LABORATORY INVESTIGATION
Muraglitazar-Eluting Bioabsorbable Vascular Stent Inhibits Neointimal Hyperplasia in Porcine Iliac Arteries Ilkka Uurto, MD, Mari Hämäläinen, PhD, Velipekka Suominen, MD, Marita Laurila, MD, Andres Kotsar, MD, Taina Isotalo, MD, Teuvo L.J. Tammela, MD, Minna Kellomäki, Dr Tech, and Juha-Pekka Salenius, MD
ABSTRACT Purpose: To evaluate the biocompatibility of a new muraglitazar-eluting polylactide copolymer stent and investigate its ability to prevent the formation of intimal hyperplasia. Materials and Methods: Ten self-expandable muraglitazar-eluting poly-96L/4D-lactic acid (PLA96) stents and 10 selfexpandable control PLA96 stents were implanted into porcine common iliac arteries. After 28 days follow-up, all stentimplanted iliac arteries were harvested and prepared for quantitative histomorphometric analysis. Results: Angiographic analysis revealed that one control PLA96 stent had occluded and one had migrated. Histomorphometric analysis demonstrated that, with the control PLA96 stent, the luminal diameter and area were decreased versus the muraglitazar-eluting PLA96 stents (means ⫾ standard error of the mean, 3.58 mm ⫾ 0.34 vs 4.16 mm ⫾ 0.14 and 9.83 mm2 ⫾ 2.41 vs 13.75 mm2 ⫾ 0.93, respectively). The control PLA96 stent induced more intimal hyperplasia than the bioactive muraglitazar-eluting PLA96 stent (557 mm ⫾ 122 vs 361 mm ⫾ 32). Vascular injury scores demonstrated only mild vascular trauma for both stents (muraglitazar-eluting, 0.68 ⫾ 0.07; control, 0.75 ⫾ 0.08). Inflammation scores also showed mild inflammation for both stents (muraglitazar-eluting, 1.05 ⫾ 0.17; control, 1.23 ⫾ 0.19). Conclusions: This new muraglitazar-eluting PLA96 stent was shown to be biocompatible with a tendency for better patency and less intimal hyperplasia compared with the control PLA96 stents.
ABBREVIATIONS PLA = polylactide, PLA96 = poly-96l/4d-lactic acid, PPAR = peroxisome proliferator–activator receptor
Vascular occlusive disease is one of the most common causes of morbidity and mortality in the Western world From the Divisions of Vascular Surgery and Urology, Department of Surgery (I.U., V.S., A.K., T.L.J.T., J.P.S.), and Department of Pathology (M.L.), Tampere University Hospital; Immunopharmacology Research Group (M.H.), University of Tampere School of Medicine and Tampere University Hospital; Biomaterials and Tissue Engineering Group, Department of Electronics and Communications Engineering (M.K.), Tampere University of Technology; BioMediTech– Institute of Biosciences and Medical Technology (M.K.), Tampere; and Department of Surgery (T.I.), Päijät-Häme Central Hospital, Lahti, Finland. Received April 18, 2014; final revision received October 1, 2014; accepted October 6, 2014. Address correspondence to I.U., Divisions of Vascular Surgery and Urology, Department of Surgery, Tampere University Hospital, P.O. Box 2000, 33521 Tampere, Finland; E-mail: ilkka.uurto@uta.fi None of the authors have identified a conflict of interest. & SIR, 2015 J Vasc Interv Radiol 2015; 26:124–130 http://dx.doi.org/10.1016/j.jvir.2014.10.005
(1). Endovascular treatment has advanced rapidly, with the latest inventions including drug-eluting balloons and stents and, most recently, bioabsorbable stents (2). Consequently, stents and balloons are no longer just passive mechanical devices but rather active and biocompatible instruments (3). However, there are some concerns regarding possible late thrombosis after the implantation of drug-eluting stents (4). In addition, it is now conceivable that all beneficial effects of drug-eluting stents could be preserved without having permanent materials embedded within the vessel wall. The idea of bioabsorbable drug-eluting stents itself is attractive (3). It would mechanically prevent recoil and negative remodeling as well as bioactively inhibit vascular smooth muscle cell proliferation, thereby preventing restenosis (5). Peroxisome proliferator–activator receptors (PPARs) belong to the nuclear receptor superfamily and consist of
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three distinct members: PPAR-α, PPAR-β/δ, and PPARγ. In addition to their metabolic effects, PPARs are also involved in the regulation of inflammatory responses (6). Muraglitazar is a dual PPAR-α/γ agonist originally developed for the treatment of type II diabetes (7). Recently, it has also been shown to possess antiinflammatory properties in vitro and in vivo (8). The aim of the present study was to evaluate the biocompatibility of a new muraglitazar-eluting biodegradable vascular stent and its ability to prevent intimal hyperplasia formation.
MATERIALS AND METHODS Stents The polymer used for the stents was medical-grade 96L/ 4D-copolymer of L-lactide and D-lactide (Purac Biochem, Gorinchem, The Netherlands). The final diameter of the fiber was 0.20 mm. The stents were formed of 16 meltspun monofilaments braided over a 6-mm-diameter mandrel. The braids were heat-treated for structural stability, and the stents were cut from the braid to the length of 15 mm each. The stents were immersion-coated with the racemic 50L/50D copolymer of L-lactide and D-lactide (Purac Biochem), into which muraglitazar was mixed. After thorough evaporation of the solvent, the stents were sterilized with more than 25 kGy of γ-irradiation by a commercial supplier. The measured molecular weight of the sterilized poly-96L/4D-lactic acid (PLA96) was 150,000 g/mol. The amount of the drug on the stents (500 mg ⫾ 70 per stent) and the thickness of the coatings were calculated by weighing the stents before and after coating. Because the mechanical properties of PLA96 stents differ quite significantly from those of bare metal stents, the pure PLA96 stent was chosen as a control stent. Its properties were similar to those of drugeluting PLA96 stents but with no bioactive agents. At the delivery device, there was a 6-mm balloon at the tip. The stent needed to be loaded onto the balloon immediately before implantation. Pulling back the sheath allowed the stent to self-expand, and the stent was then expanded with the balloon to ensure adequate implantation.
Animal Preparation and Stent Implantation All animal protocols were reviewed and approved by the institutional committee for animal research and by the eastern Finland provincial government. The study followed the recommendation guideline for the evaluation of drug-eluting stents for peripheral application as published previously (9). Ten male laboratory-bred swine were used according to the aforementioned recommendation (National Laboratory Animal Center, University of Kuopio, Kuopio, Finland), with a calculated statistical power of 0.873. Animals were 3 months old with a body weight of 25 kg each. Before the procedure,
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each animal was sedated with intramuscular azaperone (5 mg/kg) and subcutaneous atropine (0.05 mg/kg), and each was given an intramuscular dose of penicillin (750,000 IU) as antibacterial prophylaxis. The stent implantation was performed under general anesthesia with intravenous propofol (2 mg/kg). The animal was intubated and connected to a respirator. Anesthesia was maintained by propofol infusion (0.4 mL/kg/h). After systemic intravenous heparinization (150 IU/kg), the right carotid artery was surgically exposed. After arteriotomy, an 11-F sheath was introduced under visual control. The straight catheter was placed into the infrarenal aorta through a guide wire (Radiofocus Guide Wire M Standard, 0.035-inch, 150-cm; Terumo Europe, Leuven, Belgium). Preoperative angiography from the aortoiliac region was performed. The muraglitazareluting PLA96 stent was then introduced and implanted into the right common iliac artery under fluoroscopy. The same procedure was repeated with the control PLA96 stent in the left common iliac artery. The arteriotomy was closed with 6–0 sutures, and the wound was closed separately layer by layer. As antithrombotic medication, the animals received acetylsalicylic acid (250 mg/d) orally for 5 days preoperatively and until the end of follow-up and enoxaparin sodium (15 mg/d subcutaneously) from 1 day preoperatively through the third postoperative day. The animals were given buprenorphine (0.01 mg/kg subcutaneously) as analgesia for postoperative pain.
Follow-up and Analyses The animals’ health was observed continuously by specially trained laboratory animal nurses. In pigs, the intimal hyperplasia expansion rate reaches its peak at 1 month after the procedure—therefore, a follow-up of 28 days was chosen (10). After follow-up, the animals were sedated, deeply anesthetized, and heparinized (150 IU/kg) before angiography was performed and the animals were euthanized with 60 mg/kg pentobarbital. The common iliac arteries were perfusion-fixed with 10% buffered formalin at 100 mm Hg for 15 minutes and left overnight in immersion fixation with the same fixative agent. After fixation, the stent-implanted section was cut into proximal, central, and distal segments. Postimplantation angiographic findings were compared versus the angiographic findings during harvesting. The normal diameter of the stent-implanted artery and the narrowest diameter of the stent-implanted section were measured in millimeters manually with a calibrated digital ruler by one of the authors (I.U.). The percentage of decrease in luminal diameter was calculated. Stenosis was defined as an at least 50% reduction of luminal diameter. All histologic preparations were stained with hematoxylin and eosin. The histomorphometric analysis was computer-assisted (Scion Image Beta 4.0.2; Scion, Frederick, Maryland). Premeasurements were conducted
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before final measurements with relative standard error to ensure reliable results (ie, relative standard error o 10%) (11). The patency of the stent-implanted segments was evaluated by measuring the luminal diameter and area. The thicknesses of the tunica intima (ie, layer between endothelium and internal elastic membrane) and tunica media (ie, layer between internal and external elastic membrane) were measured as intima–media thickness, and the intimal/medial ratio was assessed. Intimal hyperplasia was estimated from neointimal thickness. Inflammatory response was measured according to the previously published method known as “inflammation score” (12). It estimates inflammatory cells around stent struts caused by materials and the procedure, yielding a numerical value from 0 to 3. The scoring was accomplished by a senior pathologist who was blinded to the histologic samples. Vascular trauma was assessed by means of the vascular injury score, which estimates how deep the stents struts have been embedded into the artery wall (13). Vascular inflammation and injury scoring systems are standardized methods, and were performed for every proximal, central, and distal segment of the stents.
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control PLA96 stent had occluded and one migrated. Otherwise, all stent-implanted vessels were patent. The percentages of decrease in luminal diameters were 16% for muraglitazar-eluting PLA96 stents and 39% for control PLA96 stents (Fig 1). Histomorphometric analysis was carried out for seven segments each with muraglitazar-eluting PLA96 stents and PLA96 control stents (Table). Three muraglitazareluting PLA96 stents and three control PLA96 stents were excluded from the analysis because of unsatisfactory section preparations and staining. Patency was assessed by measuring the mean luminal diameter and area. Muraglitazar-eluting PLA96 stents preserved the luminal diameter and area (4.2 mm ⫾ 0.1 and 13.8 mm2 ⫾ 0.9, respectively) better than PLA96 stents (3.6 mm ⫾ 0.3 and 9.8 mm2 ⫾ 2.4; P ¼ .15 and P ¼ .16, respectively). There were no marked differences in the intimal/medial ratio between muraglitazar-eluting and control stents (muraglitazar-eluting PLA96 stent, 0.9 ⫾ 0.1; PLA96 stent, 0.9 ⫾ 0.1; P ¼ .16). There was a tendency for the control polylactide (PLA) stent to induce more intimal hyperplasia compared with the bioactive muraglitazar-eluting PLA96 stent (PLA stent,
Statistical Analysis SPSS software (version 19.0; SPSS, Chicago, Illinois) for Windows (Microsoft, Redmond, Washington) was used for statistical analysis. A comparison of means between the two groups was carried out with the t test for independent samples. All results are presented as means ⫾ standard error of means unless otherwise stated. A P value less than .05 was considered statistically significant.
RESULTS Twenty biodegradable stents (10 muraglitazar-eluting and 10 control PLA96 stents) were implanted into the common iliac arteries of young healthy swine. Angiography was performed on all stent-implanted segments directly after implantation and before harvesting. One
Table. Histomorphometric Analysis Muraglitazar-Eluting
PLA96
PLA96 Stent
Stent
P Value
4.4 ⫾ 0.2 3.9 ⫾ 0.2
3.9 ⫾ 0.5 3.0 ⫾ 0.4
.314 .061
Mean
4.2 ⫾ 0.1
3.6 ⫾ 0.3
.145
LA (mm2) IMT (mm)
13.8 ⫾ 0.9 0.8 ⫾ 0.1
9.8 ⫾ 2.4 1.0 ⫾ 0.2
.156 .201
I/M ratio
0.8 ⫾ 0.1
0.9 ⫾ 0.1
.156
Intimal thickness (mm)
0.4 ⫾ 0.0
0.6 ⫾ 0.1
.149
Measurement LD (mm) Maximum Minimum
Values presented as means ⫾ standard error of the mean. I ¼ intima, IMT ¼ intima–media thickness, LA ¼ luminal area, LD luminal diameter, M ¼ media.
Figure 1. Angiographic images: (1) Muraglitazar-eluting PLA96 stent and (2) PLA96 stent (a) perioperative and (b) after follow-up.
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557 mm ⫾ 122; muraglitazar-eluting PLA96 stent, 361 mm ⫾ 32; P ¼ .15; Fig 2). There was also a clear trend for a more severe intimal reaction to develop at the ends of the control PLA96 stent compared with the central part of the stent (PLA stent, proximal, 538 mm ⫾ 100; central, 399 mm ⫾ 116; distal, 660 mm ⫾ 191; muraglitazar-eluting PLA96 stent, proximal, 334 mm ⫾ 59; central, 333 mm ⫾ 19; distal, 416 mm ⫾ 72; P ¼ .13, P ¼ .59, and P ¼ .26, respectively; Fig 3). Vascular injury score showed that both stents caused minimal trauma to the vessel wall (muraglitazar-eluting PLA96 stent, 0.7 ⫾ 0.1; PLA96 stent, 0.8 ⫾ 0.1; P 4 .05). There was a tendency toward lower scores at the ends of the stents compared with the central parts of the stents (PLA stent, proximal, 0.7 ⫾ 0.2; central, 1.0 ⫾ 0.1; distal, 0.6 ⫾ 0.1; muraglitazar-eluting PLA96 stent, proximal, 0.7 ⫾ 0.1; central, 0.9 ⫾ 0.1; distal, 0.6 ⫾ 0.1; P ¼ .51; Fig 4). The inflammation score demonstrated only mild inflammation related to both stent types
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(muraglitazar-eluting PLA96 stent, 1.05 ⫾ 0.17; PLA96 stent, 1.23 ⫾ 0.19; P ¼ .48). There were no clear differences between proximal, central, or distal parts of the stents, but the bioactive muraglitazar-eluting stent tended to induce less inflammation than the control stent (Fig 5).
DISCUSSION In the present study, we evaluated the effects of a bioabsorbable drug-eluting stent loaded with a dual PPARα/γ agonist on the porcine arterial vessel wall. According to our results, intimal hyperplasia was inhibited and the luminal diameter and area were better retained in vessels treated with muraglitazar-eluting PLA96 stents than in those treated with control PLA96 stents. The biocompatibility of PLA stents has been proven to be safe (14). At the same time, inflammation has been
Figure 2. Histological cross-section of (a) muraglitazar-eluting PLA96 stent and (b) PLA96 stent. (Hematoxylin and eosin staining, scale in millimeters.) (Available in color online at www.jvir.org.)
Figure 3. Mean intimal thickness (in micrometers) in the proximal, central, and distal parts of both types of stents (vertical bars represent standard error of the mean).
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Figure 4. Mean vascular injury scores in the proximal, central, and distal parts of both types of stents (vertical bars represent standard error of the mean).
Figure 5. Mean vascular inflammation scores in the proximal, central, and distal parts of both types of stents (vertical bars represent standard error of the mean).
described as a major concern regarding bioabsorbable polymeric stents (15). Polymers like PLA, with a low molecular weight, have been demonstrated to cause a more severe inflammatory reaction than polymers with a higher molecular mass (4 300 kDa) (16). This information is important because the degree of inflammation has a linear correlation with the severity of intimal hyperplasia (12). That is why the bioactive agents used in the stents should also inhibit the inflammation process. Muraglitazar is a dual PPAR-α/γ agonist with a strong γ- and a moderate α-effect (17). It has been shown to reduce hemoglobin A1C levels and improve the lipid profile in patients with type II diabetes compared with placebo, appearing to be more potent than pioglitazone (18). All major vascular and inflammatory cells, endothelial cells, vascular smooth muscle cells, and macrophages express PPAR-α and PPAR-γ, and stimulation by the agonists has been shown to have antiinflammatory effects (8). Inhibiting the phosphorylation
of cyclin-dependent kinase-2 and retinoblastoma protein as well as the inhibition of telomerase activity have been suggested as the mechanisms behind the inhibition of vascular smooth muscle cell proliferation and neointimal hyperplasia by PPAR agonists (19). The activation of inflammatory transcription factors nuclear factor–κB, C/ EPB, and STAT3, and the expression of several growth factors, adhesion molecules, and proinflammatory cytokines and enzymes have been demonstrated to be inhibited by PPAR agonists (8). Accordingly, muraglitazareluting stents inhibited intimal hyperplasia, resulting in larger luminal diameter and area in vessels than seen with the use of control PLA96 stents. Vascular trauma caused by the stent struts has also been demonstrated to correlate linearly with intimal proliferation and restenosis (14). Stents should have a sufficient radial force to prevent elastic recoil and negative remodeling in all stent-implanted segments. However, if the struts are embedded too deep into the vessel wall, they will cause vascular smooth muscle cell
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proliferation, resulting in intimal hyperplasia. In the present study, both stents caused only mild trauma, meaning that stent struts penetrated the intimal layer alone, compressing only the internal elastic lamina and medial muscle layer. Intimal hyperplasia is regarded as one of the most important factors involved in the development of restenosis after successful angioplasty and stent placement (20). Drug-eluting stents were developed to overcome this phenomenon. The initial results were encouraging, as they showed a dramatic decrease in early restenosis (21). However, subsequent studies demonstrated that the discontinuation of dual antiplatelet therapy is associated with late vessel thrombosis (4). The problem with drugeluting stents is that they effectively control neointimal formation but also delay vascular healing, especially stent endothelialization. Another recognized issue is hypersensitivity reactions induced by sirolimus-eluting stents, leading to aneurysm formation and thrombosis (22). Bioabsorbable drug-eluting stents could provide the answer to these concerns because the devices would disappear before the late adverse events could occur. PLA, which is the most used family of polymers in bioabsorbable stents, is metabolized through the Krebs cycle, in which it finally forms carbon dioxide and water. The degradation time depends on the molecular weight and composition of PLA, and has been reported to be approximately 12–18 months (23). Preclinical studies with different PLAs have proven their potential as biomaterials in vascular stents (23,24). Vogt et al (24) used a paclitaxel-eluting poly(D,L-)lactide stent in porcine coronary arteries. They showed that a bioabsorbable paclitaxel-eluting PLA stent inhibited intimal hyperplasia by 53% compared with a plain PLA stent and by 44% compared with a bare metal stent over 3 months’ follow-up. In the present study, we achieved parallel results with a muraglitazar-eluting PLA96 stent, which decreased intimal hyperplasia by 54% compared with a plain PLA96 stent after 1 month follow-up. This demonstrates the role of muraglitazar as a potent drug for the inhibition of intimal proliferation. Results of clinical studies with bioabsorbable vascular stents have been encouraging. Tamai et al (14) were the first to demonstrate their Ikagi–Tamai poly-L-lactide stent’s safety and efficacy in humans. At the moment, the most used PLA stent is the everolimus-eluting BVS stent (Abbott Laboratories, Abbott Park, Illinois) (2). The first-in-man ABSORB trial (2) demonstrated the clinical safety of the BVS stent with only one of the 30 patients developing major adverse cardiac events after 4 years of follow-up, with no late complications such as stent thrombosis (2). The second-generation version of the bioresorbable everolimus-eluting BVS stent showed that the scaffold area remained unchanged during a 12month period, but the echogenicity of the struts was decreased by 20% (25). In this cohort of 56 patients (25), the rate of major cardiac adverse events rate was 7.1%
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(four of 56). The authors concluded that these findings justify the conducting of a randomized controlled trial against the current best-standard treatment (25). The weakness of the present study was the small number of samples, which renders the statistical power of the study inadequate. However, the trends of the results were clear and positive, as the muraglitazareluting PLA stent showed marked inhibition of inflammation and intimal hyperplasia. The systemic effects of the drug or the local drug concentrations in the vessel wall, blood, or urine were not determined because there can be bias when using two different kinds of stents in the same animal. The results suggest that the radial force of the stent was higher at the middle of the stent than at the ends, which may have influenced the reaction in the vascular wall. In conclusion, we find muraglitazar a new and interesting bioactive agent that can be used in drugeluting stents. The biocompatible muraglitazar-eluting PLA96 stent demonstrated a tendency toward better patency and less intimal hyperplasia compared with the control PLA96 stents. These positive preliminary results need to be confirmed in a larger trial with more patients to improve the statistical power.
ACKNOWLEDGMENTS The authors thank Jenni Leppiniemi, MSc, Riina Nieminen, PhD, Martti Talja, MD, and Eeva Moilanen, MD, for their comprehensive contribution to this manuscript. This study was financially supported by a research grant from The Finnish Funding Agency for Innovations and Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital, Finland.
REFERENCES 1. Biancari F. Meta-analysis of the prevalence, incidence and natural history of critical limb ischemia. J Cardiovasc Surg 2013; 54:663–669. 2. Dudek D, Onuma Y, Ormiston JA, Thuesen L, Miquel-Hebert K, Serruys PW. Four-year clinical follow-up of the ABSORB everolimus-eluting bioresorbable vascular scaffold in patients with de novo coronary artery disease: the ABSORB trial. EuroIntervention 2012; 7:1060–1061. 3. Waksman S, Pakala R. Biodegradable and bioabsorbable stents. Curr Pharm Des 2010; 16:4041–4051. 4. Vorpahl M, Yazdani SK, Nakano M, et al. Pathobiology of stent thrombosis after drug-eluting stent implantation. Curr Pharm Des 2010; 16:4064–4071. 5. Rodriguez-Granillo A, Rubilar B, Rodriguez-Granillo G, Rodriguez AE. Advantages and disadvantages of biodegradable platforms in drug eluting stents. World J Cardiol 2011; 3:84–92. 6. Adeghate E, Adem A, Hasan MY, Tekes K, Kalasz H. Medicanal chemistry and action of dual and pan PPAR modulators. Open Med Chem J 2011; 5:93–98. 7. Barlocco D. Muraglitatzar. Curr Opin Investig Drugs 2005; 6:427–434. 8. Paukkeri EL, Leppänen T, Lindholm M, et al. Anti-inflammatory properties of a dual PPARgamma/alpha agonist muraglitazar in in vitro and in vivo models. Arthritis Res Ther 2013; 15:R51. 9. Schwartz RS, Edelman ER, Carter A, et al. Preclinical evaluation of drugeluting stents for peripheral applications: recommendations from an expert consensus group. Circulation 2004; 110:2498–2505. 10. Virmani R, Kolodgie FD, Farb A, Lafont A. Drug eluting stents: are human and animal studies comparable? Heart 2003; 89:133–138.
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11. Mathieu O, Cruz-Orive LM, Hoppeler H, Weibel ER. Measuring error and sampling variation in stereology: comparison of the efficiency of various methods for planar image analysis. J Microsc 1981; 121: 75–88. 12. Kornowski R, Hong MK, Tio FO, Bramwell O, Wu H, Leon MB. In-stent restenosis: contributions of inflammatory responses and arterial injury to neointimal hyperplasia. J Am Coll Cardiol 1998; 31:224–230. 13. Schwartz RS, Huber KC, Murphy JG, et al. Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model. J Am Coll Cardiol 1992; 19:267–274. 14. Tamai H, Igaki K, Kyo E, et al. Initial and 6-month results of biodegradable poly-l-lactic acid coronary stent in humans. Circulation 2000; 102: 399–404. 15. van der Giessen WJ, Lincoff AM, Schwartz RS, et al. Coronary heart disease/thromboses/myocardial-infarction: marked inflammatory sequelae to implantation of biodegradable and nonbiodegradable polymers in porcine coronary arteries. Circulation 1996; 94:1690–1697. 16. Lincoff AM, Furst JG, Ellis SG, Tuch RJ, Topol EJ. Sustained local delivery of dexamethasone by a novel intravascular eluting stent to prevent restenosis in the porcine coronary injury model. J Am Coll Cardiol 1997; 29:808–816. 17. Devasthale PV, Chen S, Jeon Y, et al. Design and synthesis of N-[(4methoxyphenoxy)carbonyl]-N-[[4-[2-(5-methyl-2-phenyl-oxazolyl)ethoxy] phenyl]methyl]glycine [muraglitazar/BMS-298585], a novel peroxisome proliferator-activated receptor alpha/gamma dual agonist with efficacious glucose and lipid-lowering activities. J Med Chem 2005; 48: 2248–2250.
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18. Rubin CJ, Viraswami-Appanna K, Fiedorek FT. Efficacy and safety of muraglitazar: a double-blind, 24-week, dose-ranging study in patients with type 2 diabetes. Diabetes Vasc Dis Res 2009; 6:205–215. 19. Zahradka P, Wright B, Fuerst M, Yurkova N, Molnar K, Taylor CG. Peroxisome proliferator-activated receptor α and у ligands differentially affect smooth muscle cell proliferation and migration. J Pharm Exp Ther 2006; 317:651–659. 20. Farb A, Weber DK, Kolodgie FD, Burke AP, Virmani R. Morphological predictors for restenosis after coronary artery stenting in humans. Circulation 2002; 105:2974–2980. 21. Sousa JE, Costa MA, Abizaid A, et al. Lack of neointimal proliferation after implantation of sirolimus-coated stents in human coronary arteries. A quantitative coronary angiography and three-dimensional intravascular ultrasound study. Circulation 2001; 103:192–195. 22. Virmani R, Guagliumi G, Farb A, et al. Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: should we be cautious? Circulation 2004; 109:701–705. 23. Hietala EM, Salminen US, Ståhls A, et al. Biodegradation of the copolymeric polylactide stent. Long-term follow- up in a rabbit aorta model. J Vasc Res 2001; 38:361–369. 24. Vogt F, Stein A, Rettemeier G, et al. Long-term assessment of a novel biodegradable paclitaxel-eluting coronary polylactide stent. Eur Heart J 2004; 25:1330–1340. 25. Serruys PW, Onuma Y, Dudek D, et al. Evaluation of the second generation of a bioresorbable everolimus-eluting vascular scaffold for the treatment of de novo coronary artery stenosis: 12-month clinical and imaging outcomes. J Am Coll Cardiol 2011; 58:1578–1588.
Muraglitazar-Eluting Bioabsorbable Vascular Stent Inhibits Neointimal Hyperplasia in Porcine Iliac Arteries Ilkka Uurto, MD, Mari Hämäläinen, PhD, Velipekka Suominen, MD, Marita Laurila, MD, Andres Kotsar, MD, Taina Isotalo, MD, Teuvo L.J. Tammela, MD, Minna Kellomäki, Dr Tech, and Juha-Pekka Salenius, MD
ABSTRACT Purpose: To evaluate the biocompatibility of a new muraglitazar-eluting polylactide copolymer stent and investigate its ability to prevent the formation of intimal hyperplasia. Materials and Methods: Ten self-expandable muraglitazar-eluting poly-96L/4D-lactic acid (PLA96) stents and 10 selfexpandable control PLA96 stents were implanted into porcine common iliac arteries. After 28 days follow-up, all stentimplanted iliac arteries were harvested and prepared for quantitative histomorphometric analysis. Results: Angiographic analysis revealed that one control PLA96 stent had occluded and one had migrated. Histomorphometric analysis demonstrated that, with the control PLA96 stent, the luminal diameter and area were decreased versus the muraglitazar-eluting PLA96 stents (means ⫾ standard error of the mean, 3.58 mm ⫾ 0.34 vs 4.16 mm ⫾ 0.14 and 9.83 mm2 ⫾ 2.41 vs 13.75 mm2 ⫾ 0.93, respectively). The control PLA96 stent induced more intimal hyperplasia than the bioactive muraglitazar-eluting PLA96 stent (557 mm ⫾ 122 vs 361 mm ⫾ 32). Vascular injury scores demonstrated only mild vascular trauma for both stents (muraglitazar-eluting, 0.68 ⫾ 0.07; control, 0.75 ⫾ 0.08). Inflammation scores also showed mild inflammation for both stents (muraglitazar-eluting, 1.05 ⫾ 0.17; control, 1.23 ⫾ 0.19). Conclusions: This new muraglitazar-eluting PLA96 stent was shown to be biocompatible with a tendency for better patency and less intimal hyperplasia compared with the control PLA96 stents.
ABBREVIATIONS PLA = polylactide, PLA96 = poly-96l/4d-lactic acid, PPAR = peroxisome proliferator–activator receptor
Vascular occlusive disease is one of the most common causes of morbidity and mortality in the Western world From the Divisions of Vascular Surgery and Urology, Department of Surgery (I.U., V.S., A.K., T.L.J.T., J.P.S.), and Department of Pathology (M.L.), Tampere University Hospital; Immunopharmacology Research Group (M.H.), University of Tampere School of Medicine and Tampere University Hospital; Biomaterials and Tissue Engineering Group, Department of Electronics and Communications Engineering (M.K.), Tampere University of Technology; BioMediTech– Institute of Biosciences and Medical Technology (M.K.), Tampere; and Department of Surgery (T.I.), Päijät-Häme Central Hospital, Lahti, Finland. Received April 18, 2014; final revision received October 1, 2014; accepted October 6, 2014. Address correspondence to I.U., Divisions of Vascular Surgery and Urology, Department of Surgery, Tampere University Hospital, P.O. Box 2000, 33521 Tampere, Finland; E-mail: ilkka.uurto@uta.fi None of the authors have identified a conflict of interest. & SIR, 2015 J Vasc Interv Radiol 2015; 26:124–130 http://dx.doi.org/10.1016/j.jvir.2014.10.005
(1). Endovascular treatment has advanced rapidly, with the latest inventions including drug-eluting balloons and stents and, most recently, bioabsorbable stents (2). Consequently, stents and balloons are no longer just passive mechanical devices but rather active and biocompatible instruments (3). However, there are some concerns regarding possible late thrombosis after the implantation of drug-eluting stents (4). In addition, it is now conceivable that all beneficial effects of drug-eluting stents could be preserved without having permanent materials embedded within the vessel wall. The idea of bioabsorbable drug-eluting stents itself is attractive (3). It would mechanically prevent recoil and negative remodeling as well as bioactively inhibit vascular smooth muscle cell proliferation, thereby preventing restenosis (5). Peroxisome proliferator–activator receptors (PPARs) belong to the nuclear receptor superfamily and consist of
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three distinct members: PPAR-α, PPAR-β/δ, and PPARγ. In addition to their metabolic effects, PPARs are also involved in the regulation of inflammatory responses (6). Muraglitazar is a dual PPAR-α/γ agonist originally developed for the treatment of type II diabetes (7). Recently, it has also been shown to possess antiinflammatory properties in vitro and in vivo (8). The aim of the present study was to evaluate the biocompatibility of a new muraglitazar-eluting biodegradable vascular stent and its ability to prevent intimal hyperplasia formation.
MATERIALS AND METHODS Stents The polymer used for the stents was medical-grade 96L/ 4D-copolymer of L-lactide and D-lactide (Purac Biochem, Gorinchem, The Netherlands). The final diameter of the fiber was 0.20 mm. The stents were formed of 16 meltspun monofilaments braided over a 6-mm-diameter mandrel. The braids were heat-treated for structural stability, and the stents were cut from the braid to the length of 15 mm each. The stents were immersion-coated with the racemic 50L/50D copolymer of L-lactide and D-lactide (Purac Biochem), into which muraglitazar was mixed. After thorough evaporation of the solvent, the stents were sterilized with more than 25 kGy of γ-irradiation by a commercial supplier. The measured molecular weight of the sterilized poly-96L/4D-lactic acid (PLA96) was 150,000 g/mol. The amount of the drug on the stents (500 mg ⫾ 70 per stent) and the thickness of the coatings were calculated by weighing the stents before and after coating. Because the mechanical properties of PLA96 stents differ quite significantly from those of bare metal stents, the pure PLA96 stent was chosen as a control stent. Its properties were similar to those of drugeluting PLA96 stents but with no bioactive agents. At the delivery device, there was a 6-mm balloon at the tip. The stent needed to be loaded onto the balloon immediately before implantation. Pulling back the sheath allowed the stent to self-expand, and the stent was then expanded with the balloon to ensure adequate implantation.
Animal Preparation and Stent Implantation All animal protocols were reviewed and approved by the institutional committee for animal research and by the eastern Finland provincial government. The study followed the recommendation guideline for the evaluation of drug-eluting stents for peripheral application as published previously (9). Ten male laboratory-bred swine were used according to the aforementioned recommendation (National Laboratory Animal Center, University of Kuopio, Kuopio, Finland), with a calculated statistical power of 0.873. Animals were 3 months old with a body weight of 25 kg each. Before the procedure,
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each animal was sedated with intramuscular azaperone (5 mg/kg) and subcutaneous atropine (0.05 mg/kg), and each was given an intramuscular dose of penicillin (750,000 IU) as antibacterial prophylaxis. The stent implantation was performed under general anesthesia with intravenous propofol (2 mg/kg). The animal was intubated and connected to a respirator. Anesthesia was maintained by propofol infusion (0.4 mL/kg/h). After systemic intravenous heparinization (150 IU/kg), the right carotid artery was surgically exposed. After arteriotomy, an 11-F sheath was introduced under visual control. The straight catheter was placed into the infrarenal aorta through a guide wire (Radiofocus Guide Wire M Standard, 0.035-inch, 150-cm; Terumo Europe, Leuven, Belgium). Preoperative angiography from the aortoiliac region was performed. The muraglitazareluting PLA96 stent was then introduced and implanted into the right common iliac artery under fluoroscopy. The same procedure was repeated with the control PLA96 stent in the left common iliac artery. The arteriotomy was closed with 6–0 sutures, and the wound was closed separately layer by layer. As antithrombotic medication, the animals received acetylsalicylic acid (250 mg/d) orally for 5 days preoperatively and until the end of follow-up and enoxaparin sodium (15 mg/d subcutaneously) from 1 day preoperatively through the third postoperative day. The animals were given buprenorphine (0.01 mg/kg subcutaneously) as analgesia for postoperative pain.
Follow-up and Analyses The animals’ health was observed continuously by specially trained laboratory animal nurses. In pigs, the intimal hyperplasia expansion rate reaches its peak at 1 month after the procedure—therefore, a follow-up of 28 days was chosen (10). After follow-up, the animals were sedated, deeply anesthetized, and heparinized (150 IU/kg) before angiography was performed and the animals were euthanized with 60 mg/kg pentobarbital. The common iliac arteries were perfusion-fixed with 10% buffered formalin at 100 mm Hg for 15 minutes and left overnight in immersion fixation with the same fixative agent. After fixation, the stent-implanted section was cut into proximal, central, and distal segments. Postimplantation angiographic findings were compared versus the angiographic findings during harvesting. The normal diameter of the stent-implanted artery and the narrowest diameter of the stent-implanted section were measured in millimeters manually with a calibrated digital ruler by one of the authors (I.U.). The percentage of decrease in luminal diameter was calculated. Stenosis was defined as an at least 50% reduction of luminal diameter. All histologic preparations were stained with hematoxylin and eosin. The histomorphometric analysis was computer-assisted (Scion Image Beta 4.0.2; Scion, Frederick, Maryland). Premeasurements were conducted
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before final measurements with relative standard error to ensure reliable results (ie, relative standard error o 10%) (11). The patency of the stent-implanted segments was evaluated by measuring the luminal diameter and area. The thicknesses of the tunica intima (ie, layer between endothelium and internal elastic membrane) and tunica media (ie, layer between internal and external elastic membrane) were measured as intima–media thickness, and the intimal/medial ratio was assessed. Intimal hyperplasia was estimated from neointimal thickness. Inflammatory response was measured according to the previously published method known as “inflammation score” (12). It estimates inflammatory cells around stent struts caused by materials and the procedure, yielding a numerical value from 0 to 3. The scoring was accomplished by a senior pathologist who was blinded to the histologic samples. Vascular trauma was assessed by means of the vascular injury score, which estimates how deep the stents struts have been embedded into the artery wall (13). Vascular inflammation and injury scoring systems are standardized methods, and were performed for every proximal, central, and distal segment of the stents.
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control PLA96 stent had occluded and one migrated. Otherwise, all stent-implanted vessels were patent. The percentages of decrease in luminal diameters were 16% for muraglitazar-eluting PLA96 stents and 39% for control PLA96 stents (Fig 1). Histomorphometric analysis was carried out for seven segments each with muraglitazar-eluting PLA96 stents and PLA96 control stents (Table). Three muraglitazareluting PLA96 stents and three control PLA96 stents were excluded from the analysis because of unsatisfactory section preparations and staining. Patency was assessed by measuring the mean luminal diameter and area. Muraglitazar-eluting PLA96 stents preserved the luminal diameter and area (4.2 mm ⫾ 0.1 and 13.8 mm2 ⫾ 0.9, respectively) better than PLA96 stents (3.6 mm ⫾ 0.3 and 9.8 mm2 ⫾ 2.4; P ¼ .15 and P ¼ .16, respectively). There were no marked differences in the intimal/medial ratio between muraglitazar-eluting and control stents (muraglitazar-eluting PLA96 stent, 0.9 ⫾ 0.1; PLA96 stent, 0.9 ⫾ 0.1; P ¼ .16). There was a tendency for the control polylactide (PLA) stent to induce more intimal hyperplasia compared with the bioactive muraglitazar-eluting PLA96 stent (PLA stent,
Statistical Analysis SPSS software (version 19.0; SPSS, Chicago, Illinois) for Windows (Microsoft, Redmond, Washington) was used for statistical analysis. A comparison of means between the two groups was carried out with the t test for independent samples. All results are presented as means ⫾ standard error of means unless otherwise stated. A P value less than .05 was considered statistically significant.
RESULTS Twenty biodegradable stents (10 muraglitazar-eluting and 10 control PLA96 stents) were implanted into the common iliac arteries of young healthy swine. Angiography was performed on all stent-implanted segments directly after implantation and before harvesting. One
Table. Histomorphometric Analysis Muraglitazar-Eluting
PLA96
PLA96 Stent
Stent
P Value
4.4 ⫾ 0.2 3.9 ⫾ 0.2
3.9 ⫾ 0.5 3.0 ⫾ 0.4
.314 .061
Mean
4.2 ⫾ 0.1
3.6 ⫾ 0.3
.145
LA (mm2) IMT (mm)
13.8 ⫾ 0.9 0.8 ⫾ 0.1
9.8 ⫾ 2.4 1.0 ⫾ 0.2
.156 .201
I/M ratio
0.8 ⫾ 0.1
0.9 ⫾ 0.1
.156
Intimal thickness (mm)
0.4 ⫾ 0.0
0.6 ⫾ 0.1
.149
Measurement LD (mm) Maximum Minimum
Values presented as means ⫾ standard error of the mean. I ¼ intima, IMT ¼ intima–media thickness, LA ¼ luminal area, LD luminal diameter, M ¼ media.
Figure 1. Angiographic images: (1) Muraglitazar-eluting PLA96 stent and (2) PLA96 stent (a) perioperative and (b) after follow-up.
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557 mm ⫾ 122; muraglitazar-eluting PLA96 stent, 361 mm ⫾ 32; P ¼ .15; Fig 2). There was also a clear trend for a more severe intimal reaction to develop at the ends of the control PLA96 stent compared with the central part of the stent (PLA stent, proximal, 538 mm ⫾ 100; central, 399 mm ⫾ 116; distal, 660 mm ⫾ 191; muraglitazar-eluting PLA96 stent, proximal, 334 mm ⫾ 59; central, 333 mm ⫾ 19; distal, 416 mm ⫾ 72; P ¼ .13, P ¼ .59, and P ¼ .26, respectively; Fig 3). Vascular injury score showed that both stents caused minimal trauma to the vessel wall (muraglitazar-eluting PLA96 stent, 0.7 ⫾ 0.1; PLA96 stent, 0.8 ⫾ 0.1; P 4 .05). There was a tendency toward lower scores at the ends of the stents compared with the central parts of the stents (PLA stent, proximal, 0.7 ⫾ 0.2; central, 1.0 ⫾ 0.1; distal, 0.6 ⫾ 0.1; muraglitazar-eluting PLA96 stent, proximal, 0.7 ⫾ 0.1; central, 0.9 ⫾ 0.1; distal, 0.6 ⫾ 0.1; P ¼ .51; Fig 4). The inflammation score demonstrated only mild inflammation related to both stent types
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(muraglitazar-eluting PLA96 stent, 1.05 ⫾ 0.17; PLA96 stent, 1.23 ⫾ 0.19; P ¼ .48). There were no clear differences between proximal, central, or distal parts of the stents, but the bioactive muraglitazar-eluting stent tended to induce less inflammation than the control stent (Fig 5).
DISCUSSION In the present study, we evaluated the effects of a bioabsorbable drug-eluting stent loaded with a dual PPARα/γ agonist on the porcine arterial vessel wall. According to our results, intimal hyperplasia was inhibited and the luminal diameter and area were better retained in vessels treated with muraglitazar-eluting PLA96 stents than in those treated with control PLA96 stents. The biocompatibility of PLA stents has been proven to be safe (14). At the same time, inflammation has been
Figure 2. Histological cross-section of (a) muraglitazar-eluting PLA96 stent and (b) PLA96 stent. (Hematoxylin and eosin staining, scale in millimeters.) (Available in color online at www.jvir.org.)
Figure 3. Mean intimal thickness (in micrometers) in the proximal, central, and distal parts of both types of stents (vertical bars represent standard error of the mean).
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Figure 4. Mean vascular injury scores in the proximal, central, and distal parts of both types of stents (vertical bars represent standard error of the mean).
Figure 5. Mean vascular inflammation scores in the proximal, central, and distal parts of both types of stents (vertical bars represent standard error of the mean).
described as a major concern regarding bioabsorbable polymeric stents (15). Polymers like PLA, with a low molecular weight, have been demonstrated to cause a more severe inflammatory reaction than polymers with a higher molecular mass (4 300 kDa) (16). This information is important because the degree of inflammation has a linear correlation with the severity of intimal hyperplasia (12). That is why the bioactive agents used in the stents should also inhibit the inflammation process. Muraglitazar is a dual PPAR-α/γ agonist with a strong γ- and a moderate α-effect (17). It has been shown to reduce hemoglobin A1C levels and improve the lipid profile in patients with type II diabetes compared with placebo, appearing to be more potent than pioglitazone (18). All major vascular and inflammatory cells, endothelial cells, vascular smooth muscle cells, and macrophages express PPAR-α and PPAR-γ, and stimulation by the agonists has been shown to have antiinflammatory effects (8). Inhibiting the phosphorylation
of cyclin-dependent kinase-2 and retinoblastoma protein as well as the inhibition of telomerase activity have been suggested as the mechanisms behind the inhibition of vascular smooth muscle cell proliferation and neointimal hyperplasia by PPAR agonists (19). The activation of inflammatory transcription factors nuclear factor–κB, C/ EPB, and STAT3, and the expression of several growth factors, adhesion molecules, and proinflammatory cytokines and enzymes have been demonstrated to be inhibited by PPAR agonists (8). Accordingly, muraglitazareluting stents inhibited intimal hyperplasia, resulting in larger luminal diameter and area in vessels than seen with the use of control PLA96 stents. Vascular trauma caused by the stent struts has also been demonstrated to correlate linearly with intimal proliferation and restenosis (14). Stents should have a sufficient radial force to prevent elastic recoil and negative remodeling in all stent-implanted segments. However, if the struts are embedded too deep into the vessel wall, they will cause vascular smooth muscle cell
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proliferation, resulting in intimal hyperplasia. In the present study, both stents caused only mild trauma, meaning that stent struts penetrated the intimal layer alone, compressing only the internal elastic lamina and medial muscle layer. Intimal hyperplasia is regarded as one of the most important factors involved in the development of restenosis after successful angioplasty and stent placement (20). Drug-eluting stents were developed to overcome this phenomenon. The initial results were encouraging, as they showed a dramatic decrease in early restenosis (21). However, subsequent studies demonstrated that the discontinuation of dual antiplatelet therapy is associated with late vessel thrombosis (4). The problem with drugeluting stents is that they effectively control neointimal formation but also delay vascular healing, especially stent endothelialization. Another recognized issue is hypersensitivity reactions induced by sirolimus-eluting stents, leading to aneurysm formation and thrombosis (22). Bioabsorbable drug-eluting stents could provide the answer to these concerns because the devices would disappear before the late adverse events could occur. PLA, which is the most used family of polymers in bioabsorbable stents, is metabolized through the Krebs cycle, in which it finally forms carbon dioxide and water. The degradation time depends on the molecular weight and composition of PLA, and has been reported to be approximately 12–18 months (23). Preclinical studies with different PLAs have proven their potential as biomaterials in vascular stents (23,24). Vogt et al (24) used a paclitaxel-eluting poly(D,L-)lactide stent in porcine coronary arteries. They showed that a bioabsorbable paclitaxel-eluting PLA stent inhibited intimal hyperplasia by 53% compared with a plain PLA stent and by 44% compared with a bare metal stent over 3 months’ follow-up. In the present study, we achieved parallel results with a muraglitazar-eluting PLA96 stent, which decreased intimal hyperplasia by 54% compared with a plain PLA96 stent after 1 month follow-up. This demonstrates the role of muraglitazar as a potent drug for the inhibition of intimal proliferation. Results of clinical studies with bioabsorbable vascular stents have been encouraging. Tamai et al (14) were the first to demonstrate their Ikagi–Tamai poly-L-lactide stent’s safety and efficacy in humans. At the moment, the most used PLA stent is the everolimus-eluting BVS stent (Abbott Laboratories, Abbott Park, Illinois) (2). The first-in-man ABSORB trial (2) demonstrated the clinical safety of the BVS stent with only one of the 30 patients developing major adverse cardiac events after 4 years of follow-up, with no late complications such as stent thrombosis (2). The second-generation version of the bioresorbable everolimus-eluting BVS stent showed that the scaffold area remained unchanged during a 12month period, but the echogenicity of the struts was decreased by 20% (25). In this cohort of 56 patients (25), the rate of major cardiac adverse events rate was 7.1%
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(four of 56). The authors concluded that these findings justify the conducting of a randomized controlled trial against the current best-standard treatment (25). The weakness of the present study was the small number of samples, which renders the statistical power of the study inadequate. However, the trends of the results were clear and positive, as the muraglitazareluting PLA stent showed marked inhibition of inflammation and intimal hyperplasia. The systemic effects of the drug or the local drug concentrations in the vessel wall, blood, or urine were not determined because there can be bias when using two different kinds of stents in the same animal. The results suggest that the radial force of the stent was higher at the middle of the stent than at the ends, which may have influenced the reaction in the vascular wall. In conclusion, we find muraglitazar a new and interesting bioactive agent that can be used in drugeluting stents. The biocompatible muraglitazar-eluting PLA96 stent demonstrated a tendency toward better patency and less intimal hyperplasia compared with the control PLA96 stents. These positive preliminary results need to be confirmed in a larger trial with more patients to improve the statistical power.
ACKNOWLEDGMENTS The authors thank Jenni Leppiniemi, MSc, Riina Nieminen, PhD, Martti Talja, MD, and Eeva Moilanen, MD, for their comprehensive contribution to this manuscript. This study was financially supported by a research grant from The Finnish Funding Agency for Innovations and Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital, Finland.
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