Title:
A 3D collagen hydrogels stem cell tissue engineered formulation basing on growth factor
phospholipid complex loaded PHBHHx nanoparticles
Authors:
Cui-Ling Donga, William R. Webba, Qiang Pengd, James Z. Tangc, Nicholas R. Forsytha, GuoQiang Chenb*, Alicia J. El Haja#
Affiliations:
a
Guy Hilton Research Centre, Institute of Science and Technology in Medicine, Keele
University, Stoke-on-Trent ST4 7QB, UK.
b
MOE Key Lab of Bioinformatics, Tsinghua-Peking University Joint Center for Life Sciences,
Department of Biological Science and Biotechnology, School of Life Science, Tsinghua
University, Beijing 100084, China.
c
Department of Pharmacy, School of Applied Sciences, University of Wolverhampton,
Wolverhampton, WV1 1SB, UK.
d
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan
University, Chengdu 610041, China.
#Corresponding
author:
Alicia J. El Haj (El Haj AJ)
Tel.: +44 01782 554605, Fax: +44 01782 747319, E-mail: a.j.el.haj@keele.ac.uk
*Co-Corresponding author:
Guo-Qiang Chen (Chen GQ)
Tel.: +86 10 62783844, Fax: +86 10 62794217, E-mail: chengq@mail.tsinghua.edu.cn
1
Abstract: This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate
and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve
growth factor sustained release for long-term stimulation of human mesenchymal stem cells
(hMSCs) proliferation/differentiation for tissue engineer application. Platelet derived growth
factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was
selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be
the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx
nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via
assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D
collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable
in terms of their mean particle size, polydispersity index and zeta potential before and after
lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release.
Furthermore, sustain released PDGF-BB was revealed to significantly promote hMSCs
proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serumfree media. Therefore, the 3D collagen matrices with locally sustained release growth factor
nanoparticles hold promise to be used for stem cell tissue engineering.
Key words: PHBHHx; Nanoparticles; Platelet derived growth factor-BB; Mesenchymal stem
cell; Collagen hydrogel
2
INTRODUCTION
Bone marrow mesenchymal stem cells (MSCs) are considered to be one of the most
promising cell types in the field of tissue engineering 1. MSCs have been shown to differentiate
into a number of cell types such as osteoblasts, chondrocytes, adipocytes, as well as muscle cells
2-4
, and it is known that culture conditions and growth factors are essential factors for the control
of their differentiation and proliferation
5,6
. Growth factors have been shown to be powerful
regulators with biological function, and their presence in MSCs is highly regulated in both time
and spatial cues
7,8
. However, the rapid inactivation and extremely short plasma half-life of the
growth factors prevent their sustained effect 9. It is therefore essential to develop a carrier system
to maintain growth factors at an effective concentration for a longer period, which potentially
can direct MSCs differentiation spatially in a 3D environment 10-11.
Among the possible strategies to achieve sufficient bioavailability of growth factors,
nanoparticle carriers made from biodegradable polymers represent an exciting approach to
improve the transport of growth factors and its subsequent initiation of a cellular response
12-14
.
Compared with other colloidal carriers, such as submicron emulsions and liposomes,
biodegradable polymeric nanoparticles offer a higher stability when they are in contact with
biological fluids 15,16. The polymeric nature of nanoparticle also protects the growth factors from
adverse external conditions and controls their release during the polymer degradation process 17.
Poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx), including its other members
of polyhydroxyalkanoates (PHA) biopolyester family, has been extensively studied as a tissue
engineering biomaterial and considered to be a very promising biomaterial due to its total
biodegradability, non-toxic, non-immunogenic degradation products and better elastic properties
18-23
. PHBHHx was ever used as a biodegradable nanoparticle carrier for sustained release of
hydrophobic drugs due to its hydrophobic ester backbone and alkane side chain
24,25
. However,
most growth factors are hydrophilic proteins and this poses a significant challenge to
encapsulating not hydrophobic protein into hydrophobic PHBHHx nanoparticles efficiently. In a
previous study, Peng et al
26
and his group developed an attractive way to improve the
liposolubility of water-soluble protein with a higher entrapment efficiency by formation of a
drug phospholipid complex, which is consistent with the results reported by Cui et al 12.
3
Thermo-sensitive collagen hydrogel is a free-flowing liquid under room temperature and
can form a non-free-flowing gel at 37oC
27
. Based on its solution to gel transition property and
favorable biological characteristics (i.e., biocompatibility, biodegradability), the thermo-sensitive
hydrogel has been widely studied as a 3D tissue engineer scaffold.
Herein, we propose to prepare the growth factor phospholipid complex loaded PHBHHx
nanoparticles to achieve growth factor sustained release for long-term stimulation of hMSCs
proliferation/differentiation in 3D collagen hydrogel cell culture systems. A growth factor,
platelet derived growth factor-BB (PDGF-BB), which has previously been demonstrated to
maintain undifferentiated proliferation of mesenchymal stem cells
28
, was used as a model
growth factor due to its ease of analysis and ready availability. PDGF-BB phospholipid complex
loaded PHBHHx nanoparticles (PDGF NPs) were fabricated, then co-embedded in collagen
hydrogel with hMSCs. The sustained release PDGF-BB from PDGF NPs was evaluated and its
effect on human MSCs proliferation was investigated via assays of Cell Counting Kit-8 (CCK-8)
and live-dead staining to cells inoculated on 2D tissue culture plate and 3D collagen gel scaffold,
respectively.
MATERIALS AND METHODS
Materials
PHBHHx [Poly(R-3-hydroxybutyrate-co-12 mol% R-3-hydroxyhexanoate)] was supplied
by Lukang Group (Shandong, China). Recombinant human PDGF-BB was purchased from
Peprotech (NJ, USA). Human PDGF-BB quantikine ELISA kit was bought from R&D systems
(UK). DMEM containing high glucose, L-glutamine, non-essential amino acids (NEAA), and
phosphate buffered saline (PBS), were purchased from Lonza (Switzerland). Rat tail collagen
type I was provided by BD Biosciences (UK). Cell Counting Kit-8 (CCK-8) was supplied by
Dojindo (Japan). Live-dead cell double staining kit, l-α-phosphatidylcholine, poloxamer 188
(F68), sodium deoxycholate, acetic acid, dimethyl sulfoxide (DMSO) and chloroform were
purchased from Sigma Aldrich (SL, USA). All other chemical reagents were analytical grade or
better (Sigma Aldrich, USA).
Fabrication of PHBHHx nanoparticles
4
PHBHHx nanoparticles were prepared based on a method reported previously 26 with some
modifications. PDGF-BB and l-α-phosphatidylcholine were co-dissolved in DMSO containing
1.25% glacial acetic acid under magnetic stirring at 30°C, then freeze-dried overnight to obtain
PDGF-BB phospholipid complex (PDGF-PLC). PHBHHx and PDGF-PLC were dissolved or
dispersed in chloroform at a weight ratio of 80:1. Subsequently, an aqueous solution containing
0.5% F68 and 0.5% sodium deoxycholate was added to the organic solution with a volume ratio
of 20:1, and followed by sonication in an ice-water bath to form a stable o/w emulsion. A
homogeneous fresh nanoparticles suspension was obtained after chloroform evaporation. Before
storage, 10% maltose was dissolved in nanoparticles suspension. The mixture was frozen at 80°C overnight followed by lyophilization for 48 h. Blank NPs and BSA NPs were prepared as
controls using the same protocol.
Physicochemical characterization of PHBHHx nanoparticles
The mean particle size, distribution and zeta potential of the resulted PHBHHx nanoparticle
suspension were studied using a Zetasizer 3000 HSA (Malvern Instruments Ltd., U.K.) at 25°C.
The particle size was evaluated based on the intensity distribution, which was evaluated by
polydispersity index (PDI). After re-dispersion in distilled water, the lyophilized PHBHHx
nanoparticle suspension was measured via the same parameters as fresh nanoparticles.
The morphology of PHBHHx nanoparticles was observed using a Scanning Electron
Microscope (Hitachi S4500) (Japan) at an accelerating voltage of 5 kV. One drop of the resulted
PDGF NPs suspension was placed on a glass surface. After an air drying process, the sample was
coated with gold using an Ion Sputter.
Encapsulation efficiency of PHBHHx nanoparticles
Briefly, 0.5 ml the resulted PDGF NPs suspension was mixed with 0.5 ml 0.05 M NaOH.
The mixture was stirred for 24 h at room temperature so that the PHBHHx nanoparticles could
be degraded by alkaline hydrolysis. The resultant sample was analyzed by ELISA kit after proper
dilution. As a result the total added amount of PDGF-BB (Wt) was obtained. Meanwhile, another
0.5 ml PDGF NPs suspension was taken, and mixed with 0.5 ml dH2O. The sample was also
analyzed by ELISA kit after proper dilution. As such the amount of non-encapsulated PDGF-BB
(Wn) was obtained. Therefore, the encapsulation efficiency could be calculated according to the
following equation:
5
EE = (Wt - Wn) / Wt × 100%
In vitro release kinetic of PDGF-BB from PDGF NPs
PDGF-BB release profile was measured using the dialysis tube (Spectrum Labs) with 100
kDa molecular weight cut off, which keeps PDGF NPs in and allows the released PDGF-BB out
of the tube. After 1 ml PDGF NPs suspension was transferred into a dialysis tube, the sample
loaded dialysis tube was soaked in 6 ml dH2O at room temperature under magnetic stirring. At
predetermined time intervals the dH2O outside the dialysis tube was withdrawn completely and
replaced with 6 ml fresh dH2O. The collected samples were diluted into 10 ml then stored at 80°C for ELISA kit analysis.
Optimization of PDGF-BB concentration for hMSCs proliferation
Primary human mesenchymal stem cells (Lonza) were employed to investigate optimal
PDGF-BB concentration to promote hMSCs proliferation in vitro. hMSCs (passage 3-5) were
plated in 96-well plates with a cell density of 4×103 cells per well, and cultured for 24 h on a full
serum medium (DMEM supplemented with 5% fetal bovine serum, 1% L-glutamine and 1%
NEAA). Then followed by 24 h culture in serum free medium (DMEM supplemented with 1%
L-glutamine and 1% NEAA). PDGF-BB was supplemented at final concentrations of 0, 5, 10,
and 25 ng/ml, respectively, with 1% NEAA and 1% L-glutamine in DMEM media. At multiple
time points, cell proliferation was evaluated using the CCK-8 assay.
Effect of sustained released PDGF-BB on hMSCs proliferation in 2D cell culture system
hMSCs were seeded in 6-well plates at the density of 5×104 cells per well, and cultured for
2 days with a full serum medium to reach about 50% confluence. Then followed by 24 h culture
in serum free medium. hMSCs were incubated in sterile PDGF NPs contained serum-free media.
Cell viability was measured using CCK-8 kit after culturing for 2 and 4 days, respectively. Cells
cultured in BSA NPs, blank NPs and no NPs contained media were used as controls.
Effect of sustained released PDGF-BB on hMSCs proliferation in cell seeded 3D collagen
hydrogels
hMSCs were seeded in T75 flasks, then cultured for 3 days with the full serum medium,
followed by 24 h culture in serum free medium. After the trypsinizing treatment, hMSCs
suspension in DMEM media, sterile 10×DMEM, sterile concentrated PDGF NPs suspension,
6
sterile 1 M NaOH, and rat tail collagen, were combined according to the BD Biosciences (UK)
manufacturer’s instructions in ice bath to produce a final concentration of 2×105 cells/ml in 0.5
mg/ml collagen gel. After mixing homogeneously by pipetting, a volume of approximately 400
µl was dispensed into 6-well plates. After incubation in 37oC for 30 min, the collagen scaffold
was covered with serum-free DMEM media, and incubated in 37oC incubator. Cell viability was
measured using CCK-8 kit after culturing 2 and 4 days, respectively. In addition, the overall cell
morphology was monitored via a live-dead cell double staining kit using Nikon Eclipse T1
microscope fluorescence microscope (Japan) at day 2 and 4. Collagen scaffolds fabricated with
BSA NPs, blank NPs and no NPs were used as controls.
Statistical analysis
All data was expressed as the mean ± standard deviation (SD). Statistical analysis was
carried out using the unpaired Student’s t-test (Microsoft, Seattle, USA). P ＜ 0.05 was
considered to be statistically significant and P＜0.01 highly significant.
RESULTS
Characterization of PHBHHx nanoparticles before and after lyophilization
The properties of PHBHHx nanoparticles before and after lyophilization were studied and
compared with their mean particle sizes, PDI, zeta potentials and encapsulation efficiencies
(Table 1). Among the three types of PHBHHx nanoparticles, mean particle size and PDI slightly
fluctuated, while PDGF NPs and BSA NPs had significantly higher zeta potential than that of the
blank NPs. In addition, the lyophilized PHBHHx nanoparticles could be dispersed in distilled
water and quickly re-formed to become a homogeneous nanoparticle suspension. Compared with
fresh nanoparticles, lyophilized nanoparticles became slightly larger having a higher PDI value
and lower zeta potential. Importantly, the lyophilization process did not lead to a significant
change in encapsulation efficiency of PDGF NPs where a slight, but non-significant decrease
from 82.97% to 74.63% was observed. These results indicated that the lyophilization process did
not lead to a big change on properties of PHBHHx nanoparticles. Meanwhile, the PHBHHx
nanoparticles, observed via SEM, were mostly spherical in shape and well distributed in size (Fig.
1).
7
In vitro release kinetic of PDGF-BB from PDGF NPs
Figure 2 showed the in vitro release profile of PDGF-BB from PDGF NPs obtained using a
dynamic dialysis method. Over the 72 h course, the cumulative amounts of released PDGF-BB
were almost 25%. Nearly 20% of encapsulated PDGF-BB was released during the initial 24 h,
and about 5% release was observed in the late 48 h. Overall, a sustained release of PDGF-BB
was achieved, and the optimal PDGF-BB concentration range could be achieved by varying of
the amount of PDGF NPs in the further cell culture studies.
Optimization of PDGF-BB concentration for hMSCs proliferation
To determine the optimal working concentration range of PDGF-BB for hMSCs
proliferation, viability of cells grown in four different concentrations (0, 5, 10 and 25 ng/ml) of
PDGF-BB media was studied after 2, 4 and 6 days incubation, respectively (Fig. 3). In all cases,
cells cultured in PDGF-BB media displayed improved viability when compared to the control
group of cells cultured without PDGF-BB. Among three different concentrations, activities of
cells cultured with 5 ng/ml and 10 ng/ml PDGF-BB were much better compared with 25 ng/ml
PDGF-BB group (Fig. 3). These results suggested that the presence of PDGF-BB promoted
hMSCs proliferation and 5-10 ng/ml had the most significant positive effect, while too high
concentration (25 ng/ml) or negative control (0 ng/ml) was not inductive for hMSCs proliferation.
Effect of sustained released PDGF-BB on hMSCs proliferation in 2D cell culture system
Cell proliferation in monolayer 2D cell culture system was analyzed by CCK-8 assay.
Primary hMSCs were used to evaluate cell proliferation in various nanoparticles containing
media. After 2 days incubation, hMSCs cultured in PDGF NPs containing media showed the
strongest increase in cell numbers compared with that in BSA NPs, blank NPs and no NPs ones
(Fig. 4). Meanwhile, cells cultured in BSA NPs and blank NPs media were similar in number
compared with the group cultured in no NPs contained media. After 4 days incubation, cell
number increased significantly in PDGF NPs media, while little increase was observed in control
groups (Fig. 4). It became clear that the presence of PDGF NPs in media improved hMSCs
proliferation.
Effect of sustained released PDGF-BB on hMSCs proliferation in cell seeded 3D collagen
hydrogels
8
After 2 and 4 days cultivation in 3D collagen scaffolds containing PDGF NPs, BSA NPs,
blank NPs or no NPs, hMSCs were stained using a live-dead fluorescent double staining kit
which is able to simultaneously stain viable cells green, and dead cells red. Although cell shapes
in hydrogels could not be clearly observed under a fluorescence microscope, more green
fluorescence was visible inside the PDGF NPs containing collagen scaffolds than that of BSA
NPs, blank NPs and no NPs containing scaffolds, respectively (Fig. 5). In addition, green
fluorescence was observed to become more intensive and widely distributed on day 4 compared
with that on day 2, especially in the PDGF NPs containing scaffolds. This phenomenon suggests
that PDGF NPs sustained release system was more effective in promoting cell proliferation than
the controls outlined above.
CCK-8 assay was employed to compare hMSCs viability in various nanoparticles
containing collagen gels. The highest cell viability was observed in PDGF NPs containing gels,
while other groups were similarly lower in viability among BSA NPs, blank NPs and no NPs
controls (Fig. 6). These results again demonstrated that sustained released PDGF-BB
significantly promoted hMSCs proliferation in serum-free media.
DISCUSSION
It is a challenge to directly encapsulate hydrophilic protein into hydrophobic PHBHHx
vehicle. Among the present encapsulation techniques used, w/o/w method represents a relatively
simple and efficient way for entrapment of hydrophilic compounds
20
. Nevertheless, the protein
loading capacity of the carriers is reduced significantly due to the rapid migration and loss of
drug into the external aqueous phase 20. Improving the liposolubility of hydrophilic protein is an
attractive way to enhance their loading efficiency. Previous studies have shown that the
lipophilicity of protein was significantly enhanced by the formation of protein phospholipid
complex, and it was successfully used to fabricate a controlled release insulin nanoparticles 26,27.
Based on this successful study, PDGF-BB was complexed with phospholipid by an anhydrous
co-solvent lyophilization procedure, which led to the high encapsulation efficiency of PDGF-BB
in the PDGF NPs system (Table 1).
To prepare PHBHHx nanoparticles loaded with growth factor, different parameters
including polymer concentrations, polymer and drug solvents, as well as preparation
9
temperatures were optimized to obtain nanoparticles with small particle sizes, low PDI value and
high zeta potential, which indicated the fine stability of the prepared nanoparticle system (Table
1). Nevertheless, it is well known that the stable system would be destroyed due to oxidation of
phospholipid, particle aggregation and/ or bacterial growth if nanoparticles were mainatianed in
a suspension for a long time. Hence, lyophilization technique was applied to maintain the longterm stability of the nanoparticle system. As shown in Table 1, nanoparticles before and after
lyophilization presented similar quality with a bit change possibly due to partial aggregation
among nanoparticles.
Generally, there are two distinct release mechanisms of encapsulated proteins from
nanoparticles or microspheres. Firstly, the initial minor rapid dissociation of surface-adsorbed
proteins; secondly, a sustained release of proteins ruled by thermodynamic equilibrium 29. Many
studies have reported that the encapsulated proteins show a significant fast release rate with an
initial burst 30,31. One potential reason is the large amount of proteins absorbed on the surface of
nanoparticles; another one is the faster degradability of materials. In this study, we have shown
that there is a high degree of slow release by diffusion through the nanoparticle skeleton of
PDGF-BB from PDGF NPs (Fig. 2). Meanwhile, a small degree of burst release was observed
potentially due to the intensive washing process and the very slow degradability of PHBHHx at
this relative short period.
It is well known that the attachment and spreading of cells can occur in serum containing
environment since there are many cell attachment and proliferation factors in serum 32. Therefore,
hMSCs were pretreated with serum free medium for 24 h before replacing the PDGF-BB
containing media. In order to study the optimal PDGF-BB concentration working for hMSCs
proliferation, serum free media was also selected with 0, 5, 10 and 25 ng/ml PDGF-BB,
respectively (Fig. 3).
In addition, since the PDGF NPs were composed of PDGF-BB, PHBHHx and l-αphosphatidylcholine, blank NPs and BSA NPs were prepared as control groups. Meanwhile,
nanoparticles free media or collagen scaffold were also selected as control groups in the cell
culture study. As expected, cell viability in PDGF NPs containing environment, tested by CCK-8
assay (Fig. 4 & 6), was better than those in all control groups, demonstrating that released
PDGF-BB from PDGF NPs improved hMSCs proliferation. More evidence was obtained by the
10
live-dead cell double staining (Fig. 5). In this study, we have also demonstrated the use of
PHBHHx nanoparticles to release growth factor in a 3D cell seeded collagen environment. The
remarkable increase in cell number and viability implied the critical role of released PDGF-BB
in hMSCs growth and proliferation in tissue engineering approaches. Using this technique, we
can begin to spatially regulate cell differentiation within a complex tissue engineered organ.
CONCLUSIONS
PDGF-BB phospholipid complex loaded biodegradable PHBHHx nanoparticles fabricated
by a solvent evaporation method was prepared for sustained release PDGF-BB in collagen
hydrogel scaffolds. Meanwhile, BSA phospholipid complex loaded nanoparticles and blank
nanoparticles were produced as controls. Physico-chemical characterization showed a small
particle size, spherical shape, as well as high cytokines encapsulation efficiency. Lyophilized
nanoparticles presented similar properties with the fresh ones. PDGF-BB released from PDGF
NPs at a slow rate showed a slight burst release. Significantly, CCK-8 assay indicated a clearly
positive effect of sustained release PDGF-BB on hMSCs proliferation regardless of whether the
inoculation was in 2D tissue culture plates or in 3D collagen scaffolds. Additional live-dead
fluorescent double staining assays further confirmed the CCK-8 results. It was concluded that
growth factor phospholipid complex loaded PHBHHx nanoparticles was a convenient and
effective approach for the sustained and controlled release of growth factors to stimulate stem
cells growth, proliferation and differentiation in 3D collagen hydrogels. Therefore, the PHBHHx
nanoparticle collagen hydrogel system is promising to serve as a 3D stem cell tissue engineering
formulation.
ACKNOWLEDGEMENTS
This research was supported by the HYANJI Scaffold Project (European Commission
Framework 7 program: SP3 PEOPLE- Grant agreement: PIRSES-GA-2008-230791).
11
References
1. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem
cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:12941301.
2. Sarkar D, Ankrum JA, Teo GSL, Carman CV, Karp JM. Cellular and extracellular
programming of cell fate through engineered intracrine-, paracrine-, and endocrine-like
mechanisms. Biomaterials 2011;32:3053-3061.
3. Park JS, Yang HN, Woo DG, Jeon SY, Park KH. Chondrogenesis of human mesenchymal
stem cells in fibrin constructs evaluated in vitro and in nude mouse and rabbit defects models.
Biomaterials 2011;32:1495-1507.
4. Pittenger MF et al. Multilineage potential of adult human mesenchymal stem cells. Science
1999;284:143-147.
5. Bianchi G, Muraglia A, Daga A, Corte G, Cancedda R. Microenvironment and stem properties
of bone marrow-derived mesenchymal cells. Wound Repair Regen 2001;9:460-466.
6. Deasy BM, QuPeterson Z, Greenberger JS, Huard J. Mechanisms of muscle stem cell
expansion with cytokines. Stem Cells 2002;20:50-60.
7. Molloy T, Wang Y, Murrell G. The roles of growth factors in tendon and ligament healing.
Sports Med 2003;33:381-394.
8. Thomopoulos S, Das R, Sakiyama ES, Silva MJ, Charlton N, Gelberman RH. bFGF and
PDGF-BB for tendon repair: controlled release and biologic activity by tendon fibroblasts in
vitro. Ann Biomed Eng 2010;38:225-234.
9. Chan BP, Fu S, Qin L, Lee K, Rolf CG, Chan K. Effects of basic fibroblast growth factor
(bFGF) on early stages of tendon healing: a rat patellar tendon model. Acta Orthop Scand
2000;71:513-518.
10. Tzeng SY, Hung BP, Grayson WL, Green JJ. Cystamine-terminated poly(beta-amino ester)s
for siRNA delivery to human mesenchymal stem cells and enhancement of osteogenic
differentiation. Biomaterials 2012;33:8142-8151.
11. Park JB, Matsuura M, Han KY, Norderyd O, Lin WL, Genco RJ, Cho MI. Periodontal
regeneration in class III furcation defects of beagle dogs using guided tissue regenerative
therapy with platelet-derived growth factor. J Periodontol 1995;66:462-477.
12. Cui F, Shi K, Zhang L, Tao A, Kawashima Y. Biodegradable nanoparticles loaded with
insulin-phospholipid complex for oral delivery: preparation, in vitro characterization and in
vivo evaluation. J Control Rel 2006;114:242-250.
12
13. Zhao J, Mi Y, Feng SS. Targeted co-delivery of docetaxel and siPlkl by herceptin conjugated
vitamin E TPGS based immunomicelles. Biomaterials 2013;34:3411-3421.
14. Yamanaka YJ, Leong KW. Engineering strategies to enhance nanoparticle-mediated oral
delivery. J Biomater Sci Pplym Ed 2008;19:1549-1570.
15. Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr
Opin Solid State Mater 2002;6:319-327.
16. Gan CW, Chien S, Feng SS. Nanomedicine: enhancement of chemotherapeutical efficacy of
docetaxel by using a biodegradable nanoparticle formulation. Curr Pharm Des 2010;16:23082320.
17. Moghimi SM. Recent developments in polymeric nanoparticle engineering and their
applications in experimental and clinicaloncology. Anticancer Agents Med Chem
2006;6:553-561.
18. Lu XY, Zhang Y, Wang L. Preparation and in vitro drug-release behavior of 5-fluorouracilloaded poly(hydroxybutyrate-co-hydroxyhexanoate) nanoparticles and microparticles. J.
Appl. Polym. Sci 2010;116:2944-2950.
19. Wang Y, Bian YZ, Wu Q, Chen GQ. Evaluation of three-dimensional scaffolds prepared
from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes
for cartilage repair in rabbits. Biomaterials 2008;29:2858-2868.
20. Williams DF. On the nature of biomaterials. Biomaterials 2009;30:5897-5909.
21. Francis L, Meng DC, Knowles JC, Roy I, Boccaccini AR. Multi-functional P(3HB)
microsphere/45S5 Bioglass (R)-based composite scaffolds for bone tissue engineering. Acta
Biomaterialia 2010;6:2773-2786.
22. Misra SK, Nazhat SN, Valappil SP, Moshrefi-Torbati M, Wood RJK, Roy I, Boccaccini AR.
Fabrication and characterization of biodegradable poly(3-hydroxybutyrate) composite
containing bioglass. Biomacromolecules 2007;8:2112-2119.
23. Philip S, Keshavarz T, Roy I. Polyhydroxyalkanoates: biodegradable polymers with a range
of applications. J Chem Technol Biotechnol 2007;82:233-247.
24. Kılıçay E, Demirbilek M, Türk M, Güven E, Hazer B, Denkbas EB. Preparation and
characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) based
nanoparticles for targeted cancer therapy. Eur J Pharm Sci 2011;44:310-320.
25. Xiong YC, Yao YC, Zhan XY, Chen GQ. Application of Polyhydroxyalkanoates
Nanoparticles as Intracellular Sustained Drug-Release Vectors. J Biomater Sci 2010;21:127140.
13
26. Peng Q, Zhang ZR, Gong T, Chen GQ, Sun X. A rapid-acting, long-acting insulin
formulation based on a phospholipid complex loaded PHBHHx nanoparticles. Biomaterials
2012;33:1583-1588.
27. Peng Q, Sun X, Gong T, Wu CY, Zhang T, Tan J, Zhang ZR. Injectable and biodegradable
thermosensitive hydrogels loaded with PHBHHx nanoparticles for the sustained and
controlled release of insulin. Acta Biomaterialis 2013;9:5063-5069.
28. Gharibi B, Ghuman MS, Hughes FJ. Akt- and Erk-mediated regulation of prolideration and
differentiation during PDGFRβ-induced MSC self-renewal. J Cell Mol Med 2012;16:27892801.
29. Shen H, Hu X, Yang F, Bei J, Wang S. Cell affinity for bFGF immobilized heparincontaining poly(lactide-co-glycolide) scaffolds. Biomaterials 2011;32:3404-3412.
30. Tabata Y, Hijikata S, Muniruzzaman M, Ikada Y. Neovascularization effect of biodegradable
gelatin microspheres incorporating basic fibroblast growth factor. J Biomater Sci Polym Ed
1999;10:79-94.
31. Liu J, Gong T, Wang C, Zhong Z, Zhang Z. Solid lipid nanoparticles loaded with insulin by
sodium cholate-phosphatidylcholine-based mixed micelles: preparation and characterization.
Int J Pharm 2007;340:153-162.
32. Underwood PA, Bean PA, Mitchell SM, Whitelock JM. Specific affinity depletion of cell
adhesion molecules and growth factors from serum. J Immunol Methods 2001;247:217-224.
14
Table 1 Characterization of PHBHHx nanoparticles before and after lyophilization
Before
lyophilization
After
lyophilization
Sample
Size (nm)
PDI
ZP (mV)
EE (%)
Blank NPs
114.1±3.9
0.244±0.030
-21±1.1
---
BSA NPs
118.9±5.1
0.243±0.037
-28.4±1.9
---
PDGF NPs
119.0±0.4
0.198±0.019
-34.3±3.3
82.97±1.83
Blank NPs
149.7±1.8
0.307±0.017
-15.6±1.1
---
BSA NPs
141.3±1.5
0.275±0.013
-19.0±2.1
---
PDGF NPs
152.7±0.8
0.216±0.021
-19.8±1.1
74.63±1.18
PHBHHx: poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); Blank NPs: empty nanoparticles;
BSA NPs: bovine serum albumin phospholipid complex loaded nanoparticles; PDGF NPs:
PDGF-BB phospholipid complex loaded nanoparticles; PDI: polydispersity index; ZP: zeta
potential; EE: entrapment efficiency; Values are mean ± s.d.; n=3.
15
Figure 1. SEM image of PHBHHx nanoparticles. Scale bar: 1.00 µm; accelerating voltage: 5kV.
16
Figure 2. In vitro release profile of PDGF-BB phospholipid complex loaded nanoparticles. Each
data presented as mean ± s.d.; n=3.
17
Figure 3. Viability of hMSCs cultured in different concentrations (0, 5, 10 & 25ng/ml) of
PDGF-BB media using CCK-8 assay. Values are mean ± s.d. *: P < 0.05 and **: P < 0.01
compared with that of 0 ng/ml PDGF-BB media; n=3.
18
Figure 4. hMSCs proliferation study cultured in sustained release 2D cell culture system. Blank
NPs: empty nanoparticles; BSA NPs: bovine serum albumin phospholipid complex loaded
nanoparticles; PDGF NPs: PDGF-BB phospholipid complex loaded nanoparticles. Values are
mean ± s.d. *: P < 0.05 and **: P < 0.01 compared with that of no NPs containing media; §: P <
0.05 and §§: P < 0.01 compared with that of blank NPs containing media; #: P < 0.05 and ##: P <
0.01 compared with that of BSA NPs containing media; n=3.
19
Day4
PDGF NPs
BSA NPs
Blank NPs
No NPs
Day2
Green: Live Red: Dead
Figure 5. Live-dead cell double staining of hMSCs cultured in sustained release 3D
cell culture system. Scale bar: 100 µm.
20
Figure 6. hMSCs proliferation study cultured in sustained release 3D cell culture system. Blank
NPs: empty nanoparticles; BSA NPs: bovine serum albumin phospholipid complex loaded
nanoparticles; PDGF NPs: PDGF-BB phospholipid complex loaded nanoparticles. Values are
mean ± s.d. *: P < 0.05 compared with that of no NPs containing collagen scaffold; §: P < 0.05
compared with that of blank NPs containing collagen scaffold; #: P < 0.05 compared with that of
BSA NPs containing collagen scaffold; n=3.
21