ABSTRACT: Objectives. We sought to test the efficacy and safety of the implantation of a stent covered with biosynthetic cellulose compared to a conventional bare-metal stent (BMS) in a rabbit iliac artery model. Background. Biosynthetic cellulose is a biocompatible film used in several fields of medicine. Stents covered with biosynthetic cellulose are devices with the potential of achieving total lesion coverage, acting as a physical barrier to the migration of smooth muscle cells from the artery wall to the arterial lumen, and capturing circulating endothelial progenitor cells that may form a functional endothelial layer. Methods and Results. Seven BMS and 7 stents covered with biosynthetic cellulose were implanted in the iliac arteries of 7 rabbits. Angiographic restudy and morphometric analysis of the specimens were performed after 4 weeks. No intrastent angiographic restenosis was observed, either with BMS or with stents covered with biosynthetic cellulose. There was also no acute or late vessel occlusion caused by stent thrombosis in either group. In the BMS and biosynthetic cellulose stented groups, respectively, mean neointimal thicknesses were 0.18 ± 0.02 mm and 0.35 ± 0.02 mm*; lumen area, 4.6 ± 0.43 mm2 and 4.04 ± 0.42 mm²; neointimal area, 0.58 ± 0.09 mm2 and 2.13 ± 0.11 mm²*; % lumen, 79.09 ± 1.6% and 58.44 ± 3.26%*; % stenosis, 10.97 ± 1.23% and 35.55 ± 3.39%* (*p Methods
Preparation of the stent covered with biosynthetic cellulose. A 16 mm-long stainless steel balloon-expandable slotted-tube stent (Curare® Coronary Stent, Translumina GmbH, Hechingen, Germany) was inserted in a tubular mold (impermeable to liquids but permeable to gases) of a slightly larger diameter filled with a culture medium inoculated with the Acetobacter xylinum. The culture medium had the following composition: peptone 5.0 g/l, yeast extracts 5.0 g/l, Na2HPO4 2.7 g/l, citric acid 1.15 g/l and glucose 20.0 g/l. By biosynthesis, a cellulose membrane juxtaposed to the inner wall of the mold was formed in loco as a perfect reproduction of the wall. The formed membrane had a width of approximately 30 µ. Membrane width depends on conditions such as temperature — kept between 15 and 32 degrees centigrade — and the time of fermentation, ranging from 48–240 hours. Once the membrane is formed, the cellulose-covered stent was removed from the mold and submitted to a chemical treatment to free it from proteins, cell fragments and other elements resulting from bacterial activity. This treatment involved immersion in sodium lauryl sulfate at 0.5–5% for a period that varied from 2–24 hours. The covered stent was then rinsed by agitation with distilled water changed 5–10 times until the sodium lauryl sulfate residues were totally eliminated. It was then treated with a sodium hydroxide solution at 0.5–5% for 2–24 hours. After that, the sodium hydroxide was neutralized by rinsing the covered stent with distilled water, which was changed 5–15 times. Controlling the rinse water Ph guaranteed the procedure. After the chemical treatment was completed, the covered stent went through a drying process in a drying chamber with filtered air to prevent contamination and the presence of solid particles in suspension in the air. During the drying process, there was retraction of the cellulosic fibers of the membrane that covered the stent (Figure 1), which resulted in better adjustment and adherence of the membrane to the structure of the stent (Figure 2). Finally the stent (16 mm long) covered with biosynthetic cellulose was submitted to gamma sterilization. Just before implantation, the stents were rehydrated in saline solution and hand-crimped on a 3.0 mm (diameter) and 18 mm (length) angioplasty balloon catheter.
Animals. The study was performed with the approval of the Committee for Animal Care at the Hospital Universitario Evangelico de Curitiba, which conforms to the “Position of the American Heart Association on Research Animal Use.” Seven New Zealand white rabbits (weight 4.5–5.9 kg) were used for these experiments. Seven stents covered with biosynthetic cellulose and 7 BMS were implanted in the iliac arteries of 7 rabbits. All animals received aspirin 50 mg/day for 3 days before the initial intervention and until they were sacrificed 28 days later.
Stent placement. All procedures were performed under general anesthesia induced by intramuscular injection of ketamine (50 mg/kg) and acepromazine (0.2 mg/kg) after premedication with xilazine (10 mg/kg). A small incision was made to expose the carotid artery, after which the uppermost level of the exposed artery was ligated. A 5 Fr introducer sheath was positioned in the carotid artery, after which nitroglycerin 0.25 mg and heparin 1000 U were administered intra-arterially. All catheters were subsequently introduced through this sheath via a 0.014 inch guidewire. The external iliac artery was used for all experiments. One stent covered with biosynthetic cellulose was implanted in the mid segment of the external iliac artery, avoiding major side branches and 1 BMS was implanted in the mid segment of the contralateral iliac artery of the rabbits. All of the stents were equal in terms of model and trademark. Stents were sized 1.1–1.2 times the reference vessel diameter and deployed with 8 atm of pressure. Angiograms of the external iliac arteries were obtained at baseline and after stent implantation. After the procedure, the carotid artery was ligated and the animals were fed a normal diet for the remainder of the study.
Follow up. Four weeks after the procedure, the animals were brought back to the catheterization laboratory for angiography, after which the animals were sacrificed with an intravenous lethal injection of barbiturate and KCL. A cannula was inserted into the lower abdominal aorta for in situ perfusion of 100 ml of 5% dextrose solution with 100 U/ml of heparin. This was followed by 5% dextrose and then submitted to pressure perfusion at 100 mmHg for 1 hour with 10% buffered formalin. Both iliac arteries were excised and fixed by immersion in buffered formalin solution. Stented arterial segments were oriented for distal and proximal ends and embedded in DDK-plast (Delaware Diamond Knives, Inc., Wilmington, Delaware).
Morphometric analysis. The stents were sectioned with a tungsten knife. Multiple 5 µm cross-sectional slices were taken from each end and from the middle of each stent. The tissues were subsequently embedded and stained with hematoxylin-eosin and elastic Van Giesen. Vessel injury and neointimal response were measured by calibrated digital microscopy. Measurements at all sections included media area, area within the internal elastic lamina, area within the external elastic lamina, lumen area and stent area (area within the stent itself). Neointimal measurement included thickness at each stent strut site and total neointimal area. The average neointimal thickness (average for all strut sites) was calculated for each section. The following calculations from histopathologic examination were derived for each stent: percent stenosis was calculated 3 ways: 1) 100 x (1 - lumen area/IEL area); 2) 100 x (1 - lumen area/proximal reference area); and 3) 100 x (1 - lumen area/distal reference area); persistent effects were calculated as neointimal area proximal reference - neointimal area mid stent. Vessel injury at each stent wire site was scored as follows: 0 = endothelium denuded; 1 = internal elastic lamina lacerated; 2 = media lacerated; 3 = external elastic lamina lacerated.
Statistical analysis. For each of 14 arterial segments in the study, the stent-to-artery ratio, mean injury score, mean neointimal thickness, lumen area, neointimal area, media area, percent media, neointima and lumen area stenosis were calculated. All data are expressed as the mean value ± standard deviation. Mean injury score, stent-to-artery ratio and mean intimal thickness were analyzed with linear regression to derive a slope and intercept mid-correlation coefficient to determine relations. Neointimal thickness at each wire site and the adjusted neointimal thickness for grades 2 and 3 injury were compared with an unpaired t-test. Lesion morphology and scores were compared at the different time intervals using analysis of variance with Sceffé F tests for multiple comparisons. Statistical significance was at the 0.05 level.
Results
Follow-up evaluation. Fourteen stents (7 BMS and 7 covered stents) were successfully implanted in 7 rabbits. Each animal received both types of stents, 1 in each iliac artery. All rabbits had patent vessels at angiography immediately after stent placement and when sacrificed at 28 days. In the cellulose stented and bare stented iliac artery segment, no intrastent angiography restenosis or thrombosis was observed (Figure 3).
Morphometric measurements. In the BMS and biosynthetic cellulose stented groups, respectively the following measurements were recorded: injury, 2.17 ± 0.09 and 1.70 ± 0.21; mean neointimal thickness, 0.18 ± 0.02 and 0.35 ± 0.02 mm (p Discussion
Biosynthetic cellulose is a polysaccharide synthesized in abundance by Acetobacter xylinum in a complex process that involves: a) the polymerization of single glucose residues into linear β(1–4) glucan chains (Figure 5); b) the extracellular secretion of these linear chains; and c) the assembly and crystallization of the glucan chains into hierarchically composed ribbons.11,16 Biosynthetic cellulose is characterized by a unique fibrillar nanostructure that determines its extraordinary physical and mechanical properties.17,18 It can be considered a slow-biodegrading material.7 The cellulose membrane presents characteristics such as an extremely thin (30 µ) semi-elastic (rehydrated), resistant (rehydrated) and nonporous membrane. It is also highly hydrophilic, inert, atoxic and biocompatible, creating a microenvironment that provides physiologic conditions for the reendothelialization process.4,6 These properties encouraged this first study to assess the response of the arterial wall to a biosynthetic cellulose-covered stent.
Several published clinical studies have used biosynthetic cellulose or cellulose-based membranes as physical barriers for tissue regeneration.2,8,9 Biosynthetic cellulose can provide total arterial coverage at the treatment site, isolating the endothelial line from the arterial lumen. Although biosynthetic cellulose is a nanoporous structure (with interconnected pores of 50–150 µm),11 it acts as a nonpermeable mechanical barrier against the two most important processes of restenosis: the migration and proliferation of smooth muscle cells from the media and the adventitia to the lumen surface,20,21,23,24 and exposure of the subendothelial extracellular matrix to circulating thrombin and platelets.25,26 In the present study, vessel injury scores were comparable in both groups, although a higher rate of intimal tissue was observed with the cellulose-covered stent, thus if one considers that the cellulose membrane acts as a mechanical barrier, the consequence of injury in the artery wall probably played no role in neointimal formation in the cellulose stent group.
This study has shown neither acute nor late vessel occlusion caused by stent thrombosis in either group, even considering that the rabbits received only a single bolus of heparin and postprocedural aspirin. Seeger et al27 suggested that hydrophilic surface modification of stainless steel stents is a promising approach to improving the hemocompatibility of endoluminal arterial devices, reducing surface platelet adhesion in vitro and initial surface platelet accumulation in vivo. Rogers et al,28 in a rabbit iliac artery injury model, assessed the effects of the stent surface material on vascular responses. After applying a thin coating of a biologically inert polymer material to the struts before implantation, they found that complete thrombosis was virtually eliminated. It is possible that aside from the fact that the stent covered with biosynthetic cellulose covers the entire lesion, the highly hydrophilic and inert property of the biosynthetic cellulose contributed to the absence of stent occlusion caused by thrombosis in our study.
The thrombogenic surface of metallic endoluminal prostheses remains a major limitation for their use in the treatment of coronary and peripheral vascular disease.27 A layer of nonocclusive thrombus is common following angioplasty, but rarely causes clinical symptoms of abrupt vessel closure.19,29,30 On the other hand, in a healthy artery, the endothelial lining regulates platelet activation and thrombus formation through the release of nitric oxide, prostacyclin and antithrombin III.31 Early endothelialization of the fibrin-rich thrombus itself, with subsequent smooth muscle cell colonization of thrombus from the lumen surface, strongly suggests that much of the neointimal volume relates to the early fibrin-rich mural thrombus.29 In the present study, the acute loss of endothelial lining and its regulatory functions secondary to endothelial coverage by the biosynthetic cellulose stent, along with an extensive surface area of the biosynthetic cellulose stent in contact with blood elements, probably led to a more extensive layer of nonocclusive thrombus at the treatment site in comparison to the BMS.
Differences in nonocclusive mural thrombus volume forming in the days and weeks after angioplasty could govern the occurrence of restenosis, as suggested by rabbit and porcine models.22 Several animal studies indicated that, independently of the stent implantation site, endothelialization is complete by 4 weeks.35,36 Klemm et al4 produced a tube-shaped cellulose structure and assessed its biosafety, biologic efficacy and potential as a substitute for blood vessels to replace part of the carotid artery of a rat. Histological observations showed that 4 weeks after implantation, the inner surface of the biosynthetic cellulose tube was covered with properly oriented endothelial cells.4 The present study shows that the stent covered with biosynthetic cellulose did not inhibit neointimal thickening when compared to the BMS. However, since the cellulose membrane acts as a barrier for cell migration from the media and adventitia, we believe that the formation of a layer of nonocclusive thrombus that had colonized at the surface of the cellulose membrane and stent struts played the most important role in neointimal formation. The prevention of thrombosis inside cellulose stents may be achieved by accelerating endothelial regeneration by natural healing through the use of vascular endothelial growth factors, endothelial cell-seeding and estrogen-loaded cellulose, which may result in less neointimal hyperplasia.
Sokolnicki et al32 recently reported that the structure of biosynthetic cellulose is effective for drug delivery. In-vivo and clinical experience suggest that it is possible to combine a biocompatible polymeric coating with an active agent used to reduce the thrombogenic potential of stents.33,34 Charpentier et al6 modified a polyester vascular graft to be used as an artificial blood vessel by coating it with biosynthetic cellulose. They also reported that this new hybrid material could be ideal for use in the creation of vascular grafts because it is hydrophilic, can prevent thrombin formation and can be coated with bioactive agents such as anticoagulant and antiproliferative compounds. It is believed that biosynthetic cellulose works as a biological and biocompatible polymer and, as such, can be coated with bioactive agents, possibly resulting in a better endovascular implant. This combination has a potential advantage over drug-eluting stents if one considers that biosynthetic cellulose works as a polymer film, allowing the drug to be uniformly loaded across the entire surface of the cellulose film and uniformly delivering the drug across the entire surface of the treatment site. On the other hand, with conventional drug-eluting stents, the polymer allows the drug to be loaded only across surface of the metal and delivers the drug only to the sites of contact between the metal and the arterial wall.
The incidence of stent restenosis and complications remains high, particularly in degenerated saphenous vein grafts (SVGs).37 The mechanical properties of a stent covered with biosynthetic cellulose acting as a physical barrier also make it suitable for use as a stent graft due to the stent’s ability to retain fragments under its structure, which prevents embolization, especially in interventions in degenerated SVGs and thrombus-laden arteries. In addition, cellulose stent grafts can be used to treat other types of lesions and complications such as aneurysms, coronary ruptures and perforations. A number of clinical studies will be necessary to prove its usefulness and applicability in these settings.
If biosynthetic cellulose proves to be effective in the stent model, then it will have to be produced on an industrial scale without damaging the environment. Due to its simple fermentation process, large-scale biosynthetic cellulose production appears to be quite feasible; however, the specific engineering details need to be elaborated. Also, additional biochemical and genetic investigations need to be conducted in order to fully understand the cellulose production process from Acetobacter.
Study limitations. One of the limitations of this study was the use of rabbits instead of pigs, though experience suggests that the coronary arteries of domestic crossbred swine and the iliac arteries of rabbits are similar in size, access and injury patterns when compared to human vessels.38 Nevertheless, the porcine coronary artery is the ideal model, while the rabbit iliac is an alternative for pharmacokinetics and tissue response evaluation.38 Rabbits are relatively inexpensive and suitable for screening, but unfortunately, they have different clotting characteristics and are therefore an inferior model for thrombosis studies.39
Conclusion
The present experimental study shows that the use of a stent covered with biosynthetic cellulose is safe, showing no adverse effects in the endovascular system up to 4 weeks after implantation in the iliac arteries of rabbits. The efficacy analysis of this device was hampered by increased intimal hyperplasia, though the stenosis was mild. Future studies will be needed to confirm this hypothesis and to evaluate its clinical applicability.
Acknowledgments. We thank the IPEM Laboratory at Hospital Universitário Evangélico de Curitiba, Brazil, for its excellent technical assistance in the animal laboratory.
From the Department of Cardiovascular Interventions, Evangelic University Hospital of Curitiba, Brazil and Experimental Interventional Laboratory, Montreal Heart Institute, Montreal, Canada.
Disclosure. Bionext Produtos Biotecnológicos Ltda., São Paulo, Brazil, provided the biosynthetic cellulose coating and financial support for this study. Dr. Ronaldo da Rocha Loures Bueno reports that he is the intellectual property holder of stents covered with biosynthetic cellulose.
Manuscript submitted November 24, 2008, provisional acceptance given February 4, 2009, final version accepted May 4, 2009.
Address for Correspondence: Ronaldo da Rocha Loures Bueno, MD, PhD, Rua Francisco Lipka 90 apto. 10, Curitiba, PR Brazil, 81.200-580. E-mail: LBueno873@terra.com.br
1. De Paola DQ, Souza MGPP. Película celulósica: Novo curativo biológico para melhoria do leito receptor de enxertia cutânea. Rev Bras Cir 1987;77:135–138.
2. Novaes Jr AB, Novaes AB. Implants placed into extraction sockets in association with membrane therapy (Gengiflex) and porous hidroxiopaite. Int J Oral Max Iplts 1992;7:536–540.
3. Mello LR, Feltrin LT, Fontes PTL, Ferraz, FAP. Duraplasty with biosynthetic cellulose: An experimental study. J Neurosurg 1997;86:143–150.
4. Klemm D, Schumann D, Udhardt U, Marsch S. Bacterial synthetized cellulose — Artificial blood vessels for microsurgery. Prog Polym Sci 2001;26:1561–1603.
5. Helenius G, Bäckdahl H, Bodin A, et al. In vivo biocompatibility of bacterial cellulose. J Biomed Mater Res 2006;76A:431–438.
6. Charpentier PA, Maguire A, Wan W. Surface modification of polyester to produce a bacterial cellulose-based vascular prosthetic device. Appl Surf Sci 2006;252:6360–6367.
7. Märtson M, Viljanto J, Hurmer T, et al. Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat. Biomaterials 1999;20:1989–1995.
8. Macedo NL, Matuda F, Macedo LG, et al. Evaluation of two membranes in guided bone tissue regeneration: Histological study in rabbits. Braz J Oral Sci 2004;3:395–400.
9. Novaes AB Jr, Novaes AB. Guided tissue regeneration versus hemisection in the treatment of furcation lesion. A clinical analysis. Braz Dent J 1993;3:99–102.
10. Watanabe K, Eto Y, Takano S, et al. A new bacterial cellulose substrate for mammalian cell culture. Cytotechnology 1993;13:107–114.
11. Czaja WK, Young DJ, Kawecki M, Brown RM Jr. The future prospects of microbial cellulose in biomedical applications. Biomacromolecules 2007;8:1–12.
12. Stefanadis C, Toutouzas K, Vlachopoulos C, et al. Autologous vein graft-coated stent for treatment of coronary artery disease. Cathet Cardiovasc Diagn 1996;38:159–170.
13. McKenna CJ, Camrud AR, Sangiorgi G, et al. Fibrin-film stenting in a porcine coronary injury model: Efficacy and safety compared with uncoated stent. J Am Coll Cardiol 1998;31:1434–1438.
14. Holmes DR, Camrud AR, Jorgenson MA, et al. Polymeric stenting in the porcine coronary artery model: Differential outcome of exogenous fibrin sleeves versus polyurethane-coated stents. J Am Coll Cardiol 1994;24:535–531.
15. Palmaz JC. Review of polymeric graft materials for endovascular applications. J Vasc Interv Radiol 1998;9:7–13.
16. Jones DWJ. Crystalline modifications of cellulose. Part V. A cristalographic study of ordered molecular arrangements. J Polym Sci 1960;42:173–188.
17. Woodcock S, Sarko A. Packing analysis of carbohydrates and polysaccharides.11.molecular and crystal structure of native ramie cellulose. Macromolecules 1980;13:1183–1187.
18. O’Sullivan AC. Cellulose: The structure slowly unraves. Cellulose 1997;4:173–207.
19. Reidy MA. Endothelial regeneration VII. Interaction of smooth muscle cells with endothelial regrowth. Lab Invest 1988;59:36–43.
20. Bauters C, Labalache J, MacFadden E, et al. Relation of coronary angioscopic findings at coronary angioplasty to angiographic restenosis. Circulation 1995;92:2473–2479.
21. Preisack MB, Karsch KR. The paradigm of restenosis following percutaneous transluminal coronary angioplasty. Eur Heart J 1993;14(Suppl I):187–192.
22. Wilesky RL, March KL, Gradus-Pizio I, et al. Vascular injury, repair, and restenosis after percutaneous transluminal angioplasty in the atherosclerotic rabbit. Circulation 1995;92:2995–3005.
23. Schwartz RS, Murphy JG, Edwards WD, et al. Restenosis after balloon angioplasty, a practical proliferative model in porcine coronary arteries. Circulation 1990;82:2190–2200.
24. Scott NA, Cipolla GD, Ross CE, et al. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury in porcine coronary arteries. Circulation 1996;93:2178–2187.
25. Strauss BH, Chisholm RJ, Keely FW, et al. Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Cir Res 1994;75:650–658.
26. Macleod DC, Stauss BH, de Jong M, et al. Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenosis lesions. J Am Coll Cardiol 1994;23:59–65.
27. Seeger JM, Ingegno MD, Bigatan E, et al. Hydrophilic surface modification of metallic endoluminal stents. J Vasc Surg 1995;22:327–336.
28. Rogers C, Edelman ER. Endovascular stent design dictates experimental restenosis and thrombosis. Circulation 1995;91:2995–3001.
29. Schwartz RS, Holmes DR, Topol EJ. The restenosis paradigm revisited: An alternative proposal for cellular mechanisms. J Am Coll Cardiol 1992;20:1284–1293.
30. McKenna CJ, Burke S, Opgenroth TJ, et al. Selective ET-A receptor antagonism reduces neointimal hyperplasia in a porcine coronary stent model. Circulation 1998;97:2551–2556.
31. Kipshidze N, Dangas G, Tsapenko M, et al. Role of the endothelium in modulating neointimal formation. J Am Coll Cardiol 2004;44:733–739.
32. Sokolnicki AM, Fisher RJ, Harrah TP, Kaplan D. Permeability of bacterial cellulose membranes. J Membr Sci 2006;272:15–27.
33. Hardhammar PA, van Beusekom HMM, Emanuelsson H, et al. Reduction in thrombotic events with heparin-coated Palmaz-Schatz stent in normal porcine coronary arteries. Circulation 1996;93:423–430.
34. Serruys PW, Emanuelsson H, van der Giessen W, et al. Heparin-coated Palmaz-Schatz stents in human coronary arteries: Early outcome of the BENESTENT II pilot study. Circulation 1996;93:412–422.
35. Asahara T, Tsurumi Y, Kearney M, et al. Accelerated restitution of endothelial integrity and endothelium dependent function following rtVEGF165 gene transfer. Circulation 1996;94:3291–1302.
36. van Belle E, Tio FO, Couffinhal T, et al. Stent endothelialization; time course, impact of local catheter delivery, feasibility of recombinant protein administration, and response to cytokine expedition. Circulation 1997;95:438–448.
37. Choussat R, Black AJ, Bossi I, et al. Long-term clinical outcome after endoluminal reconstruction of diffusely degenerated saphenous vein grafts with less-shortening wallstents. J Am Coll Cardiol 2000;362:387–395.
38. Schwartz RS, Edelman ER. Drug-eluting stents in preclinical studies. Recommended evaluation from a consensus group. Circulation 2002;106:1867–1873.
39. Nikolaychik V, Sahota H, Keelan Jr MH, et al. Influence of different stent materials on endothelialization in vivo. J Invasive Cardiol 1999;11:410–415.
