International Journal of Pharmaceutics 485 (2015) 31–40

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International Journal of Pharmaceutics

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

Injectable nanoparticle-loaded hydrogel system for local delivery of

sodium alendronate
Urszula Posadowska a , Martin Parizek b , Elena Filova b , Malgorzata Wlodarczyk-Biegun c ,
Marleen Kamperman c, Lucie Bacakova b , Elzbieta Pamula a, *
a
AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Biomaterials, al. A. Mickiewicza 30, 30-059 Krakow,
Poland
b
Academy of Sciences of the Czech Republic, Institute of Physiology, Department of Biomaterials and Tissue Engineering, Videnska 1083, 14220 Prague 4-Krc,
Czech Republic
c
Wageningen University, Laboratory of Physical Chemistry and Colloid Science, Dreijenplein 6, 6703HB, Wageningen, The Netherlands

A R T I C L E I N F O A B S T R A C T

Article history: Systemic administration of bisphosphonates, e.g. sodium alendronate (Aln) is characterized by extremely
Received 14 January 2015 low bioavailability and high toxicity. To omit aforementioned drawbacks an injectable system for the
Received in revised form 28 February 2015 intra-bone delivery of Aln based on Aln-loaded nanoparticles (NPs-Aln) suspended in a hydrogel matrix
Accepted 2 March 2015
(gellan gum, GG) was developed. Aln was encapsulated in poly(lactide-co-glycolide) (PLGA 85:15) by
Available online 4 March 2015
solid–oil–water emulsification. Drug release tests showed that within 25 days all the encapsulated drug
was released from NPs-Aln and the release rate was highest at the beginning and decreased with time. In
Keywords:
contrast, by suspending NPs-Aln in a GG matrix, the release rate was significantly lower and more
Sodium alendronate
Injectability
constant in time. The GG–NPs-Aln system was engineered to be easily injectable and was able to
Gellan gum reassemble its structure after extrusion as shown by rheological measurements. In vitro studies showed
PLGA that the GG–NPs-Aln was cytocompatible with MG-63 osteoblast-like cells and it inhibited RANKL-
Nanoparticles mediated osteoclastic differentiation of RAW 264.7 cells. The injectability, the sustained local delivery of
Osteoporosis small doses of Aln and the biological activity render the GG–NPs-Aln system promising for the local
treatment of osteoporosis and other bone tissue disorders.
ã 2015 Published by Elsevier B.V.

1. Introduction
Bisphosphonates, e.g. sodium alendronate (Aln), are one of the
most important classes of drugs for the treatment of bone tissue
pathologies. It is known that the Aln prevents hydroxyapatite loss
and inhibits osteoclast-mediated bone resorption (Hayden et al.,
2014). This drug is mainly used for the treatment of osteoporosis,
bone metastasis and Paget’s disease (Ezzati Nazhad Dolatabadi
et al., 2014). More recently it has been proposed as adjuvant
analgesic in combination with opioids in the management of
cancer pain symptoms (Mitra and Jones, 2012) and as a bone
antitumour agent (Cornelis et al., 2014). However, its systemic
application beside low bioavailability (<1% when administered
orally) evokes numerous side effects such as fever, esophageal
erosions, ulcers and problems with gastrointestinal tract (Marshall
et al., 2000).
The side effects and limitations of oral administration may be
overcome by local delivery of Aln. So far, there have been few
reports in the literature describing systems for local bisphosph-
onates administration; they were based on hydroxyapatite micro-

Abbreviations: Aln, sodium alendronate; NPs-Aln, sodium alendronate loaded nanoparticles; NPs, nanoparticles; GG, gellan gum; GG–NPs- Aln, injectable nanoparticle-
loaded hydrogel system; PLGA, poly(lactide-co-glycolide); FDA, Food and Drug Administration; PVA, polyvinyl alcohol; OPA, O-phthaldialdehyde; PBS, phosphate buffered
saline; DMEM, Dulbecco’s modified Eagle’s Minimum Essential Medium; RPMI, Roswell Park Memorial Institute (Medium); FBS, fetal bovine serum; MG-63, osteoblast-like
cells; RAW 264.7, murine monocyte-like cells (macrophages); RANKL, receptor activator of nuclear factor kappa-B ligand; TRAP, tartrate-resistant acid phosphatase; RTCA,
real-time cell analyzer; UHQ-water, ultra high quality water; Zr(Acac)4, zirconium acetylacetonate; S/O/W, solid–oil–water emulsification; DLS, dynamic light scattering; %EE,
encapsulation efficiency; %LE, loading efficiency; AFM, atomic force microscopy; FMAX, maximal extrusion force; MWCO, molecular weight cut-off; G0 , storage modulus; G00 ,
loss modulus.
* Corresponding author. Tel.: +48 12 617 44 48; fax: +48 12 617 33 71.
E-mail addresses: uposadow@agh.edu.pl (U. Posadowska), parizek@biomed.cas.cz (M. Parizek), filova@biomed.cas.cz (E. Filova), gosia.wlodarczyk-biegun@wur.nl
(M. Wlodarczyk-Biegun), marleen.kamperman@wur.nl (M. Kamperman), lucy@biomed.cas.cz (L. Bacakova), epamula@agh.edu.pl (E. Pamula).

http://dx.doi.org/10.1016/j.ijpharm.2015.03.003
0378-5173/ ã 2015 Published by Elsevier B.V.
32 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40
spheres (Lee et al., 2013), calcium phosphate microspheres (Kim
et al., 2010), poly(b-hydroxybutyrate-co-b-hydroxyvalerate)–hy-
droxyapatite composites (Huang et al., 2009) or carbopol gels
(Reddy et al., 2005). Attempts to coat titanium implants with
bisphosphonates to improve osteointegration have also been
described (Harmankaya et al., 2013). Nevertheless the approach of
local delivery of Aln is still poorly explored.
There is a need to develop a local delivery system for Aln which
will be easy to handle by the surgeon, and will remain at the
targeted area of the affected tissue. We hypothesize that such a
system will assure local controlled delivery of small doses of the
drug, so systemic side effects typical for oral Aln administration
will be reduced. Moreover, the system will be administered via
injection so it will be less-invasive and traumatic than typical
surgical intervention performed to fill in the defect or treat affected
area with bone graft. To this end, we developed an injectable
material consisting of Aln-loaded nanoparticles suspended in a
hydrogel matrix.
A resorbable polymer, poly(lactide-co-glycolide) (PLGA), was
chosen to encapsulate Aln because this FDA-approved copolymer
has a tunable degradation rate (i.e., by molar ratio of lactide to
glycolide, chain structure and molecular weight) and can be easily
processed into nanocarriers using emulsification (Shive and
Anderson, 1997). As a hydrogel matrix gellan gum (GG) was used.
GG is an anionic polysaccharide produced by bacteria
(Sphingomonas elodea) during aerobic fermentation (Doner and
Douds, 1995). GG is widely applied in the food and pharmaceutical
industries (Babu et al., 2010) and more recently it has been
proposed for cartilage (Oliveira et al., 2010) and bone (Douglas
et al., 2014) regeneration. Further advantages of GG are low cost,
biodegradability and low toxicity of its degradation products, as
well as the fact that it is not animal-derived, avoiding regulatory
concerns (Gantar et al., 2014).
The aim of this study was first to manufacture and characterize
PLGA nanoparticles loaded with Aln. In the second step, drug
delivery system based on Aln-loaded nanoparticles and GG
hydrogel was produced and characterized in terms of its
mechanical properties (rheology), surgical handiness (injectability,
swelling) and Aln release kinetics. Finally, to confirm the feasibility
of the developed system in the treatment of bone tissue defects
and diseases, in vitro tests in contact with osteoblast-like cells and
osteoclast-like cells were conducted.
2. Materials and methods
2.1. Materials and chemicals
Sodium alendronate (Aln) was a gift from Polpharma S.A.
Poland (batch no.: 504041227). Polyvinyl alcohol (PVA, Mowiol1
4-88, Mn = 31,000 Da) and gellan gum (GelzanTM CM, classified as
low-acyl gellan by the manufacturer) were bought from Sigma–
Aldrich, Poland. Dichloromethane, methanol, 2-mercaptoethanol,
isopropanol, orthophthaldialdehyde (OPA), phosphate buffered
saline (PBS) buffer (concentrate), calcium chloride, potassium
hydroxide and borate acid were all of analytical grade and were
purchased from POCh, Poland. Chemicals used for biological tests
included acid phosphatase, TRAP (leukocyte tartrate resistant acid
phosphatase kit, Sigma–Aldrich), LIVE/DEAD1 viability/cytotoxic-
ity assay for mammalian cells (calcein-AM and ethidium homo-
dimer-1, Invitrogen, Molecular Probes) and RANKL (receptor
activator of nuclear factor kappa-B ligand, Sigma–Aldrich, USA).
Cell culture media (DMEM, Dulbecco’s modified Eagle’s Minimum
Essential Medium; RPMI-1640) were purchased from Sigma–
Aldrich, USA. Osteoblast-like MG-63 cells were obtained from the
European Collection of Cell Cultures, Salisbury, UK; and RAW
264.7 murine monocytes were obtained from ATCC, TIB-71, USA.
Ultra high quality water (UHQ-water) was produced in an UHQ-PS
purification system (Elga, UK). Poly(lactide-co-glycolide) (PLGA,
85:15, Mn = 100 kDa, d = 1.9) was synthesized by a ring-opening
polymerization in bulk at 100  C using a biocompatible Zr(Acac)4
initiator (Dobrzynski et al., 2001) and kindly provided by the
Centre of Polymer and Carbon Materials of the Polish Academy of
Sciences in Zabrze, Poland.
2.2. Nanoparticle fabrication
PLGA nanoparticles were fabricated using a double emulsifi-
cation/solvent evaporation technique. A portion of 10 mg of Aln
powder (solid – S) was emulsified in a 1.67% w/v PLGA solution in
dichloromethane (6 ml, oil – O), forming the primary emulsion (S/
O). The primary emulsion was then introduced dropwise into an
aqueous solution with a stabilizer, PVA (4% w/v, 20 ml) on ice
during ultrasonication (3 min, 40% of the cycle, Sonics Vibra
CellTM, USA) forming the secondary emulsion (S/O/water).
Afterwards, the emulsion was stirred at 500 rpm overnight in
order to evaporate dichloromethane. The dispersion of nano-
particles was centrifuged (14,000 rpm, 4  C, 20 min) and washed
three times with UHQ-water. The obtained nanoparticles were
freeze-dried for 48 h and then stored at 4  C. Before further
analyses the particles were redispersed in UHQ-water using
ultrasound.
2.3. Size and zeta potential of nanoparticles
The size as well as the polydispersity index of empty and Aln-
loaded nanoparticles (NPs, NPs-Aln) were determined by dynamic
light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instru-
ments). The size of the nanoparticles was determined according to
the Stokes–Einstein equation. The zeta potential was measured by
a laser Doppler electrophoresis method using the same apparatus
and calculated using the Smoluchowski equation based on
electrophoretic mobility data.
2.4. Drug encapsulation and loading efficiencies
In order to assess the amount of encapsulated drug, the
supernatant obtained after particles centrifugation was collected.
Aln content was examined spectrophotometrically (UV-VIS Cecil
CE 2502, Corston UK) at 332 nm with the use of an OPA test (Anhalt,
1977). Encapsulation efficiency (%EE) and loading efficiency (%LE)
were calculated according to formulae (1) and (2), respectively:
mass of Aln in nanoparticles
%EE ¼  100% (1)
initial mass of Aln in the system
mass of Aln in nanoparticles
%LE ¼  100% (2)
mass of nanoparticles
2.5. Morphology of nanoparticles
Morphology evaluation of the unloaded NPs and NPs-Aln was
performed by atomic force microscopy (AFM). A drop of
nanoparticles suspension (0.1% w/v) was placed on a micro-
scopic glass slide (Thermoscientific, Menzel-Glaser, Germany)
and the solvent was allowed to evaporate for 12 h at room
temperature. Topographic images were recorded in contact
mode AFM (Explorer, Thermomicroscopes, spring constant
k = 0.02 N/m, image size 5 mm  5 mm). All the images were
processed using SPMLab6.02 software provided by the AFM
manufacturer.
 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40
2.6. Fabrication of nanoparticle-loaded hydrogel system
GG powder was dissolved in UHQ-water at an initial
concentration of 1.4% w/v at 90  C. After the temperature of the
GG hydrogel was decreased to 60  C, a portion of the NPs-Aln
suspension (10% w/v) was added. To adjust the injectability, the
matrices were cross-linked by addition of 6% w/v CaCl2 solution.
The final GG–NPs-Aln samples contained 0.7% w/v GG, 1% w/v
nanoparticles and 0.3% w/v CaCl2. As a reference, GG cross-linked
hydrogels containing 0.7% w/v GG and 0.3% w/v CaCl2 were also
prepared.
2.7. Injectability of nanoparticle-loaded hydrogel system
GG–NP-Aln samples were characterized in terms of injectability
by a method described elsewhere (Ghadiri et al., 2013). The
maximal force needed to extrude 1 ml of sample from a 2 ml
syringe equipped with a standard needle (18 G, 0.85 mm and
1.45 mm of inner and outer diameter respectively, Becton Dick-
inson) was measured in compression mode on a universal testing
machine (Zwick 1435, Germany). The crosshead speed during the
test was 50 mm/min.
2.8. Swelling experiments
To analyze the swelling properties of the hydrogel-based
systems, samples of known weight were immersed in a sealed
glass container filled with phosphate buffer (PBS; pH 7.4) (Kim
et al., 2007) for 24 h at 37  C. Then the samples were taken out
from PBS and their mass was determined again. The swelling
degree was calculated using a gravimetric method according to
the formula (3) :
ðmass after swelling  initial massÞ
Swelling degree ¼ 100%
initial mass
(3)
2.9. Rheological measurements
To characterize the rheological properties, tests were performed
on GG–NPs-Aln system and GG hydrogel (Physica MCR 501
ðimpedance at individual time interval  background impedanceÞ
Cell index ¼
Rheometer, Anton Paar, Graz, Austria; Couette CC10/T200 coaxial
geometry with a bob diameter 10.002 mm, cup diameter
10.845 mm). For each sample a layer of paraffin oil was applied to
avoid evaporation. Gelation was analyzed by measuring storage and
loss moduli (G0 and G00 ) as a function of time during application of
oscillatory sinusoidal deformation (0–30 min, frequency f = 1 Hz,
strain c = 0.01%). During the second time interval (30–60 min) gels
were broken by increasing the strain (c = 0.01–100%, f = 1 Hz). In the
third time interval (60–90 min), the strain was reduced and
recovery of the gel was analyzed (frequency f = 1 Hz, strain
c = 0.01%). All measurements were performed at 37  C.
2.10. Drug release studies
The release profiles of Aln from NPs-Aln suspended in PBS
buffer as well as from GG–NPs-Aln system were analyzed. Dialysis
33
bags (ZelluTransRoth, MWCO 12 kDa) were filled with 1 ml of
sample, end sealed and immediately immersed in the vials with
20 ml of PBS at pH 7.2 under continuous stirring (50 rpm) at 37  C.
At predetermined time intervals, 2 ml of buffer was collected and
the amount of drug was quantified using an OPA assay as described
elsewhere (Anhalt, 1977).
2.11. In vitro tests with osteoblast-like and osteoclast- like cells
Cellular response of osteoblast-like and osteoclast-like cells
was studied in contact with extracts from GG–NPs-Aln system,
which were prepared in two culture media (DMEM and RPMI) at
37  C for 72 h, in conditions similar to drug release studies.
Osteoblast-like MG-63 cells were cultured in DMEM (supple-
mented with 10% fetal bovine serum (FBS), gentamicin 40 mg/ml)
and RAW 264.7 monocyte-like cells were cultured in RPMI
(supplemented with 10% FBS, gentamicin 40 mg/ml) at 37  C in a
humidified air atmosphere containing 5% of CO2. A real-time cell
analyzer (RTCA, xCELLigence, Roche Applied Science, Mannheim,
Germany and ACEA Biosciences, San Diego, California) was used
to evaluate the survival and growth of cells in the prepared
extracts continuously, during a 7 day time span. RTCA is an
impedance-based system used for dynamic and label-free
monitoring of adherent cells attachment, spreading and prolifer-
ation. As adherent cells spread and proliferate on the bottom of
the e-plate covered with gold electrodes, cell membranes act as
an insulator blocking detection of electric current by the
electrodes (Peper et al., 2014). First, 162 ml of cell culture medium
as control samples, i.e. pure DMEM or RPMI, and the extracts from
GG–NPs-Aln system in DMEM or RPMI were poured into the wells
of a 96-well sensory E plate (E-Plate 96, BioTech a.s., Prague, CR,
Cat. No. 05232368001), and background impedance was measured
in each well. After that, 18 ml FBS were added into each well.
Subsequently, a dense suspension of cells (5  103 in 20 ml of
DMEM for MG-63 or of RPMI for RAW 264.7) was added to the
wells in triplicates. To elicit osteoclastic differentiation of RAW
264.7 monocyte-like cells, 100 ng/ml of RANKL was used. RAW
264.7 cells in RPMI without RANKL were also cultured. The
impedance was measured every 30 min. Cell index values
(reflecting cell attachment, spreading and proliferation) were
calculated automatically by the instrument according to the
formula (4):
(4)
15V
To determine the viability of cells on day 1 and 7 cells were
stained using LIVE/DEAD1 viability/cytotoxicity assay according to
manufacturer’s protocol. Briefly, cells were incubated with calcein-
AM and ethidium homodimer-1 (2 and 4 mM in PBS, respectively)
for 10 min at 37  C. Calcein-AM, metabolized by esterase activity of
living cells, emitted green fluorescence, and ethidium homodimer-
1, which penetrated into dead cells through their damaged
membrane, produced red fluorescence. After incubation of cell
with these dyes, microphotographs were taken under an
epifluorescence microscope (Olympus IX-51 with DP-70 digital
camera, Japan).
To determine the differentiation of RAW 264.7 cells towards
osteoclasts, the cells after 7 days of culture were stained by using
a TRAP staining kit according to the manufacturer’s protocol.
Briefly, the cells were fixed in citrate/acetone solution and
incubated in solutions of acetate, naphtol AS-BI phosphoric acid
34 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40
and Fast Garnet GBC Salt with or without tartrate for 1 h at 37  C
in dark. After rinsing with deionized water, the cells were
counterstained with acid hematoxylin. Afterwards microphoto-
graphs were taken under IX 51 Olympus microscope equipped
with DP70 digital camera.
2.12. Statistics
The results were presented as a mean  standard error of the
mean (S.E.M.) from 3 individual measurements (n = 3). The
statistical analyses were performed using t-test and significant
differences were assumed at ***p < 0.001, **p < 0.01 and *p < 0.05.
3. Results
3.1. Properties of nanoparticles
Nanoparticles were formulated with an average diameter of ca.
270 nm and ca. 230 nm for empty and Aln-loaded batches,
respectively (Table 1). The polydispersity was low (0.2 and
0.3 for Aln-loaded and empty nanoparticles, respectively), which
corresponded to a narrow size distribution (Fig. 1A and B). Laser
electrophoresis demonstrated a negative zeta potential in the
range of 28 and 30 mV (Table 1). The solubilization efficiency
and loading efficiencies of Aln in the nanoparticles were ca. 70%
and 5%, respectively. AFM pictures (Fig. 1C and D) revealed that the
nanoparticles were spherical and had smooth morphology.
3.2. Properties of nanoparticle-loaded hydrogel system
Injectability tests of the systems (Fig. 2A) showed that the
measured extrusion force, FMAX, using the standard 18 G needle for
both GG and GG–NPs-Aln was lower than 10 N. For GG hydrogels
the force was 8.1 1.1 N and it increased to 9.5  1.0 N upon
nanoparticles addition (no significant difference was found
according to t-test). It is worth noting that for non-cross-linked
GG hydrogels (without Ca2+) the extrusion force was significantly
lower, i.e. 0.81  0.06 N (data not shown). For GG–NPs-Aln systems
swelling was significantly lower (42.0  3.5%) than for GG hydro-
gels (105  2.3%) (Fig. 2B).
The elastic as well as the viscous response of GG and GG–NPs-
Aln were analyzed by measuring storage (G0 ) and loss (G00 ) moduli,
respectively (Fig. 3). In the first time interval (0–30 min) the elastic
response dominated the viscous behavior for both GG and GG–
NPs-Aln. For GG a G0 of 8.5 kPa was reached (Fig. 3A) whereas G00
reached 0.5 kPa (Fig. 3B). For GG–NPs-Aln G0 and G00 were found
7.4 kPa (Fig. 3A) and 0.5 kPa (Fig. 3B), respectively. During breaking
of the hydrogels by strain sweep deformation (s = 0.01–100%) G0
and G00 decreased continuously; G0 decreased to 10 Pa, while G00
decreased to 80 Pa. Fig. 3C and D shows G0 and G00 moduli as a
function of strain during the breaking test up to 10%. It is apparent
that both the drop in G0 and in G00 was faster for GG–NPs-Aln than
for GG hydrogel. During the last interval (60–90 min) both GG and
GG–NPs-Aln restored the initial values of G0 and G00 ; they reached
6.8 kPa and 0.4 kPa for GG hydrogel and 7.1 kPa and 0.5 kPa for the
GG–NPs-Aln injectable system, respectively.
Table 1
Physico-chemical analysis of empty and Aln-loaded PLGA nanoparticles.
Nanoparticles DH Pdl
(nm)
NPs (empty) 267  7 0.290  0.029
NPs-Aln 234  4 0.195  0.006
DH – hydrodynamic diameter, Pdl – polydispersity index, z – zeta potential, %EE – encapsulation efficiency, %LE – loading efficiency.
3.3. Drug release studies
An initial burst release phase measured after day 1 was 17% and
2% for NPs-Aln and GG–NPs-Aln respectively (Fig. 1A and B). For
NPs-Aln the release of Aln was the highest within the first 5 days of
the experiment (200 mg of Aln), it decreased to 120 mg in the 2nd
5-day time interval, followed by 80 mg for the 3rd and 4th 5-day
time intervals and finally it diminished to 10 mg in the last analyzed
5-day time interval (i.e., days 20–25 of the experiment) (Fig. 4A).
For GG–NPs- Aln the release rate was slower and constant in time
(linearity of zero-order kinetics); the system delivered 16  3 mg of
Aln within each 5-day interval (Fig. 4B). In total, after 25 days, 95%
and 8% of the drug was released from NPs-Aln and GG–NPs-Aln,
respectively. After 30 days, the release curves of NPs-Aln leveled off
while the injectable GG–NPs-Aln system still showed the ability to
deliver drug with constant doses.
3.4. In vitro studies
MG-63 osteoblasts-like cells had normal spindle-shaped
morphology in both pure DMEM and extracts of GG–NPs-Aln
(DMEM/GG–NPs-Aln) (Fig. 5A). The LIVE/DEAD1 viability/cyto-
toxicity assay showed that the initial attachment of MG-63 cells
and cell densities achieved in both media were similar on days 1
and 7. A waste majority of cells was viable, i.e. stained in green, and
only few cells were dead, i.e. stained in red, in both DMEM and
DMEM/GG–NPs-Aln extract (Fig. 5A). In accordance with these
results, the continuous impedance- based measurement showed
overlapping curves of MG-63 cells in both DMEM and DMEM/GG–
NPs-Aln extract during a 7-day culture (Fig. 5B).
RAW 264.7 macrophages cultured in pure RPMI displayed
mostly a round morphology, they partially and weakly adhered to
the surface of culture dish (Fig. 6A). The microscopic observations
after RANKL addition (Fig. 6B) showed that the cells formed
aggregates, which can be considered as a tendency to fuse and to
form multinucleated cells, and some of them were spread having
polygonal or spindle-shaped morphology, which are indicators of
their osteoclastic differentiation (Steinberg and Hiken, 2007;
Araujo et al., 2009). In tested conditions, i.e. when culture was
performed in GG–NPs-Aln extracts (Fig. 6C), the cells were mostly
round, the spread cells were less numerous and the cell
morphology was similar to that of cells cultured in pure RPMI.
The presence and activity of TRAP, i.e. an important marker of
osteoclastic differentiation, manifested with brown-reddish color,
was most apparent in RAW 264.7 cells cultured in the medium
with RANKL and least apparent in cells in GG–NPs-Aln extracts. In
addition, in RPMI + RANKL medium, RAW 264.7 macrophages
reached higher cell population density in comparison with cells in
pure RPMI and GG–NPs-Aln extracts.
Microscopic observation went along with impedance-based
curves (Fig. 6D). For RAW 264.7 cells cultured in RPMI with
addition of RANKL cell index was the highest suggesting RAW
264.7 osteoclastic differentiation (i.e., generation of impedance by
cell aggregation and spreading), and also proliferation (i.e.,
generation of impedance by occupancy of the well bottom by a
higher number of cells). In the case of RAW 264.7 cells cultured in
z %EE %LE
(mV) (%) (%)
30.5  0.6 – –
27.7  0.2 70.3  4.9 5.1  0.6
 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40 35

Fig. 1. Histograms presenting size distribution of empty (A) and Aln-loaded nanoparticles (B) obtained using Malvern Zetasizer NanoZS. AFM visualization of empty (C) and
Aln-loaded nanoparticles (D) (Explorer, Thermomicroscopes).

RPMI + RANKL/GG–NPs- Aln extracts the cell index reached lower
values which were similar as for RAW cells cultured in RPMI.
4. Discussion
We aimed at manufacturing a new type of Aln delivery system
for the local treatment of osteoporosis and other bone diseases.
The system, intended to be administered inside affected bone
tissue as a minimally invasive and handy injection, was based on
hydrogel containing Aln encapsulated within PLGA nanoparticles.
Our hypothesis was that the system would assure local and
sustained release of drug, which is known to inhibit osteoclasts
maturation. In the study NPs-Aln were obtained and their
physicochemical properties were assessed. Then, NPs-Aln were
suspended in GG hydrogels and the hydrogel matrix was cross-
linked with calcium ions to provide structural integrity suitable for

Fig. 2. Extrusion force (A) and swelling (B) of the cross-linked hydrogel (GG) and nanoparticles loaded system (GG–NPs-Aln). Significant difference ***p < 0.001 according to
t-test.
36 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40

Fig. 3. Rheological characteristics of the cross-linked hydrogel (GG) and nanoparticles loaded system (GG–NPs-Aln). Storage modulus, G0 , (A) and loss modulus, G00 , (B) during
assembling (0–30 min, f = 1 Hz, strain, s = 0.01%), breaking (30–60 min, f = 1 Hz, s = 0.01–100%) and reassembling (60–90 min, f = 1 Hz, s = 0.01%). G0 (C) and G00 (D) as a function
of strain (f = 1 Hz, s = 0.01–10%) plotted on a logarithmic scale.

injection. Afterwards, the kinetics of the drug release was
measured. Lastly, to assess biological activity of developed system
cellular response of bone-forming and bone-resorbing cells was
evaluated.
4.1. Properties of Aln-loaded PLGA nanoparticles
Aln-loaded PLGA nanoparticles were successively formulated
according to a solid/oil/water (S/O/W) method, which enabled
encapsulation of 70% of the drug introduced in the system, and in
the final nanoparticles the drug loading efficiency was equal to
5%.
The average diameter of NPs-Aln was 230 nm with a narrow size
distribution as reflected by the polydispersity index (below 0.2).
NPs-Aln were spherical as shown by AFM observations and
demonstrated a negative zeta potential (ca. 28 mV), which can be
attributed to the presence of the ionized carboxyl groups of PLGA
on the particles’ surface (Le Broc-Ryckewaert et al., 2013).

Fig. 4. Aln release curves from drug-loaded nanoparticles (A) and gellan gum based injectable system (B). Cumulative curves (black squares and lines) as well as doses
released from 1 ml of NPs-Aln suspension (1% w/v) or 1 ml of GG–NPs-Aln system during 5-day intervals (boxes) are shown.
 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40 37

Fig. 5. Morphology of osteoblast-like MG-63 cells cultured in DMEM/GG–NPs-Aln extracts and in DMEM on day 1 and 7 (A). The living cells were stained with LIVE/DEAD1
viability/cytotoxicity assay and observed under Olympus IX51 epifluorescence microscope equipped with DP70 digital camera; objective 10, scale bar = 200 mm. Cell index
of MG-63 cells cultured in DMEM or in DMEM/GG–NPs-Aln extracts during a 7-day culture, measured using xCELLigence system real-time cell analyzer (B). (For interpretation
of the references to color in text, the reader is referred to the web version of this article.)

Interestingly, unloaded nanoparticles were slightly bigger (ca.
270 nm) and less uniform (polydispersity ca. 0.3) than NPs-Aln. The
dispersion of Aln powder in the oily phase in the first step of the S/
O/W emulsification may have resulted in solid Aln nuclei on which,
during the second step, polymer chains precipitated more easily
thus creating smaller and more uniform nanoparticles. As a result,
the process of nanoparticle formation became more controlled
when Aln powder was introduced to the system.
4.2. Properties of injectable GG–NPs-Aln system
Although there are many evidences that PLGA nanoparticles are
potent drug delivery vehicles it was also reported that they are
often instable, agglomerate and are prone to phagocytosis in vivo
(Li et al., 2014; Kohane, 2007). Therefore it may be advantageous to
process nanoparticles into implantable or preferably injectable
forms (for example by suspending them in hydrogels). In this
study, GG hydrogel was used as a matrix to suspend NPs-Aln. GG
forms a double helix out of two left-handed chains. Three pairs of
such double helices build an associate that constitutes a gel. The
structure exposes carboxylic groups on the periphery that are
prone to interact with bivalent positive ions (e.g. Ca2+), which
physically crosslink the hydrogel and thus increase its stiffness
(García et al., 2011). When enriched with drug loaded nano-
particles (e.g. NPs-Aln), functionality of the GG hydrogel expands
towards therapeutic injections.
In order to determine the handiness of such hydrogel-based
injectable system, the extrusion force from the syringe was
determined. Suspending NPs-Aln at a concentration of 1% w/v, i.e.
higher than the GG matrix itself (0.7% w/v) was found not to
significantly affect its injectability (see Fig. 2A). A slight but not
statistically significant increase in extrusion force (FMAX 1 N)
possibly resulted from an increase in viscosity of the system due to
friction arising between nanoparticles and GG chains. Still, the
obtained values (9.5  1.0 N) were lower than the injectability of
commercially available medical products (e.g. Radiesse1 – a
dermal filler, FMAX 27 N) (Ji et al., 2012) and were in the range of the
injectability of tricalcium phosphates pastes proposed for bone-
grafting applications (5.4  1.1 N–18.5  1.7 N) (Montufar et al.,
2013).
Swelling of the GG–NPs-Aln system was found to be lower (42%)
than that of GG (105%) (see Fig. 2B). Presumably this can be related
either to the hydrophobic character of the PLGA nanoparticles that
possibly repelled water molecules or to the location of the NPs-Aln
within the matrix. It is known that swelling occurs when water
molecules fill the pores within the hydrogel matrix (Gantar et al.,
2014). Par analogy it can be speculated that NPs-Aln are located
within the pores of the hydrogel and occupy available space.
Nevertheless, from the application point of view and hydrogel
structural integrity, a low extent of swelling is beneficial.
In order to characterize the GG–NPs-Aln system prior, during and
after injection the rheological tests were divided in three stages:
gelation (assembling), breaking (injection) and reassembling (self-
healing). It was found that both the GG–NPs-Aln system and the
reference cross-linked GG hydrogel behave elastically predominant-
ly (G0 > G00 ) (see Fig. 3A and B, time 0–30 min). Addition of NPs-Aln
into GG resulted in a very small decrease in G0 but it did not influence
G00 . This may be ascribed to the larger distance between the GG
38 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40

Fig. 6. Morphology of RAW 264.7 cells (non-differentiated macrophages) in RPMI medium (A), morphology of cells differentiating towards osteoclasts in RPMI + RANKL; red
arrows indicate cell fusion (B), morphology of cells cultured in RPMI + RANKL/GG–NPs- Aln extracts (C) after staining cells for TRAP (objective 20, scale bar = 100 mm). Cell
index of RAW 264.7 cells cultured in RPMI or in RPMI + RANKL or RPMI + RANKL/GG–NPs- Aln extracts during a 7-day culture, measured using xCELLigence system real-time
cell analyzer (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

chains due to the presence of NPs-Aln which possibly hindered the
formation of calcium mediated cross-links.
To simulate the injection process, the GG–NPs-Aln gel was
broken by applying a high shear rate (see Fig. 3A and B, time
30–60 min), which transformed GG–NPs-Aln into a flowing sol
(both G0 and G00 decreased drastically and G00 became higher than
G0 ). The drop in both moduli (G0 , G00 ) was due to structural changes
within the gel network: possibly cross-links destruction or an
alignment of hydrogel chains occurred as shown in other studies
(Morais et al., 2013). The G' of the GG–NPs-Aln system dropped
much faster than the G0 of GG when strain deformations were
applied (see Fig. 3C and D), which suggests that nanoparticle
addition caused the system to become weaker than GG. In general,
the viscosity drop upon shearing of the GG–NPs-Aln showed that
the system proposed would be suitable and handy to extrude.
The structural integrity of both GG and GG–NPs-Aln was
immediately recovered after decreasing the strain back to 0.01%
(see Fig. 3A and B, time 60–90 min). Both G0 and G00 were restored to
their initial values, i.e. G0 = 7.1 kPa, G00 = 0.5 kPa, which indicates gel
reformation. The self-healing behavior may be ascribed to
attractive electrostatic interactions that can be reformed after
being broken. A similar behavior, i.e. electrostatically-driven
immediate structure restoration after elimination of the shear,
was previously described by Wang et al. for colloidal gels obtained
from oppositely charged gelatin nanospheres (Wang et al., 2011).
The rheological tests showed that GG–NPs-Aln system behaved
predominantly elastically and displayed self-healing behavior, i.e.
the ability to restore its structure upon destruction for example
during injection.
4.3. Drug release from NPs-Aln and injectable GG–NPs-Aln system
In our studies the controlled release of small doses of Aln was
considered a key criterion in selecting the compositions for intra-
bone drug administration. The nanoparticles with encapsulated
Aln were produced from PLGA 85:15 which is known to degrade
more slowly than PLGA containing equal molar ratio of lactide and
glycolide, i.e. 50:50. The latter is most often used for encapsulation
of different types of drugs (Fredenberg et al., 2011; Mundargi et al.,
2008). Thus by using PLGA 85:15 of relatively high molecular
weight (Mn = 100 kDa, Mw = 190 kDa) we intended to slow down
hydrolytic degradation of the polymer matrix, reduce the burst
release and obtain retarded and sustained delivery of the
encapsulated drug. Our approach resulted in an initial burst
release of 17% after 1 day and all the encapsulated drug was
released during 25 days. It is probable that the initial burst release
from NPs-Aln was associated with Aln adsorbed on the nanoparti-
cle surfaces, while in the following stage Aln was released upon
hydrolytic degradation of PLGA. It was found, however, that NPs-
Aln did not assure a constant drug release rate: within the first 5-
day time interval 200 mg of Aln was released, while in the fifth
analyzed 5-day time interval only 10 mg of Aln was released from a
1 ml of NPs-Aln suspension (see Fig. 4A).
In contrast, by using GG–Aln-NPs system controlled delivery of
Aln was obtained: the system delivered 16  3 mg of Aln within
each 5-day interval up to day 25, which cumulatively accounted to
8% of total drug entrapped within the system (see Fig. 4B). In our
system drug release was retarded due to the suspension of the NPs
in a GG hydrogel matrix. It is well known that the diffusion rate of
molecules through a hydrogel depends mainly on: the amount of
liquid in the hydrogel, the distance between polymer chains (the
mesh size), the structural flexibility (Bertz et al., 2013) and their
size and charge (Golmohamadi and Wilkinson, 2013). We
hypothesize that for the GG–NPs-Aln system degradation of
nanoparticles and thus drug release was retarded mainly due to
lower availability of free water molecules within the hydrogel as
compared with the situation when the nanoparticles were freely
suspended in aqueous media (e.g. PBS). As a result hydrolytic
degradation of PLGA nanoparticles was hindered and less drug was
released from NPs-Aln. However the hydrogel was not an obstacle
to drug release because both Aln and GG are negatively charged
thus drug transport through the gel structure might be even
enhanced. The diffusion of a negatively charged probe (Oregon 2C)
through a negatively charged hydrogel (alginate) was already
described by Golmohamadi and Wilkinson (2013), who found that
there is depletion of anionic probes within the polyanion structure.
 U. Posadowska et al. / International Journal of Pharmaceutics 485 (2015) 31–40
4.4. Biological effects of injectable GG–NPs-Aln
Real time cell analysis together with viability tests and
microscopic observations were used to assess the behavior of
osteoblast-like cells and the efficiency of osteoclastic differen-
tiation of macrophages in contact with extracts from GG–NPs-
Aln system. In general, differentiation of macrophages occurs in
the presence of the recombinant RANKL factor, which causes
cell fusion and transformation into multinucleated osteoclast-
like cells (Steinberg and Hiken, 2007). We found that RANKL-
stimulated RAW 264.7 cells tended to fuse, exhibited a flattened
morphology, were positively stained for TRAP and their
normalized cell index was the highest (see Fig. 6), which are
indicators of osteoclastic differentiation (Steinberg and Hiken,
2007; Araujo et al., 2009). When such cells were cultured in
GG–NPs-Aln extracts they were round, poorly spread and
behaved as non-differentiated macrophages. The curves plotting
cell index of RANKL- stimulated RAW 264.7 in contact with
GG–NPs-Aln extracts and cells cultured under control con-
ditions (i.e. in pure RPMI medium) overlapped. Evidently,
alendronate-induced blockage of RANKL-stimulated cytoskeleton
organization of monocytes for osteoclasts (Vaananen, 2005) took
place, causing an impedance drop for cultures in GG–NPs-Aln
extracts. The results obtained are in accordance with the study
performed by Kim et al. (2010) who showed that Aln-loaded
drug delivery systems reduced osteoclastic differentiation. So, it
can be concluded that injectable GG–NPs-Aln systems down-
regulated osteoclasts formation.
The real time cell analysis of MG-63 cells show that no change
in cell index was found between reference conditions and GG–NPs-
Aln extracts. Moreover, the morphology as well as the viability of
the cells did not differ (see Fig. 5). It altogether indicates no
negative impact of GG–NPs-Aln on model bone-forming cells. Thus
injectable GG–NPs-Aln system was found to inhibit osteoclasts
differentiation and at the same time not to affect osteoblasts
functions. This is a proof that drug released from the developed
system retained its biological activity.
5. Conclusion
Our system based on Aln-loaded PLGA nanoparticles suspended
in gellan gum hydrogel matrix: (i) was found to be injectable and
restored its elastic structure after extrusion, (ii) assured local and
uniform drug delivery, and (iii) inhibited osteoclasts differentia-
tion without affecting osteoblasts functions. The results show the
potential of our approach for the local treatment of osteoporosis
and other bone tissue pathologies.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgments
The authors would like to thank to Professor Piotr Dobrzynski
(Center of Polymer and Carbon Materials, Polish Academy of
Sciences) for providing us with degradable copolymer, Polpharma
S.A. for providing sodium alendronate and Dr. Lukasz Zych
(Department of Ceramics and Refractories, Faculty of Materials
Science and Ceramics) for the access to the Zetasizer. Polish
National Science Centre (Grant no: 2012/05/B/ST8/00129) and
Ministry of Health of the Czech Republic (Grant No. NT13297-4/
2012) provided financial support to this project.
39
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