Materials & Design 209 (2021) 109962

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Materials & Design

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

Co-electrospinning polycaprolactone/gelatin membrane as a tunable

drug delivery system for bone tissue regeneration
Mohammed A. Al-Baadani, Kendrick Hii Ru Yie, Abdullrahman M. Al-Bishari, Bilal A. Alshobi,
Zixin Zhou, Kai Fang, Binwei Dai, Yiding Shen, Jianfeng Ma ⇑, Jinsong Liu ⇑, Xinkun Shen ⇑
School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou 325027, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 PCL and Gel synergistically provide Schematic illustration of the PCL/Gel membrane prepared by co-electrospinning technique for bone tis-
the mechanical properties and sue engineering. PCL was utilized to provide sufficient mechanical strength for enhancing overall integ-
bioactivity. rity of the membranes. These co-electrospun membranes could slow-release hydrophobic drugs (loading
 Co-electrospun membrane can slowly in PCL nanofibers) and controlled-release hydrophilic drugs (loading in Gel nanofibers). In addition, the
release both hydrophilic and combination of the natural polymers could enhance the biocompatibility of these membranes and per-
hydrophobic drugs. form their respective functions in the bone healing process.
 Regulating PCL content can result in a
time-tunable release of hydrophilic
drugs.
 PCL controls drug release by
inhibiting the dissolution/diffusion of
Gel fibers.

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

Article history: The rising popularity of co-electrospinning technique in the field of biomaterial has enabled the possibil-
Received 14 January 2021 ity of combining various polymers, in which they provide the mechanical properties and the bioactivity
Revised 3 June 2021 for each other. Herein, a series of polycaprolactone/gelatin (PCL/Gel) composite membranes were pre-
Accepted 5 July 2021
pared by co-electrospinning, and their biocompatibility and drug-controlled release ability were evalu-
Available online 07 July 2021
ated in detail for the first time. The addition of Gel significantly enhanced the adhesion and
differentiation of osteoblasts, while the addition of PCL significantly improved the mechanical properties.
Keywords:
Moreover, the in vitro degradation and drug release results showed that by adjust the PCL fibers amount
Co-electrospinning
PCL/Gel fibers
and size, the degradation of Gel and the release profile of hydrophilic drugs/proteins could be effectively
Controlled drug release controlled: in the low PCL groups (PCL/Gel-1 & PCL/Gel-4), the Gel nano-fibers dissolved rapidly, resulting
Osteogenesis in an early explosive release within 1 week; however, with the increase of PCL content, the drug release
rate decreased gradually, and the total release period could be extended to over 2 weeks (PCL/Gel-2, PCL/
Gel-3 & PCL/Gel-5) or more (PCL/Gel-6). Therefore, the preparation of a controllable drug delivery system
with good biological activity by co-electrospinning of PCL and Gel could provide an effective strategy for
bone regeneration.
Ó 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding author at: 268# Xueyuan West Road, Lucheng District, Wenzhou City, Zhejiang Province, China.
E-mail addresses: dentistmacn@aliyun.com (J. Ma), jinsong0719@wmu.edu.cn (J. Liu), 20111902015@cqu.edu.cn (X. Shen).

https://doi.org/10.1016/j.matdes.2021.109962
0264-1275/Ó 2021 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al.
1. Introduction
Bone healing is a very complex regenerative process and new
strategies for bone tissue engineering are desperately required
with the ever-rising incidence of traumatic injuries, bone disor-
ders, and bone defects. Comprehensive advancement in nanotech-
nology have contributed to the use of membrane scaffolds for
tissue engineering [1]. Bio-composite membrane of electrospin-
ning nanofibers mimic the bone’s natural structure and the nanos-
cale components of the extracellular matrix (ECM), preserving the
ability to organize cells and induce cell signaling [2,3]. Moreover,
the benefits of these membranes include large surface area, diam-
eter tunability, excellent porosity, mechanical stability, ease of use,
and the ability to encapsulate various bioactive agents to serve as a
carrier [4,5].
A variety of biodegradable polymers has been exploited and uti-
lized in electrospinning to fabricate fiber membranes varying in
diameters (nanometers or microns) through parametric tuning,
which includes polymer concentration, polymer flow rate, the
applied voltage, and the needle-collector distance [6]. There are
two forms of polymers, natural and synthetic polymers that also
naturally exhibits different constitution (hydrophobicity and
hydrophilicity). The nature of these polymers play a crucial role
in the integrity and drug loading capacity of the fabricated mem-
branes [7,8]. Natural polymer such as the collagen-derived gelatin
(Gel), is hydrophilic in nature. Aside from being biodegradable and
biocompatible, previous study have proved its effectiveness in
enhancing cell proliferation and differentiation [4]. In contrast,
synthetic polymers such as polycaprolactone (PCL), is hydrophobic
in nature which is beneficial when trying to attain a prolonged
drug release [9]. Apart from biodegradable and biocompatible, it
has been demonstrated to possess excellent mechanical features
and tunability to give a wide range of physical properties [10].
However, the use of solitary PCL-fiber membranes is not feasible
due to various limitations. It has been proved that PCL-fiber mem-
branes have extremely slow degradation rate because of its
hydrophobicity [11]. In addition, it may decrease or inhibit cell
adhesion and proliferation due to their constitution, semi-
crystalline structure and lack of natural bioactive properties
[12,13].
Blended electrospinning technique containing both hydropho-
bic and hydrophilic polymers have attracted much attention in
recent years. Many studies have been conducted to study its poten-
tial usage in drug delivery and in improving the biocompatibility of
the membranes [14,15]. However, the uncontrolled burst-release
of drugs, cases of incomplete drug release and membrane cytotox-
icity are some of the concerns that needs to be considered [16]. A
recent study has claimed that the drug release profile is influenced
by the degradation rate of the membranes as well as the diffusion
rate of the drugs [17]. In addition, the drug release behavior, which
is perceived to be one of the more significant variables, has always
received the most interest, especially in terms of the rate of initial
release and the subsequent sustained release. As a consequence,
most single-fiber electrospinning approaches for single drug deliv-
ery are not feasible for clinical application. Therefore, a specialized
system has been designed which utilizes dual drug delivery for an
intended duration at specific sites to mitigate adverse side effects
and improve the therapeutic potency of administered drugs.
To comply with the circumstances and needs of each individual
patient in cases requiring bone regeneration, the enablement of a
well-regulated, time-tunable delivery of bioactive cargos or drugs
is vital [4,18]. For instance, immediately following an implant sur-
gery, the exposed surgical site becomes more susceptible and vul-
nerable to bacterial infections, hence the rapid administration of
antibacterial drugs is crucial for the mitigation of sepsis-
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Materials & Design 209 (2021) 109962
related implant failure [25]. In addition, a slow and sustained
release of osteogenic factors along with cell recruitment drugs
within the first week is essential and would be ideal for bone repair
or regeneration purposes [4]. With that being said, the fabrication
of a local multi-drug therapy with a predictable, controllable drug
release profile is highly essential in bone tissue reconstruction and
implant surgery for the treatment and mitigation of bacterial infec-
tions, and also to enhance early-stage cell recruitment [19,20]. In
this study, we had utilized the co-electrospinning technique to rec-
tify the a forementioned issues. This subsidiary technique involves
the simultaneous electrospinning of two different polymers that
would interlay one another, resulting in the synthesis of a dual-
polymer membrane. The resultant composite films are expected
to possess improved physical, chemical, and mechanical properties
[21]. More importantly, in regards to the specific nature of the
polymers (hydrophobicity and hydrophilicity) degradation rates,
the loading of various drugs or bioactive components could be
achieved and their release behaviors could be tuned [22].
Based on our understanding of the degradation behavior of the
polymers, we hypothesized that increasing the PCL concentration
of the co-electrospinning membranes will inherently protect the
drug-loaded Gel, subsequently allowing for the controlled release
of bioactive substances from the Gel nanofibers. Our objective in
the present study is to improve the mechanical properties and
the biocompatibility of the electrospun membranes through co-
electrospinning of Gel and PCL polymers, and to further utilize
them as drug-carriers (hydrophilic and hydrophobic drugs), with
the intent to obtain a tunable drug delivery system.
2. Materials and methods
2.1. Materials and reagents
PCL (Mw = 80,000) was purchased from Sigma-Aldrich (MO,
USA). Gel, vancomycin hydrochloride (Van), simvastatin (Sim),
and 2,2,2-Trifluoroethanol (TFEA) were obtained from Aladdin Bio-
chemical Technology Co., Ltd (Shanghai, China). Fluorescein
isothiocyanate-bovine serum albumin (FITC-BSA) was provided
by MeilunbioVR (Dalian, China). Cell Counting Kit-8 (CCK-8), 5-br
omo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BICP/
NBP) Alkaline Phosphatase Color Development Kit, Bicinchoninic
Acid (BCA) Kit, and p-Nitrophenyl Phosphate Assay Kit were pur-
chased from Beyotime Biotechnology Co., Ltd. (Jiangsu, China). Ali-
zarin red stain was purchased from (Solarbio, China). All of the
above materials were used without additional purification.
2.2. Electrospinning procedures
To select the appropriate polymer concentration and electro-
spinning parameters for our study, the conventional electrospin-
ning method was used to prepare a single PCL and Gel
membrane separately. Briefly, PCL (6, 10 and 15 wt%) was dis-
solved in TFEA solution, and Gel (10, 15 and 20 wt%) was dissolved
in a mixture of deionized water and TFEA solutions (v/v = 4/1). The
parameters were set as the followings: the applied voltage was
20 kV; the flow rate was 1 mL/h; and the distance gap from the
needle to the drum collector was 15 cm. The collector was covered
with an aluminum foil sheet (10 cm  15 cm), and the collector’s
rotational speed was set at 300 rpm. During sample preparation,
the temperature and relative humidity were 25–35 °C and 35%
50%, respectively. Afterward, the co-electrospinning method was
used to fabricate the PCL/Gel micro/nano-sized fiber membranes
using two separate needles and pump-feds, as shown in Fig. 1A.
The PCL/Gel co-electrospinning membranes were collected from
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al.
the aluminum foil and then vacuum dried for two days at ambient
temperature to remove residual solvent. To prepare the target
samples, the Gel (20 wt%) and PCL (10 & 15 wt%) were electrospun
at a flow rate of 1 to 3 mL/h, respectively: PCL/Gel-1 (20 wt% Gel,
1 mL/h; 10 wt% PCL, 1 mL/h), PCL/Gel-2 (20 wt% Gel, 1 mL/h;
10 wt% PCL, 2 mL/h), PCL/Gel-3 (20 wt% Gel, 1 mL/h; 10 wt% PCL,
3 mL/h), PCL/Gel-4 (20 wt% Gel, 1 mL/h; 15 wt% PCL, 1 mL/h),
PCL/Gel-5 (20 wt% Gel, 1 mL/h; 15 wt% PCL, 2 mL/h), and PCL/
Gel-6 (20 wt% Gel, 1 mL/h; 15 wt% PCL, 3 mL/h). The applied volt-
age and distance were also 20 kV and 15 cm, respectively.
2.3. Characterization PCL/Gel membrane
To evaluate the structure of PCL/Gel fibers and size distribution,
different PCL/Gel samples were observed using scanning electron
microscopy (SEM, Zeiss AURlGA FlB, Germany) at 10 kV voltage.
Different membranes were placed on the SEM sample plate by con-
ductive tape and sprayed with gold for 45 s (Leica EM ACE600). The
distribution and average diameter of PCL and Gel fibers were then
analyzed by measuring 150 random fibers from ten SEM images
with Image J software. The detailed algorithm was shown in
Fig. S1. The zero-shear viscosity of PCL solution was measured by
rheometer DHR-2 with a cone-plate configuration at 20.5 °C. The
water contact angles of all samples were measured to evaluate
the wettability of different co-electrospinning membranes using
a Contact Angle device (WCA, Model 200, Future Scientific, China).
For the water contact angle detection, 5 mL of deionized water was
placed on different membrane surfaces. The image was taken at
Fig. 1. (A) Schematic illustration of the PCL/Gel membrane prepared by co-electrospinning technique; (B) SEM images of different PCL/Gel co-electrospinning membranes at
various magnifications; and (C) the statistics of fiber size in different co-electrospinning membranes.
3
Materials & Design 209 (2021) 109962
different time (0, 10, 25 and 50 s) at ambient temperature accord-
ing to previous study [21].
2.4. Mechanical properties
The stress–strain curves were measured to evaluate the
mechanical properties of Gel and PCL/Gel co-electrospinning mem-
branes. Before measurement, the samples were cut into rectangles
(3 cm  6 cm) and vacuum dried for 24 h at ambient temperature.
Each group had four duplicate samples. The dry state was recorded
using a Testing Machine (4465, Instron, USA) with a cross-head
pulling speed of 1 mm/min. The ultimate strain, tensile strength
and Young’s modulus were calculated in static mode.
2.5. Cell adhesion, morphology and viability
For in vitro cell experiments, all drug-free PCL/Gel samples were
cut into squares (1.5 cm  1.5 cm) and sterilized under ultraviolet
(UV) irradiation for 2 h. Each group had six duplicate samples in all
the cell experiments. Fluorescence observation and CCK-8 assays
were carried out to determine the cell adhesion, morphology and
viability of MC3T3-E1 cells on different PCL/Gel membranes.
Briefly, cells were first cultured at an initial density of 1  104
cells/cm2 with a-MEM medium supplemented with 10% FBS at
37 °C under 5% CO2 atmosphere. For cell adhesion or morphology
detection, the MC3T3-E1 cells were fixed with 4% paraformalde-
hyde after culturing for 2 or 24 h. Then, the cytoskeleton and/or
nuclei were stained with phalloidin (40 min, green-fluorescent)
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al.
and DAPI (10 min, blue-fluorescent) solution, and observed using
an inverted fluorescence microscope (FM, OLYMPUS IX81, Japan).
For the CCK-8 assay, the culture medium was removed after cultur-
ing for 4 and 7 d, and 300 lL of fresh medium supplemented with
10% CCK-8 was re-added to each well. After further incubated for
2 h, 200 lL of mixture solution was collected and recorded at
450 nm using a microplate reader (Spectra Max M5, Molecular
Devices, USA).
2.6. Alkaline phosphatase (ALP) activity
To evaluate the osteogenic differentiation capacity of MC3T3-E1
cells grown on PCL/Gel membranes, the ALP activity was mea-
sured. After culturing for 4 and 7 d, the cells were lysed by 1% tri-
ton X-100 solution on ice for 40 min. The BCA protein and ALP
assay kits were then used to detect the protein concentration (at
562 nm) and ALP activity (at 520 nm). Moreover, BICP/NBP ALP
color development kit was used to observe the ALP staining on
the membrane for more confirmation.
2.7. Mineralization
Alizarin red staining was used to evaluate the mineralization
matrix of MC3T3-E1 cells grown on different PCL/Gel membranes.
After culturing for 14 d, the samples were washed three times with
PBS, fixed with 4% paraformaldehyde for 40 min, and then stained
with alizarin red solution for 30 min at ambient temperature.
Afterward, the samples were washed several times with deionized
water and imaged under the stereomicroscope. Afterward, the
stained nodules were dissolved and extracted by 10% cetylpyri-
dinium chloride for 30 min and recorded at 540 nm using a spec-
trophotometric microplate reader (Bio-Rad 680, USA).
2.8. Drug loading and release
In the past decade, many studies have shown that the topical
use of Van (small molecule, hydrophilic drug) and Sim (small mole-
cule, hydrophobic drug) can significantly improve the antibacterial
and osteogenic activities of biomaterials [23,24] In addition, FITC-
BSA (macromolecule, hydrophilic protein), like many growth fac-
tors, is also widely used as a model drug to study the sustained-
release properties of protein drugs in biomaterials [25]. Therefore,
Van, FITC-BSA, Sim were used as different model drugs to evaluate
the drug release behaviors from different PCL/Gel membranes.
Before electrospinning, Sim (10 mg/mL) was dissolved in the TFEA
solution containing PCL (10 wt%, 15 wt%), while FITC-BSA (10 ug/
mL) or Van (3 mg/mL) were dissolved in the mixture solution of
deionized water and TFEA (v/v = 4/1) containing Gel (20 wt%). Ref-
erence to the preparation of drug-free films (part 2.2), different
membranes loaded with specific model drugs were prepared. In
order to study the chemical structure of drug-loaded films, three
kinds of membranes (PCL/Gel-Van, PCL/Gel-BSA and PCL/Gel-Sim)
were prepared by using PCL/Gel-5. Before measurements, the dif-
ferent samples were mixed with potassium bromide (KBr) at a
ratio of 1:20. The mixture of KBr and nanofibers was then com-
pressed into sheets under 20 MPa conditions and analysed by a
Fourier transform infrared spectroscopy (FTIR, Thermo scientific,
Nicolet 6700, USA).
To determine the loading efficiency, different co-
electrospinning membranes were collected from the aluminum
foil, vacuum dried for 24 h at 40 °C, and then weighed one by
one. The theoretical loading dose of three model drugs in 100 mg
co-electrospinning membranes was obtained by theoretical calcu-
lation: theoretical dose = total weight of the loaded drug / total
weight of the co-electrospinning membrane  100 mg. In addition,
after fully dissolving the drug-loaded films (100 mg) in TFEA solu-
4
Materials & Design 209 (2021) 109962
tion, the actual loading dose of three drugs could be directly mea-
sured. The Sim and Van were detected using UV–Visible
spectrophotometer (Thermo ScientificTM, Nanodrop 2000 and
2000c, USA) at 280 and 238 nm wavelengths, respectively
[26,27]. The FITC-BSA was measured by monitoring the fluores-
cence intensity of emission wavelength at 535 nm and excitation
wavelength at 485 nm using spectrophotometric microplate reader
(Bio-Rad 680, USA) [28]. Finally, the drug loading efficiency in dif-
ferent samples was calculated by calculating the ratio of the actual
dose to the theoretical dose.
Next, for drug release determination, samples (100 mg) were
soaked in 5 mL of phosphate-buffered saline (PBS) and incubated
at 37 °C. At each interval time point (FITC-BSA/Van: 0, 0.5, 1, 24,
48, 120, 168, 216, 288 and 336 h; Sim: 0, 1, 2, 3, 6, 9, 12, 15, 18,
21, 24 and 27 d), 1 mL of the solution was collected and replaced
by adding isovolumic fresh PBS solution. The release behavior of
FITC-BSA and Van was only investigated within 336 h because of
the sensitivity limitation of fluorescence/UV detection and fluores-
cence quenching characteristics. The drug release percentage at
different time points was obtained by calculating the ratio of the
drug release content to the actual total dose mentioned above.
Each of the release experiments was repeated four times.
2.9. Mathematical model for drug release
The release kinetics of Van, FITC-BSA and Sim were analysed by
four mathematical models: Zeroth-order (equation: y = kt), First-
order [equation: y = a(1-e-bt)], Higuchi (equation: y = kt1/2), and
Ritger-Peppas (equation: y = ktn). All the release curves were fitted
with linear or nonlinear curve by Origin software (version 8.0). In
Ritger-Peppas analysis, the value of n can reflect the drug release
pattern: n  0.45 and n  0.89 are considered as the Fick diffusion
mechanism and skeleton corrosion mechanism, respectively; while
0.45 < n < 0.89 indicates that the drug release mechanism is mixed,
including Fick diffusion and skeleton corrosion. In addition, n can
also reflect the kinetics of drug release: when n greater than 0.66,
the drug is released mainly with zero-order kinetics, and when
n = 1, the drug release exhibits complete zero-order kinetics.
2.10. Membrane degradation
The PCL/Gel co-electrospinning membrane’s degradation behav-
ior was evaluated in vitro by soaking the samples (1.5 cm  1.5 cm) in
2 mL of PBS solution at 37 °C. Each group had four duplicate samples.
The PBS was changed every 3 d. After that, the samples were col-
lected and rinsed with deionized water. All samples were weighed
by a high precision balance (Al204 Analytical Balance, Mettler
Toledo) before and after soaking [29]. Furthermore, the morphology
of soaked samples was characterized via SEM.
2.11. Statistical analysis
All data were expressed as mean ± standard deviation (SD). Sta-
tistical analysis was conducted by Student-Newman-Keuls (SNK)
multiple comparison test and one-way analysis of variance
(ANOVA) using Origin (version 8.0). The confidence levels were
taken to be 95% (*p < 0.05) and 99% (**p < 0.01).
3. Results
3.1. Characterization of PCL/Gel co-electrospinning membrane
A series of individual PCL and Gel membranes were prepared
through conventional electrospinning technique to obtain the suit-
able parameters prior to synthesizing an optimal membrane. Their
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al.
surface morphology was shown in Fig. S2. It was observed that the
individual Gel or PCL membranes were irregularly formed and con-
tained many bead-shaped defects at low concentrations. By
increasing the concentrations of Gel to 20% and PCL to 10%/15%,
the obtained fibers become more uniform with an average size of
0.35 ± 0.07 lm, 0.99 ± 0.3 lm, and 1.5 ± 0.3 lm, respectively
(Fig. S2 & S3). The increasing concentrations of PCL also bring a sig-
nificant change on the PCL solution viscosity, as shown in Fig. S4.
After assembling the PCL and Gel by co-electrospinning with
two separate needles (Fig. 1A), hierarchical micro-nanofibrous
structures was successfully fabricated. As shown in Fig. 1B, well-
defined, uniform, continuous, interconnected and randomly-
oriented micro/nano fibers were formed with no bead-shaped
defects. The analyzed results (Fig. 1C) further showed that a higher
PCL concentration and flow rate led to an increased PCL fiber size,
1 ± 0.3 lm, 1.6 ± 0.3 lm, 1.9 ± 0.3 lm in PCL/Gel-1, PCL/Gel-2, PCL/
Gel-3, and 1.5 ± 0.3 lm, 1.8 ± 0.3 lm, 2.1 ± 0.3 lm in PCL/Gel-4,
PCL/Gel-5, PCL/Gel-6, respectively. However, the Gel fiber size
was around 0.35 in all co-electrospinning membranes.
Then, the chemical bonds of three drugs (FITC-BSA, Van, Sim),
two single fibers (PCL, Gel) and four co-electrospinning films
(PCL/Gel-5, PCL/Gel-Van, PCL/Gel-FITC-BSA, PCL/Gel-Sim) were
analyzed by FTIR. As shown in Fig. S5, the pure PCL infrared spec-
trum was defined by stretching vibration at 1734 cm 1 (C@O) and
1170 cm 1 (CAOAC). Gelatin peaks appear at approximately
1678 cm 1 (amide I, mainly C@O stretching vibrations) and
1541 cm 1 (amide II, the coupling of bending vibrate of NAH and
stretching vibrate of CAN) [30]. Van’s spectrum showed character-
istic peaks at 1658 cm 1 (C@O), 1500 cm 1 (C@C), 1231 cm 1 (phe-
nolic hydroxyl), and 1093.52 cm 1 (RAOAR) [31]. The pure Sim
characteristic peaks showed a specific band at 1692 cm 1 (C@O)
and 1272 cm 1 (CAOAC) [32]. The FTIR peaks of pure FITC-BSA
could be seen at 1652 cm 1 for amide I and 1539 cm 1 for amide
II bands [33]. The spectrums of the drug-loaded co-electrospun
fibers were similar to those of the individual polymers, exhibiting
no new characteristic peaks. This could be attributed to the small
amount of drugs loaded in the polymers or maybe were shifted
or replaced by the polymers’ peaks [34].
3.2. Mechanical properties
The stress–strain measurements were carried out to investigate
the mechanical properties of different membranes. As shown in
Table S1, The Gel nanofiber membrane showed to possess a good
Young’s Modulus elasticity (21.6 ± 4.0 MPa) compared to other
co-electrospinning (PCL/Gel-1 to PCL/Gel-6 groups) where the
Young’s Modulus was significantly decreased 6.67 ± 0.36,
8.49 ± 0.31, 9.56 ± 0.34, 6.49 ± 0.35, 8.19 ± 0.34, and 11.67 ± 0.3
MPa, respectively (Fig. 2B). However, the Gel nanofiber membrane
showed a significantly weaker tensile strength and ultimate strain
(0.15 ± 0.07 & 0.8 ± 0.04; Fig.S6 & Table S1) compared with other
co-electrospun membranes (Fig. 2C,D). The stress–strain curve
(Fig. 2A) illustrated that all samples had a direct relationship to
the amount of PCL; in other words, the increase in PCL concentra-
tion within the membrane displayed distinct effects on the co-
electrospun membrane in terms of mechanical performance. Fur-
thermore, a general trend is observed in (Fig. 2C, D) showing a
decrease in tensile strength and the ultimate strain with the
increasing fiber size. PCL/Gel-3 membrane (average PCL fiber size
1.9 ± 0.3 lm) significantly increased the tensile strength and ulti-
mate strain (elongation at break) compared to other groups.
3.3. Cell adhesion, morphology and viability
The fluorescence staining of the nucleus and cytoskeleton were
used to evaluate the initial adhesion and morphology of MC3T3-E1
5
Materials & Design 209 (2021) 109962
cells seeded on different PCL/Gel co-electrospinning membranes.
From Fig. 3A & B, we could observe an obvious difference in the
number of cells adhesion, and it was in the order of PCL/Gel-5 > P
CL/Gel-2 > PCL/Gel-3 > PCL/Gel-6 > PCL/Gel-1 > PCL/Gel-4. The PCL/
Gel-5 membrane showed a significantly higher (p < 0.05) initial cell
adhesion compared to PCL/Gel-1, PCL/Gel-4, and PCL/Gel-6. In
addition, MC3T3-E1 cells displayed more protruded filamentous
pseudopodia on PCL/Gel-2, PCL/Gel-3 and PCL/Gel-5 than those
in other groups (Fig. 3C).
The CCK-8 assay was carried out to measure the cell viability
after 3 and 7 d of culture and shown in Fig. 3D. It was confirmed
that the MC3T3-E1 cells on different PCL/Gel co-electrospinning
membranes, after incubation for 3 d, had no significant differences
in cell viability. While after incubation for 7 d, the viability of
MC3T3-E1 cells cultured on the PCL/Gel-5 sample was higher
(p < 0.01) than those cultured on the other groups.
3.4. Osteogenic differentiation
The staining and quantitative analysis of ALP activity and
matrix mineralization were carried out to evaluate the osteogenic
capacity of MC3T3-E1 cells on the PCL/Gel co-electrospinning
membrane. The quantitative analysis results of ALP (Fig. 4B)
demonstrated no significant difference after four days of incuba-
tion. However, after seven days, the ALP activity in the PCL/Gel-5
group was higher (p < 0.05 or 0.01) than the other groups, and
the PCL/Gel-4 and PCL/Gel-1 groups had the lowest ALP activity.
The related stain images further confirmed that the ALP stain on
PCL/Gel-5 had a higher density and a broader region than other
groups (Fig. 4A). Moreover, the calcium deposits quantified
through alizarin red staining (Fig. 4C) also showed that the cells
on the PCL/Gel-5 had highest degree of mineralization compared
to the other groups followed by PCL/Gel-6 and PCL/Gel-2 (Fig. 4A).
3.5. In vitro degradation
The degradation of Gel nanofibers was shown in (Fig. S7A, B).
We found that the Gel membranes would turn into a gel-like struc-
ture and be completely dissolved in PBS after 24 h. This made it
impossible to determine its mass. However, the PCL/Gel co-
electrospinning membranes still maintained their membrane
structure after two weeks. After 14 d, the mass loss of PCL/Gel-1,
PCL/Gel-2, PCL/Gel-3, PCL/Gel-4, PCL/Gel-5, and PCL/Gel-6 mem-
branes were calculated and determined at 68%, 52%, 29%, 51%,
43%, and 25% mass loss, respectively (Fig. 5B). Additionally, SEM
images of PCL/Gel co-electrospinning fibers after 24 h and 7 d of
incubation in PBS (Fig. 5A). It showed that the structural integrity
of the PCL/Gel co-electrospun membrane were highly dependent
on the concentration of PCL. Through SEM, we could see that the
Gel fibers have been partially covered by PCL fibers. This partial
covering limited the direct contact between the water and the
inner Gel fibers.
To further investigate the reasoning behind the difference in
degradation rates of various PCL/Gel membranes, we had utilized
the water contact angle measurement. The water contact angle
analysis displayed different degrees with the absorption time
(Fig. S8A). The water contact angle result (Fig. S8B) demonstrated
a different water absorption resistance behavior of the PCL/Gel
membranes over time. As can be seen, the water absorption resis-
tance of the membrane becomes stronger as the amount and con-
centration of PCL increased.
3.6. Drug release
During electrospinning, the loading efficiency of the drugs
depends on their solubility in polymer solution. If the drug is com-
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al. Materials & Design 209 (2021) 109962

Fig. 2. Mechanical Characterization of co-electrospinning membranes with different PCL concentrations and flow rates: (A) representative stress–strain curves of co-
electrospinning membranes recorded from a tensile test; the Young’s modulus (B), tensile strength (C) and ultimate strain (D) calculated from the stress–strain curves. Error
bars represent mean ± SD for n = 6, *p < 0.05, **p < 0.01.

Fig. 3. Early cell adhesion, morphology, and cell viability on PCL/Gel co-electrospinning membranes: (A) fluorescent staining images of MC3T3-E1 nucleus after culture for
2 h; (B) quantitative analysis of MC3T3-E1 cells attached to different PCL/Gel membranes after 2 h; (C) fluorescent staining images of MC3T3-E1 cells after 24 h (blue: nucleus,
green: cytoskeleton); (D) cell viability evaluated by CCK-8 assay. Error bars represent mean ± SD for n = 6, *p < 0.05, **p < 0.01. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)

pletely dissolved and does not volatilize, 100% loading efficiency
can be ensured. In this study, the drug loading efficiency calculated
by fluorescence intensity and UV-analysis was 96.8%, 98.7%, and
98.4% for BSA, Van, and Sim, respectively.
Hydrophilic drugs (FITC-BSA & Van) were used as a model drugs
in this study and were loaded into Gel of the different PCL/Gel
membranes. As shown in Fig. S7D, after incubation for 1 h, the
release curve of the single Gel nanofibers samples showed a rapid
initial burst release, where approximately 85% of the Van had been
rapidly released in the first hour, and more than 98% of Van was
released after 24 h. All co-electrospinning groups displayed an
increasing drug release rates with the decrease in the amount
and size of PCL fibers. In addition, all PCL/Gel membranes exhibits
a two-stage release profile (Fig. 6B). The PCL/Gel-1 and PCL/Gel-4

6
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al. Materials & Design 209 (2021) 109962

Fig. 4. Effects of the PCL/Gel co-electrospinning membranes on the osteogenic differentiation of MC3T3-E1 cells: (A) representative staining images of ALP and mineralized
nodules after 7 or 14 d; quantitative analysis of ALP activity (B) and mineralization (C). Error bars represent mean ± SD for n = 6, *p < 0.05 and **p < 0.01.

Fig. 5. The mass loss data and the SEM images to observe the progressive changes in the PCL/Gel co-electrospinning membrane’s surface morphology after immersion for
different time: (A) SEM images representing membrane architecture of PCL/Gel after 1 and 7 d; B) The weight loss of PCL/Gel co-electrospinning membrane after immersion
in PBS for 14 d. Error bars represent mean ± SD for n = 6, *p < 0.05, **p < 0.01.

co-electrospun membranes showed an initial burst release during
the first two days, where almost more than 65 ± 4% of the Van
has released in the first stage (<120 h), followed by a continuous
release profile until approximately 95 ± 6% of drugs released in 9
d. While PCL/Gel-2 membrane showed a burst release and
57 ± 3% of the drugs were released in stage one (<120 h), and the
remaining drugs were released in stage two in a sustained manner
(120–336 h). The PCL/Gel-3 and PCL/Gel-5 membranes showed a
minimal burst release where 42 ± 7% of the drug was released in
stage one (<120 h), followed by slow and sustained release during
stage two (120–336 h). However, the PCL/Gel-6 membrane dis-
played a more controlled release profile in the first stage of initial

7
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al. Materials & Design 209 (2021) 109962

Fig. 6. The representative release process of the PCL/Gel co-electrospinning drug delivery system: FITC-BSA (A) and Van (B) were accumulatively released from PCL/Gel and
measured at different time in PBS solution. Noted from the release curves, the drugs released from the Gel fiber within the PCL/Gel co-electrospinning depend on the PCL
amount; C) Schematic illustration of the Gel degradation after immersed in H2O and its impact on the drug release process in three types of PCL/Gel co-electrospinning
membranes.

release, where approximately 28 ± 3% of Van were released, fol-
lowed by a 80 ± 5% of Van was released in stage two (120–336 h).
Secondly, we evaluated the in vitro release of FITC-BSA from sin-
gle Gel nanofibers and PCL/Gel co-electrospinning membranes by
monitoring the fluorescence intensity. The FITC-BSA release profile
from single Gel nanofibers was shown in Fig. S7C. As illustrated,
almost 85% of the FITC-BSA was released in the first 2 d. Whereas,
the PCL/Gel co-electrospinning membranes released FITC-BSA in a
time-controlled manner dependent on the amount of PCL, with a
slow and sustained release profile for 9 d to over 14 d (Fig. 6A).
The release curve of FITC-BSA showed no signs of plateauing, sug-
gesting an extended release duration. However, unlike Van, the
release of FITC-BSA from the co-electrospun membranes only
occured in one stage.
In order to investigate the release profile of PCL-loaded
hydrophobic drug (Sim), we had selected PCL/Gel-5 for this inves-
tigation and compared it to the single PCL fiber (Fig. S9). The
release profile of Sim showed similar trends where the release con-
tinued for 28 d. Notably, after 9 d, the PCL/Gel group showed a
slight increase in the release curve (~10%) compared to single
PCL membrane. This sudden burst of release may be attributed to
8
the degradation of Gel, leaving a intra-fiber spaces facilitating the
direct contact of water to with more PCL fiber surfaces.
4. Discussion
The ever-rising incidence of bone-related disorders over the
years has piloted the advancement of guided tissue regeneration
membranes (GTRm) technology. In contrast to the traditional elec-
trospinning technique, a single co-electrospinning membrane
could be synthesized using multiple polymers and are endowed
with their special characteristics and traits [38]. Inspired, by the
nature of the extracellular matrix, we had designed a hierarchical
micro-nano PCL/Gel membrane for an in situ guided tissue regen-
eration equipped with drug-loading properties as well as the intent
to achieve a controlled and sequential release of drugs.
Hierarchical micro-nanofibrous structures were successfully
fabricated through co-electrospinning method. We observed the
apparent differences in the average diameter of the PCL/Gel fibers
among the groups. The difference in the sizes could be attributed to
the viscosity, concentrations, and flow rate of the polymer solution.
According to several studies, the aforementioned parameters
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al.
determines the amount of solute being pushed out from the needle
during the electrospinning process and hence, are essential in con-
trolling the resultant fiber sizes [35,36]. In our study, it is also
worth mentioning that in order to eliminate the possible flow
obstruction of Gel from the needle during electrospinning, the
addition of TFEA into the Gel solution was essential. The outcome
was in line with a previously reported study [37].
Another essential feature for GTRm to be considered as ideal is
its mechanical stability and integrity. According to a previous
study, bone tissue regeneration will occur for at least 4 to 6 weeks,
thus GTRm should be more resistant to degradation throughout
this process [11]. In addition, these membranes should be ade-
quately stable to serve as a barrier for unwanted cell migration
to facilitate a smooth bone regeneration process [11]. As is known,
natural polymers (e.g., gelatin), when used alone are more prone to
degradation compared to its synthetic counterpart [43]. Therefore,
to address this issue, the integration of PCL with Gel was expected
to enhance the mechanical stability and integrity of the mem-
branes. The co-electrospinning PCL/Gel membranes could serve
as a mechanical barrier isolates the defect’s internal and external
side, preventing large numbers of cells from flowing into the mus-
cle fascial space and external soft tissue invasion, which could
compromise the bone regeneration process. The porous fibrous
PCL/Gel co-electrospinning membrane could also slowly pass
endogenous cytokines and growth factors. Furthermore, the tensile
strength and ultimate strain also play an important determiner for
the degradation behaviour of the fibers [4]. According to our obser-
vation, both the tensile strength and ultimate strain of the PCL
fibers within the PCL/Gel membrane showed an inverse correlation
with its fiber size. This outcome could be attributed to the extreme
interlaying and entanglement of the PCL/Gel membrane fibers.
Wong et al. and Lim et al. also reported an increase in the PCL fiber
diameter leads to decreased PCL membrane’s ultimate strain
[38,39]. Based on these findings, we verified that mechanical char-
acterization findings showed that all the PCL/Gel co-
electrospinning membrane had outstanding mechanical strength
depending on the concentration of PCL presence in the membranes
compared to the single gel nanofiber membranes.
It has been established that the combination of both natural and
synthetic polymers in the membranes would further enhance cell
function and tissue regeneration [25]. Equipped with a high surface
area and highly intricate biochemical components, these highly
hierarchical structures facilitates the increased absorption of gly-
coprotein molecules (fibronectin and vitronectin) which aids in cell
adhesion as well as promoting osteoblastic differentiation [40].
Also, the addition of Gel would bring about anti-inflammatory
effects and are conducive to angiogenesis, facilitating bone repair
along with the prevention of fibrotic scarring [41,42]. The assem-
blies constitute a physical connecting bridge between the cellular
actin backbone to the ECM that allows cells to substrate the sensa-
tion of different physical factors, including matrix rigidity, physical
size, geometry, external matrix composition, and the surface’s
topology and molecular polarity. These changed their ability to
grow and differentiate [43]. Based on the obtained results, the
PCL/Gel co-electrospinning membrane with hierarchically micro-
nanofibers structures was beneficial to the promotion of bone tis-
sue regeneration.
We had evaluated the impact of the varying ratio of PCL poly-
mers on the degradation rate of Gel nanofibers within the co-
electrospinning membranes (Fig. 5). Water contact analysis obser-
vations showed an increase in the hydrophobicity of the mem-
brane with the increasing PCL fiber size resulting in a prolonged
water absorption. This phenomenon may be caused by the strong
PCL intra-fiber interaction and its water-repelling effect preventing
the contact between water and Gel, consequently slowing Gel
degradation and the initial drug release. We had also utilized
9
Materials & Design 209 (2021) 109962
SEM imaging to verify the complex interlaying structure of PCL/
Gel membrane. From our observation we could see the intercala-
tion of PCL and Gel fibers within the complex hierarchical fibrous
membrane. However, unlike ‘‘full encapsulation”, PCL only serve
as a partial barrier in limiting the amount of direct contact of water
with Gel, resulting in the delaying of Gel degradation. Together
with the observations obtained from the water contact angle anal-
ysis (Fig. S8), we theorized that degradation of Gel first began with
the more exposed Gel fibers located at specific sites on the mem-
brane, gradually spreading to the inner fibers. From Fig. 5, we could
observe the formation of grooves on the PCL fibers, which may be
left behind by degraded Gel. Moreover, Gel fibers will lose its struc-
tural integrity following the degradation by moisture causing it to
transform into a jelly-like substance which will adhere and accu-
mulate around the PCL fiber. This verifies that the size and amount
of PCL fiber added directly correlates to the slowing of Gel nanofi-
bers degradation rate.
Van and FITC-BSA were used as the model hydrophilic drugs to
evaluate their release profile from Gel. It is also worth mentioning
that the concentration of PCL within the membrane plays a crucial
determiner for the release profile of both Van and FITC-BSA. From
Fig. 6, it was obvious that the different groups of PCL/Gel co-
electrospinning membranes exhibited varying rates of a slow and
sustained release of both Van and FITC-BSA. In contrast, the indi-
vidual Gel membranes displayed a rapid burst release, which could
be linked to its high degradation rate in water due to its hydrophi-
lic nature. Different mathematical formulas (Table S2) were
applied for the in-depth understanding of the release kinetics at
every stage for both Van and FITC-BSA. In general, the release
curves of Van displayed a two-stage release mechanism, with dif-
ferent rates of burst release at different time points. At the first
stage of release, we theorized that it was during the swelling per-
iod that the drugs were being released. Drug diffuses out from the
swollen regions of the fibers into the inter-fiber spaces and then
into the external medium, which was in accordance to Fick’s law
of diffusion. However, the high amount of PCL in PCL/Gel-6 group
limits the swelling of Gel fibers, hence affecting the release of
drugs, causing them to be released via two release mechanism.
The drug release profile involves a standard diffusion as well as
fiber dissolution-associated release. This follows the non-Fickian
law of diffusion. Stage two release occurs due to the persistent
swelling of both Gel and PCL fibers, resulting in the alteration of
fiber morphology and size, ultimately causing the Gel and PCL
fibers to fuse. Based on the all the observations made above, we
could verify that PCL plays an important role in the resultant drug
release by indirectly regulating the Gel degradation through a
‘shielding’ effect. Therefore, to better understand the mechanics
behind stage two release, we had further used different mathemat-
ical formula (Table S2) to evaluate the release profile of the loaded
drugs from Gel with the varying concentrations of PCL within the
PCL/Gel membrane. As depicted from Fig. 6B, we could see that
an equal ratio of PCL to Gel (1:1) resulted in another burst release
of drugs abiding Fick’s law of diffusion. This could be due to the
fact that Gel was more exposed to the surrounding medium, signif-
icantly increasing the contact between water and Gel, increasing
its swelling and ultimately resulting in a rapid release rate of
Van. Gel transfer to the gel-like structure but failed to block the
inter-fiber spaces and the pores still that remain. The drug contin-
ues to diffusing, and Fick’s diffusion law was fitted. In contrast,
with the increase in PCL concentration (2: 1, PCL: Gel), some of
the Gel were still protected by the PCL fibers while the rest would
completely lose their shape and transform into a jelly-like sub-
stance. At this period the drug release would abide by the non-
Fickian diffusion law, involving the mixed type of release mecha-
nism, diffusion and dissolution. When the amount of PCL was
increased to (3: 1, PCL: Gel), most of the Gel fibers would still
M.A. Al-Baadani, K. Hii Ru Yie, A.M. Al-Bishari et al.
maintain their structure due to the ‘shielding’ effect of the higher
concentration of PCL. However, with time, the PCL fibers would
also begin to swell and undergo degradation resulting the PCL to
alter its shape, and further limiting the contact between the exter-
nal medium with the inner Gel fibers. This results in a significant
decrease of Van as well as its sustained release until the complete
degradation of the membrane, as can be seen in Fig. 6B, the release
kinetics to explain this phenomenon would be in line with the
zero-order kinetics.
Unlike Van, the release of FITC-BSA only occurred in one stage.
The molecular weight of FITC-BSA is similar to various growth fac-
tors, notably the VEGF [25]. For this reason, we assume that this
could influence the release profile of FITC-BSA. Consistent to the
mechanism previously explained in Van release, dependent on
the concentration and size of the PCL fibers, it would provide a
‘shielding’ effect for Gel against degradation by the external med-
ium to mitigate rapid release of FITC-BSA. Hence, we could corrob-
orate to the fact that the degradation of gel nanofibers and its
subsequent release could be manipulated through the regulation
of PCL concentration within the PCL/Gel membrane.
In this study, the prepared PCL/Gel co-electrospinning mem-
branes could be feasible as drug carriers endowed with tunable
drug release profiles ranging from initial rapid release (phase
one) to slow prolong release (phase-two). The collated results pro-
vide enough proof for the potential application of these mem-
branes in various biomedical applications. For instance, in a
situation where it requires a short-term rapid release of drugs
(e.g., potential bacterial infection after surgery) [44], a membrane
equipped with initial rapid release (stage one), PCL/Gel-1 or PCL/
Gel-4, would be a good choice. Another example where it requires
a rapid but sustained release of over a week [e.g., growth factors
(SDF-1, SEW2871) or a peptide (Substance P) for cell recruitment]
[45–47], PCL/Gel-2 or PCL/Gel-5 would be a suitable choice. Lastly,
when there is a need for a slow and sustained release of drugs for
over two weeks (e.g., growth factors, bone regeneration drugs)
[18], PCL/Gel-3 or PCL/Gel-6 would be an excellent choice. Further-
more, hydrophobic drugs could also be incorporated into PCL to
achieve prolonged sustained release profiles of over 27 days to
improve the bone regeneration process [48].
5. Conclusion
In the present research, a series of GTRm consisting of hydro-
philic Gel and hydrophobic PCL fibers were successfully fabricated
using the co-electrospinning technique. A number of parameters
including concentration and the flow rate of the PCL and Gel were
tuned to fabricate an optimal hierarchical micro-nanofiber mem-
brane with enhanced mechanical properties, biocompatibility and
controlled drug release properties. We demonstrated that PCL
fibers had enough mechanical strength to shield the membrane
and maintain its integrity until new bone formed. By incorporating
Gel into the process have proved to enhance the biocompatibility
of the membranes and simultaneously preserve its osteogenic
osteoinductive abilities. In addition, the effect of PCL in the mem-
brane could indirectly regulate the degradation of Gel, facilitating
drug release with tunable release profiles: 1) short-term rapid
release for two days to a week; 2) rapid initial release with sus-
tained release for five days to over two weeks; 3) slow, prolonged
release for over two weeks. In summation, the preparation of a
PCL/Gel-based co-electrospinning membranes with a tunable ini-
tial release of drugs from hydrophilic polymer and a subsequent
sequential release from a hydrophobic polymer could serve as a
model in the design of a more complex multifunctional co-
polymers-based drug delivery systems.
10
Materials & Design 209 (2021) 109962
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
This work was funded by National Natural Science Foundation
of China (82071170 and 81701016), Zhejiang Provincial Science
and Technology Project for Public Welfare (LY21H180006 and
LGF21H140004), Key Technological Innovation Projects of Wen-
zhou (ZY2019009), Wenzhou Public Welfare Science and Technol-
ogy Project (Y20190099), Wenzhou Medical University Basic
Scientific Research Operating Expenses (KYYW201905).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.matdes.2021.109962.
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