Journal of Biomaterials and Nanobiotechnology, 2011, 2, 500-509
doi:10.4236/jbnb.2011.225061 Published Online December 2011 (
Copyright © 2011 SciRes. JBNB
Low-Molecular-Weight Heparin and
Protamine-Based Polyelectrolyte Nano Complexes
for Protein Delivery (A Review Article)
Masayuki Ishihara1*, Satoko Kishimoto1,2, Megumi Takikawa3, Yasutaka Mori1,4, Shingo Nakamura5,
Masanori Fujita1
1Research Institute, National Defense Medical College, Tokorozawa, Japan; 2Research Fellow of the Japan Society for the Promotion
of Science, Tokyo, Japan; 3Department of Plastic Surgery, National Defense Medical College, Tokorozawa, Japan; 4Aeromedical
Laboratory, Japan Air Self-Defense Force, Sayama, Japan; 5Department of Surgery, National Defense Medical College, Tokoro-
zawa, Japan.
E-mail: *
Received October 1st, 2011; revised November 14th, 2011; accepted November 28th, 2011.
We produced low-molecular-weight heparin/protamine micro (nano) particles (LMW-H/P MPs·NPs) as a carrier for
heparin-binding growth factors (GFs), such as fibroblast growth factor (FGF)-2 and various GFs in platelet-rich
plasma (PRP). A mixture of LMW-H (MW: approximately 5000 Da, 6.4 mg/ml) and protamine (MW: approximately
3000 Da, 10 mg/ml) at a ratio of 7:3 (vol:vol) yields a dispersion of micro (nano) particles (200 nm - 3 µm in diameter).
The diluted LMW-H solution in saline (0.32 mg/ml) mixed with diluted protamine (0.5 mg/ml) at a ratio at 7:3 (vol:vol)
resulted in soluble nanoparticles (approximately 100 nm in diameter). The generated NPs could be then stabilized by
adding 2 mg/ml dextran (MW: 178-217 kDa) and remained soluble after lyophilization of dialyzed LMW-H/P NPs so-
lution. The LMW-H/P MPs·NPs adsorb GFs, control their release, protect GFs and activate their biological activities.
Furthermore, administration of GFs-containing F/P MPs·NPs exhibited significantly higher inductions of vasculariza-
tion and fibrous tissue formation in vivo than GFs alone. LMW-H/P MPs·NPs can also efficiently bind to tissue culture
plates and retain the binding of GFs. The LMW-H/P MPs·NP-coated matrix with various GFs or cytokines provided
novel biomaterials that could control cellular activity such as proliferation and differentiation. Thus, LMW-H/P
MPs·NPs are an excellent carrier for GFs and are a functional coating matrix for various kinds of cell cultures.
Keywords: Polyelectrolyte Complexes, Nanoparticles, Heparin-Binding Growth Factors, Platelet-Rich Plasma, Drug
1. Introduction
Polyelectrolyte complexes (PECs) are produced by elec-
trostatic interactions between oppositely charged polye-
lectrolytes. When this interaction occurs at non-equiva-
lent ratios, nonstoichiometric PECs are produced, caus-
ing each PECs to carry an excess charge. Proteins inter-
act with both synthetic and natural PECs [1,2]. These
binding characteristics, along with a simple preparation,
allow PECs to be an excellent model for studying the in
vivo behavior of charged biopolymers as well as having
potential applications in medicine and biotechnology [3].
Reported data indicate that polyanions and polycations
can bind to proteins below and above their isoelectric
points, respectively. These interactions can result in so-
luble complexes, complex coacervation and/or the for-
mation of amorphous precipitates [1,2]. Main aspects
studied by different authors are compositions of PECs
obtained under various experimental conditions, such as
the strength and position of ionic sites, charge density,
and rigidity of polymer chains as well as chemical prop-
erties such as solubility, pH, temperature, and concentra-
tion [3].
Those electrostatic interactions are also important be-
cause of their similarity to biological systems [4]. Inter-
actions between proteins and nucleic acids, for example,
play a role in the transcription process [1]. DNA/chitosan
PECs [5], chitosan/chondroitin sulfate PECs and chito-
san/hyaluronate PECs [6] were described as gene and
drug micro-carriers. Moreover, PECs that are insoluble
Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes 501
for Protein Delivery (A Review Article)
also have potential applications as membranes, micro-
capsules, micro (nano) particles, and scaffolds for tissue
engineering [7].
Basic protamine molecules complexed with acidic mo-
lecules such as heparin form a microcomplexes through
ionic interactions. We previously have reported the a
low-molecular-weight heparin/protamine micro (nano)
particles (LMW-H/P MPs·NPs) which we originally pre-
pared as PECs [8,9]. LMW-H/P MPs·NPs are specifi-
cally bound to FGF-2 and other various heparin-binding
growth factors (GFs) in platelet-rich plasma (PRP) [10]
through interaction between LMW-H molecules in LMW-
H/P MPs·NPs and those GFs. LMW-H/P MPs·NPs are
able to protect those GFs from heat and proteolytic inac-
tivation and to enhance those biological activities. Since
those GFs-containing LMW-H/P MPs·NPs are 200 nm -
3 µm in diameter, those can be easily injected [8-10].
Moreover, the GFs-containing the LMW-H/P MPs·NPs
showed a substantial effect to induce vascularization and
fibrous tissue formations due to stabilizating, activating,
and gradually releasing GFs molecules from GFs-con-
taining the LMW-H/P MPs·NPs [8-10].
In this review articles, we described on the LMW-H/P
MPs·NPs which we originally prepared as PECs, its
characterizations and its potential medical applications as
carriers for GFs such as FGF-2 and GFs in PRP. Fur-
thermore, as a coating matrix, LMW-H/P MPs·NPs were
efficiently bound to tissue culture plates. With the ability
of LMW-H/P MPs·NPs to retain GFs, the LMW-H/P
MPs·NPs could serve as a useful coating matrix for cul-
ture of various types of cells.
2. Preparation of LMW-H/P MPs·NPs
Heparin interacts with a variety of functional proteins,
including heparin-binding growth factors (GFs), cyto-
kines, extracellular matrix components, and adhesion
molecules [11-13]. Thus, heparin may be useful as a the-
rapeutic agent in various pathological conditions that
involve functional proteins. However, high-dose heparin
cannot be used because of the excessive risk of bleeding
[14]. In contrast, low-molecular-weight heparin (LMW-
H, MW: approximately 5000 Da) which has much lower
anti-coagulant activity, has pharmacological and practi-
cal advantages compared with native heparin. The lower
protein binding activity of LMW-H produces a low, sta-
ble, and predictable anticoagulant response, thereby by-
passing the need for laboratory monitoring of drug levels
to adjust the dosage [14]. In addition, one or two subcu-
taneous injections per day are sufficient to maintain
therapeutic concentrations because of its longer plasma
half-life [14].
On the other hand, protamine, a purified mixture of
proteins obtained from fish sperm, neutralizes heparin
and LMW-H by forming a stable complex that lacks an-
ticoagulant activity [15]. Protamine is also in clinical use
to reverse the anticoagulant activity of heparin following
cardiopulmonary bypass as well as in cases of heparin-
induced bleeding [16]. We previously prepared water-
insoluble particles (>10 µm in diameter) by mixing non-
anticoagulant heparin with chitosan. We then mixed fuc-
oidan with chitosan and investigated the ability of the
resulting insoluble fucoidan/chitosan microparticles to
protect fibroblast growth factor-2 (FGF-2) activity [17,
18]. We also prepared water-insoluble micro (nano) par-
ticles (200 nm - 3 µm in diameter) by mixing LMW-H
(6.4 mg/ml) with protamine (10 mg/ml) at a ratio of 7:3
(vol:vol), and reported the ability of the resulting in-
jectable low-molecular-weight heparin/protamine micro
(nano) particles (LMW-H/P MPs·NPs) to protect FGF-2
activity (see Figure 1) [8,9]. Furthermore, GFs released
from platelets that were involved in cell proliferation,
migration, and angiogenesis were able to adsorb onto
LMW-H/P MPs·NPs [10].
In order to produce of the nanoparticles, equally di-
luted LMW-H and protamine (100-fold, 50-fold, and
20-fold diluted) were mixed in a ratio at 7:3 (vol:vol) in
this study (see Figure 2). The diameter of generated
LMW-H/P NPs by mixing 100-fold, 50-fold, and 20-fold
diluted protamine to equally diluted LMW-H in the ratio
of 3:7 (vol:vol) were 84.6 ± 26.8, 95.0 ± 27.0, and 112.5
± 46.1 nm, respectively (see Figure 2) [9]. And no mi-
croparticles (>1 µm in diameter) were observed in the
mixtures. In contrast, generations of small amount of
microparticles (approximately 1 µm in diameter) were
observed by mixing of 10-fold diluted protamine (1
mg/ml) to PMW-H (0.64 mg/ml) in the ratio of 3:7
(vol/vol). When non-diluted protamine (10 mg/ml) was
added to non-diluted LMW-H (6.4 mg/ml) up to ratio of
3:7 (vol:vol), maximal LMW-H/P MPs (200 nm - 3 µm
in diameter) was produced and the high turbidity was
observed. When 10-fold concentrated protamine (100
mg/ml) was added to the equally 10-fold concentrated
LMW-H (64 mg/ml) up to ratio of 3:7 (vol:vol), mixtures
of larger LMW-H/P MPs (3 - 10 µm in diameter) and
larger cotton-like precipitates (>10 µm) were immedi-
ately generated and those products were insoluble [9]. An
illustration on generations of micro/nanoparticles and
insoluble precipitates by mixing protamine to LMW-H in
various concentrations was shown in Figure 3.
Cotton-like compounds were generated after lyophili-
zations of both LMW-H/P MPs·NPs (200 nm - 3 µm in
diameter) and LMW-H/P NPs (approximately 100 nm in
diameter) solutions without dextran, and they were
hardly re-soluble in water. However, both the freeze-
Copyright © 2011 SciRes. JBNB
Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes
for Protein Delivery (A Review Article)
Copyright © 2011 SciRes. JBNB
Figure 1. Preparation of LMW-H/P MPs·NPs.
Figure 2. LMW-H/P NPs by mixing diluted protamine and LMW-H.
Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes 503
for Protein Delivery (A Review Article)
Figure 3. Generation of LMW-H/P MPs·NPs as PECs.
dried LMW-H/P MPs·NPs were easily dessolved in wa-
ter by adding 0.5% and 0.2% dextran, respectively, be-
fore their lyophilizations. In addition, aggregation of
LMW-H/P NPs in solution to LMW-H/P MPs was pro-
hibited in the presence of dextran [9]. Thus, the addition
of dextran is effective to stabilize the LMW-H/P MPs·NPs
and to prepare stable and resoluble freeze-dry LMW-H/P
As a coating matrix, LMW-H/P MPs·NPs were effi-
ciently bound to tissue culture plates. With the ability of
LMW-H/P MPs·NPs to adsorb and retain GFs, the
LMW-H/P MPs·NPs could be very useful in various
types of cell culture as a coating matrix. Human micro-
vascular endothelial cells and human dermal fibroblast
cells well adhered to LMW-H/P MPs·NPs-coated tissue
culture plates [19] and grew optimally in low fetal bovine
serum (FBS) (1% - 2%) medium supplemented with
FGF-2 (5 ng/ml). This protocol could make it possible to
use low autologous human serum (1% - 2%) for the cul-
turing of human bone marrow-derived mesenchymal
stem cells (BMSCs) and human adipose-derived stromal
cells (ASCs) [20]. Furthermore, CD34+ hematopoietic
progenitor cells (CD34+ cells) derived from human bone
marrow exhibited a comparatively higher proliferation on
LMW-H/P MPs·NPs-coated plates in hematopoietic pro-
genitor growth medium (HPGM) supplemented with
appropriate cytokines than those on uncoated plates
3. Protein-Delivery Micro (Nano) Particles
3.1. FGF-2 Containing LMW-H/P MPs·NPs
FGF-2 binds heparin with high affinity (Kd of 8.6 × 109
M). The polysaccharides can prolong the biological half-
life of FGF-2 as well as protect FGF-2 from heat, acid,
and proteolytic inactivation [22]. Similarly, the LMW-
H/P MPs·NPs have high affinity for FGF-2 (Kd = 2.4 ×
109 M) [8], and this interaction of FGF-2 with the
LMW-H/P MPs·NPs can substantially prolong the bio-
logical half-life time of FGF-2. The protection of FGF-2
against heat inactivation and trypsin degradation by the
LMW-H/P MPs was effective in a concentration-depen-
dent manner [8]. FGF-2 molecules were released in vitro
from the FGF-2-containing LMW-H/P MPs·NPs with
half-releasing time of about 6 days. Those results dem-
onstrated that FGF-2 molecules are bound and stabi-
lized on the LMW-H/P MPs·NPs, and that the FGF-2
molecules incorporated into the LMW-H/P MPs·NPs will
be gradually released upon biodegradation of the hy-
drogel in vivo.
When the FGF-2-containing LMW-H/P MPs·NPs were
subcutaneously injected into the backs of mice, neovas-
cularization was induced near the injection site after 3
days. Neovascularization induced by the FGF-2-con-
taining LMW-H/P MPs·NPs reached a maximum at 1
week, after which a slight decrease in the neovasculari-
zation rate occurred. No significant vascularization was
Copyright © 2011 SciRes. JBNB
Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes
for Protein Delivery (A Review Article)
observed after either the injection of FGF-2 alone or the
LMW-H/P MPs·NPs alone [8].
Another study demonstrated advanced fat survival and
capillary formation in FGF-2-containing LMW-H/P
MPs·NPs-assist subdivided free fat-grafting groups in
rats [23]. Furthermore, our study demonstrated the ability
of FGF-2-containing LMW-H/P MPs·NPs to induce both
arteriogenesis and angiogenesis in rabbit models of
ischemic limbs (see Figure 4) [24]. The primary conclu-
sion is that FGF-2-containing LMW-H/P MPs·NPs-treat-
ment effectively induces the development of collateral
vessels, which can provide sufficient blood flow to the
pre-existing vascular network in ischemic tissue. Since
all components used in the FGF-2-containing LMW-H/P
MPs·NPs are also used clinically, we feel safety in a
clinical setting is probable [24].
3.2. Platelet-Rich Plasma (PRP) Containing
PRP contains a high concentration of thrombocytes (plate-
lets). When the platelets are activated, various GFs and
other bioactive proteins in α-granules of platelets are
released and those proteins augment tissue repair and
regeneration processes [10,25,26]. Platelets contain over
20 GFs, including platelet-derived growth factors (PDGFs),
FGFs, hepatocyte growth factor (HGF), transforming
growth factors (TGFs), and vascular endothelial growth
factors (VEGFs), almost all of which are known to bind
to heparin and to LMW-H/P MPs·NPs. Recent studies
suggest that GFs in PRP not only influence the viability
of transferred cells but may also play bioactive roles in
the regulation of proliferation and differentiation in
various types of cells [10]. Any treatment aiming to
mimic the critical aspects of the natural biological proc-
ess should not be limited to the provision of a single GF,
but rather should release multiple GFs at an optimized
ratio, at a physiological dose and in a specific spatio-
temporal pattern. Those results indicated that the LMW-
H/P MPs·NPs also activate the platelets to release the
GFs, and that in turn the released GFs from the platelets
can be immobilized, be stabilized, and be activated on
the LMW-H/P MPs·NPs [10].
The GFs in PRP are stably bound to LMW-H/P MPs
in vivo. The GFs adsorbed onto LMW-H/P MPs·NPs
may be gradually diffused and released upon biodegrada-
tion of LMW-H/P MPs·NPs. When PRP-containing
LMW-H/P MPs·NPs were subcutaneously injected into
the backs of mice, significantly higher neovascularization
and granulation tissue with enhanced filtration of in-
flammatory cells were observed compared with the mouse
Figure 4. Quantification of visible collateral arteries under angiographic viewing on day 28.
Copyright © 2011 SciRes. JBNB
Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes 505
for Protein Delivery (A Review Article)
groups injected with PRP alone, LMW-H/P MPs·NPs
alone, and the control [18]. Compared to either PRP
alone or LMW-H/P MPs·NPs alone, locally administered
PRP-containing LMW-H/P MPs·NPs augmented the
wound bed and substantially increased viability of rat
dorsal paired pedicle skin flaps [27]. The improved flap
survival was noted if PRP-containing LMW-H/P MPs·NPs
was administered 2 days before the flap elevation [27].
PRP-containing LMW-H/P MPs·NPs may thus represent
a promising new biomaterial for improving skin flaps,
particularly in the field of reconstructive surgery.
Clinical research was performed using autologous
PRP-containing LMW-H/P MPs·NPs and PRP alone in
26 patients with thin hair (including 10 women) [28].
Hair growth and thickening following administration of
both PRP-containing LMW-H/P MPs·NPs and PRP alone
was observed in all patients compared with the control,
but PRP-containing LMW-H/P MPs·NPs appeared to
provide the most substantial change in the hair (see Fig-
ure 5) [28]. Because of the use of autologous materials,
this method using PRP-containing LMW-H/P MPs·NPs
is simpler, cheaper, and has no side effect compared with
conventional methods.
4. Cell Culture System Using
Microparticle-Coated Plates
4.1. Various Types of Cell Cultures Using
LMW-H/P MPs·NPs-Coated Plates
The LMW-H/P MPs·NPs are able to attach to polymeric
surfaces such as plastic and glass. The LMW-H/P
MPs·NPs generate a stable paste-like coating through
complete drying. It is probable that polypeptides, such as
FGF-2, interleukin (IL)-3, and granulocyte/macrophage-
colony stimulating factor (GM-CSF) once bound to the
LMW-H/P MPs·NPs-coated plates, are gradually re-
leased from the coated surface in vitro with a half-life of
4 - 6 days [19]. Furthermore, LMW-H/P MPs·NPs- coat-
ing could optimally stimulate growth of human mi-
cro-vascular endothelial cells (hMVECs) and human
dermal fibroblast cells (hDFCs) in low FBS (1% - 2%)-
DMEM with FGF-2 and growth of hematopoietic cell
line (TF-1) with IL-3 and GM-CSF (see Figure 6) [19].
Heparin and heparinoids bind various GFs and cyto-
kines including FGFs, HGF, VEGF, heparin-binding
epidermal growth factor (HBEGF), PDGF, TGF-β,
GM-CSF, interleukins (i.e., IL-1, IL-2, IL-3, IL-4, IL-6,
Figure 5. PRP-containing LMW-H/P MPs·NPs treatment for Alopecia Areata.
Copyright © 2011 SciRes. JBNB
Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes
for Protein Delivery (A Review Article)
Figure 6. Preparation of LMW-H/P MPs·NPs-coated plates.
IL-7, and IL-8), interferon γ, and macrophage inflamma-
tory protein-1 [11-13]. These GFs and cytokines can po-
tentially be immobilized on the LMW-H/P MPs·NPs-
coated plates. Actually, in addition to FGF-2, IL-3 and
GM-CSF described above, we have already observed that
FGF-1, HGF, HBEGF, TGF-β, human stem cell factor
(SCF), thrombopoietin (Tpo), and Flt-3 ligand (Flt-3)
could be efficiently immobilized on the LMW-H/P
MPs·NPs-coated plates [21]. Furthermore, the bound GFs
to the LMW-H/P MPs·NPs-coated plates appeared to
enhance and to stabilize those biological activities. Thus,
LMW-H/P MPs·NPs-coating provides an excellent bio-
material to immobilize and retain GFs and cytokines for
optimal growth of various types of cells with low (no)
serum medium (see Figure 6).
4.2. Proliferation of BMSCs and ASCs on
LMW-H/P MPs·NPs-Coated Plates
Cell-based therapies such as tissue engineering will
benefit from a source of autologous multipotent stem
cells, including bone marrow-derived mesenchymal stem
cells (BMSCs) and adipose tissue-derived stromal cells
(ASCs). There are two stem cell lineages in bone marrow
cell populations, i.e., hematopoietic cells (HCs) and
BMSCs. The BMSCs and ASCs are multipotential, indi-
cating that in culture [29,30] or after in vivo implantation
these cells can differentiate into a variety of cell types
including osteoblasts, chondrocytes, adipocytes, myo-
blasts [31], and neuronal cells [32]. Furthermore, cul-
tured ASCs secreted significant amounts of angiogenic
growth factors such as FGF-2, HGF, PDGF, and VEGF
at levels that are bioactive [33]. Thus, LMW-H/P MPs·NPs
may serve as an effective matrix for cultures of BMSCs
and ASCs. The safe and effective expansions of BMSCs
and ASCs represent a promising option for tissue engi-
neering strategies.
Most protocols for the expansion of BMSCs and ASCs
include high concentrations (10% - 20%) of animal se-
rum such as FBS as a nutritional supplement. In some
cell cultures, this involves multiple doses of FBS, which
raises concerns over possible contamination as well as
immunological reactions caused by medium-derived FBS
proteins, sialic acid derivatives, etc. [34,35]. Patients
may experience problems when undergoing autologous
cell-based therapies if a serum other than an autologous
serum is used during the culturing of the cells. However,
it would be difficult to obtain large amounts of autolo-
gous serum from the patient for large-scale autologous
cell culture [20]. It should be noted that the growth of
cultured BMSCs or ASCs on LMW-H/P MPs·NP-coated
plates in combination with FGF-2 and FBS (1% - 2%)
was significantly stimulated, and similar stimulation was
Copyright © 2011 SciRes. JBNB
Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes 507
for Protein Delivery (A Review Article)
observed in those cultured cells on LMW-H/P MPs·NPs-
coated plates with FGF-2 and 1% - 2% human serum
(HS) prepared from adult bloods instead of FBS.
4.3. Proliferation of CD34+ Hematopoietic
Progenitor Cells (CD34+ HCs) on LMW-H/P
MPs·NPs-Coated Plates
Hematopoietic progenitor cells proliferate and mature in
semi-solid media when stimulated by exogenous hema-
topoietic cell growth factors (HCGFs) such as SCF, Tpo,
Flt-3, IL-3, and GM-CSF [36,37]. These cells also pro-
liferate in association with bone marrow-derived stromal
cells (BMSCs) [38,39], although biologically active
amounts of HCGFs cannot be detected in stromal culture
supernatants [38,39]. It is possible that HCGFs are syn-
thesized by the stromal cells but remain bound to the
stromal cells and/or their extracellular matrix. In fact, it
was demonstrated that both natural and recombinant
HCGFs, such as IL-3 and GM-CSF, could be adsorbed
by heparan sulfate, which is the major sulfated glycosa-
minoglycan of bone marrow stroma [38,39]. Serum-free
medium supplemented with large amounts of SCF, Tpo,
and Flt-3 was reported for expansion of CD34+ HCs [40,
41]. Although such medium is commercially avail- able
(HPGM, Lonza Japan Corp., Tokyo, Japan), it is prohibi-
tively expensive. We demonstrated that recombinant
HCGFs such as SCF, Tpo, and Flt-3 were immobilized
onto LMW-H/P MPs·NPs-coated plates, and the immobi-
lized cytokines were stabilized, were activated, and were
gradually released into the medium. Those cytokines,
once bound, can be presented in the biologically active
form to hematopoietic progenitor cells [38,39]. Further-
more, only one-fourth of the concentration of the cyto-
kines recommended by the manufacture was required for
maximal expansion of CD34+ HCs on the LMW-H/P
MPs·NPs-coated plates. These findings may have impor-
tant implications for the use of heparinoid as an artificial
matrix for ex vivo expansion of hematopoietic progenitor
cells with adequate cytokines. The LMW-H/P MPs·NPs-
coating matrix in the presence of lower concentrations of
SCF, Tpo, and Flt-3 is a convenient and safe material for
stable expansion of CD34+ HCs using HPGM without
any animal serum.
5. Conclusions
It is recognized in polymer chemistry that positively and
negatively charged polymers interact ionically [17].
Through these ionic interactions, basic protamine mole-
cules can bind with acidic molecules (LMW-H) to form
micro (nano) particle complexes. We previously reported
that GF-containing LMW-H/P MPs·NPs, which are 200
nm - 3 μm in diameter, can be easily injected [8-10]. Fur-
thermore, the LMW-H/P MPs·NPs were observed on the
protection of FGF-2 and GFs in PRP activity from heat
and proteolytic inactivation. These results indicate that
LMW-H/P MPs·NPs may serve as an effective microcar-
rier for various GFs, particularly for the local application
of GFs. GFs-containing LMW-H/P MPs·NPs show a
substantial effect to induce vascularization and fibrous
tissue formation because of stabilization, activation, and
gradual release of GF molecules from GFs-containing
LMW-H/P MPs·NPs [8-10].
The presented method for the optimal proliferation and
differentiation of ASCs and BMSCs on LMW-H/P
MPs·NPs-coated plates in low concentration human se-
rum medium (1% - 2%) supplemented with FGF-2 (5
ng/ml). No animal serum is required in the culture of
those cell types. The bound GFs to the LMW-H/P
MPs·NPs-coated plates appeared to enhance and to stabi-
lize those biological activities. The proliferated cells
maintained their potential to differentiate into adipocytes
and osteoblasts [20,33]. Furthermore, the LMW-H/P
MPs·NPs-coating matrix in the presence of lower con-
centrations of SCF, Tpo, and Flt-3 were convenient ma-
terials for stable expansion of CD34+ HCs using HPGM
without any animal serum. These results suggest a prom-
ising cell source, particularly for the preparation of large
amounts of ASCs, BMSCs, or CD34+ HCs required for
cell-based therapies in several clinical fields.
LMW-H, protamine, several GFs and cytokines, and
autologous PRP are already in clinical use. Since auto-
logous ASCs, BMSCs, or CD34+ HCs are available, the
clinical safety of LMW-H/P MPs·NPs as protein-carrier
is possible. Furthermore, ASCs, BMSCs, or CD34+ HCs
can be efficiently expanded as cell sources for regenera-
tive medicines with the use of LMW-H/P MPs·NPs-
coated plates as a matrix without animal serum or feeder
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