CC BY
32
УДК: 617.3:[678.09+678.011]
EFFECT OF ELECTRON-BEAM IRRADIATION ON ELECTROSPINNING PRODUCED SCAFFOLDS
Alena Olegovna STEPANOVA12, Mikhail Vasilevich KOROBEYNIKOV3, Aleksandr Sergeevich YUNOSHEV4, Pavel Petrovich LAKTIONOV1,2
1 Novosibirsk Research Institute of Circulation Pathology of Academician E.N. Meshalkinща Minzdrav of Russia
630055, Novosibirsk, Rechkunovskaya str., 15
2 Institute of Chemical Biology and Fundamental Medicine of SB RAS, Novosibirsk, Russia 630090, Novosibirsk, Lavrent'ev ave., 8
3 Institute of Nuclear Physics, Novosibirsk, Russia 630090, Novosibirsk, Lavrent'ev ave., 11
4 Insitute of Hydrodynamics, Novosibirsk, Russia 630090, Novosibirsk, Lavrent'ev ave., 15
The influence of electron beam irradiation on the stiffness, stability and biocompatibility of electrospun scaffolds, produced from Nylon 6, polycaprolactone (PCL) and poly(DL-lactic-co-glicolic acid) (PLGA) was investigated. It was shown that irradiation of the matrices with the electron beam generated by the electron beam accelerator ILA-6 (2.2 MeV, 400 mA, 10 Hz, «Radiochemical technologies Ltd») can potentially be used for sterilization of the matrices (irradiation by the dose of 25 kGy does not interfere with the mechanical properties of all studied matrices). Irradiation of the matrices produced from PCL by the dose of 100 kGy increases the proportional limit of the material and allowed us to introduce durable regions into the vascular grafts by irradiating the graft through the template with open areas. Electron beam irradiation of the matrices does not influence over the capacity of the matrices to support adhesion and viability of primary human endotheliocytes at the surfaces of these matrices, as it was shown using Alamar Blue assay.
Keywords: electron beam irradiation, electrospinning, PCL, PLGA, Nylon 6.
One of the promising technologies nowadays for production scaffolds for tissue engineering is elec-trospinning. The method is based on stretching of the thin and ultrathin fibers from the polymer solution in a strong electric field. Thus, nonwoven 3D matrices of different types of fibers with different fiber orientations are easily been obtained. The method allows the production of different variants of fibers, including fibers from two or more solutions, coaxial fibers, fibers containing a variety of low and high molecular weight biologically active substances [2]. Electrospinning produced scaffolds
have been shown to be used for different tissues displacement [4], but modification of their properties, such as stiffness, stability, sterilization, is still needed.
The cross-linking of polymers through electron beam irradiation (EBI) in technology changes a thermoplastic material into a thermoset. This technology is widely used also for modification of mechanical properties of plastic products, by generating free radicals, which lead to polymer chain degradation, or intermolecular crosslink formation [5]. So far, this technology suggests treatment of the end
Stepanova A.O. - junior researcher of laboratory for molecular medicine and laboratory of biomedical technologies,
e-mail: lebedeva@niboch.nsc.ru
Korobeinikov M.V. - candidate of technical sciences
Yunoshev A.S. - candidate ofphysical mathematical sciences, head of laboratory for high speed processes of the department for explosive processes
Laktionov P.P. - candidate of biological sciences, leading researcher, head of laboratory for molecular medicine, head of laboratory for biomedical technologies, e-mail: lakt@niboch.nsc.ru
product, widely used for sterilization of medical products in a simple and low-cost way.
We evaluated potentialities of electron beam irradiation for the modification of scaffolds produced by electrospinning that are intended to be used for tissue engineering.
MATERIAL AND METHODS
Materials
Poly(s-caprolactone) (PCL), Nylon 6 and 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP), Phosphate buffer saline (PBS), gelatine were obtained from Sigma-Aldrich Co. (USA), 50:50 poly(DL-lactide-co--glicolide) (PLGA, inherent viscosity 1.11 dL/g in HFIP) was purchased from Lactel Asorbable Polymers (Pelham, AL, USA). Isolve's Modified Dulbecco's medium (IMDM) (phenol red free), Fetal bovine serum (FBS), penicillin/streptomycin antibiotics, were obtained from Gibco, Life Technologies Co (USA).
Scaffold preparation
Electrospun scaffolds were fabricated using Nylon 6, as the nonbiodegradable polymer, PCL, as the slowly biodegradable polymer and PLGA, as the rapidly biodegradable polymer. Briefly, each polymer Nylon 6, PCL and PLGA was dissolved in HFIP at total concentrations of 10 %, 7 % and 15 % respectively. The polymer solutions were individually electrospun using electrospinning setup NF-103 (MECC, Japan). The solutions were delivered through a 22G blunt tip needle connected to a 5 mL syringe. The spinning conditions for Nylon 6 were 1.5 mL/hour flow rate, 20 kV applied voltage, 190 mm air gap; for PCL - 1.5 mL/hour flow rate, 23 kV applied voltage, 190 mm air gap; for PLGA -1.5 mL/hour flow rate, 18.4 kV applied voltage, 200 mm air gap. The samples were collected on a custom-designed mandrill collector with an outer diameter of 6 cm. The rotation speed of the collector was 300 rpm. The thickness of all samples was 150 (m. After electrospinning all scaffolds were additionally dried in room conditions for day. Additionally, tubes with inner diameter 1.7 mm and wall thickness 150 (im were prepared using a stainless steel 180 mm rod collector rotating 300 rpm.
Electron beam irradiation
All samples were irradiated by electron beam accelerator ILA-6 (2.2 MeV, 400 mA, 10 Hz, «Radiochemical technologies Ltd») in the Institute of nuclear physic SB RAS (Novosibirsk, Russia) at doses 25, 50, 100, 150 and 200 kGy. Before irradiation, every sample was cut in rectangular specimens 1 x 4 cm, suitable for tensile strength testing, and packaged in a polyethylene zip-lock bag.
Tensile test
The tensile strength of electrospun nonirradiated and irradiated polymer scaffolds was characterized using the Zwick/Roell Z100 (Gemany) testing machine in Institute of hydrodynamic SB RAS (Novosibirsk, Russia) with a deformation speed of 10 mm/min. The mechanical characteristics of the samples were obtained from the stress-strain diagram.
Scanning electron microscopy
Fiber morphologies of irradiated and nonirradi-ated Nylon 6, PCL and PLGA electrospun samples were examined by scanning electron microscopy (SEM; Model JSM-6460 LV, Jeol, Japan) at acceleration voltages of 20 kV and 25 kV. The samples were sputter coated with gold to achieve the layer 10 A. The average diameters and standard deviations were calculated from 20 separate fibers per image. The pore size was calculated from 20 separate holes between fibers on the image.
Differential scanning calorimetry (DSC)
DSC data were obtained with Calorimeter setup DSC 550 (Russia) in the Institute of solid state chemistry and mechanochemistry SB RAS (Novosibirsk, Russia) in a nitrogen atmosphere.
Degradation rate of matrices in PBS solution 10 mm diameter discs were prepared with cutting from nonirradiated and irradiated with a dose 100 kGy scaffolds. The discs were replaced into the well with 250 (l of phosphate buffer saline (PBS) of 48 well microplate and incubated at 37°C for 5, 7, 14, 21, and 28 days. After the incubation, the discs were removed from the well, rinsed with water, dried and examined by SEM.
In vitro cell culture analysis
10 mm diameter discs were obtained from non-irradiated and irradiated with dose from 0 to 150 kGy scaffolds. Discs were wetted in PBS and placed into the well of 48 well microplates. Every disc was fixed close to the bottom by a special plastic ring with outer diameter 10 mm and wall thickness 2 mm. After that, 100 (l of IMDM culture medium with 10 % serum was added. Human umbilical vein endothelial cells (HUVEC) were obtained and cultured as described previously [1]. The matrices were incubated at 37°C for 2 hours, after that 2 x 104 of HUVEC cells in 100 (l of IMDM culture medium with 10 % serum were added and incubated in 37°C, 6 % CO2 for next 24 hours. Control cells were incubated in wells of 48 well microplates equipped with plastic rings. After the incubation 20 (l of AlamarBlue reagent was added into the each well.
Table
Average diameter and pore size of electrospun scaffolds
Polymer Fiber diameter, цт Pore size, ^m
PLGA 2.05 ± 0.12 6.6 ± 0.22
PCL 1.6 ± 0.40 7.0 ± 0.57
Nylon 6 0.8 ± 0.21 3.0 ± 0.37
Fig. 1. SEM Scaffolds morphology before and after irradiation
After the incubation in CO2 incubator for 6 hours, OD of the culture medium was assessed at 570/620 nm.
RESULTS AND DISCUSSION
Characterization of morphology of Nylon 6, PCL and PLGA scaffolds
The structure of the obtained scaffolds was investigated by scanning electron microscopy. All
samples consist of uniform fibers (Fig. 1). The average diameters for Nylon 6, PCL and PLGA fibers are presented in the Table.
The matrices were irradiated at doses from 0250 kGy with irradiation increment 25 kGy Considering heat capacity of PCL as 1600 J/kg-°C, and its melting temperature as 60 °C (other polymers have higher Tm) the irradiation power necessary to melt PCL could be estimated as C x m x dT = Q, C x x dT = Q/m (J/kg = C). To melt PCL the material should be irradiated with the energy Q/m = 1.6 x x103 x 0.6 x 102 = 96 kGy. As far as we treated the scaffolds with discrete dose (~1 min intervals and in a ventilation box) the scaffolds are not apt to be melted at least at irradiation dozes lower than 200 kGy. Actually, SEM demonstrated that scaffolds do not change their morphology and microstructure after irradiation (Fig. 1).
Tensile strength
Before and after irradiation the samples were cut into rectangular specimens of size 10 x 40 mm, and tested on a mechanical testing machine. Stressstrain diagrams demonstrate that EBI scaffolds made of Nylon 6 and PLGA decrease elastic region and strength in plastic region. On the contrary, EBI increased the strength in the area of elastic region for PCL scaffolds along with irradiation dose. At the dose of 100 kGy, the stiffness in the area of elastic region (proportional limit) increased almost twice from 1.2 ± 0.2 MPa to 2.1 ± 0.3 MPa. It
о p
•e
•Й
's-£
................. 50 kGy
25 kGy
-г
30 40 Extension, mm
20 30 Extension, mm
0 kGy 25 kGy 50 kGy
100 kGy 150 kGy 200 kGy
0 10 20
Extension, mm
Fig. 2. Typical stress-strain diagrams of 3D matrices PLGA (a), PCL (b), and Nylon 6 (c). All matrices were tested in the fiber laying direction in triplicate
8
w
I
w
2000 H 10000
-1000-
-2000
20
"••100 kGy
.......50 kGy
0 kGy
70
120 170
T, °c
T
i
800060004000! 20001 0; -2000-I -4000-6000-8000-10 000
220 270
40
b
........................100 kGy
nwov --25 kGy
...............................................................50 kGy
M__________200 kGy
T
-1-1—
90 140
T, °c
25 kGy
S 1200-
w
Ф 1000° -800-и -600-^00-
т,° с
Fig. 3. Differential scanning spectroscopy of irradiated and nonirradiated electrospun scaffolds, produced from Nylon 6 (a), PCL (b) and PLGA (c)
should be also noted that the EBI in a dose 25 kGy, which is used for sterilization of medical products, does not interfere with stiffness of the matrices and can be used also for sterilization of electrospinning produced scaffolds (Fig. 2).
Differential scanning spectroscopy
We considered that irradiation would interfere with the crystallinity of polymers, but the data of Differential Scanning Calorimetry did not demonstrate strong effects. It is possible to form interconnections between polymer aggregates that can be generated during electrospinning due to spinodal decomposition of polymer solution [3] (Fig. 3).
Introducing the stiffness zones into tubular elec-trospun construction.
The illumination of the material through the perforating template allowed us to obtain more stiffness areas in vascular grafts produced by electrospinning from PCL. Tubular structures with inner diameter 1.7 mm and wall thickness 200 ^m were irradiated through a steel grid as shown on Figure 4 (a, b). After incubation of the tube in the solution of fluorescently labeled albumin, protein adsorption occurred mainly in the irradiated areas. Thus, irradiation led to modification of the irradiated surfaces. Vascular grafts with stiffeners have twice as great elastic deformation limit and kinked resistance, ~1.5 times lower bend radius. Cyclic hydrodynamic
load (106 cycles of 0/200 mmHg) (data not shown) did not interfere with the mechanical properties and did not lead to disaggregation of the vascular graft in the interzone areas, demonstrating the efficacy of the approach for the introduction of webbings into electrospun produced goods.
In vitro degradation of irradiated and nonirradi-ated electrospun matrices
It was shown that EBI decreased the stiffness of the PLGA and Nylon 6 matrices. Therefore, we evaluated the influence of EBI on the degradation rate of the matrices in vitro. The structure of PCL
Fig. 4. Introduction of the stiffeners into the small diameter vascular graft. Bottom (A) and side (B) views of the duralumin templates used for the production of the electron beam irradiated regions in the vascular grafts. (C) Irradiated (light) and not irradiated (dark) areas of the graft, obtained after its incubation with the solution of the fluorescein labeled BSA (fluorescent image obtained using the SteReo Discovery V12)
5 days
7 days
14 days
21 days
28 days
5 days 7 days 14 days 21 days 28 days
Fig. 5. Degradation rate of the untreated and irradiated PLGA matrix
120 n
100-
80
I 60
u д
40
20-
0
iii
ii
il
il
il
Irradiation dose, kGy 0 Control
25 50 100150 PCL
0 25 50 100150 PCL+ 10% gelatin
Fig. 6. Viability of endothelial cells cultivated on the surface of different irradiated and nonirradiated matrices
and nylon matrices was not significantly changed after incubation in PBS at 37 °C for 7 weeks (data not shown). However, PLGA matrices were shown to degrade faster in compared to non-irradiated scaffold (Fig. 5). Thus, EBI could be used for decreasing the degradation rate, not only for PLGA but also for other similar polymers.
Cell viability test
To evaluate the cell viability, HUVEC cells were cultivated on the surface of tissue culture plastic as a control and on the surface of irradiated matrices of PCL and PCL with 10% of gelatin in fiber. The cell viability was measured by cell viability reagent Alamar Blue (Life Technology production). It was noticed that the cells that were grown on the surface of the irradiated and nonirradiated matrices had no significance (Fig. 6).
CONCLUSIONS
Using electrospinning nonwoven scaffolds from Nylon 6, PCL and PLGA were fabricated. It was shown that EBI of the matrices produced from PCL by a dose of 100 kGy increases the proportional limit of the material and allowed us to introduce a durable re-
gion into the vascular grafts by irradiating the graft through the template with open areas. In contrast, EBI decreases the stability of PLGA scaffolds and could be used for increasing the degradation rate of polyester scaffolds. Electron beam irradiation was not shown to interfere with biocompatibility of scaffolds and could be used for scaffold sterilization.
ACKNOWLEDGMENTS
This work was supported by a Russian Science Foundation grant (№ 14-15-00493).
REFERENCES
1. Cherepanova A.V., Bushuev A.V., Vlassov V.V., Laktionov P.P. Cell-surface-bound DNA inhibits po-ly(I:C)-activated IL-6 and IL-8 production in human primary endothelial cells and fibroblasts // Circulating Nucleic Acids in Plasma and Serum. Ed. P.B. Gahan. Springer, 2011. 207-211.
2. Khorshidi S., Solouk A., Mirzadeh H. et al. A review of key challenges of electrospun scaffolds for tissue-engineering applications // J. Tissue Eng. Regen. Med. 2015. [Epub ahead of print].
3. Madurantakam P.A., Cost C.P., Simpson D.G., Bowlin G.L. Science of nanofibrous scaffold fabrica-
tion: strategies for next generation tissue-engineering scaffolds // Nanomedicine. 2009. 4. (2). 193-206.
4. Pham Q.P., Sharma U., Mikos A.G. Electro-spinning of polymeric nanofibers for tissue engineering applications: a review // Tissue Eng. 2006. 12. (5). 1197-1211.
5. Wu J., SoucekM.D., CakmakM. Effect of electron beam radiation on tensile and viscoelastic properties of styrenic block copolymers // Polym. Eng. Sci. 2014. 54. (12). 2979-2988.
ВЛИЯНИЕ ОБЛУЧЕНИЯ ПУЧКОМ ЭЛЕКТРОНОВ НА ФИЗИКО-ХИМИЧЕСКИЕ ХАРАКТЕРИСТИКИ МАТРИЦ, ИЗГОТОВЛЕННЫХ МЕТОДОМ ЭЛЕКТРОСПИННИНГА
Алёна Олеговна СТЕПАНОВА1'2', Михаил Васильевич КОРОБЕЙНИКОВ3, Александр Сергеевич ЮНОШЕВ4, Павел Петрович ЛАКТИОНОВ1,2
1 Новосибирский НИИ патологии кровообращения им. академика Е.Н. Мешалкина Минздрава России
630055, г. Новосибирск, ул. Речкуновская, 15
2 ФГБУНИнститут химической биологии и фундаментальной медицины СО РАН 630090, г. Новосибирск, пр. Академика Лаврентьева, 8
3 ФГБУН Институт ядерной физики им. Г.И. Будкера СО РАН 630090, г. Новосибирск, пр. Академика Лаврентьева, 11
4 ФГБУН Институт гидродинамики им. М.А. Лаврентьева СО РАН 630090, г. Новосибирск, пр. Академика Лаврентьева, 15
Исследовано влияние облучения пучком электронов на прочность, стабильность и биосовместимость матриц, изготовленных методом электроспиннинга, из нейлона 6, поликапролактона (PCL) и сополимера молочной и гликолевой кислот (PLGA). Показано, что облучение матриц электронным пучком, генерируемым ускорителем электронов ИЛУ-6 (2,2 МэВ, 400 мА, 10 Гц, ФГБУН Институт ядерной физики СО РАН) потенциально может быть использовано для их стерилизации (доза облучения 25 кГр не влияет на механические свойства всех исследованных матриц). Облучение матриц, изготовленных из PCL, в дозе 100 кГр увеличивает предел упругой деформации материала и позволяет ввести прочные участки в структуру протезов сосудов, облучая трансплантаты через шаблон с открытыми участками. Электронно-лучевое облучение матриц не влияет на их способность поддерживать адгезию и жизнеспособность первичных эндотелиальных клеток человека на поверхности этих матриц, как это было показано с помощью анализа на жизнеспособность Alamar Blue.
Ключевые слова: облучение пучком электронов, электроспиннинг, PCL, PLGA, Nylon 6.
Степанова А.О. - младший научный сотрудник лаборатории молекулярной медицины, лаборатории биомедицинских технологий центра новых технологий, e-mail: lebedeva@niboch.nsc.ru Коробейников М.В. - к.тех.н.
Юношев А. С. - к.ф.-м.н., зав. лабораторией высокоскоростных процессов отдела взрывных процессов. Лактионов П.П. - к.б.н., в.н.с., зав. лабораторией молекулярной медицины, зав. лабораторией биомедицинских технологий центра новых технологий, e-mail: lakt@niboch.nsc.ru
