The journal of Nuclear Science and Technology - Volume 10, Number 3, September 2020

ISSN 1810-5408

Nuclear Science

and Technology

Volume 10, Number 3, September 2020

Published by

VIETNAM ATOMIC ENERGY SOCIETY

VIETNAM ATOMIC ENERGY INSTITUTE

NUCLEAR SCIENCE AND TECHNOLOGY

Volume 10, Number 3, September 2020

Editorial Board

Editor-in-chief

Tran Huu Phat (VINATOM)

Executive Editors

Vuong Huu Tan (VARANS)

Tran Chi Thanh (VINATOM)

Cao Dinh Thanh (VINATOM)

Hoang Anh Tuan (VAEA)

Editors

Nguyen Kien Cuong (VINATOM)

Nguyen Nhi Dien (VINATOM)

Nguyen Thi Kim Dung (VINATOM)

Ho Manh Dung (VINATOM)

Nguyen Nam Giang (VINATOM)

Trinh Van Giap (VINATOM)

Le Ngoc Ha (108 Military Central Hospital)

Phan Son Hai (VINATOM)

Le Hong Khiem (IOP)

Dao Tien Khoa (VINATOM)

Tran Hoai Nam (Duy Tan University)

Dang Duc Nhan (VINATOM)

Nguyen Hao Quang (VINATOM)

Nguyen Mong Sinh (VINATOM)

Tran Duc Thiep (IOP)

Dang Quang Thieu (VINATOM)

Le Ba Thuan (VINATOM)

Le Huy Ham (VAAS)

Nguyen Quoc Hien (VINATOM)

Le Van Hong (VINATOM)

Nguyen Trung Tinh (VARANS)

Tran Ngoc Toan (VINATOM)

Duong Thanh Tung (VINATOM)

Nguyen Nu Hoai Vi (VARANS)

Nguyen Tuan Khai (VARANS)

Pham Dinh Khang (HUST)

Science Secretary

Hoang Sy Than (VINATOM)

....................................................................................................................................................................................................................

(VINATOM).

Pusblished by

Vietnam Atomic Energy Society, 59 Ly Thuong Kiet, Hanoi, Vietnam

Tel: 84-24-39420463 Fax: 84-24-39424133 Email: [email protected]

Vietnam Atomic Energy Institute, 59 Ly Thuong Kiet, Hanoi, Vietnam

Tel: 84-24-39420463 Fax: 84-24-39422625 Email: [email protected]

....................................................................................................................................................................................................................

Contents

Simulation of atmospheric radiocesium (¹³⁷Cs) from Fukushima nuclear accident

using FLEXPART-WRF driven by ERA5 reanalysis data

Kieu Ngoc Dung, Nguyen Hao Quang, Hoang Huu Duc, Nguyen Thị Hang, Nguyen 01

Thi Thoa, Nguyen Quang Trung……………………………………………...……………..

Radiation-crosslinked scaffolds from gelatin/CM-chitin and gelatin/CM-chitosan

hydrogels for adipose-derived stem cell culture

Dang Van Phu, Nguyen Quoc Hien, Le Anh Quoc, Nguyen Ngoc Duy, Phan Phuoc 13

Thang, Nguyen Thi Ly, Nguyen Thi Kim Lan, Tran Le Bao Ha…………………………

The growth promotion effect of microelement fertilizers containing low molecular

weight chitosan and xanthan on radish (Raphanus sativus L.)

Tran Minh Quynh, Nguyen Van Binh, Duong Kim Thoa, Le Thi Minh Luong...……... 22

Comparison and evaluation for the dose distribution and physical characteristics

between two AAA and AXB algorithms in Eclipse v13.6 software on treatment

plans in regions heterogenous densities at 108 Military Central Hospital

Hoang Huu Thai, Nguyen Tien Đat, Nguyen Thi Van Anh, Pham Hong Lam, Bui 31

Duy Linh, Le Manh Duc, Pham Quang Trung……………………...……………………..

Design of a FPGA-based controller for power and period measurement in the start

range of Dalat Nuclear Research Reactor

Vo Van Tai, Nguyen Van Kien, Nguyen Nhi Dien, Trinh Dinh Hai, Le Van Diep...…… 41

Experimental investigation of hydrodynamic phenomena in vertical-upward

adiabatic two-phase flow conditions

Anh Tuan Dinh, Tuan Nam Pham, Tu Oanh Nguyen…………......……………………...

49

Nuclear Science and Technology, Vol.10, No. 3 (2020), pp. 01-12

Simulation of atmospheric radiocesium (¹³⁷Cs) from Fukushima

nuclear accident using FLEXPART-WRF driven by ERA5

reanalysis data

Kieu Ngoc Dung^1,*, Nguyen Hao Quang², Hoang Huu Duc³, Nguyen Thị Hang¹,

Nguyen Thi Thoa³, Nguyen Quang Trung⁴

¹National Committee for Search and Rescue of Vietnam, Long Bien, Ha Noi, Vietnam

²Vietnam Atomic Energy Institute, 59 Ly Thuong Kiet, Ha Noi, Vietnam

³Military Environmental Chemistry Institute, An Khanh, Hoai Duc, Ha Noi, Vietnam

⁴Vietnam Institute of Meteorology, Hydrology and Climate change, Ha Noi, Vietnam

*Corresponding author, E-mail: [email protected]

(Received 19 August 2020, accepted 18 November 2020)

Abstract: This study investigates short-range atmospheric transport of radiocesium (¹³⁷Cs) after

Fukushima nuclear accident using the Weather Research and Forecasting (WRF) model and the

Lagrangian particle dispersion FLEXPART-WRF model. The most up-to-date ERA5 reanalysis

dataset is used as initial and boundary condition for the WRF model for every hour. Four experiments

were carried out to examine the sensitivity of simulation results to micro-physics parameterizations in

the WRF model with two configured domains of 5 km and 1 km horizontal resolution. Compared with

observation at Futaba and Naraha station, all experiments reproduce reasonably the variation of ¹³⁷Cs

concentration from 11/03 to 26/03/2011. Statistical verification as shown in Taylor diagrams

highlights noticeable sensitivity of simulation results to different micro-physics choices. Three

configurations of the WRF model are also recommended for further study based on their better

performance among all.

Keywords: ¹³⁷Cs dispersion, Fukushima Daiichi nuclear power plant, FLEXPART–WRF model,

ERA5 reanalysis data, Futaba, Naraha.

The FDNPP consists of six units that

were strongly impacted by the earthquake and

tsunami, leading to a serious nuclear accident,

radioactive substances were released from the

plant area and released into soil, water and air

environments (Fig. 2). The most serious is that

radioactive materials are released into the air,

they will be spreaded under different weather

conditions and can be fell in continent and sea

areas that is very far from the accident site.

I. INTRODUCITON

The massive earthquake in Japan occurred

at 14:46 JST on 11/03/2011, with a magnitude of

9.0 [1] that caused heavy damage to infrastructure

along the east coast. It was followed by the

inundations of tsunami that caused power outages

and flooding in a large residential and industrial

area. This event had a major impact on five

nuclear power plants along Japan's northeast coast,

Higashidori, Onagawa, Fukushima Daiichi,

Fukushima Daini and Tokai Daini. Fig. 1 shows

the epicenter of the earthquake was far from

Fukushima Daiichi nuclear power plant (FDNPP)

180km in the northeast and Onagawa NPP 130km

in the east [2,3].

A number of computational models have

been used to study radioactive contamination

in the vicinity of the FDNPP from the

15/03/2011 [4], the regional simulation [5-8],

and global scale simulation [9-11]. The results

SIMULATION OF ATMOSPHERIC RADIOCESIUM (¹³⁷Cs) FROM FUKUSHIMA NUCLEAR ACCIDENT…

of these models are quite consistent in

reproducing high radioactive deposition at the

center of Fukushima prefecture. However,

these models have not yet accurately assessed

the radioactive matter deposition in the vicinity

of the factory, especially at radioactive

monitoring stations within a radius of 10 km.

Especially, the European Regional

Weather Forecast Center (ECMWF) provides

high resolution global forecasts with a

frequency of twice a day at 00 UTC and 12

UTC,

used

innovative

4D-Var

data

assimilation system with 91 different pressure

levels [16]. Recently, the ECMWF created a

new ERA5 reanalysis data with horizontal

resolution of 31 km and 137 different pressure

levels. In addition, the land surface and ocean

surface data are provided, including

Currently, the rapid development of

numerical dynamical weather model as well

as of particle dispersion models allows

high-resolution simulation of atmospheric

radionuclides [12]. An important factor for

these simulation is global meteorological

datasets driven the regional models.

precipitation, temperature at

atmospheric radiation [16].

2

m

and

In this study, the most up-to-date ERA5

reanalysis dataset will be used as initial and

boundary condition for a numerical weather

prediction model. Then, simulation output of

this weather model will force a particle

dispersion model, in order to simulate the

transport of radiocesium (¹³⁷Cs) after the

Fukushima nuclear accident. In addition,

different experiments are carried out to

evaluate the sensitivity of simulation results to

physics choices of the atmospheric model. The

sensitivity can be evaluated by discrepancies

among outputs of experiments, as analyzed in

the following sections.

Implementing

Multiscale

Global

model,

Environmental

the Canadian

Meteorological Center (CMC) provides

analysis data with horizontal resolution of

approximate 33 km and at 80 pressure levels

[13].

The

National

Center

for

Environmental Prediction (NCEP) runs the

global data assimilation system (GDAS)

four times per day (i.e. 00, 06, 12, and 18

UTC) to provide prediction issues [14]. The

UK Met Office gives a forecast data 6

hourly at horizontal resolution of 25km with

70 pressure levels [15].

(a)

(b)

Fig. 1. (a) The epicenter of the massive earthquake in Japan on 11/03/2011 [2] and (b) the satellite image of

the FDNPP site [3]

2

KIEU NGOC DUNG et al.

solver which implements flux-form equations

II. EXPERIMENT DESIGN

with variables that have conservation

properties and a terrain-following mass vertical

coordinate, is used in this study (Fig. 2). The

LPDM applied in this study is FLEXPART-

WRF model which is modified version of the

FLEXPART model to works with the WRF

model [12, 18]. The FLEXPART-WRF model

differs from its preceding versions in that it has

novel turbulence scheme for the convective

boundary layer [12].

In this study, the WRF model and a

Lagrangian particle dispersion model (LPDM)

are used to simulate short-range atmospheric

transport of radionuclides (i.e. ¹³⁷Cs). Figure 2

depicts the process to carry out experiments, in

form of a flow-chart. The WRF model is a

numerical dynamical atmospheric simulation

model, governed by compressible and non-

hydrostatic Euler equations [17]. The

Advanced Research WRF (ARW) dynamics

Fig. 2. Flow-chart of simulation processes in this study, implemented the WRF-ARW atmospheric model and

the FLEXPART-WRF dispersion model

(a)

(b)

Fig. 3. Simulation domains of the WRF model (a) with horizontal resolutions of 5 km (d01 – blue rectangle)

and 1 km (d02 – black rectangle) and (b) location of Futaba, Naraha station as well as Fukushima Daiichi

Nuclear Power Plant (FD1NPP)

3

SIMULATION OF ATMOSPHERIC RADIOCESIUM (¹³⁷Cs) FROM FUKUSHIMA NUCLEAR ACCIDENT…

The WRF model provides spatial and

temporal meteorological variables as forcing

for the FLEXPART-WRF to simulate the

spread of radionuclides. Two domains of the

WRF model are configured in this study with

horizontal resolutions of 05 km and 01 km

(Fig. 3.). Topography of the vicinity of the

FNPP is very complicated, with coastal lines

on the East and surrounded by mountains on

the West. The computational domain with a

high-resolution of 01 km inside the 100 km

vicinity of the plant (domain d02) and 05 km

for the outside of 100km region (domain d01)

is expected to reasonably capture the spead of

radiocesium plumes. The WRF model runs

with 51 vertical levels of the atmosphere and

04 soil layers of 10, 30, 60, 100 cm thick. The

ERA5 reanalysis data is used as initial and

boundary conditions for the WRF model with

hourly update timestep. Simulation time is

from 21:00 UTC on March 11, 2011 to 01:00

UTC on March 26, 2011.

to calculate the soil temperature and moisture

content in soil layers, taking into account snow

cover and freezing processes in the soil [19].

Yonsei University (YSU) planetary boundary

layer scheme [20], Dudhia short-wave

radiation scheme [21] and Rapid Radiative

Transfer Model (RRTM) long-wave radiation

scheme [22] are used in this research.

Radiation schemes are updated with time steps

of 5 minutes and 1 minute for 05 km and 01

km-resolution domains, respectively.

The sensitivity of the atmospheric

radionuclide simulation to different micro-

physics options will be investigated with

four experiments (Table I). Different

microphysical options will yield different

moisture variables, depending on the phase

transitions and interactions of water and ice

particles. The Kessler scheme is suitable for

a warm cloud consisting of water vapor,

water droplets and raindrops, and the other

processes including the generation, fall and

evaporation of raindrops [23]. The WSM 6-

class scheme includes snowfall and other

related processes and the phase transition of

ice [24]. This scheme is suitable for dealing

with grids that contain clouds while other

processes are similar to WSM 3-class

scheme [25]. Thompson’s scheme assumed

snow size distribution depends on both

temperature and ice water content and is

represented as a sum of exponential and

gamma distributions [26].

Physical processes are parameterized in

the WRF model include (1) micro-physics, (2)

cumulus parameterization, (3) planetary

boundary layer, (4) land surface model, and (5)

radiation

processes.

The

cumulus

parameterization is only valid for coarse grid

resolution (i.e. greater than 10 km) as

convective assumptions will be violated for

finer resolution. Therefore, in this study, the

convection parameterization schemes are not

activated. The Noah surface scheme was used

Table I. List of experiments in this study

Experiment

name

No.

Microphysics

Description

Reference

1

Exp 1

Kessler scheme

Warm rain (i.e. no ice or

idealized case)

Kessler (1969)

2

3

4

Exp 2

Exp 3

Exp 4

WRF Single-Moment (WSM)

3-class

Simple ice (3 arrays)

Hong et al., (2004)

WSM 6-class graupel scheme

Cloud scale, single moment (6

arrays, graupel)

Hong and Lim

(2006)

Thompson graupel scheme

Double moment (8-13 arrays)

Thompson et al.,

(2004)

4

KIEU NGOC DUNG et al.

In order to run a dispersion model, a

after the Fukushima accident are used [28]. In

addition to comparison on time-series plot and

map of concentrations, the Taylor diagram are

used to compare simulation results [29]. This

specific emission source is required to

indentify. The source term of the ¹³⁷Cs

radioactive nuclide released from the reactor

area by time and position is determined based

on the analysis report of Katata et al., (2015)

[27]. To evaluate simulation results, the time-

series analysis of atmospheric radiocesium at

two monitoring sites (i.e. Futuba and Naraha)

diagram provides

a

concise statistical

verification of how well simulation match

observation, in terms of Pearson’s correlation

coefficient, root-mean-square difference, and

the ratio of standard deviations [29].

10¹⁵

10¹⁴

10¹³

10¹²

10¹¹

KATATA

0

50

100 150 200 250 300 350 400 450 500

Time,h

Fig. 4. The source term of ¹³⁷Cs over the time after the accident,

retrieved from [27]

of wind barbs in Fig. 5a is thinned with factor

III. RESULTS AND DISCUSSIONS

A. Simulation of meteorological conditions

of 10. It means that the WRF model can

provide much more details of atmospheric

circulation over study area.

With coarse resolution of approximately

31 km, the ERA5 reanalysis data can not

reproduce meteorological variables over

complex terrain of Japanese region. The WRF

model can downscale dynamically to finner

mesh resolutions (i.e. 05 km and 01 km in this

study). Fig. 5 shows that the WRF model can

maintain well the spatial pattern of geo-

potential height over Japanese region from the

forcing ERA5 data. The high pressure system

located at the North of Japan as well as the

pressure gradient followed Northwest-

Southeast axis are reproduced well in the WRF

model (Fig. 5). It’s worthy to note that, for

facilitating “eyeball” verification, the number

Because precipitation is an important

factor for the wet deposition of radioactive

material in plumes, simulated precipitation

should be compared with observed data and

other previous published data. Fig. 6 presents

accumulated simulated precipitation from the

WRF model in Exp 1, from 09:00 to 15:00 on

March 15, 2011. Rainfall amount and intensity

in this case is highly similar to simulation

results from Katata et al., (2015) [27]. Heavy

rain, from 5 mm to 10 mm per 6 hours,

occurred over broad area around Fukushima

region. Due to the impacts of earthquake and

tsunami, almost all the meteorological

5

SIMULATION OF ATMOSPHERIC RADIOCESIUM (¹³⁷Cs) FROM FUKUSHIMA NUCLEAR ACCIDENT…

observation equipments were inoperable after

the nuclear accident. Therefore, it’s difficult to

obtain good quality meteorological observation

in this case. Large-scale meteorological

information during the occurrence of

radioactive material emissions into the air was

presented in the 2013 World Meteorological

Organization (WMO) report by previous

research groups such as Kinoshita et al., (2011)

[30], Stohl et al., (2012) [10] and Sugiyama et

al., (2012) [31]. In fact, rain occured over the

north area of Fukushima prefecture from 17:00

JST March 15 to 04:00 JST on the March 16

[30]. On the 20 to 22 March, sustainable low

pressure caused moderate rainfall in the

vicinity of Tokyo.

(a)

(b)

Fig. 5. Simulation of geo-potential height (shaded color) and wind field (barbs) on level of 850 mb, at 12h00

UTC 15/03/2011 from the WRF model in Exp 1 (a), in comparison with the ERA5 reanalysis data (b).

Notice: Factor of 10 is applied to thin the number of wind barbs in (a)

Fig. 6. Accumulated simulated precipitation from the WRF model in experiment Exp 1, from 09:00 to 15:00

on March 15, 2011

6

KIEU NGOC DUNG et al.

resolution. In this paper, high resolution of

B. Evaluation of atmospheric radiocesium

01 km is very suitable for considering the

geographical location of Futaba station, as

well as other neighboring stations (e.g.

Naraha station). Calculation results of the

concentration of atmospheric radiocesium

¹³⁷Cs for every hour at Futaba and Naraha

station are displayed in Fig. 7 and Fig. 8,

respectively. The observation data displayed

in these figures are retrieved from Tsuruta et

al., (2011) [28].

The Futaba station, an observatory

station in the town of Futaba, is very close to

the Fukushima NPP. The distance between

Futaba town and the plant is only about 3.2

km, where was severely affected by both

earthquakes, tsunamis and the effects of

radiation [28]. For the other researches of

global radioactive dispersions, the vicinity

areas of the plant are often not taken into

account, because of the limitation of the grid

Fig. 7. Hourly accumulated concentration of ¹³⁷Cs at Futaba station from observation (red dashed line) and

Exp 4 (blue solid line) with range of simulation results from all experiment (shaded light blue)

From Fig. 7 and Fig. 8, it can be seen

that simulation results have a good agreement

with the observed data, especially from 12 to

14/03/2011 at Futaba station and from 15 to

16/03/2011 at Naraha station. Peak values of

¹³⁷Cs concentration on 12 and 19/03/2011 at

Futaba station are reproduced well in all

experiments. Peak values on 15, 16 and

19/03/2011 at Naraha station are also captured

well by the FLEXPART-WRF model. The

range of simulated values from four

experiment (i.e. shaded light blue area in

Fig.7 and Fig. 8) can be recorgnized,

especially for concentrations of less than 10²

Bq.m^-3per hour. The uncertainty in simulation

or the sensitivity of calculation results to

different micro-physics option is more clear in

the case of Futaba station than in Naraha

station. This can be seen on simulated range

of 13-14/03/2011 and 19-21/03/2011 in Fig.

7. The Exp 4 was displayed due its better

performance, in comparison with others

experiments, which is confirmed by statistical

verification shown in Fig. 9.

7

SIMULATION OF ATMOSPHERIC RADIOCESIUM (¹³⁷Cs) FROM FUKUSHIMA NUCLEAR ACCIDENT…

Fig. 8. Hourly accumulated concentration of ¹³⁷Cs at Naraha station from observation (red dashed line) and

Exp 4 (blue solid line) with range of simulation results from all experiment (shaded light blue)

(a)

(b)

Fig. 9. Taylor diagram compares simulation results from 04 experiments using Pearson correlation

coefficient and Normalized Standard Deviation for (a) Futaba and (b) Naraha station. Observation value is

depicted by black star

Fig. 9 demonstrates high sensitivity of

simulation results to different micro-physics

options of the WRF model. Scatter of

experiment’s points on the Taylor diagram

highlights the significant variations of not

only correlation coefficients (CC) but also

standard deviations (σ) of simulated

atmospheric radiocesium retrieved from

four experiments. For example, at Futaba

station, simulation result from experiment

Exp 1 has CC value of 0.28 and normalized

σ of 0.48. While respective verification

metrics for Exp 4 are 0.77 and 0.36 which

means better capture of hourly observed

8

KIEU NGOC DUNG et al.

release of ¹³⁷Cs air concentration. At Naraha

maps explain the peak of concentration

shown in Fig. 7 and Fig. 8. At level of 100

m, atmospheric radionuclide propagated to

the North on 12/03/2011 which plumes

station, the higher CC values can be seen, in

comparison with simulation results at

station Futaba (i.e. 0.92 for Exp 4 or 0.89

for Exp 1). Based on this Taylor diagram,

spreaded

widely

to

Southwest

on

the experiment Exp

3

show worse

15/03/2011. Smaller plumes in both area

and intensity blowed along coastal line to

the South are simulated on 19/03/2011.

These results show a similarity to the results

of Tsuyoshi et al., (2015) [1] in which

different horizontal grid resolutions are used

to calculate radioactivity concentration on

15/03/2011.

simulation results than Exp 1, Exp 2 and

Exp 4. Therefore, the configuration of Exp

1, Exp 2 or Exp 4 can be recommended for

further study in the future.

From Fig. 10, dispersion plume of

137Cs concentration at 100 m can be seen

for three different days. These distrubition

(a)

(b)

(c)

Fig. 10. Local-scale spatial distributions of accumulated concentrations of 137Cs at 100 meter from Exp 4

retrieved (a) from 00 UTC 12 to 00 UTC 13/03/2011, (b) from 00 UTC 15 to 00 UTC 16/03/2011 and (c)

from 00 UTC 19 to 00 UTC 20/03/2011. Unit: Bq.m-3

configured with two domains of 05 km and 01

km. Both meteorological conditions and

dispersion of atmospheric radiocesium (¹³⁷Cs)

are evaluated. In comparison with observation

at Futaba and Naraha station, all experiments

captured reasonably the variation of ¹³⁷Cs

concentration from 11/03 to 26/03/2011.

Analysis on Taylor diagram confirm the

noticeable sensitivity of simulation results to

four selected micro-physics parameterizations.

The configurations of Exp 1, Exp 2 and Exp 4

IV. CONCLUSIONS

This study investigates short-range

atmospheric transport of radionuclides after

Fukushima nuclear accident using a numerical

weather model and a Lagrangian particle

dispersion model. Four different experiments

were carried out using the FLEXPART-WRF

model coupled with the WRF model. The

ERA5 reanalysis data is used as initial and

boundary conditions for the WRF model with

hourly update time step. The WRF model is

9

SIMULATION OF ATMOSPHERIC RADIOCESIUM (¹³⁷Cs) FROM FUKUSHIMA NUCLEAR ACCIDENT…

[5]. Morino et al., “Atmospheric behavior,

deposition, and budget of radioactive materials

from the Fukushima Daiichi nuclear power

plant in March 2011”, Geophysical Research

Letters, VOL. 38, L00G11, 2011.

are recommended for further study due to their

better performance among all.

ACKNOWLEDGEMENT

This article is supported by the project

titled “Study the effects of the floating nuclear

power plant on the sea and the nuclear power

plants on Hainan Island on Vietnam’s marine

environment by modelling and developing

response plans for a nuclear accident occurred

over the sea”, project code: KC.AT, in the

framework of technical research program,

nuclear safety to ensure combat readiness for

the Army in the 2016-2020 period. This article

is also supported by the project titled “Study

and calculating the spread of atmospheric

radionuclides and modernization of stations

for radioactive analysis” (PACT-1).

[6]. Yasunari et al., “Cesium-137 deposition and

contamination of Japanese soils due to the

Fukushima

nuclear

accident”,

PNAS

December 6, 108 (49) 19530-19534, 2011.

[7]. Katata et al., “Atmospheric discharge and

dispersion of radionuclides during the Fukushima

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Source term estimation and local-scale

atmospheric dispersion in early phase of the

accident”,

Journal

of

Environmental

Radioactivity 109, 103-113, 2012.

[8]. Le Petit et al., “Analysis of radionuclide

releases from the Fukushima Dai-ichi nuclear

power plant accident part I”, Pure Appl.

Geophys. , 2012.

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12

Nuclear Science and Technology, Vol.10, No. 3 (2020), pp. 13-21

Radiation-crosslinked scaffolds from gelatin/CM-chitin and

gelatin/CM-chitosan hydrogels for adipose-derived stem cell culture

Dang Van Phu^1,*, Nguyen Quoc Hien¹, Le Anh Quoc², Nguyen Ngoc Duy¹, Phan Phuoc Thang¹,

Nguyen Thi Ly¹, Nguyen Thi Kim Lan¹, Tran Le Bao Ha²

¹Research and Development Center for Radiation Technology, Vietnam Atomic Energy Institute,

202A, Street 11, Linh Xuan Ward, Thu Duc District, Ho Chi Minh City

²Faculty of Biology and Biotechnology, University of Science, Vietnam National University Ho Chi Minh City,

227-Nguyen Van Cu, Ward 4, District 5, Ho Chi Minh City

Email: [email protected]

(Received 06 October 2020, accepted 30 October 2020)

Abstract: Radiation technology has been providing a useful tool to modify polymers involving the

radiation crosslinking of hydrogels. In the present work, two kinds of hydrogels composed gelatin and

carboxymethyl chitosan (gelatin/CM-chitosan), and carboxymethyl chitin (gelatin/CM-chitin) were

prepared by the radiation crosslinking. The resulting hydrogels were freeze-dried, sterilized under

gamma-ray at a sterilization dose of 25 kGy, and characterized to be utilized as the scaffolds for

culturing adipose-derived stem cells (hADSCs). The physicochemical properties, biodegradability,

and cytotoxicity of these scaffolds were also investigated. The results indicated that both CM-chitosan

and CM-chitin much improved the swelling capacity, porosity, and pore size of the gelatin-derived

scaffolds. The swelling degree of gelatin/CM-chitosan and gelatin/CM-chitin scaffolds was about 7-9

g/g after 6 hrs immerging in PBS, and their porosity was about 70-73% with pore size ranging from

100 to 300 mm. The results also revealed that the compressive modulus of gelatin/CM-chitosan and

gelatin/CM-chitin was 45.6 and 66.4 kPa, respectively, which were suitable for soft tissue engineering.

Both scaffolds were enzymatically biodegradable by collagenase and non-cytotoxic for hADSCs with

an RGR of ~97%. Thus, the resultant scaffolds can be suitably utilized for culturing hADSC in

practice to regenerate soft tissues.

practice, the hydrogel-scaffold materials in

tissue engineering ought to meet several design

criteria to mimic ECM, which is dependent on

the cultured source cells and the targeting

tissues. Alternatively, in the soft tissue-

regeneration technique from the adult

mesenchymal stem cells, a biomaterial scaffold

should be owned to the functional

specifications as follows: the hydrophilic 3D-

microporous structure, porosity >70% and pore

size ~100 – 300 m, swelling degree ~6 – 12

g/g, compressive strength ~1 – 80 kPa,

admissible biodegradability, non-cytotoxicity,

and providing a good platform for cell

adhesion, function, differentiation, and

transplantation [3, 5-8]. In the last couple of

years, the human adipose-derived stem cells

I. INTRODUCTION

Tissue engineering is a quickly enlarging

interdisciplinary field that attempts to provide

solutions for the regeneration of defected or

damaged tissues. Designing of biologically

active scaffolds with optimal characteristics is

one of the most important factors for

successful tissue engineering [1]. Recently,

hydrogel has been acquired substantial

attraction as an immense potential material for

engineered tissue scaffolds due to their unique

compositional and structural similarities to the

natural extracellular matrix (ECM), which is an

indispensable component to ensure the survival,

communication,

proliferation,

and

differentiation of the cells in tissues [2-4]. In

RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN…

(hADSCs), one exciting kind of the adult

gelatin on the features of hybrid scaffolds are

still lacking. This work characterizes the

scaffolds derived from gelatin/CM-chitosan

and gelatin/CM-chitin hydrogels obtained by

radiation-crosslinking in combination with

freeze-drying and radiation sterilization for

further applications.

mesenchymal stem cell sources copiously

found in the perivascular area of adipose

tissues, have been examined for application in

regeneration-tissue therapy due to their

abundance, multipotent nature, facile isolation,

and non-controversiality [7, 9]. In terms of

conventional technology, the polymer-based

mesenchymal stem cell culture scaffolds are

hydrophilic hydrogels in the form with a 3D

microporous structure, which were prepared by

chemical crosslinking [3, 8, 9], ionizing

radiation followed by freeze-drying treatment

[2, 10-12]. Among them, the radiation-

crosslinking approach is recognized as a

potential way with some advantages such as no

chemicals to initiate the reaction and the

product sterilized at the same time [1, 13]. The

crosslinking reaction in polymer solution

occurred through the recombination of free

radical macromolecules, which are produced

by the indirect and direct effect of radiation

interaction [10, 12]. The radiation-crosslinked

gelatin is a promising cell culture scaffold due

to its chemical resemblances to the ECM [11].

Despite the advantages, poorly mechanical and

physicochemical properties, and enzymatic

degradation beyond controllability have

restricted its applications and levered the

production of gelatin-based composite

hydrogels [11, 12]. Because of the many

inherent useful bio-properties of CM-chitosan

and CM-chitin, natural polysaccharide

derivatives of modified chitosan/chitin, it has

been considered as the leading materials to

make hydrogels for biomedical applications.

So far, several novel hybrid scaffolds have

been developed from a combination of

gelatin/CM-chitosan [2, 11, 12] as well as

gelatin/CM-chitin [4, 5] for the adult

mesenchymal stem cell tissue engineering.

However, studies on the effect of CM-

chitosan and CM-chitin in mixture with

II. MATERIALS AND METHODS

A. Materials

Three polymers including gelatin (CAS

9000-70-8, type A, source from porcine skin

with bloom ~300, an average molecular weight

~100.000 Da, and pI 8-9), carboxymethyl-

chitosan (CAS 83512-85-0, a deacetylation

degree of ~95%, a molecular weight ~60.000

Da, and a substitute degree of 0.87), and

carboxymethyl-chitin (CAS 52108-64-2, a

deacetylation degree of ~95%, a molecular

weight ~110.000 Da, and a substitute degree of

0.62) were purchased from Sigma-Aldrich,

Merck, Germany. PBS buffer (pH 7.4) was

supplied by Elabscience, USA. The tetrazolium

dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide was bought from

Sigma Aldrich, USA. Dulbecco’s Modified

Eagle’s Medium (DMEM)/F12 cell culture

medium was purchased from Gibco Co., USA.

The collagenase enzyme, type I (activity 2

U/ml) was a product of Sigma Aldrich, USA.

Human adipose-derived stem cells (hADSCs)

in the 4^thgeneration were provided by the

Faculty of Biology and Biotechnology,

University of Science – Vietnam National

University Ho Chi Minh City.

B. Methods

Preparation and sterilization of

scaffolds: The solutions with a total polymer

concentration 15% (w/v) of gelatin and both

composites of gelatin/carboxymethyl-chitosan

(G/CM-chitosan) and gelatin/carboxymethyl-

14

DANG VAN PHU et al.

chitin (G/CM-chitin) with the weight ratio of

magnification of 1000 for post sputter-coated

samples. The pore sizes and morphological

shapes of porous scaffolds were also

determined using SEM images. The porosity of

the scaffolds was measured by the liquid

supplanted method [2, 3]. Briefly, the porosity

is conventionally equal to the volume of

trapped ethanol in the porous scaffolds. The

scaffold samples were put into a measuring

cylinder containing an exact volume of

absolute ethanol (V1). After 24 h of soaking,

read the mixture volume of ethanol with

immersed scaffolds (V2). Afterward, the

scaffold samples were taken out and the

residual ethanol volume was determined (V3).

The porosity (P, %) was calculated by the Eq.

2 as follows [2]:

8.5/1.5 were prepared by completely dissolving

in distilled water at 50C for 2 h. The solutions

were poured into Petri dishes (diameter 10 cm,

thickness 1 cm) and cooled to solidify in

pseudo-gel at room temperature. The pseudo-

gels were irradiated at a dose of ~35 kGy to

obtain hydrogels with crosslinked bonds [10,

11]. The hydrogels were cut into cubic

specimens 101010 mm³in size and

lyophilized (freeze-drying) to create porous

scaffolds [5]. Afterward, the porous scaffolds

were subjected to -irradiation with ~25 kGy

for sterilization [1].

Swelling in PBS and mechanical

property of scaffolds: Swelling behavior of

porous scaffolds in PBS (0.1 M, pH 7.4) at

37C as a function of immersion-time was

P (%) = (V1 − V3) × 100/(V2 − V3)

(2)

evaluated by

calculated using Eq. (1) as follows [3, 13]:

a

weighting method and

Biodegradation and cytotoxicity tests:

Enzymatic degradation of the scaffolds was

evaluated through the weight loss after

immerging in PBS solution containing

colagenase. Briefly, scaffold sample of about

40 – 60 mg (Wo) was incubated in 5 ml

enzyme solution, which was prepared by

diluting stock collagenase in PBS (0.1 M, pH

7.4) to an activity concentration of 2 U/ml. The

biodegradation of scaffolds was carried out in

vitro at 37C for a period of 21 h. At destined

time interludes, the remainder of the samples

was removed to dry by freeze-drying and

weighed (W1). The biodegradability was

figured out using Eq. 3 [3, 11].

Swelling degree (g/g) = Ws/Wd

(1)

Where Ws and Wd are respectively the

weights of swollen scaffolds and dried

scaffolds.

The compressive modulus, a parameter

representing the mechanical property of

scaffolds, was measured using a texture tester

(INSTRON 5582, USA) equipped with a 1000

N load cell. To determine the compress

modulus, scaffolds were swollen in PBS for 24

h and cut into cylinders (diameter 10 mm,

height 10 mm) and applied a constant stain rate

of 60 mm/min [5, 11].

Biodegradability (%) = (Wo − W1) ×100/Wo

(3)

Porosity and pore size determination:

The porous scaffolds in the dry state were cut

into cubic pieces with the sizes of 101010

mm³for measuring porosity. Microscopic

structural morphology of the scaffold was

observed by the scanning electron microscopy

(S-4800, HITACHI, Japan) with an

Cytotoxicity of the scaffolds was

appraised by an indirect contact method

through extracts and MTT assay according to

ISO 10993-5:2009 [11]. The scaffold

extraction was prepared by incubating the

scaffolds in the DMEM/F12 medium

containing 10% FBS and 1% specific antibody

accelerating voltage of 10 kV and

a

15

RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN…

with a ratio of 3 cm²/ml at 37C for 24 h, then

centrifuged to obtain extraction media. The

hADSCs with a density of 10⁵cells/ml were

incubated into the 96-well culture plate at 37C,

5% CO₂for 24 h. After that, the culture

medium in the cell-incubated wells was

replaced with the extraction media and then

incubated for 24 h. The cells cultured with the

DMEM/F12 medium set out as the negative

control group and latex rubber extraction that

was a cytotoxic compound was as a positive

control group [9]. The relative growth rate

(RGR, %) of the cells in treatments was

determined using the MTT assay at 24 h and

calculated according to Eq. 4 [4]. The

absorbance of MTT-dyed cell suspensions was

measured at the wavelength of 570 nm using a

spectroscopy equipped microplate reader.

penetration, the infiltration of culturing cells,

and the excretion of secretions [3]. Fig. 1

describes the swelling of the porous scaffolds

in the PBS solution. The swelling degree of

the gelatin scaffold was the lowest (about 4.8

g/g) in comparison to the G/CM-chitin (about

7.0 g/g) and the G/CM-chitosan (around 9.1

g/g). These differences may be caused by

inconformity in crosslinking density and the

number of hydrophilic groups in the scaffold,

particularly the lower the crosslinking density

and the higher the hydrophilic groups, the

higher the swelling degree. Yang et al. [11]

substantiated that CM-chitosan in a mixture of

gelatin/CM-chitosan was able to improve the

swelling degree of irradiation-crosslinked

gelatin hydrogels based on reducing the

crosslinking density and increasing the

hydrophilic groups. In addition, Zhao et al.

[13] supported that the irradiation-crosslinked

extent of CM-chitin was higher than that of

CM-chitosan, which could also be the basis

for the explanation of the difference in

swelling of the G/CM-chitosan and G/CM-

chitin (Fig. 1).

RGR (%) = As ×100/Anc

(4)

Where As and Anc are the optical

density of the test sample and the negative

control, respectively. The samples with an

RGR value higher than 70% are considered

non-cytotoxicity or quality [4].

In the more notable worth, the CM-

chitosan and CM-chitin not only ameliorated

to the swelling proportion but also improved

the mechanical property of the resulted

scaffolds from G/CM-chitosan and G/CM-

chitin. The compressive modulus or stiffness

of scaffolds plays a significant role in the

regulation of stem cell fate as well as

adhesion, migration, and differentiation. The

crosslinking density of the hydrogels is the

main element to affect scaffold stiffness [6].

Fig. 2 shows the compressive modulus of the

prepared scaffolds. The attained results

indicated that the compressive modulus of the

gelatin scaffold (about 167.5 kPa) was

effectively diminished to 45.6 kPa and 66.4

kPa for G/CM-chitosan and G/CM-chitin

scaffolds, respectively. The stiffness of the

III. RESULTS AND DISCUSSION

In these experiments, the polymeric

formulation and the radiation-crosslinking dose

were applied based on the findings as reported

in the previous paper [10]. And the porous

scaffolds from gelatin, gelatin/CM-chitin and

gelatin/CM-chitosan hydrogels prepared by

radiation-crosslinking in combination with

freeze-drying

and

gamma

irradiation

sterilization were characterized.

A. Swelling in PBS and mechanical property

of scaffolds:

The swelling behavior and compressive

modulus of scaffolds are the principal

parameters. The swelling ability of scaffolds

is closely relative to the essential nutrition

16

DANG VAN PHU et al.

G/CM-chitosan and G/CM-chitin scaffolds

requirements in the soft tissue engineering (10

was believed to be appropriate for the scaffold

– 75 kPa in compressive modulus) [6, 11].

10

8

200

1. Gelatin

160

2. G/CM-chitosan

3. G/CM-chitin

120

6

4

80

40

0

G/CM-chitosan

G/CM-chitin

Gelatin

2

0

2

6

1

0

4

8

10 12

2

3

Time, h

Scaffolds

Fig. 1. Swelling degree of scaffolds in phosphate

Fig. 2. Compressive modulus of

buffer solution as a function time

the different scaffolds

B. Porosity and pore size of scaffolds:

Table I. The porosity and pore size of the scaffolds.

Scaffolds

Porosity, %

50.0^b 3.7

73.3^a 4.2

70.6^a 5.8

Pore size, m

300 – 450

Gelatin

G/CM-chitosan

G/CM-chitin

100 – 250

120 – 300

In a column, the means followed by the same letter are not different significantly (P <0.05).

The scaffolds with high porosity and

adequate pore size are required to provide a

microenvironment for cell-cell and cell-

surrounding interactions. The interactions

influence an attachment, migration, and

penetration of cellular in-growth as well as

pore size in the range of 100 – 300 m

with thin pore walls and abundance of

interconnects. Furthermore, these scaffolds

had a significantly higher porosity (around

70 – 73%) than that of the gelatin one

(about 50%). From these results, it was

perspicuous that CM-chitin and CM-

chitosan in blend with gelatin had a

vascularization

in

them

[8].

The

microstructural properties, ascertained

from SEM images such as the pore sizes

and porosity of the produced scaffolds,

were audibly manifested in Table 1 and Fig.

3. The SEM images revealed that the

gelatin scaffold possessed a large pore size

(300 – 450 m), thick walls, and almost no

interconnecting channels among the pores.

Meanwhile, both the scaffolds of the

G/CM-chitosan and G/CM-chitin owned a

satisfactory

effect

on

the

porous

microstructural properties of the gelatin

scaffold, which could also be mainly due to

the change in a crosslinking behavior.

More specifically, the porous properties of

G/CM-chitosan and G/CM-chitin scaffolds

were adequately assumed to culture the

hADSCs

and

generate

soft

tissue

engineering [3, 5, 7].

17