Abstract
One of the major challenges in developing appropriate vascular substitutes is to produce a graft that adapts to the biological and mechanical conditions at the application or implantation site. One approach is the use of tissue engineered electrospun grafts pre-seeded with autologous cells. However, bending stresses during in vivo applications could lead to kinking of the graft which may result in life-threatening stenosis. The aim of this study was to develop an electrospun vascular graft consisting of biodegradable polymers which can reduce or prevent kinking, due to their higher flexibility. In order to improve the bendability of the grafts, various electrospinning collectors were designed using six different patterns. Subsequently, the grafts were examined for scaffold morphology, mechanical strength and bendability. Scaffolds spun on a collector structured with a v-shaped thread (flank angle of 120°) showed a homogenous and reproducible fiber deposition as compared to the unstructured reference sample. The results of the tensile tests were comparable to the unstructured reference sample, supporting the first observation. Studies on bendability were performed using a custom made flow-bending test setup. It was shown that the flow through the v-shaped grafts was reduced to less than 45 % of the reference value even after bending the graft to an angle of 140°. In contrast, the flow through an unstructured graft was reduced to more than 50 % after bending to an angle of 55°. The presented data demonstrate the need for optimizing the bendability of the commonly used electrospun vascular grafts. Using of macroscopic v-shaped collectors is a promising solution to overcome the issue of graft kinking.
1 Introduction
Currently, atherosclerosis is the leading cause of death in adults [1]. The treatment depends on how far the disease has progressed, but a surgical vessel replacement often remains as least therapeutically option [2]. The availability of preferably autologous vascular grafts is limited and often not possible [3]. Therefore, artificial vascular grafts are needed [4]. The requirements for these grafts are manifold. A low occlusion rate, high biocompatibility, availability and sterilization name just a few of these requirements [5? ]. Early approaches (e.g. use of glass or polymer blend like Vinyon “N”) proved to be ineffective, which lead to development of new synthetic materials such as polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) which are commonly used nowadays [7? ]. The patency rate of these materials decreases with increasing implantation time. This increases the risk of restenosis, since these materials are intended to remain inside the human body for a whole lifetime [5]. A more promising approach is the use of biodegradable polymers such as polycaprolactone (PCL). They serve as raw material for the fabrication of a matrix of nanofibers that is seeded with autologous cells. Upon implantation the graft will start to degrade, being resorbed by the human body, in turn leaving a vascular replacement consisting of autologous tissue [4]. Among other methods, electrospinning is settled to focus for manufacturing these grafts. A fundamental drawback of PCL-based vascular prostheses are their stiffness that causes the lumen to collapse under bending stress. Pleating of grafts make it possible to reduce kinking behavior of vascular prostheses (Figure 1) [9, 10].
The aim of this study is to develop an electrospun vascular graft to overcome the issue of kinking. In addition, a test method is designed for a standardized evaluation of the fabricated grafts.
Commonly used synthetic vascular prosthesis. (A) unstructured, (B) pleated, (C) stiffened by orbiting spiral. The pleated and stiffened graft prevent kinking by their specific structure while the unstructured graft is dependent on the flexibility of the compositing material.
2 Material and methods
2.1 Scaffold fabrication
Custom-made collectors were designed and manufactured to modify the macroscopic structure of electrospun vascular grafts in order to avoid kinking. The design is based on ISO-metric-, ISO-trapezoidal- and pipe-threads [11–14]. The tooth geometries are modified and a selection of six different threads (thd.) were manufactured (Figure 2). Electrospun scaffolds were fabricated with a custom-made setup. PCL (Sigma-Aldrich, MW 80,000) was dissolved in 2,2,2-trifluoroethanol (TFE, ABCR) with a final concentration of 170 mg/ml. Scaffolds were spun with a voltage of 20 kV for 15 min at a rotation speed of 1000 rpm.
Selected threads for the electrospinning collectors. The outer diameter is set to ϕ8 mm for all designs.
2.2 Assessment of scaffold morphology
Samples were analyzed with regard to fiber deposition on the corresponding collector using a scanning electron microscope (SEM: Hitachi S-3700N). One objective of the study was to measure depth placement of fibers in the root diameter of the thread, which indicates whether the fiber deposition is homogeneous and without the formation of large air pockets and wall weakening. Samples were sputtered with gold-palladium in argon environment (with Emitech SC7620). The images were acquired with a magnification of 50x at 15 kV.
2.3 Uniaxial tensile testing
For mechanical characterization, the samples are loaded until they break. In standard tensile tests, parameters like force and moving distance are recorded to calculate stress and strain, after determination of the cross section of the sample [15, 16]. Controlled rupture is assured by a predetermined breaking point. In case of the macroscopic structured scaffolds it was not possible to determine a defined cross section. That is why force at break and strain at break were determined instead. Tubular samples with a free length of 25 mm were embedded into a sample holder on both ends with a two-component silicone (Elastosil M 4601). The tensile tests were performed at a constant speed of 40 mm/min using a 500 N loading cell (Instron 5565A).
2.4 Flow-bending test method
The measurement is based on DIN EN 13868 and ISO/DIS 7198. In these standards bending test methods for hoses and circular geometries with a single lumen have been described. According to them, test conditions have to mimic physiological conditions at the application site as close as possible. Furthermore, a kink is defined as a reduction to 50 % of the original straight flow by kinking (Figure 3) [17, 18].
Bending test methods for hoses according to DIN EN 13868 and ISO/DIS 7198.
Due to the length of the electrospun scaffolds of approximately 60 mm, the given test procedures were applied to the separate samples [17]. Consequently a bending test-bench, adapted to the sample geometry, was designed. It allowed a steady adjustment of the bending angle. The Test medium was pre-heated pure water to 37 °C. All samples were analyzed in triplicates for 61 s while measuring the mass flow. All experiments started with a bending position of 0° to determine the initial and undisturbed mass flow. After this, the bending angle was increased gradually until the flow rate was reduced to more than 50 % of the initial mass flow. The method has not been described in detail, while it is on patent examination.
3 Results and discussion
3.1 Fiber deposition
Various patterns were observed for deposition of the fibers on the collectors. While some achieved well-shaped tooth root, others could not even partially penetrate it. Those shapes of tooth roots which are not or only partially penetrated, have clamped and axially extending fiber bundles between their teeth. This effect is known as gap spinning and industrially used [19]. Only with an expanded flank angle, can this effect be decreased, which is demonstrated by 120° v-thread with only few inhomogeneities in fiber deposition (Figure 4).
3.2 Force at break
Compared to the reference sample
3.3 Flow measurement
The reference sample is characterized by early failure. While a bend of 50° occur in
SEM images of representative samples show homgeneous fiber deposition on the tooth head (A) whereas fibers between two tooths were mainly deposited via gap-spinning. Furthermore, the cross-sectional view of the grafts demonstrates an increasing fiber penetration to the tooth root with increasing flank angle: (C) 30° v-thread, (D) round thread, (E) 90° v-thread.
Results of tensile test. It shows the reference sample (ref.), 30-, 60-, 90-, 120- v-, round- (rd.) and trapezoidal thread (tzd). Error bars indicate maximum and minimum values obtained from three technical replicates (n=3).
The frequently progressive increase of the values can be explained by kinking of the grafts. This phenomenon occurs primarily on rigid and unstructured samples – such as reference sample. If the structure is affected in this way, the failure history is correspondingly progressive. Due to the spontaneous occurrence of the folds, prediction of fold-occurrence is difficult. This raises the question of whether the folding, which occurs earlier than 50 % reduction of the flow, is an additional or exclusively criterion for failure. Since it can only be determined optically, this criterion is difficult to measure. The 90° sample reveals a clear and long-lasting shape retention compared to other samples.
Results of flow measurement. The overview is reduced to bending angles of 50, 90 and 140°. Three samples were measured at each bar and each of these with three measurements. For error indicator, the minimum and maximum value is shown.
For further research 90° - and 120° v-thread is determined. In order to achieve objective assessment, an evaluation table with multiple criteria was prepared. In this, tensile strength, elongation, homogeneity and fiber deposition in the tooth root, total flexibility and at 90°, stability, feel and reproducibility is assessed individually. The criteria were weighted different to create a ranking.
4 Conclusion and outlook
The aim of this study was to develop an electrospun vascular graft consisting of biodegradable polymers, with increased flexibility to avoid kinking. Furthermore, a method had to be developed to evaluate the non-kinking approaches. In order to improve the flexibility of the grafts, six geometrically different thread structures have been designed and manufactured as collectors for electrospinning. Samples were prepared to compare the fiber deposition, mechanical properties and bendability. SEM images were acquired as an optical evaluation criterion. Furthermore, the samples were validated with tensile and flow-bending tests. Analysis of SEM images revealed that the v-thread with an angle of 120° lead to a graft with the most homogenous fiber deposition, as compared to all other structured collectors. With the use of the developed flow-bending test rig, it was possible to achieve a bending of 140° for the 60°, 90° and 120° grafts without exceeding the limit of 50%-flow-reduction.
Taking into account the results of the flow measurement und further criteria, the v-thread with an included angle of 120° is best suited. The developed flow-bending test setup will serve as a standardized method to assess the bendability in the future.
With process optimization, a better fiber deposition can be achieved. The actuating variables of the manufacturing process offer a wide range of possibilities. Another aspect that will be investigated as an essential basis before clinical application is the behavior of seeded cells. Preliminary results has already been published [20]. The inner structuring of the vascular prostheses could be detrimental to cell proliferation due to the fact, that endothelial cells require mechanical stimulation of blood flow for developing a native morphology and optimal growth conditions. In the first step, the colonization of a sterile sample can be done with a bioreactor. A seeded vascular prosthesis is probably changing the properties. Therefor a new mechanical consideration, particularly for bending tests, is required.
Author's Statement
Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animals use.
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