Volume 7, Issue 2, February – 2022 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

A Study on the Crystallization of Hydroxyapatite on

Hydroxyethyl Cellulose Sponges
[1]Fathima Shahithaa, [2] Ang Pei Xinb, [3]Mohammed Al-Sibania,
[4] Ab. Rahim, Mohd Hasbib, [5] Ahmed Al Harrasic, [6]Asiya Obaid Abdallah Al Saadia

The College of Arts and Sciences, Department of Biological Sciences and Chemistry, University of Nizwa Initial Campus, Birkat
Al Mouz, P.O. Box 33, PC 616, Nizwa, Sultanate of Oman, bFaculty of Industrial Sciences & Technology, Universiti Malaysia
Pahang, Lebuhraya Tun, Razak, 26300 Gambang, Kuantan, Pahang, Malaysia, bNatural and Medical Sciences Research Center,
University of Nizwa Initial Campus, Birkat Al Mouz, P.O. Box 33, PC 616, Nizwa, Sultanate of Oman.

Abstract:- In this research, highly porous hydroxyethyl other bone transport methods. These are some of the common
cellulose (HEC) sponges with different concentrations standard treatments, but they are having some inherent
were prepared and cross linked by which the HEC sponges limitation as well. For instance, the defect size and viability of
were changed from soluble into insoluble from in water the host bed will affect the bone grafting as it is avascular and
which also enhanced its biodegradability. After the cross- dependent on diffusion. Besides, it may be problematic
linking process, hydroxyapatite (HA) crystals were formed especially in large defects as the body may reabsorb the grafts
on the HEC sponges through the crystallization process by before the osteogenesis is completed. For vascularized bone
using 20X simulated body fluid solution (SBF) solution, transplant, highly specialized technical expertise is needed for
and the product was characterized by using FTIR, optical this sophisticated operation, the operating time needs to be
microscopy, FESEM with EDX, and XRD. From the FTIR prolonged, and the bone transplanted may be not large enough
results obtained, the presence of acetal group at the to match the big defect of the bone for immediate
wavenumbers around 1022.20-1025.83 cm-1 had showed biomechanical stability (Philips and Nather, 2001).
the cross-linking process was succeeded. FTIR results after Furthermore, the donor tissue for autografts is always scarce,
the crystallization showed peak of PO43- and CO32- which and significant morbidity of the donor site which usually
indicate the presence of HA crystals. This result was associated with infection, hematoma and pain may be occurred.
confirmed by using XRD and FESEM-EDX. There were
extra peaks shown in the sample after crystallization Hence, bone tissue engineering is developed and applied
compared to the sample before crystallization for the XRD as an alternative way for the traditional bone replacement
analysis. This can proved the existence of the HA crystals. method (Choong et al., 2011). The highly porous scaffold
Further confirmation was done by using FESEM-EDX, biomaterial is mainly used in the tissue engineering.
and the element composition of HA crystals was revealed, Biomaterial can be defined as implanted materials which can
which consisted of C, O, P and Ca. The morphology and replace or treat any tissues, organ or function of our body
the size of the HA crystals was determined by using (Hollinger, 2012). It is used as a temporary matrix for the
FESEM. From the results obtained, it showed that HA growth of bone which offers a specific environment and
crystals synthesized were in round shape, and the size of structure for tissue development. Tissue engineering has
the HA crystals increased when the concentration of the utilized the growth factors, scaffolds and mesenchymal stem
HEC sponges increased. This is due to the polar hydroxyl cells for the bone regeneration process. Bone scaffolds
side groups of the HEC molecules tend to interact with produced with various compositions and structures has been
Ca2+ during the HA crystallization process. The developed successfully by scientists in different methods
hydroxyapatite coated HEC sponges were highly stable in (Matassi et al., 2011). The scaffold will combine with the body
water and can be a promising scaffold material. cell to help in regenerating the damaged tissues (O'Brien,
2011).
Keywords:- Hydroxyethyl cellulose, biomaterial,
hydroxyapatite, freeze drying, simulated body fluid Hydroxyappatite (HA) with the chemical formula of
Ca10(PO4)6(OH)2 is acted as the most significant biomineral
I. INTRODUCTION and inorganic component in the bone tissue of the vertebrates
(Chen et al., 2012). The high resemblance of HA with the
Nowadays, intense research is being carried out to inorganic component of bone matrix has encouraged the
develop scaffolds for tissue engineering in an environmentally researchers to study on the synthetic HA. HA has high
friendly way by green chemistry approach. According to Burg biocompatibility with the soft tissues like gums, muscle and
et al. (2000), the development of bone tissue engineering as an skin. This characteristic makes it to be a suitable material for
alternative has brought some advantages compared to the the orthopedic and dental implants compared to traditional
traditional ways of bone replacement such as allografts and allografts and metallic implants. Synthetic HA has been
autografts, vascularised grafts of the fibula or iliac crest and applied widely in the repairment and augmentation of bone,

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Volume 7, Issue 2, February – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
other than as the implant’s coating and fillers in bone or teeth II. EXPERIMENTAL SECTION
(Zhou and Lee, 2011).
A. Materials
Bones and teeth are basically made up of HA and Hydroxyethyl cellulose (HEC) was purchased from
collagen fibers, which are organic/inorganic composites. Nacalai Tesque, Japan. Glutaraldehyde, grade II, 25% in H2O
Therefore, in bone tissue engineering, HA/polymer was purchased from Sigma-Aldrich, USA. Phosphoric acid
composites are applied extensively as they are chemically was purchased from R&M Chemical, UK. Analytical reagent
similar to the natural bone. The interaction and bonding grade acetone was purchased from Bendosen. Among the
between inorganic and organic phases have to be strong in chemicals used for preparing the SBF solution, sodium
order to possess a good mechanical property. In the living chloride (NaCl) was purchased from Merck KGaA, Germany,
organisms, the bone cell and tissues starting from nano-blocks calcium chloride dehydrate, (CaCl2.2H2O) was purchased
can combine to form self-assembled biomaterials under a from Fisher Chemical, UK, sodium bicarbonate (NaHCO3),
controlled organic matrix. Therefore, in the process of potassium chloride (KCl), magnesium chloride hexahydrate
synthesizing the synthetic HA, a matrix mediated (MgCl2.6H2O) and sodium dihydrogen phosphate
mineralization is very important as it can mimic the structure (NaH2PO4.H2O) were purchased from Sigma-Aldrich, USA.
of the natural bone and the environment of the growth of Deionized water was used throughout the research for
natural bone tissues (Li et al., 2010). Organic matrix can help preparing all the solutions and cleaning the glassware.
in controlling the HA formation include the nucleation and
crystal growth as well as the morphology of HA (Sinha et al., B. Experimental
2009).
 Preparation of the Hydroxyethyl Cellulose Solution
HEC is a type of non-ionic polymer which is soluble in Prior to the experiment, all the glassware were washed
water. In order to strengthen the mechanical properties, the and cleaned with soap and deionized water to prevent
structure of HEC sponges is modified by cross-linking to contamination of the sample. The HEC solution with five
remove the water soluble characteristics. There are numerous different concentrations: 1 wt%, 3 wt%, 5 wt%, 7 wt% and 10
vitro studies had been done on the affection and wt% were prepared by dissolving 1 g, 3 g, 5 g, 7 g and 10 g
biocompatibility of the HA within the synthetic polymers like HEC powder with 100 mL of deionized water in five separated
HEC on the biological function of bone-associated cells, and beakers. Deionized water was used as the solvent it has no
positive results had been obtained. These proved that the charge, therefore it will not react with the HEC powders. The
mineralization of HA on the polymeric substrates like HEC solutions in the beakers were then left to stir overnight by
will aid in bone matrix synthesis (Chalal et al., 2014). The using magnetic stirrers in order to obtain homogeneous
simulated body fluid (SBF) is often used for the formation of solutions.
HA on biomaterial. SBF is an electrolyte solution which
mimics the human blood plasma, as it has the inorganic  Preparation of the Hydroxyethyl Cellulose Sponges
composition that similar to the human blood plasma. In this After the homogeneous HEC solutions (1 wt%, 3 wt%, 5
study, HA crystal was formed by using the 20x SBF which has wt%, 7wt% and 10 wt%) were obtained, the HEC sponges
20 times calcium and phosphate concentration compared to the were prepared through the freeze drying process. Freeze
conventional SBF. drying is a process of removing frozen solvents from a
material through sublimation and desorption. Before the
The formation of the HA on the HEC sponges by using samples were placed into the freeze dryer, the five beakers
the SBF solution is related to the theory of crystallization. contained with HEC solution were put into a freezer with the
There are two steps involved in the crystallization process, temperature of -80°C for five days until the samples were
which are nucleation and crystal growth. The porous structures completely turned into solid form. In fact, one day of freezing
of the HEC sponges provide numerous pores for the growth of is enough for the sample to turn solid, but the longer the time
HA crystals on it. When this product is applied for the in vitro of freezing, the better the condition for the next step. The
or in vivo tests, the porous structure of HEC sponges and HA samples should to be completely frozen before placing under
will permit the flow of extra cellular fluid through the inner vacuum in the freeze dryer, to prevent the unfrozen part
structure of this biomaterial. Consequently, high expands outside of the container. The beakers were placed
osteoconductivity, mechanical interlocking for firm fixation of slanting on the wall inside the freezer, in order to increase the
material will be created. Other than these, the adhesion surface area of the sample which can help in the freeze dry
between the natural and synthetic bone tissue will be enhanced process later on. After five days, the samples were taken out
through the formation of the apatite layer (Chavan et al., from the freezer and put into the freeze dryer for four days to
2010). Hence, this will be an excellent and potential remove the water contained in the samples. HEC sponges were
biomaterial for the bone tissue engineering. then formed.

In this research, hydroxyapatite is formed through the  Cross-linking of Hydroxyethyl Cellulose Sponges
process of crystallization on the hydroxyethyl cellulose (HEC) The purpose of carry out the cross-linking process is to
sponges of varying concentrations. remove the water solution characteristic of the HEC sponges.
The HEC sponges should have good integrity in the aqueous
solution so that it is practicable in tissue engineering. Cross-
linking process was carried out by using the glutaraldehyde

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ISSN No:-2456-2165
which acted as the cross-linking agent, acetone and phosphoric  Crystallization of Hydroxyapatite on Hydroxyethyl
acid which is the acid catalyst in the reaction. Firstly, HEC Cellulose Sponges
sponges in five different concentrations were cut into small The SBF solution prepared previously was used for the
pieces and put into five different petri dishes. Then, 500 μL crystallisation of hydtoxyapatite. Before the scaffold
glutaraldehyde, 3mL acetone and 5-8 drops of phosphoric acid mineralization, 100 mL of the stock SBF solution was
were added into each petri dish with the help of micropipette transferred into a beaker. After that, 0.168 g of NaHCO3 was
and pipette. All the HEC sponges must be completely added to increase the pH value to pH 6.5 (Tas and Bhaduri,
immersed in the chemical solution. Otherwise, the amount of 2004) and dissolved completely. The HEC sponges with the
glutaraldehyde, acetone and phosphoric acid must be concentration of 1 wt%, 3 wt%, 5 wt%, 7wt% and 10 wt% in
increased by two times. The petri dishes containing the HEC small pieces which had cross linked previously were put in the
sponges with the cross-linking chemicals were then left for 24 tissue culture testplate. Then 3 mL of SBF solution was
hours at room temperature. After 24 hours, the cross-linking pipetted and added into each hole which contained 1 piece of
reaction was stop by rinsing the HEC sponges with the HEC sponges. The samples must be fully immersed in the SBF
deionized water. The solubility of the HEC sponge was solution. The tissue culture testplate was then covered with
checked by immersing it in the beaker containing deionized aluminium foil to prevent contamination of samples and left
water. for 48 hours at room temperature. After 48 hours, the samples
were rinsed with deionized water to remove the salt residue
 Preparation of 20× Simulated Body Fluid Solution for and left to dry in room temperature before proceeding to the
Crystallization of Hydroxyapatite on Hydroxyethyl characterization.
Cellulose Sponges
20× SBF solution was used to form the HA crystal on the In fact, there are two precaution steps that should be
HEC sponges in this reasearch. The SBF solution was followed during the preparation of SBF solution. Firstly,
prepared based on Tas and Bhaduri (2004) and Mavis et al. plastic container should be used instead of glass container and
(2009). There are several chemical needed to prepare the SBF make sure that there is no scratch on the surface of plastic
solution, including sodium chloride, potassium chloride, container. This is due to apatite nucleation may be induced at
calcium chloride dehydrate, magnesium chloride hexahydrate, the surface of a container or the edges of scratches. Secondly,
sodium dihydrogen phosphate and sodium bicarbonate. The the reagents used should not be dissolved at the same time.
amount needed is listed in the Table 3.1 below. In order to One reagent should be dissolved completely before
prepare 500 mL of 20× SBF solution, the first five chemicals proceeding with the next reagents (Kokubo and Takadama,
listed in the table was measured and dissolved in 400 mL 2006).
deionized water. The solution was stirred continuously using
a glass rod until all the chemical was dissolved completely in C. Characterization of hydroxyapatite on hydroxyethyl
the deionized water. Then, the volume of the stock solution cellulose
was topped up to 500 mL and stored at room temperature in a
capped glass bottle.  Fourier Transform Infrared (FTIR) Spectroscopy
Fourier Transform Infrared (FTIR) spectroscopy was
Table 1: Reagents for preparing 500 mL of 20× SBF solution used to determine the functional group presented in the
samples before cross-linking, after cross-linking and after
Order Reagent Amount Molarity crystallization. Attenuated Total Reflection (ATR) technique
(g) (mM) was used in this research. This was done by using Perkin
1 NaCl 58.443 2000 Elmer spectrum 100 spectrophotometer in the range of 700
cm-1 to 4000 cm-1, with resolution of 4 cm-1 and 10 scans per
2 KCl 0.373 10
sample. The crystal area was cleaned and background was
3 CaCl2.2H2O 3.675 50 collected before the analysis. Then, small pieces of HEC
4 MgCl2.6H2O 1.016 10 sample before cross-linking, after cross-linking and after
5 NaH2PO4.H2O 0.250 7.24 crystallization were placed directly onto the small crystal area
6 NaHCO3 0.840 20 to determine the FTIR spectrum.
Source: Chalal et al. (2013)
 Field Emission Scanning Electron Microscopy (FESEM)
SBF is supersaturated with respect to apatite. JEOL Field Emission Scanning Electron Microscopy
Therefore, any small mistake that happened during the (FESEM) was used to determine the morphology and size of
preparation of SBF may lead to the formation of apatite the HEC pores and HA crystal formed on the HEC sponges. It
precipitate in the solution. SBF solution should be colourless is operated under vacuum condition. Before the sample
and transparent. When there is precipitate observed in the analysis, sample preparation was done by coating a thin layer
solution, the SBF solution preparation is considered as fail and of platinum on the sample which was in a very dry condition.
should be discarded and restart with clean glassware (Kokubo This is due to HEC sponges is a non-conductive material and
and Takadama, 2006). it was done by using a sputter coater in order to provide a
current conductive surface on the sample. Besides, FESEM
was worked with Energy Dispersive X-Ray (EDX) to
determine the elemental composition of HA crystal on the
HEC sponges.Ca/P ration was determined by using EDX as

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well.

 Optical Microscopy
Optical microscopy was performed to observe the
surface morphology of the HEC sponges and the HA crystal
formed on the HEC sponges. Highest resolution of Nikon
E100 microscope 1280 × 960 was used in this analysis, and it
provided the magnification to 4, 10, 40 or 100 times. This type
of microscope operates by using the visible light and a system
of lenses to magnify the image of the small sample. Besides,
Dino Eyes Software was installed and used in the computer
that connected to the microscope, in order to capture the image
of the HEC sponges and HA crystals on it. The sample was put
on the microscope glass slide and placed on the stage. Then Fig 4.2: HEC sponges formed after freeze drying process
the light intensity and the magnification of the lenses were
adjusted to obtain the best image of morphology of samples. B. CROSS-LINKING OF HEC SPONGES
Since the optical microscopy is performed by the light passing In order to fabricate a scaffold with good integrity for the
through the sample, the sample have to be as thin as possible application in tissue engineering, cross-linking process was
in order to obtain a better result. done on the HEC sponges to remove the water-soluble
property. After immersing the HEC sponges in the mixture of
 X-Ray Diffraction (XRD) glutaraldehyde, acetone and phosphoric acid with a fixed
X-Ray Diffraction (XRD) was conducted to determine amount for 24 hours (Figure 4.3), the HEC sponges were
the presence of HA crystal on the HEC sponges. Rigaku washed by deionized water and the water solubility of HEC
Miniflex II Diffractometer was used in this analysis with sponges was tested by immersing in the deionized water. The
CuKα (wavelength = 1.54 Å) as the x-ray source. It was result showed that HEC sponges after cross-linking was not
generated at 30 kV and 15 mA in 2θ range of 3 ° to 60 °, with soluble in deionized water (Figure 4.4). This proves that the
the scan rate of 1 degree per minute. 1 cm × 1 cm HEC sponges water-soluble ability of the HEC was successfully removed
before crystallization and after crystallization of HA were used through the cross-linking process. The removal of the water-
for the soluble ability of the HEC is very important to ensure that it
will not dissolve in the SBF solution during the incubation.
III. RESULTS AND DISCUSSION

A. HEC SPONGES
During the preparation of 1 wt%, 3 wt%, 5 wt%, 7 wt%
and 10 wt% of HEC solutions, homogeneous solutions were
formed after the stirring, Figure 4.1. The viscosity and colour
intensity of the HEC solutions increased when the
concentration increased. This is due to there are more particles
presented in the HEC solution of higher concentration.

HEC sponges were produced through the freeze drying

process (Figure 4.2). The sizes of the HEC sponges formed
were different according to the concentration of the HEC
solution. The lower the concentration of the HEC solution, the Fig 4.3: HEC sponges immersed in cross-linking agents
smaller the size of the HEC sponges formed. This is because
of there are more water contained in the HEC solution of low
concentration, and more water molecules will be removed
during the freeze drying process.

Fig 4.1: HEC solutions after stirring with concentration

Fig 4.4: HEC sponges insoluble in water after cross-linking
of 1 wt%, 3 wt%, 5 wt%, 7 wt% and 10 wt% from left to
right

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C. CRYSTALLIZATION OF HYDROXYAPATITE ON HEC
SPONGES
The formation of HA crystal was done on the HEC
sponges which had been cross-linked previously. Five
different concentrations of HEC sponges of in pieces which
had been cross-linked were immersed into the 20× SBF
solution in cell culture plate for 48 hours as shown in figure
4.5. Nominal ion concentrations of SBF compare with human
blood plasma is given in table 2. The SBF solution that
prepared was a clear solution without any precipitation. After
48 hours, some white precipitate can be seen in the solution
and the surface of HEC sponges as shown in figure 4.6. This
“precipitate” is actually the white HA crystals which formed
in the SBF solution. The HEC sponges were then remove from
the SBF solution and the salt residue was rinsed off with Fig 4.6: Immersion of HEC sponges in 20× SBF solution
deionized water. The sample was then left to dry under normal (after 48 hours)
room temperature in the desiccators before the
characterization part. D. Fourier Transform Infrared (FTIR) Spectroscopy Study
ATR-FTIR was used to determine the functional groups
Table 2. Nominal ion concentrations of SBF compare with that presented in the HEC sponges. Figure 4.7 shows FTIR
human blood plasma spectra of the combination (1 wt%, 3 wt%, 5wt%, 7 wt% and
Ion concentration(mM) 10 wt%) of HEC sponges before the cross-linking. From the
Ion Blood Plasma SBF result obtained, there is no much difference of the spectrum
Na+ 142.0 142.0 between the 1 wt%, 3 wt%, 5wt%, 7 wt% and 10 wt% of HEC
K+ 5.0 5.0 sponges. The broad peaks with the wavenumbers 3380.57 cm-
Mg2+ 1.5 1.5
1
to 3384.79 cm-1 indicate the –OH stretching vibration. Due
Ca2+ 2.5 2.5 to the presence of hydroxyl group, HEC sponges are soluble
Cl- 103.0 147.8 in water. Therefore, cross-linking was carried out to remove
HCO3- 27.0 4.2 the hydroxyl group. The peaks at 2880.02-2881.62 cm-1
HPO42- 1.0 1.0 represent the C-H stretching vibration. The peaks around
SO42- 0.5 0.5 1642.84-1649.05 cm-1 are corresponding to the bending mode
pH 7.2-7.4 7.40 of the water absorbed naturally (Azzaoui, 2015). Peaks
appeared at 1354.80-1356.54 cm-1 represent the C-H vibration.
The sharp peaks at 1052.94-1054.02 cm-1 indicate the C-O-C
stretching vibration in ether group or C-O stretch in primary
alcohol. The small peaks at wavenumbers around 885.20-
886.01 cm-1 represent the C-C stretching vibration.

Fig 4.5: Immersion of HEC sponges in 20× SBF solution

(beginning)

Fig 4.7: Combined FTIR spectra of (a) 1 wt% (b) 3 wt% (c) 5
wt% (d) 7 wt% (e) 10 wt% HEC sponges before cross-
linking

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The combined FTIR spectra of different concentrations 872.01-889.67 cm-1. The carbonate ion indicates that it is B-
of HEC sponges after cross-linking are shown in figure 4.8. type carbonate apatite (CO3 substituting PO4) (Chalal et al.,
There is no much difference of the spectrum between the 1 2014). It may be caused by the absorption of CO2 from the
wt%, 3 wt%, 5wt%, 7 wt% and 10 wt% of HEC sponges after atmosphere to the HA particles.
cross-linked. From the spectrum obtained, the broad peaks
with the wavenumbers 3369.12 cm-1 to 3390.66 cm-1 indicate
the –OH stretching vibration. It is lower compared to the -OH
spectrum of the sample before cross-linking. This shows that
the water soluble characteristic has been reduced after cross-
linked with glutaraldehyde. The peaks at 2881.80-2925.96 cm-
1
represent the C-H stretching vibration. The peaks around
1644.52-1649.86 cm-1 are corresponding to the bending mode
of the water absorbed naturally (Azzaoui, 2015). The presence
of this peak may be due to the water molecule that still
contained in the sample and not fully dried after washing with
deionized water. The peaks around 1456.18-1458.51 cm-1
indicate the CH2 or CH3 in aliphatic compounds are stronger
compared to the samples before cross-linking. This is due to
the formation of acetal group after the cross-linking. Peaks
appeared at 1355.86-1358.31 cm-1 represent the C-H vibration.
The sharp peaks at 1053.90-1056.72 cm-1 indicate the C-O-C
stretching vibration in ether group or C-O stretch in primary
alcohol. The presence of acetal group due to the reaction Fig 4.9: Combined FTIR spectra of (a) 1 wt% (b) 3 wt% (c) 5
between HEC and glutaraldehyde has contributed to the peaks wt% (d) 7 wt% (e) 10 wt% HEC sponges after crystallization
of HA
at the wavenumber around 1022.20-1025.83 cm-1. The small
peaks at wavenumbers around 888.15-889.70 cm-1 represent
E. Optical Microscopy
the C-C stretching vibration., (Azzaoui 2015) (Khairy
M.Tohamy 2018). Optical microscopy was done on the 5 different
concentrations of HEC sponges before cross-linking, after
cross-linking and after crystallization of HA. These were done
by using 10X magnification. However, since the resolution of
optical microscope is quite poor, characterization by using
FESEM was done to observe the morphology of HA crystals
as well.

Figure 4.10 (a), (b), (c), (d) and (e) show the morphology
of HEC sponges before the crystallization step. The pores of
the HEC sponges can be clearly seen. However, there is a
difficulty to measure the size of the pores with optical
microscope. Therefore, the actual effect of the HEC
concentration on the pore size cannot be determined.
Theoretically, HEC sponges with higher concentration will
have smaller pore size. This is due to the HEC sponges with
higher concentration is more viscous and the pore wall tend to
be thicker and more homogeneous. Therefore, the morphology
of the sponge’s surface will be denser (Ikeda et al., 2014). This
Fig 4.8: Combined FTIR spectra of (a) 1 wt% (b) 3 wt% (c) 5 is a vital factor to a successful scaffold in tissue engineering.
wt% (d) 7 wt% (e) 10 wt% HEC sponges after cross-linking The pore structures of the HEC sponges did not affected after
the cross-linking step. The space in the pores was utilized for
Figure 4.9 shows the combined FTIR spectra of HEC the cell growth as well as metabolic wastes and nutrients-
sponges after crystallization of HA. From the spectra exchange between the scaffold and the environment (Zulkifli
obtained, O-H stretching vibrations are shown in the broad et al., 2013).
peaks at the wavenumbers 3378.50-3379.68 cm-1. The peaks
at 2881.44-2930.50 cm-1 and 1355.35-1357.00 cm-1 are Figure 4.11 (a), (b), (c), (d) and (e) reveal the optical
attributed to the C-H stretching vibration. Besides, H2O band microscopy images of the HEC sponges after coated with the
is observed at the peak with the wavenumbers around 1638.28- HA crystals. HA crystals formation was done by immersing
1642.62 cm-1 which may be the water molecule associated with the cross-linked HEC sponges into the 20× SBF solution for
the HA particles. The peaks at 1050.46-1052.84 cm-1 show the 48 hours. It showed that the HA crystals formed on the HEC
PO43- . These can prove the presence of HA on the HEC sponges with higher concentration is larger in size compared
sponges. Other than these, CO32- can be observed at the weak to the HA crystals formed on HEC sponges with lower
band with the wavenumbers 1413.78-1421.04 cm-1 and concentration. However, the exact size and shape of the

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crystals cannot be determined by using optical microscope due to the surface of the HEC sponges has been covered by the HA
to its poor resolution and magnification. Therefore, further crystals and the pores were blocked by the HA crystals. The
analysis was done by using the FESEM. results revealed that the HA crystals formed were in round
shapes. The average size the HA crystals formed on 1 wt%, 3
wt%, 5 wt% and 10 wt% HEC sponges are around 0.10-0.13
μm, 0.18-0.21 μm , 0.19–0.23 μm and 0.53–0.69 μm
respectively. The size of the HA crystals were increasing with
the concentration of the HEC sponges.

Zhang et al. (2009) stated that the crystal growth in HEC

1 wt % 3 wt % is strongly depends on the interaction between the HEC
molecules and the crystal surface. HEC with higher
concentration may lead to the interaction between the HEC
molecules and crystal surface sufficient to lower the crystal
surface energy as well as to stabilize the crystals in metastable
phase. The difference in concentration of HEC will affect the
morphology and size of the crystals formed. When the HEC
concentration increased, there is less space between the HEC
5 wt % 7 wt %
molecules and become entangled or aggregated. This will alter
the interaction between the HEC molecules and HA crystals.
Polar hydroxyl side groups of the HEC molecules tend to
interact with Ca2+ during the HA crystallization process, to
create the nucleation sites. The interface energy is then
reduced and eases the formation of metastable phase.

10 wt %
Fig 4.10: Optical microscopy image of (a) 1wt% (b) 3wt%
(c) 5wt% and (e) 10 wt% HEC sponges before cross-linking

Fig 4.12: FESEM image of HA on 3 wt% HEC sponges after

48 hours incubation in 20× SBF solution

Other than these, EDX was performed to determine the

Fig 4.11: Optical microscopy image of HA crystallization on mineral phase and elemental composition of HA crystals
(a) 1 wt% (b) 3 wt%, (c) 5 wt% and (e) 10 wt% HEC sponges
formed on the HEC sponges. Figure 4.13 shows the FESEM-
EDX image of 10 wt% HEC sponges after the incubation in
F. Field Emission Scanning Electron Microscopy (FESEM)
the 20× SBF solution for 48 hours. Table 3, reveals the element
FESEM was done to determine the morphology of the
which consisted in the HA-coated HEC sponges and their
HA crystals. Figure 12 shows the FESEM images of HA respective weight percentage and atomic percentage. There are
crystals formed on 1 wt%, 3 wt%, 5 wt% and 10 wt% of HEC four main elements in the mineralized HEC sponges, which
sponges respectively. From the images obtained, the pores of are carbon (C), oxygen (O), phosphorus (P) and calcium (Ca).
the HEC sponges cannot be seen clearly, and this may be due C and O could be from the HEC while P and Ca could be from

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Volume 7, Issue 2, February – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
the mineral phase. The percentage of C and O are higher than
Ca and P may be due to the area selected for the EDX analysis
is not fully covered with the HA crystals. From the EDX data,
Ca/P ratio was calculated and 1.37 was obtained. This result is
comparable with the study done by Cengiz et al. (2008) which
showed the Ca/P ratio of 1.34 after the HA crystallization
process by using SBF solution. Normal human bone has a Ca/P
ratio of 1.67.

Fig 4.14: Combined XRD pattern of (a) 1 wt% (b) 3 wt% (c)
5 wt% (d) 7 wt% (e) 10 wt% HEC sponges before
crystallization

From the results obtained, the amorphous hump and the

first two peaks are same as the x-ray pattern shown in pure
HEC sponges. Other than these, there are extra peaks
compared to pure HEC sponges, which appeared at 29.42 °,
47.58 ° and 48.65 ° (hydroxyapatite) for the HA crystals on 1
wt% HEC. However, the intensity of the peaks are quite low,
and this may be due to the HA crystals formation are not fully
Fig 4.13: FESEM-EDX image of 10 wt% HEC sponges after on the surface of the sample but inside the pores of HEC
48 hours incubation in 20× SBF solution sponges. The extra peaks were shown at 29.11 °
(hydroxyapatite, 210), 31.74 ° (hydroxyapatite, 211) and 31.97
Table 3: Composition of elements of HA crystals on HEC ° (hydroxyapatite, 000) for HA crystals on 3 wt% HEC
sponges sponges, 32.12 ° (hydroxyapatite), 45.84 ° (hydroxyapatite,
Element Weight % Atomic % 211) and 56.85 ° (hydroxyapatite, 313) for HA crystals on 5
Carbon, C 27.42 36.76 wt% HEC sponges, 29.20 ° (hydroxyapatite, 120), 31.91 °
Oxygen, O 55.13 55.48 (hydroxyapatite, 000), 45.64 ° (hydroxyapatite, 203) and
Phosphorus, P 6.28 3.27 56.64 ° (hydroxyapatite, 114) for HA crystals on 7 wt% HEC
sponges, 31.86 ° (hydroxyapatite, 211), 45.67 °
Calcium, Ca 11.16 4.48
(hydroxyapatite, 203) and 56.52 ° (hydroxyapatite, 500) for
Totals 100 100 HA crystals on 10 wt% HEC sponges. These results prove the
existence of the HA crystals on the HEC sponges after 48
G. X-Ray Diffraction (XRD) hours crystallization process by using SBF solution (Abdallah
XRD analysis was done to determine the presence of the 2011) (Mirta 2012). The further confirmation of the presence
HA crystals on the HEC sponges. Figure 4.14, show the x-ray of HA crystals was done by using FESEM-EDX. It can be seen
diffractograms of combined, 1 wt%, 3 wt%, 5 wt%, 7 wt% and that there are more peaks and the intensity of the peaks are
10 wt% HEC sponges. It can be clearly seen that there is not increased when the concentration of the HEC sponges
much differences between these 5 x-ray diffractogram. From increased. This may be because of the HA crystals formed are
the results obtained, most of them show amorphous hump at more when the HEC concentration increased.
14.67 °, 15.03 °, 14.29 ° and 14.66 ° for 1 wt%, 3 wt%, 7 wt%
and 10 wt% HEC sponges. Other than these, there are two IV. CONCLUSION
sharp peaks at 21.42 °and 23.75 ° for 1 wt% HEC sponges,
20.34 ° and 21.49 ° for 3 wt% HEC sponges, 21.44 ° and 23.74 Highly porous and stable, HEC sponges with different
° for 5 wt% HEC sponges, 21.24 ° and 23.56 ° for 7 wt% HEC concentrations, 1 wt%, 3 wt%, 5 wt%, 7 wt% and 10 wt% were
sponges, 21.38 ° and 23.67 ° for 10 wt% HEC sponges. prepared in a green chemistry approach. The HEC sponges
were successfully coated with hydroxyapatite (HA) crystals
using the 20× SBF solution and characterized. The optical and
SEM images showed that there is no change of the
morphology of the pores after the cross-linking process. From
the FTIR results obtained, the presence of acetal group at the
wavenumbers around 1022.20-1025.83 cm-1 had showed the
cross-linking process was succeeded. FTIR results after the
crystallization showed peak of PO43- and CO32- which indicate

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Volume 7, Issue 2, February – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
the presence of HA crystals. This result was confirmed by chitosan sponge as a tissue engineering scaffold. BioMed
using XRD and FESEM-EDX. The element composition of Research International. 2014.
HA crystals was revealed, which consisted of C, O, P and Ca. [10]. Khairy M.Tohamy, MostafaMabrouk, Islam E.Soliman,
The morphology and the size of the HA crystals was Hanan H.Beherei and Mohamed, A.Aboelnasr, 2018,
determined by using FESEM. From the results obtained, it Novel alginate/hydroxyethyl cellulose/hydroxyapatite
showed that HA crystals synthesized were in round shape, and composite scaffold for bone regeneration: In vitro cell
the size of the HA crystals increased when the concentration viability and proliferation of human mesenchymal stem
of the HEC sponges increased. This is due to the polar cells. International Journal of Biological
hydroxyl side groups of the HEC molecules tend to interact Macromolecules, 112, 448-460.
with Ca2+ during the HA crystallization process. This highly [11]. Kokubo, T. and Takadama, H. 2006. How useful is SBF
porous and stable HEC sponges coated with hydroxyapatite in predicting in vivo bone bioactivity?. Biomaterials.
crystals are a promising biomaterial for bone tissue 27(15): 2907-2915.
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ACKNOWLEDGEMENT chitosan–pectin polyelectrolyte complex network.
Materials Science and Engineering: C. 30(6): 795-803.
The authors acknowledge the support from the College [13]. Matassi, F., Nistri, L., Paez, D.C. and Innocenti, M.
of Arts and sciences, University of Nizwa and TRC, sultanate 2011. New biomaterials for bone regeneration. Clinical
of Oman. Cases in Mineral and Bone Metabolism. 8(1): 21-24.
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