Tissue engineering is known to be a promising approach to solve issues in medical treatment related to the limited availability of donor tissue [1, 2]. The process is based on the seeding of scaffolds with autologous human cells. One of the greatest challenges in fabricating scaffolds for tissue engineering deals with the imitation of the morphology of native structures . Most tissues consist of multiple tissue layers and every corresponding cell type needs a specific structure or material to grow. Such complex multilayered structures can be created by electrospinning. Electrospinning is an effective and adequate way of producing tailor-made scaffolds from polymeric biomaterials. There are two commonly used setups: single and coaxial electro-spinning. Compared to single electrospinning the coaxial approach uses two cannulaes adjusted coaxially in each other . This is usually done with the use of custom-made nozzles. A typical coaxial electrospinning setup is made up of two coaxially arranged spinnerets, two reservoirs with pumps, a high voltage supply and a grounded collector see Figure 1 [3, 9].
The described setup has a lot of benefits regarding the fabrication of multi-layered structures due to the coaxial arrangement of the spinnerets and the independent loops for the inner and outer fluids. Furthermore, coaxial electrospinning allows the fabrication multi-fiber structures and enables the use of polymer solutions with high evaporation rates by ’coating’ the solution stream in additionally solvent. It is also possible to produce core-shell fibres . This can be used for drug delivery systems by controlling the rate of diffusion of the drug releasing core fibre with the shell fibre. The aim of this study is to explore new applications for the coaxial electrospinng and to investigate different parameters on the process. More precisely the in-process adjustment of polymer concentration and the relationship between fiber diameter and polymer concentration.
1.1 Design of the coaxial nozzle
Different types of nozzles are used and described in the literature [3, 4]. The design of a new nozzle concept aimed to optimize parameters like easy and reproducible coaxial adjustment of the spinnerets, a small dead volume and a fast assembly and cleaning process. The new nozzle is built up of four parts, each made of stainless steel. They are connected via metric threads between the outer cylinder and tip as well as the outer cylinder and top screw nut (Figure 2).
The adjustment is realised by four screws on the extent. It is possible to loosen the tip and the top screw nut to clean the nozzle, without changing the positions of the spinnerets. The dead volume is defined by the gap between tip and inner cylinder and the length of the blunt cannulaes pressed in the inner cylinder. Two holes in the top screw are acting as the solution supply. Cannulaes can be stung directly through a sealing into the press fitted blunt cannulaes. The handling and cleaning remains easy because of the small dead volume and the modular concept of the nozzle.
2 Material and methods
2.1 Scaffold fabrication
ε−polycaprolactone (PCL) was purchased from Sigma-Aldrich and the solvent 2,2,2-trifluorethanol from ABCR GmbH (Karlsruhe, Germany).
Two polymer reservoirs were connected to the coaxial nozzle. The inner fluid had a concentration of 100 mg/ml, 200 mg/ml being used as the outer fluid. The flow rates have been calculated on the following formula:
Concentrations of 100, 125, 150, 175 and 200 mg/ml were used as references. The influence of polymer concentration fiber diameter was investigated. The assumption was, that a higher polymer solution would result in a bigger fiber diameter . In addition, the influence of rotation velocity on the level of anisotropy was observed. Referring to the literature, fiber alignment increases with rotation velocity [5–8]. Scaffolds were spun using a custom-made setup consisting of a high voltage supply, two syringe pumps and a rotating drum collector. Both, single and coaxial setup samples were spun with a collector-emitter distance of 250 mm, voltage of 25 kV, a temperature of 25°C, a humidity between 50% and 64% and a flow rate of 4 ml/h overall. Reference measurements were recorded for 100, 125, 150, 175 and 200 mg/ml and also for the coaxial tests. Furthermore, the collectors’ rotation speed was set to 0, 500 and 1000 rpm. For the coaxial tests, the flow rates were calculated according to [E1] and are displayed in Table 1.
Samples have been sputter coated and several images have been taken with a SEM (magnifications of 500x, 1000x, 2000x, 8000x). Carl Zeiss AxioVision software has been used to measure fiber diameters.
3 Results and discussion
The results of the fiber diameter analysis of the single setup are shown in Figure 3. The graph depicts the fiber diameter in relation to the concentration and the rotation speed of the collector. The mean values are located between 0.4 μm for 100 mg/ml at 1000 rpm and 3.3 μm for 200 mg/ml at 0000 rpm. For samples with a rotating velocity, fiber diameter increased from 0.7 μm to 3.3 μm with increasing polymer concentration.
The other groups showed mean values between 0.4 μm and 3.3 μm for 0500 rpm and between 0.4 μm and 2.9 μm for 1000 rpm. Not only polymer concentration had an influence on fiber diameter. In addition rotating velocity influenced the fibers. The higher the speed, the thinner the fibers, e.g. 0.73 μm at 0000 rpm and 100 mg/ml, 0.42 μm at 0500 rpm and 0.41 μm at 1000 rpm.
In addition, a relationship between collectors’ rotation velocity and the quantity of aligned fibers could be observed. To investigate the level of anisotropy SEM-images were prepared (Figure 6). It was shown, that the anisotropy does not only depend on the rotation speed but also on the fiber diameter.
The influence of the collectors’ speed decreases with increasing fiber diameter. From this, it follows that the rotational speed has to be increased along with the fiber diameter in order to achieve comparable levels of anisotropy through the different values of polymer concentration, but also that the concentration has to be increased to achieve the desired diameter.
The analysis of the coaxial tests has shown the same correlation between the fiber diameter and polymer concentration. Compared to the single setup results, there are differences in the absolute values and the gradient, but the trend is still noticeable (Figure 5). The mean values for the coaxial tests are located between 1 μm and 1.5 μm.
The distribution of thick and thin fibers in some samples (Figure 6) stands out from the other results. This occurred at the coaxial tests with 125 mg/ml at 0000 rpm, diameters between 0.29 and 1.49 μm have been measured.
Tissue engineering is dependent on the affinity of different human cells to several fiber diameters and pore sizes. To serve these needs the production of scaffolds with the right properties are indispensable. The results of this study demonstrate a solution for in-process adjustment of polymer concentration. It is not longer necessary to change solutions during the process. Based on this, the fabrication scaffolds with distributions of fibers with different diameters or multi-layered structures can be rapidly realized. The fiber diameter has been successfully influenced by the concentration of the used polymer solutions. The higher the concentration the bigger the diameter of the fibers. The level of anisotropy of the fibers has been regulated by the rotational speed of the collector. While the level has been shown to increase with increasing rotation velocity, an opposing effect of the polymer concentration has also been observed. Only for samples with 100 and 125 mg/ml polymer concentration a meaningful change in the amount of aligned fibers has been observed. At higher concentrations such as 150, 175 and 200 mg/ml the level of anisotropy has been lower than at the 100 and 125 mg/ml samples.
This study presented promising tools and methods to modify the micro- and macroscopic structures of electrospun scaffolds. These methods can be used to tailor scaffolds’ properties to specific applications in the field of tissue engineering.
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About the article
Published Online: 2015-09-12
Published in Print: 2015-09-01
Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent has been obtained from all individuals included in this study. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.