European Polymer Journal 119 (2019) 426–437

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European Polymer Journal

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

3D bio-printing of levan/polycaprolactone/gelatin blends for bone tissue T

engineering: Characterization of the cellular behavior
Busra Tugce Duymaza,b, Fatma Betul Erdilera,b, Tugba Alana,b, Mehmet Onur Aydogdua,c,
Ahmet Talat Inana,d, Nazmi Ekrena,e, Muhammet Uzunf, Yesim Muge Sahing,h, Erdi Bulush,
Faik Nüzhet Oktara,b, Sinem Selvin Selvib,i, Ebru ToksoyOnerb,i, Osman Kilica,j,
⁎
Muge Sennaroglu Bostank, Mehmet Sayip Erogluk,l, , Oguzhan Gunduza,m
a
Center for Nanotechnology & Biomaterials Application and Research at Marmara University, Goztepe Campus, 34722 Istanbul, Turkey
b
Department of Bioengineering, Faculty of Engineering, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey
c
Department of Metallurgical and Materials Engineering, Master of Science, Institute of Pure and Applied Sciences, Marmara University, 34722 Istanbul, Turkey
d
Department of Mechanical Engineering, Faculty of Technology, Marmara University, 34722 Istanbul, Turkey
e
Department of Electrical and Electronics Engineering, Faculty of Technology, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey
f
Department of Textile Engineering, Faculty of Technology, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey
g
Biomedical Engineering Department, Faculty of Engineering & Architecture, Istanbul Arel Univ., Istanbul, Turkey
h
ArelPOTKAM (Polymer Technologies and Composite A & R Center), Istanbul Arel University, Turkey
i
IBSB, Department of Bioengineering, Faculty of Engineering, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey
j
Department of Electrical and Electronics Engineering, Faculty of Engineering, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey
k
Department of Chemical Engineering, Faculty of Engineering, Marmara University, Istanbul, Turkey
l
TUBITAK-UME, Chemistry Group Laboratories, PO Box 54, 41471 Gebze, Kocaeli, Turkey
m
Department of Metallurgy and Materials Engineering, Faculty of Technology, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: Poly(ε-caprolactone) (PCL), gelatin (GT) and different concentrations of low molecular weight Halomonas levan
Halomonas levan (HLh) were combined and examined to develop physical networks serving as tissue scaffolds to promote cell
3D printing adhesion for biocompatibility. Three-dimensional bioprinting technique (3D bioprinting) was employed during
Tissue engineering manufacturing the test samples and their comprehensive characterization was performed to investigate the
physicochemical properties and biocompatibility. Physical properties of the printing materials such as viscosity,
surface tension, and density were measured to determine optimal parameters for 3D bioprinting. The scanning
electron microscope (SEM) was used to observe the morphological structure of scaffolds. Fourier-Transform
Infrared Spectroscopy (FT-IR) and differential scanning calorimetry (DSC) were used to identify the interactions
between the components. In-vitro cell culture assays using standard human osteoblast (Hob) cells showed in-
creased biocompatibility of the printing materials with increasing HLh content. Thus, the formulations including
the HLh are expected to be a good candidate for the production of 3D printed materials.

1. Introduction
Organ dysfunction is a common health problem that originated from
diseases, traumas, infections and aging factors in tissues or organs.
Although these problems have been decreasing by organ or tissue
transplantation [1], restrictions by certain limitations such as lack of
organ donation and tissue rejection induced by the immune system of
the host have been significant obstacles [2]. Regenerative medicine and
tissue engineering are the alternative fields to biomedical sciences,
which replaces the lost or damaged tissues by restoring or improving
the tissue functions [3]. These fields are based on the accumulation of
body cells on porous 3D scaffold materials made of natural or bio-
compatible synthetic polymers, which require the contributions of
biochemical and physicochemical factors enabling tissue growth
through cell proliferation [4]. The 3D printing technique is used in
various industries, and tissue engineering is focused on material com-
binations to produce controlled scaffolds providing cell growth on de-
fected tissues [5–7]. Cell-seeded scaffolds can act as a template for
tissue formation [8]. Furthermore, surface reorganization and porosity
of the scaffolds are expected to promote cell adhesion, migration, cell

⁎
Corresponding author at: Department of Chemical Engineering, Faculty of Engineering, Marmara University, Goztepe, 34722 Istanbul, Turkey.
E-mail addresses: mehmet.eroglu@marmara.edu.tr (M.S. Eroglu), ucemogu@ucl.ac.uk (O. Gunduz).

https://doi.org/10.1016/j.eurpolymj.2019.08.015
Received 9 July 2019; Received in revised form 8 August 2019; Accepted 13 August 2019
Available online 14 August 2019
0014-3057/ © 2019 Elsevier Ltd. All rights reserved.
B.T. Duymaz, et al.
differentiation, and proliferation. Additionally, materials used for
scaffold construction should possess the suitable level of biodegradation
to prevent losing the scaffold form with extremely rapid biodegradation
and should provide enough time for cells to produce their extracellular
matrix (ECM) while their presence on the scaffolds [3,9].
Bone tissue is known for self-repairing ability. However, large bone
defects cannot be entirely treated without external operations such as
autografts or allografts. Today, bone tissue engineering is focused on
techniques to synthesize bone-like scaffolds to preserve or enhance its
functions [10]. Fibrous structure, porosity, pore size and modifications
of selected polymer scaffolds are important to become a good candidate
for bone tissue engineering [11]. Pore size higher than 300 µm is re-
ported to provide bone formation and enhance vascularisation.
Poly(ε-caprolactone) (PCL) is a biodegradable and biocompatible
semi-crystalline synthetic polyester, which is mostly used to be 3D bone
scaffold material due to its high mechanical strength and relatively low
melting temperature. The high pore volume of PCL scaffolds provides
enhanced bone regeneration and increased strength and biocompat-
ibility on HOb cells. On the other hand, due to its slow degradation
capability, PCL appears more promising over the other biodegradable
equivalents [12,13].
Gelatin (GT) is a naturally originated material consisting of small
peptides formed by hydrolysis of collagen fibers. Gelatin can promote
wound repair and has remarkable biocompatibility, which overcomes
some limitations of using synthetic polymeric materials in bioprinting
[14].
Halomonas levan (HL) is a β (2 → 6) - linked fructan type poly-
saccharide, produced by various microorganisms and plants. It is a
water-soluble, non-toxic, biocompatible and strongly adhesive poly-
saccharide. Halophilic Halomonas smyrnensis AAD6T is classified as the
first levan producing extremophile, which provides unsterile and low-
cost production. Antioxidant, anticoagulant and heparin mimetic ac-
tivity together with peptide and protein-based drug delivery properties
of HL were reported [15]. In our previous study, improved bio-
compatibility and cell proliferation at the HL/N-isopropyl acrylamide
(PNIPAm) hydrogel surface was observed with increasing HL portion,
which was attributed to the enhancement of HL on the hydrogel surface
at close to body temperature [8]. In our separate study, we observed
that HL induced mouse fibroblast L929 cell viability and proliferation
when included in poly(ethylene oxide)/chitosan (PEO/Ch) blend films
[16]. Additionally, HL suppressed the PEO crystallinity via hydrogen
bonding formed between hydroxyl groups of HL and etheric C-O-C
groups of PEO, which resulted in more amorphous, and thus, more
homogeneous and flexible blend films [16]. HL is a high molecular
weight (4,200 kD) fructan [8], which could result in viscosity problems
during the 3D bioprinting process. Therefore, low molecular weight
Halomonas levan (HLh) was prepared by nitrous acid hydrolysis of HL.
It is noteworthy that, the presence of HLh in the scaffold compositions
results in a more homogeneous and flexible structure with induced Hob
cell proliferation. Considering the hydrogen bonding formed between
PCL, GT, and HLh, the scaffold matrix is expected to keep their uniform
3D shape for an extended period of time with more bio-availability as
reported in the previous studies explaining the characteristics of Poly
(ethylene oxide) blends with sodium alginate [17,18].
The present study aims to develop highly biocompatible 3D scaf-
folds with improved biocompatibility and physical properties. Different
combinations of PCL, GT, and HLh were used for this purpose. Although
many articles concerning PCL/GT based formulations for 3D printing
are available in the literature [19,20], to the best of our knowledge, this
is the first study reporting 3D printing results of HLh containing PCL
and GT scaffold formulations. Including the high bioadhesive HLh into
the 3D printing compositions is expected to provide increasing bioa-
vailability of the scaffolds. Additionally, the hydroxymethyl groups of
HLh can interact with carbonyl groups of PCL and primary amine
groups of GT, forming more stable physical networks in 3D bioprinting
[21]. The initial results of the mechanical properties of the developed
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European Polymer Journal 119 (2019) 426–437
scaffolds are very promising for future development. The scaffolds were
characterized by their surface tension, viscosity, and density properties.
Moreover, their morphological, thermal and chemical properties along
with in-vitro cell culture, using HOb cells for the modeling of bone
tissue, were studied.
2. Materials and methods
2.1. Materials
Dimethyl sulfoxide (DMSO), Dichloromethane (DCM) and poly(ε-
polycaprolactone) (PCL, Molecular weight 80,000 g/mol), and gelatin
(GT, Type A, Gel strength ~ 300 g Bloom) were obtained from
Sigma–Aldrich (St. Louis, MO, USA). Halomonas levan (HL) was mi-
crobially produced by Halomonas smyrnensis bioreactor cultures and
purified as described [22]. Sodium nitrite (NaNO2) and sodium nitrate
(NaNO3) were supplied from JT Baker. Acetic acid was purchased from
Sigma-Aldrich. Sodium carbonate (Na2CO3) was supplied from Riedel-
deHaen. For cell culture experiments; WST-1(4-[3-(4-iodophenyl)-2-(4-
nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) was obtained
from Roche Applied Science(Germany). DMEM(Dulbecco’s Modified
Eagle Medium), Trypsin, Fetal Bovine Serum (FBS) and Penicillin-
Streptomycin were purchased from PAN Biotech (UK Ltd). HOb cell line
was kindly provided by Gamze Torun Kose (Yeditepe University,
Turkey). DAPI (4′,6-diamidino-2-phenylindole) was purchased from
AppliChem(Germany), Trypan Blue, Phosphate Buffered Saline(PBS)
tablets were obtained from MP Biomedicals.
2.2. Instrumentation
Scanning electron microscope (SEM) images of the printed samples
at different magnifications were obtained using a Zeiss EVO MA10
(Germany) SEM instrument which was operated at 20 kV acceleration
voltage. Samples were sputter-coated with a thin layer of gold. Melting
behavior and temperature of pure PCL and the blend samples were
comparatively determined by differential scanning calorimetry (DSC)
using Perkin-Elmer Thermal Analyzer System equipped with Jude DSC
system and Pyris software. Approximately 5–7 mg of each sample was
tested with two successive heating and cooling cycle under an argon
atmosphere (200 ml.min−1 flow rate). Heating and cooling rates were
10 °C/min and 5 °C/min, respectively. 2D 13C–1H NMR spectra were
recorded using a Varian 600 MHz NMR instrument at room temperature
in D2O. Molecular weight determination was performed using gel per-
meation chromatography-light scattering system (GPC-LS) equipped
with Perkin Elmer 200 high-pressure pump, serial connected column
system (Ultrahydrogel 120 + Ultrahydrogel 250 + Ultrahydrogel
500 + Ultrahydrogel 100), Wyatt Optilab differential refractometer
(654 nm) and Dawn Heleos multi-angle light-scattering detector.
Mobile phase was 0.1 M NaNO3 solution in 2% acetic acid/water (v/v)
mixture with a flow rate of 1.0 ml/min. A JASCO FT-IR-4700
Spectrometer (USA) was used to determine intermolecular interactions
between the components. FT-IR analyses were performed between 400
and 4000 cm−1. Mechanical testing of the scaffolds was conducted
using a universal testing machine (Devotrans, Turkey) with a cross-
head speed of 1.0 mm/min. The surface area of the samples was mea-
sured using a digital clipper (500, Mitutoyo, USA).
2.3. Preparation of the hydrolyzed Halomonas levan (HLh)
Halomonas levan (HL) was obtained from halophilic Halomonas
smyrnensis AAD6 T cultures according to the method described in our
previous study [8]. Low molecular weight levan (HLh) was obtained by
the acid hydrolysis of high molecular weight HL (4,200 kDa) under mild
condition. HL (3.0 g) was dissolved in 150 ml of 2% acetic acid/water
(v/v) solution. To this solution, NaNO2 (0.5 g dissolved in 5 ml water)
was added dropwise under vigorous stirring, which was continued for
B.T. Duymaz, et al. European Polymer Journal 119 (2019) 426–437

Table 1
Compositions, 3D printing conditions and mechanical properties of the samples.
Compositions (wt%) 3D printing conditions Mechanical properties

Sample no PCL GT HLh Print head travel speed (mm/s) Build plate temperature (°C) Nozzle diameter (mm) Compressive strength (MPa)

S1 20 0.5 0 100 45 0.4 35.20 ± 0.2

S2 20 0.5 0.2 150 55 0.4 33.12 ± 0.3
S3 20 0.5 1.2 150 47 0.4 32.93 ± 0.4
S4 20 0.5 1.6 130 55 0.4 30.54 ± 1.2

one day at 60 °C and then additional two days at 25 °C. The solution was
neutralized with 1.0 M of Na2CO3 and dialyzed against ultrapure water
using SpectraPor®Dialysis Membrane cut of 2 kDa. The product was
freeze-dried (at −60 °C and 0.001 atm), which yielded low molecular
weight levan (HLh) as a white solid (2.1 g, yield 70%). The molecular
weight of HL and HLh was determined using GPC-LS and NMR spec-
troscopy was used to determine if any structural change after hydrolysis
of HL [8].
2.4. Preparation of solutions and production of bone scaffolds
Different compositions were prepared to determine the optimum
concentration for bone tissue printing. Four different scaffold compo-
sitions were prepared from stock solutions of PCL/GT (20:0.5 wt%) in a
mixture of DCM/DMSO (60:40 wt%). PCL/GT and HLh were mixed at
desired proportions given in Table 1 and then stirred at room tem-
perature for 24 h. The compositions were printed by using a modified
3D printer (Ultimaker® 2+, Netherlands), which utilized a fused de-
position modeling (FDM) system. It included a computer-aided design
(CAD) technology using a heatable build-plat. A digital syringe pump
was used to feed the solutions. Nozzle diameter was 0.4 mm, the build-
plate temperature was varied between 45 and 55 °C, and print head
travel speed was kept between at 100–150 mm.s−1. The 3D bioprinting
compositions and their printing parameters are given in Table 1.
2.5. Solution characterization
The density of the PCL/GT and the PCL/GT/HLh solutions at dif-
ferent concentrations were measured using a standard 10 ml pycn-
ometer. The surface tension of the solutions was determined using a
Sigma 700, DYNE, tensiometer (UK). The solution viscosity of the
samples was determined using a Brookfield type viscometer
(Brookfield, DV-E, Massachusetts, USA). Measurements were performed
at 25 °C.
2.6. Biodegradation studies
10 mg of each dry scaffold sample was placed in a glass test tube
containing 10 ml of PBS solution (pH 7.0). The test tubes were in-
cubated in a thermostatic oven at 37 °C. After 1, 3, 5 and 7 days in-
cubation, samples were removed from the test tubes, quickly blotted
with a filter paper and weighted to calculate water absorption capa-
cities using Eq. (1). Subsequently, each swollen sample was dried and
weighed again to calculate the biodegradability using Eq. (2).
Ww − Wr
Waterabsorption (%) = × 100
Wr
W0 − Wr
Weightloss (%) = × 100
W0
where Ww is the weight of the swollen sample, Wr is the weight of the
dry sample after degradation and W0 is the initial weight of the dry
sample.
2.7. Biocompatibility of the scaffolds
The WST-1 viability test was performed using HOb cell line for 24,
48 and 72 h. Before seeding, the cells were cultured in DMEM complete
medium (10% FBS, 1% penicillin-streptomycin) at 37 °C in 5%CO2(6–8
passages). Sterilization was provided under UV light for each side,
followed by incubation in 70% ethanol for 30 min and 2% penicillin-
streptomycin/PBS for 2 h. The scaffolds were placed into 24 well plates
and pre-saturated with DMEM complete medium for 2 h. After the
Trypsinization process, cells were dyed by trypan blue and counted by
creating a colorimetric difference since the dye used is meant to be
absorbed only by the cells. HOb cells were seeded on the scaffolds at the
density of 1.0 × 104 and incubated for 24, 48, and 72 h at 5%CO2.
WST-1 test reagent was added onto wells and incubated at a dark place
for 37 °C and 5% CO2 for 2 h. The absorbance was measured at 405 nm.
Adhesion of HOb cells on scaffolds was determined with fluorescence
microscopy. Scaffolds with cells and control wells were incubated for
24, 48, and 72 h, and fixation was performed with 4% PFA/PBS solu-
tion and then washed with PBS for 2 times. Cells on the scaffold samples
were observed under a Leica DMLB2, (Leica Microsystems, Wetzlar,
Germany) fluorescent microscopy.
2.8. Statistical analysis of the biocompatibility tests
The statistical analysis was determined by Graph Pad V 5.0 prims
program. The statistically significant mean differences between scaf-
folds and controls were estimated by One-Way ANOVA with Tukey’s
method. The confidence interval (CI) for all data was assumed to be
95% and p-values below 0.05 were regarded as significant.
3. Results and discussion
3.1. Characterization of hydrolysed Halomonas levan (HLh)
HLh was prepared by acid hydrolysis of native Halomonas levan
(HL). After hydrolysis, the number average molecular weight (Mn) re-
markably reduced from 4.247 × 106 g/mol to 1.051 × 105 g/mol with
better water solubility. The specific refractive index (dn/dc) of HLh in
0.1 M NaNO3 in 2% (v/v) acetic acid/water solution at 25 °C was de-
termined to be 0.1370 ± 0.0028 ml/g. GPC-LS chromatograms (Fig.
S1) and molecular weight values (Table S1) of HL and HLh are given in
the supporting file. To determine if any structural change that occurred
in hydrolysis, NMR characterization of HL and HLh was performed.
Fig. 1A and B show 1H NMR spectra of HL and HLh. Fructose rings of
HLh showed similar 1H NMR signals at the same shift values observed
for HL at (δ, ppm): H1(3.54, 3.62), H3(4.04), H4 (3.96), H5(3.81),
H6(3.42, 3.75). For further characterization, the 2D 13C–1H NMR cor-
(1) relation spectrum of HLh was recorded (Fig. 1C). 13C NMR signals of
HLh (δ, ppm); C1(59.8), C2(1 0 4), C3(76.2), C4(75.0), C5(80.1)
C6(63.3) and corresponding 1H NMR signals (δ, ppm); H1(3.54, 3.62),
H3(4.04), H4(3.96), H5(3.81), H6(3.42, 3.75) are observed. Since C2
(2)
carbon is quaternary, the corresponding 1H NMR signal was not ob-
served in the spectrum. NMR characterization showed that the nitrous
acid hydrolysis of HL did not cause any decomposition in fructose rings
of HL, while reducing the molecular weight.

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Fig. 1. 1H NMR spectrum of native Halomonas levan (A); 1H NMR spectrum of hydrolysed Halomonas levan (B); 1H–13C correlated NMR spectrum of hydrolysed
Halomonas levan (C).

3.2. SEM-XSEM interconnectivity of the fabricated 3D scaffolds were displayed in this

figure. The HLh portion of the scaffolds increased by 0.2, 1.20, and
Fig. 2 shows the production setup, SEM and XSEM images of the 3D 1.60 wt% to optimize the printing parameters to obtain smoother bio-
printed scaffolds. The structural integrity, porosity, and printing process and better scaffold features. The viscosity of printing

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B.T. Duymaz, et al.
Fig. 1. (continued)
materials is a particularly important parameter. Lower viscosity results
in deformation and collapse in printed scaffolds. On the other hand,
higher viscosity generally results in an undesirable defect in scaffold
and nozzle jam during printing, which leads to morphological differ-
ences between the scaffolds. In our formulations, while the percentages
of PCL and GT were kept constant, the viscosity of the printing material
increased with increasing HLh portion. As shown in Fig. 2, the scaffold
formulation containing 1.20 wt% of HLh concentration showed uniform
shape with dense appearance and pores compared to the others.
Moreover, the addition of HLh into the PCL/GT solution exhibited finer
and ordered scaffold structures as well as an increment in the number of
pores. As reported in a previous study [23], a ternary composite na-
nofibrous scaffold from PCL/GT/chitosan was fabricated for tissue en-
gineering applications. SEM images showed that the fiber morphology
of the scaffolds was found to be influenced by the concentrations of
PCL/GT/chitosan in the polymer solution. Increasing the PCL and de-
creasing the GT concentrations were suggested to obtain better mor-
phology with uniform structure [23]. In the present study, similar re-
sults were obtained consistent with the outcome of the previous study
[23]. The higher concentration of PCL compared to GT in the PCL/GT
scaffold provided better results such as morphological integrity and
improved solidification while bioprinting as well as the smooth bio-
printing phase.
3.3. Mechanical properties
The ultimate compressive strength of a sample means the load ap-
plied per unit area at which the sample will fail in compression. The
compressive strength value of a scaffold should be close to the value of
the native bone to maintain a certain level of mechanical compatibility.
The mechanical properties of pure PCL scaffolds produced by different
methods were reported in various studies. The compressive modulus,
resulting in a wide range of compressive strength (14.9 MPa −
215 MPa), is believed to be directly related to the production method
and the morphology of the scaffolds [24]. It has also been reported that
the compressive strength of the pure and bulk PCL was 38.7 MPa [25].
Tissue scaffolds were also manufactured using low molecular weight
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European Polymer Journal 119 (2019) 426–437
PCL with a compressive strength value of 21.5 MPa. However, me-
chanical properties can be improved by using higher molecular weight
polymer [24]. The compressive strength of sample-S1 (Table 1), con-
sisting of PCL and GT, was measured to be 35.20 MPa. It was reported
that the GT incorporation into the PCL increased the flexibility of PCL/
GT blend [26]. However, the compressive strength gradually decreased
as the HLh was included in the formulation. It was measured to be
30.54 MPa for the formulation containing 1.6 wt% of HLh (sample-S4).
This could be due to the more brittle nature of HLh than PCL and GT
[27]. Additionally, the average compressive strength value of the
human femur cortical bone was reported to be between 96.35 and
143.35 MPa for female and 122.86 to 161.44 MPa for male [28].
3.4. FT-IR
Fig. 3 shows the FT-IR spectra of native components and the scaffold
sample-S3. In the FT-IR spectrum of pure PCL, the characteristic ab-
sorption bands at 1749 cm−1 and 1185 cm−1 are due to the carbonyl
stretching and axial symmetric stretching of CeOeC groups, respec-
tively. The peaks in the range of 2850–3000 cm−1 were assigned to
different CeH vibrational stretching absorption of PCL [29]. In the FT-
IR spectrum of pure GT, the bands at 1636 cm−1 and 1524 cm−1 are
due to the characteristic peptide amide-1 and amide-2 absorptions,
respectively [26]. In the FT-IR spectrum of pure HLh, a broad absorp-
tion peak observed between 3200 cm−1–3500 cm−1 is due to the OeH
stretching of OH and CH2eOH groups of fructose rings. The strong
absorption peak at 1120 cm−1 was attributed to the CeOeC symmetric
bending vibration of the fructose ring of HLh [8,30]. FT-IR spectra of
the scaffold formulations exhibited the characteristic absorption peaks
of the pure components. As an example, in the FT-IR spectrum of the
sample-S3, which contains 1.2% of HLh, the peaks of PCL at 1749 cm−1
and 1185 cm−1 shifted to 1720 cm−1 and 1165 cm−1, respectively.
Moreover, the CeOeC band of HLh and the amide I band of GT shifted
to 1106 cm−1 and 1626 cm−1, respectively. These remarkable shifts of
characteristic absorption peaks of the pure components could be con-
sidered as evidence of their energetic interactions in scaffold formula-
tions. This confirmed the hydrogen bond formation between scaffold
B.T. Duymaz, et al. European Polymer Journal 119 (2019) 426–437

Fig. 2. (a) Schematic representation of scaffolds and 3d printing system with surface and cross-sectional SEM microscope of sample-S1 (b, b1); sample-S2 (c, c1);
sample-S3 (d, d1); sample-S4 (e, e1) with high and low magnifications for each sample respectively, showing the morphological structure of the scaffolds and the
porous structure of it (f) with the mechanical properties of the scaffolds.

components, resulting in physical crosslinking leading a more di-
mensionally stable 3-D printing scaffold.
3.5. DSC
DSC curves of pure PCL and sample-S3 were recorded to determine
the interaction of the blend components (Fig. 4). Successive heating and
cooling cycle was used for the removal of possible impurities and the
elimination of possible error arising from PCL crystallization condition.
Although sample-S3 and pure PCL displayed the same melting tem-
perature (approx. 54 °C), they exhibited different melting behavior.
While sample-S3 had a narrow and sharp endotherm, pure PCL ex-
hibited relatively wide melting endotherm. HLh and GT do not have a
melting temperature at around 54 °C. By considering the different
melting behavior could be due to the hindrance of the segmental
movement of PCL chains in sample-S3 to some extent, which was
probably due to the hydrogen bonding formed between the components
in the presence of GT and HLh [17]. This was another confirmation of
the intermolecular interactions of the components and, thus, the for-
mation of physical networks between the components, which provided
more dimensionally stable 3D printing scaffolds with an effective
bioavailability.
3.6. Solution characterization
Surface tension, viscosity, and density of the printing solutions are
displayed in Fig. 5. The solution viscosity of the samples-S2-S4 was
found to be higher than that of the sample-S1. Surface tension had a

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Fig. 3. FT-IR spectra of native components and sample-S3.

high value for sample-S1 and sample-S2 solutions, which was in the
range of 47.56–84.50 mN.m−1 and decreased with the increasing of
HLh content (for sample-S3 and sample-S4). The density was measured
between the range of 1.08–1.16 g.ml−1 and increased with HLh con-
tent. It can be concluded that as the HLh concentration increases the
viscosity of the sample solutions increases to some extent. Sample-S4,
which has a higher amount of HLh presented the highest viscous
structure, which is not considered to be ideal for the printing process.
Because of the evaporation of the solvent during the printing process, it
was very difficult to use highly viscous printing solutions. The density
of the solutions also increased with the increase of HLh. Pore size is an
important parameter since the pores in the scaffold provide cell mi-
gration and efficient exchange of nutrients or wastes between the
scaffold and its environment [31]. According to the measurements in
Fig. 6a1-a4, the macrostructure of scaffolds becomes arranged with an
increasing HLh portion. This was probably due to the effective hy-
drogen bonding capability of HLh with PCL and GT. As a result of ob-
servations, 1.1 ± 0.05 mm (ranging from 1.00 to 1.30 mm) diameters
for sample-S3 have a more uniform pore size distribution. In a previous
study, PCL/GT nanofiber membranes were prepared at different PCL/
GT ratios, and the average pore sizes of the membranes were found to
be remarkably less than L929 fibroblast cells (in the range of 1–3 µm)
[32]. Much smaller membrane pore sizes than L929 fibroblast cells
might seem favorable, which allows the cell to proliferate on the
membrane surface rather than inside of the membrane. However, this
can create limitations for the area whereas cells can find a place to
attach. Our pore sizes are found to be bigger compared to the previous
studies [32] due to the nature of the selected production method and
yet they can provide a suitable environment for proper proliferation.
Since the scaffold is biodegradable, either guiding cells to inside of the

Fig. 4. DSC thermograms of (a) sample-S3 and (b) pure PCL.

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Fig. 5. Solution properties of blends; (a) surface tension, (b) viscosity and (c)
density.
porous membrane or having them on the surface of the membrane does
not change anything and this versatility increases the value of the
scaffolds.
3.7. Water absorption and biodegradability
The water absorption percentage of the scaffolds was calculated
using Eq. (1), and a graph was drawn as a function of degradation time
(Fig. 6b). The absorption rate varied depending on the content of HLh
during the incubation period. The maximum water absorption rate was
observed after the first day of incubation for each scaffold samples and,
among them, the sample-S4 scaffold had the highest absorption rate.
After one day, water absorption started to decrease. After three days,
sample-S2 and sample-S4 scaffolds showed nearly a linear slope until
the end of 7 days of incubation, which indicated that these scaffolds
reached to a water absorption equilibrium. However, the water ab-
sorption of the sample-S3 scaffold dropped severely at the end of 5 days
and increased slightly at the end of 7 days. Physically formed hydrogels
of biomaterials are spontaneously formed networks via intermolecular
interaction of the components. In the course of network formation,
while most of the polymer chains join the network in gel form, others
remain in sol form as a result of chain irregularities such as physical
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European Polymer Journal 119 (2019) 426–437
entanglements and cyclization. Therefore, some differences in both
rates of swelling and equilibrium swelling values of the networks can be
observed [33,34]. Despite the decreasing water absorption of these
scaffolds, the sample-S1 scaffold started to increase its water absorption
rate after 3 days of the incubation period. As a result of the biode-
gradation tests, similar to the previous studies, it has been observed that
the degradation of samples decreased the water absorption behavior of
the samples [35]. Fig. 6c shows the weight loss of the scaffold samples
at different time intervals (1, 3, 5, and 7 days) in PBS solution
(pH = 7.4) at 37 °C. On 1, 3, 5, and 7 days, the samples were in-
vestigated for any apparent weight loss of scaffolds in the PBS solution.
During the first day of incubation, all of the scaffolds had a negligible
amount of weight loss. After 3 days of incubation, the samples-S1 and
S2 scaffolds showed slightly fast degradation compared to others. After
5 days of the incubation period, the sample-S3 scaffold started to de-
grade drastically and kept losing weight after day 7. Other scaffold
samples were degraded very slowly and exhibited a slight linear slope
throughout the seven days of study. The weight losses were found to be
10.18%, 7.18%, 46.54%, and 3.45% according to the Eq. (2) for the
samples-S1, S2, S3, and S4, respectively after 7 days of incubation.
In a previous study, PCL/GT biocomposite scaffolds were fabricated
by varying the ratios of PCL/GT concentrations for nerve tissue en-
gineering. PCL/GT nanofibers with a ratio of 50:50 was displayed the
fastest degradation rate in PBS over 2 weeks, and biodegradability of
PCL/ GT nanofibrous scaffolds increased by increasing the GT content
[36].
3.8. Cell proliferation and viability
As can be seen in Fig. 7, the cell proliferation and viability of HOb
cells on 3D printed scaffolds were determined for 24, 48, and 72 h.
Viability results of samples-S1, S2, S3 and S4 were 86.18, 89.76, 88.54,
and 91.11% at 24 h, 82.77, 80.63, 81.93, and 81.29% at 48 h whereas,
77.76, 84.79, 84.62, and 90.19% at 72 h, respectively (Fig. 7a1-a3).
Among the synthetic biocompatible polymers, PCL has been widely
used as a porous scaffold material for tissue engineering particularly
bone and cartilage tissue repairing [19,37,38]. On the second day, all
viability results decreased and at 72 h, increased aside from sample-S1.
Viability results of the scaffold samples consisting of HLh indicate that
after 48 h and viability increased. Additionally, chemicals could be
released from scaffolds that can affect cell viabilities during the first
48 h and cells could proliferate after adjusting the new environment. On
the other hand, viability proceeds decreasing through 24–72 h on the
sample-S1 scaffold. Notably, the results show that HLh enhances the
viability of more than PCL and GT. Additionally, DMSO/DCM solution,
which used as a solvent for the scaffold samples, might have toxic ef-
fects on cells. DMSO is generally used as a freezing agent in cell culture
and it has a toxic impact on concentrations above 10% at room tem-
perature [39]. Cells on the scaffolds tolerate these cytotoxic effects with
its high biocompatible and proliferation properties. In previous studies,
microspheres composed of GT and hydroxyapatite were produced with
uniform morphology by a wet-chemical method. Cell viability tests of
microspheres were performed by MTT test with HOb cell line G-292.
The materials were diluted into 1:4 and 1:1 with culture medium and
only 1% and 2% cell damage were observed, respectively. This result
illustrates that the gelatine-hydroxyapatite blend was non-toxic for
bone tissue engineering [40]. In another study, 5 and 10% (w/v) PCL
coated 3D-printed scaffolds were prepared to enhance the osteogenic
potential and mechanical strength. In vitro tests of 3D scaffolds, con-
sisting of calcium sulfate-based hydroxyapatite were performed by MTS
assay with the MG-63 cell line. On the first day, there were no differ-
ences in cell proliferation results between 5% PCL coated and uncoated
scaffolds. On the 10% PCL coated scaffolds, proliferation was higher
than other scaffolds and cell growth was increased rapidly until day 5.
On day 15, 5% of PCL coated scaffolds were the highest for cell growth.
These results indicated that PCL coating did not cause toxicity [41]. In
B.T. Duymaz, et al.
Fig. 6. Pore size distribution of (a1) sample-S1, (a2) Sample-S2, (a3) Sample-S3, (a4) Sample-S4 scaffolds. Weight loss (b) and water absorption (c) graphs of scaffolds.
our previous study, we synthesized HLh based PNIPAM hydrogels and
performed their viability tests with mouse fibroblast L929 for 24, 48,
and 72 h. Viability increased with increasing HLh concentration, where
the result is consistent with previous studies [8]. Morphology of ad-
herent cells on controls and scaffolds were also determined by fluor-
escent microscopy, and cell nuclei were observed after DAPI staining on
24, 48, and 72 h of culture time (Fig. 7b1-b3). The scaffold samples and
control cells were seeded on the glass in well plates, where the cell
population was observed to increase with increasing culture time. After
24 h adherence of cells on sample-S1 and sample-S4 were found to be
significantly higher than the other scaffolds but less than the control
group. At 48 h and 72 h, cell adherence on the sample-S1 scaffold de-
creased, which was consistent with the viability results. Cell adherence
increased with increasing concentration of HLh and at 48 h, the number
of adherent cells decreased on all scaffolds, which was also consistent
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European Polymer Journal 119 (2019) 426–437
with the viability results. Adherent cells increased after 48 h in HLh
scaffolds and maximum adherent cells were observed at the sample-S4
scaffold, which was also consistent with viability result at 72 h. Cells
were highly aggregate on some specific areas, which were considered as
high HLh concentration spots on scaffolds. Scaffolds were floating over
the culture medium due to their lightweight and it was the main pro-
blem on cell culture studies. The cells were not able to contact with the
scaffolds completely, which was observed on fluorescent microscopy
images (Fig. 7b1-b3), but the intolerable amount of toxic chemicals
were not released from scaffolds.
Adhesion of HOb cells on the scaffolds was observed at 24, 48, and
72 h (Fig. 7c1i-c4ii) of culture by using SEM to examine in depth the
biocompatibility of these scaffolds. The SEM images indicated that HOb
cells adhered tightly to the scaffold surface and grew actively by
showing typical morphological features of osteoblast. Additionally, the
B.T. Duymaz, et al. European Polymer Journal 119 (2019) 426–437

Fig. 7. HOb cell viability results after (a1) 24, (a2) 48 and (a3) 72 h of cultivation on scaffolds with fluorescent microscopy images of HOb cells after (b1) 24, (b2) 48,
and (b3) 72 h of cultivation on control, along with bioprinted scaffolds with SEM images of (c1, c1i, c1ii) sample-S1, (c2, c2i, c2ii) sample-S2, (c3, c3i, c3ii) sample-S3,
and (c4, c4i, c4ii) sample-S4 after cell culture test.

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cells were proven to be worthy of vitality and fast proliferation at the
end of 72 h, which indicated the biocompatibility of these scaffolds. The
distribution of pores and toughness of the scaffold affected the mor-
phology of the cells on the scaffold. The SEM images showed that the
cells were randomly distributed on the surface and inside of the scaf-
folds. The cell microfilaments, which are the thinnest filaments of the
cell skeleton, were shown as linkages to the scaffold material. When
comparing the scaffolds, the ones with HLh had more uniform pore size
distribution and shape. The addition of HLh to the PCL/GT solution
increased its compatibility and had a significant effect on cell growth of
the scaffold. It is notable that the increasing HLh portion of the com-
positions increased the number of cells on the scaffold and showed
better proliferation rates. In our previous study, we showed that HL
enhanced the biocompatible, cell attachment and proliferation prop-
erties of polyethylene oxide/chitosan/HL polymer blend films, and HL
increased the viability [16]. These results indicated that the scaffolds
have excellent biocompatibility. Moreover, adequate growing of cells
on the scaffold reveals that there is no toxic substance release to the
medium.
4. Conclusion
The 3D scaffolds of the PCL/GT and PCL/GT/HLh blends were
successfully fabricated using novel 3D bioprinting formulations. The
effect of HLh content on the properties of the polymer solution and 3D
scaffolds was comprehensively tested and analyzed. The increasing HLh
ratio in PCL/GT composite formulation led to an increase in density and
viscosity. The surface tension of the solution decreased as well as the
compressive strength of the scaffolds. The obtained scaffolds had uni-
form pore size with homogeneous distribution and shape with dense
appearance. The hydrogen bonding between the scaffold components
was observed, which resulted in more dimensional stability of scaffolds.
PCL/GT/HLh scaffold formulations provided better proliferation to
cells than PCL/GT scaffold. These results indicated that the scaffold has
good biocompatibility and potential for the preparation of effective Hob
cell scaffolds.
Acknowledgment
This study has been funded by BAPKO, Marmara University, Turkey;
grant no: SAG-B-090217-0036 and Fen-C-YLP-120917-0549; grand no:
FEN-E-130515-0175.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.eurpolymj.2019.08.015.
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