surrounding tissue. Because of such unique structural design, Triplex® may be used instantly in urgent cases and minimizes bleeding from needle holes or blood vessels. In addition, because they do not employ biodegradable materials, Triplex® grafts are expected to reduce the inflammatory reaction associated with degradation and absorption of biodegradable materials. We conducted an animal implant study in order to demonstrate the effects of the Triplex® graft on inflammatory reactivity of the
implanted body and the process of tissue healing, and performed histopathological analyses at 4 or 26 weeks after implantation. This article reports the results of the study.
MATERIALS AND METHODS The Triplex® vascular graft has a crimped, three-layered structure (Fig. 1). The middle layer is made of non-biodegradable styrene elastomer resin, and the inner and outer layers are made of a knitted matrix of polyester fibres, a porous matrix having high connectivity with the body tissue. In other words, the Triplex® graft has a uniquely designed structure, in which an elastomer middle layer having blood-sealing ability is sandwiched between the two knitted matrices of polyester fibres.
METHODS The samples tested in the study In this animal study, the following samples were used: (i) Triplex®: 12 straight grafts (ϕ 8 mm, length 8 cm).
© The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.
T. Morota and S. Takamoto / Interactive CardioVascular and Thoracic Surgery
557
Figure 1: Triplex®: Three-layer structure; the middle layer is made of nonbiodegradable styrene elastomer, and the inner and outer layers are made of knitted polyester fibres.
(ii) Control graft: 12 Hemashield Gold woven straight grafts (ϕ 8 mm, length 8 cm).
Animal studies Twenty-four dogs weighing 10–14 kg were used in the study. All animals were cared for in accordance with the guidelines approved by the Terumo animal experiment committee. Animals were fasted for 24 h prior to implantation and treated with atropine sulphate (Mitsubishi Tanabe Pharma Corporation) 0.05 mg/kg (intramuscularly) as an anaesthetic preconditioning. Subsequently, ketamine (Fujita Pharmaceutical Co., Ltd) 25 mg/kg was administered intravenously for initial anaesthesia induction, and then a bronchial catheter (Terumo Corporation) was intubated for inhalation anaesthesia. For inhalation anaesthesia, 1–2% mixed gas of nitrous oxide, oxygen (nitrous oxide vs oxygen = 1:1), and halothane (Takeda Pharmaceutical Company Limited) was used, while controlled respiration was maintained. A 22-G Surflow indwelling needle (Terumo Corporation) was deployed in the cephalic vein, during which operation, Solulact (Terumo Corporation) 250 ml was infused continuously, and Cefamezin (Fujisawa Pharmaceutical Co., Ltd), an antibiotic, was administered intravenously at several times to infuse 250 mg in total. The left breast of each animal was shaved and disinfected, and a thoracotomy was performed at the sixth intercostal space. The thoracic aorta was exposed, and intercostal arteries that branch from the aorta were ligated while the thoracic aorta long enough for a graft implant was held. Just before the operation of a vascular graft transplant, heparin (Shimizu Pharmaceutical Co., Ltd) 70 U/ kg was administered intravenously in the animal. Following installation of a bypass using a polyvinyl chloride tube of internal diameter 2.1 mm between the right carotid artery and the right femoral artery, the thoracic aorta was blocked. Then, a part of the thoracic aorta was resected so that a vascular graft could be implanted. After checking that the vascular graft was 6.0–8.0-cm long, an end-to-end anastomosis was performed with the continuous suture technique using a 5-0 polypropylene suture (BEAR Medic Corporation). When the suture procedure was completed, clamping of the aorta was released to restore blood flow. Following reperfusion in the thoracic aorta, haemostasis of the graft anastomoses and wall was assured, and the chest was closed. Upon closing of the chest, a 12-Fr trocar catheter (Terumo Corporation) was deployed in the thoracic cavity and drainage was performed. When the skin was sutured, ampicillin (Kawasaki Mitaka Seiyaku K.K.) 150 mg was administered hypodermically. The trocar catheter was removed at the closing of the chest. After the closing of the chest, the bypass was removed, and the right carotid artery and the right femoral artery were sutured.
After the given period of vascular graft deployment, Nembutal (Dainippon Sumitomo Pharma Co., Ltd) 25 mg/kg was administered intravenously as an anaesthetic. The cervical region of the animal was shaved and disinfected, a skin incision was performed and the common carotid artery was exposed. After 300 U/Kg of heparin was administered intravenously and the patency of the vascular graft was confirmed, the animal was euthanized under anaesthesia by blood removal from the common carotid artery. After euthanasia, a thoracotomy was performed to observe the state of tissue healing as well as to detect any abnormality macroscopically. Prior to the removal of the specimen, observation of the vascular graft was performed with regard to false aneurysm, infection, seroma and the state of the surrounding tissue. Subsequently, the vascular graft was taken out from the body together with 1.0 cm or longer anastomotic vessels and the surrounding tissue. The distal side anastomotic vessel was longer than the proximal side anastomotic vessel. After the specimen was rinsed of blood in sterile physiological saline, it was cut open longitudinally for observation of the inside of the vascular graft in terms of the state of thrombus adhesion and appearance. The collected specimen was fixed in 10% buffered formalin. For each of the proximal side anastomotic vessel, the distal side anastomotic vessel and the central part of the vascular graft, a paraffin section was prepared according to the conventional procedure, hematoxylin-eosin-stained, Azan-stained and observed histologically under the light microscope with regard to thrombus formation on the inner and outer layers of the vascular graft, the degrees of pseudointima formation and encapsulation, and tissue reaction of the surrounding tissue.
RESULTS Results of the 4-week implant study At the study conducted 4 weeks after implantation, the patency of the vascular graft was confirmed in all 12 animals that underwent implantation. The results of the histopathological analyses of the collected specimens are summarized in Table 1 and Fig. 2. As given in Table 1 and Fig. 2, the degrees of encapsulation and neutrophil and lymphocytic infiltrations were mild in the animals implanted with Triplex® grafts, whereas significant inflammations such as foreign-body reaction, neutrophil and lymphocytic infiltrations were often observed in the animals implanted with control grafts at 4 weeks. Also, as given in Table 1, a thick thrombus formation was observed in most of the animals implanted with the Triplex® graft, although the findings for the other specimens were comparable with those for the specimens of the control graft. As demonstrated by the results described above, at 4 weeks after implantation, the degree of pseudointima formation was comparable between the animals implanted with Triplex® grafts and those implanted with control grafts (Fig. 2).
Results of the 26-week implant study At the study conducted 26 weeks after the implant, the patency of the vascular graft was confirmed in all 12 animals that underwent
ORIGINAL ARTICLE
Histopathological analyses
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T. Morota and S. Takamoto / Interactive CardioVascular and Thoracic Surgery
Table 1: The results of the 4-week study 4-Week reaction
Triplex®
Grade
±
Encapsulation Foreign-body reaction Neutrophil infiltration Lymphocytic infiltration Deposition of yellowish brown pigment 4-Week healing Grade Thickness of the proximal thrombus Thickness of the central thrombus Thickness of the distal thrombus Thickness of the proximal pseudointima Thickness of the central pseudointima Thickness of the distal pseudointima
1 1 3 – 1 3 2
Collagen-coated graft +
++
+++
2 4
3 1
1 1 1
2 2
1
± 5 3 2 6 4 6
±
+
1
++
1
2
+++
1 1
5 5 5
1
1 Triplex® +
+
+++
–
1 6 5
2 3
2 Collagen-coated graft ± + ++ 6 6 6
+++
1
6
±: Minor; +: Mild; ++: Moderate; +++: Severe; –: Negative; ±: Minor; +: Mild; ++: Moderate; +++: Severe.
Figure 2: Histological findings with Triplex® (left) and collagen-coated grafts (right) after 4 weeks of implantation (hematoxylin-eosin stain ×200): the degrees of encapsulation and neutrophil and lymphocytic infiltrations were mild in the animals implanted with Triplex® grafts (left), whereas significant inflammations such as foreignbody reaction, neutrophil infiltration and lymphocytic infiltration were often observed in the animals implanted with the control grafts (right).
implant except one Triplex®-implanted animal immediately sacrificed because of postoperative infection. The results of the histopathological analyses of the collected specimens are summarized in Tables 1 and 2 and Fig. 3. As given in Table 1, although significant lymphocytic infiltration was seen in one animal implanted with a Triplex® graft and significant neutrophil infiltration was seen in one animal implanted with a control graft, inflammatory reactivity was comparable between the animals implanted with Triplex® grafts and those implanted with control grafts. Compared with the results of the 4-week study, significant numbers of immature mesenchymal cells, fibroblasts and collagen fibres were observed at 26 weeks after implantation, and foreignbody reactions found in animals implanted with the control graft in the 4-week implant study was reduced in this 26-week implant study. Thus, the xenobiotic property and the degree of encapsulation were comparable between animals implanted with Triplex® grafts and those implanted with control grafts. In both the animals implanted with Triplex® and control grafts, similar findings were observed regarding tissue healing, although some differences were found with regard to thrombus adhesion and pseudointima, as given in Table 2.
DISCUSSION The properties required for large diameter vascular grafts used in the thoracic region or the abdominal region include low blood permeability from the vascular graft and operability that allows easy suturing during the operation as well as postoperative histocompatibility and long-term patency [1–4]. Unlike conventional vascular grafts, Triplex® has a unique threelayered structure that comprises a non-porous middle layer and porous inner and outer layers, without the use of bioabsorbable materials such as collagen and gelatin, and some publications show this graft to have excellent performance in clinical use [5–7]. On this occasion, in order to demonstrate tissue healing facilitated by this uniquely structured Triplex® graft, we conducted a study on thoracic aorta implantation in dogs using collagencoated vascular grafts as the control grafts. Four or 26 weeks after canine thoracic aortas were replaced by the grafts, they were taken out, and microscopic specimens were prepared to analyse the state of tissue healing. As a result, at 4 weeks after implantation, the degree of pseudointima formation and thickness of pseudointima from both the proximal and distal side anastomotic vessels were comparable
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T. Morota and S. Takamoto / Interactive CardioVascular and Thoracic Surgery
26-Week reaction
Triplex®
Grade
±
+
Encapsulation Foreign-body reaction Neutrophil infiltration Lymphocytic infiltration Deposition of yellowish brown pigment 26-Week healing Grade Thickness of the proximal thrombus Thickness of the central thrombus Thickness of the distal thrombus Thickness of the proximal pseudointima Thickness of the central pseudointima Thickness of the distal pseudointima
4
1 5
ORIGINAL ARTICLE
Table 2: The results of the 26-week study Collagen-coated graft ++
+++
±
+
++
2 1
3 3
1 2
+++
1 1
1
– 5
±
Triplex® +
2
4 2 1
1 2
3
1
2
2
++
+++
– 2 6
1 5
2
2
1
± 2 2
Collagen-coated graft + ++ 1 1
3 2 3
1 1 2
+++ 1 1 2 3 1
±: Minor; +: Mild; ++: Moderate; +++: Severe; −: Negative; ±: Minor; +: Mild; ++: Moderate; +++: Severe.
Figure 3: Histological findings with Triplex® (left), and collagen-coated graft (right) after 26 weeks of implantation (Azan stain ×25).
between the Triplex® grafts and the control collagen-coated vascular grafts. Meanwhile, although only mild neutrophil infiltration and foreign-body reactions were found in the Triplex® grafts in the spaces between the vascular graft fibres and tissues neighbouring the vascular graft, in collagen-coated vascular grafts, significant acute inflammatory reactions such as neutrophil, foreign-body giant cell and lymphocyte infiltrations were found. It is speculated that those reactions were caused by hydrolysis of the polyester fibre. The collagen coating of the vascular graft is considered to be absorbed in 12 weeks. Because the difference of inflammation results between the two grafts was seen during the period of collagen absorption, we assume that the inflammatory reactions found in collagen-coated vascular grafts were associated with collagen absorption. There have been many reports that indicate the clinical effects of coated vascular graft use on continuous postoperative pyrexia and effusion of exudate [8, 9]. Because these abnormal reactions are not observed in the conventional vascular grafts that require preclotting, they are considered to be reactions to the foreign proteins (bovine-derived collagen or gelatin) used for the coated vascular grafts as the coating material [10].
In the longer implant period—the 26-week implant study— the findings on inflammation and tissue healing were comparable between the Triplex grafts and the collagen-coated vascular grafts. Generally, in an implant study for the canine aorta, the tissue reactions such as pseudointima formation and thrombus adhesion are seen in the inner layer of the vascular graft as observed in this implant study. The degree of tissue reactions varies because of the structure of the vascular graft material, the structure of the vascular graft surface and the effect of porosity. In particular, it was reported that the porosity of vascular grafts had an impact on tissue ingrowth from the outside to the inside of the vascular graft, and that the vascular grafts of high porosity were superior to the vascular grafts of low porosity in pseudointima formation [11, 12]. For the vascular grafts used in this study, the Triplex® grafts have only the inner and outer layers of porous material and thus do not allow tissue ingrowth from the outside to the inside of the graft, whereas the control grafts have a structure that allows tissue ingrowth from the outside to the inside of the graft. We assume that the difference in the state of tissue healing seen between the Triplex® grafts and the control grafts would be caused by early
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T. Morota and S. Takamoto / Interactive CardioVascular and Thoracic Surgery
absorption of the coating material in the control grafts and subsequent ingrowth of cells and tissues into the porous matrix. Therefore, we conducted an implant study using these grafts that replaced the thoracic aorta in dogs, in order to see if there is any difference in tissue healing between these two grafts with a different structure. In the 4-week implant study, foreign-body reactions that might be associated with absorption of the coating material were observed in the collagen-coated vascular grafts; however, the other findings were similar to those found in the 26-week implant study. There are many factors that can influence an inflammatory reaction such as the structure, the material, toxicity and compliance, and an inflammatory reaction was noted in the Hemashield graft in this study at 4 weeks. We consider that the inflammatory reaction due to Dacron at 4 weeks may have greatly exceeded that by other inflammatory factors. The results suggest that ingrowth of cells or tissues from the outside to the inside of the vascular graft has a minor impact on pseudointima formation. Ingrowth of cells or tissues from the anastomotic line has a very important role for pseudointima formation.
CONCLUSION (i) In the 4-week animal implant study, although significant inflammation and xenobiotic properties were observed in the control grafts, the degree of pseudointima formation from both the proximal and distal side anastomotic vessels were comparable between the Triplex® grafts and the control grafts. (ii) In the 26-week animal implant study, the degree of pseudointima formation and inflammation were comparable between the Triplex® grafts and the control grafts. The state of tissue healing was also comparable. (iii) With the results described above, it was experimentally demonstrated that with Triplex® vascular graft the postoperative tissue healing process is similar to that of the conventional vascular grafts. (iv) Triplex® is expected to play its role as a clinically beneficial next-generation vascular graft.
Limitations of the study There are limitations with regard to showing evidence in the long term because of the difference between humans and dogs, and also because of the limited observation periods of 4 and 26 weeks.
FUNDING This study was partly supported by funding from Terumo Corporation. Conflict of interest: Terumo Co. provided the room, equipment, supplies including drugs and artificial grafts, and animals for the experiment.
REFERENCES [1] Oshima T, Yamada T, Nakahara H, Tanabe S, Irie Y, Katayama Y et al. Sealed versus unsealed Dacron knitted prosthesis: comparison of intraoperative blood loss and inflammatory response. J Artif Organs 1994;23: 825–8.
[2] Nishina T, Okabayashi H, Shimada I, Ohno N, Minatoya K, Kameyama T. Experience of Gelseal Triaxial for abdominal aortic aneurysm. J Artif Organs 1994;23:829–31. [3] Shoji Y, Itoh T, Hata M, Sadahiro M, Niibori K, Miura M et al. Early and late clinical results of thoracic aortic replacement using gelatin-impregnated woven Dacron graft. J Jpn Soc Vasc Surg 1997;6:417–22. [4] Adachi H, Yamaguchi A, Murata S, Kamio H, Okada M, Mizuhara A et al. Results of replacement of the aorta by sealed graft and future outlook. J Jpn Soc Vasc Surg 1997;6:393–8. [5] Tamura A, Yamaguchi A, Yuri K, Noguchi K, Naito K, Nagano K et al. Clinical experience with a new vascular graft free from biodegradable material. Interact CardioVasc Thorac Surg 2011;12:758–61. [6] Tabata M, Shimokawa T, Fukui T, Manabe S, Sato Y, Takanashi S. New uncoated vascular prosthesis reduces mediastinal tube drainage after thoracic aortic surgery. Ann Thorac Surg 2011;91:899–902. [7] Takamoto S, Yasuda K, Tabayashi K, Kyo S, Miyata T, Kazui T et al. Clinical experience with Termumo large diameter graft (Triplex®)—result of a multicenter clinical trial. Jpn J Cardiovasc Surg 2007;36:253–60. [8] Yamamoto K, Noishiki Y, Kosuge T, Ishii M, Ichikawa Y, Imoto K et al. Disadvantages of Gelseal graft as a vascular prosthesis. J Assoc Thorac Surg 1993;46:762–5. [9] Aizaki M, Ishikawa S, Hamada Y, Tsuda K, Otaki Y, Ichikawa H et al. Clinical experience of a knitted Dacron prosthesis impregnated with gelatin. J Artif Organs 1994;23:1000–2. [10] Hadama T, Mori Y, Shigemitsu O, Miyamoto S. Postoperative dilation of gelation impregnated knitted Dacron prosthesis and technical contrivance as reversed-end insertion anastmosis. Jpn Soc Vasc Surg 1997;6: 399–404. [11] Zarins CK, Chengpei Xu, Hisham S, Bassiouny SG. Intimal Hyperplasia, Hai Movici’s, Vascular Surgery 5th edn. Blackwell Science 2004; chapter 10:164–75. [12] Haverich A, Oelert H, Maatz W, Borst HG. Histopathological evaluation of woven and knitted Dacron grafts for right ventricular conduits. A comparative experimental study. Ann Thorac Surg 1984;37:404–11. [13] ISO. 7198 Cardiovascular Implants—tubular Vascular Prostheses. 1st edn. International Organization for Standardization 1998, pp. 42–43. [14] FDA. Guidance for the Preparation of Research and Applications for Vascular Graft Prostheses, US Department of Health and Human Services 1993;p52–55. [15] Adachi H. The History of Aortic Aneurysm Operation. Artificial Vascular Graft: Illustrated Surgery for Aortic Aneurysm, Kanehara & Co., Ltd; 1999; 3–8, 25–28. [16] Nishina T, Okabayashi H, Shimada H, Ohno N, Minatoya K, Kameyama T. Experience of Hemashield graft for abdominal aortic aneurysma. J Artif Organs 1995;24:838–40. [17] Kuwabara M, Onitsuka T, Nakamura K, Araki K, Yano M, Koga Y. Clinical experience with coated grafts and inflammatory response. J Artif Organs 1995;24:129–31. [18] Ohshima O, Yamada T, Nakahara H, Tanabe S, Irie Y, Katayama Y et al. Sealed versus unsealed Dacron knitted prosthesis: comparison of intraoperative blood loss and inflammatory response. J Artif Organs 1994;23: 825–8. [19] Landau O, Haddad M, Landau M, Lerner’ E, Stelman E, Livn E et al. Rejection of a gelatin-impregnated Dacron graft. Cardiovasc Surg 1993;1: 389–91. [20] Ishikawa S, Ohtaki A, Takahashi T, Sato Y, Suzuki M, Koyano T et al. Non-infective high fever after replacement of thoracic aorta using collagen-impregnated Dacron prosthesis. J Cardiovasc Surg 1995;36: 143–5. [21] Adachi H, Ino T, Ide H, Mizuhara A, Yamaguchi A, Kawahito et al. Replacement of thoracic aorta using gelatin impregnated knitted Dacron graft and results of follow up. J Artif Organs 1994;23:1029–32. [22] Sako H, Hadama T, Mori Y, Shigemitsu O, Kimura T, Miyamoto S et al. Postoperative dilation of a prosthetic graft for the thoracic aortic aneurysm. Jpn Soc Vasc Surg 1995;4:45–9. [23] Utoh J, Goto H, Hirata T, Hara M, Kitamura N, Miyauchi Y. Clinical controversies concerning sealed vascular prostheses: a comparison of Gelseal and Hemashield. Jpn Soc Vasc Surg 1997;6:385–92. [24] Shoji Y, Itoh T, Hata M, Sadahiro M, Niibori K, Miura M et al. Early and late clinical results of thoracic aortic replacement using gelatin-impregnated woven Dacron graft. Jpn Soc Vasc Surg 1997;6:417–22. [25] Goldstein LJ, Davies RR, Rizzo JA, Davila JJ, Cooperberg MR, Shaw RK et al. Stroke in surgery of the thoracic aorta: incidence, impact, etiology, and prevention. J Thorac Cardiovasc Surg 2001;122:935–45.
Interactive CardioVascular and Thoracic Surgery 18 (2014) 556–561 doi:10.1093/icvts/ivt544 Advance Access publication 27 January 2014
Healing process of a novel zero-porosity vascular graft Tetsuro Morota* and Shinishi Takamoto* Department of Cardiothoracic Surgery, The University of Tokyo, Tokyo, Japan * Corresponding author. Department of Cardiothoracic Surgery, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan. Tel: +81-3-38155411 (T.M.)/ +81-3-38629111 (S.T.); fax: +81-3-58008654 (T.M.)/+81-3-38629233 (S.T.); e-mail: [email protected] (T.M.); [email protected] (S.T.) Received 31 July 2013; received in revised form 25 September 2013; accepted 21 October 2013
Abstract OBJECTIVES: We report on a study of thoracic aortic replacement in a canine model in order to demonstrate experimentally the process of tissue healing facilitated by the uniquely structured Triplex® graft. METHODS: Twelve Triplex® grafts were used in this study with 12 collagen-coated vascular grafts (Hemashield®, Boston Scientific, Natick, MA, USA) as the controls in 24 dogs; the grafts were taken out 4 or 26 weeks after implantation and were analysed histopathologically. RESULTS: The results demonstrate, at 4 weeks after implantation, that the degree of pseudointima formation was comparable between the Triplex® grafts and the control grafts, although significant inflammatory reactivity was observed in the control grafts. After 26 weeks of implantation, significant lymphocytic infiltration was found in one animal treated with a Triplex® graft and significant neutrophil infiltration was found in one animal implanted with a control graft. In the other animals implanted with Triplex® or control grafts, the observed inflammatory reactions were similar. Specifically, in both animals implanted with Triplex® or control grafts, significant numbers of immature mesenchymal cells, fibroblasts and collagen fibres were observed at 26 weeks after implantation, and foreign-body reactions found in animals implanted with the control graft at 4 weeks after implantation were not observed at 26 weeks after implantation. Thus, the xenobiotic property and the degree of encapsulation were comparable between both the animals implanted with the Triplex® graft and those treated with the control graft. CONCLUSIONS: Our study demonstrated that Triplex® vascular grafts, which have a unique structure that conventional grafts do not possess, induced mild inflammation in the acute phase after the implant compared with the control grafts, and contributed to tissue healing comparable with the control graft 26 weeks after implantation. Keywords: Vascular graft • Sealed graft • Zero-porosity • Less leakage • Tissue healing • Animal implantation study
INTRODUCTION In recent years, macroangiopathy including aortic aneurysm and dissection is increasing. For these disorders, a treatment method using vascular grafts has been established, in which polyester grafts are employed in many cases; especially in the thoracic region, polyester vascular grafts coated with some bioabsorbable material are typically employed. The newly developed vascular graft, Triplex® (Terumo corporation), has a unique three-layered structure that comprises a middle layer of an elastomer non-porous matrix without using bioabsorbable materials such as collagen and gelatin. Also, for the inner and outer layers, Triplex® incorporates a porous matrix in order to facilitate cell infiltration and enhances its connectivity with the surrounding tissue. Because of such unique structural design, Triplex® may be used instantly in urgent cases and minimizes bleeding from needle holes or blood vessels. In addition, because they do not employ biodegradable materials, Triplex® grafts are expected to reduce the inflammatory reaction associated with degradation and absorption of biodegradable materials. We conducted an animal implant study in order to demonstrate the effects of the Triplex® graft on inflammatory reactivity of the
implanted body and the process of tissue healing, and performed histopathological analyses at 4 or 26 weeks after implantation. This article reports the results of the study.
MATERIALS AND METHODS The Triplex® vascular graft has a crimped, three-layered structure (Fig. 1). The middle layer is made of non-biodegradable styrene elastomer resin, and the inner and outer layers are made of a knitted matrix of polyester fibres, a porous matrix having high connectivity with the body tissue. In other words, the Triplex® graft has a uniquely designed structure, in which an elastomer middle layer having blood-sealing ability is sandwiched between the two knitted matrices of polyester fibres.
METHODS The samples tested in the study In this animal study, the following samples were used: (i) Triplex®: 12 straight grafts (ϕ 8 mm, length 8 cm).
© The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.
T. Morota and S. Takamoto / Interactive CardioVascular and Thoracic Surgery
557
Figure 1: Triplex®: Three-layer structure; the middle layer is made of nonbiodegradable styrene elastomer, and the inner and outer layers are made of knitted polyester fibres.
(ii) Control graft: 12 Hemashield Gold woven straight grafts (ϕ 8 mm, length 8 cm).
Animal studies Twenty-four dogs weighing 10–14 kg were used in the study. All animals were cared for in accordance with the guidelines approved by the Terumo animal experiment committee. Animals were fasted for 24 h prior to implantation and treated with atropine sulphate (Mitsubishi Tanabe Pharma Corporation) 0.05 mg/kg (intramuscularly) as an anaesthetic preconditioning. Subsequently, ketamine (Fujita Pharmaceutical Co., Ltd) 25 mg/kg was administered intravenously for initial anaesthesia induction, and then a bronchial catheter (Terumo Corporation) was intubated for inhalation anaesthesia. For inhalation anaesthesia, 1–2% mixed gas of nitrous oxide, oxygen (nitrous oxide vs oxygen = 1:1), and halothane (Takeda Pharmaceutical Company Limited) was used, while controlled respiration was maintained. A 22-G Surflow indwelling needle (Terumo Corporation) was deployed in the cephalic vein, during which operation, Solulact (Terumo Corporation) 250 ml was infused continuously, and Cefamezin (Fujisawa Pharmaceutical Co., Ltd), an antibiotic, was administered intravenously at several times to infuse 250 mg in total. The left breast of each animal was shaved and disinfected, and a thoracotomy was performed at the sixth intercostal space. The thoracic aorta was exposed, and intercostal arteries that branch from the aorta were ligated while the thoracic aorta long enough for a graft implant was held. Just before the operation of a vascular graft transplant, heparin (Shimizu Pharmaceutical Co., Ltd) 70 U/ kg was administered intravenously in the animal. Following installation of a bypass using a polyvinyl chloride tube of internal diameter 2.1 mm between the right carotid artery and the right femoral artery, the thoracic aorta was blocked. Then, a part of the thoracic aorta was resected so that a vascular graft could be implanted. After checking that the vascular graft was 6.0–8.0-cm long, an end-to-end anastomosis was performed with the continuous suture technique using a 5-0 polypropylene suture (BEAR Medic Corporation). When the suture procedure was completed, clamping of the aorta was released to restore blood flow. Following reperfusion in the thoracic aorta, haemostasis of the graft anastomoses and wall was assured, and the chest was closed. Upon closing of the chest, a 12-Fr trocar catheter (Terumo Corporation) was deployed in the thoracic cavity and drainage was performed. When the skin was sutured, ampicillin (Kawasaki Mitaka Seiyaku K.K.) 150 mg was administered hypodermically. The trocar catheter was removed at the closing of the chest. After the closing of the chest, the bypass was removed, and the right carotid artery and the right femoral artery were sutured.
After the given period of vascular graft deployment, Nembutal (Dainippon Sumitomo Pharma Co., Ltd) 25 mg/kg was administered intravenously as an anaesthetic. The cervical region of the animal was shaved and disinfected, a skin incision was performed and the common carotid artery was exposed. After 300 U/Kg of heparin was administered intravenously and the patency of the vascular graft was confirmed, the animal was euthanized under anaesthesia by blood removal from the common carotid artery. After euthanasia, a thoracotomy was performed to observe the state of tissue healing as well as to detect any abnormality macroscopically. Prior to the removal of the specimen, observation of the vascular graft was performed with regard to false aneurysm, infection, seroma and the state of the surrounding tissue. Subsequently, the vascular graft was taken out from the body together with 1.0 cm or longer anastomotic vessels and the surrounding tissue. The distal side anastomotic vessel was longer than the proximal side anastomotic vessel. After the specimen was rinsed of blood in sterile physiological saline, it was cut open longitudinally for observation of the inside of the vascular graft in terms of the state of thrombus adhesion and appearance. The collected specimen was fixed in 10% buffered formalin. For each of the proximal side anastomotic vessel, the distal side anastomotic vessel and the central part of the vascular graft, a paraffin section was prepared according to the conventional procedure, hematoxylin-eosin-stained, Azan-stained and observed histologically under the light microscope with regard to thrombus formation on the inner and outer layers of the vascular graft, the degrees of pseudointima formation and encapsulation, and tissue reaction of the surrounding tissue.
RESULTS Results of the 4-week implant study At the study conducted 4 weeks after implantation, the patency of the vascular graft was confirmed in all 12 animals that underwent implantation. The results of the histopathological analyses of the collected specimens are summarized in Table 1 and Fig. 2. As given in Table 1 and Fig. 2, the degrees of encapsulation and neutrophil and lymphocytic infiltrations were mild in the animals implanted with Triplex® grafts, whereas significant inflammations such as foreign-body reaction, neutrophil and lymphocytic infiltrations were often observed in the animals implanted with control grafts at 4 weeks. Also, as given in Table 1, a thick thrombus formation was observed in most of the animals implanted with the Triplex® graft, although the findings for the other specimens were comparable with those for the specimens of the control graft. As demonstrated by the results described above, at 4 weeks after implantation, the degree of pseudointima formation was comparable between the animals implanted with Triplex® grafts and those implanted with control grafts (Fig. 2).
Results of the 26-week implant study At the study conducted 26 weeks after the implant, the patency of the vascular graft was confirmed in all 12 animals that underwent
ORIGINAL ARTICLE
Histopathological analyses
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Table 1: The results of the 4-week study 4-Week reaction
Triplex®
Grade
±
Encapsulation Foreign-body reaction Neutrophil infiltration Lymphocytic infiltration Deposition of yellowish brown pigment 4-Week healing Grade Thickness of the proximal thrombus Thickness of the central thrombus Thickness of the distal thrombus Thickness of the proximal pseudointima Thickness of the central pseudointima Thickness of the distal pseudointima
1 1 3 – 1 3 2
Collagen-coated graft +
++
+++
2 4
3 1
1 1 1
2 2
1
± 5 3 2 6 4 6
±
+
1
++
1
2
+++
1 1
5 5 5
1
1 Triplex® +
+
+++
–
1 6 5
2 3
2 Collagen-coated graft ± + ++ 6 6 6
+++
1
6
±: Minor; +: Mild; ++: Moderate; +++: Severe; –: Negative; ±: Minor; +: Mild; ++: Moderate; +++: Severe.
Figure 2: Histological findings with Triplex® (left) and collagen-coated grafts (right) after 4 weeks of implantation (hematoxylin-eosin stain ×200): the degrees of encapsulation and neutrophil and lymphocytic infiltrations were mild in the animals implanted with Triplex® grafts (left), whereas significant inflammations such as foreignbody reaction, neutrophil infiltration and lymphocytic infiltration were often observed in the animals implanted with the control grafts (right).
implant except one Triplex®-implanted animal immediately sacrificed because of postoperative infection. The results of the histopathological analyses of the collected specimens are summarized in Tables 1 and 2 and Fig. 3. As given in Table 1, although significant lymphocytic infiltration was seen in one animal implanted with a Triplex® graft and significant neutrophil infiltration was seen in one animal implanted with a control graft, inflammatory reactivity was comparable between the animals implanted with Triplex® grafts and those implanted with control grafts. Compared with the results of the 4-week study, significant numbers of immature mesenchymal cells, fibroblasts and collagen fibres were observed at 26 weeks after implantation, and foreignbody reactions found in animals implanted with the control graft in the 4-week implant study was reduced in this 26-week implant study. Thus, the xenobiotic property and the degree of encapsulation were comparable between animals implanted with Triplex® grafts and those implanted with control grafts. In both the animals implanted with Triplex® and control grafts, similar findings were observed regarding tissue healing, although some differences were found with regard to thrombus adhesion and pseudointima, as given in Table 2.
DISCUSSION The properties required for large diameter vascular grafts used in the thoracic region or the abdominal region include low blood permeability from the vascular graft and operability that allows easy suturing during the operation as well as postoperative histocompatibility and long-term patency [1–4]. Unlike conventional vascular grafts, Triplex® has a unique threelayered structure that comprises a non-porous middle layer and porous inner and outer layers, without the use of bioabsorbable materials such as collagen and gelatin, and some publications show this graft to have excellent performance in clinical use [5–7]. On this occasion, in order to demonstrate tissue healing facilitated by this uniquely structured Triplex® graft, we conducted a study on thoracic aorta implantation in dogs using collagencoated vascular grafts as the control grafts. Four or 26 weeks after canine thoracic aortas were replaced by the grafts, they were taken out, and microscopic specimens were prepared to analyse the state of tissue healing. As a result, at 4 weeks after implantation, the degree of pseudointima formation and thickness of pseudointima from both the proximal and distal side anastomotic vessels were comparable
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26-Week reaction
Triplex®
Grade
±
+
Encapsulation Foreign-body reaction Neutrophil infiltration Lymphocytic infiltration Deposition of yellowish brown pigment 26-Week healing Grade Thickness of the proximal thrombus Thickness of the central thrombus Thickness of the distal thrombus Thickness of the proximal pseudointima Thickness of the central pseudointima Thickness of the distal pseudointima
4
1 5
ORIGINAL ARTICLE
Table 2: The results of the 26-week study Collagen-coated graft ++
+++
±
+
++
2 1
3 3
1 2
+++
1 1
1
– 5
±
Triplex® +
2
4 2 1
1 2
3
1
2
2
++
+++
– 2 6
1 5
2
2
1
± 2 2
Collagen-coated graft + ++ 1 1
3 2 3
1 1 2
+++ 1 1 2 3 1
±: Minor; +: Mild; ++: Moderate; +++: Severe; −: Negative; ±: Minor; +: Mild; ++: Moderate; +++: Severe.
Figure 3: Histological findings with Triplex® (left), and collagen-coated graft (right) after 26 weeks of implantation (Azan stain ×25).
between the Triplex® grafts and the control collagen-coated vascular grafts. Meanwhile, although only mild neutrophil infiltration and foreign-body reactions were found in the Triplex® grafts in the spaces between the vascular graft fibres and tissues neighbouring the vascular graft, in collagen-coated vascular grafts, significant acute inflammatory reactions such as neutrophil, foreign-body giant cell and lymphocyte infiltrations were found. It is speculated that those reactions were caused by hydrolysis of the polyester fibre. The collagen coating of the vascular graft is considered to be absorbed in 12 weeks. Because the difference of inflammation results between the two grafts was seen during the period of collagen absorption, we assume that the inflammatory reactions found in collagen-coated vascular grafts were associated with collagen absorption. There have been many reports that indicate the clinical effects of coated vascular graft use on continuous postoperative pyrexia and effusion of exudate [8, 9]. Because these abnormal reactions are not observed in the conventional vascular grafts that require preclotting, they are considered to be reactions to the foreign proteins (bovine-derived collagen or gelatin) used for the coated vascular grafts as the coating material [10].
In the longer implant period—the 26-week implant study— the findings on inflammation and tissue healing were comparable between the Triplex grafts and the collagen-coated vascular grafts. Generally, in an implant study for the canine aorta, the tissue reactions such as pseudointima formation and thrombus adhesion are seen in the inner layer of the vascular graft as observed in this implant study. The degree of tissue reactions varies because of the structure of the vascular graft material, the structure of the vascular graft surface and the effect of porosity. In particular, it was reported that the porosity of vascular grafts had an impact on tissue ingrowth from the outside to the inside of the vascular graft, and that the vascular grafts of high porosity were superior to the vascular grafts of low porosity in pseudointima formation [11, 12]. For the vascular grafts used in this study, the Triplex® grafts have only the inner and outer layers of porous material and thus do not allow tissue ingrowth from the outside to the inside of the graft, whereas the control grafts have a structure that allows tissue ingrowth from the outside to the inside of the graft. We assume that the difference in the state of tissue healing seen between the Triplex® grafts and the control grafts would be caused by early
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absorption of the coating material in the control grafts and subsequent ingrowth of cells and tissues into the porous matrix. Therefore, we conducted an implant study using these grafts that replaced the thoracic aorta in dogs, in order to see if there is any difference in tissue healing between these two grafts with a different structure. In the 4-week implant study, foreign-body reactions that might be associated with absorption of the coating material were observed in the collagen-coated vascular grafts; however, the other findings were similar to those found in the 26-week implant study. There are many factors that can influence an inflammatory reaction such as the structure, the material, toxicity and compliance, and an inflammatory reaction was noted in the Hemashield graft in this study at 4 weeks. We consider that the inflammatory reaction due to Dacron at 4 weeks may have greatly exceeded that by other inflammatory factors. The results suggest that ingrowth of cells or tissues from the outside to the inside of the vascular graft has a minor impact on pseudointima formation. Ingrowth of cells or tissues from the anastomotic line has a very important role for pseudointima formation.
CONCLUSION (i) In the 4-week animal implant study, although significant inflammation and xenobiotic properties were observed in the control grafts, the degree of pseudointima formation from both the proximal and distal side anastomotic vessels were comparable between the Triplex® grafts and the control grafts. (ii) In the 26-week animal implant study, the degree of pseudointima formation and inflammation were comparable between the Triplex® grafts and the control grafts. The state of tissue healing was also comparable. (iii) With the results described above, it was experimentally demonstrated that with Triplex® vascular graft the postoperative tissue healing process is similar to that of the conventional vascular grafts. (iv) Triplex® is expected to play its role as a clinically beneficial next-generation vascular graft.
Limitations of the study There are limitations with regard to showing evidence in the long term because of the difference between humans and dogs, and also because of the limited observation periods of 4 and 26 weeks.
FUNDING This study was partly supported by funding from Terumo Corporation. Conflict of interest: Terumo Co. provided the room, equipment, supplies including drugs and artificial grafts, and animals for the experiment.
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