Composite vascular grafts formed by micro-imprinting and electrospinning exhibited improved mechanical properties relative to those formed by electrospinning alone. The three-layered composite grafts mimic the three-layered structure of natural blood vessels. The middle layer is made by micro-imprinting poly-p-dioxanone (PPDO), while the inner and outer layers are electrospun mixtures of chitosan and polyvinyl alcohol. The graft morphology is characterized with scanning electron microscopy. For constant graft thicknesses, the PPDO increases the mechanical strength. Cells cultivated on the vascular grafts adhere and proliferate better because of the natural, biological chitosan in the inner and outer layers. Overall, the composite scaffolds could be good candidates for blood vessel repair.

Cardiovascular disease is one of the leading causes of death worldwide.1 Tissue-engineered vascular grafting is a promising solution that would provide patients with an alternative source of vascular replacements, thereby circumventing the shortage of autologous cells and avoiding diseased vessels.2 To better assist cell engraftment and proliferation, surrogate scaffolds must be bioengineered to be as similar as possible to the native histo-architecture of the damaged vessel.3 The main challenge in tissue engineering is to fabricate customizable and biodegradable scaffolds that mimic the components and structural aspects of native extracellular matrices.4 

Electrospinning has important applications in biomedicine and offers unique advantages in the preparation of vascular grafts, such as the high surface-area-to-volume ratio of nanofibers and the biomimicry of extracellular matrix structure and functions.5 However, the structure of these vascular grafts is not maintained because of poor mechanical properties,6 and their non-woven fabrics pattern.7 Since ideal vascular grafts should have robust biocompatibility and mechanical properties, natural materials have been blended with synthetic polymers.8 However, despite a number of efforts with various blending systems, as well as different process parameters and post-processing treatments, the mechanical properties of electrospun fibrillar matrices are still poor.3 We address the issue of mechanical strength by forming composites in the vascular scaffold.

A three-layered composite graft was prepared for the first time. The middle layer of the graft was obtained by micro-imprinting poly-p-dioxanone (PPDO), while the inner and outer layers were formed from electrospun chitosan and polyvinyl alcohol (PVA). The mechanical properties of the composite vascular grafts were better than those of grafts prepared through electrospinning alone. Cell proliferation rates on the chitosan–PVA fibers were higher when the cells did not contact the PPDO polymers. The results indicate that the composite vascular grafts made of chitosan-PVA and PPDO might be suitable candidates for vascular repair applications.

1. Materials

Granular PPDO (Evonik R ö HM Limited, by Share Ltd, Germany) is an aliphatic polyester-polyether with excellent biodegradability, biocompatibility and bio-absorbability. It has been approved by the U.S. FDA for use as a base material for absorbable sutures (trade name PDS) and also has potential applications in orthopedics and drug delivery.9 PPDO is chemically stable, histocompatible, pliable, and exhibits an intermediate rate of degradation.10 

2. Procedure

The micro-imprinting apparatus is depicted in Figure 1. A vascular graft mold cavity is obtained by micro-milling on a five-axis machining center (DMU80) that is highly accurate and rigid. A hole for mounting a thermocouple is also formed.

The detailed procedure of micro-imprinting is shown in Figure 2, and a finished sample that will become a middle layer in the composite is shown in Figure 3. The mold cavity is open-ended and has the advantage of a sliding-lock structure where the diameter of the vascular grafts can be adjusted.11 

1. Starting materials

PVA (grades JP233, degree of polymerization 3500, alcoholysis degree of 88%, Kuraray Company of Japan, Ltd.) was dissolved in hot water at 8 wt %. This solution was heated to boiling and stirred until the PVA was completely dissolved. Chitosan (viscosity-average molecular weight Mη = 112 × 105, degree of deacetylation 82.5%, Zhejiang Golden-Shell Biochemical Co., Ltd.) was dissolved in a 10% acetic acid solution. The PVA and chitosan solutions were then mixed with a respective volume ratio of 2:1 and stirred.12 

2. Electrospinning apparatus

Equipment for electrospinning layers for the vascular graft composites is shown in Figure 4. It included a moving platform, a high-voltage electric field control subsystem, Taylor cone monitoring, and a feeding subsystem. The moving platform had four-axis motion (X, Y, Z, and U) via a precision multi-axis controller control card. The electric field control subsystem included a high-voltage DC power supply (DW-P503-1AC, Dong Wen, Tianjin), which provided 0–50 kV. The Taylor cone monitoring subsystem was used to monitor the entire electrospinning process, and included a high-speed CCD camera (CMLN-13S2M, PointGrey), a light source, and custom detection software. The feeding subsystem was a syringe pump (TJ-3A/W0109-1B, USA Lange). The movement, voltage, feeding, and online monitoring system were automatically controlled by custom PC software.13 

3. Procedure

The micro-imprinted middle layer described above (Figure 3) was fixed on the electrospinning collection device shown in Figure 4. To fabricate the inner and outer layers of the composite graft, the mixed solution of chitosan-PVA was placed in the injector and both layers were electrospun with 3 ml of solution. The solutions were placed in a 5-ml plastic syringe with a blunt-ended needle, and the syringe was attached to a micro-pump that dispensed the solution at a rate of 20 μl/min. A voltage of 15 kV was applied across the needle and the grounded collector (a 6-mm-diameter mandrel with a rotating speed of 300 rpm). The inner layer of the graft required 3 h of spinning. Similarly, the outer layer was fabricated after the scaffold was removed from the roller and re-attached with the other side exposed. Photographs of three-layered composite vascular grafts are shown in Figure 5. These are designated Type I scaffolds, whereas those from the control group prepared by electrospinning only (with other process parameters unchanged) are Type II scaffolds.

The composite scaffold was soaked in alcohol for 1 h and then washed three times with phosphate-buffered saline solution to remove the alcohol. Excess solution within the scaffold was removed by extensive suction. Rat fibroblast cells were harvested, suspended in a culture medium at a density of 5.0 × 107 cells/ml, and then 0.05-ml quantities were seeded on the composite scaffolds to form cell–scaffold constructs. The cell–scaffold constructs were placed in culture dishes. After 4 h of incubation to allow the cells to adhere, 8 ml of growth medium was added. The constructs were thus cultured in vitro, with the media changed twice a week.14 

The number of cells on the scaffold was detected by DNA assays after 7, 14 and 21 days. Briefly, in the assays, the cell-scaffold constructs were crushed and subjected to repeated freezing and thawing to release the DNA. DNA quantification was performed (n = 9 per group per time point) with Hoechst 33258 dye (Sigma-Aldrich) following the manufacturer’s protocol.

The morphology of the composite graft structures was imaged with scanning electron microscopy (SEM SU1510, Analysis and Testing Center, Shanghai University). As shown in Figure 6, the outer layer consists of evenly distributed chitosan-PVA fibers with uniform diameters and good porosity. This simulated extracellular matrix pattern works well for cell growth. A SEM micrograph of a cross-section of the three-layered composite is shown in Figure 7. The middle PPDO layer is 0.2 mm thick and the inner and outer chitosan-PVA layer thicknesses are each 0.05 mm. Sample thicknesses were measured with a micrometer with a precision of ±0.01 mm. Overall, the composite vascular grafts mimic the three-layered structure of natural vessels and have good mechanical properties as well as biocompatibility.

Tensile strengths were measured for 10 scaffold samples from each of the Type I and Type II groups, and average values were calculated. The tensile and radial strengths are listed in Table I; the thicknesses of both scaffolds are approximately 0.3 mm. Clearly, the Type I scaffolds have greater tensile and radial strengths relative to those of the Type II scaffolds. Thus the Type I middle layer of PPDO formed by micro-imprinting had a strong positive effect on the mechanical strength of the scaffold. The corresponding chitosan-PVA middle layer of Type II scaffolds does not have the density of that obtained through micro-imprinting PPDO. Thus, relative to the Type II grafts, the Type I composite grafts have greatly improved mechanical properties and make the its degradation much more controllable. Furthermore, because the Type I inner and outer layers are still obtained through electrospinning, they have the simulated extracellular matrix that favors cell proliferation.

The composite scaffolds are designed to provide a suitable environment for cell growth, proliferation and differentiation. Because fibroblasts can be easily cultivated and grown quickly, they are good candidates for evaluating scaffold biocompatibility. Therefore, rat fibroblasts were grown to evaluate cell attachment and proliferation on the inner and outer layers of the composite scaffolds. Figure 8 is a SEM micrograph acquired after 7 days of cell growth. Cells on the composite scaffolds proliferated promptly, most likely because of biological functional groups introduced by the chitosan. The results also suggest that natural hydrophilic surfaces may be more suitable for cell growth, consistent with the good biocompatibility of the composite scaffold. The micro-imprinted middle layer provides good mechanical support.

To assess cell proliferation, Hoechst 33258 optical densities (OD) that are proportional to the number of cells were acquired. The OD values fluctuated greatly (Figure 9). The number of cells on the composite scaffolds increased slightly by the 7th day, increased significantly by the 14th day, and then tapered off by the 21st day. The composite scaffold was found to promote cell growth because of its three-dimensional structure, micro-pores, mechanical properties, and surface morphology.

A composite vascular graft was prepared for the first time through micro-imprinting and electrospinning techniques. It consisted of a chitosan-PVA inner and outer layer, and a PPDO middle layer. The procedure for composite fabrication can avoid defects that result from single processes. The composite vascular graft had a three-layered structure that mimics a natural vessel, it had good mechanical properties, and it was biocompatible. This composite forming process also makes controlling the degradation of the composite vascular graft more easily. In conclusion, composite vascular grafts could be better candidates for blood vessel repair.

This study is partly supported by the National Natural Science Foundation of China (51475281, 51375292), and the National Youth Foundation of China (51105239).

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