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Department of Surgery, Medical University of Vienna, Vienna, AustriaCentre for Biomedical Research, Medical University of Vienna, Vienna, AustriaLudwig Boltzmann Cluster for Cardiovascular Research, Medical University of Vienna, Vienna, Austria
Centre for Biomedical Research, Medical University of Vienna, Vienna, AustriaLudwig Boltzmann Cluster for Cardiovascular Research, Medical University of Vienna, Vienna, AustriaAustrian Cluster for Tissue Regeneration, Vienna, Austria
Biodegradable materials for in situ vascular tissue engineering could meet the increasing clinical demand for sufficient synthetic small diameter vascular substitutes in aortocoronary bypass and peripheral vascular surgery. The aim of this study was to design a new degradable thermoplastic polycarbonate urethane (dPCU) with improved biocompatibility and optimal biomechanical properties. Electrospun conduits made from dPCU were evaluated in short and long term follow up and compared with expanded polytetrafluoroethylene (ePTFE) controls.
Both conduits were investigated prior to implantation to assess their biocompatibility and inflammatory potential via real time polymerase chain reaction using a macrophage culture. dPCU grafts (n = 28) and ePTFE controls (n = 28) were then implanted into the infrarenal abdominal aorta of Sprague–Dawley rats. After seven days, one, six, and 12 months, grafts were analysed by histology and immunohistochemistry (IHC) and assessed biomechanically.
Anti-inflammatory signalling was upregulated in dPCU conduits and increased significantly over time in vitro. dPCU and ePTFE grafts offered excellent long and short term patency rates (92.9% in both groups at 12 months) in the rat model without dilatation or aneurysm formation. In comparison to ePTFE, dPCU grafts showed transmural ingrowth of vascular specific cells resulting in a structured neovessel formation around the graft. The graft material was slowly reduced, while the compliance of the neovessel increased over time.
The newly designed dPCU grafts have the potential to be safely applied for in situ vascular tissue engineering applications. The degradable substitutes showed good in vivo performance and revealed desirable characteristics such as biomechanical stability, non-thrombogenicity, and minimal inflammatory response after long term implantation.
This study provides long term results of a novel, degradable, small diameter vascular graft in a rat model. The Degradable polycarbonate urethane (dPCU) exhibits appropriate biomechanical properties and improved biocompatibility with reduced secondary inflammation. dPCU conduits promoted rapid complete endothelialisation, increased and sustained transmural cellular ingrowth, proliferation of cells and microvessel formation with minimal inflammatory response.
Autologous saphenous vein, internal mammary artery (IMA), and the radial artery (RA) are currently the gold standard for small diameter vascular reconstructions. The limited availability of autologous graft material is aggravated by prior use or insufficient graft quality. These shortages are becoming more frequent as our ageing society suffers from metabolic syndrome, arteriosclerosis, and varicosities. However, successful long term replacement of small diameter vascular grafts (SDVG) by a synthetic ready to use material remains challenging.
Currently used synthetic materials such as expanded polytetrafluoroethylene (ePTFE) and Dacron perform well as large diameter conduits. In grafts with 2–6 mm inner diameter suitable for peripheral arterial or coronary artery revascularisation, patency is very low because of high thrombogenicity, intimal hyperplasia (IH), inflammatory processes, or graft infection.
After implantation, inflammatory cells migrate onto the graft and are important mediators of graft remodelling, switching from the initial pro-inflammatory signal to tissue remodelling and repair. Monocytes/macrophages are present until degradation and produce cell proliferation and remodelling factors such as interleukin (IL)-6, IL-10, and matrix metalloproteases. Long term presence of synthetic material may lead to extended foreign body reaction and fibrosis/scar tissue formation.
the ideal ready to use material should be designed to match the native vessel in compliance, dimensions, haemodynamic factors, anti-thrombotic and anti-inflammatory (non-toxic) qualities, and should promote fast endothelial cell adherence.
From the investigated synthetic materials, non-degradable electrospun polycarbonate urethane (PCU) has shown promise to meet the criteria. Thermoplastic PCUs are block copolymers with hard and soft segments (semi)crystalline and amorphous domains, consisting of (poly)carbonate based precursors, which form physically cross linked networks with superior mechanical properties. Non-degradable, thermoplastic PCU with various modifications has been tested in small and large animal models with sound results, and pilot studies have been performed successfully in human arteriovenous fistulae.
For this reason, the present study group aimed to further improve the qualities of PCUs by design and synthesis of a material with specific biodegradability for in situ vascular tissue engineering. The advantage of a biodegradable approach includes continuous remodelling of graft until an endogenous neovessel has formed. Prerequisites for degradable materials are adequate biomechanical stability until sufficient vascular specific, host dependent, tissue formation has occurred to avoid leakage, rupture, and aneurysm formation.
The advantage of this newly synthesised thermoplastic degradable polycarbonate urethane (dPCU) is its slow degradation rate by the use of cleavable carbonate based chain extenders, resulting in controlled graft reduction without development of acidic or toxic byproducts. The biocompatibility of the polyurethanes has been improved by the use of aliphatic rather than aromatic organic compounds.
The aim of the study was to characterise the graft material with regard to biomechanical properties and biocompatibility and further analyse the short and long term performance of this new synthesised dPCU in a rodent model.
Material and biomechanical testing
A pre-polymer method was used to synthesise the thermoplastic polyurethane polymer.
In contrast to the previously described polymers, the dPCU in this study was based on poly (hexamethylene carbonate), hexamethylene diisocyanate (HMDI), and bis(3-hydroxypropyl) carbonate. An average molecular weight of 860 Da for poly (hexamethylene carbonate) (which corresponds to a typical chain length in thermoplastic polyurethanes) and a molar ratio of the above mentioned components of 1:2:1 resulted in a good trade off between tensile strength and toughness of the material.
dPCU grafts were fabricated by the electrospinning method and characterised morphologically and mechanically by scanning electron microscopy (SEM; JEOL JSM-5400, Japan) and micro-computed tomography (μCT) (μCT-35, SCANCO Medical, Zurich, Switzerland).
Additionally, dPCU's fibre distribution was studied by atomic force microscopy (AFM) using a JPK NanoWizard III (JPK Instruments AG, Germany) connected to an inverted optical microscope (Axio Observer Z1, Zeiss). AFM silicon nitride probes (DNP-S10, Bruker, USA) were applied as cantilevers with a spring constant of 0.3 N/m.
The suture retention of dPCU was tested prior to implantation using a polypropylene thread (7/0 Prolene, BV 176–8, Ethicon, USA) with a tensile tester (Messphysik Beta 10–2,5, Messphysik Materials Testing GmbH, Altenmarkt, Austria).
The maximum tensile force and compliance in the physiological range were investigated before and after six and 12 months of implantation and compared with ePTFE (n = 4 per group and time, details in supplements) using a uniaxial BOSE ElectroForce LM1 test bench system (Bose Corp. MN, USA).
Before proceeding with any in vivo or in vitro experiment samples were sterilised with ethylene oxide.
In vitro evaluation by macrophage markers and inflammatory cytokines
All experiments involving animals or animal tissues were conducted in compliance with European and national legislation and were approved by the Austrian Federal Ministry of Education, Science and Research (reference number: BMWF-66.009/0097-II/3b/2013). The animals’ care was in accordance with institutional guidelines.
Macrophages were isolated from adult male Sprague–Dawley rats (350–400 g, Centre for Biomedical Research, Vienna, Austria) by a peritoneal lavage technique as described elsewhere.
Isolated macrophages at a density of 2 × 105 cells/well (24 well plate) were seeded on the luminal side of the grafts (10 mm* 1.8 mm) and incubated for one, three, seven, and 21 days. All experiments were repeated independently three times (n = 3 per time point).
Real time polymerase chain reaction (RT-PCR) was used to identify expression of CD68 for pan-macrophages, CCR7 and CD80 for pro-inflammatory (M1) and CD163 and CD206 for anti-inflammatory (M2) macrophage markers. The ratio of CD80/CD163 gene expression was calculated as an indicator of M1/M2 response. Furthermore, pro-inflammatory cytokines IL-1α and tumour necrosis factor (TNF)-α and anti-inflammatory IL-10 were studied in the presence of the dPCU and ePTFE. RT-PCR was performed using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) via QIAcube system (Qiagen).
Data were analysed using LightCycler Software (LightCycler Software Version 3.5, Roche, Basel, Switzerland).
In vivo evaluation
Electrospun dPCU and ePTFE prostheses were implanted as interposition grafts into the infrarenal aorta of 56 inbred Sprague–Dawley rats (male, body weight 300–400 g) (n = 7 per time point and group) using an operating microscope (Zeiss OPMI 9-FC, Zeiss, Germany).
The group allocation was randomised. Prostheses were anastomosed end to end using an interrupted suture technique (Monosof 10/0, Tyco, Norwalk, CT, USA) by one experienced surgeon. Neither anti-coagulation nor anti-platelet drugs were administered to the animals. Grafts remained in situ for seven days, one, six, or 12 months (dPCU n = 7, ePTFE n = 7, for each time point, respectively). In the six month and 12 month implantation groups, digital subtraction angiography was performed using 1 mL/kg iopamidol (Jopamiro®, 300 mg/mL, Bracco, Vienna, Austria).
Histology, immunohistochemistry (IHC), and immunofluorescence
Histological samples were obtained from all time points, processed and evaluated regarding endothelialisation (von Willebrand Factor, vWF), cell proliferation and invasion (Ki67, haematoxylin eosin (HE), smooth muscle actin (SMA), calponin, vimentin, collagen), and inflammatory potential (ED1/CD68, ED2/CD163) (detailed information in the Supplementary material). To assess occurrence of degradation, stenosis, or aneurysm formation, five consecutive histological slide samples from both anastomotic sites and midgraft regions were evaluated regarding their respective inner diameter and wall thicknesses. Samples after seven days implantation were excluded from statistical evaluation regarding quantitative histological parameters, because of difficult tissue fixation on the graft.
Median (quartile) values or mean and standard deviation, if applicable, were given to describe continuous variables, and absolute numbers and percentages were used to describe categorical variables. The number of microvessels, foreign body giant cells, SMA, calponin, ED1, ED2 positive cells in the media, and absolute numbers of cells within the graft were set in relation to the graft area. Differences in continuous variables between different time points and materials were tested using analysis of variance (ANOVA), Scheffé’s post hoc analysis, and the two sample t test. Non-normally distributed variables were compared by the Wilcoxon rank sum test. Correlations of continuous variables were characterised using the Spearman correlation coefficient. Patency rates were calculated using the Kaplan–Meier analysis. All p values are results of two sided tests and p values < .05 were considered to be statistically significant. SPSS software version 24.0 (IBM corporations Inc. 1989–2016; Armonk, NY, USA) was used for statistical analyses.
Fabricated grafts had the following characteristics: fibre diameter: 1.66 ± 0.77 μm; porosity: 54.9 ± 1.5%; pore size: 3.8 ± 1 μm; inner diameter: 1.8 ± 0.1 mm; wall thickness: 84 ± 12 μm; graft length: 18 mm (Fig. 1). ePTFE grafts (inner diameter: 1.5 mm; wall thickness: 100 μm; graft length: 18 mm; internodal distance: 5–25 μm, Zeus, Orangeburg, SC, USA) were used as controls.
Surgical handling and biomechanics
dPCU grafts were easy to handle and remained open by their inherent wall tension, whereas ePTFE grafts, similar in wall thickness, collapsed prior to implantation. Markedly improved suturability was observed in dPCU (suture retention dPCU 1.87 ± 0.17 N vs. ePTFE 0.09 ± 0.01 N, p < .001). The ultimate tensile force was higher in dPCU grafts than in ePTFE throughout the experiment (prior implantation: 3.79 ± 0.64 vs. 1.49 ± 0.26 N, p < .001; after six months: 4.79 ± 1.13 N vs. 2.05 ± 0.49 N, p < .001; and after one year: 5.02 ± 0.13 N vs. 1.98 ± 0.36 N, p < .001). The compliance of dPCU was similar to that of ePTFE grafts before (3.19 ± 0.75%/100 mmHg vs. 3.50 ± 1.4%/100 mm Hg, n.s.) and after 180 days of implantation (3.72 ± 0.95%/100 mmHg vs. 3.54 ± 0.89%/100 mmHg, n.s). After one year the compliance increased significantly in dPCU compared with baseline, but remained unaffected in ePTFE (dPCU 5.0 ± 1.62%/100 mmHg, p = .026; ePTFE 3.66 ± 1.13%/100 mmHg, n.s.).
In vitro testing
In vitro macrophage culture showed a predominant pro-inflammatory response within the first three days in both materials. After seven days there was a clear transition from M1 pro-inflammatory (CD80) to M2 anti-inflammatory (CD163) macrophages in dPCU. In ePTFE the pro-inflammatory response decreased significantly; however, the transition of M1 to M2 did not occur in these grafts. In detail, pro-inflammatory signalling in ePTFE was significantly higher regarding CCR7 (d3 p < .001) and CD80 (d3,7,21 p < .001). By contrast, anti-inflammatory signalling was upregulated and increased significantly over time in dPCU (p < .001), while there was only a moderate increase in ePTFE (n.s.). After 21 days CD163 and CD206 expression was significantly higher in dPCU in comparison to ePTFE (p < .001).
In ePTFE, pro-inflammatory cytokines were significantly increased at an early phase, but downregulated significantly at later stages (IL-1α d1 p < .001; TNF-α d3 p < .001). IL-10 as anti-inflammatory cytokine was increased significantly in dPCU (d1 p = .037, d3 p = .004, and d21 p < .0001) (Fig. 2, Supplementary tables).
In vivo results
In one dPCU animal, occlusion occurred after 100 days because of intimal hyperplasia, and two rats died after early thrombosis within the first week in the ePTFE group. Therefore, patency was 92.9% for dPCU and ePTFE according to Kaplan–Meier estimates (Fig. 7, Table S5). At days seven, 30, 180, and 365, the individual patency was 100%, 100%, 92.9%, and 92.9% for dPCU and 100%, 92.9%, 92.9%, and 92.9% for ePTFE, respectively.
Peri-operative mortality was 8.9% because of bleeding and complications from anaesthesia and these animals were not considered in further calculations. In all animals reaching the endpoints of six and 12 months respectively, angiography showed a patent graft without signs of aneurysm or stenosis. The inner diameter according to digital subtraction angiography and histology did not change significantly over time. On longitudinal opening of the samples, all grafts were patent, displaying a shiny regular luminal surface without signs of plaque, thrombus, or calcification (Fig. 3).
A decrease in average graft wall thickness was observed after 12 months, but was not significant compared with pre-operative values (84 ± 12 μm vs. 59.1 ± 16.7 μm, n.s.). Full endothelialisation of all dPCU grafts was accomplished within 30 days, while overall 14.3% of ePTFE had no coverage (p = .05) or were only partially covered (17.9%, p = .02). Small areas of intimal hyperplasia [median 12 cell layers (7; 13)] were found in proximity to the anastomosis in three dPCU grafts, while IH covering the entire length of the graft was only found in one dPCU sample. By contrast, in eight ePTFE grafts [median of 11 cell layers (8; 17)] IH covered the total graft surface. The total number of cells within the dPCU graft remained stable over time (p = .58), in ePTFE conduits cell numbers decreased (p = .03). Overall, more cells migrated into dPCU than ePTFE grafts (437.2 ± 257.9 cells/mm2vs. 110.7 ± 121.6 cells/mm;
p < .001). Proliferation remained constant in dPCU, while it started at a higher level in ePTFE (Ki67, 35.4 ± 34.3 cells/mm2vs. 636.3 ± 436.7 cells/mm2 after 30 days, p = .001) and dropped significantly over time (p < .001), until there were no significant differences between materials at six (p = .2) and 12 months (p = .17). Within the first month, neovessel formation with the regular composition of (single layer) endothelium, intermediate graft, neomedia, and adventitia was visible in all dPCU grafts (Fig. 4). SMCs aligned, forming the typical layer of the media. Furthermore, colocalisation of SMA and calponin was present at six and 12 months (r = .75, p = .005) (Fig. 5). The total number of vasa vasorum was higher in dPCU grafts (dPCU: 67.0 ± 66.9 vessels/mm,
p = .01) (Fig. 6). There were few signs of inflammation in either graft, although overall pan-macrophage infiltration was significantly higher in ePTFE grafts (151.6 ± 138.9 cells/mm2vs. 370.2 ± 355 cells/mm,
p = .02). In vivo ED1 and ED2 positive cells or foreign body giant cells were exceedingly rare and did not differ between grafts.
The present study comprised an investigation of a slowly biodegradable small diameter vascular conduit from a newly designed dPCU regarding its in vivo characteristics and in vitro biocompatibility behaviour. The graft is highly promising, as it showed superiority in surgical handling, biomechanical properties, and endothelialisation, and lower in vitro inflammatory potential compared with the control ePTFE material.
Non-availability of autologous veins or arteries may be a limitation for performing peripheral or coronary revascularisation. Ready made, off the shelf SDVG have yet to reach adequate performance. The first commercially available and CE certified, non-degradable PCU prosthesis (AVflo™) for haemodialysis showed 56% primary and 82% secondary patency rate in 12 patients after 24 months.
resulting in a compliance mismatch and shear stress at the anastomosis. In this study, the dimensions of the graft were adjusted to be as close as possible to those of the host vessel. In the left anterior descending (LAD) arteries of healthy volunteers, the luminal diameter was 2.2 ± 0.6 mm, as measured by high resolution transthoracic echocardiography.
However, a marginally smaller diameter was chosen to avoid mismatch to the rat's aorta. The outstanding properties of dPCU made possible matching to the host dimensions and biomechanical performance, which reflects the excellent overall performance and superiority in surgical handling of this graft. Remarkably, the compliance of dPCU increased significantly after 12 months of implantation and was comparable or somewhat inferior to that of human LAD or IMA, but superior to that of other synthetic grafts.
As expected, because of the slow degrading nature of the material with a calculated full degradation after two years, there was no significant decrease in wall thickness. Nonetheless, the structural thinning of the material by bulk degradation and concomitant regeneration of tissue may reflect in increasing compliance over time. In the absence of anticoagulant therapy, the patency rate of dPCU was 92.9% at a follow up of six to 12 months compared with the previously mentioned plasma heparin treated PCU and POSS-PCU (86% and 64%, respectively).
The present study shows that, despite the slow degradation and persistent presence of foreign material, infiltration of macrophages at the interface and into the graft was exceedingly rare. No acidic degradation byproducts were expected,
followed by a shift towards anti-inflammatory M2 positive macrophages and cytokines to promote healing and remodelling. In the ePTFE group, the pro-inflammatory response remained upregulated. Successful graft endothelialisation determines short and long term patency and is host dependent.
Here, a significantly quicker, full endothelialisation of the dPCU grafts was observed. Endothelialisation occurred mainly through transanastomotic ingrowth and additionally by means of fallout endothelialisation as described by Pennel et al.
dPCU provided a more liveable environment for cells, expressed by the significantly higher level of cells in the graft and at the interface. After only one month the vessel anatomy was partially restored, showing over time an increasingly organised neomedia and collagen rich neo-adventitia accompanied by a high number of microvessels. Despite the presence of collagen, no evidence was found for elastin formation as in the native aorta. Kurobe et al. detected formation of minimal amounts of elastin surrounding their electrospun polylactic acid grafts in a mouse model.
Therefore, it may be hypothesised that slow degradation without inflammatory stimulus as found in the present material encourages ordered cell alignment and tissue regeneration.
Small rodent models may have limitations regarding their immunological response, cardiovascular physiology, and haemostasis mechanisms compared with humans. However, these models are an essential tool in vascular graft development when limitations are seriously considered. Results obtained from this study have to be confirmed in a long term large animal model.
In this study, dPCU grafts acted as temporary scaffolds for the newly forming artery and supported formation of a structured smooth muscle cell layer, microvessel formation, and full endothelialisation. The main issue of slow degrading polymers perpetuating an extended foreign body reaction has not been confirmed in dPCU. Slow degrading and almost inert dPCU conduits appear to be a promising, safe, and long term approach for in situ tissue engineering of small diameter vessels.
conflict of Interest
The authors declare no conflict of interest.
This work was supported by the Ludwig-Boltzmann-Cluster for Cardiovascular Research and the Austria Wirtschaftservice (PRIZE).
We would like to thank all supporting staff of the Centre of Biomedical Research, Claudia Höchsmann for her meticulous support with the immunohistochemistry, Dr Jagoba Iturri for the AFM imaging, and Matthew Di Franco for the μCT imaging and proof-reading.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
Synthetic grafts are associated with excellent long term patency when they are used to replace large diameter arteries where the flow is high and the resistance is low. Conversely, their performance is disappointing when they are used to bypass small diameter arteries such as the coronary and infragenicular vessels. The construction of an artificial blood vessel with biomechanical properties identical to those of native vessels, including the ability to contract, to secrete, to heal, and even to grow, has been elusive.
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