Materials Science and Engineering C 68 (2016) 163–171

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Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Ovine tendon collagen: Extraction, characterisation and fabrication of

thin lms for tissue engineering applications
M.B. Fauzi a, Y. Lokanathan a, B.S. Aminuddin a,b, B.H.I. Ruszymah a,c, S.R. Chowdhury a,⁎
a
Tissue Engineering Centre, UKM Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia
b
Ear, Nose & Throat Consultant Clinic, Ampang Puteri Specialist Hospital, Taman Dato Ahmad Razali, 68000 Ampang, Selangor, Malaysia
c
Department of Physiology, UKM Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Collagen is the most abundant extracellular matrix (ECM) protein in the human body, thus widely used in tissue
Received 13 February 2016 engineering and subsequent clinical applications. This study aimed to extract collagen from ovine (Ovis aries)
Received in revised form 14 April 2016 Achilles tendon (OTC), and to evaluate its physicochemical properties and its potential to fabricate thin lm
Accepted 23 May 2016
with collagen brils in a random or aligned orientation. Acid-solubilized protein was extracted from ovine Achil-
Available online 24 May 2016
les tendon using 0.35 M acetic acid, and 80% of extracted protein was measured as collagen. SDS-PAGE and mass
Keywords:
spectrometry analysis revealed the presence of alpha 1 and alpha 2 chain of collagen type I (col I). Further anal-
Ovine collagen ysis with Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray
Collagen lm spectroscopy (EDS) conrms the presence of triple helix structure of col I, similar to commercially available rat
Collagen type I tail col I. Drying the OTC solution at 37°C resulted in formation of a thin lm with randomly orientated collagen
Tissue engineering brils (random collagen lm; RCF). Introduction of unidirectional mechanical intervention using a platform rock-
Biocompatibility er prior to drying facilitated the fabrication of a lm with aligned orientation of collagen bril (aligned collagen
Cell alignment lm; ACF). It was shown that both RCF and ACF signicantly enhanced human dermal broblast (HDF) attach-
Unidirectional alignment
ment and proliferation than that on plastic surface. Moreover, cells were distributed randomly on RCF, but
aligned with the direction of mechanical intervention on ACF. In conclusion, ovine tendon could be an alternative
source of col I to fabricate scaffold for tissue engineering applications.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Scaffolds that mimic the microenvironment of native tissues are an
essential component in tissue engineering applications [1,2]. The prop-
erties of scaffolds, such as biomaterial composition, architecture and
mechanical strength determine the efciency of cellular interaction,
successful integration with the host tissue and functional effectiveness
in regeneration [3]. Natural, synthetic and composite biomaterials are
extensively used in fabricating scaffolds for tissue engineering applica-
tions [1]. However, natural biomaterials containing extracellular matrix
(ECM) components are preferred over synthetic materials as they pro-
vide advantages regarding their biological properties and other related
bio-mechanistic factors [4].
Among the natural biomaterials, collagen is widely used due to its
excellent biocompatibility and biodegradability, low immunogenicity
and superior versatility for fabricating scaffolds in different forms
⁎ Corresponding author at: Tissue Engineering Centre, Universiti Kebangsaan Malaysia
Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur,
Malaysia.
E-mail addresses: shiplu@ppukm.ukm.edu.my, shiplu56@gmail.com
(S.R. Chowdhury).
(sheets, sponges, gels and nanobres) [5]. Collagen is the major extra-
cellular matrix protein in humans, and is thus in high demand in the tis-
sue engineering, regenerative medicine and cosmetic industries [6].
Collagen from animal sources has been extensively studied and is com-
monly used in various applications [7,8]. Due to its abundance, collagen
has been isolated from various mammalian (bovine, porcine, goat and
rat) and non-mammalian (sh, amphibian and sea plant) sources [7–
11]. Collagen isolated from different sources differs in terms of molecu-
lar organisation, stability and immunogenicity, which affect the func-
tionality of the engineered tissue [9]. Recombinant collagen has
emerged as an alternative source as it is biocompatible and mimics
the natural micro-environment [12]. However, the absence of post-
translational modications on recombinant collagen affects normal bio-
logical functions upon implantation [13]. Thus, careful selection of the
collagen source is important for the fabrication of scaffolds and the sub-
sequent clinical outcome.
There are 29 different types of collagen that have been described in
the literatures; nine of them are commonly available, including collagen
types I, II, III, IV, V, VII, IX, XI and XII [14,15]. Among them, collagen type I
(col I) comprises about 90% of the total collagen of the human body, and
is found abundantly in skin, tendon, bone, ligament and cornea [16,17].
Col I is most often used in the fabrication of scaffolds for tissue

http://dx.doi.org/10.1016/j.msec.2016.05.109
0928-4931/© 2016 Elsevier B.V. All rights reserved.
164 M.B. Fauzi et al. / Materials Science and Engineering C 68 (2016) 163–171
engineering applications, especially for the aforementioned tissues [6].
Similarly, in other mammalians such as bovine, porcine, ovine etc., col
I is abundant in skin and tendon, and commonly isolated from these tis-
sues. Skin mostly contains col I and col III, whereas tendon contains
mostly col I, a small portion of elastin and proteoglycan and some inor-
ganic components [18]. Col I from these tissues isolated via acid extrac-
tion using acetic or citric acid [19], enzymatic digestion using pepsin
[20], neutral salt extraction using disodium phosphate [21], organic ex-
traction using urea [22] and combinations of these methods.
In the native state, the orientation and distribution of col I brils
varies between tissues, and have an effect on cellular organisation. Al-
terations in the structure may cause functional defects [23,24]. For ex-
ample, collagen brils are randomly distributed in the dermal layer of
normal skin. A uniaxial alignment of collagen brils in the dermal
layer results in scar formation and, in severe cases, keloid formation
[25,26]. In contrast, collagen brils in the corneal stroma need to be
aligned, and alterations from this cause blindness [27]. Thus, consider-
ation should be given to the organisation of collagen brils during the
fabrication of scaffolds for tissue engineering applications. The fabrica-
tion of collagen scaffolds normally produces a random distribution of
collagen brils. However, the application of an external intervention
such as mechanical, electrical or magnetic stimulation can align collagen
brils in the scaffold [28,29]. These methods include the application of a
static magnetic eld to dilute collagen gels [30], running an electrical
current through a collagen solution [31], shear ow deposition using a
micro-uidic device [32], ow processing [33], electrospinning of a col-
lagen solution with conductive organic and non-organic solvents [34,
35], spin coating [36], micro-patterning of surfaces [37], stretching of
the collagen lm on a PDMS substrate [38] or even combined tech-
niques such as electrospinning associated with a magnetic eld [29]. Al-
though these methods have excellent potential for aligning collagen
brils, specied preparations and tedious techniques limit their exten-
sive application in tissue engineering and cell biology. Moreover, the ap-
plication of extreme conditions such strong magnetic [39] and electric
elds as well as the use of organic solvents will likely to lead to collagen
denaturation [40].
In our previous studies, ovine (Ovis aries) tendon collagen (OTC) was
extracted, and its biocompatibility towards human dermal broblasts
(HDF) was shown [41,42]. However, the physico-chemical properties
of the collagen were not tested. The aims of the current study were to
characterise and identify the major constituents in the isolated OTC.
Furthermore, an attempt was made to fabricate collagen lms with
aligned collagen brils using simple mechanical intervention, and to
elucidate its effect on HDF attachment, proliferation and alignment.
2. Materials & methods
2.1. Collagen extraction and purication from ovine tendon
Crude tendon was cleaned of fascia and muscle tissues and freeze-
dried for 48 h. The dried tendon was cut into small pieces and dissolved
in 0.35 M acetic acid (Analar, USA) at 4 °C for 24–48 h to extract colla-
gen. Sodium chloride (0.05 g/ml; Sigma, USA) was added to the collagen
solution and incubated at 4 °C for 24–48 h, followed by centrifugation at
10,000 rpm for 45 min. The collagen pellet was then dialysed for 72 h
using dialysis tube (molecular weight cut off = 14 kDa) (Sigma) with
alternate change of disodium hydrogen phosphate solution (Na2HPO4;
0.2 M) and phosphate buffer saline (PBS; 1x) as dialysis buffer every
12 h. Dialyzed collagen was freeze-dried for 24–48 h and re-dissolved
in 0.35 M acetic acid.
2.2. Total protein and total collagen quantication
Quantication of total protein in the OTC solution was performed by
the bicinchoninic acid (BCA) assay kit (Sigma) according to the
manufacturer's protocol. In brief, BCA working solution was prepared
by mixing of reagent A and reagent B. Then, 12.5 l of collagen solution
was mixed with 100 l of BCA working solution, followed by incubation
at 37 °C for 30 min. The absorbance was recorded at 562 nm using a
spectrophotometer. Bovine serum albumin (Sigma) was used as the
standard.
The concentration of total collagen in OTC solution was determined
with the collagen Sircol assay (Biocolor, UK), performed according to
the manufacturer's instructions. Bovine collagen (Biocolor, UK) was
used as the standard. Briey, 1 ml of Sircol dye was added to an equal
volume of standard and test samples. The solution was shaken gently
for 30 min and centrifuged at 12,000 rpm for 10 min. The pellet was
re-suspended in 0.75 ml of ice-cold acid salt wash reagent (contains
acetic acid, sodium chloride and surfactants) followed by centrifugation
at 12,000 rpm for 10 min. The pellet was then mixed with 0.2 ml of alkali
reagent (contain of 0.5 M sodium hydroxide) and absorbance was mea-
sured at 555 nm using a spectrophotometer.
2.3. Identication of protein in the collagen solution
Protein identication in the OTC solution was executed via 1D SDS-
PAGE and MALDI-TOF/TOF mass spectrometry. 1D SDS-PAGE was per-
formed using 5% acrylamide gel at a constant current 400 mA, 150 V
on miniVE apparatus (GE, USA) for 2 h. Later, the gel was stained with
Coomassie blue and an image of the gel was captured using
ImageScanner (GE, USA). The protein bands from the SDS gel were cut
separately and analysed on a MALDI-TOF/TOF mass spectrometer
using mascot sequence matching software (Matrix Science) with the
Ludwig NR Database.
2.4. Chemical characterisation of OTC
Fourier transform infrared spectrometry (FTIR), X-ray diffraction
(XRD) and energy dispersive x-ray spectroscopy (EDS) were used for
the chemical characterisation of OTC. FTIR, XRD and EDS analyses
were conducted using thin lms of air-dried OTC. To analyse the chem-
ical compound, FTIR spectra were recorded from 4000 to 650 cm− 1
using a Perkin Elmer Spectrum 400 FT-Infrared (IR) Spectrometer
with Spotlight 400 Imaging System (PerkinElmer, USA). The graph con-
taining different peaks was analysed with references. The OTC lm was
also tested using X-ray diffraction (XRD; Bruker D8 Advance, USA) with
a setting of 0.02° step size and a scan rate of 1°/min with CuK radiation
( = 1.54 nm). The OTC lm was scanned using a Phenom ProX micro-
scope (Phenom, Netherland) with EDS to obtain the percentage of ele-
ments available in OTC. Data were analysed using integrated software
(ProSuite-Element Identication). For both analyses, rat tail col I
(Sigma) was used as the control.
2.5. Fabrication of OTC lms
Simple mechanical intervention by means of directional movement
was applied using a platform rocker to fabricate the aligned collagen
lm (ACF). In brief, the collagen solution was poured into 12-well cul-
ture plates (1.5 ml/well) and placed on a unidirectional rocker for 6 h
followed by drying at 37 °C in an incubator. The random collagen lm
(RCF) was fabricated without applying any mechanical intervention.
Collagen lms, both ACF and RCF, were sterilised via subsequent wash-
ing with sterile phosphate buffer saline (PBS; Sigma) and 70% ethanol
(Analar). Prior to use for cell culture, OTC lms were washed with a
large amount of PBS and air-dried in a biosafety cabinet.
2.6. Assessment of the collagen orientation in OTC lms
Dried lms were observed under differential interference contrast
(DIC) to visualise collagen bril orientation using A1R confocal laser
scanning microscope (CLSM; Nikon, Japan). The orientation of collagen
brils in OTC lms was also visualised via scanning electron microscopy
 M.B. Fauzi et al. / Materials Science and Engineering C 68 (2016) 163–171
(SEM) using Ultra-High-Resolution FE-SEM SU8020 series (Hitachi,
Japan). Dried OTC lms were coated with platinum for 30 s using Quo-
rum Q150PS (Rowaco, Sweden) sputter coater prior to viewing. Addi-
tionally, OTC lms were stained with picro sirius red (Polyscience,
USA) and observed under polarized light to detect the orientation of
col I brils. In brief, lms were hydrated with distilled water, immersed
in phosphomolybdic acid solution for 2 min and rinsed with distilled
water. Subsequently, lms were placed in picro sirius red for 60 min
and 0.1 N hydrochloric acid for 2 min. Films were then soaked in 70%
ethanol for 45 s. Finally, lms were dehydrated with xylene, air-dried
and mounted for observation.
2.7. Mechanical testing
OTC lms were cut into an appropriate size (1 cm × 3 cm) for me-
chanical testing. Instron 8874 (Instron, USA) tted with a 50 N load
transducer at a crosshead velocity of 0.05 mm/min was used to evaluate
the tensile strain and Young's modulus of the OTC lms. The toughness
of the samples was obtained from area under stress-strain curves [43].
2.8. Human skin cell harvest & culture
Redundant skin tissues were obtained during abdominoplasty from
six consenting patients and processed within 24 h as described else-
where [41,44]. Skin samples (3 cm2) were minced into small pieces
and digested with 0.6% collagenase type I (Worthington, USA) for 5 h
at 37 °C in a shaker incubator, followed by treatment with trypsin-
EDTA (Lonza, USA) for 10 min. The cell suspension was then centrifuged
and resuspended with co-culture medium, containing an equivalent
mixture of Epilife (Gibco/BRL, USA) and F12:DMEM (1:1; Gibco/BRL)
supplemented with 10% FBS (Biowest, USA). Skin cells, including both
keratinocytes and broblasts, were seeded in three wells of a 6-well
polystyrene plate (tissue culture treated; Greiner, Germany) and main-
tained at 37 °C in a 5% CO2 incubator, with medium changes every 2–
3 days. After skin cells reached 70–80% conuence, differential
trypsinisation was performed to dissociate human dermal broblasts
(HDF). HDF were seeded in 75 cm2 culture asks with F12: DMEM con-
taining 10% FBS for continuous culture. HDF at passage 3 were seeded
onto the OTC lms at a density of 1000 cells/cm2. Cells were cultured
for 24 h to measure the cell attachment and until day 7 for analysis of
growth rate. Images of HDF seeded on top of all scaffolds were captured
at ve different locations to calculate the number of attached cells. Cell
attachment and growth rate were analysed using image analysis and
calculated by the following formulae.
Total cell attached  100
Cell attachment ð%Þ ¼
Initial cell seeding
 
‐1
Growth rate h
ln ðFinal cell concentration=Initial cell concentrationÞ
¼
ðFinal culture time‐Initial culture timeÞ
2.9. Live and dead cell staining
Live and dead cell staining (Life Technologies, USA) was used to an-
alyse the viability of HDF on OTC lms according to the manufacturer's
instructions. Briey, live and dead stain solution was prepared with
2 mM calcein AM and 4 mM EthD-1 in PBS as the working solution.
HDF on OTC lms and plastic surface were incubated with the working
solution for 30 min. Cells were then washed with PBS and observed
using A1R CLSM (Nikon).
165
2.10. Detection of HDF orientation
To detect the cellular orientation on OTC lms, HDF were stained
with phalloidin. HDF cultured on OTC lms were xed with 4% parafor-
maldehyde (Sigma) for 1 h at room temperature. Cells were
permeabilised using Triton-X (Sigma) followed by blocking non-specif-
ic binding sites with 10% goat serum (Sigma) for 1 h at 37 °C. Cells were
then stained with DAPI (Invitrogen, USA) for visualisation of the nucleus
and phalloidin (Biotium, USA) to visualise lamentous actin (F-actin).
Images were captured using A1RCLSM (Nikon). The angle of cell align-
ment was measured using NIS-Element Software (Nikon).
Scanning electron microscopy (SEM; FEI, USA) was also performed
to observe HDF morphology and orientation on the OTC lms. OTC
lms cultured with HDF were xed with 4% paraformaldehyde over-
night. Fixed samples were dehydrated by immersion in a series of etha-
nol solutions (30%, 50%, 70%, and 100%; 10 min each). Finally, samples
were allowed to dry overnight in a critical point drying machine.
Dried samples were sputter-coated with nanogold prior to SEM
observation.
2.11. Statistical analysis
The statistical analysis was performed using SPSS v20.0 software.
One-way analysis of variance (ANOVA) was used to compare multiple
groups. P b 0.05 was considered signicant.
3. Results
3.1. Characterisation of ovine collagen
Isolation of collagen from ovine tendon using acetic acid resulted in a
mixture of collagenous and non-collagenous proteins. As shown in Fig.
1a, the major proportion of the proteins was collagen (1.2 mg/ml), i.e.
80% of the total protein content (1.5 mg/ml). For further identication,
extracted collagen was analysed via SDS-PAGE and MALDI-TOF/TOF.
Electrophoretic separation of OTC along with commercial rat tail col I
showed that isolated OTC shared a similar band pattern as the control
col I (Fig. 1b). There were four clear bands, which were identied as
gamma (), beta (), alpha 1 (1) and alpha 2 (2). Analysis of the
bands excised from SDS-PAGE gel using MALDI-TOF//TOF mass spec-
trometry produced highly signicant matches to collagen I alpha I (for
the 1 and  bands) and collagen I alpha 2 (for the 2 band) (Fig. 1c).
However, no match was detected for the  band.
Chemical characterisation of OTC was performed via FTIR, XRD and
EDS analysis in comparison with rat tail col I (control). As shown in
Fig. 2a, the IR spectrum of OTC showed absorbance peaks for NH
stretching (3302 cm− 1), CH2 asymmetrical stretching (2923 cm− 1),
amide I (1632 cm− 1), amide II (1548 cm− 1) and amide III
(1237 cm− 1). The XRD patterns of OTC and rat tail col I was showed
in the Fig. 2b. Both collagens were displayed no denaturation of colla-
gen, with the existence of a distinct peak located at 2θ (2Theta) = 7°.
A broad diffuse peak at about 2θ = 20° indicated the characteristic in-
terchain spacing of the collagen triple helix. Elemental analysis via
EDS detected three major elements in OTC, including carbon (C =
50.17 ± 0.32%), nitrogen (N = 21.07 ± 0.52%) and oxygen (O =
27.19 ± 2.92%), which closely corresponds to rat tail col I (Fig. 2c). Alto-
gether, these results indicate that col I was the most abundant protein in
the OTC solution.
3.2. Properties of the collagen lms
OTC lms were fabricated with or without mechanical intervention,
denoted as aligned collagen lm (ACF) and random collagen lm (RCF),
respectively; both lms appeared as translucent, soft lms with a
smooth surface (Fig. 3a). Observation of collagen brils under DIC (Fig.
3b), SEM (Fig. 3c) and polarized light (Fig. 3d; picro sirius red staining)
166 M.B. Fauzi et al. / Materials Science and Engineering C 68 (2016) 163–171

Fig. 1. a) Total protein and collagen extracted from ovine tendon via the bicinchoninic assay and Sircol collagen assay, respectively. b) SDS-PAGE analysis of extracted collagen: lane (1)
protein molecular weight marker, (2) col I from rat tail, (3) col from ovine tendon. c) MALDI-TOF/TOF mass spectrometer analysis of gamma, alpha-1, alpha-2 and beta bands excised from
the gel. Specic peptide sequences were compared to the Mascot database to determine the signicant hits of each available band.

revealed that collagen brils were distributed in random and aligned
orientations on the RCF and ACF, respectively. However, changes in
the alignment of the collagen brils did not signicantly affect the me-
chanical properties such as tensile strain and Young's modulus (Fig.
3e). In addition, the toughness of the ACF (~70 kJ/m3) was higher com-
pared to the RCF (~ 60 kJ/m3), but no signicant difference was
observed.
demonstrated the presence of prominent actin stress bres, cells on
the polystyrene surface possessed a high number of lopodia at both
day 1 and 7 (Fig. 5a and 5b). As expected, HDF on ACF demonstrated
an aligned orientation along the direction of mechanical intervention.
It was also noted that actin stress bres were aligned in a similar direc-
tion. In contrast, HDF on the polystyrene surface and RCF were random-
ly distributed (Fig. 5c).

3.3. Biocompatibility of OTC lms 4. Discussion

HDF were cultured on polystyrene, RCF and ACF surfaces. It was
found that HDF cultured on RCF and ACF were viable (Fig. 4a), indicating
no cytotoxic effect. Moreover, the HDF attachment and growth rate
were signicantly higher on OTC lms compared to polystyrene surface.
However, no differences in HDF attachment and growth rate were ob-
served on RCF versus ACF (Fig. 4b and 4c).
3.4. Morphological features of HDF on collagen lms
HDF cultured on OTC lms (ACF and RCF) and polystyrene surface
showed distinct morphological features. As shown in Fig. 5, HDF on
the polystyrene surface showed a at and spread morphology, whereas
cells on OTC lms demonstrated an elongated, thin and less spread out
morphology. Although HDF on the polystyrene surface and OTC lms
Type I collagen (col I) is the major constituent of different tissues
such as skin, tendon, bone, ligament and cornea [16,17]. It has been
used extensively for biomedical applications in medicine, cosmetology,
pharmacology and tissue engineering. Most of the col I for biomedical
applications is extracted from mammalian sources, especially bovine
or porcine skin and tendon as they have the most similar properties
with human [23]. However, the potential of transmitting pathogens
(BSE; bovine spongiform encephalopathy, TSE; transmissible
spongiform encephalopathy and FMD; foot and mouth disease) [19,
20] and religious issues limit the use of col I from these sources. These
collagens also have the trace of immunogenicity in the clinical applica-
tions [45]. In our previous study, collagen has been extracted from the
ovine tendon, which is more acceptable across cultures and religions,
and has less susceptibility towards infection with transmitting
 M.B. Fauzi et al. / Materials Science and Engineering C 68 (2016) 163–171 167

Fig. 2. Chemical structural of col I via Fourier transform infrared (FTIR) (a), X-ray diffraction (XRD) analysis (b) and col I elements analysis via energy dispersive X-ray spectrometry (c).
Major elements in col I are of carbon, oxygen and nitrogen.

pathogens [41,42]. Moreover, in our ongoing experiment no immuno-
genicity of OTC was detected in in vitro and in vivo model (data not
shown). In the current study, we have shown that collagen extracted
from ovine tendon mainly consists of col I, which is 80% of the total pro-
tein. It is suitable for the fabrication of random and aligned collagen
lms that can regulate cellular orientation.
Acetic acid extraction is commonly used for col I from tendon as this
method is simple, efcient and results in high yields [9]. However, the
concentration of acetic acid used varies among studies [46]. In this
study, a low concentration of acetic acid (0.35 M) was used together
with salt-leaching and dialysis to extract collagen from ovine tendon.
The concentration of total collagen in the extract was 1.2 mg/ml,
168 M.B. Fauzi et al. / Materials Science and Engineering C 68 (2016) 163–171

Fig. 3. Characterisation of RCF and ACF. (a) Gross appearance of OTC lm. (b) Microscopic appearances of OTC lm; RCF and ACF in DIC. (c) Scanning electron microscopy of RCF and ACF,
(Scale bar = 2 m). (d) Orientation of OTC brils in RCF and ACF via polarized microscopy. (Scale bar = 50 m). (e) Mechanical testing of OTC lm (RCF and ACF); n = 6.

Fig. 4. a) Biocompatibility analysis of OTC I towards human dermal broblasts (HDF) via live (green) and dead (red) cell staining. b) Percentage of cell attachment for HDF seeded on
plastic, RCF and ACF at 2 h, c) growth rate of HDF from day 1 to day 7, * Represents signicantly lower cell attachment and growth rate compared to RCF and ACF (p b 0.05). (Bar = 50 m).
 M.B. Fauzi et al. / Materials Science and Engineering C 68 (2016) 163–171 169

Fig. 5. Immunochemical (a) and SEM analysis (b) of the HDF growth pattern and cell morphology seeded on plastic, RCF and ACF at day 1 and 7. White two headed arrow represented the
direction of OTC type I lm fabricated via uni-directional intervention. The evaluation of HDF growth deviation at ±90° from 0° (c). (Bar = 50 m).

which comprised 80% of the total protein content. A comparative study
with rat tail col I via SDS-PAGE analysis showed similar band patterns;
the bands were identied as two alpha chains (1 and 2), and the sec-
ondary and tertiary structures of beta and gamma chain, respectively.
MALDI-TOF/TOF spectrometry with the Mascot database showed
signicant hits for the alpha 1, alpha 2 and gamma chain of col I, but
no signicant hits for the beta chain. FTIR and EDS analysis of OTC was
comparable with that of rat tail col I. FTIR analysis with mid-infrared
wave number range (4000–650 cm−1) shown the presence of typical
peaks of amide I, II and III for collagen. However previous study
170 M.B. Fauzi et al. / Materials Science and Engineering C 68 (2016) 163–171
shown that it is not suitable to distinguish col I from other types of col-
lagen [47]. But, FTIR data can provide valuable information on the struc-
tural properties of isolated collagen from ovine tendon. The FTIR peaks
at 1632 cm−1 indicate that OTC attribute -sheet and triple helix struc-
ture. In addition, the ratio of the peak intensity at 1450 cm−1 to that at
1235 cm−1, which indicates the triple helical content in a collagen sam-
ple [47], was also comparable with rat tail collagen. This result was sup-
ported by the XRD analysis, which attributed a diffused peak at about
2θ = 20° indicating the presence of triple helical structure of col I in
OTC [48–50]. In addition, the broad diffraction peaks implying that the
collagen were poorly crystalized. Together all these results conrm
that the extracted collagen from ovine tendon contains mostly col I.
Collagen can be easily modied to fabricate various types of scaffold
such as hydrogels, lms, sponges and microspheres [51–53]. Collagen
sponges have been demonstrated to be favourable for human dermal -
broblast (HDF) attachment and spread [27,41]. Collagen lms are used
in bone, cornea, skin and vascular tissue engineering, as well as in
drug delivery [54]. In this study, the OTC lms were fabricated via air
drying, and were found to be thin and transparent. The col I brils
were randomly distributed; therefore this lm is suitable for use in
skin tissue engineering where a random distribution of collagen brils
is necessary to reduce scarring [37]. In several tissues such as ligaments
and tendons, collagen brils are highly aligned, which is essential for tis-
sue functionality [55]. In this study, we employed a simple mechanical
intervention by means of the uniaxial movement of the collagen solu-
tion using a platform rocker to align col I brils. As expected, col I brils
were found to be aligned in the OTC lms along the direction of
movement.
Cellular compatibility with a biomaterial is a key factor determining
whether a scaffold is suitable for tissue engineering applications. Col I
has been shown to promote the attachment and proliferation of various
cell types [56,57]. Consistent with these studies, HDF showed signi-
cantly higher attachment and growth on OTC lms compared to cells
grown on polystyrene surface; however, no differences were observed
between RCF and ACF. The cellular morphology observations suggest
that HDF were elongated on all surfaces, but were comparatively thin-
ner on OTC lms compared to the polystyrene surface. Expectedly,
HDF were oriented randomly in RCF, whereas uniaxial orientation in
the direction of the collagen brils was observed in ACF. Thus, modica-
tions to the orientation of collagen brils in lms, which directly affects
cellular alignment, will provide the opportunity to fabricate scaffolds
depending on the tissue requirements. In the future, in vitro and in
vivo experiments need to be performed to elucidate the importance of
random and aligned collagen lms in developing functional tissue
substitutes.
5. Conclusion
Collagen extracted from ovine tendon contains mainly col I, which
has similar physico-chemical properties as commercially available rat
tail col I. Thus, ovine tendon has the potential to be a safer source of
col I due to its greater availability and wide acceptability. Collagen
lms fabricated from OTC were shown to increase HDF attachment
and growth. The random distribution collagen brils led to a random
orientation of the attached cells. Introduction of uniaxial mechanical in-
tervention facilitated the fabrication of lms with uniaxially aligned col-
lagen brils, leading cells to orient in the direction of movement
without altering growth and morphological properties. These collagen
lms, both RCF and ACF, have great potential to be used as scaffolds
for the fabrication of different tissue substitutes.
Conict of interest
The authors declare that they have no conict of interest.
Acknowledgement
This work was supported by the [Ministry of Science, Technology
and Innovation (MOSTI)] under Grant [02-01-02-SF0964]; [Universiti
Kebangsaan Malaysia] under Grant [Arus Perdana: AP-2013-015]; [Fun-
damental Grant UKM Medical Centre] under Grant [FF-2014-370].
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