Journal of Polymer Research (2020) 27: 169

https://doi.org/10.1007/s10965-020-02112-6

MANUSCRIPT

Reinforcement of hydroxyethyl cellulose / poly (vinyl alcohol)

with cellulose nanocrystal as a bone tissue engineering scaffold
Nor Sarahtul Nadirah Hairol Nizan 1 & Farah Hanani Zulkifli 1

Received: 13 August 2019 / Accepted: 6 April 2020 / Published online: 20 May 2020
# The Polymer Society, Taipei 2020

Abstract
In this present work, a porous three-dimensional (3D) scaffold of HEC/PVA and HEC/PVA/CNC were successfully fabricated by
freeze-drying technique. HEC (5 wt%) and PVA (15 wt%) were dissolved and blended at a ratio of 50:50 and incorporated with
CNC (3 wt%) as nanofillers to obtain a highly porous scaffolds. The morphology, chemical and thermal properties of scaffolds were
characterized by SEM, ATR-FTIR, and TGA. Meanwhile, cytotoxicity studies on both porous scaffold biomaterials were carried
out by utilizing human fetal osteoblast (hFOB) cells using MTT assays. Incorporated HEC/PVA with CNC were exhibited superior
functionality which resulted in decreasing average pore size from ~54.1 μm to ~33.4 μm. There were slightly changes in the
chemical structure as determined by FTIR spectra. Thermal studies revealed that the melting temperatures of HEC/PVA/CNC
scaffold were slightly shifted to a higher value. It was observed that hFOB cells were able to attach and spread on both scaffolds and
supported the cell adhesion and proliferation. Due to its biocompatible and biodegradable properties, these newly developed highly
porous scaffolds may provide a promising alternative scaffolding matrix for bone tissue engineering regeneration.

Keywords Freeze-dry . Biomaterials . Scaffold . Cellulose nanocrystal . Bone tissue engineering

Introduction
Orthopedic medication becomes one of the major health is-
sues for senior citizens across the world. Besides the incre-
ment of aging population, lifestyle and rise in prevalence of
trauma, and fracture has driven the tremendous interest among
researcher to acknowledge the best medicals practices to treat
patients with bone injury [1]. Nowadays, the emerging of
tissue engineering has surpassed autograft, allograft and xeno-
graft approaches by replace, repair or restore the tissue func-
tionality through mimicking the native extracellular matrix
(ECM) in human body. ECM consists of fibrous collagen
and proteoglycans. Proteoglycans are made of proteins and
polysaccharide chains known as glycosaminoglycans
(GAGs). Tissue engineering promises to solve problems in
organ transplantation by utilized the application of chemical,
biological and engineering principles in replacing or repairing
* Farah Hanani Zulkifli
farahhanani@ump.edu.my
1
Faculty of Industrial Sciences & Technology, Universiti Malaysia
Pahang, Lebuhraya Tun Razak, 26300 Gambang Kuantan, Pahang,
Malaysia
the living tissues like bone using biomaterials, cells, and
growth factors, alone or in combination [2]. It is worth to
mention that as a result of various contributions from expertise
and researchers to the new processing methodologies, design
principles and surgical techniques in the last 50 years, today,
people gain the benefit of many excellent biomaterials and
surgical implants that improved the quality of lives of many
people each year around the world. In recent years, nanotech-
nology, biomimetics and tissue engineering concepts are gen-
erating a new phase of challenges in the development of bio-
materials at nanoscale levels.
Scaffolds, which made from synthetic or/and natural poly-
mers, are commonly serves as temporary support for the cell
growth in a three-dimensional (3D) platform for assistance of
new tissue regeneration [3]. An ideal scaffold should be bio-
degrade parallel with the integration of redevelopment tissue
as these criteria become the main key factors for designation
and fabrication of biomimetic scaffolds in tissue engineering
applications. Other than that, an excellent scaffold should pro-
mote desirable physiological responses without loss of bioac-
tivity and able to deliver bioactive molecules in a controlled
fashion to accelerate healing. In tissue engineering, scaffold
plays an important role as artificial ECM in which they should
mimic the structure and biological functions of natural ECM
169 Page 2 of 9
as closely as possible to create conducive living substrates that
will induce cells to function naturally by promoting cell adhe-
sion while maintaining their differentiated function without
hindering the proliferation [4].
Although there is a wide range of biomaterials used as
scaffolds, only a few biopolymers closely mimic the chemical
structure of ECM by having polysaccharide chains in them
[5]. HEC is a non- ionic with β (1 → 4) glycosidic linkage
held together with H-bonds and it is biocompatible water sol-
uble polysaccharide material with protective colloidal action.
It is less expensive and commonly used in various pharma-
ceutical compositions, wound dressing and wound healing
applications [6]. Previous studies has shown that the blended
of HEC and PVA provide better platform for cell proliferation
[7]. PVA is a polyhydroxy water soluble polymer that used
water as the only solvent. It has become a considerable choice
due to its excellent biocompability, mechanical properties and
widely used in biomedical applications which include drug
delivery reservoirs, orthopaedic stabilization splints, and
blood contacting material [8] [9].
Nanofillers has been widely applied as additive to be uni-
formly distributed within polymer matrices in order to in-
crease the performance of a material [10]. Cellulose
nanocrystals (CNCs) are defect-free, rod-like crystalline resi-
dues obtained from acid hydrolysis process from wide variety
of natural sources such as wood, grass stalks, bacteria and alga
[11]. CNCs have attracted great attention due to their high
aspect ratio (i.e., ~3 to 5 nm wide and ~50 to 500 nm in length)
and high crystallinity index (~54 to 88%) [12]. Combination
of HEC with other hydrogel like PVA improves the mechan-
ical properties such as Young’s modulus and elongation at
break value [13]. In general studies, CNCs are used in order
to improve various mechanical and barrier properties of bio-
polymers [14].
The fabrication of biopolymeric scaffolds that could mimic
the natural ECM structure has been extensively explored for
decades. Preparation of porous scaffolds involved some
methods such as gas-forming foam, freeze-drying and
electrospinning [4]. Lyophilization or known as freeze-
drying technique has been widely used to fabricate polymer
scaffolds with high porosity and pore interconnectivity as long
as to improve the long-term stability of polymeric nanoparti-
cles for many applications by avoiding their instability in sus-
pension [15]. Even though the dehydration process that takes
place may induce stresses to nanoparticles, this technique has
been considered as an excellent technique to efficiently pre-
vent aggregation and preserve the re-dispersibility of nanopar-
ticles [16]. Other advantages of using freeze-drying method
were it is very inexpensive and only requires basic laboratory
equipment.
The aim of this study are to synthesis and fabricate the
HEC/PVA incorporated with CNC by using freeze-drying
technique and its performance as potential substrate for bone
J Polym Res (2020) 27: 169
tissue engineering. Up to now, there is no report on the effect
of CNC within HEC/PVA polymer matrices has been con-
ducted. Herein, the morphology, chemical and thermal prop-
erties of scaffolds will be characterized by SEM, ATR-FTIR
and TGA. Next, the cell studies will be carried out to investi-
gate the biocompatibility of the scaffolds by utilized the hFOB
cells for bone tissue engineering applications.
Experimental
Materials
HEC and analytical reagent grade glutaraldehyde (GA) solu-
tion (25%) were purchased from Merck-Schuchardt,
Germany. PVA (molecular weight, Mw of 95,000 g mol−1)
was obtained from ACROS, New Jersey, USA. Meanwhile,
phosphate buffer saline (PBS) was purchased from Gibco Life
Technologies, USA. Human fetal osteoblast (hFOB), SV4D
large T antigen transfected and all materials used for cell cul-
ture study were supplied from American Type Culture
Collection (ATCC), USA. All the chemicals were of highest
purity and used without further purification. Note that, all the
solutions were prepared using only deionized water.
Preparation of aqueous polymeric solution
HEC solution with a concentration of 5 wt% was prepared by
dissolving 5 g of HEC powder in 100 ml of Millipore water
and stirred for 12 h at room temperature until a clear solution
was obtained with a slight increase in viscosity. Meanwhile,
PVA solution with a concentration of 15 wt% was prepared by
dissolving 15 g of PVA granules in 100 ml of Millipore water
and stirred at 80 °C for 2 h and stirred continuously for another
12 h at room temperature. HEC was then blended in PVA
solution with ratios 50:50 and then stirred overnight to get a
homogeneous mixture. For ternary blend system, cellulose
suspension with concentration of 3 wt% was added into the
HEC/PVA solution and stirred until completely blended.
Freeze-drying
The scaffolds were fabricated by pouring the aqueous
polymeric solutions into falcon tube and kept in a deep
freezer at −80 °C for 24 h and followed by lyophilized
them in a freeze-dryer (FreeZone 6 Liter Benchtop Freeze
Dry System, Labconco), at −50 °C for 72 h. Thereafter,
the produced scaffolds underwent crosslinking via heat
treatment at 80 °C in 30 min and kept in dry cabinet for
further use.
J Polym Res (2020) 27: 169
Characterizations of the scaffolds
Scanning electron microscope (SEM) study
The morphology of the scaffolds was examined using SEM
(FEI QUANTA 450) at an accelerating voltage of 15 kV. To
obtain sharp and clear images, the scaffolds were fractured in
the liquid nitrogen and sputter coated with a thin layer of
platinum in double 30 s consecutive cycles at 45 mA. The
average pore size was analyzed using image visualization soft-
ware, ImageJ.
Porosity study
The porosity of scaffolds was measured by using liquid dis-
placement method. All samples were cut 1 cm × 1 cm × 1 cm,
weighed and then immersed in deionized water as the dis-
placement liquid for 30 min in a falcon tube. This method is
based on the known volume before immersed of sample (V1)
in a falcon tube, the unknown volume after 30 min immersed
sample (V2) and the volume after removing of impregnated
sample (V3).
The porosity of the sample can be calculated by the equa-
tion below;
V1 −V3
Porosity ð100%Þ ¼  100%
V2 −V3
Attenuated total reflection-Fourier transform infrared
(ATR-FTIR) study
Structural analysis of the scaffolds was carried out using FTIR
spectrophotometer from Spectrum One, (Perkin–Elmer, USA)
coupled with ATR. ATR-FTIR spectroscopic analysis of scaf-
folds was performed on over a range of 700 to 4000 cm−1 at a
resolution of 2 cm−1 with 10 scans per sample.
Thermogravimetric analysis (TGA) study
Thermal degradation of 5 mg of each sample was analysed
under a nitrogen flow (purge gas) at 10 °C min−1 (heating rate)
using Toledo STAR-1 (Mettler, Switzerland). The test tem-
peratures were ranged from 30 to 750 °C.
Cell culture studies
Cell expansion and seeding
Firstly, both scaffolds were punched into small disks with
10 mm diameter, soaked in 100% ethanol for 24 h, and then
sterilized under UV light for 3 h and subsequently immersed
in cell culture medium, Dulbecco’s modified Eagle’s medium
Page 3 of 9 169
(DMEM) solution. Then, the scaffolds were kept in incubator
at 37 °C in 5% carbon dioxide (CO2) humidified atmosphere.
All changes were observed including the pH of media before
and after five days in the incubator. Finally, the scaffolds were
washed thrice with sterile PBS and transferred into a 24-well
tissue culture plate.
Human fetal osteoblast cell (hFOB) cell lines were propa-
gated in Dulbecco’s modified Eagle’s medium (DMEM) in a
cell culture flask, all supplemented with 10% fetal bovine
serum and 5% penicillin/ streptomycin and maintained at
37 °C in 5% carbon dioxide (CO2) humidified atmosphere.
Subculture of hFOB cells were carried out by passaged the
cells when they have reached 80–90% confluence of the sur-
face area of a flask. DMEM solutions were removed and
TrypLE solutions were added and swirled across the adhered
cells to ensure it reaches all cells so that the cells will detached.
The detachments of the cells were checked by using micro-
scope after being incubated for 5 min. Once the cells were
detached, the cell suspensions were transferred into 15 mL
tube and centrifuged at 800 rpm for 5 min. TrypLE solution
was removed and fresh DMEM solution containing serum
was then added to inactivate the TrypLE solution in the cell
suspension. Finally, the aliquot of cell suspensions were
placed into a new flask containing 5 mL of DMEM solution
and stored in the incubator.
Cell proliferation study
hFOB cells were distributed in 24-well cell culture
plates containing scaffolds at a concentration of approx-
imately 10 × 103 cells/well in each different well with
DMEM solution. The proliferation test was observed
via MTT assay test by measuring the reduction of the
yellow tetrazolium salt to purple formazan crystals by
dehydrogenase enzymes secreted from the mitochondria
of metabolically active cells. The amount of the purple
formazan crystals formed is proportional to the number
of viable cells. For this study, 150 μL of Dimethyl
sulfoxide (DMSO) solvent was added into each of the
cell-scaffolds to dissolve the MTT formazon crystal.
After that, the well plate was placed in a NanoQuant
micro plate reader (infinite M200PRO) to detect biolog-
ical, chemical or physical events of samples by using
emitting light. The absorbance was recorded at 570 nm
with background 630 nm representing the number of
viable cells was measured. For data analysis, the cell
proliferation is calculated using the following formula:


Asample −Ablank −ðAcontrol −Ablank Þ
Cell viability ð%Þ ¼
ðAcontrol −Ablank Þ
where Asample = absorbance at 570 nm of the sample,
Acontrol = absorbance at 570 nm of the positive control
169 Page 4 of 9
which is cells in complete medium incubated in the
absence of the scaffolds and Ablank = background absor-
bance of the blank wells. An average of triplicate read-
ing was calculated for each sample.
Cell morphology study
hFOB cells grown on scaffolds were rinsed twice with PBS
and fixed in 3% GA for 30 min. Thereafter, the scaffolds were
dehydrated with increasing concentrations of alcohol (20, 40,
60, 80 and 100%). The samples were air dried by keeping the
samples in a fume hood. Lastly, the morphology of cell-
scaffolds were observed using SEM at an accelerating voltage
of 10 kV.
Results and discussion
Morphology of scaffolds
The morphology and pore size of three-dimensional porous
scaffolds were examined by SEM and ImageJ respectively. In
tissue engineering, the scaffolds should have high porosity
and sufficient distribution of pore size. Figure 1 shows the
image of CNC suspension which is spherical with diameter
of ~30 nm. Meanwhile, Fig. 2 shows the SEM images of (a)
HEC/PVA and (b) HEC/PVA/CNC scaffolds.
Fig. 1 FESEM micrographs of CNC nanoparticle at (a) low
magnification (50,000 ×) and (b) high magnification (130,000 ×)
J Polym Res (2020) 27: 169
Both scaffolds showed highly porous structure with pore
channels and interconnected pore structures. Adding CNC in
the blended polymer solution increased the reaction sites (–
OH groups), hence the structure become more porous. The
average pore size for HEC/PVA scaffolds and HEC/PVA/
CNC was ~54.1 μm and ~33.4 μm, respectively. The average
pore size of HEC/PVA/CNC scaffolds decreased due to the
greater influence of the cross-linking mechanism among –OH
groups of HEC, PVA and CNC.
It is worth mentioning that an ideal scaffold should have (i)
macro- (pore size >100 μm) and microporosity (pore size
<20 μm); (ii) interconnected open porosity for successful dif-
fusion of essential nutrients and oxygen for cell survivability
[17].
Porosity study
Porosity is one of the important features to evaluate the scaf-
folds properties for bone tissue engineering. It is well-known
that HEC, PVA and CNC are water-soluble biopolymers,
which are not favorable for cell culture studies. Hence,
cross-linking these materials is a must to decrease their solu-
bility as well as to give mechanical strength for cell culture
studies. In this study, porosity measurements of cross-linking
scaffolds were completed by using liquid displacement meth-
od with deionized water as the displacement liquid.
As illustrated in Fig. 3, all porous scaffolds show high
porosity with percentage value above 77.5% at a maximum
of 99.6% for pure HEC freeze-dried scaffolds. The results
pointed that porosity of scaffold containing CNC decreases
with the addition of CNC content. The morphology of 3D
scaffold structure should possess high porosity to support
the cell.
ATR-FTIR study
ATR-FTIR spectroscopy was conducted to enlighten the pres-
ence of HEC, PVA, and CNC and figure out the interaction
occurs among them. FTIR spectra in the 4000 to 700 cm-1
spectral range of pure HEC, PVA, and CNC, HEC/PVA and
HEC/PVA/CNC scaffolds were illustrated in Fig. 4.
From the results, the similar strong broad bond was obtain-
ed by all samples between 3320.28 and 3370.13 cm−1 region
due to the O-H stretching vibration and C-H stretching ap-
peared between 2898.15 and 2923.08 cm −1. Pure HEC
showed peak absorption at 1644 cm−1indicates the O-H vibra-
tion of absorbed water. CH2-symmetrical bending appeared at
1050.10 cm−1. Similar peak absorptions also reported which
were assigned at 2909 and 1438 cm−1 [18]. For pure PVA, the
absorption spectra presented specific bands in 1665.04
cm−1(C=C stretching), 1420.38 cm−1(O-H bending) of alco-
hol, 1094.97 cm−1(C-O stretching) of secondary alcohol and
855.65 cm−1(C-H bending). Meanwhile, for pure CNC, the
J Polym Res (2020) 27: 169
Fig. 2 SEM images of (a) HEC/PVA and (b) HEC/PVA/CNC scaffolds
absorption at 2898.15 cm−1refers to the aliphatic saturated C-
H stretching, associated with methylene groups in cellulose.
Ring stretching vibrations (C=C stretching) occurs at 1576.92
cm−1. At FTIR spectra of 1410.73 cm−1, O-H bending occurs
and C-O stretching of alcohol at 1043.45 cm−1.
Referring to previous studies, a few characteristic peaks
were reported in the neat PVA systems which are at
3339 cm−1 (O-H stretching) from inter and intra- molecular
hydrogen bonding, 2919 cm−1 (C-H stretching), 1719 cm−1
(C=O stretching), 1251 cm−1 (C-O bending) and 1071 cm−1
(C-O stretching) can be related to the crystallinity of the sys-
tem [19].
From Fig. 4, it is clearly can be seen that the FTIR spectra
for HEC/PVA/CNC scaffolds showed the combination of ma-
jor peaks of HEC, PVA and CNC. The absorption peaks of
HEC/PVA were assigned at 839.01, 1070.04, 1417.38,
2906.46 and 3320.28 cm−1, while for HEC/PVA/CNC at
Fig. 3 Porosity of CNC, HEC and PVA alone, HEC/PVA and HEC/
PVA/CNC scaffolds
Page 5 of 9 169
839.03, 1078.35, 1425.69, 2914.77 and 3328.59 cm−1. The
ratio of absorption intensities of O-H stretching increased as
well as shifted to left with the addition of CNC. The broad
pattern observed at ~3320 cm−1 indicates stretching vibrations
of the hydroxyl groups due to the intramolecular and intermo-
lecular hydrogen bands of the OH groups of PVA, HEC and
CNC. The peaks at ~2900 cm−1are due to CH2 stretching
vibrations. The presence of peaks at ~1420 cm-1 indicates
presence of O-H bending. At FTIR spectra of ~1070 cm−1,
C-O stretching were assigned and C=C bending were assigned
at ~839 cm−1 . After all, it is found that by adding CNC to the
blended scaffolds, the absorption peaks increased but has no
significant effect on the overall infrared spectra. The intensity
of the absorption band increased due to the substantial intro-
duction of –OH groups from CNC [2].
TGA measurements
The porous scaffolds of HEC/PVA and HEC/PVA/CNC were
analyzed using TGA to compare the degradation characteris-
tics among them. The derivative thermogravimetric analysis
Fig. 4 ATR-FTIR analysis for (a) CNC, (b) PVA, (c) HEC alone and (d)
HEC/PVA and (e) HEC/PVA/CNC scaffolds
169 Page 6 of 9 J Polym Res (2020) 27: 169

Fig. 5 TGA and DTA curve for (a) HEC/PVA and (b) HEC/PVA/CNC scaffolds

(DTA) illustrates the detection of maximum weight loss rate
for the components. The thermograms and DTA curves of
scaffolds were represented in Fig. 5. The details of the decom-
position step and percentage mass loss were summarized in
Table 1.
From the results, both curves exhibited three major weight
losses. The first stage of weight loss which is ~4% starting
from 25 °C to 197 °C due to the presence of a thermal process
where the removal of absorbed water occurs. At second de-
composition stage, there was more significant weight loss
range from 72.0 to 78.90% at 197 °C until 378 °C, proving
the degradation reaction of C-OH groups in PVA where the
side chain of PVA decomposed, loss of CO2 in HEC and
degradation of CNC [20]. The temperature was shifted to
higher values when CNC is added. The onset temperature of
CNC was reported at slightly lower temperature (~200 °C)
due to higher surface area [21].
The weight loss for third stage of decomposition starts from
387 °C to 494 °C was due to the by-products generated by
PVA during thermal degradation process attributed to decom-
position of the main chain of PVA. It can be seen that the
thermal stability increased and the total weight loss of scaf-
folds decreased when CNC is added. After all, adding CNC
into blended polymer of HEC/PVA will enhance the
degradation of HEC and PVA polymer and hence give effects
to the thermostability of the scaffolds.
Cell scaffold interaction study
Cell proliferation on scaffolds
Cell adhesion and proliferation are important criterion for
evaluating the biocompatibility of biomaterials. Figure 6 pre-
sents the proliferation of hFOB cells cultured on scaffolds as
determined by MTT assay. The cell viability of HEC/PVA/
CNC scaffold shows the higher numbers of cells compare to
HEC/PVA scaffold. The cell viability on both scaffolds in-
creased significantly from 1 to 7 days but there is some reduc-
tion in cell numbers at 3 days. This might be due to the cell
cultured is dependent on temperature during incubation peri-
od. It is worth to mention that hFOB cells proliferate rapidly
with a doubling time of ~36 h at permissive temperature
(33.5 °C). hFOB cells cultured at the restrictive temperature
(39.5 °C) do not proliferate, whereas cells cultured at 38.0 °C
proliferate very slowly, with a doubling time of more than
96 h. When the cells are switched back to 33.5 °C, prolifera-
tion resumes and attains a rate similar to those grown contin-
uously at 33.5 °C [22].

Table 1 TGA and DTA for both

scaffolds Sample Region of decomposition Temperature (°C) Weight loss (%)

Tstart Tend Tpeak Partial Total

HEC /PVA 1st 26 175 46 4.100 98.43

2nd 175 378 304 72.00
3rd 378 465 400 22.33
HEC/PVA/CNC 1st 25 197 75 3.860 94.77
2nd 197 360 280 78.90
3rd 360 494 412 12.01
J Polym Res (2020) 27: 169
Fig. 6 Graph showing hFOB cell viability after 1, 3, 5 and 7 days of
incubation for HEC/PVA and HEC/PVA/CNC scaffolds
From the results, it can be said that adding CNC to HEC/
PVA blended polymer will make the scaffolds become more
comparable to promote cell adhesion with hFOB cell. This
could be attributed to the rougher surfaces with smaller pore
size, whereas the lower amount of cells on HEC/PVA was
attributed to the bigger pore size, where the cell suspension
was more easily deposited to the bottom of the culture plate
through the hole.
Cell morphology study of hFOB cell
hFOB cells cultured for 3 and 7 days on the samples were also
observed by SEM. Figure 7 and Fig. 8 present the SEM im-
ages of cells cultured on the samples at 3 and 7 days. Round
shape morphology of osteoblast cells adhered on the surface
of the all scaffolds. It is proven that the cells exhibited slightly
rough surface for HEC/PVA/CNC scaffolds. After 3 days of
cell culture, it was observed that cells get adhered to the
Fig. 7 SEM micrographs of hFOB cells attached on HEC/PVA scaffolds at (a) day 3 and (b) day 7
Page 7 of 9 169
scaffold and have polygonal morphology with the cell mem-
brane being somewhat flattened onto the rough surface of the
scaffold, created by HEC, PVA and CNC particles. At 7 days
of cell culture, more flat osteoblast cells were produced indi-
cating that the osteoblast cell proliferation had begun. SEM
image of osteoblast cell-scaffolds depicts that osteoblast cells
have spread all around the porous structure of the scaffold and
covered almost the entire scaffold’s surface at 7 days. The
cells on the HEC/PVA/CNC scaffolds spread better than on
HEC/PVA scaffolds as the incubation time increased.
After all, HEC/PVA/CNC scaffold demonstrates greater
response towards hFOB cells with highest number of viable
cells where the cells endorse good biocompatibility, non-
toxicity and could be potential substrates for bone tissue en-
gineering. Overall, both freeze-dried scaffolds show excellent
cell attachment, adherence and proliferation. Incorporated
CNC with HEC/PVA blended polymer gives a great influence
towards scaffolds especially in tissue engineering.
Conclusions
The research and development of work brought in important
results, which would eventually help the industry to develop
new biocompatible scaffolds that offer rapid healing response
for tissue engineering applications. In this study, 3D highly
porous and well-interconnected scaffolds were fabricated suc-
cessfully by freeze-drying technique and subsequently
crosslinking via heat treatment. The SEM images displayed
interconnected porous structure ranging from ~33.4 μm to
~54.1 μm. All scaffolds exhibited porosity above 77% which
revealed their suitability in cell-scaffold environment. The Tg
values were found in the range of HEC, PVA and CNC pure
scaffolds, emphasizing the miscibility of all polymers. The
Young’s modulus of HEC/PVA which varied from 14.7–
22.46 MPa indicated better mechanical properties of blend
polymers compared to single polymer. In vitro cell culture
with hFOB cells demonstrated that the incorporation of
169 Page 8 of 9
Fig. 8 SEM micrographs of hFOB cells attached on HEC/PVA/CNC at (a) day 3 and (b) day 7
CNC did not bring any cytotoxicity to the scaffolds and could
promote cell proliferation and cell adhesion. The HEC/PVA/
CNC scaffolds showed the most positive response towards
bone cells compare to the HEC/PVA scaffold. In conclusion,
scaffolds containing CNC showed an improvement in proper-
ties, thus promising a great potential for application, in tissue
engineering, ideally in bone cell culture.
As for the recommendation for further research, the studies
of HEC/PVA and HEC/PVA/CNC scaffolds can be extended
to different cell growth works such as mesenchymal stem cells
and endothelial cells. In addition, multiple types of proteins
like chitosan, gelatine or silk fibroin incorporated with these
blended polymers could be explored as potential scaffolds for
tissue engineering.
Acknowledgements The authors thank Universiti Malaysia Pahang for
the financial support under the RDU 1703266, RDU 1901117 and
PGRS1903192.
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