Tingting Yang, Han Xu, Congrui Zhao, Di Tang, Fan Mu, Hongjiang Lu, Zhoufeng Rao, Shufang Wang

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials for Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071,China

Keywords:Poly (butylene succinate)Gelatin Co-electrospinning Degradation Vascular graft

A B S T R A C T In this study, a series of poly (butylene succinate) (PBSU)/gelatin composites were prepared by electrospinning. The morphology, physicochemical analysis, biomechanical properties, biocompatibility, and biodegradability of the materials were evaluated. The results showed that the ultimate tensile stress of the vascular PBSU/gelatin grafts at (95/5), (90/10), (85/15), and (80/20) was (4.17 ± 0.54) MPa,(3.81 ± 0.44) MPa, 2.94 ± 0.69 MPa and 2.11 ± 0.72 MPa respectively, and the burst pressure was (282.7 ± 22.3) kPa, (295.3 ± 3.9) kPa, (306.8 ± 13.9) kPa and (307.6 ± 9.0) kPa respectively, which met the requirements of tissue-engineered blood vessels. Furthermore, the addition of gelatin improved the hydrophilicity and degradation properties of PBSU, thus enhancing cell adhesion and promoting the inward growth of vascular smooth muscle cells.In summary,the research in this paper provides a useful reference for the preparation and optimization of vascular scaffolds.

1. Introduction

Cardiovascular disease, such as atherosclerotic cardiovascular disease(CVD),has become a global serious threat to human health and life [1]. Autogenous blood vessels are usually the main source of materials for vascular repair, however, their use is usually limited by the pathophysiological conditions of patients[2].Therefore,attention has turned to formulate manmade implantable grafts.Tissue-engineered vascular grafts (TEVGs) provide an alternative source of vascular grafts [3]. Ideal artificial vascular grafts need excellent biocompatibility, with special emphasis on excellent mechanical properties and proper biodegradability that meet the requirements of vascular biomechanics and physiology. Besides,it should also serve as a suitable matrix for endothelial cell adhesion, proliferation and migration, thereby promoting vascularization [4-6]. Therefore, suitable materials are the key to construction of artificial vascular grafts.

Poly (butylene succinate) (PBSU) is a biodegradable aliphatic polyester, synthesized by the condensation reaction of succinic acid (SA) and 1,4-butanediol (BDO) [7]. Compared with other polymers, PBSU has the advantages of low melting point (around 114-118 °C), high chemical resistance, easy processing, and no biological toxicity of degradation products [8-10]. PBSU and its copolymers have attracted wide attention from scientists due to their excellent properties. At present,their applications have been extended to many fields, such as medical treatment, agriculture,flame retardant, and packaging [11-13]. However, the present researches are still focused on the primary stage, such as material synthesis, mechanical properties and modification. Based on this,this study attempts to research on the potential application of PBSU in artificial vessels. However, there are some defects in synthetic materials, such as strong hydrophobicity and slow degradation rate [10]. To improve the properties of PBSU material and make it a better graft for artificial blood vessel, coelectrospinning with natural biodegradable materials was introduced.

Gelatin,the hydrolyzed derivative of collagen,is primarily composed of subunits of native collagen α-chains. It also contains higher molecule weight fractions with multiple crosslinks (β- and γ-chains) [14]. The molecular weight of gelatin is usually 15,000-2,500,000,and the change of molecular weight has a great influence on the functionality of gelatin. Besides, one of the main features of this water-soluble protein is its thermo-responsive character, undergoing a reversible sol-gel transition when cooled upper its critical solution temperature (25-35 °C) [15]. Gelatin is rich in sources, has excellent biocompatibility, rapid biodegradability,and non-immunogenicity[15].It can promote cell adhesion and proliferation, such as endothelial cells (ECs). As a potential biomedical material, gelatin is often used for drug loading, hemostatic materials and artificial skin [16-18].

Gelatin has been widely used in the field of tissue engineering because of its excellent properties.Lianget al.[19]developed a series of injectable antimicrobial conductive hydrogels based on glycidyl methacrylate functionalized quaternized chitosan (QCSG),gelatin methacrylate (GM), and graphene oxide (GO). Subsequent experiments verified that the composite antibacterial hydrogel has good biocompatibility, inherent antibacterial property in vivo and in vitro,and has good wound healing effect in repairing infectious skin tissue defects in mice. Tondneviset al. [20] used gelatin and single-walled carbon nanotubes to physico-chemically and biologically modulate polyurethane nanofibers, and the addition of gelatin and single-walled carbon nanotubes increased the hydrophilicity,elongation at break and conductivity of electrospun nanofibers. Biological evaluation demonstrated that nanofibrous surface was covered by confluent and dense layer of both myocardial myoblast and endothelial cells, which is crucial for cardiovascular tissue engineering. The use of PBSU/gelatin composites has also been reported previously.Chenget al.prepared a kind of gelatin coated PBSU electrospun membrane and used it to immobilize thrombin. The results showed that the material had high enzyme activity retention rate and hemostatic performance,and could provide a very promising alternative to the existing hemostatic materials used in first aid [21].

In this study, we attempted to research on the potential application of PBSU/gelatin composite as vascular grafts. To integrate the advantages of different biomaterials into a graft, different amounts of gelatin were introduced through co-electrospinning with PBSU. In this way, PBSU’s excellent mechanical performance can maintain, and the addition of gelatin can improve the hydrophilicity and degradation performance of PBSU,thus improve the adhesion of endothelial cells and promote the ingrowth of vascular smooth muscle cells.

2. Materials and Methods

2.1. Materials

Poly (butylene succinate) (PBSU) pellets (Mw= 190,000-230,0 00)was purchased from HeXing chemical industry(Anqing,China),Gelatin(Type A)and lipase from porcine pancreas were purchased from LianXing biotech company(Tianjin,China).Methanol,chloroform and Hexafluoroisopropanol (HFIP) were obtained from Tianjin Chemical Reagent Company (Tianjin, China).

2.2. Fabrication of nanofiber materials

The hybrid PBSU/gelatin nanofiber materials were fabricated by two-nozzle electrospinning approach.Five different ratios of PBSU/gelatin materials were prepared by changing the feeding rates of PBSU and gelatin solutions. The parameters of electrospinning are listed in Table 1.Fibers were deposited on the collector to form a non-woven film, or on a rotating stainless steel collector(diameter = 2 mm) to form a tubular graft. The collected scaffolds were placed in a vacuum oven for 48 h to evaporate remaining solvent.

Table 1 The parameters of electrospinning

To crosslink the gelatin in the hybrid materials,the dried materials were placed in ethanol solution of EDC overnight in the ice bath (EDC concentration for P/G = 95/5, 90/10, 85/15 and 80/20 were 0.01917 g·ml-1, 0.03834 g·ml-1, 0.05751 g·ml-1, and 0.07668 g·ml-1respectively). After the crosslinking was finished,the films were washed with ethanol repeatedly and dried at room temperature.

2.3. Morphology characterization

To investigate the micromorphology of prepared materials, the obtained nanofiber films were cut into 0.5 cm×0.5 cm and pasted on the sample stage.After sputter-coated with gold,the fiber diameter and morphology of the obtained films were observed by a FEI quanta 200 scanning electron microscope (SEM). Image J software was used to measure the pore sizes of each group of films.

2.4. Contact angle measurement

Each material was cut into a rectangular slice(3 cm×1 cm)and pasted on a glass slide.The contact angles of the prepared material were determined on a contact angle measurement instrument(HARKE-SPCA, Beijing Harke Experimental Instrument Company,Beijing,China).Six parallel samples were measured in each group,and images were recorded using a color CCD camera.

2.5. Tensile mechanical test

The electrospinning nanofiber films were cut into rectangular slices(3 cm×1 cm)and horizontally mounted on two mechanical gripping units of the universal materials testing machine. Forceelongation data were recorded at a crosshead speed of 2 mm·min-1with a load of 100 kg, and the stress-strain curves were obtained.The films were tested under dry conditions.Six parallel films were measured in each group.Maximum stress and elongation at break were calculated from the stress-strain curves out of six parallel measurements.

2.6. Burst pressure test

The tubular grafts were cut into 2 cm long (n= 3). One end of the graft was connected to the catheter with the same diameter.The graft was then filled with vaseline through the needle of a catheter. The other end of the graft was secured with 3-0 silk suture. Pressurized CO2was introduced into the system and the pressure was increased at a constant rate until the graft ruptured.The burst pressure was read by the pressure recorder at which the graft ruptured.

2.7. Fibroblast proliferation

The electrospun films were cut into circular slices of 1 cm diameter and then put into 48-well plates respectively. All samples in the 48-well plates were sterilized overnight by ultraviolet irradiation. 3T3 fibroblasts were seeded in 48-well plates with 300 μl DMEM (which contained 10% fetal bovine serum (FBS) and 1%penicillin/streptomycin), and placed in a 37 °C incubator containing 5% CO2; cell density was 1 × 104cells per well plate. The cell viability of all cells on electrospun films was evaluated by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)assay after the 1st, 3rd, and 5th day of being cultured. For this assay, 50 μl MTT solution (5 mg·ml-1) was added into 48-well plates with samples. After being cultured in an incubator at 37 °C(5%CO2)for 4 h,the MTT solution was removed and 300 μl DMSO solution was added. Plates maintained shaking 30 min at 37 °C.Then 50 μl solutions were taken from each well to absorbance test.The absorbance of each sample was measuring at 490 nm using a 96-well microplate reader. The absorbance is proportional to the number of 3T3 fibroblasts,which can characterize the proliferation of 3T3 fibroblasts on the surface of the materials.

2.8. Enzymatic degradation of materials

The materials are cut into rectangles (3 cm × 1 cm) for the enzymatic degradation experiment. The lipase degradation solution (1 mg·ml-1) was prepared. The prepared materials were put into a 50 ml test tube. 30 ml of the enzyme degradation solution was added into the test tube and soaked the material.The materials were labeled and incubated at the physiological temperature(37 °C). The degradation solution was changed once a week. In the process of incubation,the film material was regularly removed and treated with 90% ethanol, then washed with distilled water,then dried to constant weight in a vacuum.Weighing and calculating the rate of weight loss of the material.The micromorphology of the electrospun fibers was observed under the scanning electron microscope (SEM).

Fig.1. Morphology of PBSU/gelatin hybrid vascular grafts(SEM:a.P/G=95/5;b.P/G=90/10;c.P/G=85/15;d.P/G=80/20;e.Fluorescence image:red:gelatin;green:PBSU;f. tubular graft).

2.9. Endothelial cells (ECs) adhesion

Endothelial cells (2 × 104cells) stained with 1,1-diododecyl-3,3,3,3-tetramethylindocarbocyanine-5,5-disulfonic acid (DiI) dye were seeded on each piece of prepared materials in a 48-well plate,and the total volume of culture medium was 300 μl. After 4 h at 37°C,the electrospun films were rinsed twice with PBS to remove the unattached cell.The adhesion of endothelial cells was observed by fluorescence microscope. The number of adhered cells was counted using Image J software.

2.10. Vascular smooth muscle cells (VSMCs) infiltration

Vascular smooth muscle cells (5 × 104cells) stained with DiI dye were seed on materials, and the total volume of culture medium was 400 μl. The cells were cultured at 37 °C and 5% CO2, and the culture medium was changed every 24 h.After 1,4,and 7 days,the medium was removed, and films were rinsed with PBS. The samples were fixed overnight with 4% paraformaldehyde, dehydrated with 30% sucrose solution, and then embedded in OCT for frozen section. The sample was sliced with 10 μm thick, stored at-20 °C, and examined under a fluorescence microscope.

2.11. Statistical analysis

Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS andT-test was used to analyze the statistical significance.A value ofP＜0.05 was considered to be significant andP＜0.01 very significant.

3. Results and Discussions

3.1. Construction of PBSU/gelatin hybrid vascular grafts

Four different mass ratios of gelatin(PBSU/gelatin=95/5,90/10,85/15, and 80/20) were introduced into the final product by coelectrospinning with PBSU solution in this study. SEM images showed that two types of fibers coexisted with different fiber diameters (Fig. 1a-d). The thick fibers were attributed to PBSU,and the thin fibers were attributed to gelatin because the solution conductivity and molecular weight of PBSU were far higher than those of gelatin. The pore sizes of the four groups of films were(1.7207 ± 0.7043) μm, (1.9207 ± 0.5255) μm, (1.9847 ± 0.4508)μm and(2.2968±0.5532)μm,respectively,with no significant difference. In order to characterize the composition and relative distribution of the two types of fibers more precisely, fluorescent dye was used to stain the two types of fibers. Gelatin fibers (red)and PBSU fibers (green) were distributed randomly and uniformly(Fig. 1e). Furthermore, the inner diameter of tubular grafts was 1.5 mm (Fig. 1f).

Fig. 2. Water contact angle of different ratio of PBSU/gelatin vascular grafts (n = 6,**P ＜0.01).

Fig. 3. Tensile properties of prepared materials (n = 6, *P ＜0.05).

3.2. Hydrophilicity of PBSU/gelatin hybrid vascular grafts

The hydrophilicity of prepared materials was measured by the water contact angles. The results (Fig. 2) showed that the contact angles of P/G = 100/0, 95/5, 90/10, 85/15, and 80/20 were(107 ± 0.8)°, (58 ± 3.2)°, (46 ± 1.1)°, (36 ± 4.5)°, and (22 ± 2.2)°respectively, which decreased as the ratio of gelatin increases.The results demonstrated that the introduction of gelatin improved the hydrophilicity of nanofiber films.

Fig. 4. Burst pressures of prepared materials.

3.3. Mechanical properties

3.3.1. Tensile test

Tensile tests were conducted to determine whether the mechanical strength of prepared materials was conducive to a vascular graft. Typical stress-strain curves of prepared grafts under tensile loading were showed in Fig. 3a. Ultimate tensile stress of P/G = 100/0, 95/5, 90/10, 85/15 and 80/20 were (4.11 ± 0.38)MPa, (4.17 ± 0.54) MPa, (3.81 ± 0.44) MPa, (2.94 ± 0.69) MPa and(2.11 ± 0.72) MPa, which decreased gradually (shown in Fig. 3b).PBSU is a flexible polymer with a high level of toughness, which has a linear backbone structure without any side groups or branch chains. After the incorporation of gelatin into the grafts, the mechanical properties decreased, which might be due to the relatively low mechanical strength of gelatin. Given that the ultimate tensile stress of the adventitial layer of the coronary arteries was about(1.4±0.6)MPa[22],all the prepared grafts met the requirement for vascular implantation. In the meanwhile, the ultimate strain was stably increasing with the ratio of gelatin(Fig.3c).Overall, the ultimate strain of these grafts was comparable to the human coronary artery (45%-99%) [23]. In terms of elastic modulus,all the grafts except P/G=80/20 were higher than that of radial arteries (2.68 ± 1.8) MPa [24], which were showed in the Fig. 3d.The grafts of P/G = 100/0, 95/5, 90/10, 85/15, and 80/20 had modulus values of (16.28 ± 1.13) MPa, (15.75 ± 0.58) MPa,(13.29 ± 0.98) MPa, (12.48 ± 1.74) MPa, and (8.564 ± 2.1) MPa respectively. Although the mechanical strength of gelatin is weak,PBSU/gelatin materials showed excellent mechanical properties in our designed ratios.

3.3.2. Burst pressure test

The burst pressure is one of the most important parameters which determine the safety of a vascular graft for implantation[25]. In this study, all the prepared materials were soaked in PBS for 20 min before testing. The burst pressure results were shown in Fig. 4. The burst pressures of P/G = 95/5, 90/10, 85/15 and 80/20 were (282.7 ± 22.3) kPa, (295.3 ± 3.9) kPa, (306.8 ± 13.9)kPa and (307.6 ± 9.0) kPa. Given that burst pressure of saphenous vein and mammary artery were 166-332 kPa and 266-425 kPa respectively [26], the prepared PBSU/gelatin vascular grafts could meet the burst pressure requirements of implantation.

Fig.5. MTT assay of fibroblast proliferation on different ratio of PBSU/gelatin(n=6,*P ＜0.05).

Fig. 6. Curve of weight remaining rate of films with different ratio of gelatin.

3.4. Cytocompatibility of PBSU/gelatin hybrid vascular grafts

The MTT assay of 3T3 fibroblasts was utilized to evaluate cytocompatibility of PBSU/gelatin hybrid vascular grafts(Fig.5).It was observed that 3T3 fibroblasts proliferated slowly at the beginning of culture.Cell numbers on P/G=100/0 and P/G= 95/5 films were more than that on other films.This might be ascribed to the excellent hydrophilicity of films, which was disadvantageous for cell adhesion. However, significant differences between groups could be found as the proliferation rates increased during the period from day 3 to day 5. Statistically, proliferation rate of 3T3 fibroblasts on the PBSU/gelatin films were enhanced significantly when compared to that on the PBSU film and TCPS.As the culturing time continued, proliferation rates of 3T3 fibroblasts on the grafts were significantly accelerated with the gelatin introduced. The reason might be that gelatin had excellent biocompatibility, which could promote cell proliferation. In addition, the dissolution of part of gelatin provided a certain space for the cell proliferation.

Fig. 7. SEM image of enzymatic degraded films with different ratio of gelatin.

3.5. Enzymatic degradation

To evaluate the biodegradability of the PBSU/gelatin materials in a short time interval, enzymatic degradation was performed.Compared with P/G = 100/0, the mass remaining rate of P/G = 95/5, 90/10, 85/15, and 80/20 were greatly reduced (Figs. 6 and 7). In the same time interval, the mass loss increased with the ratio of gelatin in the final product (Fig. 6). After 24 days, the mass remaining rate of P/G = 100/0 was (59.7 ± 15.89)%, and the mass remaining rate of P/G = 80/20 was just (43.9 ± 9.18)%.

The morphology of the polymer films was investigated using SEM. The SEM images of polymer films taken before and after enzymatic hydrolysis are presented in Fig. 7. The SEM image of P/G = 100/0 revealed homogeneous and smooth nanofibers. On the contrary,fibers of P/G=95/5,90/10,85/15,and 80/20 appeared irregularities, which became deeper with the increasing ratio of gelatin. Then holes, cracks and channels formed with an exposure time of enzymatic degradation and the ratio of gelation increased.

Fig. 8. Adhesion of endothelial cells (a. P/G = 100/0; b. P/G = 95/5; c. P/G = 90/10; d. P/G = 85/15; e. P/G = 80/20; f. histogram of cell number) (n = 6, *P ＜0.05).

3.6. Endothelial cells (ECs) adhesion

Fig. 8 showed that the numbers of ECs were positively associated with the ratio of gelatin. There were more adhered cells on PBSU/gelatin films, which might be due to the hydrophilicity of films that had a positive effect on adhesion and proliferation. Furthermore, gelatin has been reported to improve the adhesion and proliferation of ECs, thus favors the regeneration of microvessels[27,28].

3.7. Infiltration of vascular smooth muscle cells (VSMCs)

To evaluate the effect of PBSU/gelatin graft on cell infiltrationin vitro,VSMCs were seeded on the surface of nanofiber films with different ratios of gelatin and cultured for up to 7 days (Fig. 9). In accordance with our expectation, gelatin in the films were found to enlarge the pore size and porosity because of fast degradation compared to PBSU, yielding a significantly enhanced cell infiltration throughout the grafts, as confirmed by fluorescent images.As shown in Fig. 9, the depth of cell infiltration on the P/G = 80/20 and 85/15 films were significantly greater than others.In contrast, no obvious cell infiltration was found on the P/G = 100/0 nanofiber films during the entire culture period,although an increase in cell population was confirmed (Fig. 4).The enhanced cell infiltration might be attributed to the large pores and grooves from the fast degradation and dissolution of gelatin, which caused fewer impediments to cell migration into the grafts. In addition, the improved cell infiltration contributes to the significant cell proliferation on the PBSU/gelatins grafts.

Fig. 9. Ingrowth of vascular smooth muscle cells (a. P/G = 100/0; b. P/G = 95/5;c. P/G = 90/10; d. P/G = 85/15; e. P/G = 80/20, scale bar: 200 μm).

4. Conclusions

In our study, PBSU/gelatin films and grafts were successfully fabricated by co-electrospinning.The films and grafts showed outstanding hydrophilicity, mechanical properties and degradation property. It was found to be positive for cell growth.Furthermore,ECs adhesion and VSMCs infiltration could also be regulated by the introduction and degradation of gelatin. Such materials in our study might be promising grafts for vascular tissue engineering.Thein vivoperformance of co-electrospun PBSU/gelatin materials as potential vascular grafts needs further examination.

Acknowledgements

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by National Natural Science Foundation of China (31870966, 81800931, 81901062), National Key Research Development Program of China (2020YFA0803701,2017YFC1103504), and Tianjin Science Foundation(20YFZCSY01020).