Rhee et al
Trop J Pharm Res, February 2017; 16(2): 271
Tropical Journal of Pharmaceutical Research February 2017; 16 (2): 271-278
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
All rights reserved.
Available online at http://www.tjpr.org
http://dx.doi.org/10.4314/tjpr.v16i2.3
Original Research Article
Bone regeneration potential of sub-microfibrous
membranes with osteogenic induction of rBMSC for tissue
engineering
Yun-Hee Rhee
1
, Seong-Hwa Oh
2
, Hyun-Ju Lim
3
, Jang-In Shin
1
, Chan Kim
4
and
Chung-Hun Oh
1,2,3
*
1
Department of Oral Physiology, College of Dentistry,
2
Department of Nanobiomedical Science & WCU Research Center,
3
Department of Medical Laser, Graduate school, Dankook University, Cheonan, 330-714,
4
AMOMEDI Co. Ltd, New Materials
Research Center, 597-2 Wonsanri, Hasungmyun, Kimpo, Kyungkido, 415-887, Republic of Korea
*For correspondence: Email: choh@dankook.ac.kr; Tel: +82-41-550-1918; Fax: +82-41-559-7906
Received: 18 October 2016 Revised accepted: 13 January 2017
Abstract
Purpose: To examine the biocompatibility and osteoinductive potential of sub-microfibrous membranes
with cells in vitro and in vivo.
Methods: Polylactic acid (PLA) and poly-ε-caprolactone (PCL) were blended at various volume ratios
(PLA:PCL = 100:0, 70:30, 50:50, 30:70 and 0:100) and each membrane form was prepared by
electrospinning. Cell viability, biocompatibility, and bone regeneration were measured.
Results: The membranes from the PLA/PCL blends prepared by an electrospinning process showed a
range of diameter distribution ranging from 1,580 to 550 nm. The cells of 100 % PCL membrane
(smallest diameter) exhibited significantly higher adhesion and proliferation than those of the other
membranes. Among the membranes from PLA/PCL blends, PCL membrane showed weak inflammatory
changes in the early stages of implantation without acute or chronic inflammation. PCL membranes with
osteogenically-induced cells successfully stimulated new bone formation in a rate calvarial defect
model.
Conclusion: The results indicate that biodegradable PCL sub-microfibrous membrane produced by
electrospinning process seems to have excellent biocompatibility, and may be used as a scaffold for
bone tissue engineering.
Keywords: Biocompatibility, Hard tissue, Biomaterial availability, Bone remodeling, Polylactic acid,
Poly-ε-caprolactone, Osteoinductive potential, Sub-microfibrous membranes
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INTRODUCTION
Fibrous membranes of biodegradable polymers
fabricated by an electrospinning technique have
been developed for medical applications such as
vascular graft [1], skin substitution [2], nerve
regeneration [3], bone tissue engineering [4], and
guided bone regeneration for dental surgery [5].
Many studies have been devoted to evaluating
the suitability of these applications for in vitro and
in vivo models [6]. Biodegradable substance,
such as poly (lactic acid) (PLA), Poly-ε-
caprolactone (PCL), poly (glycolic acid) (PGA),
and their copolymers, are regarded as the ideal
materials for medical applications.
To develop an ideal biomaterial, we need to take
into account not only its biocompatibility but also
the balance between the biodegradation rate of
exogenous polymers and the repair rate of the
Rhee et al
Trop J Pharm Res, February 2017; 16(2): 272
home tissue. Biodegradation rate is stimulated by
penetration, attachment, growth, and
differentiation of functional cells, but is inhibited
by inflammatory responses and tissue rejection
reactions. For optimal tissue engineering of
damaged tissue, the biodegradation of the
implanted scaffold, by host enzymatic and
hydrolytic activities, must be followed by tissue
replacement and functional restoration such as
mechanical strength and original cellular
activities.
Some studies have focused on the
biodegradability [7], physic-chemical properties
[8] and in vitro biocompatibility [2] of aliphatic
polyesters and aliphatic polyester blends. In this
work, two synthetic polymers were selected;
poly-ε-caprolactone (PCL) and poly-lactic acid
(PLA). The polymers were blended at different
ratios and were subjected to electrospinning. To
examine biocompatibility of these membranes,
human mesenchymal stem cells (hMSCs) were
grown on the PCL/PLA composite membranes,
and the tissue responses were examined in the
subcutaneous tissues of mice. To assess the
efficiency of bone formation, we evaluated the
bone hilling activity stimulated by bone marrow-
derived stromal cells (BMCs) with or without
osteogenic induction on the membranes in a rat
calvarial defect model.
EXPERIMENTAL
Preparation and characterization of fibrous
membrane
PLA (Sigma–Aldrich USA) and PCL (Sigma–
Aldrich, USA) were separately dissolved in 2,2,2-
trifluoroethanol (TFE, Sigma–Aldrich). The
volume ratio of PLA and PCL in the mixed
solutions was either 100:0, 70:30, 50:50, 30:70
or 0:100. The mixed solutions were stirred for 24
h. Each solutions were applied into the 10ml
syringe to electrospinning. An 15 kV voltage was
applied and the tip to collector distance was
maintained at 10 cm. The fibrous membranes
were dried under vacuum to evaporate the
solvent. The morphology of the samples was
examined by scanning electron microscopy
(SEM), and the diameter of the fibers was
calculated from the SEM images.
In vitro viability test
Human mesenchymal stem cells (hMSC) were
purchased from Lonza (Walkerville, USA) and
maintained in Dulbecco’s modified Eagle’s
medium (DMEM) containing 4.5 g/ml D-glucose,
10 % FBS, and 10 U/ml penicillin-streptomycin.
The proliferation of hMSCs on fibrous
membranes was evaluated using a MTS assay
(Promega, USA). Briefly, hMSCs were seeded at
a density of 2 × 10
5
cells/cm
2
on various types of
fibrous membranes in a 12-well plate at 0 days.
The cells were cultured in the growth medium.
Every 24 h, an MTS solution was added to each
well, incubated for 30 min, and measured using a
microplate reader (Molecular Devices, USA) at
450 nm. The proliferation of hMSCs was
observed by confocal microscopy. The cells were
processed, as described in the MTS assay. All
membranes were fixed with chilled ethanol every
24 h, and 50 μg/ml propidium iodide solution was
added to each well. The membranes with the
cells were incubated for 15 min and washed with
PBS briefly. Each time the number of cells in the
fibrous membranes was counted and
photographed by LSM510 confocal microscopy
(Carl Zeiss, Switzerland).
In vivo biocompatibility test
The animal protocol used in this study was
reviewed and approved based on ethical
procedures and scientific care by the Dankook
University-Institutional Animal Care and Use
Committee (DKU-IACUC). To implant the fibrous
membranes, incisions were cut on the dorsal
section of each mouse and the fibrous
membranes were and implanted into the
incisions. The incisions were then sutured. After
two weeks, the mice were sacrificed, and the
entire graft was harvested with surrounding
tissue. The samples were fixed with 4 %
paraformaldehyde and were subjected to
Hematoxylin-Eosin staining.
Assessment of bone regeneration in calvarial
defects
To evaluate whether bone marrow-derived
stromal cells (BMCs) with osteoinduction
participate in in vivo bone formation, a calvarial
bone defect model was used for this assay in 8-
week-old Sprague-Dawley (SD) rats. BMCs were
induced in osteogenic media for 7 days after
growth in DMEM for 7 days. The osteogenic
media consisted of 50 mM L-ascorbic 2-
phosphate, 10 mM glycerol-2-phosphate
disodium, and 1 μM dexamethasone, and the
medium was replaced every 3 days.
Under general anesthesia, induced by an
intramuscular injection of ketamine/rumpun (80
and 10 mg/kg), the external cortical plates were
removed using a 5-mm trephine bur with saline
irrigation [9] [10]. One asymmetrical defect was
made on the right side of the midline of each rat.
Extreme care was taken not to harm the brain
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Trop J Pharm Res, February 2017; 16(2): 273
membranes. The bone defects were washed with
normal saline, and membranes were then
applied to the defects.
Animals were divided into three groups: Group 1,
PCL membrane only (n=3); Group 2, PCL
membrane with non-induced rBMCs (n=3);
Group 3, PCL membrane with osteogenic
induction of rBMCs (n=3); No
immunosuppressants were used. After 4 weeks,
the rats were euthanized, and the transplanted
constructs were dissected carefully and taken out
of the surrounding soft tissue.
Microradiographs of the skull were performed
using an Image Station FX (Kodak, Rochester,
NY) for 6 s at 12.5 kVp. After the radiographs
had been taken, the defected calvaria samples
were de-calcified with 20 % EDTA. The samples
were then dehydrated in a graded series of
ethanol and embedded in paraffin. The center of
the defect area was sectioned to a 4 μm
thickness, stained with Masson’s Trichrome, and
photographed using an optical microscope
(Olympus BX51, Olympus, Miami, FL, USA).
Statistical analysis
All results are presented as mean and stabdard
deviation (SD) and were evaluated using one-
way analysis of variation (ANOVA) and also
Tukey’s analysis for pair wise comparison.
Differences were considered significant at p <
0.05.
RESULTS
Structural morphology of the fibrous
membrane
As shown in Figure 1, fibrous membranes
produced from different volume ratios of PLA and
PCL by an electrospinning technique led to a
range of diameter distributions. The three-
dimensional fibrous structures with polygonal,
interconnected pores and randomly oriented
fibers were evaluated for their diameter range.
The diameters of homogenous PLA fabrications
was determined to be 1,580 ± 232 nm (Figure 1A
and F), PLA: PCL=3:7 835 ± 123 nm (Fig. 1B
and G), PLA: PCL = 5:5 842±97 nm (Fig. 1C and
H), PLA: PCL = 3:7 743 ± 173 nm (Figure 1D
and I), and the homogenous PCL 558 ± 142 nm
(Figure 1E and J).
Viability of hMSC on the fibrous membrane
The viability of the hMSCs on the fibrous
membrane was examined using an MTS assay
and PI staining (Fig. 2). On day 0, the hMSCs
were seeded onto the various types of
membrane, and their attachment and
proliferation were observed after 7 days. As
shown in Fig. 2a, the hMSCs were showed
exponential growth patterns, without any
cytotoxicity, for all types of membranes until day
4. Cells on PCL fibrous membranes showed a
significantly higher viability than those on any of
the other materials, and were in a relatively
quiescent state throughout the 7 day period after
seeding, compared to those of other membranes.
In vivo biocompatibility
Histological analysis by optical pathological
microscopy was performed to make a
comparison of the infiltration to various types of
membrane. The pore size of the fibrous
membranes is too small for cells to penetrate in
vitro. However in vivo cellular infiltration was
observed in the subcutaneous tissue of mice
examined in week 2. As shown in Figure 3,
histological examination of subcutaneous
implantation with fibrous membranes revealed an
infiltration of histiocytes and lymphocytes in the
area of the margin and center, respectively.
There was no evidence of acute or chronic
inflammation in any groups. Our results showed
that hypertrophy of lining histiocytes and
infiltration of lymphocytes in the membranes
gradually decreased with increasing ratio of PCL.
However, the difference was not determined to
be statistically significant (p > 0.5). For this
reason, PCL seems to be a suitable candidate as
a degradable fibrous membrane for use in
biomaterials.
Bone healing in rat calvarial defect model
Bone regeneration effects were evaluated in the
rat calvarial defect model by Soft x-ray and
histological staining after four weeks. From the
Soft x-ray observation (Figure 4B, implantation of
fibrous membranes with non-induced rBMSC
(group 2) to calvarial defected rats showed
limited bone formation at the periphery, as did
membrane only group (group 1). In contrast,
implantation of the membrane with osteogenic
induction of rBMSC (group 3) showed a
significantly greater extent of bone healing, both
on the inside and peripheral regions of the
defect, compared to other groups. The
quantitative analysis of bone density was
consistent with Soft X-ray images. The relative
bone regeneration area of group 3 was 0.73 ±
0.08 mm, which was significantly greater than
that of group 2 (0.36 ± 0.07 mm) and group 1
(0.28 ± 0.03 mm).
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Trop J Pharm Res, February 2017; 16(2): 274
Figure 1: Ultrastructural morphology and fiber diameter distributions of PLA/PCL composites fabricated by the
electrospinning technique. Microphotographs were made by SEM. (A) PLA 100 %, (B) PLA:PCL = 70:30, (C)
PLA:PCL=50:50, (D) PLA:PCL=70:30, (E) PCL 100 %
Figure 2: Cell proliferation on various PLA/PCL blended membranes. The proliferation of hMSC on the
nanofibrous membrane was evaluated by an MTS assay and confirmed by PI staining. The hMSCs were seeded
at a density of 2 × 10
5
cells/cm
2
on various types of nanofibrous membranes put in a 12-well plate. The cells were
cultured in growth medium. MTS assay and PI staining were performed every 24 h for 4 days. Each time, the
viable cells on the nanofibrous membranes were assessed by a microplate reader and photographed by confocal
microscopy. The data is expressed as the mean ± SD and the significance was determined at *p < 0.1, **p < 0.05
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Trop J Pharm Res, February 2017; 16(2): 275
Figure 3: Histological analysis of the tissue adjacent to the nanofibrous membrane implanted subcutaneously in
mice. The mice were sacrificed 2 weeks after various types of nanofibrous membranes were implanted. Multiple
mononuclear elements, chiefly macrophages, and rare lymphocytes adhere to the membrane. Mild acute
responses were observed in the PCL membrane. Some membranes exhibited fibrosis in the adjacent tissue. The
slit under the membrane was probably due to artificial retraction of the tissue
Figure 4: Photographs and soft X-rays of calvarial defects Bone marrow-derived stromal cells were isolated and
maintained for passage three, and then plated onto 1cm × 1cm of a square nanofibrous membrane at a density
of 2×10
4
cells/sheet. The cells were cultured with/without differentiation induction medium for 7 days. One
asymmetrical defect was made on the right side of the midline in each rat. The PCL membrane with the cells was
placed over the defects, and after 4 weeks, the rats were euthanized, and the transplanted constructs were
dissected carefully and free from the surrounding soft tissue. Photographs (A) and microradiographs (B) of the
skull were taken using Image station FX