Materials Science Forum Submitted: 2020-08-23

ISSN: 1662-9752, Vol. 1025, pp 185-190 Revised: 2020-10-27

doi:10.4028/www.scientific.net/MSF.1025.185 Accepted: 2020-10-27
© 2021 Trans Tech Publications Ltd, Switzerland Online: 2021-03-30

Biomineralization of Hydroxyethyl Cellulose/Sodium Alginate

for Bone Tissue Application
Etdal Bakhiet1,a, Nur Fatini Ilyana Mohamat Jauhari1,b
and Farah Hanani Zulkifli1,c *
1
Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Tun
Razak, 26300 Gambang, Kuantan, Pahang Darul Makmur, Malaysia
a
ebakhiet19@aol.com, bfatini96.fi@gmail.com, c,*farahhanani@ump.edu,my

Keywords: biomineralize, tissue engineering, biomaterial, calcium phosphate, simulated body

fluid.

Abstract. The aim of this research is to synthesis biopolymeric materials from hydroxyethyl
cellulose (HEC) (5 wt. %) blended with sodium alginate (SA) (10 wt. %) at 1:1 ratio fabricated by
using freeze-drying technique. The HES/SA was treated with simulated body fluid (SBF) by
immersion technique through the depositing of calcium phosphate on the scaffold’s surfaces. All
scaffolds were characterizing by using field emission electron microscope (FESEM), attenuated
total reflectance-Fourier infrared transform (ATR-FTIR), and thermogravimetric analysis (TGA).
The FESEM images results displayed interconnected porous structure with diameter ranging from
40 to 400 μm with average apatite diameter in range of 95 nm – 148 nm. The ATR-FTIR results
exhibit possible interactions between hydroxyl groups of HEC, SA and apatite groups of the
scaffolds. The TGA results showed four different regions of mass losses, represents the amorphous
transition temperature and water disposal, side-chain bond breaking, pyrolysis of SA and
dihydroxylation behaviour of calcium phosphate, respectively. Cell-scaffolds interaction
demonstrated that human fetal osteoblast (hFOB) cells differentiated and spread well on scaffolds
with better cell proliferation and attachment was more prominent on HEC/SA treated with SBF.
Since these biocompatible and biodegradable scaffolds showed promising results, these scaffolds
could be adopted for the design of next-generation tissue-engineered bone grafts.

Introduction
The emergence of tissue engineering has revolutionized the conventional autograft, allograft and
xenograft techniques in order to remain, restore or/and improve tissue functions. Numerous studies
have been done by utilizing the biopolymer materials that fabricated via various techniques such as
electrospinning, freeze-drying, solvent casting, phase template and many others. Through these
techniques, scaffolds characteristics can be modified to produce an ideal scaffold that possesses
good biodegradability, biocompatibility and mechanical properties for tissue regeneration [1].
Nowadays, bone graft tissue engineering has succeeded in bringing an alternative solution for
patients experience bone diseases including osteoporosis and bone fracture due to aging, trauma and
accidents. Many available bone grafts are currently available in market, however there are exhibit
several limitations such as high cost, environmental toxicity, inappropriate biodegradable rate, and
weak mechanical properties [2]. Furthermore, beside those properties, a good scaffold must implicit
a fit immune reaction to prevent inflammatory response that could reduce healing consumption and
cause rejection which are still lacking in many findings. Therefore, the development of new bone
tissue scaffold is vital by not only provide adequate mechanical properties but also great capacity
for vascularization in the body.
Many researches have confirmed that biocomposite materials could offer significant
improvements that can help to overcome some of the inherent of scaffold materials. Previous study
showed that, the use of hydroxyethyl cellulose improved the percentages of porosity but lowered
the mechanical strength [3]. However the addition of HEC with other polymers proved to boost the

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186 Industrial Science and Technology II

bioactivity, mechanical properties and helped in protein adsorption, thus increased the cell
availability and proliferation bone tissue engineering applications [4].
By considering, there are various parameters could be altered based on the targeted tissue
formation and functionality, former studies by Zhou et al. modified the collagen network surface,
coated with hydroxyapatite by biomimetic mineralization and resulted in the increment of the
mechanical strength [5]. The incorporation of hydroxyapatite was proved could enhance the
stability, however the optimized concentration remained indistinct and issue related with less
interconnected pore structure and porosity has limits the potential of fabricated scaffolds [6]. Due to
these gaps, a profound study is stimulated by taking the advantageous of the formation of calcium
phosphate by surface modification that may improve the biocompatibility and strength of scaffolds,
thus increase the potential as one of candidate as bone tissue grafts.

Materials and Methods

Raw materials. SA, HEC (Mw = 250,000) and glutaraldehyde (GA) solution (25 %) were
purchased from Merck-Schuchardt, Germany. Phosphate buffer saline (PBS) was purchased from
Gibco Life Technologies, USA and Dulbecco’s Modified Eagle Medium (DMEM) was purchased
from Life Technologies, USA. Human fetal osteoblast (hFOB) 1.19 (ATCC® CRL¬11372™),
SV4D large T-antigen transfected was supplied from American Type Culture Collection (ATCC),
USA for cell culture studies part. All chemicals were characterized by analytical purity and applied
without further treatment. All the solutions were prepared using Millipore water.
Synthesis of HEC/SA. Hydroxyethyl cellulose (HEC) solution of 5 wt% was prepared by
dissolving 5 g of HEC powder in 100 ml of Millipore water at room temperature, the solution was
then stirred for a period of 2 hours. Solution of sodium alginate (SA), 10 wt% was prepared by
dissolving 10 g of SA powder in 100 ml of Millipore water at room temperature; the solution was
then stirred and blended for 2 hours. HEC/SA blended solution was formulated by mixing HEC
with SA solution at weight ratios of HEC/SA, 1:1. Solution were stirred overnight to achieve
homogeneous solutions.
Freeze-drying. The basic principle of freeze-drying essentially involves the following phases, (i)
freezing, (ii) primary drying, and (iii) secondary drying. During the freezing process, the solution is
frozen at temperatures ranges between -80 to -50 °C, followed by a primary stage. In the primary
drying stage, the frozen samples undergo a sublimation process where the frozen water in the
materials sublimates by lowering the pressure and controlling the temperature. Subsequently, in the
secondary drying stage, the samples undergo an adsorption process to remove the bound water
molecules by increasing the temperature higher than in primary drying, thus breaking the bound
molecules.
Crosslinking process. The cross-linking process is a chemical and physical interaction which
link one polymer to another via a covalent or an ionic bond. A ratio of 24 ml, 4 ml and 0.4 ml of
acetone, glutaraldehyde, and phosphoric acid were used, respectively to carry out the cross-linking
process. The scaffolds were crosslinked via vaporization technique by placed both scaffolds and
crosslinker in the desiccator for 24 h. Thereafter, the immersion testing was done to ensure its
stability of scaffolds in water.
Surface modification with Simulated Body Fluid (SBF). The simulated body fluid was
prepared by the adding 900 ml of Millipore water to sodium chloride, potassium chloride, calcium
dehydrates, magnesium chloride hexahydrate and dihydrogen monohydrate phosphate with amount
of 58.448 g, 0.373 g, 3.575 g, 1.016 g, and 1.140 g, respectively. Lastly, 0.840 g of sodium
hydrogen carbonate were added to a total mixture of 1000ml. Scaffolds were immersed in SBF at
37 °C as early reported by Kokubo et al. (1990) and was carried out to obtain a coating of mineral
crystals on the surface of scaffolds [7]. Samples were immersed in SBF solution for up to 7 days in
the beaker. After day 7, the scaffold samples were isolated from the solution, rinsed with distilled
water and dehydrated in an oven at 40 °C until constant weight.
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Characterization technique. The morphological, chemical, and thermal analysis of the prepared
scaffolds were investigated by using different techniques of field emission scanning electron
microscopy (FESEM), attenuated reflectance transmission-Fourier transforms infrared spectroscopy
(ATR-FTIR) and thermogravimetric analysis (TGA), respectively. The in-vitro biocompatibility of
the materials is conducted through cell viability (staining) and cell proliferation analysis (MTT
assays) by utilizing the hFOB cells.

Results and Discussions

Scanning electron microscope. The morphological surface of scaffolds is one of the crucial
factors that influence the cell attachment, proliferation and differentiation. As shown in Fig. 1 (a)
exhibit interconnected microstructural with a lamellate shape morphology with diameter in a range
of 40 – 400 µm. The structure of morphological scaffolds was found to mimic the trabeculae of
spongy bone which contains bone cells like osteocyte, osteoblast, osteogenic cell and osteoclast.

Figure 1: SEM image of (a) HEC/SA, and (b) HEC/SA (SBF treated) at (i) low and (ii) high
magnification.

Fig. 1 b (i) and (ii) show the FESEM images after the scaffolds immersed in the SBF for 24
hours. From these images, we can observe the apatite layers were uniformly coated on the scaffolds
surface with average particle diameter is 95 nm- 148nm. It is worth mentioning, this zero-
dimensional spherical shape apatite particles increased the surface area of the scaffolds, and
provided more osteoinductive surface along with the formation of new osteoblasts and bone
ingrowth simultaneously. A sequence of biochemical interactions lead to the formation of calcium
phosphate or apatite layer on the scaffolds surface which can be attributed to the surface
modification during immersion by inducing both Ca2+ and ions on the surface of scaffolds [8]
Attenuated total reflectance – Fourier Transform Infrared. ATR-FTIR spectroscopic
technique have been used to determine the chemical interaction and bonding between the polymers
of scaffolds. Herein, the ATR-FTIR spectra of the investigated scaffolds are as showed in Fig. 2.
All spectra showed the characteristic O-H bands, within the range of 3340 cm-1 - 3365 cm-1.
However, it is worthy to note that the HEC/SA showed a slight shift to lower wave number (3365
cm-1) in comparison to HEC/SA treated with SBF which are 3340 cm-1. This could be due to the
development of hybrid nano scaffolds which possess higher hydrophilic properties as well as Ca2+
ions reaction with O-H. As stated, the greater intensity and shifting of -OH absorption band is
evident for the scaffolds without SBF process which suggest that the prepared composite scaffold
showed more hydrophilic behaviour.
188 Industrial Science and Technology II

Figure 2: FTIR spectra for (a) HEC/SA and (b) HEC/SA (SBF) scaffolds.
On the other hand, C-H assigned to methylene stretching vibrational group were shifted to higher
wave number for HEC/SA and HEC/SA (SBF), which were at 2824 cm-1 and 2924 cm-1,
respectively. The carbonyl (C=O) for scaffold without immersion in (SBF) has a higher wavelength
at1628 cm-1 if compared to scaffold without immersion was assigned to bending vibrations of
strongly adsorbed water.The C-O bands which appeared at wavelengths 799 and 882 cm-1 indicates
the presence of well crystallized hydroxyapatite contents which also suggests the occurrence of
chemical interactions between the scaffold components which ultimately influences the overall
physicochemical, mechanical properties, and in vitro bioactivity of the prepared scaffolds.
Thermogravimetric Analysis. TGA was done to investigate the thermal behaviour of the
prepared scaffolds. Fig.3 shows that the first region was attributed to the loss of moisture content as
well as the amorphous transition temperature with initiated temperature in range of 74 °C to 80 °C
which corresponded to 5 – 22 % weight loss. It is worth noting, HEC/SA (SBF) had affected the
low weight loss of percentage due to increase of -OH bonding between HEC and SA towards
apatite group structure. In the 2nd region, HEC/SA soaked in SBF showed major weight loss at 67%
and 45%, respectively, while HEC/SA shows corresponded weight loss to approximately 16 %.
This region was attributed to decomposition of polysaccharide network due to the breaking of C-H
bond with onset temperature in the range of 200 °C to 230 °C. Further weight loss was observed in
3rd decomposition region varying from 264°C to 350 °C with a weight loss percentage of up to 32%
(more prominent in HEC/SA) and therefore verified the existence of a chemical degradation process
resulting from bond scission (carbon-carbon bonds) in the polymeric bone of HEC. Minor weight
loss around 10% in scaffolds treated with SBF was attributed to the pyrolysis of calcium alginate
and depolymerization of cellulose [9]
(a) (b)
Weight loss (%)

Weight loss (%)

Temperature (°C)
Temperature (°C)

Figure 3: TGA graph for (a) HEC/SA and (b) HEC/SA (SBF) scaffolds.
 Materials Science Forum Vol. 1025 189

Cell-scaffold viability study. The MTT assay was carried out for 3 and 7 days and the
proliferation results are as in Fig. 4. All scaffolds show significant increase (p≤ 0.05) from day 3 to
day 7 due to the increase in the number of cell attachment and penetration through the porous
scaffolds. On day 3, all scaffolds presented positive cell-scaffold interaction with more prominent
results observed for HEC/SA (SBF) scaffold (absorbance index at 0.35). This adhesion and
differentiation of hFOB cells might be triggered by the calcium phosphate platform which matches
the alkaline phosphatase gene expressed by the cells. The biocompatibility of the cells-scaffold was
further confirmed by cells imaging (Fig. 5) in which the microplate contains cell was observed via
inverted light microscope. The results of the analysed cells indicated that the cell was well attached
with the scaffold which is a major sign of viable cells. All wells revealed the growth of bone cells
prolonged with time of 3 and 7 days incubation period with minor dead cells (dark blue) which was
clearly observed. The micrograph shown that cells extension with increase in spreading and
elongated morphology, signifying cells proliferation and indicating a major sign of viable cells.

Conclusion
The results reveal that the SEM images for scaffolds displayed interconnected porous structures
ranging from 40 to 400 μm. The immersion of scaffolds into SBF has depositing apatite layer on the
surface of scaffold with the particle size in range of 95 nm - 148nm. ATR-FTIR provided
information of chemical bonds, assigning significant fundamental frequencies of the group
-1
which appeared at wavelengths 799 and 882 cm and indicated the existence of well crystallized
hydroxyapatite contents which also suggests the occurrence of chemical interactions between the
scaffold. The TGA results showed four different regions of mass losses representing the amorphous
transition temperature and water disposal, (C-H) bond breaking, pyrolysis and dihydroxylation
behaviour of hydroxyapatite respectively for each stage. MTT assay that was carried out for 3 and 7
days indicated positive increment of absorbance index cell viability prolonged with times with more
prominent effect on HEC/SA (SBF) scaffolds. The immersion of HEC/SA in SBF is proven to be a
better option for synthesizing scaffolds for bone tissue engineering.

HEC/SA (3 days) HEC/SA (SBF) (3 days)

HEC/SA (7 days) HEC/SA (SBF) (7 days)

Figure 4: MTT assay for 3 and7 days for all Figure 5: Micrograph for 3 and 7 days
scaffolds. incubation for (a) HEC/SA and (b) HEC/SA
(SBF) at 400× magnification.

Acknowledgement
The authors are thankful to the Universiti Malaysia Pahang and The Ministry of Higher Education,
Malaysia for providing financial support from PGRS1903192 and RDU1901117
(FRGS/1/2019/WAB13/UMP/02/).
190 Industrial Science and Technology II

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