Elsevier

Biomaterials

Volume 33, Issue 1, January 2012, Pages 38-47
Biomaterials

Long term performance of polycaprolactone vascular grafts in a rat abdominal aorta replacement model

https://doi.org/10.1016/j.biomaterials.2011.09.024Get rights and content

Abstract

In the active field of vascular graft research, polycaprolactone is often used because of its good mechanical strength and its biocompatibility. It is easily processed into micro and nano-fibers by electrospinning to form a porous, cell-friendly scaffold. However, long term in vivo performance of polycaprolactone vascular grafts had yet to be investigated. In this study, polycaprolactone micro and nano-fiber based vascular grafts were evaluated in the rat abdominal aorta replacement model for 1.5, 3, 6, 12, and 18 months (n = 3 for each time point). The grafts were evaluated for patency, thrombosis, compliance, tissue regeneration, and material degradation. Results show excellent structural integrity throughout the study, with no aneurysmal dilation, and perfect patency with no thrombosis and limited intimal hyperplasia. Endothelialization, cell invasion, and neovascularization of the graft wall rapidly increased until 6 months, but at 12 and 18 months, a cellular regression is observed. On the medium term, chondroid metaplasia takes place in the intimal hyperplasia layers, which contributes to calcification of the grafts. This study presents issues with degradable vascular grafts that cannot be identified with short implantation times or in vitro studies. Such findings should allow for better design of next generation vascular grafts.

Introduction

Creating clinically acceptable small diameter vascular prosthesis (<6 mm) as alternatives to autologous arterial or venous vascular substitutes is currently the subject of intense research. In order to ensure long term patency and structural integrity, the ideal vascular graft should have: an anti-thrombotic surface, resistance to aneurysms, and no complications such as intimal hyperplasia or calcifications. Four main strategies are being explored to create such vascular grafts [1]. The first is based on biostable synthetic materials, which offer excellent mechanical resistance on the long term. An example is ePFTE, that is widely used in the clinic for large diameter vessel replacements, but fails due to thrombosis when used for small diameter applications [2]. In addition, the residency of the material on the long term exposes the patient to chronic foreign body reaction with intimal hyperplasia, graft calcification, and a risk of infection [3]. The second strategy is to use biodegradable synthetic materials. After implantation, the synthetic material progressively degrades and should be replaced by autologous cells, which secrete an extracellular matrix (ECM) and promote neo-angiogenesis. This approach aims to combine the practical advantages of synthetic materials (availability, manufacturing, sterilization, storage, ease of implantation) and the excellent long term performance of natural tissues. The third strategy is to seed cells in the synthetic or natural graft scaffold before implantation to improve its performance [4]. For example, growing a confluent endothelial layer in vitro on the luminal surface would reduce the risk of thrombosis upon implantation. However, this strategy requires bioreactors for cell maturation, grafts are not readily available, and cell source can be an issue, which all complicate use in the clinical setting. The fourth strategy is fully natural tissue engineered vascular grafts. Cells and ECM elements are cultured in bioreactors to form vital mature vascular substitutes [5]. These grafts have an optimal biocompatibility since they can be fully derived from the patient's tissues and a good biofunctionality since they mimic natural vessels in terms of cell types and ECM. However, obtaining adequate mechanical strength with these constructs is challenging and their cost intensive production can take months, which is not compatible with wide clinical application and emergencies. The emerging field of bioprinting should also be mentioned as a possible fabrication method for the next generation of cell seeded vascular grafts [6].

Of the four strategies described above, only the first has reached the clinic. The only clinically approved alternatives to autologous grafts for revascularization procedures are ePTFE and Dacron® synthetic biostable prostheses, both of which have high failure rates for small diameter vessel replacements [7]. In research, many other materials are being evaluated, but this is generally limited to an in vitro proof of concept (mechanical strength, cell survival) or short term in vivo studies (less than 6 months). Such studies generally show interesting preliminary results, but do not reveal the clinical potential of the materials for vascular applications: the absence of complications in vivo on the long term needs to be demonstrated.

Several studies have evaluated next generation vascular grafts in vivo for 12 months or more. L'Heureux et al. developed fully natural tissue engineered vessels [5], which have been tested in 10 patients as a hemodialysis access [8]. These vessels perform well, but their main limitation is the manufacturing time, which exceeds 3 months. Shinoka et al. have implanted biodegradable synthetic grafts, preseeded with autologous bone marrow cells, in 25 pediatric patients as large diameter, low pressure extracardiac cavopulmonary conduits [9]. The 6 year follow-ups show very promising results with no mortalities, but several cases of stenosis and one case of thrombosis [10]. Yokota et al. have tested a collagen – polyglycolic-acid – poly-l-lactic acid construct in a canine carotid model, which shows good in situ tissue regeneration and patency up to 12 months [11]. Cummings et al. who evaluated an autologous vascular cell seeded biodegradable synthetic graft (polyglicolic-acid/poly-4-hydroxybutyrate) up to 24 months as a pulmonary artery replacement in the lamb model [12]. These studies are extremely valuable since they provide important information about the clinical potential of the different strategies for designing the next generation of vascular grafts.

Polycaprolactone (PCL) is an interesting material for vascular applications because of its excellent mechanical properties, slow degradation rate, and good biocompatibility. An easy way to process PCL into a porous scaffold is electrospinning that uses an electrical field to produce polymeric micro and nano-fibers. Over the last 5 years, a number of studies have been published on new vascular grafts based on electrospun PCL [13], [14], [15], [16], [17], [18]. However, the long term performance of electrospun PCL vascular grafts in vivo has never been evaluated in detail, which is the driving force for the work presented here. A good knowledge of the long term issues linked to regenerating arteries is the key to a better design of next generation small diameter vascular grafts.

In a previous feasibility study by our group, electrospun PCL grafts were evaluated in the rat model up to 6 months [18]. This provided insight on the tissue response to such a scaffold and the appropriateness of the material as a vascular prosthesis. These medium term implantations showed promising results such as rapid and stable endothelialization, no thrombosis, good cell invasion, and limited intimal hyperplasia. This led us to conduct a long term study up to 18 months (maximum time in the rat model because of aging), the results of which are presented here. As in our 6 months study, patency, structural integrity, polymer degradation, and vascular remodeling were evaluated. In addition, further in vitro characterization of the graft was performed and new investigation tools such as high-resolution ultrasound for in vivo graft compliance measurement and micro computed tomography (MicroCT) for volume quantification of calcified areas were also used.

Section snippets

Prosthesis preparation

2 mm inner diameter vascular prosthesis was prepared by electrospinning a solution of 15% PCL (Mw 80,000 Da, Sigma, Germany) in CHCl3/EtOH (70% v:v). The spinning conditions were as follows: 20 cm needle collector distance, 20 kV voltage, 12 ml/h flow rate, and 6 min spin time. A detailed description of the electrospinning setup has been previously published [19]. All PCL grafts were sterilized by gamma-irradiation (25 kGy) prior to implantation. Average fiber size was measured from scanning

Graft characterization and implantation

The grafts have a wall thickness of 650 ± 15 μm and an average fiber diameter of 2.2 ± 0.6 μm (Fig. 1A). The fiber diameters range from submicron to several microns. 20% of the diameters are below 1 μm, 60% range between 1 and 3.5 μm, and 20% are larger than 3.5 μm, up to 7 μm. The grafts are therefore a random mix of micro and nano-fibers, which could be manufactured with a good reproducibility. The longitudinal stress and strain to rupture are 4.1 ± 0.5 MPa and 1092 ± 28%. Burst pressure was

In vitro characterization and implantation

Following the ANSI guidelines [20], a complete in vitro characterization of the prosthesis was performed. The mechanical properties of these PCL grafts are largely superior to natural arteries and clinically recommended values, and are therefore appropriate for implantation. Because of the high porosity, pressurized water leaks through the graft wall. However, the blood leakage (even for heparinized blood) is significantly lower because of the higher viscosity and the coagulation factors of

Conclusion

The long term evaluation of PCL micro and nano-fiber based vascular grafts in the rat abdominal aorta model provides important insight on the promises and challenges of degradable vascular grafts and gives valuable lessons for future research in the field. This study shows that electrospun PCL make an excellent scaffold for shelf ready vascular graft applications in terms of patency, mechanical properties, and rapid endothelialization, but insufficient regeneration of the vascular wall on the

Acknowledgments

The authors would like to thank Jean-Pierre Giliberto for his assistance with the animal surgeries, Marie-Claude Reymond for her help with the SEM, Xavier Montet for the microCT scans and quantifications, Denis Jabaudon for making available his high-resolution ultrasound equipment, and Patricia Gindre and Unn Lutzen for histological preparations. The authors would also like to acknowledge the support, in part, of the Swiss National Science Foundation (FNS – Subside FN 320030-119822).

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