Abstract
Bone defects in patients entail the microenvironment that needs to
boost the functions of stem cells (e.g., proliferation, migration, and
differentiation) while alleviating severe inflammation induced by high
oxidative stress. Biomaterials can help to shift the microenvironment
by regulating these multiple events. Here we report multifunctional
composite hydrogels composed of photo-responsive Gelatin Methacryloyl
(GelMA) and dendrimer (G3)-functionalized nanoceria (G3@nCe).
Incorporation of G3@nCe into GelMA could enhance the mechanical
properties of hydrogels and their enzymatic ability to clear reactive
oxygen species (ROS). The G3@nCe/GelMA hydrogels supported the focal
adhesion of mesenchymal stem cells (MSCs) and further increased their
proliferation and migration ability (vs. pristine GelMA and nCe/GelMA).
Moreover, the osteogenic differentiation of MSCs was significantly
stimulated upon the G3@nCe/GelMA hydrogels. Importantly, the capacity
of G3@nCe/GelMA hydrogels to scavenge extracellular ROS enabled MSCs to
survive against H[2]O[2]-induced high oxidative stress. Transcriptome
analysis by RNA sequencing identified the genes upregulated and the
signalling pathways activated by G3@nCe/GelMA that are associated with
cell growth, migration, osteogenesis, and ROS-metabolic process. When
implanted subcutaneously, the hydrogels exhibited excellent tissue
integration with a sign of material degradation while the inflammatory
response was minimal. Furthermore, G3@nCe/GelMA hydrogels demonstrated
effective bone regeneration capacity in a rat critical-sized bone
defect model, possibly due to an orchestrated capacity of enhancing
cell proliferation, motility and osteogenesis while alleviating
oxidative stress.
Keywords: Bone regeneration, Dendrimer, Nanoceria, Hydrogel, Stem cell
activation, ROS scavenging
Graphical abstract
[33]Image 1
[34]Open in a new tab
1. Introduction
Bone defects occasioned by an infection, trauma or enduring
inflammation can result in a serious burden on a patient's physical and
mental health. Repair and regeneration of critical bone defects is a
challenging process that remains a serious clinical task [[35]1,[36]2].
Bone biomaterials are important in bone defects because they function
as a link between native bone tissues, and cells and also steer the
functional regeneration of bone tissues [[37]3]. To facilitate the new
bone formation, tissue engineering based on scaffolds and stem cells
has also been derived as an alternative approach [[38]4,[39]5]. Being
the most favourable biomatrices, hydrogels possess immense scientific
interest. Apart from acting as a cell-friendly microenvironment, the
inner porous morphology of hydrogels maintains and facilitates nutrient
and gas exchange which supports endogenous cell growth and could act as
a delivery platform for the controlled release of several bioactive
molecules [[40][6], [41][7], [42][8]]. Naturally derived gelatin-based
Gelatin Methacryloyl (GelMA) hydrogel is one such candidate widely used
in various tissue regeneration applications due to its versatile cell
supportiveness, controllable properties, and enzymatic disintegration
[[43][9], [44][10], [45][11]]. Although GelMA hydrogels have likenesses
with native bone extracellular matrix (ECM), they lack osteogenic
factors or other inorganic components that could endorse bone
mineralization, hence GelMA hydrogels in their native form are not
suitable for in situ bone regeneration [[46]12]. Moreover, the native
bone comprises of multifaceted structure (micro/nanoscale) which guides
cell growth and differentiation.
Several exertions have been done to modulate the degradation tendency,
and mechanical performance as well as advance biological
functionalities of microscale GelMA, especially by engineering using
bioactive nanoscale materials [[47]12,[48]13]. For example, Z. Wu
et al. engineered GelMA using lithium-modified bioglass for functional
bone regeneration under diabetic conditions [[49]14]. Here GelMA
regulated cellular activities such as cell adhesion and proliferation
while the nanomaterial integrated supported osteogenesis, angiogenesis,
and immunomodulation. Halloysite (HNT)-incorporated GelMA hydrogels
were recently reported by Huang et al. to promote bone regeneration
[[50]15]. The study showed that the HNT-based composite improved bone
cell proliferation and differentiation in vitro and enhanced bone
regeneration in a rat calvarial defect model in vivo. The researchers
attributed the improved bone regeneration to the unique structure and
properties of HNTs, which can serve as bioactive and biocompatible
platforms for engineering bones.
Similarly, Q. Ou and colleagues used nanosilver-incorporated HNT to
engineer GelMA hydrogels to form a hydrogel with superior bioactivity
[[51]16]. Moreover, the incorporation of nanomaterials rendered GelMA
hydrogel with addition properties such as antibacterial activity, and
immunomodulatory property which supported regeneration of infected bone
tissues. In another study, ultrathin nano silicates were incorporated
into GelMA to design a highly stiff GelMA hydrogel which could support
osteogenesis even in the absence of osteoinductive factors due to the
supreme bioactivity of nanomaterial incorporated [[52]17]. In some
cases, GelMA hydrogel has been directly functionalized with drugs such
as alendronate, without the use of nanomaterials, for supporting
osteogenesis. Liu et al. conducted a study using this approach
[[53]18]. Although such hydrogels have bioactive properties due to the
modulation of the physiochemical properties of GelMA, their
applications in bone tissue engineering are limited by factors such as
the need for multiple functionalization steps, concerns over
biodegradability, and high cost. All these studies point out that bone
tissue engineering (BTE) using GelMA hydrogels requires engineered
nanomaterials as functional units, which should greatly improve the
physicochemical properties, osteogenic capacity, biocompatibility etc.,
and renders other superior properties to the hydrogel [[54]12].
While numerous GelMA nanocomposites have shown promise in BTE , many of
them lack ROS scavenging properties, which can restrict their
effectiveness to only osteogenic properties. As a result, it is crucial
to integrate ROS scavenging properties into pristine GelMA, while
maintaining osteogenic potential to enhance their efficiency in
regenerating bone tissues. ROS-responsive GelMA hydrogels can reduce
oxidative stress, providing a better microenvironment for cell
proliferation and differentiation [[55]19]. Over the years, nanoceria
(nCe) incorporated GelMA hydrogels have been extensively studied for
their potential applications in regenerative medicine. The nCe has
emerged as a strong candidate in biomedical research because of its
exceptional multi-enzymatic properties owing to the ability to shift
between oxidation states (Ce^3+ & Ce^4+) by an auto-sequential redox
cycle [[56]20,[57]21]. Since bioactive and biocompatible, it is one of
the potent candidates for handling health conditions related to
oxidative stress and is also known to be efficacious in a wide range of
inflammatory diseases as well as in cancer therapy [[58][21], [59][22],
[60][23]]. Moreover, numerous studies have already demonstrated the
therapeutic potential of nCe in BTE [[61][24], [62][25], [63][26]].
Previous studies using nCe and GelMA have primarily relied on the
intrinsic antioxidant property of nCe to achieve targeted regenerative
potential, especially for soft tissues like skin [[64]27,[65]28].
However, nCe alone may not provide the necessary microenvironmental
cues when it comes to hard tissues like bones. In recent
years,bioinspired materials based on dendrimers also have lately
attracted extensive scientific interest because of their unique
biocompatibility, chelation with proteins, and provision of a cell
microenvironment that benefits tissue regeneration [[66][29], [67][30],
[68][31]]. Dendrimer-engineered nanomaterials are very desirable to be
deployed in the development of functional biomaterials for bone
regeneration because of their highly defined chemical structure in
nanometer dimension and globular form [[69]32,[70]33].
To address the existing limitations, we propose an innovative approach
to develop a hybrid nanomaterial based on nCe, which is surface
functionalized with macromolecular structures called dendrimers (G3).
For this, first, we synthesized nCe and functionalized it with 3rd
generation polyamidoamine (PAMAM) dendrimer to generate G3@nCe with nCe
as the core and amine groups (-NH[2]) from G3 as the periphery
[[71]34,[72]35]. The resulting G3@nCe retains the intrinsic properties
of nCe, including ROS responsiveness, crystallinity, and stability,
while also providing additional features to enhance bioactivity. The
combination of superior bioactivity and antioxidant property of G3@nCe
can lead to synergistic effects, where the properties of each component
enhance the properties of the other. When incorporated into the GelMA
matrix by photogelation, G3@nCe led to enhanced interfacial
interactions and stability of resultant hydrogels characterized by
improved mechanical properties and swelling kinetics, because of their
cationic nature and symmetrical geometry. The immobilization capacity
of dendrimers enables G3@nCe to function as a “nano-reservoir” that can
immobilize and release a range of bioactive molecules (including growth
factors and ECM proteins) in a controlled and sustained manner,
providing superior bioactivity compared to nCe. Moreover, their unique
architecture allows for high surface area which can enhance the
stability and activity of immobilized proteins. Also due to the
cationic nature, dendrimers interact with negatively charged cell
membranes, enhancing their cellular uptake. The increased cellular
uptake of G3@nCe leads to its better distribution and localization
within the targeted cells or tissues.
For this reason, G3@nCe/GelMA served as a bioactive platform which
offers superior microenvironmental cues for modulating cellular
responses (proliferation, migration, and osteogenic differentiation)
compared to pristine GelMA and nCe/GelMA. G3@nCe/GelMA also has
antioxidant properties which benefit cells under oxidative stress. The
transcriptome analysis by RNA sequencing revealed that G3@nCe/GelMA
upregulated various genes related to cell growth, migration,
osteogenesis, and ROS metabolic process and revealed the involvement of
the canonical Wnt signalling pathway in stimulating osteogenesis. We
believe that the design of G3@nCe/GelMA hydrogel that can successfully
orchestrate cellular responses will meet multiple requirements to
ensure the successful restoration of bone defects and could provide
insightful ideas for developing future bone biomaterials. In addition,
our findings suggest that this approach to engineer GelMA hydrogels
using dendrimer-functionalized nanomaterials is not only universal but
also can be extended to other tissue platforms, providing a unique
opportunity to impart new biological properties and functions to
regenerate diseased and injured tissues.
2. Experimental section
2.1. Synthesis of GelMA
GelMA was made according to previous reports with slight modifications
[[73]36]. Briefly, a 10% (w/v) uniform gelatin solution was made by
dissolving gelatin (Bovine skin type B, Bloom 300, Sigma Aldrich) in
distilled water (DW) at 50°C. Then gradually add 0.6 ml of methacrylic
anhydride (MA, 94%, Sigma Aldrich) per gram of gelatin and continue
stirring for 1 h. This mixture was then centrifugated at 3500 rpm for
2 min and purified by dialyzing against warm DW for 7 days using
12–14 kDa tubular dialysis membrane (CelluSep, Regenerated Cellulose
Tubular Membrane (T4) MFPI). The pH of the solution was then adjusted
to 7.4 using 1 molar (1 M) sodium bicarbonate (NaHCO[3], Sigma
Aldrich) before being snap-frozen in liquid nitrogen and lyophilized
for 1 week (ilshin Lab Co. Ltd, Korea). The freeze-dried GelMA
prepolymer was stored at −20°C before use. Further characterization of
GelMA was done using Fourier transform infrared spectroscopy (FTIR,
Varian 640-IR, Australia). The degree of methacrylation (DOM) was found
by comparing the free –NH[2] groups in GelMA before and after
functionalization using ninhydrin assay following a slightly modified
protocol [[74]36]. For this gelatin and GelMA solutions were made in DW
at 50°C until the solutions become clear. Ninhydrin reagent (Sigma
Aldrich) was made in sodium citrate monobasic (Sigma Aldrich) or
glycerol (Sigma Aldrich) mixture and made into a final concentration of
2.5 mg/ml. Then 50 μl of gelatin or GelMA was mixed with 950 μl of
ninhydrin in an Eppendorf tube and warmed in a water bath for 15 min.
DW is used as the blank solution. After 45 min of cooling, the
absorbance was measured at 570 nm with a plate reader (Molecular
Devices, USA). A linear regression line was plotted from the gelatin
dilution series using the average absorbance value. The absorbance
value at 570 nm corresponds to the concentration of amine groups in
free form. The decrease in free amine concentration is due to the
successful methacryloyl substitution. The average absorbance value of
GelMA corresponds to A% gelatin concentration on the standard curve.
Then the DOM was calculated as (100-A) %.
2.2. Synthesis of G3@nCe
For the synthesis of G3@nCe, first, nCe was made using a hydrothermal
process with slight modifications from previous reports
[[75]37,[76]38]. In brief, 2.6 g of cerium (III) nitrate hexahydrate
(Ce(NO[3])[3]·6H[2]O, 99%, Sigma Aldrich) was dissolved in 60 ml DW
and pH was adjusted to basic (8.0) using ammonium hydroxide solution
(NH[4]OH, 28.0–30.0%, Sigma Aldrich). Separately 22 mg of
Hexadecyltrimethylammonium bromide (CTAB, 98%, Sigma Aldrich) was
dissolved in DW and used as a surfactant. To obtain hydrothermally
processed nCe, the Teflon vessel was transferred to an autoclave and
thermal-treated at 140°C for 24 h. The unreacted surfactants were
removed completely by repeated washing and drying at 450°C for 3 h.
Before functionalizing with G3, first, the surface of nCe was modified
independently by a carboxylation reaction. For this, a known amount of
nCe was treated with citric acid (99.5%, Sigma Aldrich). After that,
the pH was adjusted to 5.0 with 1 M sodium hydroxide (NaOH, 97.0%,
Sigma Aldrich). The obtained carboxylated nCe were thoroughly washed
using DW and dried in a freeze-dryer. For G3 conjugation, a calculated
amount of carboxylated nCe was dispersed in DW by ultrasonication and
an equimolar mixture of N-(3-dimethylamino-propyl)-N′-ethylcarbodiimide
hydrochloride (EDC, 98% Sigma Aldrich), and N-hydroxysuccinimide (NHS,
98% Sigma Aldrich) was added to the solution by continuous stirring and
allowed stirring for 30 min for activation. Finally, the PAMAM
dendrimer ethylenediamine core, generation 3.0 solution (20 wt% in
methanol, Sigma Aldrich) was gradually added to the mixture and allowed
to stir for 24 h at room temperature (RT). The obtained G3@nCe was
then washed with DW several times to remove impurities, then
freeze-dried and stored in a vacuum desiccator.
2.3. Characterization of G3@nCe
The nanosized G3@nCe was first observed using Transmission electron
microscopy (TEM, JEOL-7100). The characteristic infrared spectra were
then analyzed using FTIR. The X-ray diffraction (XRD) measurements were
performed to analyze the changes in the crystalline structure of nCe
after functionalization using a Rigaku Ultima IV powder diffractometer
with Cu-Kα radiation. The Zeta potential of particles was determined
with the Malvern Zetasizer device (ZEN3600; Malvern). The surface
chemistry of G3@nCe was investigated using X-ray photoelectron
spectroscopy (XPS, ESCA2000, Thermo VG, U.K.). MagicPlot software was
used to deconvolve the peaks of Ce (3d) orbital satellites. Further
experiments were carried out using bovine serum albumin (BSA) as the
model protein to confirm the protein immobilizing capacity of G3@nCe.
For the test, different amounts of BSA were well dispersed in 1 ml of
PBS along with a calculated amount of G3@nCe by sonication. After that,
the mixture was placed in a 37°C water bath for 24 h. The
nanoparticle-BSA dispersion was centrifuged at 15,000 rpm for 5 min,
and the absorbance values of BSA in the supernatant at an absorbance
maximum of 280 nm were measured using a UV–Vis spectrophotometer
(Biochrom UV libra S22), and the corresponding loading quantity was
determined using the BSA standard curve.
The antioxidant effects of synthesized G3@nCe were then confirmed using
various assays. First, the peroxidase-mimic catalytic activity was
investigated using the redox chemistry between
3,3′,5,5′-tetramethylbenzidine (TMB, ≥99%, Sigma Aldrich) and hydrogen
peroxide (H[2]O[2]; 30 wt% in H[2]O, Sigma Aldrich). The TMB solution
was prepared using acetate buffer solution (pH 4.1) and added with
1 mM H[2]O[2]. A known volume of G3@nCe at various concentrations was
added to the prepared TMB solution and incubated for 30 min at RT.
Following that, a broad wavelength scan of UV–vis spectroscopy (Cary
Varian UV, USA) was used to record the representative absorbance peak
at 652 nm. The superoxide anion (O[2]^.) radical scavenging capacity
was determined using the superoxide dismutase (SOD) assay kit (Cell
Biolabs, Inc.). Following the manufacturer's protocol, a specific
volume of various concentrations of G3@nCe was added to the working
solution for 1 h at 37°C. The absorbance of the solution was measured
at 490 nm after 1 h using a microplate reader (Varioskan LUX, Thermo
Scientific). The SOD inhibition percentage was calculated according to
the protocol. The ability of G3@nCe to bleach the stable
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used to assess its
DPPH radical scavenging capacity (DPPH Assay kit, Dojindo). According
to the manufacturer's protocol, a definite volume of various
concentrations of G3@nCe was incubated with a working solution for
30 min in the dark at 25°C. After 30 min, the absorbance of the
mixture was measured using a microplate reader at 517 nm. The
inhibition percentage of DPPH was calculated by following the protocol.
The autocatalytic or self-regeneration property of G3@nCe was evaluated
based on the shift in the UV spectrum of G3@nCe under continuous
exposure to H[2]O[2]. An equimolar amount of H[2]O[2] and G3@nCe were
used for the tests. The UV absorbance curves were plotted before and
after adding H[2]O[2]. After adding H[2]O[2], G3@nCe was kept under
dark conditions and the UV absorbance curves were observed on the 7th
and 14th days. The spectrum shifts from lower to higher value over time
confirms the autocatalytic property of G3@nCe.
2.4. Fabrication of G3@nCe/GelMA hydrogel
The G3@nCe/GelMA hydrogels were fabricated following a UV
polymerization. Specifically, 10% GelMA (w/v) was uniformly mixed in DW
and added with 0.5% (w/v) of photoinitiator
2-hydroxy-1-[4-(2-hydroxyethoxy)- phenyl]-2-methyl-1-propanone
(Irgacure-2959, 98% Sigma Aldrich) which is dissolved in warm DW
beforehand. G3@nCe was then mixed with the GelMA prepolymer solution at
a final concentration of 500 μg/ml with proper sonication to prevent
particle agglomeration. The mixture was then transferred to a
rectangular-shaped Teflon mould and crosslinked using a UV light
(Omnicure S2000, Lumen Dynamics, Canada) for 5 min. The resultant
hydrogels were punched out from the mould using a biopsy punch and kept
in PBS for further material characterization. Hydrogels intended for
cell culture were directly formed inside cell culture plates under
sterile conditions and were additionally incubated in PBS and growth
media to remove the unreacted photo-initiator. For in vivo studies the
hydrogel samples were formed in sterile Teflon blocks under a clean
bench and punched to a suitable size using a biopsy punch. The punched
hydrogel discs are then washed gently with PBS and kept in sterile
conditions until implantation.
2.5. Characterization of G3@nCe/GelMA hydrogel
Firstly, we examined the microarchitecture and element composition of
G3@nCe/GelMA hydrogels using a scanning electron microscope (JEOL-SEM
3000, Hitachi, Japan) at a 10 keV operating voltage and an ultradry
EDS detector to observe the porous morphology and chemical composition
(Thermo Fisher, USA). The hydrogels were freeze-dried and
frozen-fractured in liquid nitrogen for this purpose, and the
cross-sections were observed after sputter-coating with Pt (IB-3 Eiko,
Japan). Next, the stress versus strain curves and respective
compressive modulus values of G3@nCe/GelMA hydrogels were also
measured. A single-column testing system was also used to measure the
results (3344, Instron, USA). Each hydrogel sample was subjected to a
uniform compression load (10 mm diameter x 4 mm height) at a constant
strain of 15% and a deformation rate of 0.1 mm/min using a static load
of 10 N.. To evaluate the swelling performance of hydrogel constructs,
disc-shaped hydrogel constructs (10 mm × 4 mm) were prepared and
directly soaked in DW, 1X PBS, and minimum essential medium (αMEM) as
growth medium before being incubated at 37°C in an incubator for 24 h.
Later constructs were gently blotted with KimWipe to remove residual
solutions, and weight values were recorded. The swollen constructs were
then lyophilized after being frozen at −80°C. The hydrogel swelling
ratio was calculated using the formula:
[MATH: Percentswelling=Wswollen-Wdry/Wdry×100% :MATH]
where W[s][wollen] and W[d][ry] are the mass of swollen and dry
hydrogels respectively. Next, the hydroxyapatite-forming ability of
G3@nCe/GelMA hydrogels was tested using simulated body fluid (2x SBF)
at 37°C. All the required chemicals used for preparing SBF were
obtained from Sigma Aldrich and used directly without additional
purification. For mimicking acellular mineralization, the fabricated
hydrogel constructs (10 mm × 4 mm) were immersed at 37°C for 3
days. The apatite-like granule formation on hydrogel constructs was
then observed using SEM (JEOL JSM 6510, Japan).
2.6. Adhesion, proliferation, and migration of rMSCs
For in vitro studies, rat bone marrow mesenchymal stem cells (rMSCs)
were harvested and maintained according to the procedures described in
a previous study [[77]39]. Cells sustained at 4−5 passages were used
for all experiments. The optimal G3@nCe/GelMA hydrogels needed for in
vitro studies were first chosen by performing a cytotoxicity check
using cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Inc.)
by culturing 2 × 10^4 rMSCs on top of various hydrogels using α-MEM
media (HyClone, USA) supplemented with 10% fetal bovine serum (FBS,
Corning, USA) and 1% penicillin/streptomycin antibiotic (PS, Gibco,
USA) for 24 h. The early responses of rMSCs with the hydrogels were
assessed by analyzing the expression of focal adhesion protein
vinculin. For this study, 1 × 10^4 rMSCs were seeded on hydrogel
samples in 24-well plates and cells were fixed with 4% paraformaldehyde
solution (PFA, Tech & Innovation, South Korea) after 6 h of culture,
followed by permeabilization of cell membrane with 0.05% Triton-X
(Sigma Aldrich) for 10 min and blocking with 1% BSA (Solmate) for
30 min at RT. Each step involved twice washing samples using phosphate
buffer solution (PBS, Tech and Innovation, Korea). Finally, the cells
are marked with anti-vinculin (Abcam, USA) primary antibody and stained
using TRITC-conjugated secondary antibody (Santacruz, USA), followed by
staining AlexaFluor-488 conjugated Phalloidin for cytoskeleton and
4,6-diamidino-2-phenylindole (DAPI, Thermofisher, USA) for nuclei.
Cells were imaged using a confocal laser scanning microscope (ZEISS-LSM
700, Germany).
After confirming the focal adhesion, the proliferation of rMSC on
hydrogel samples was evaluated at different time points. For this,
hydrogel samples were first made inside 24 well plates and 1 × 10^4
rMSCs were seeded on each sample and cultured for up to 5 days. At
different culturing times, the proliferation of cells was measured
using CCK-8 by measuring mean optical density (OD) at 450 nm using a
microplate reader (Molecular Devices). The cell morphology was then
examined using a fluorescence microscope (Olympus IX71, Japan) by
staining the cytoskeleton with Phalloidin and the nuclei with DAPI. To
confirm the rMSC proliferation, immunofluorescence of the Ki67
proliferation marker was performed. For this brief, 3 × 10^4 rMSCs
were cultured with hydrogels in 24-well plates for 24 h. Following
washing and fixing, cells were stained with rabbit anti-human Ki67
(Abcam, USA) overnight at 4°C and then with goat anti-rabbit Alexa 488
(Abcam, USA) at RT for 2 h. The nuclei were stained with DAPI, the
cell nuclei were detected using a fluorescence microscope, and the
number of Ki67-positive cells was quantified using ImageJ. For
confirming the directional behaviour of rMSCs under the influence of
G3@nCe/GelMA, an in vitro wound healing assay was performed. Initially,
a 2-well silicone insert (Ibidi) is attached inside a 24-well plate and
a 70 μl suspension of 3 × 10^5 cells was seeded to attain a
confluent layer of rMSC after 24 h. Then carefully remove the insert
using forceps and washed with PBS to remove the debris. The complete
media is then replaced by serum-free media and culture with hydrogels
for up to 24 h. After live staining at different time points, the
fluorescent images were obtained and quantified the wound closure by
using ImageJ. All experiments were performed in triplicate.
2.7. Osteogenic differentiation of rMSCs
To assess the role of G3@nCe/GelMA in promoting osteogenesis, we
performed osteogenic differentiation of rMSCs and their relative gene
expressions were analyzed using quantitative real-time polymerase chain
reaction (qRT-PCR). A suspension of 5 × 10^4 rMSCs was seeded on each
sample and cultured with differentiation media (DM) composed of αMEM,
100 nM dexamethasone, 10 mM glycerophosphate, and 50 g/ml ascorbic
acid for 3 and 7 days. After extracting the differentiated cells from
hydrogels, Total mRNA was collected with a Ribospin kit (GeneAll,
Korea) following the manufacturer's protocol. First, the cDNA was
synthesized using AccuPower PCR premix (Bioneer, Korea) and the reverse
transcription was executed with the help of a thermal cycler (HID
Veriti Thermal Cycler, Applied Biosystems). For qRT-PCR, SensiMi SYBR
Hi-ROX kit (Bioline) with added MgCl[2] (Bioline) was used and the
reaction was executed using StepOne plus software (Applied Biosystems).
The change in the fold of the osteogenic genes was determined by the
comparative Ct method (2^−ΔΔCt) and normalized to GAPDH (housekeeping
gene). The expression of bone-associated genes such as collagen type I
(Col-I), Runt-related transcription factor-2 (RunX-2), alkaline
phosphatase (ALP) and osteopontin (OPN) was confirmed in this way. The
primers used are listed in [78]Table S1.
Following qRT-PCR, rMSC differentiation and mineralization were
confirmed using alkaline phosphatase (ALP) and alizarin red staining
(ARS). For this rMSCs were seeded on hydrogels at a density of
3 × 10^4 cells per well and cultured using DM and staining was
conducted on the 7th and 14th day. The cells were washed with PBS and
fixed with 4% PFA for 30 min at RT before using an ALP staining kit
(Sigma, FAST BCIP/NBT tablet) according to the manufacturer's
instructions. The fixed cells were stained with ALP solution for 1 h
at 37°C. The optical images were obtained after removing the excess
stain. For ARS, the fixed cells were stained at RT for 2 h using 1%
alizarin-red solution (Sigma Aldrich) with a pH of 4.2. The cells were
then thoroughly washed with PBS and dried at RT. For quantification,
the calcium deposits on hydrogels from the 14th day of osteogenic
differentiation were dissolved in 10% cetylpyridinium chloride (CPC,
Sigma Aldrich), and then the mean OD value of the solution was measured
at 562 nm using an iMark microplate reader (Bio-Rad, USA). Next, the
expression of RunX2 was analyzed by immunostaining on the 7th day of
osteogenic differentiation. For this, cells were first fixed with 4%
PFA and marked with RunX2 antibody (Santacruz, USA) at 4°C overnight
and then further stained with TRITC-conjugated secondary antibody
(Santacruz, USA) at RT for 2 h. Actin was stained using Phalloidin and
nuclei using DAPI and the relative expression of RunX2 was quantified
using ImageJ.
2.8. Oxidative stress assay and intracellular ROS production
For confirming the ROS scavenging effects of hydrogels under high ROS
conditions, first, we performed an oxidative stress assay using
H[2]O[2]. For this brief, 3 × 10^4 rMSCs were seeded and cultured
with hydrogels. Then, H[2]O[2] of various concentrations (0.1 mM,
0.5 mM and 1 mM) were introduced and cultured for 24 h to challenge
the rMSCs under a pathologic oxidative stress microenvironment. Then
CCK-8 assay was performed to quantify the survival of rMSCs. Following
this, the live and dead cells were marked using a calcein AM and
ethidium homodimer-1-based kit (Thermo Fisher Scientific, USA).
Further, the intracellular ROS levels in rMSC under oxidative stress
conditions were then analyzed using the Image-iT live green ROS
detection kit (Invitrogen, USA). For this, briefly, 3 × 10^4 cells
per well were first seeded and cultured with hydrogels for 24 h. After
aspirating media and hydrogels, high oxidative stress conditions were
enabled by treating 0.1 mM H[2]O[2] for 3 h. After carefully
removing H[2]O[2], cells were gently washed with warm Hanks Balanced
Salt Solution (HBSS with Ca/Mg, Welgene) and labelled with enough
25 μM carboxy-2′,7′-dichlorodihydrofluorescein diacetate
(Carboxy-H[2]DCFDA) working solution at 37°C in dark for 30 min. The
mean fluorescence values were then calculated using a plate reader (Ex.
495 nm; Em. 529 nm). For imaging, the fluorescently stained samples
were mounted in warm HBSS and imaged immediately. For performing and
confirming osteogenic differentiation of rMSC on G3@nCe/GelMA hydrogels
under ROS conditions, the same steps were followed as mentioned in the
previous section with an additional induction of oxidative stress
conditions using 0.05 mM H[2]O[2] during differentiation.
2.9. QuantSeq 3′ mRNA-sequencing and data analysis
For QuantSeq 3′ mRNA sequencing the 5 × 10^4 rMSCs were cultured on
hydrogels for 24 h afterwards the total RNA was collected using
Ribospin-II (Geneall, Korea) based on manufacturers' instructions. The
Agilent TapeStation 4000 system (Agilent Technologies) was used to
evaluate the quality of isolated RNA, and the ND-2000 Spectrophotometer
(Thermo Inc., USA) was used to quantify the RNA amount. The QuantSeq
3′mRNA-Seq Library Prep Kit (Lexogen, Inc.) was used to build libraries
for samples. After total RNA preparation, reverse transcription was
carried out using an oligo-dT primer that had an Illumina-compatible
sequence at its 5′ end. A random primer with an Illumina-compatible
linker sequence at its 5′end launched second strand synthesis after the
RNA template had degraded. By using magnetic beads, the double-stranded
library was made completely free of reaction by-products. The entire
adaptor sequences necessary for cluster creation were added to the
library through amplification. Using NextSeq 550 (Illumina, Inc., USA),
single-end 75 sequencing for high-throughput sequencing was carried out
and QuantSeq 3′mRNA-Seq reads were aligned using Bowtie2 analyzing tool
[[79]40]. For aligning to the genome and transcriptome, Bowtie2 indices
were either produced from the representative transcript sequences or
the genome assembly sequence. The transcripts were collated, abundances
calculated, and differential gene expression was discovered using the
alignment file. Using coverage in Bedtools counts from single and
multiple alignments were used to discover which genes were
differentially expressed [[80]41]. The TMM + CPM normalization method
was used with EdgeR within R (R Development Core Team, 2020) using
Bioconductor to process the Read Count data [[81]42]. Gene
classification was based on searches in the Medline and Database for
Annotation, Visualization, and Integrated Discovery (DAVID) databases
respectively [[82]43]. The Excel-based differentially expressed gene
analysis tool (ExDEGA, ExDEGA Graphic Plus, Ebiogen Inc., Korea) was
used for data analysis and visualization. The transcription factor
enrichment analysis (TFEA) was done by orthogonal omics integration
using ChIP-X Enrichment Analysis Version 3 (ChEA3) [[83]44] based on
the submitted gene sets and the top 15 enriched transcription factors
(TF) were considered.
2.10. Biocompatibility of G3@nCe/GelMA hydrogel
The in vivo biocompatibility of G3@nCe/GelMA hydrogel was evaluated by
performing blood and tissue compatibility studies using SD rats. The
Animal Care and Use Committee at Dankook University, Republic of Korea
approved all animal experiments (Approval no: DKU-18-032). First, the
blood compatibility of hydrogels was analyzed using an in vitro
haemolysis assay. For this, blood was collected from male Sprague
Dawley (SD) rats (5 weeks old) in a sampling tube without coagulation.
Then red blood cells (RBC) were obtained by centrifuging whole blood at
3000 rpm for 5 min and repeatedly washing with PBS 3 times. The pure
RBCs obtained were diluted to 5% (v/v). Then 500 μl of hydrogel and
500 μl of 5% RBCs were added into a 2 ml mini centrifuge tube and
mixed uniformly and kept at 37°C for 1 h. Then the samples are
centrifuged at 3000 rpm for 10 min. A microplate reader was used to
measure the absorbance of the clear supernatant at 540 nm. The
negative control is PBS, and the positive control is 0.1% Triton-X. All
tests were performed three times, and the percentage of hemolysis was
calculated using the formula:
[MATH:
Haemolysis(%)=AS-AP/AT-AP×100% :MATH]
where A[S][,] A[P], A[T] are the absorbance values of the sample, PBS,
and Triton-X respectively.
In vivo, tissue compatibility and immune responses were then assessed
using subcutaneous implantation of hydrogels in male SD rats (5 weeks
old). All rats were first sedated by an intramuscular injection of
Xylazine (10 mg/kg body weight) and Ketamine HCl (80 mg/kg body
weight). Hydrogel constructs of a specific size (10 mm × 3 mm) were
made and sterilized using UV irradiation before use. The dorsal skin
was clean-shaven and sterilized with ethanol/povidone-iodine rub and an
incision (2 cm) was made to form subcutaneous pouches. The
experimental groups were randomly allocated, and the hydrogel
constructs were implanted in each rat (n = 5 per group). Following
implantation, the incision was sutured using a polypropylene suture
(Prolene, B. Braun, Germany). For examining the impact of implantation
in host tissues, rats were sacrificed following 2 and 4 weeks of
implantation and the hydrogel-integrated tissue samples were retrieved.
This time point was chosen for the study because shorter periods of
implantation might not be sufficient to observe the complete range of
tissue reactions and will not provide information about the long-term
biocompatibility of the material. Tissue samples were fixed in neutral
buffered formalin (10% NBF) before being dehydrated in a series of
ethanol solutions. After embedding the samples in paraffin, thin slices
(5 μm) were cut with a microtome (Leica, USA) and stained with
hematoxylin and eosin (H&E) to confirm tissue responses. The
involvement of monocytes/macrophages and host response to implanted
hydrogel constructs at implantation sites was then assessed through
immunohistochemistry (IHC) of CD68 and CD3 (Santacruz, USA). All
samples were imaged with a confocal microscope (CLSM; Zeiss LSM 700,
Germany) and were quantified using ImageJ.
2.11. Implantation of G3@nCe/GelMA hydrogels in critical-sized bone defects
12 weeks aged healthy male SD rats (SJ Bio, Korea) were used for
inducing critical-sized calvaria bone defects in vivo. The Animal Care
and Use Committee at Dankook University, Republic of Korea approved all
animal experiments (Approval no: DKU-18-032). Rats were sustained in an
optimum environment and maintained under sedation throughout surgery by
an intramuscular administration of ketamine and xylazine. The hydrogel
constructs that exactly fit the defects was prepared and sterilized by
UV before use. The dorsal skin above the cranium was clean-shaven and
sterilized with ethanol/povidone-iodine rub. Then, using a surgical
blade, a linear sagittal midline skin cut was made over the skull, and
the incised skin flap was opened to properly locate the defect site.
Then, under sterile saline solution flow, 5 mm diameter critically
sized circular full thickness calvarial bone defects were created at
the centre of each parietal bone by drilling. The experimental groups
were randomly allocated, and 2 hydrogel constructs were implanted per
animal (n = 5 per group). Pristine GelMA hydrogel was used as the
control group. Following implantation, the defects were first sewed
with an absorbable suture (4-0 Vicryl, Germany) inside and then with a
non-absorbable suture (Dafilon, B. Braun, Germany) outside. The animals
were then sustained in separate cages in an optimum environment and
observed to see any signs of inflammation or infection. The animals
were sacrificed 12 weeks after the operation to collect bone samples
from the defect site and surrounding bone.
After being fixed in 10% NBF for 24 h at RT, the specimens were
prepared for micro-computed tomography (μCT), histology, and
immunohistochemistry. The first μCT scan was done to check for neo-bone
formation. A μCT scanning machine was used to scan all the specimens
(Skyscan, Belgium). The reconstructed images were formed and were used
to examine the neo-bone formation over the region of interest such as
volume over total volume (BV/TV) and bone surface density (BSD) using
CTAn Skyscan software. 3D images were produced and visualized using
software (CTvol Skyscan software). The harvested samples were prepared
for histology after μCT analysis. NBF-fixed bone samples were
decalcified (RapidCal, BBC Chemical Co, USA), dehydrated with a series
of ethanol solutions, and then embedded in paraffin for slicing. For
histology, 5 mm thick tissue sections were prepared at the central
region of the circular defects using a microtome (Leica, Germany), and
the tissue slices were stained with H&E staining for new bone formation
and then Masson-Trichome staining (MT) to check collagen deposition.
For IHC, tissue samples were stained with primary antibodies related to
bone formation in vivo (Col-1, OCN, OPN, and CD31) overnight at 4°C,
followed by 2 h of treatment with Alexa Flour 594-conjugated secondary
antibodies at RT. The nuclei were then counterstained with DAPI
(Invitrogen) and images were captured using a confocal laser scanning
microscope. ImageJ was used to quantify relative expressions to compare
groups.
2.12. Statistical analysis
GraphPad Prism software was used to generate all statistical analyses
(version 8.4.3). All data are presented as mean standard deviation
(SD). ANOVA or the Student's t-test were used to assess significant
differences between groups, and any ∗p value less than 0.05 was
considered statistically significant.
3. Results
3.1. Physicochemical characterization of G3@nCe
G3@nCe was synthesized using nCe and PAMAM dendrimer following a
reaction involving carboxylation and EDC-NHS activation as indicated in
([84]Fig. 1a). For this nCe was first synthesized by a hydrothermal
reaction following the previous studies. The TEM images of nCe before
and after functionalization displayed a cube-like morphology
([85]Fig. 1b). The particle size distribution was also calculated based
on the TEM images ([86]Fig. 1c). This variation in particle size
distribution is owing to the surface functionalization of nCe with G3.
It was observed that after the surface functionalization of nCe, its
zeta potential increased significantly from +12.01 mV to +30.8 mV
([87]Fig. 1d). Next, we performed the XPS analysis of G3@nCe to
validate the elemental composition and chemical bonding. [88]Fig. 1e
shows the full-width XPS curves which provide detailed information on
the chemical composition of G3@nCe. Out of the major peaks we focused
on the Ce3d and N1s peaks as it is more relevant to the proposed
nanomaterial. [89]Fig. 1f verified the distribution of Ce^3+ and Ce^4+
valence states in G3@nCe and the percentage of Ce^3+ and Ce^4+ in
G3@nCe was found to be 16.42% and 83.57% respectively ([90]Fig. S1).
[91]Fig. 1g shows the characteristic N1s peak in G3@nCe owing to the
presence of nitrogen-containing NH[2] groups from G3, which is absent
in nCe. The XRD peaks of both nCe and G3@nCe showed similar
characteristic peaks indicating a cubic fluorite phase (JCPDS card no:
81–0792) ([92]Fig. 1h). XRD results also confirm that the surface
functionalization doesn't affect the crystal structure of nCe. The FTIR
spectra showed a characteristic C–N stretching peak in G3@nCe at
1075 cm^−1. Similar peaks were absent in nCe, indicating the existence
of plentiful amine groups in G3@nCe. The characteristic peak
corresponding to Ce–O was observed at 670 cm^−1 ([93]Fig. 1i).
Fig. 1.
[94]Fig. 1
[95]Open in a new tab
Synthesis and characterization of G3@nCe (a) Schematic illustration of
the steps involved in the synthesis of G3@nCe. (b) TEM images showing
the cubical particle morphology. Scale bar: 20 nm. (c) Size
distribution curves obtained from representative TEM images (d) Zeta
potential measurement. (e) Full-width XPS analysis showing
characteristic peaks in G3@nCe. (f) XPS analysis at Ce3d region. (g)
XPS analysis at N1s region. (h) Characterization by XRD. (i) FTIR
spectra of G3@nCe showing characteristic peaks. (j) The protein loading
capacity of G3@nCe evaluated using BSA as a model protein. (k)
Oxidase-like activity of G3@nCe evaluated using TMB assay at 652 nm.
(l) Superoxide radical scavenging assay. (m) DPPH radical scavenging
assay. Data reported as mean ± SD (n = 3).
The delocalization or loading of biomolecules on implantable
biomaterials is crucial for improving their bioactivity
[[96]45,[97]46]. Being abundant in plasma, we used albumin as the model
protein for our protein loading studies. [98]Fig. 1j shows the amount
of BSA loaded by G3@nCe and nCe. The G3@nCe showed a significantly
higher amount of protein loading at all concentrations compared to nCe.
Being highly cationic G3@nCe is expected to retain the attached
proteins while the non-covalently attached proteins may readily desorb
from the surface in the case of nCe [[99]34]. The presence of
detrimental ROS in the tissue microenvironment leads to the impairment
of biological functions and holds a negative role in bone tissue
remodelling. Designing biomatrices with superior ROS scavenging
properties is hence considered to be a good approach to solving such
problems [[100]47]. So next we evaluated the multi-enzymatic properties
of nanoparticles using the oxidase-like activity assay, SOD radical
scavenging assay, DPPH radical scavenging assay ([101]Fig. 1k-m), and
self-regeneration assay ([102]Fig. S2). In all assays, G3@nCe also
exhibited radical scavenging properties like nCe confirming that
surface functionalization does not interfere with the innate ROS
scavenging properties of nCe.
3.2. Physicochemical characterization of G3@nCe/GelMA hydrogel
GelMA was prepared by introducing the methacrylate groups on the
gelatin chain following a chemical reaction between amine functional
groups in gelatin with MA [[103]9,[104]12]. The successful
functionalization was further confirmed by performing FTIR
([105]Fig. S3b) and the DOM is calculated to be 84% by ninhydrin assay
([106]Fig. S3c). Next, we prepared G3@nCe/GelMA hydrogels with a
combination of 10% (w/v) GelMA polymer and 500 μg/ml of G3@nCe
([107]Fig. 2a). This concentration of nanoparticles was chosen for the
study as it is the optimal amount at which cells exhibited no
observable toxicity compared to pristine GelMA ([108]Fig. S4).
[109]Fig. 2b shows the images of G3@nCe/GelMA before and after UV
cross-linking and the images of different hydrogel groups after
photogelation. Following fabrication, the hydrogel samples were
characterized by FTIR ([110]Fig. 2c). The stretching vibrations of
hydroxy groups in GelMA were observed at 3200-3600 cm^−1 and the
stretching bonds of C Created by potrace 1.16, written by Peter
Selinger 2001-2019 O at 1626 cm^−1. N–H deformation bonds appeared at
1242 cm^−1 and 1529 cm^−1. In the case of G3@nCe/GelMA, an additional
peak was observed at 835 cm^−1. Since most of the characteristic peaks
from G3@nCe overlapped with GelMA peaks, it was not easily
distinguishable. Next, we experimented with the mechanical properties
of the G3@nCe/GelMA hydrogel by evaluating the stress versus strain
curves and the compressive modulus.
Fig. 2.
[111]Fig. 2
[112]Open in a new tab
Physicochemical characterization of G3@nCe/GelMA hydrogel. (a)
Graphical illustration showing the photo-encapsulation of G3@nCe into
GelMA hydrogel and existing supramolecular interactions between GelMA
and G3@nCe. (b) Images showing G3@nCe/GelMA before and after
cross-linking and images of different hydrogel groups after
photogelation. (c) FTIR spectra of hydrogels showing characteristic
peaks. (d) Stress versus strain curves of hydrogels under compression.
(e) Compressive modulus measured for various hydrogels. (f) Swelling
behaviour of hydrogels in PBS, DW and growth media. (g) SEM images
showing honeycomb-like morphology by the cross-section of freeze-dried
hydrogels. Scale bar: 100 μm. (h) Elemental mapping of freeze-dried
hydrogel performed using EDS. The presence of ceria detected in both
G3@nCe/GelMA and nCe/GelMA are also shown in the EDS graph. Data
reported as mean ± SD (n = 3; ∗p < 0.05, ∗∗p < 0.01,
∗∗∗p < 0.001).
It is recognized that stem cells tend to differentiate towards
osteogenic lineage under the influence of a stiff microenvironment
[[113]48,[114]49]. Pristine GelMA is known to have inferior mechanical
properties when compared to its composites; therefore improving the
mechanical performances of GelMA by incorporating various nanomaterials
provides it with remarkable properties for bone tissue regeneration
[[115]12]. [116]Fig. 2d displays stress versus strain curves of various
hydrogels under compression mode and the compressive modulus of GelMA,
nCe/GelMA, and G3@nCe/GelMA was observed to be 12.44 kPa, 13.04 kPa
and 16.10 kPa respectively ([117]Fig. 2e). Remarkably, the addition of
nCe didn't much improve the mechanical characteristics of GelMA, while
the addition of G3@nCe resulted in an improved compressive modulus
compared to pristine GelMA. It is expected that the supramolecular
interactions that arise amongst the carboxyl groups in GelMA and
abundant amine groups in G3@nCe are responsible for the better
integration and mechanical stability of hydrogels [[118][50],
[119][51], [120][52], [121][53]]. A. El-Fiqi et al. and X. Ding et al.
also observed similar interactions after incorporating surface-aminated
nanomaterials in biopolymers [[122]54,[123]55]. Following this, we
evaluated the swelling kinetics of hydrogels using 3 different
representative solutions (DW, PBS and Growth Media). An ideal tissue
engineering scaffold should maintain a suitable amount of water to bear
a resemblance to the native microenvironment, which benefits normal
cell functions and tissue metabolism after implantation of the hydrogel
[[124]56]. There were no significant differences in the swelling
kinetics between the GelMA and nCe/GelMA, but there was a decrease in
the swelling behaviour of G3@nCe/GelMA hydrogels in all three solutions
analyzed ([125]Fig. 2f). For implantable hydrogels, this type of
controlled swelling is particularly desired because it prevents the
hydrogel from expanding from the boundaries of the trauma and
separating from the implantation site in clinical applications
[[126]51].
Next, we analyzed the interior morphology of freeze-dried G3@nCe/GelMA
hydrogels using SEM and elemental composition by EDS. The porous nature
of hydrogels is also known to influence cell fate. Hydrogels exhibiting
a porous microstructure facilitate efficient nutrient and fluid
exchange that encourages stem cell adhesion and proliferation
[[127]49]. The uniformly distributed microsized pores in G3@nCe/GelMA
suggest the creation of a porous, interconnected network without any
observable aggregation of nanoparticles ([128]Fig. 2g). However, the
pore size distribution among the hydrogel groups showed no discernible
changes. The EDS analysis of the freeze-dried G3@nCe/GelMA hydrogels
confirmed the successful loading of G3@nCe in the GelMA matrix
([129]Fig. 2h). Together with the morphological analysis and
hygroscopic behaviour of hydrogels, it is expected that GelMA
engineered using G3@nCe could result in bioactive hydrogels that serve
as a promising biomaterial for engineering bones [[130]57,[131]58].
Biomatrices that support bone regeneration must essentially mimic the
composition of bones for better regenerative efficiency [[132]59]. The
hydroxyapatite formation ability of the hydrogels was then evaluated by
incubating hydrogels in SBF for 3 days. SEM images revealed the
formation of spherical nodules of apatite deposited on hydrogel
surfaces ([133]Fig. S5). G3@n Ce/GelMA hydrogels showed relatively
higher deposition of hydroxyapatite among other groups due to the
presence of abundant –NH[2] groups that exert strong electrostatic
interactions with phosphate and calcium ions in SBF and could behave as
nucleation regions for the mineralization of hydroxyapatite nodules
[[134]60,[135]61].
3.3. G3@nCe/GelMA facilitates rMSC adhesion, proliferation, and migration
Cell adhesion is the initial step in cell-hydrogel interaction, where
the cells attach to the substrate through specific binding interactions
between cell-surface receptors and ligands present on the hydrogel
surface [[136]62]. The capability to enhance early cell attachment and
their consequent growth is considered an important prerequisite while
engineering various biomaterials for bone tissue regeneration
[[137]63]. So, we analyzed the initial response of cells on hydrogels
and their focal adhesion was confirmed using immunofluorescence of
vinculin protein at 6 h ([138]Fig. 3a). Hydrogels integrated with
amine-terminated nanomaterials are known to influence initial cell
adhesion and proliferation by interacting with the negatively charged
cell membrane. All such cationic surfaces also functioned as protein
immobilization anchor points, which is also advantageous for cell
migration [[139]64]. After the rMSC has firmly attached to hydrogel,
the proliferation rate was examined by CCK-8 assay for up to 5 days
([140]Fig. 3b). Subsequently, the morphology of proliferating cells was
observed by fluorescence staining. G3@nCe/GelMA showed a significantly
higher number of cells almost confluent over the hydrogels on the 5th
day indicating their cell proliferative effects ([141]Fig. 3c). This
was further confirmed using the immunostaining of the Ki67
proliferation marker. The results indicated that more Ki67-positive
nuclei were observed in the case of G3@nCe/GelMA followed by nCe/GelMA
and GelMA ([142]Fig. S6). It is anticipated that G3@nCe/GelMA hydrogels
deliver the nutrients and growth factors required for cells by
sequestration and sustained release, which stimulates the ECM
remodelling process, leading to the formation of a more favourable
microenvironment for cell proliferation and migration [[143]65].
Fig. 3.
[144]Fig. 3
[145]Open in a new tab
Adhesion, proliferation, and migration of rMSCs promoted by
G3@nCe/GelMA hydrogel. (a) Immunofluorescence images of vinculin
expression in rMSCs at 6 h. Scale bar: 50 μm. (b) The proliferation
of rMSCs evaluated up to 5 days of culture using CCK-8 assay. (c)
Indicative fluorescent images of actin and nuclei of rMSCs after
culturing up to 5 days. Scale bar: 200 μm. (d) Representative images
from the migration assay using rMSCs up to 24 h. Scale bar: 500 μm.
(e) The quantitative analysis of wound closure rate using ImageJ. All
data expressed as mean ± SD (n = 3; ∗p < 0.05, ∗∗p < 0.01,
∗∗∗∗p < 0.0001).
The MSC migration in the initial stage of bone formation is very
crucial because MSCs must first migrate to the bone surface before they
can take part in bone formation. Hence cell migration is another key
event involved in bone formation and bone disease treatment. So, we
experimented with the directional behaviour or migration ability of
rMSCs under the influence of hydrogels using a wound healing assay
([146]Fig. 3d). All hydrogel groups exhibited cell migration over time.
G3@nCe/GelMA hydrogel enhanced rMSC migration profoundly compared to
pristine GelMA and nCe/GelMA at both 12 h and 24 h. We expect that in
addition to the effects of various signalling molecules and ECM
components, the physicochemical cues provided might have also triggered
the stimulation of cell migration by G3@nCe/GelMA. It was also observed
that the migration of cells between nCe/GelMA and pristine GelMA were
also significantly different ([147]Fig. 3e). We believe that because
both nCe and G3@nCe are ROS-responsive, alternate ROS-related
signalling pathways also played a role in the case of nCe/GelMA at
24 h [[148]66]. The differences in hydrogel composition, stiffness,
and the presence of signalling molecules can also play a role in
activating alternative signalling pathways suggesting that the
signalling pathways regulating various cellular processes may be
differentially activated [[149]67]. Overall, our findings suggest that
the G3@nCe/GelMA hydrogels may provide an adequate microenvironment for
cell adhesion, proliferation, and migration, which is beneficial for
bone therapeutics.
3.4. G3@nCe/GelMA promotes osteogenic differentiation of rMSCs
It is a fact that the intrinsic osteogenic function of the majority of
hydrogels employed in tissue regeneration is insufficient, resulting in
a limited therapeutic effect in clinical settings [[150]68,[151]69].
The successful implantation of hydrogels relies on their interaction
with bone tissues. Understanding the molecular and cellular level
events in the immediate microenvironment of bone progenitor cells is
therefore essential. Hence to confirm the favourable osteogenic
activity exhibited by G3@nCe/GelMA hydrogel on rMSCs, we performed
osteogenic differentiation by supplementing DM to cells cultured on
hydrogels. To confirm the differentiation of rMSCs to osteogenic
lineage, the first PCR was performed on days 3 and 7 to estimate the
relative expression of osteogenic genes. The analysis of bone-related
genes such as Runx2, Col-I, ALP and OPN showed higher expression in
G3@nCe/GelMA compared to pristine GelMA and nCe/GelMA. The Runx2 and
Col-I being early markers of osteogenic differentiation, upregulated
during the early timepoint of differentiation, while ALP and OPN
expressed more during the late phase. ([152]Fig. 4a). ALP and ARS
staining, which are key osteogenic markers for osteogenesis of rMSCs
were then carried out at 7 and 14 days of osteogenic differentiation to
further ascertain the potential of G3@nCe/GelMA in promoting bone
formation. G3@nCe/GelMA group possessed the highest ALP activity
compared to nCe/GelMA and pristine GelMA at both 7 and 14 days of
osteogenic differentiation ([153]Fig. 4b). The same trend was observed
in the case of ARS staining also. ARS staining showed mineralized
nodules in all groups whereas denser bright red mineralized nodules
were found in G3@nCe/GelMA ([154]Fig. 4c). The relative quantification
of the mineralized nodules on the 14th day of differentiation using CPC
further confirmed that G3@nCe/GelMA possessed a higher rate of
bio-mineralization suggesting an enhanced osteogenic potential of
G3@nCe/GelMA ([155]Fig. 4d).
Fig. 4.
[156]Fig. 4
[157]Open in a new tab
Osteogenic differentiation of rMSCs promoted by G3@nCe/GelMA hydrogel
(a) Relative gene expression by qRT-PCR for confirming osteogenic
differentiation of rMSCs cultured on G3@nCe/GelMA hydrogels. (b) ALP
activity of rMSCs at 7th and 14th day of differentiation. Scale bar:
200 μm and (c) Mineralization of rMSCs during differentiation observed
by ARS. Scale bar: 200 μm. (d) Colorimetric estimation of
mineralization at the 14th day of differentiation using CPC (562 nm).
All data reported as mean ± SD (n = 3; ∗p < 0.05, ∗∗p < 0.01,
∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).
To further confirm our observation, the expression of RunX2, a crucial
transcription factor that is only expressed in mineralized tissues was
done using immunofluorescence. As expected, the relative expression of
RunX2 was observed to be higher in G3@nCe/GelMA compared to nCe/GelMA
and pristine GelMA groups ([158]Fig. S7). These findings indicate that
G3@nCe/GelMA hydrogel matrix stimulated rMSCs by promoting the
secretion of growth factors and other signalling molecules to
differentiate towards the osteogenic lineage that involves a complex
interplay of microenvironmental cues and signalling pathways. Although
pristine GelMA already has some advantages for providing a suitable
microenvironment for preosteoblast cells, the osteogenic
microenvironment provided by G3@nCe/GelMA must be more effective and
suitable for successful applications in bone therapeutics as they mimic
the micro/nano composition of native bone as well as supports the
dynamic mineralization process of the bone matrix.
3.5. G3@nCe/GelMA rescues rMSCs from H[2]O[2]-induced oxidative stress
As there is an enhanced ROS level in the microenvironment of
critical-sized bone defects, an efficient strategy for bone
regeneration must comprise a fast and responsive elimination of extreme
levels of ROS [[159]70]. Inorganic nanomaterials like nCe are well
known to be ROS-scavenging materials with supreme surface catalytic
activity. The self-regenerative oxidation states of nCe allow them to
behave like biological enzymes, making them one of the versatile
ROS-responsive nanomaterials used in tissue engineering
[[160]22,[161]23]. [162]Fig. 5a illustrates a graphical outline of the
experiment depicting the antioxidant nature of G3@nCe/GelMA under
culture conditions. The in vitro ROS scavenging effects of G3@nCe/GelMA
hydrogel was demonstrated using a cellular oxidative stress method by
treating rMSCs at various concentration of H[2]O[2] (0.1 mM, 0.5 mM,
and 1 mM). G3@nCe/GelMA was then introduced along with several
concentrations of H[2]O[2,] and the survival of cells was quantified by
CCK-8 assay ([163]Fig. 5b) and imaged by Live/Dead staining
([164]Fig. 5c). When initially cells were treated with
0.1 mM H[2]O[2], the number of live cells was significantly
maintained in the case of G3@nCe/GelMA and nCe/GelMA while suppressed
in the case of pristine GelMA. As the concentration was increased to
0.5 mM, cell viability was more repressed and finally at 1 mM, most
of the cells were dead in pristine GelMA, while G3@nCe/GelMA and
nCe/GelMA, still maintained a significant number of viable cells.
Consequently, the intracellular ROS levels in rMSCs after treatment
with hydrogels were detected by fluorescent signals and quantified by
fluorescence measurement. The rMSCs cultured with G3@nCe/GelMA and
nCe/GelMA significantly reduced the ROS levels by scavenging
detrimental ROS induced by H[2]O[2], hence exhibiting weak green
fluorescence, while pristine GelMA lacking antioxidant activity showed
strong green fluorescence signals indicating higher intracellular ROS
levels ([165]Fig. 5d). The intracellular ROS production in rMSCs
quantified using DCF fluorescence also confirms the same
([166]Fig. 5e). These findings also rule out the possibility that
intracellular ROS levels in cells cultured with G3@nCe/GelMA and
nCe/GelMA were comparable due to similar antioxidant properties.
Fig. 5.
[167]Fig. 5
[168]Open in a new tab
Survivability, intracellular ROS production, and osteogenic
differentiation of rMSCs under H[2]O[2]-induced oxidative stress
conditions. (a) A graphical illustration depicting the antioxidant
nature G3@nCe/GelMA under culture conditions. (b) Cell survivability
under the influence of hydrogels measured using CCK-8 assay after
exposing rMSCs to various concentrations of H[2]O[2]. (c)
Representative live/dead images of rMSCs after being exposed to various
concentrations of H[2]O[2]. Scale bar: 100 μm. (d) Intracellular ROS
production observed using fluorescence staining (e) Intracellular ROS
production in rMSCs quantified using DCF fluorescence. Scale bar:
200 μm. (f) The influence of G3@nCe/GelMA in the osteogenic
differentiation of rMSCs under oxidative stress was confirmed using ALP
and ARS. Scale bar: 200 μm. (g) Colourimetric estimation of
mineralization using CPC (562 nm). Data reported as mean ± SD
(n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).
It is also well-known that antioxidant biomaterials are also known for
inducing the differentiation of osteoblast progenitor cells by
modulating oxidative stress [[169]47]. So further we evaluated the
effect of G3@nCe/GelMA on the differentiation of rMSCs to osteogenic
lineage by evaluating the ALP and ARS expression under the oxidative
stress microenvironment. As shown in ([170]Fig. 5f), G3@nCe/GelMA and
nCe/GelMA maintained the osteogenic differentiation capacity of rMSCs
under H[2]O[2]-induced oxidative stress conditions up to 14 days of
differentiation, while pristine GelMA showed reduced differentiation
capacity as evident from fewer ALP positive area and reduced calcium
deposition. [171]Fig. 5g indicates the colourimetric estimation of
mineralization using CPC at 562 nm. Therefore, it is indicated that
H[2]O[2] reduced the differentiation capacity of rMSCs on GelMA while
G3@nCe/GelMA and nCe/GelMA hydrogels improved the process of osteogenic
differentiation by scavenging and neutralizing ROS which is beneficial
for bone regeneration. Overall, the findings suggest that G3@nCe/GelMA
protects preosteoblast cells from oxidative stress injury and is
beneficial for bone formation under harsh conditions.
3.6. QuantSeq 3′ mRNA-sequencing to identify functions and signalling
pathways activated by G3@nCe/GelMA
Transcriptome analysis (a total of 17,048 genes) was carried out to
identify the important signalling pathways and biological or molecular
processes through which G3@nCe/GelMA hydrogel controls cellular
functions and promotes the formation of new bone. [172]Fig. 6a
indicates the distance-based clustering analysis from global
transcriptional changes in GelMA, nCe/GelMA and G3@nCe/GelMA groups
from the combinative comparison. The differentially expressed genes
(DEGs) of rMSCs on different hydrogel groups were broadly detected
between groups (577 upregulated genes in G3@nCe/GelMA vs GelMA, 485
upregulated genes in nCe/GelMA vs GelMA and 216 co-upregulated genes in
G3@nCe/GelMA vs GelMA and nCe/GelMA vs GelMA), as shown in the Venn
diagram ([173]Fig. 6b). The cluster 1 and 2 gene sets were used for
Gene Ontology (GO) and pathway enrichment analysis by DAVID. As an
outcome, the top 30 enriched GO terms, including biological process
(BP), molecular functions (MF), KEGG pathway, Reactome pathway, and
Wikipathway, were displayed ([174]Fig. S8), wherein DEGs of cluster 1,
co-expressed in G3@nCe/GelMA and nCe/GelMA owing to the presence of
ceria, were revealed to be primarily rich in cell proliferation
(mitotic cell cycle, intracellular protein transport, regulation of
mitotic cell cycle, regulation of mitotic nuclear division),
osteogenesis (skeletal system morphogenesis, positive regulation of
canonical Wnt signalling pathway), and ROS-metabolic process (chaperone
binding) terms ([175]Fig. 6c). Functional annotation clustering for
cluster 1 using DAVID analysis were confirmed based on above key
biological terms ([176]Fig. S9). Canonical Wnt signalling is an
important regulatory pathway in the bone formation process, regulating
a variety of biological processes related to stem cell function such as
proliferation, migration, differentiation, and so on [[177]71,[178]72].
The Wnt signalling-related genes like Aspm, Csnk1g1 were significantly
upregulated in G3@nCe/GelMA. Additionally, the transcription factor
enrichment analysis (TFEA) of cluster 1 DEGs using Chip-X enrichment
analysis tool (ENCODE ChipSeq library) also disclosed possible
involvement of Transcription factor 7-like 2 (TCF7L2), a key effector
to promote osteogenesis through canonical Wnt-signalling pathway
[[179]73,[180]74] ([181]Fig. 6d). In-depth mechanistic studies
accounting for the TCF7L2 related Wnt-signalling dependent bone
regenerative potential of G3@nCe/GelMA would be thoroughly explored in
future.
Fig. 6.
[182]Fig. 6
[183]Open in a new tab
Transcriptome analysis reveals the key functions and signalling
pathways activated by G3@nCe/GelMA. (a) The distance-based clustering
analysis from global transcriptional changes in GelMA, nCe/GelMA and
G3@nCe/GelMA groups with 1.5 fold change and over 2 log[2] values from
the combinative comparison. (b) Venn diagram showing the comparison
between differential gene expression across 2 key comparisons
(nCe/GelMA vs GelMA and G3@nCe/GelMA vs GelMA) based on co-up or –down
and contra-regulated genes. (c) Among the top 30 enriched GO or pathway
using up-regulated genes from cluster 1 (mainly due to the presence of
nanoceria) using DAVID (BP, MF, KEGG, Reactome and Wikipathway), major
terms related to cell proliferation, osteogenesis and ROS-related
processes were disclosed. (d) Transcription factor enrichment (TFE) of
cluster 1 was analyzed using ChEA3, a web-based tool indicating the
enrichment of TCF7L2, a key transcription factor involved in Wnt
signalling pathway. (e) Heat map showing DEGs in G3@nCe/GelMA,
nCe/GelMA, and pristine GelMA from specific GO terms related to cell
proliferation, migration, osteogenesis, and ROS-metabolic process.
The individual heat maps from specific GO terms also displayed
differentially expressed genes in all groups; especially those key
genes involved in cell proliferation (Hgf, Csf1, Cited2, Fzd6, Atf2
etc.), osteogenesis (Gli2, Dhx36, Bmpr2, Lef1, Vcan Gpm6b etc.) and
migration (Ttbk2, Rock1, Aspm, Nck2, etc.) were significantly
up-regulated in G3@nCe/GelMA as compared to nCe/GelMA and pristine
GelMA. The ROS-metabolism-related genes such as Nox1, Atp7a were highly
co-upregulated in G3@nCe/GelMA and nCe/GelMA while Mt3, Ucp2, Sod3,
Duoxa1 genes got downregulated, indicating the role of ceria in
regulating ROS that encourages bone regeneration ([184]Fig. 6e). The
entire gene list from heatmap and their respective fold change values
are also given ([185]Fig. S10). When the top 30 enriched GO terms from
cluster 2 were analyzed by the DAVID database, cell proliferation,
osteogenesis, and ROS-related terms were similarly detected owing to
the presence of dendrimer on ceria ([186]Fig. S11). The TFEA of cluster
2 DEGs using Chip-X also further revealed the possible involvement of
TCF7L2 ([187]Fig. S12). Taken all, G3@nCe/GelMA hydrogels would act as
a therapeutic hydrogel that synergistically regulates stem cell
function (proliferation and differentiation) and modulates ROS
processes for efficient bone regeneration.
3.7. Biocompatibility implies the tissue healing capacity of G3@nCe/GelMA
To ensure the safety, biocompatibility, and functionality of an
implantable hydrogel, it may be essential to have a thorough grasp of
how the immune system responds to it. The clinical applications using
conventional hydrogel-based materials are generally limited due to the
reason that they are often not properly integrated into in vivo, which
leads to insistent inflammatory responses in the organism
[[188]6,[189]75]. First, we used an in vitro hemolysis assay to assess
hydrogel biocompatibility. The haemolysis ratio of all hydrogel samples
falls below the critical safe haemolysis ratio for biomaterials,
allowing them to be used in clinical studies ([190]Fig. S13). Further,
we implanted hydrogel constructs in rat subcutaneous sites and examined
the resultant immune responses evoked by them with the local tissues
([191]Fig. 7a). The results revealed that G3@nCe/GelMA hydrogels
displayed tissue compatibility with observance of some residual
hydrogels ([192]Fig. S14). Being highly bioactive and biocompatible we
expect that the residual hydrogels would minimize any potential
negative effects. Histology analyses using high-magnification images
revealed that neither implanted hydrogel elicited significant
inflammatory responses and had less deposition of a fibrous collagenous
capsule, indicating its tissue biocompatibility ([193]Fig. 7b).
Fig. 7.
[194]Fig. 7
[195]Open in a new tab
Subcutaneous implantation of G3@nCe/GelMA hydrogels in vivo. (a)
Schematic illustration showing the steps involved in subcutaneous
implantation of G3@nCe/GelMA hydrogel. (b) Histological analysis of
explanted tissue samples by H&E staining. Scale bar: 100 μm. (c)
Expression of CD68; pan macrophage marker (green) and CD3; pan T-cell
marker (red) in the interface of hydrogel and tissue after 2 weeks of
implantation by immunofluorescence. Scale bar: 100 μm. (d)
Quantitative analysis of CD68 and CD3 expression at 2 weeks. (e)
Relative expression of CD68 (green) and CD3 (red) inflammatory markers
in the interface of hydrogel and tissue after 4 weeks of implantation.
Scale bar: 100 μm. (f) Quantitative analysis of CD68 and CD3
expression at 4 weeks.. All data expressed as mean ± SD (n = 5;
∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (For interpretation of
the references to colour in this figure legend, the reader is referred