PCL small diameter nanofibrous conduit for biomedical application

Materials Science & Engineering C 110 (2020) 110692

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Electrospun PET/PCL small diameter nanofibrous conduit for biomedical application ⁎

Maryam Rahmati Nejada, Maryam Yousefzadeha, , Atefeh Soloukb, a b

T

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Textile Engineering Department, Amirkabir University of Technology (Tehran Polytechnic), Tehran 1591634311, Iran Biomedical Engineering Department, Amirkabir University of Technology (Tehran Polytechnic), Tehran 1591634311, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: PET/PCL nanofibers Vascular graft Mechanical properties Nanofibrous conduit electrospinning

In recent years, the mortality rate caused by cardiovascular diseases has increased dramatically around the world. Tissue engineering is considered as a novel and efficient approach to offer a substituent of engineered tissues for defective body tissues. For this purpose, fabrication of the scaffold that resembles the physical and mechanical properties of natural body vessels, and culturing appropriate cells seems to be a promising approach. Due to the fibrous structure of the vascular wall, the nanofibrous scaffold produced by electrospinning could be a proper choice for vascular tissue engineering. One of the main properties of artificial vessels is its mechanical properties consistency with the native one in order to mimic its natural characteristics. To do so, in present study two biocompatible polymers, polyethylene terephthalate (PET) and polycaprolactone (PCL) with different blend ratio were electrospun into a tubular nanofibrous structure with 6 mm internal diameter and the mechanical properties such as tensile strength, modulus, compliance, bursting pressure, elastic recovery, and suture retention were investigated. The results revealed that PET/PCL (1:3) had better similar properties with the reported natural one as its longitudinal and transverse tensile strength was about 9.47 and 6.38 MPa, respectively. The longitudinal strain at break, compliance, bursting pressure, and suture retention were 205.88 ± 51.12%, 4.19 ± 0.78%/100 mmHg, 6378.76 ± 2159.20 mmHg, and 287.73 ± 13.10 gmf, respectively. The elasticity of this studied sample was 60.21 ± 12.49% as it was relieved, and this may be a good candidate for the artificial vessel in this size, as the MTT test confirmed its appropriate substrate for cell culture.

1. Introduction According to the World Health Organization report, 17.9 million deaths occur annually due to cardiovascular disease, which is expected to reach 23.4 million in 2030 [1]. Cardiovascular disease and atherosclerosis are generally due to the blockage of blood vessels. As a result, it reduces the intake of oxygen and nutrients, and blood flow decreases, causing tissue damage [2]. Most commonly, autograft surgery is done to treat this disease. If it is not possible to use the patient's vessels for autograft, another alternative treatment is artificial vessels. Tissue engineering aimed at the substitution of engineered/regenerated tissues for defective body tissues is considered a novel and efficient approach to addressing this issue. For this purpose, fabricating a scaffold with physical and mechanical properties similar to natural vessels, and culturing appropriate cells on it is required [3]. Until now, various methods like electrospinning [4–6], molding [7], bio-printing [8], freeze-drying [9], solvent casting [10], and phase separation [11] or a combination of these methods [12,13] have been

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utilized to produce tissue-engineered vascular grafts. Among these methods, electrospinning is considered as an effective method to produce micro and nano-structured fibrous substrates that can imitate the behavior of the extracellular matrix of the natural tissue [14]. The electrospinning technique can also be used to produce vascular tubes in a suitable diameter and size. In order to mimic the extracellular matrix, the porosity, pore size, and fiber orientation can be controlled in this process [15–17]. The interconnected pores in the electrospun scaffolds encourage the cells to attach, penetrate, and grow. Due to these interconnected pores, there is the possibility of the exchange of nutrients and waste [18]. According to the results of previous researches, biosynthetic polymers are suitable for the production of artificial blood vessels and can be used to make porous layers nearly similar to the physical and mechanical properties of the native blood vessels. The most popular ones are made of expanded polytetrafluoroethylene (PTFE, Teflon®) and polyethylene terephthalate (PET, Dacron®) which were approved by the American Food and Drug Administration (FDA) [19,20].

Corresponding authors. E-mail addresses: [email protected] (M. Rahmati Nejad), [email protected] (M. Yousefzadeh), [email protected] (A. Solouk).

https://doi.org/10.1016/j.msec.2020.110692 Received 8 October 2019; Received in revised form 23 January 2020; Accepted 23 January 2020 Available online 24 January 2020 0928-4931/ © 2020 Elsevier B.V. All rights reserved.

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properties close to the natural vessels. Considering the mentioned properties of PET and PCL polymers, in this study, to enhance the elasticity and compliance of small diameter nanofibrous conduit made of polyethylene terephthalate, the PET polymer was mixed with PCL one in different blend ratios, and after that the physical and mechanical properties were studied. In general, longitudinal strength has been reported in the manuscripts, and few have focused on mechanical properties in the radial direction, which is the major determinant of its clinical success. Longitudinal mechanical properties alone cannot be a good option for comparing the mechanical properties of natural vessels. Also, research on the cyclic performance of produced grafts that display material malleability has rarely been discussed. In this work, both subjects were examined and the results were investigated.

Mismatch of mechanical properties, narrowing or blockage, and a high rate of thrombosis are some of the major problems in small diameter artificial vessels that should be considered in the process of producing these grafts. The PET is a thermoplastic and semi-crystalline polymer. It is widely used in medical applications and tissue engineering due to its favorable properties such as biocompatibility, high mechanical strength, low degradability, low toxicity, and being lightweight [21]. However, the lack of compliance of vascular grafts made of PET limits its uses in the production of small-diameter vascular (inner diameter < 6 mm) [19]. Researches have shown that 40% of artificial vascular grafts have completely failed within five years due to thrombus formation and anastomotic intimal hyperplasia. There is a relationship between the mechanical properties and the severity of thrombosis. Compliance is a crucial factor in determining the success of graft [22]. Many attempts have been made to design vascular grafts with proper compliance at pulsed pressures, which is the ability and capacity of the vascular graft to extend in the circumferential direction to respond to the pulsed blood flow pressures [23,24]. Therefore, the artificial vascular grafts must especially have a combination of requirements such as proper mechanical properties, compliance, suture ability, blood sealing, and antithrombogenicity [2,25,26]. Generally, a polymer alone cannot provide all of the desired properties. A mixture of two or more polymers can be used to take advantage of each one. Polycaprolactone (PCL) is a biocompatible, biodegradable, and good elastic polymer with a low degradation rate when used alone, or in a mixture of polymers when it is used to modify the brittle polymers like PLA [27]. Madhavan and his co-workers [28] used a blend of polyurea, poly (serinol hexamethylene urea) (PSHU), and PCL with different ratios for the production of artificial vessels. The results of mechanical tests showed that by increasing the amount of PCL in the electrospinning solution, the elasticity of the scaffold was close to the mammalian arteries. Guo and his colleagues [29] produced a high strength scaffold with a mixture of two polymers of polycaprolactone and polyurethane compared with pure polyurethane and found that by adding a certain amount of PCL, in addition to dramatically improving its mechanical properties, biocompatible scaffolds were produced with high porosity and cyclic performance. Also, many materials have been used to produce small diameter conduit such as PET/polyurethane (PU) [30], PU/PCL [31], PCL/poly (lactic-co-glycolic acid) (PLGA) [32], poly(L-lactic acid) (PLLA)/segmented poly(ester urethane) (SPEU) [5], and polyurethane namely tecophilic (TP)/gelatin (gel) [33] to enhance its properties. It was shown adding PU into PET solution could improve the mechanical properties compare to pure PET nanofibrous conduit. The production of biphasic scaffolds from the PCL and PU showed that mechanical properties such as the tensile strength, suture retention, compliance, and burst pressure were improved compared to the pure samples. The results of mechanical and biodegradability tests of PCL/PLGA blends showed that adding PLGA (10%) into PCL solution in addition to obtaining appropriate mechanical properties and increasing the rate of biodegradability promoted cell infiltration. The researchers also produced vascular grafts with very similar properties to natural vessels using PLLA polymer that resembled the mechanical properties of collagen and SPEU polymer that was close to the elastin properties. Adding gelatin to the TP solution have been improved cell adhesion and provided biomechanical

2. Materials and methods 2.1. Materials Polyethylene terephthalate (PET, MW = 24,000 g/mol) was purchased from Shahid Tondgooyan Petrochemical Company (Iran). Polycaprolactone (PCL, MW = 80,000 g/mol) was received by Merck (USA). Dichloromethane (DCM) andrifluoroacetic acid (TFA) and other chemical materials were derived from pure analytical reagents, which were purchased from Merck chemical company (Germany) and used as received.

2.2. Tubular nanofibrous structure preparation First, five different electrospinning solutions were prepared. The pure PET solution was made according to an optimum condition that was found in our previous work [34,35]. The PET solution in a concentration of 12 wt%, was prepared by dissolving the polymer in DCM/ TFA (1:1) and stirring it for about 2 h. The 14 wt% PCL solution was made by adding it in the same mixed solvents system. To obtain the blend solution of PET/PCL, the PET was initially dissolved in DCM/TFA (12 wt%) for about 1 h and then the PCL (14 wt%) was added and stirred more to obtain a homogeneous transparent solution at a ratio of 1:3, 1:1, and 3:1. The electrospun conduit was produced using a custom-made electrospinning apparatus equipped with a stainless steel mandrel rotating collector with a 6 mm diameter. To this order, the polymer solution was loaded into a syringe and delivered to a stainless steel 22G blunt needle. The best electrospinning condition resulted after optimization of the process parameters as listed in Table 1, and the scheme of the electrospinning apparatus is shown in Fig. 1. To create a layer with uniform thickness, the syringe pump had a traverse in the longitudinal direction. The electrospinning process was carried out for 8 h to obtain a 400 μm thick electrospun tube, which was in the range of the native vessel's wall thickness [36]. In order to remove the graft from the mandrel, the collector was placed in the open air for 24 h to completely remove the solvent, thereby the surface adhesion was reduced. Then, it was cut to the appropriate length with a surgical blade and removed with the use of forceps.

Table 1 The optimum electrospinning condition for producing tubular nanofibrous PET, PCL and, PET/PCL. Polymer

Polymer concentration (%)

Voltage (kV)

Needle to collector distance (cm)

Flow rate (mL/h)

Collector speed (m/min)

PET PET/PCL (3:1) PET/PCL (1:1) PET/PCL (1:3) PCL

12 12 12 12 14

14 20 20 20 16

15 15 15 15 10

0.3 0.3 0.3 0.3 0.3

4 4 4 4 4

2

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Fig. 1. Schematic of the process for producing small diameter nanofibrous conduit.

2.3.3.2. Tensile strength and modulus. The produced conduit should provide mechanical properties similar to the host blood vessel. The appropriate tensile strength of the vessel is essential to provide mechanical stability, since the vascular graft scaffold experiences long-term hydrodynamic stress. The longitudinal and radial tensile properties of the samples were measured. Three specimens were prepared in 10 × 5 mm2 dimensions, both in axial and radial directions. The strain rate of 10 mm/min and 50 N load cell were applied during the test until the sample failed. The ultimate tensile strength, strain at break, and Young's modulus were obtained using the test data.

2.3. Characterization 2.3.1. Morphological analysis A digital microscope with a magnification of 900× (AM4515, DinoLite, Taiwan) was used for pre-assessment of nanofibers to determine the optimum electrospinning condition. In order to study the morphological features of electrospun scaffolds, the samples were coated by sputtering a thin layer of Au (SC7620, Quorum Technologies, UK) and then the scanning electron microscope (SEM, AIS-2100, Seron technology, Korea) was used to evaluate the uniformity, diameter, pore size, and porosity of the nanofibrous scaffolds by using ImageJ software (1.46r, USA). To quantify the surface porosity of the scaffolds, each grayscale SEM image (magnification 5 μm) was converted to a black/white one by selecting an appropriate threshold value. Then, the ratio of the number of white pixels to the total was calculated, which indicated the surface porosity [37]. To measure the size of the pores, an ellipse was fitted in each one, and the larger diameter was measured as pore size [23]. This measurement was conducted in 30 pores (n = 30).

2.3.3.3. Elastic recovery. Considering the increase in blood flow pressure inside the vascular graft, the vessel wall should have appropriate elastic recovery. To compare the elastic recovery of the electrospun conduit, the samples were cut in 10 × 5 mm2 dimensions. The specimens were placed between two clamps of a uniaxial tensile system. The strain rate was set as 10 mm/min, and the samples were undergoing 50% of the total strain. Loading-unloading curves were plotted in terms of stress-strain in one cycle. The elastic recovery of the sample was calculated from Eq. (1):

2.3.2. Water contact angle measurement To measure the hydrophilicity of the samples, the static water contact angle (WCA) of the drop deionized water with the sample surface was measured at room temperature by using a goniometer (SSCDC318P, Sony, Japan). For this purpose, using a micro-syringe, 4 μL droplets of distilled water was dropped on the surface samples and repeated three times in each sample (n = 3). For each, the photograph was taken after 5 s, 1, 3, and 5 min to measure the WCA using the ImageJ software.

L − L0 × 100⎞ Elastic recovery (%) = 100 − ⎛ 2 ⎝ L1 − L0 ⎠ ⎜

⎟

(1)

where L0, L1, and L2 are the initial length, the extended length, and the final length after releasing the stress, respectively [38]. 2.3.3.4. Compliance. Compliance is one of the most important factors in the production of vascular substitutes. Compliance mismatch between the vascular prosthesis and its host artery can result in a loss of patency and is detrimental to graft performance. The compliance of tubular scaffolds was determined by the same uniaxial tensile testing system as described earlier. The electrospun rings were cut in a width of 5 mm from the tube. The ring was placed between two hooks, one being fixed and the other moving with a rate of 10 mm/min, and then it was drawn until the ring ruptured (Fig. 2). Using stress-strain curves and Eqs. (2) to (6), compliance of the samples in the physiological range of blood pressure (80–120 mmHg) was computed.

2.3.3. Mechanical characterization 2.3.3.1. Tensile preconditioning. Regarding the fact that the blood flow is pulsed and the wall of the vessel is under pulsed pressure, which can affect its mechanical properties, before each test, tensile preconditioning was carried out. In this test, the samples were cut in an effective length of 10 mm and a width of 5 mm and were placed between two clamps of a uniaxial tensile testing machine (Instron, TMSM, UK). The strain rate was 10 mm/min, and the load cell was 50 N. Considering the total tensile strength and strain of electrospun scaffolds, 10% of total elongation was applied, and then removed for 15 consecutive cycles [29]. To compare the lost energy of the samples, the integral of the loading-unloading curve in terms of stress-strain was calculated for the first cycle, and the cyclic curves were compared in these 15 periods.

A 0 = 2·W·t

(2)

F A0

(3)

σ=

L 0 = πR 0 3

(4)

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Fig. 2. The photographs and schema of compliance and bursting test and sample preparation.

A0 R L = = A R0 L0

poured into each well, and placed in an incubator for 4 h. After this time, the solution on the cells was removed, and synthesized purple crystals were dissolved by adding isopropanol. For better dissolution of the precipitation, the plate was placed on a shaker for 15 min. After that, the concentration of the dissolved material in isopropanol was calculated using a STAT FAX 2100 (USA) device at a wavelength of 545 nm. Wells with more cells show higher optical density (OD) than wells with lower cell numbers. Thus, toxicity and cell viability were calculated using Eqs. (8) and (9) [39].

(5) R2 − R1

Compliance (%) R1 = × 10 4 100 mmHg P2 − P1

(6)

where, A0 and A are the surfaces under stress, W is the width of the sample, t is the thickness, L0 is the initial length, L is the length after the stretching, P1 and P2 are applied pressure, and R1 and R2 are the internal radius at pressures P1 and P2 [33]. 2.3.3.5. Bursting pressure. The vessels should withstand being exposed to physiologic vascular environments that include high pressure and blood flow. Using the results of the tensile strength test of the ring samples, the theoretical bursting pressure of the conduits was calculated as in Eq. (7).

σ t P b = UTS R0

mean OD of sample ⎞ Toxicity% = ⎛1 − × 100 mean OD of control ⎠ ⎝

(8)

Viability (%) = 100 − Toxicity (%)

(9)

2.5. Biodegradability In order to investigate the biodegradability of the electrospun scaffolds, the samples were placed in phosphate-buffered saline (PBS) solution with a pH of 7.4. The PBS solution in the degradability test can be used to simulate the fluid body. Then the plates were placed in an incubator at 37 °C under carbon dioxide gas. After 1, 3, 7, 14, 21, and 30 days, the samples were removed from the incubator and weighed after washing with deionized water and drying. The degradation of mass loss (%) was calculated using Eq. (11) [40].

(7)

where, σUTS, t, and R0 are ultimate tensile strength, thickness, and initial internal radius of the electrospun tube, respectively [33]. 2.3.4. Suture retention The vascular graft would be joint to the natural artery, so it must have high strength suture retention. Suture retention testing for nanofibrous conduits was performed according to ISO 7198:2016. It was measured by a uniaxial tensile system with ETHICON coated Vicryl 5-0 suture thread. Specimens were cut to obtain rectangular strips (length 20 mm, width 5 mm) and one end of the sample was gripped on the bottom clamp of the tensile machine. The suture was inserted at a 2 mm distance from the top edge of the electrospun strip to form a half loop. The other end of the suture thread was attached to the top clamp in contact with 50 N load cell. The suture was pulled at a rate of 50 mm/ min until it had pulled through the graft. The test was repeated two more times on the same sample, and the force needed to tear the scaffold was measured.

Degradation test (%) = [(W0 − W1)/W0] × 100

(10)

where, W0 is the initial weight, and W1 is the weight of the degraded sample. Also, the pH of the solution was measured. The morphology of the samples was demonstrated using the SEM technique after a month. 2.6. Statistical analysis Each test was repeated at least three times, and the results were reported as mean ± 95% confidence interval. To investigate the statistical difference of the obtained results, the one-way ANOVA and Duncan's post-hoc analysis was performed mostly at an accuracy of 95%.

2.4. MTT assay To evaluate the cytotoxicity of the prepared samples, the MTT colorimetric assay was performed. In this study, 10,000 endothelial cells of human umbilical vein (HUVEC) with 100 μL of culture medium were poured into each well of the cell culture plate and then placed in an incubator at 37 °C for 24 h. After ensuring the adhesion of the cells to the internal wall of the cells, the culture medium was removed properly. Next, 90 μL of the extract of each sample and 10 μL of fetal bovine serum were added to wells, and the cells were aged for 24 h. Then, the culture medium was removed, and 100 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 0.5 mg/mL) was

3. Results 3.1. Morphological characterization and porosity evaluation An initial study of the nanofibers structure was carried out using a digital microscope to evaluate the samples in terms of uniformity and the presence of beads and determine the optimum electrospinning condition. Samples of microscopic images are shown in Fig. 3a. In optimum conditions as described in Table 1, uniform and beadless 4

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Fig. 3. (a) The digital microscope images of pre-assessing the electrospinning conditions (scale bar: 100 μm, 370×), SEM images (scale bar: 5 μm) and nanofibers diameter histogram of (b) PET, (c) PET/PCL (3:1), (d) PET/PCL (1:1), (e) PET/PCL (1:3), (f) PCL.

of 60–90% [42–44]. Porosity measurements indicate that the porosity of the electrospun samples is in the appropriate range for cell proliferation.

nanofibers were spun, while in other conditions, many beads were presented in the structure of nanofibers or nanofibers had not been generated, and electrospraying was carried out. The morphology of the prepared nanofibrous conduits in optimal conditions was investigated by SEM. Fig. 3b–f illustrates the SEM images of the produced scaffolds and their nanofibers diameter distribution histograms. As can be seen in the SEM images, all of the nanofibers have a smooth surface, and there are no beads in their structure. Also, all the selected samples have a uniform and randomly oriented nanofiber structure. The histograms of nanofibers diameter reveal a uniform distribution of diameters in all samples. According to statistical analysis, the PET/ PCL (1:1) with an average nanofiber diameter of 494 ± 130 nm possessed the highest diameter due to the viscosity and electrospinning conditions (Table 2). The porosity and pore size of the electrospun samples was calculated by image analysis of the SEM micrographs such it is illustrated in Fig. 4 and results are reported in Table 2. Both of these factors are crucial for the diffusion of nutrients and waste, gas exchange, tissue regeneration, and are a substantial factor in the cell proliferation rate. Researchers have found that with increasing fiber diameter, pore size and porosity increase [41]. According to the results, increasing the diameter of the nanofibers had led to an increase in pore size and porosity. Typically, appropriate porosity for cell proliferation is in the range

3.2. Hydrophilicity evaluation The water contact angle of the samples as a proper indicator for evaluating hydrophilicity of a surface have been measured according to the images, as shown in Fig. 5. For all samples, after 5 s, water contact angles were higher than 90°. After 5 min, WCA of all the samples was still higher than 76°. According to statistical analysis PET/PCL (1:1) and PET/PCL (1:3) were more hydrophilic scaffolds than PET and PCL (p-value < 0.05). PET and PCL did not show a statistically significant difference with the PET/PCL (3:1) sample (p > 0.05). In order to investigate the rate of water diffusion into the scaffold, the gradient of WCA over the studied time was calculated as a dynamic change of the WCA parameter. 3.3. Mechanical properties of the nanofibrous conduit The vascular graft would be affected by pulsed pressures. Repeating these conditions could affect its mechanical properties. Thus, before any mechanical test, the samples were subjected to periodic forces in 15 cycles. The stress-strain curves of the samples are shown in Fig. 6. In this test, the energy reduction in the first cycle was calculated by measuring the inside area of the load-unloading curve in terms of stress-

Table 2 The results of nanofibers diameter, scaffolds porosity, and WCA (Duncan's post-hoc analysis grouping as a, b, and c). PET Tube thickness (μm) Nanofibers diameter (nm) Porosity (%) Pore size (μm) Initial WCA (°) Dynamic changes of WCA (°/s)

419.4 ± 30.17 362 ± 91c 75.3 ± 14.7b 1.4 ± 0.1d 103.6 ± 3.5a 0.033

PET/PCL (3:1) a

PET/PCL (1:1) a

424.2 ± 27.6 450 ± 118b 83.7 ± 14.9a,b 2.6 ± 0.3b 99.1 ± 1.9a 0.035

5

PET/PCL (1:3) a

413.4 ± 36.1 494 ± 130a 89.2 ± 6.4a 3.1 ± 0.3a 94.3 ± 9.5b,c 0.500

a

395.4 ± 8.4 433 ± 114b 84.9 ± 16.7a,b 2.8 ± 0.3b 89.9 ± 1.4c 0.412

PCL 407.8 ± 26.3a 372 ± 91c 74.7 ± 15.2b 1.9 ± 0.1d 99.5 ± 10.2a 0.063

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Fig. 4. The image processing and manual precise pores measurement (a) raw SEM, (b) black/white image and surface pores measurement.

the linearity of the PCL chains, which makes it possible to traverse and regain more after deformation than the PET sample. The prepared nanofibrous conduit must resemble the behavior of native vessels against blood flow and expansions and contractions caused by it. The study of the compliance as an appropriate indicator for investigation of this feature was carried out, and the results are reported in Table 3. Despite the exclusive properties of PET for producing vascular grafts, low compliance of its products is the main drawback. According to the results, the compliance of the PET sample is 1.76 ± 0.67%/100 mmHg. PET/PCL (1:1) and PET/PCL (1:3) (pvalue = 0.651 > 0.05) possess the most compliance among the samples without any statistically significant difference. Considering the results of bursting strength, in which all the samples had the same wall thickness and inner diameter, the PET/PCL (1:3) had the most bursting strength (6378.76 ± 2159.20 gmf) due to high radial strength. The results of the suture retention strength test are also presented in Table 3. The PET/PCL (1:3) exhibited the highest suture retention strength with a significant difference with the other samples (pvalue < 0.05). Also, there was no significant difference between PET and PCL samples (p-value = 0.214 > 0.05). The rupture pattern of suture (Fig. 8) reveals that no delamination occurs during applying a tensile force to the suture. Also, the orientation of fiber and the texture did not change during rupture.

strain, and the results are reported in Table 3. According to the results, the PET polymer hysteresis was more than other samples. Since vascular graft is in tension along with the longitudinal and radial directions, this test was performed in both directions in order to measure the mechanical properties of the scaffolds. Fig. 7a–b shows the stress-strain graph of 5 studied samples in the longitudinal and radial directions. Using stress-strain graphs data, ultimate tensile strength, Young's modulus, and strain at break of samples were calculated as reported in Table 3. According to the results, comparing the PET and PCL, the PET samples had a higher strain, and PCL had higher strength. In both groups of samples in the longitudinal and radial direction, it is observed that the PET/PCL (1:3) had the highest strength and the PET/PCL (3:1) sample had the highest strain value, and these two values were higher than the corresponding values in the pure PET and PCL samples. The strength of the PET/PCL (1:3) sample had increased by 40.5% compared to the PCL sample, and the strain at the break had increased by 179.5% compared to PET. Also, these results demonstrated that for all samples, the strength values in the longitudinal direction were higher than the values of strength in the radial direction. In the production of artificial vessels, cycle performance is essential for the elastic expansion and retraction of vessels. In this study, to measure the elasticity of the samples, loading-unloading was performed at a strain of 50%, and comparison is made between the studied groups as illustrated in Fig. 6f. The elastic recovery percentage of samples was calculated by Eq. (1) and reported in Table 3. According to the results of the elastic recovery test, with increasing the amount of PCL in the composition, the elastic recovery of the sample has been increased. The highest percentage of elastic recovery is related to PET/PCL (1:3) and PCL specimens. These two samples did not have a statistically significant difference in elastic recovery. The reason for this may be due to

3.4. MTT assay In this study, in order to determine cytotoxicity, MTT assay was used for the purpose of cell viability. Fig. 9 and Table 4 illustrate the viability of HUVEC cells on the control and produced nanofibrous scaffolds.

Fig. 5. Water contact angle of (a) PET, (b) PET/PCL (3:1), (c) PET/PCL (1:1), (d) PET/PCL (1:3), (e) PCL at time 5 and 300 s, and dynamic changes of water contact angle recorded for 300 s. 6

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Fig. 6. (a–e) Loading-unloading curves of nanofibrous samples in 15 cycles at 10% of final strain, (f) in one cycle at 50% strain. Table 3 The results of mechanical properties of the nanofibrous conduits. PET Tensile strength (MPa) Longitudinal Yang modulus (MPa) Strain at break (%) Tensile strength (MPa) Transvers Yang modulus (MPa) Strain at break (%) Energy loss (%) Elastic recovery (%) Compliance (%/100 mmHg) Bursting strength (mmHg) Suture retention (gmf)

4.46 39.33 153 ± 3.39 17.93 172.68 1.73 14.75 1.76 3391.67 133.06

PET/PCL (3:1) c

± 1.08 ± 6.77a 52.87b ± 0.60b ± 5.83a ± 52.87c ± 0.4a ± 7.52e ± 0.67c ± 596.38c ± 5.36b

3.62 24.13 429.54 2.95 5.74 463.62 1.61 24.45 2.67 2953.43 108.70

± ± ± ± ± ± ± ± ± ± ±

PET/PCL (1:1) c

0.81 2.89b 249.45a 0.04b 0.50b,c 249.45a 0.19a,b 1.03d 0.54b 45.79c 4.01c

PET/PCL(1:3) c

4.66 ± 1.93 11.41 ± 4.85c 374.01 ± 169.77a 3.16 ± 1.86b 4.55 ± 1.02c 371.05 ± 169.77b 1.6 ± 0.04a,b 31.31 ± 2.26c 4.34 ± 1.09a 3157.82 ± 1865.29c 118 ± 2.17c

9.47 8.82 205.88 6.38 7.28 157.22 1.17 60.21 4.19 6378.76 287.73

± ± ± ± ± ± ± ± ± ± ±

PCL a

0.7 0.77c 51.12b 2.16a 1.20b 12.50c 0.24c 12.49a 0.78a 2159.20a 13.10a

6.74 10.70 62.56 4.82 5.90 92.78 1.22 53 ± 3.91 4819.39 137.05

± 2.21b ± 1.24d ± 19.16b ± 0.78a ± 0.98c ± 19.16c ± 0.09b,c 11.43b ± 1.52a ± 776.85b ± 5.31b

nanofibrous conduits do not go under degradation and they are biostable at least during 30 days of evaluation. Finally, SEM images were prepared for the qualitative evaluation of the samples. According to Fig. 9, no apparent degradation is observed in this period.

3.5. Biodegradability To determine the degradability of electrospun scaffolds, the samples were removed after 1, 3, 5, 7, 21, and 30 days from the incubator and then weighed. The results indicated that after one month, the weights of samples were not changed. Also, the pH of the samples was measured at all time intervals and as expected, during this time, no significant change in pH was observed (p-value < 0.03). It seems that the blended

Fig. 7. Stress-strain graphs of nanofibrous samples in (a) the longitudinal, and (b) the radial directions (NV: native vessel). 7

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Fig. 8. Suture retention failure photo and SEM images by different magnifications.

4. Discussion

PET/PCL with different ratios were prepared, and after determining the optimum electrospinning condition, tubular nanofibrous scaffolds with a fiber diameter of 360–495 nm were produced. Using image analysis of the SEM micrographs, Physical and chemical properties of a surface, particularly its hydrophilicity, have a significant impact on the biological interactions like cell adherence, proliferation, and migration. Hydrophilicity is dependent on surface energy affected by chemical compounds of the surface and its morphology. Due to the hydrophobic nature of both PET and

Studies have shown that elastic properties and compliance mismatch between the synthetic graft and host natural artery lead to anastomotic rupture, reduced distal perfusion, flow induced shear stress and subsequently pathogenesis of distal anastomotic intimal hyperplasia (DAIH). Therefore, the production of a vascular graft, which has high compliance with the natural artery in the structure and mechanical properties, is important. To reach this goal, electrospinning solvents of

Fig. 9. SEM images of nanofibrous scaffolds (a) PET, (b) PET/PCL (3:1), (c) PET/PCL (1:1), (d) PET/PCL (1:3), and (e) PCL after 30 days in biodegradability test. 8

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To investigate the mechanical properties of the produced samples, the tensile strength test was performed. According to the results, it can be seen that produced samples such as natural arteries have anisotropic properties [50]. The strength of the samples in the longitudinal direction was higher than the strength in the transverse direction. Despite the random orientation of nanofibers, the results differed in both longitudinal and transverse directions, which indicates that the mechanical properties of an electrospun scaffold are influenced by its morphology. In fact, there are a number of nanofibers in the direction of loading, the mechanical test results of which are a function of their number [51–53]. The tensile strength of the produced samples was compared by the native vessels. According to the reports, the strength of the various native vessels is 1.5 to 8 MPa [33,50]. Therefore, it can be concluded that all of the prepared samples in this study in both longitudinal and radial directions have the same strength as the native vessels. Conduit made of the blend PET/PCL has shown good tensile strength compared to natural vessels and commercial vascular grafts. For example, the tensile strength of vascular grafts made of PLLA/SPEU, PET/PU, and TP/Gel is reported as 2.56 ± 0.28, 2.89, and 6.5 ± 0.9 MPa, respectively [5,30,33]. In this study, in the PET/PCL (3:1) sample, the appropriate tensile strength was obtained in both longitudinal and radial directions. The highest reported elastic modulus for native vessels is 12 MPa [50]. As shown in Table 3, the PET sample has a much higher elastic modulus than native vessels; however, by increasing the PCL percentage, the elastic modulus of the samples decreased. In the longitudinal direction, the PET/PCL (1:1), PET/PCL (1:3), and PCL with values of elastic modulus 11.41 ± 4.85, 8.82 ± 0.77 and 10.70 ± 1.24, respectively, and in the radial direction the values of the elastic modulus 4.55 ± 3.91, 7.28 ± 1.20 and 5.90 ± 0.98, respectively, have a high correlation with the native vessel. These samples are similar to femoral arteries in terms of strength and elastic modulus. The elastic modulus of

Table 4 The results of the MTT assay and biodegradability test of nanofibrous conduits.

OD Viability (%) Toxicity (%) Weight change (μg) pH change

PET

PET/PCL (3:1)

PET/PCL (1:1)

PET/PCL (1:3)

PCL

Control

0.33 93.30 6.7 0

0.30 84.40 15.60 0

0.29 81.25 18.75 0

0.31 87.39 12.61 0

0.35 97.86 2.14 0

0.36 100 0 –

0.03

0.16

0.2

0.06

0.13

PCL [45,46] and not the presence of a hydrophilic functional group in their chemical structures, hydrophobicity of the final produced grafts is expected. According to previous studies, the water contact angle of electrospun PET is 126.5 ± 4.4°, and the PET film range is between 7080° [46]. Nevertheless, the surface roughness arising from the nanofibrous structure leads to the greater hydrophobic property and consequently more water contact angles. The difference in the water contact angle of the samples maybe is due to the difference in porosity and fiber diameter. However, the water contact angle of electrospun grafts is within the appropriate range of 50–90°, which is the best condition for efficient cell adhesion [47]. Before any mechanical test, 15 cycles of preconditioning were applied to each sample. As shown in Fig. 6, there is an obvious hysteresis in the first cycle, but from cycles 2–15, there is no significant change in the mechanical properties of the samples. As a result, it seems that after the first load, the mechanical properties of the samples remain constant, which is consistent with previous studies [48,49]. By calculating hysteresis in the first cycle, it is observed that the PET polymer hysteresis is more than PET/PCL (1:3) and PCL. This means that these samples show lower energy consumption than the pure PET sample by applying cyclic loads. The mechanical properties are summarized in Table 3 and Fig. 10.

Fig. 10. Comparing mechanical properties of fabricated samples with native and commercial arteries. 9

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improves the mechanical properties such as strength, compliance, bursting, and suture retention strength that were in the range of the ones presented by natural vessels. PET/PCL (1:3) with high strength in longitudinal and radial directions, high elastic recovery, and higher compliance than pure PET conduit can be a good candidate for artificial vessels.

the woven grafts made of Dacron has been reported as 800–900 MPa [19]. The grafts produced in this study are not as hard as commercial grafts, and their elastic modulus is close to the natural vessels. Concerning the results of the compliance test, it seems that the combination of these two polymers results in higher compliance in comparison with the PET graft. Using Von Mises theory, Castillo-Cruz et al. [54] introduced Eq. (11) to calculate radial compliance.

C=

2r 2i (1 − v 2) E(r 20 − r 2i )

CRediT authorship contribution statement (11)

Maryam Rahmati Nejad: Methodology, Formal analysis, Investigation, Resources. Maryam Yousefzadeh: Conceptualization, Methodology, Investigation, Supervision. Atefeh Solouk: Conceptualization, Methodology, Investigation, Supervision.

where, E refers to elastic modulus, ν refers to Poisson's ratio, and r0 and r refer to radial before and after applying the force, respectively. According to this equation, less modulus leads to more compliance. Accordingly, the PET sample with the highest modulus possesses the lowest compliance (17.93 ± 5.83). In the previous research, the compliance of the Saphenous vein is reported as 4.4 ± 0.8 [24]. Therefore, the compliance of the PET/PCL 1:1 and PET/PCL 1:3 are more in line with the Saphenous vein. Compliance of commercial woven and knitted Dacron and PTFE grafts, have been reported 1.9, 2.3, and 1.6 (%/100 mmHg), respectively [19]. The compliance of the produced samples in this study was closer to the natural vascular grafts than the commercial samples. As the value of the modulus is correlated to the compliance value, the results of the bursting strength test show that the bursting strength is correlated to tensile strength. The sample with the highest tensile strength has the highest bursting strength. It seems that PET/PCL (1:3) with low initial modulus and high tensile strength, which results in higher compliance and higher bursting strength, is a good option for producing a vascular graft. The bursting strength of all samples is in the same range as that of Saphenous vein 1680 ± 310 [24]. In previous researches, in the PLLA/SPEU, PCL/PLGA, and TP/gel blends maximum value reported for bursting pressure is 1775.21 ± 57.5, 1703.15 ± 261.36, and, 2633 ± 383 mmHg, respectively [5,32,33]. The results of this study showed that the samples had good bursting strength compared to natural and artificial grafts. In previous studies, the suture retention strength of the internal mammary artery, Saphenous vein and Carotid artery have been reported as 138 ± 50, 196 ± 2 and, 199 ± 112 (g), respectively [26]. In the, PLLA/SPEU, PCL/PLGA, and TP/gel blends maximum value reported for suture retention strength is 249.83 ± 27.53, 177.43 ± 0.3, and 183.54 ± 5.09 gf, respectively [5,32,33]. The PET/PCL (1:3) possesses more suture retention strength compared with the natural and artificial vessels. The rest of the samples also have the suture retention strength close to this value in the internal mammary artery. MTT assay can be helpful for biological studies and gives an evaluation of compatibility and non-toxicity of materials used in medical applications. According to the results, all of the samples showed cell viability above 80%. These results suggested that the produced PET/ PCL nanofibrous scaffolds have no harmful effects on HUVECs viability. The degradability of the samples was investigated for 30 days. In addition to weight variations during 1, 3, 7, 14, and 30 days, the pH of the solution was also measured. In the production of tissue engineering scaffolds, the unchanged and excessive amount of pH during the destruction process is an important feature. In this study, none of the samples showed a significant change in pH (p-value > 0.05), indicating that they did not produce acidic products or inappropriate pH for the cells.

Declaration of competing interest None. Acknowledgments This work was partially supported by Iran National Elites Foundation, Grant award 15/64553. References [1] World health statistics 2018: monitoring health for the SDGs, sustainable development goals, https://apps.who.int/iris/handle/10665/272596. [2] H.Y. Mi, X. Jing, Z.T. Li, Y.J. Lin, J.A. Thomson, L.S. Turng, Fabrication and modification of wavy multicomponent vascular grafts with biomimetic mechanical properties, antithrombogenicity, and enhanced endothelial cell affinity, J. Biomed. Matter. Res. B 107 (2019) 2397–2408, https://doi.org/10.1016/j.msec.2018.12. 126. [3] T. Hoffman, A. Khademhosseini, R.S. Langer, Chasing the paradigm: clinical translation of 25 years of tissue engineering, Tissue Eng. 25 (2019) 1–24, https:// doi.org/10.1089/ten.tea.2019.0032. [4] H.-Y. Mi, X. Jing, E. Yu, X. Wang, Q. Li, L.-S. Turng, Manipulating the structure and mechanical properties of thermoplastic polyurethane/polycaprolactone hybrid small diameter vascular scaffolds fabricated via electrospinning using an assembled rotating collector, J. Mech. Behav. Biomed. Mater. 78 (2018) 433–441, https://doi. org/10.1016/j.jmbbm.2017.11.046. [5] F. Montini-Ballarin, D. Calvo, P.C. Caracciolo, F. Rojo, P.M. Frontini, G.A. Abraham, G. V. Guinea, Mechanical behavior of bilayered small-diameter nanofibrous structures as biomimetic vascular grafts, J. Mech. Behav. Biomed. Mater. 60 (2016) 220–233, https://doi.org/10.1016/j.jmbbm.2016.01.025. [6] H. Du, L. Tao, W. Wang, D. Liu, Q. Zhang, P. Sun, S. Yang, C. He, Enhanced biocompatibility of poly (L-lactide-co-epsilon-caprolactone) electrospun vascular grafts via self-assembly modification, Mater. Sci. Eng. C 100 (2019) 845–854, https://doi. org/10.1016/j.msec.2019.03.063. [7] V.A. Kumar, J.M. Caves, C.A. Haller, E. Dai, L. Liu, S. Grainger, E.L. Chaikof, Acellular vascular grafts generated from collagen and elastin analogs, Acta Biomater. 9 (2013) 8067–8074, https://doi.org/10.1016/j.actbio.2013.05.024. [8] C. Norotte, F.S. Marga, L.E. Niklason, G. Forgacs, Scaffold-free vascular tissue engineering using bioprinting, Biomaterials 30 (2009) 5910–5917, https://doi.org/ 10.1016/j.biomaterials.2009.06.034. [9] M. Koens, K. Faraj, R. Wismans, J. Van der Vliet, A. Krasznai, V. Cuijpers, J. Jansen, W. Daamen, T. Van Kuppevelt, Controlled fabrication of triple layered and molecularly defined collagen/elastin vascular grafts resembling the native blood vessel, Acta Biomater. 6 (2010) 4666–4674, https://doi.org/10.1016/j.actbio.2010.06. 038. [10] S. Asadpour, H. Yeganeh, J. Ai, H. Ghanbari, A novel polyurethane modified with biomacromolecules for small-diameter vascular graft applications, J. Mater. Sci. 53 (2018) 9913–9927, https://doi.org/10.1007/s10853-018-2321-5. [11] W. Wang, W. Nie, X. Zhou, W. Feng, L. Chen, Q. Zhang, Z. You, Q. Shi, C. Peng, C. He, Fabrication of heterogeneous porous bilayered nanofibrous vascular grafts by two-step phase separation technique, Acta Biomater. 79 (2018) 168–181, https:// doi.org/10.1016/j.actbio.2018.08.014. [12] Z. Li, X. Li, T. Xu, L. Zhang, Vascular endothelial growth factor immobilized on mussel-inspired three-dimensional bilayered scaffold for artificial vascular graft application: in vitro and in vivo evaluations, Appl. Sci. 9 (2019) 333–344, https:// doi.org/10.1016/j.jcis.2018.11.039. [13] S.K. Norouzi, A. Shamloo, Bilayered heparinized vascular graft fabricated by combining electrospinning and freeze drying methods, Mater. Sci. Eng. C 94 (2019) 1067–1076, https://doi.org/10.1016/j.msec.2018.10.016. [14] J. Liu, Y. Qin, Y. Wu, Z. Sun, B. Li, H. Jing, C. Zhang, C. Li, X. Leng, Z. Wang, The surrounding tissue contributes to smooth muscle cells’ regeneration and vascularization of small diameter vascular grafts, Biomater. Sci. 7 (2019) 914–925, https:// doi.org/10.1039/C8BM01277F. [15] A. Salifu, B. Nury, C. Lekakou, Electrospinning of nanocomposite fibrillar tubular

5. Conclusion In summary, we produced biocompatible PET/PCL tubular nanofibrous scaffolds with different blend ratios and a broad range of mechanical properties by the electrospinning method. The results reported in this study demonstrated that the presence of PCL in the blend 10

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