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Improvement of in vitro osteogenesis and anti-infection properties by GelMA

scaffold containing levofloxacin nanoparticles and strontium microspheres
for osteomyelitis

Article  in  Journal of Materials Science · July 2022

DOI: 10.1007/s10853-022-07456-6

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J Mater Sci (2022) 57:13603–13619

Materials
M A T E R I A L S Ffor
O R Llife
I F E Ssciences
CIENCES

Improvement of in vitro osteogenesis and anti-infection

properties by GelMA scaffold containing levofloxacin
nanoparticles and strontium microspheres
for osteomyelitis
Elham Jamshidifar1, Mehdi Esfandyari-Manesh2,*, Hamidreza Motasadizadeh3,
Sara Naderizadeh4, Alaleh Yourdkhani1, Nasrin Samadi5, and Rassoul Dinarvand1,2,6,*

1
Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
2
Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
3
Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
4
School of Engineering and Materials Science, Queen Mary University of London, London, UK
5
Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
6
Leicester School of Pharmacy, De Montfort University, Leicester, UK

Received: 6 March 2022 ABSTRACT

Accepted: 17 June 2022 Biomaterials that have capacities to simultaneously induce bone regeneration
Published online: and kill bacteria are in high demand because bone defects face risks of severe
9 July 2022 infection in clinical therapy. The aim of this study was to investigate the use of
gelatin methacryloyl (GelMA), alginate, gelatin hydrogels containing levo-
Ó The Author(s), under floxacin loaded poly (lactic-co-glycolic acid) nanoparticles (LEV-PLGA NPs) as
exclusive licence to Springer antibacterial agent and strontium loaded PLGA microspheres (Sr-PLGA
Science+Business Media, LLC, microspheres) as osteoinductive agent, intended for improving the treatment of
part of Springer Nature 2022 osteomyelitis. Nanoparticles and microspheres were prepared using oil-in-wa-
ter (o/w) and water-in-oil-in-water (w/o/w) emulsion method, respectively.
Then GelMA-alginate-gelatin scaffold loaded with both LEV-PLGA NPs and Sr-
PLGA microspheres, was prepared by UV radiation crosslinking. The
mechanical strength of the GelMA scaffold was increased to 0.1 MPa by intro-
ducing gelatin and alginate into the GelMA hydrogel. The highest cumulative
drug release from the LEV-PLGA-loaded hydrogels reached 64% over 25 days.
The LEV-PLGA-NPs had an effective antibacterial response against Escherichia
coli, Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, the perfor-
mance in terms of cell viability had no adverse influence upon the absence of
cytotoxicity, as indicated in tests carried out using normal adult human
fibroblast cells. The presence of Sr-PLGA microspheres led to upregulation of

Handling Editor: Annela M. Seddon.

Address correspondence to E-mail: mehdiesfandyari@gmail.com; dinarvand@tums.ac.ir

https://doi.org/10.1007/s10853-022-07456-6
13604 J Mater Sci (2022) 57:13603–13619

RUNX2, Osteonectin, and Osteocalcin genes about 10, 9, and 10 times higher than
control group in osteogenic differentiation of MC3T3 cells, respectively. Alka-
line Phosphatase activity was 59 u/l on day 21. It could therefore be concluded
that scaffold might be considered as a potential useful biomaterial for the
treatment of osteomyelitis with antibacterial properties.

Introduction
Osteomyelitis is a bone defect caused by the growth
infectious microorganisms in the bone. This disease
can be caused by open surgery, fracture or spreading
infection through the bloodstream and nearby tissues
[1]–[3]. Osteomyelitis, especially the chronic type, has
a chronic clinical course, long periods of extinction
and recurrence. Therefore, designing a continuous
and long-term antibiotics releasing system would be
crucial in the treatment of this disease. Following this
issue, finding an effective approach to regenerate the
bone structure remains a significant challenge in the
osteomyelitis treatment [4, 5].
Designing a bi-functionalized drug delivery system
based on releasing antibiotics and bone regenerated
materials simultaneously can be helpful in
osteomyelitis treatment [6]–[8]. Numerous studies
have been conducted on incorporating materials into
scaffolds showing considerable improvement in bone
regeneration [9]–[11]. Furthermore, many attempts
have been made to introduce antibiotics into these
structures to treat the infected bone tissues in recent
years [12][13].
Escherichia coli, Staphylococcus aureus and Pseu-
domonas aeruginosa are the main microorganisms
causing bone infection. Various antibiotics can kill
these bacteria, such as Vancomycin, Levofloxacin,
Gentamycin, Ciprofloxacin, etc. [14]–[17]. Levo-
floxacin (LEV) is one of the antibiotics, which is
effective against both Gram-positive and Gram-neg-
ative bacteria [18]–[21]. High quantity of antibiotics,
along with burst and long-term release, may cause
toxicity in the tissues. However, designing an effi-
cient system with controlled local drug delivery
activity can resolve this issue [22]–[24].
Moreover, incorporating other bioactive materials
such as Strontium (Sr) into the designed structure can
help bone regeneration [13, 25]–[28]. Strontium is an
inorganic osteoinductive ion, which can prevent bone
tissue breakdown by inhibiting the osteoblast cells
[29, 30]. Many studies have shown that adding Sr into
scaffolds not only can improve the mechanical
properties of scaffolds but also can regenerate the
bone tissue by modulating the cell’s metabolism
[31, 32]. Although Sr is very beneficial to be used in
the osteogenesis systems, but long-term use of this
material can cause issues in different body organs,
such as heart and kidney. Therefore, fabrication of a
local drug delivery systems with controlled drug
release is highly beneficial for bone regeneration
[33, 34].
Poly lactic-co-glycolic acid ( PLGA) is a
biodegradable and biocompatible polymer, used for
controlled release applications in drug delivery sys-
tems [35, 36]. Nanoparticles and microspheres can
improve the drug efficacy in most cases due to the
loading of drug molecules into polymeric structures,
preventing the drug decomposition, improving the
drug stability and controlling drug release [37]–[39].
Fabrication of scaffolds can be a good choice for
treating osteomyelitis disease, providing a 3D struc-
ture for cell proliferation and improving bone
regeneration [40, 41]. A wide range of materials such
as polymers, ceramics, metals and composite, are
used in scaffold designing, which each of them has
different properties [42, 43]. One of the critical factors
in designing bone scaffolds is their biodegradation
rate, corresponding to the bone regeneration rate
[44, 45]. Among different scaffolds, biopolymer-
based ones have more advantages such as low cost,
abundance, biocompatibility, and biodegradation
[43, 46].
Recently, hydrogel-based scaffolds have played an
important role in tissue engineering and drug-deliv-
ery systems due to the high absorption of water and
biologic fluids, which can enhance the bone regen-
eration [47]–[49]. They can be divided into different
groups such as natural, synthetic and hybrid based
on the constituent polymers, and each of them can
provide different properties. GelMA is an example of
hybrid hydrogel scaffolds. It is based on methacry-
lated gelatin, which benefits from bioactivity
J Mater Sci (2022) 57:13603–13619 13605

properties and physicochemical characteristics of antibacterial capabilities, respectively. Physicochem-

gelatin and methacrylic anhydride simultaneously
[47, 50, 51].
In this study, to improve the properties of the
hydrogel scaffold a mixture of GelMA, gelatin, and
alginate was used (Fig. 1). Then Sr-PLGA micro-
spheres and LEV-PLGA NPs were loaded into the
scaffold hydrogel to provide the osteogenesis and
ical properties of different categories of the materials
were studied in the related parts through various
analyses. Antibacterial activity, drug-release behavior
cytotoxicity, and osteon differentiation of osteoblast
MC3T3 cells in the final scaffolds were also
evaluated.

Figure 1 Graphical
representation of osteoblastic
cell laden GelMA-alginate-
gelatin scaffold containing
levofloxacin-PLGA
nanoparticles for anti-infection
and Strontium-PLGA
microspheres for bone
regeneration. Fabrication of
hydrogel ink, nanoparticles,
microspheres, and scaffold,
osteogenesis test, antibacterial
test, and uses for osteomyelitis
treatment.
13606
Materials and methods
Materials
Poly lactic-co-glycolic acid (PLGA, Resomer RG 504
H) was purchased from Evonik, Germany.
Methacrylic anhydride, Gelatin extracted from
bovine skin, Dextran, Strontium chloride and Levo-
floxacin were from Sigma-Aldrich, USA. Polyvinyl
alcohol (PVA) with molecular weight
20,000–30,000 Da was obtained from Acros Organics,
USA. Dulbecco’s modified Eagle’s medium (DMEM),
Fetal bovine serum (FBS) and Trypsin–EDTA were
purchased from Gibco, England. 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT)
was bought from Sigma-Aldrich, USA. BHI Broth,
Mueller Hinton Broth and Mueller Hinton agar were
obtained from Merck, Darmstadt, Germany. DNA,
RNA, and alkaline phosphate assay kits were pro-
vided by Pars tous, GeneAllÒ RiboEx and Abcam,
respectively. Taq DNA Polymerase 2 9 master mix
red and Real Q plus 2 9 master mix green were
purchased from AMPLIQON. All other chemicals
and solvents used in this work were obtained from
Sigma-Aldrich, USA and used as received.
The materials related to the cell culture were
DMEM (Dulbecco’s Modified Eagle’s Medium), FBS
(Fetal Bovine Serum), Trypsin–EDTA were pur-
chased from Gibco, England and MC3T3 cells, which
were provided by Pasteur Institute of Iran.
Preparation of Gelatin- Methacryloyl
(GelMA)
First, 5 g gelatin was fully dissolved in 50 ml phos-
phate buffer saline (pH = 7.4) and stirred at 55 °C for
20 min. Then, 5 ml methacrylic anhydride was added
into the gelatin solution under magnetic stirring at
1200 rpm and the reaction proceeded for 3 h. In order
to separate the unreacted methacrylic anhydride, the
solution was centrifuged (Sigma 3K30, Germany) for
2 min at 3500 RCF at R.T. To stop the reaction, the
obtained supernatant was diluted with PBS and
stirred for 15 min at 50 °C. After filtration, the solu-
tion was dialyzed against water (membrane 14 KDa)
for 5–7 days at 37 °C under stirring at 300 rpm. At
the end of the process, the pH was set at 7–7.4.
Finally, to obtain the solid product, the solution was
lyophilized at - 70 °C (Lyotrap Plus, LTE Scientific
J Mater Sci (2022) 57:13603–13619
Ltd, UK) and the produced GelMA was kept at 4 °C
for further investigations.
Preparation of levofloxacin PLGA
nanoparticles
Poly lactic-co-glycolic acid (PLGA) nanoparticles
containing Lev were prepared through oil-in-water
(o/w) emulsification followed by solvent evapora-
tion. First, 10 mg of LEV powder and 100 mg of
PLGA were dissolved in 2 ml dichloromethane.
Separately, 1 g of PVA powder was dissolved in
100 ml water. To prepare the nanoparticles, PLGA/
LEV solution was added to 10 ml PVA solution,
under sonication (Ultra Sonic, Techno-gaz, Tecna 6)
in 3 min, until the final emulsion was obtained. The
emulsion was stirred for 4 h at 700 rpm, followed by
centrifugation for 30 min at 20,000 rpm at 4 °C to
evaporate the solvent completely. The nanoparticles
were collected from the precipitate, rinsed with water
for 3 times, lyophilized for 24 h and kept at 4 °C for
further investigations. The supernatant from the first
run of the centrifugation was used to estimate the
loading capacity and encapsulation efficiency.
Preparation of Strontium PLGA
microspheres
The PLGA microspheres containing Sr were prepared
via water-in-oil-in-water (w/o/w) emulsification
followed by solvent evaporation. First, the aqueous
solution was prepared by dissolving 50 mg of dex-
tran and 100 mg ammonium bicarbonate in 1 ml
water, followed by adding 40 mg of Sr. Separately,
250 mg PLGA was added into 3 ml dichloromethane
to prepare the organic solution. To fabricate the
microspheres, 1 ml of aqueous solution was added to
the 3 ml PLGA solution, under sonication in 3 min, to
obtain the initial water-in-oil emulsion. Then, the
initial oil-in-water emulsion was added slowly into
200 ml PVA solution 0.4% under stirring, and the
second water-in-oil emulsion was produced. Then,
the solution was stirred for 4 h at 700 rpm, until the
solvent was evaporated completely. The final emul-
sion was centrifuged for 10 min at 10,000 rpm, and
microspheres were collected from the precipitate,
rinsed with water 3 times, lyophilized at - 20 °C and
kept in 4 °C for further studies.
J Mater Sci (2022) 57:13603–13619 13607

Preparation of GelMA-alginate-gelatin
hydrogel scaffold containing nanoparticles
and microspheres
First, 5 mg gelatin powder, 40 mg alginate and 70 mg
GelMA were added into 1 ml PBS and stirred for
30 min at 40 °C, followed by a few minutes of vor-
texing (VELP, Scientifica) to obtain a uniform solu-
tion. Then, 80 ll of photoinitiator was added to the
solution and vortexed again. Finally, the mixture was
exposed to UV radiation for 5 min to form the
crosslinked hydrogel structure. The hydrogel was
freeze-dried for 48 h to obtain the solid product. The
final hydrogel contains gelatin, alginate, GelMA and
photoinitiator with 0.5, 4, 7 and 0.5 (w/v) %,
respectively. To fabricate the hydrogel scaffold con-
taining nanoparticles and microspheres, 10 mg of
LEV-PLGA NPs and 11 mg of Sr-PLGA microspheres
were added into the GelMA-alginate-gelatin mixture,
vortexed for 2 min and exposed to the UV radiation.
Characterizations
test was done under the same conditions under a
0.5 mm/min rate. The amount of LEV loaded in
PLGA nanoparticles was evaluated using UV–Vis
spectroscopy (S-3100, Scinco, Korea), where LEV
shows a distinct absorption peak.
The encapsulation efficiency, loading capacity and
production yield were calculated through the fol-
lowing equations:
Encapsulation efficiencyð%Þ
total amount of drug  amount of unloaded drug
¼
total amount of drug
 100
Loading capacityð%Þ
total amount of drug  amount of unloaded drug
¼
weight of particles
 100
Production yieldð%Þ
Weight of particles
¼
 100
Theoretical weight particles + drug

The morphology of LEV-PLGA NPs, Sr-PLGA

microspheres and the hydrogel scaffold were exam-
Levofloxacin release behavior
ined through scanning electron microscopy (SEM)
The release behavior of LEV was studied via the
(TESCAN MIRA3 LMU, Czech Republic). Dynamic
dialysis technique. The freeze-dried nanoparticles
light scattering (DLS) (Malvern, Worcestershire, UK)
were dispersed into 1 ml PBS and were placed into
was utilized to measure of particle size. The chemical
the dialysis bags (MW 14 KDa), which was placed in
structure of GelMA was studied through the nuclear
2 ml PBS with pH = 7 and was then transferred in the
magnetic resonance (NMR) technique (Varian Unity
incubator (Heidolph, Incubator 1000-Unimax 10Fo,
plus 400 spectrometer, USA) and the H1 NMR spec-
Germany) with 37° C and shacked steadily at 70 rpm.
trum of synthesized GelMA in deuterated water was
Levofloxacin releasing from the dialysis bag into the
obtained. To confirm the formation of GelMA and
external solution was investigated at certain times.
loading of LEV into the PLGA nanoparticles, Fourier
2 ml of the sample was removed. At the same time, a
Transform Infrared (FTIR) (Nicolet Magna 550-FT
similar amount of fresh PBS was added instead in
spectrometer, SpectraLab Scientific Inc., Canada)
order to keep the sink condition constant. The con-
spectroscopy was used and the measurements were
centration of released LEV was measured using a
done for pure materials and the synthesized ones in
UV–Vis spectrophotometer at 300 nm and the
transmission mode in the range from 400 to
released concentration was evaluated for each certain
4000 cm-1. DSC thermograms were obtained using a
time. In the end, the cumulative amount of LEV
METTLER TOLEDO DSC823e instrument from 25 to
release from the LEV-PLGA NPs was calculated
300 °C at a heating rate of 10° C/min. ICP-OES
through the following equation:
(Inductively Coupled Plasma Optical Emission
Spectroscopy) technique (ICPS-7500, Shimadzu, Cumulative drug relaese ð%Þ

Japan) was used to estimate Sr loading capacity into weight of drug released mg
¼
 100ð%Þ
the microspheres. To evaluate the variation in weight of drug in the mat mg
mechanical properties of the scaffold, the compres-
sive modulus was calculated. Briefly, three samples
of each category were prepared, and the compression
13608
Antibacterial studies and evaluation of MIC
and MBC of levofloxacin
To culture the bacteria, 20 ml of BHI broth was added
into the vials containing lyophilized bacteria such as
S. aureus (ATCC 6538), E. coli (ATCC 8739) and P.
aeruginosa (ATCC 9027) and incubated at 37° C for
4 h. Then, 200 ll of these solutions were spread on
agar plates in parallel lines in several directions and
incubated for 24 h at 37° C, until the bacteria colonies
were formed.
Antibacterial analysis of the hydrogel scaffold was
performed through the disk-diffusion method. First,
an average number of bacteria were investigated
through turbidity measurement of each bacterial
suspension using the standard solution (Mc. Farland
0.5), which contains 1.5 9 108 CFU/ml bacteria. 4–5
colonies of different bacteria were added to the saline
solution and the turbidity was compared with the
standard solution to understand the turbidity of the
bacterial solutions. This comparison could be done
with eyes, turbidimeter or spectrophotometer.
The minimum inhibiting concentration (MIC) and
minimum bactericidal concentration (MBC) of LEV
were measured through serial dilution technique, in
which bacteria were exposed to serial dilution of the
antibacterial drug. In this method, MIC is considered
the lowest concentration of antibiotic, which prevents
bacterial growth in laboratory conditions. To achieve
these results, various antibiotic dilutions in the range
of 0.25- 512 lg/ml were prepared in the Mueller-
Hinton broth. Then, 100 ll of each dilution was fitted
into a 96-wells tissue culture plate and 100 ll of the
freshly grown bacteria in Mueller- Hinton broth (0.5
McFarland) was added into each well. The plate was
incubated at 37° C for 16–18 h and the turbidity of the
wells was investigated. Some wells were considered
control and negative control wells showed bacterial
growth after incubation, which confirms the validity
of the experiments.
To understand the MBC, the lowest concentration
of the antimicrobial agent that kills the bacteria,
100 ll of the well’s solution without turbidity of the
bacteria growth was cultured on blood agar and
incubated at 37° C for 16–18 h.
Cell viability by MTT method
MTT (3-(4,5-Dimethylthiozole-2-yl)-2,5-diphenylte-
trazolium bromide) is used in cycle performance and
J Mater Sci (2022) 57:13603–13619
cytotoxicity studies. Briefly, 25 ll of MTT solution
(4 mg/ml) in PBS was added into the 96-wells tissue
culture plate and incubated for 3 h at 37° C. To dis-
solve the purple formazan crystals, 10 ll DMSO was
added to the wells. Finally, the solution absorbance
was determined at 540 nm. The results were com-
pared to the performance of the control cells, which
had not been manipulated.
Determination of produced alkaline
phosphatase by osteoblast cells MC3T3
The sterilized scaffolds were placed into the 24-wells
tissue culture plate and 5 9 103 MC3T3 osteoblast
cells were added into the wells, then the volume was
reached 2 ml with culture medium. Plates were
incubated for 7, 14 and 21 h at 37 °C. Then, the cul-
ture medium was removed after 7, 14 and 21 days
and the cells were lysed to achieve cell lysis. 20 ll of
the cell’s supernatant was mixed with paranitrile
phosphatase and diethanolamine (4:1 ratio) to eval-
uate the alkaline phosphatase production and the
optical absorbance was measured at 405 nm by
spectrophotometer.
Cell differentiation via real time polymerase
chain reaction (RT-PCR) method
The process for RT-PCR analysis is described here,
briefly. First, MC3T3 cells and nanoparticles were
incubated and RNA extraction was done after 48 h.
The amount and purity of the extracted RNA were
studied through spectroscopy measurements
(Eppendorf, US). RNA concentration and optical
absorbance ratio were measured at 260 and 280 nm.
Revert AidTM First Strand cDNA synthesis kit (Fer-
mentas, USA) was used to fabricate cDNA. A group
of genes such as RUNX2, Osteocalcin and Osteonectin
were selected for this study.
Based on different primers used in this study, RT-
PCR was done using iCycler Thermal Cycler (Bio-
Rad, Hercules, CA, USA). Data processing was done
through DDCt method and the ratio between miR-145
(as target gene) and snord (as reference gene) was
calculated by rest software.
J Mater Sci (2022) 57:13603–13619
Results and discussion
Chemical characterization of synthesized
GelMA
To confirm the formation of GelMA, the pure gelatin
and synthesized GelMA were chemically character-
ized by FTIR spectroscopy (Fig. 2a). The characteris-
tic peak for gelatin at 1522 cm-1 was ascribed to the
second amide bond. However, this peak for GelMA
was shifted to 1534 cm-1. The other peaks regarding
the O–H, N–H and C-H functional groups were
located at 3283, 3066 and 2934 cm-1, respectively and
they were found in both pure gelatin and synthesized
GelMA. GelMA had a strong peak of C=O at
1638 cm-1. The double carbonyl stretching peaks for
methacrylic anhydride fall at 1751 and 1782 cm-1,
these peaks were disappeared in GelMA by forma-
tion of amide bonds. The presence of the main
functional groups of the gelatin in synthesized
GelMA with shifts in some peak position can confirm
the rearrangement in the chemical structure and the
formation of GelMA.
To go further, the chemical structure of the syn-
thesized GelMA was studied through H1 NMR
analysis and compared to pure gelatin (Fig. 2b). By
comparing these two spectra, it can be seen that there
is a new peak at 1.8 ppm for GelMA, which cannot be
Figure 2 a FTIR spectra of gelatin, methacrylic anhydride, and synthesized GelMA in the range of 400- 4000 cm-1, b H1 NMR spectrum
of the synthesized GelMA.
13609
found for pure gelatin. It indicated that the methyl
methacrylate group was successfully bonded to the
gelatin structure. Furthermore, the peaks at 5.3 and
5.5 ppm are ascribed to the acrylic protons of the
methacrylate functional group. These two analyses
confirmed that GelMA was synthesized through the
chemical reaction between gelatin and methacrylate
anhydride.
Characterization of LEV-PLGA
nanoparticles
Particle size is one of the most critical factors to
control drug release and prevent burst release. The
size distribution and zeta potential of the LEV-PLGA
NPs were analyzed through DLS measurements and
the results are shown here. The particles size indi-
cates a narrow size distribution at 327 nm with PdI
about 0.114, which confirms the formation of uniform
nanoparticles (Fig. 3a). The zeta potential of these
nanoparticles was measured around - 5.22 mV,
which was caused by carboxylate groups in PLGA
and suggests the presence of stable dispersions
(Fig. 3b). However, drug loading inside the structure
does not significantly impact the zeta potential values
of the nanoparticles. The morphology of the
nanoparticles was studied through FESEM analysis
and the results are shown in Fig. 3c. The obtained
13610
Figure 3 a Particle size distribution, b zeta potential, and c FESEM imaging of LEV-PLGA NPs.
particles size from FESEM analysis was around
170 nm, smaller than the size values from DLS mea-
surements. This difference in the size values is due to
the drying of the samples for FESEM experiments
rather than measuring the hydrodynamic diameter in
DLS analysis. The images display the semi-spherical
morphology for the nanoparticles, with no aggrega-
tion among them.
The encapsulation yield and loading capacity of
LEV into the PLGA nanoparticles were measured
around 73% and 8%, respectively.
FTIR spectroscopy was used to determine the
loading of the drug into the nanoparticles and the
results are shown in the Fig. 4a. Main peaks of PLGA
were ascribed to C–O and C=O stretching modes at
1084 and 1749 cm-1. On the other hand, LEV showed
the characteristic peaks of acidic C=O, carbonyl C=O
and acidic C–O stretching modes at 1720, 1615 and
1286 cm-1, and there is a peak at 1440 cm-1, which is
related to the C-H aromatic bonds. The appearance of
the new peak at 1617 cm-1 for LEV-PLGA NPs,
which is attributed to the carbonyl C=O stretching
mode of LEV and decreasing in the intensities of
PLGA main peaks at 1084 and 1749 cm-1 in the LEV-
PLGA NPs, can confirm the successfully loading of
LEV into the PLGA nanoparticles [52].
Moreover, DSC analysis was performed to confirm
the loading of LEV into the PLGA nanoparticles. The
J Mater Sci (2022) 57:13603–13619
results are shown in Fig. 4b. The sharp endothermic
peak around 130° C is attributed to the melting point
of LEV. The glass transition peak for PLGA is pre-
sented at around 60° C, which shows this polymer’s
amorphous characteristic. The same peaks can be
seen in the thermogram attributed to the physical
mixture of LEV and PLGA. However, the absence of
LEV melting peak in the LEV-PLGA NPs thermo-
gram suggests the amorphous and non-crystalline
structure of LEV in the LEV-PLGA NPs, together
with homogenous dispersion of the drug into the
PLGA nanoparticles. Furthermore, the shifting of the
PLGA peak in the LEV-PLGA NPs, toward the lower
temperature was due to the LEV loading into the
PLGA nanoparticles.
Characterization of PLGA microspheres
The particle size and morphology of the micro-
spheres were analyzed through SEM imaging and the
results are shown in Fig. 5. The microspheres show
the semi-spherical morphology with a porous surface
and the size was measured around 200–300 lm.
Loading of Sr into the PLGA microspheres was
studied through ICP-OES analysis and the Sr loading
amount was measured around 4.5%.
J Mater Sci (2022) 57:13603–13619 13611

(a) (b)

Exo
PLGA-LEV NPs
1617 PLGA- LEV
C=O,
PLGA
carbonyl
Transmittance, %

LEV

Heat flow, mW
1720
C=O, acidic 1286
C-O, acidic
1615 1440
C=O, carbonyl C-H

LEV-PLGA NPs
LEV 1749 1084
PLGA NPs C=O C-O

3500 3000 2500 2000 1500 1000 0 20 40 60 80 100 120 140

Wavenumber, cm-1 Temperature, °C

Figure 4 a FTIR spectra and b DSC thermograms of PLGA NPs, LEV and LEV-PLGA NPs.

Mechanical property is another essential factor in

bone scaffolding, divided into two categories (ac-
cording to the tissue type), such as cortical and can-
cellous [53]. Scaffolds originating from natural
polymers have poor mechanical properties compared
to synthetic ones. The mechanical properties of
GelMA and GelMA-gelatin-alginate hydrogel scaf-
folds were determined under compression test and
compression modulus for these scaffolds were
obtained around 0.047 and 0.1 MPa, respectively
(Fig. 6c). These results show that the mechanical
properties of the GelMA scaffold were improved by
300µm adding gelatin and alginate into the GelMA hydrogel
(p-value \ 0.01). As alginate is a tough polysaccha-
ride, adding this polymer into the structure can
Figure 5 SEM image of PLGA microspheres.
improve the mechanical properties [51].
Morphology and mechanical properties
of hydrogel scaffold Drug release evaluation

One of the essential parameters in bone scaffolding is
having interconnected pores of appropriate size.
High surface to volume ratio (high porosity), pres-
ence of the uniform porosity and connection among
them cause the nutrients release, easing the cells
migration and thus accelerating the growth of the
target tissue on the scaffold. So, the morphology of
the GelMA-gelatin-alginate hydrogel scaffold with
and without PLGA microspheres was studied
through SEM imaging. The corresponding images
show the porous structure in both cases, which is
needed for cell growth and migration (Fig. 6a and b).
As osteomyelitis disease, especially the chronic type,
has a prolonged clinical course and long periods of
extinction and recurrence, the design of continuous
and long-term antibiotics release systems is crucial in
treating this disease. So, the LEV release behavior
from PLGA NPs and GelMA-gelatin-alginate hydro-
gel scaffold were determined on different days in
saline phosphate medium (pH = 7.4). The results are
shown in Fig. 7. It can be seen that the LEV release
profile from PLGA nanoparticles is slow and steady
and the released amount was calculated about 73%
after 25 days, which was due to the drug encapsu-
lation inside the structure and hydrophobic
13612 J Mater Sci (2022) 57:13603–13619

(a) (b)

300µm 300µm

(c) 0.14
**
0.12
Young modulus, MPa

0.10

0.08

0.06

0.04

0.02

0.00
GelMA GelMA/G/A

Figure 6 SEM images of GelMA-gelatin-alginate hydrogel scaffold a without PLGA and b with PLGA microspheres. c GelMA and
GelMA-gelatin-alginate hydrogel scaffolds young’s modulus.

interactions between the drug and PLGA. The
cumulative released LEV amount from GelMA-ge-
latin-alginate hydrogel scaffold was obtained about
64% after 25 days, indicating the reduction in the
initial release, improvement in continuous release
and the duration of LEV release compared to the
PLGA nanoparticles. In general, the results showed
continuous and long-term antibiotic release behavior,
which can be helpful for the treatment of
osteomyelitis. Also GelMA-gelatin-alginate hydrogel
scaffold and PLGA nanoparticles completely degra-
ded after 5 and 9 weeks incubation in the release
medium, respectively.
Antibacterial analysis for GelMA-gelatin-
alginate hydrogel scaffold
Reviewing the literature showed that nanoparticles
and scaffolds have considerable antibacterial prop-
erties toward the important bacteria in osteomyelitis
disease, including S. aureus, E. coli and P. aeruginosa
[54]. The antibacterial activity of the GelMA-gelatin-
alginate hydrogel scaffold was studied by evaluation
of MIC (minimum inhibition concentration) and MBC
(minimum bactericidal concentration) values for LEV
antibiotic and the LEV- PLGA NPs against S. aureus,
E. coli, and P. aeruginosa. The results are shown in
Table 1. The results indicated that in 1, 0.5 and 2 lg/
ml of LEV and 2, 1 and 4 lg/ml of LEV- PLGA NPs,
S. aureus, E. coli, and P. aeruginosa were inhibited,
J Mater Sci (2022) 57:13603–13619 13613

(a) (b)

(c) (d)

Figure 7 LEV release profiles from PLGA and GelMA-gelatin-

alginate hydrogel scaffold.

Table 1 MIC and MBC values for LEV and LEV- PLGA NPs (e) (f )
(lg/ml)

MIC MBC

LEV PLGA-LEV LEV PLGA-LEV

S. aureus 1 2 2 4
E. coli 0.5 1 1 4
P. aeruginosa 2 4 4 8

Figure 8 Disk diffusion images of hydrogel scaffold (control

samples) toward a S. aureus, c P. aeruginosa, e E. coli and with
respectively and these amounts were considered as LEV antibiotic toward b S. aureus, d P. aeruginosa, f E. coli.
MIC values. 100 ll of the non-showed turbidity
content was placed onto the blood agar plates and Table 2 Inhibition disk diameter values of LEV-loaded hydrogel
incubated overnight at 37 °C to understand the MBC by using diffusion technique against S. aureus, E. coli and P.
aeruginosa
values. The findings showed that in 2, 1 and 4 lg/ml
of LEV and 4, 4 and 8 lg/ml of LEV- PLGA NPs, S. Bacteria Inhibition disk diameter (mm)
aureus, E. coli, and P. aeruginosa were sacrificed. These
S. aureus E. coli P. Aeruginosa
results confirm that the MIC and MBC values for
LEV-loaded nanoparticles were higher than the free LEV-loaded hydrogel 37 ± 1 41 ± 2 34 ± 2
LEV. PLGA-LEV nanoparticles had a regulated
release of LEV for up to 4 weeks under conventional
incubation settings and had a MIC and MBC higher
antibacterial behavior. However, the LEV-loaded
than that of free LEV.
hydrogel showed a considerable antibacterial char-
The antibacterial property of the GelMA-gelatin-
acteristic against all three different types of bacteria.
alginate hydrogel scaffold with and without LEV
The most significant inhibition disk is attributed to
antibiotic against S. aureus, E. coli, and P. aeruginosa
E. coli, S. aureus and P. aeruginosa, respectively, cor-
was studied using the disk diffusion technique. The
responding with the related MIC and MBC values.
images related to the drug diffusion and the inhibi-
The obtained results for MIC, MBC and disk diffu-
tion disk diameter toward different bacteria are
sion showed that the nanoparticles and hydrogel
reported in the Fig. 8 and Table 2. The results indi-
scaffold formation techniques did not have any
cated that the un-loaded hydrogel does not have any
adverse effects on the antibacterial characteristics of
13614
the LEV and the antibiotic released from the scaffold
can keep its antibacterial properties.
Cytotoxicity investigation of GelMA-
gelatin-alginate hydrogel scaffold
MTT test is a reliable method to study the cellular
responses and biocompatibility of the GelMA-algi-
nate-gelatin hydrogel scaffolds. The toxicity effect of
the hydrogel scaffold and the scaffold containing
different concentrations of Sr, such as 3 mM, 6 mM,
12 mM, and 24 mM, was studied through MTT
analysis in 7, 14, and 21 days. According to the
standard of biological evaluation of medical devices,
the minimum survival rate approved by the bio-
compatibility of medical devices for laboratory cyto-
toxicity (ISO 10993–5: 2009) is 70%. The obtained
results showed that all the scaffolds used in this
study have an activity of 70%. There was no cyto-
toxicity in the presence of GelMA-alginate-gelatin
hydrogel scaffolds. The biocompatibility of all scaf-
folds in 24 h has been confirmed compared to the
control samples. Also, many studies have shown that
using Sr in low concentrations can cause osteogenesis
by osteoblast cells and simultaneously prevent bone
resorption by osteoclast cells. However, using stron-
tium in high concentrations can decrease cell viabil-
ity, which agrees with the results from
biocompatibility analysis [55]. The cytotoxicity of the
hydrogel scaffolds containing 3, 6, 12, and 24 mM of
Sr on osteoblast cells was obtained around 100, 83, 81,
and 80%, respectively. According to the results, the
hydrogel loaded with 3 mM Sr was selected for ALP
and gene expression tests.
Evaluation of alkaline phosphatase
produced by osteoblast MC3T3 cells
Alkaline phosphatase enzyme is one hydrolyzing
enzyme that can be naturally found in all tissues in
the body, especially the liver, kidneys, and bones
(osteoblasts). In bones, this enzyme is involved in
matrix calcification and protein production. In the
process of matrix bone production by osteoblast cells,
the activity of this enzyme is significantly increased,
so this material can be used as a marker to detect the
osteoblast cells activity in the bone production pro-
cess. Some studies have shown that Sr-loaded scaf-
folds can increase alkaline phosphatase production
by osteoblast cells [55]. The amount of alkaline
J Mater Sci (2022) 57:13603–13619
phosphatase produced by osteoblast MC3T3 cells
was determined using the direct method in 7, 14 and
21 days and the results are shown in Fig. 9. The
produced amount in day 7 did not show any signif-
icant difference for various groups. However, the
hydrogel scaffold with and without Sr indicates dif-
ferent behavior concerning the control sample at day
14 (P \ 0.05 and P \ 0.01, respectively). Furthermore,
the hydrogel scaffold with and without Sr compared
to the control sample and Sr-loaded hydrogel scaffold
and unloaded scaffold reflect different behavior at
day 21 (P \ 0.05, P \ 0.05 and P \ 0.001,
respectively).
Differentiation study of osteoblast MC3T3
cells via RT-PCR analysis
One of the best techniques to evaluate the function of
the cells is gene expression through the PCR method.
RUNX2 is an osteoblast-particular protein gene,
which plays a vital role in osteoblast differentiation,
chondrocyte maturation, bone production and
regeneration. Osteocalcin is a K-vitamin dependant
protein, a bone formation marker and one of the most
common non-collagenous bone proteins. Osteonectin
is a bone matrix protein, which has an important role
in bone matrix mineralization. To understand the
effect of strontium on bone regeneration through RT-
PCR analysis, particular genes from osteoblast cells
such as RUNX2, Osteonectin and Osteocalcin were
used. The gene expression profile for RUNX2 at dif-
ferent times, such as 7, 14 and 21 days, was studied,
80
Control ***
** *
70 GelMA
GelMA-Sr * *
60
ALP U/L Protein
50
40
30
20
10
0
D-7 D-14 D-21
Figure 9 Alkaline phosphatase produced by MC3T3 cells on day
7, 14 and 21.
J Mater Sci (2022) 57:13603–13619 13615

and the results are shown in Fig. 10a. The findings at day 21 (P \ 0.001, P \ 0.01 and P \ 0.001,
show a significant difference for hydrogel scaffold respectively).
with and without Sr compared to the control sample A similar analysis was performed for Osteocalcin
at different periods (P \ 0.05 and P \ 0.01 at day 7, gene at different periods and the results are dis-
P \ 0.05 and P \ 0.001 at day 14, P \ 0.05 and played in Fig. 10c. The gene expression did not show
P \ 0.001 at day 21, respectively). any considerable difference at day 7. However, the
The exact process was done for the Osteonectin gene behavior was different for Sr-loaded hydrogel scaf-
at different periods and the results are displayed in folds with respect to the control sample and scaffold
Fig. 10b. The gene expression did not show any without Sr compared to the control sample (P \ 0.05,
considerable difference at day 7. However, the P \ 0.05, respectively) and at day 21 (P \ 0.001,
behavior was different for hydrogel scaffolds with P \ 0.01, respectively).
and without Sr compared to the control sample and The results indicate that the scaffolds containing Sr
between scaffolds with and without Sr at day 14 have bone regeneration characteristics, which are
(P \ 0.001, P \ 0.05 and P \ 0.001, respectively) and confirmed by increasing RUNX2, Osteonectin and
Osteocalcin into 10, 9, and 10 times with respect to the

(a) (b)
30 30
Control *** Ratio of changes in Osteonectin expression Control
Ratio of changes in RUNX2 expression

GelMA GelMA ***

*
25 GelMA-Sr 25 GelMA-Sr **
***
20 20
***

15 *** 15 *

* **
10 10
**
5 * 5

0 0
D-7 D-14 D-21 D-7 D-14 D-21

(c)
30
Ratio of changes in Osteocalcin expression

Control
GelMA
25 GelMA-Sr

20
***

15 * **

*
10

0
D-7 D-14 D-21

Figure 10 Gene expression of hydrogel scaffold with and without Sr on day 7, 14, and 21 with a RUNX2, b Osteonectin, and
c Osteocalcin.
13616
control sample. Various studies have shown that the
presence of Sr in the scaffolds can cause the incre-
ment in bone-specific gene expression, and thus bone
regeneration [55, 56].
Conclusions
A multi-functionalized drug delivery system based
on biodegradable GelMA scaffold was developed for
osteomyelitis treatment. It was based on GelMA-al-
ginate-gelatin hydrogel containing LEV nanoparticles
for anti-infection and Sr microspheres for bone
regeneration. The morphology of LEV-loaded PLGA
nanoparticles was spherical, and the mean particle
size was approximately 237 nm. The microspheres
had 200–300 lm particle size with an interconnected
porous structure. SEM images confirmed that the -
porous structures of GelMA scaffold allowed MC3T3
cell seeded on the scaffold to migrate. The long-term
antibiotic release was achieved by encapsulation of
LEV into PLGA nanoparticles. Antibacterial charac-
teristics toward the specific bacteria in osteomyelitis
such as S. aureus, E. coli, and P. aeruginosa were 4, 4,
and 8 lg/ml, respectively. The results indicate an
effective antibacterial ability in the final scaffold.
MC3T3 cell proliferation in the Sr loaded hydrogel
was also significantly improved. The MTT and PCR
results indicated the biocompatibility and the osteo-
genesis capability of the fabricated scaffold, which
confirms its impact in drug delivery and
osteomyelitis treatment.
Acknowledgements
This project was sponsored by a grant awarded by
Nanotechnology Research Centre, Tehran University
of Medical Sciences (Grant Number 99341151263).
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