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Generation of scaffold incorporated with nanobioglass encapsulated in
chitosan/chondroitin sulfate complex for bone tissue engineering
Bhisham Narayan Singh
a
, Vivek Veeresh
b
, Sarada Prasanna Mallick
c
, Shivam Sinha
b
,
Amit Rastogi
b
, Pradeep Srivastava
a,
⁎
a
School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi 221005, India
b
Department of Orthopedics, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
c
Department of Biotechnology, Koneru Lakshmaiah University, Guntur 522502, India
abstractarticle info
Article history:
Received 11 November 2019
Received in revised form 11 February 2020
Accepted 15 February 2020
Available online 18 February 2020
Over the past decade, various composite materials fabricated using natural or synthetic biopolymers incorpo-
rated with bioceramic have been widely investigated for the regeneration of segmental bone defect. In the pres-
ent study, nano-bioglass incorporated osteoconductive composite scaffolds were fabricated through
polyelectrolyte complexation/phase separation and resuspension of separated complex in gelatin matrix. Devel-
oped scaffoldexhibits controlledbioreactivity, minimize abrupt pH rise (~7.8), optimal swellingbehavior (2.6+–
3.1) and enhances mechanical strength (0.62 ± 0.18 MPa) under wet condition. Moreover, in-vitro cell study
shows that thefabricated scaffoldprovide suitable template for cellularattachment, spreading, biomineralization
and collagen based matrix deposition. Also, the developed scaffold was evaluated for biocompatibility and bone
tissue regeneration potential through implantation in non-union segmental bone defect created in rabbit animal
model. The obtained histological analysis indicates strong potential of the composite scaffold for bone tissue re-
generation, vascularizationand reconstructionof defects. Thus, the developed compositescaffold might be a suit-
able biomaterial for bone tissue engineering applications.
© 2020 Elsevier B.V. All rights reserved.
Keywords:
Chitosan
Bioglass
Bone tissue engineering
Scaffold
1. Introduction
Bone tissues are remodeled continuously throughout the life time
and possess superior regenerative potential [1]. However, large size
bone defect or non-union critical bone fracture requires surgical inter-
vention and mostly autologous bone tissue were used to restore the
bone tissue at the site of critical defect. Autologous bone graft consid-
ered as gold standard treatment method but possess various clinical
challenges such as lack of availability and donor site morbidity [2,3].
Apart from autologous bone graft various metallic implant of stainless
steel, titanium and cobalt-chromium alloys are also used widely. How-
ever, metallic implants possess certain significant limitations such as
stress shielding, repeated surgical interventions and thereby develop-
ment of suitable biodegradable graft is widely considered by various re-
searchers worldwide [3]. Also, in the last few decades demands for bone
restoration materials increases drastically with increment in human
population. Moreover, tissue engineering and biomaterial science is
widely considered to develop regenerative graft for the restoration of
bone defects. Tissue engineering aims to combine biomaterial, cells
and bioactive biomolecules to restore or improve biological functions
of damaged/diseased tissues. In natural tissues, cells were programmed
to interact with protein and other biological molecule of nanoscale
structure associated with extracellular matrix to modulate cellular be-
havior and its development. Thus, nanotechnology together with
other microfabrication technologies facilitates fabrication of tissue sub-
stitutes with nano to micron scale topographical features to influence
wide range of cellular function during tissue regeneration [4,5]. More-
over, surface with nanometer topographical features favors availability
of amino acid and proteins for cell adhesion [6]. Also, bone cells adhe-
sion over composite scaffold/implant can be further enhanced by de-
creasing particle size below 100 nm. In last few decades, novel
technique of siRNA application to accelerate bone regeneration was
also investigated widely and nanotechnology wasused to develop suit-
able vehicle for siRNA delivery to facilitate bone tissue regeneration [7].
Thereby, nanotechnology plays vital role in addressing various issues
associated with development of suitable tissue substitutes with en-
hanced tissue regeneration potential. Various biomaterials including
natural and synthetic biopolymers, bioceramics and various cells
sources such as mesenchymal stem cells, autologous osteoblast etc.
were investigated to develop potential bone tissue graft [8–10]. Al-
though, biomaterials intended to fabricate three dimensional scaffolds
need to be biocompatible, biodegradable, optimal physico-chemical
properties and produces non-toxic degraded product [11]. Among
International Journal of Biological Macromolecules 153 (2020) 1–16
⁎Corresponding author at: IIT BHU, Varanasi, India.
E-mail address: pksrivastava.bce@iitbhu.ac.in (P. Srivastava).
https://doi.org/10.1016/j.ijbiomac.2020.02.173
0141-8130/© 2020 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: http://www.elsevier.com/locate/ijbiomac
various biopolymers, chitosan (CH) is considered as one of the most
suitable biopolymer for biomedical application due to its various advan-
tageous properties such as biocompatibility, minimal inflammatory re-
action, hydrophilicity, positively charged at physiological pH, favors
cell adhesion and wound healing [12,13]. Furthermore, previously it
has been also observed that chitosan based paste containing hydroxy-
apatite possess osteoconductive properties and thus might be suitable
biomaterial for bone tissue graft generation [14]. However, chitosan
has certain limitation such as excessive swelling behavior, poor me-
chanical strength/structural stability in hydrated condition, poor bioac-
tivity and low osteoconductivity [15]. Thereby, chitosan based scaffold
for bone tissue engineering application need to be further modify to
overcome the limitation associated with the chitosan.
The primary concerns associated with chitosan based scaffold is ex-
cessive swelling behavior and poor structural integrity under hydrated
condition and this could be overcome up to certain extent through poly-
electrolyte complexation (PEC) of chitosan with other anionic biopoly-
mer. Also, cationic chitosan can form water insoluble PEC with anionic
biopolymers [16]. Chondroitin sulfate (CS), an anionic polysaccharide
was found to be suitable biomaterial for tissue reconstruction due to
its anti-inflammatory activity and ability to enhance regeneration abil-
ity of bone defect or injury [17,18]. Moreover, CS play important role
in tissue mineralization due to its ability to sequester calcium ion at
mineralization foci as postulated earlier and control crystal growth
while calcification of bone tissue [19]. Thereby, we utilized both chito-
san and chondroitin sulfate to develop PEC, which was further utilized
to fabricate scaffold with controlled swelling behavior. Apart from
PEC, we also used gelatin as one of component to provide structural
framework of the developed scaffold and act as binder to integrate de-
veloped PEC as a solid 3D structure. Gelatin is obtained through the par-
tial hydrolysis of collagen and shows significantly lower
immunogenicity in comparison to collagen. Also, gelatin provides suit-
able RGD motifs for cell adhesion, migration and proliferation [20].
Moreover, gelatin, collagen and collagen with other biopolymers such
as polyglycolic acid, silk etc. were investigated widely and considered
as a suitable biomaterial for tissue engineering applications [21]. How-
ever, polymeric scaffold shows poor mechanical properties and fails to
promote load bearing tissue regeneration. Thus, for an ideal scaffold
for bone tissue engineering applications an inorganic bioactive ceramic
need to be considered for the fabrication of high strength porous com-
posite scaffold with ability to mimic extracellular matrix of bone tissue
and possess ability to support bone tissue regeneration. Among various
bioceramic, nano-bioactive glass (nBG) shows superior bioreactivity
and facilitates hydroxyapatite deposition under physiological condition.
Also, as the bioglass reacts with physiological fluids it leads to the re-
lease of various vital ions which favors bone tissue regeneration and
subsequently enhances vascularization [22]. Previously, we reported
fabrication of CH/CS/nBG based composite scaffold as a filler biomaterial
with limited wet strength (0.28 MPa) for trabecular bone tissue regen-
eration [23]. However, the proposed method allows maximum incorpo-
ration of 8% (w/v) of nBG, as further increment in bioglass limits
chitosan solubility and thereby fails to fabricate scaffold with higher
nBG content. Similarly, chitosan/hydroxypropyl methylcellulose/
bioglass/zinc oxide based composite scaffold was also reported with
compressive strength of 0.45 MPa in dry state [24]. Thus, considering
of biopolymer of natural origin with suitable mechanistic approach
may overcome the limitation such as poor structural or mechanical sta-
bility associated with using naturalbiopolymers. Also, bioglass wasused
widely to fabricate composite scaffolds but higher bioreactivity leads to
the uncontrolled apatite deposition and abrupt rises in surrounding pH
[25]. As, the bioglass come in contact with water or physiological fluid
abrupt release of bioglass ions were occur and affect functioning of
Na/K or Ca ion pump. Suchphenomenon will ultimately leads to protein
denaturation or reduce the cellular metabolic activity or even leads to
cells deatheither through apoptosis or necrosis [26]. Thereby, precondi-
tioning of various bioglass based composite scaffold through various
approaches were suggested such as incubation of scaffolds in culture
medium, buffer, simulated body fluid etc. for certain time period de-
pending upon the composition of the scaffolds. In the present study
we attempt to fabricate novel composite scaffold incorporated with
nBG encapsulated within PEC. Also, combination of polysaccharides
used to fabricate PEC and further binding of PEC with protein to gener-
ate 3D matrix was observed to play vital role in enhancement of struc-
tural, mechanical and biological properties of the composite scaffold.
The developed scaffold favors optimal pH rise of the surrounding envi-
ronment and controlled bioreactivity. Also, the developed scaffolds
were characterized for various properties such as physico-chemical,
mechanical and bioreactivity. The cell supportive properties such as
metabolic activity, viability, cell attachment, spreading, biomineraliza-
tion, collagen type I deposition etc. of the scaffolds were evaluated
through in-vitro cell studies. The acellular scaffold was also evaluated
for biocompatibility and bonetissue regeneration potential through im-
plantation in a non-union segmental defect created in rabbit animal
model.
2. Materials
Ethanol, Sodium carbonate (Na
2
CO
3
), acetic acid (98%), Dimethyl
sulfoxide (DMSO) and Nitric acid were procured from Merck, India. Chi-
tosan (Medium molecular weight, ~90% Deacetylated Degree, Viscosity
(20 °C) 150–500 m·Pas) was purchased from Sisco Research Laborato-
ries Pvt. Ltd. Diamidino-2-phenyindole (DAPI), Dulbecco's Modified
Eagle Medium (DMEM), Phosphate buffer solution, Fetal bovine serum
(FBS), Trypsin (0.25%), Alexa-Fluor 488 conjugated phalloidin, and An-
tibiotic –antimycotic solution were purchased from Invitrogen, USA.
Triethyl phosphate (TEP), Tetraethyl orthosilicate (TEOS), Bovine
serum albumin (BSA), Alizarin red S (ARS) solution, Paraformaldehyde,
Chondroitin-4-sulfate (Molecular weight 5000–50,000 Da), Triton X-
100, 4′6-MTT assay kit, ammonium hydroxide, Cetylpyridinium chlo-
ride (CPC), and Calcium nitrate tetrahydrate (Ca (NO
3
)
2
·4H
2
O) were
purchased from Himedia. SIGMAFAST™p-nitrophenyl phosphate
(pNPP) tablets were purchased from Sigma Aldrich. Collagen type I
mouse monoclonal antibody was procured from Santa Cruz Biotechnol-
ogy. FITC-conjugated secondary antibody was purchased from Abcam.
3. Methods
3.1. Nano-bioactive glass synthesis
Nano-bioactive glass particles were synthesized through acid medi-
ated sol-gel method. Nano-bioactive glass (nBG) with molar composi-
tion of 60% SiO
2
, 36% CaO, and 4% P
2
O
5
was synthesized by dissolving
TEOS (4.53 ml) in water (pH b3) followed by adding 3.002 g of calcium
nitrate tetrahydrate and 0.195 ml of triethyl phosphate. Then, the ob-
tained clear sol was kept in an oven at 60 °C for gelation followed by
aging for next 3 days. Finally, obtained transparent glass was stabilized
through calcinations at 600 °C for 2 h [27,28].
3.2. Generation of composite scaffold
Porous composite scaffold of chitosan (CH, 2% (w/v)), chondroitin
sulfate (CS, 2.5% (w/v)), gelatin (G, 20% (w/v)) and nano-bioactive
glass (nBG, 4% w/v, 8% w/v & 12% w/v) was fabricated through resus-
pension of phase separated polyelectrolyte complex of CH, CS, and
nBG in gelatin followed by freeze drying of the obtained composite so-
lutions.Freeze dried scaffold wascross-linkedfirstly in 3wt% EDC + NHS
[2:1 (w/w)] in [95:5 v/v (Ethanol:water)] solution for 12 h then 0.5%
glutaraldehyde solution was added in 1:1 ratio followed by incubation
for another 12 h. The obtained scaffold was washed repeatedly with dis-
tilled water, dried and used for further studies.
2B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
3.3. Morphological, structural and functional characterization
Field emission scanning electron microscope (FE-SEM, Quanta 200 F,
USA), Transmission electron microscope (TEM-FEI, Tecnai G
2
20 Twin,
Czech Republic) and particle size analyzer (Malvern Zeta sizer) were
used to characterize synthesized nano-bioactive glass and scaffolds.
Moreover, the average pore size was determined from obtained
FESEM images of scaffold using Image J software. At least 25 different
pores were selected from each FESEM images to measure pore size dis-
tribution using Image J tools. For particle size analysis, 0.01 mg of nBG
powder was suspended in 20 ml of water followed by ultrasonication
and analyzed using particle size analyzer. The structural properties of
nBG and scaffolds were evaluated using X-ray diffractometer (XRD,
Rigaku-Ultima IV, USA). Furthermore, nBG and scaffold samples were
mixed with potassium bromide (KBr) and then pelletized using a hy-
draulic press. Finally, pelletized samples were evaluated for functional
characteristic using Fourier transform infrared spectroscopy (FTIR-
8400S, Shimadzu, Japan).
3.4. Evaluation of scaffold porosity, strength, biodegradability and water
uptake behavior
Scaffold porosity was estimated through liquid displacement
method [29].Wherein, small piece of scaffold was immersed into a mea-
suring cylindrical vessel containing absolute ethanol (V
1
). Thereafter,
volume of ethanol containing scaffold in measuring vessel was deter-
mined as V
2
and the volume of solid framework of scaffold was mea-
sured as V
2
−V
1
. Then the scaffold was removed and the residual
volume of ethanol was measured as V
3
. The void volume of scaffold oc-
cupied by absorbed ethanol was determined as V
1
−V
3
. Finally, poros-
ity (ԑ) of the composite scaffold was determined as ԑ=(V
1
−V
3
)/
(V
2
−V
3
).
The compressive strength of hydrated scaffold was determined
using Texture analyser (CT3, Brookfield, USA). Scaffolds were cut in to
cylindrical structure (10 mm height and 10 mm diameter) and soaked
in PBS till the scaffolds were completely hydrated. Then the hydrated
sample was compressed to approximately 40% of their original height
using 100 N load cell withcrosshead speed of 1 mm/min. Finally, the ob-
tained values were represented as mean ± standard error. Water up-
take behavior of scaffold was determined by soaking pre-weighed
(W
d
) sample in PBS for certain time interval. After certain time of
soaking, samples were removed, wiped with tissue paper and weighed
to measure wet weight of the scaffold samples (W
t
). Finally, water up-
take ratio of the scaffold samples were determined by dividing the
weight differences between scaffold sample before and after soaking
in PBS by the original weight of the scaffold sample.
Water uptake ratio ¼Wt−Wd=Wd
½
3.5. In-vitro mineralization
Apatite forming abilities of nanobioglass and composite scaffolds
were evaluated using simulated body fluid (SBF). Simulated body fluid
used for in-vitro mineralization study was prepared according to
method proposed by Kokubo et al. [30]. Simulated body fluid was pre-
pared by dissolving NaCl, NaHCO
3
, KCL, K
2
HPO
4
·3H
2
O, MgCl
2
·6H
2
O,
CaCl
2
,andNa
2
SO
4
in double distilled water. It was then buffered at
pH 7.25 using trishydroxymethyl aminomethane and HCl at 36.5 °C.
Thereafter, nanobioglass powder (0.3 g/200 ml) and scaffold samples
(1 cm
3
/50 ml) were soaked separately in SBF at room temperature for
7 days. After 7 days of SBF treatment nBG and scaffold samples were re-
moved and washed repeatedly with distilled water. Obtained samples
were then washed with ethanol and dried at room temperature. Finally,
samples were analyzed using FESEM, XRD & FTIR to evaluate in-vitro
apatite forming abilities of nBG and scaffolds.
3.6. In-vitro cell study
Biological properties of scaffolds were evaluated using MG-63
human osteoblast cells provided by National Center for Cell Science
(NCCS) India. The obtained cells were cultured and maintained in com-
plete media (DMEM supplemented with 10% FBS and 100 U/ml
penicillin-streptomycin) at 37 °C, 5% CO
2
and 95% humidity. Cultured
cells were detached upon attending around 70% of the confluence
using 0.25% trypsin at 37 °C. The detached cells were resuspended in
completemedia and then centrifuged at 300gfor 10 min. Finally, the ob-
tained cells were used for cell seeding (10
5
cells/cm
2
) over the scaffolds
to evaluate their various cells supportive properties.
3.7. Evaluation of cellular metabolic activity, viability and morphology over
scaffold
Metabolic activity of osteoblast cells over the developed scaffolds
were evaluated using the colorimetric MTT assay. For MTT assay, man-
ufacturer assay protocol was adopted and finally absorbance was re-
corded at 570 nm. Cellular viability was assessed using Calcein AM
and Ethidium homodimer-1 solution. Cultured cell-scaffolds construct
for 24 h were washed twice with PBS and then incubated with live/
dead staining solution for 60 min. Finally, images of stained samples
were collected using fluorescence microscopy (Olympus BX51, Japan).
Cells attachment, spreading and morphological feature over the cul-
tured scaffold fixed with 2.5% glutaraldehyde and dehydrated with se-
ries of ethanol gradient solution was investigated on day 7 and 14
using scanning electron microscopy (EVO|18, ZEISS, USA). Also, stability
of scaffold architecture was investigated after culturing cells for two
weeks over the scaffolds using scanning electron microscopy.
3.8. Evaluation of ALP activity and biomineralization
Alkaline phosphatase activity was evaluated to investigate osteo-
genic activity of cultured cells over the composite scaffold. On day 7
and 14, cultured samples were evaluated using SIGMAFAST™pNPP tab-
let as per the manufacturer protocol and finally, absorbance was re-
corded at 405 nm using spectrophotometer. Biomineralization
potential of scaffold was assessed through Alizarin red S (ARS) assay.
Cultured constructs were washed and then fixed with 2.5% glutaralde-
hyde solution. Fixed samples were washed with PBS and then incubated
in 1 ml alizarin red solution (0.5 g of ARS in 25 ml Milli-Q water,
pH 4.1–4.3) for 1 h at 37 °C, washed repeatedly with distilled water
and images were collected using inverted phase contrast microscopy.
Quantitative evaluation of deposited apatite like calcium nodules was
done using cetylpyridinium chloride (CPC). The ARS stained samples
were incubated in 500 μl CPC solution for 1 h. Thereafter, CPC solution
containing ARS stain desorbed from stained samples was aspirated
and absorbance was measured at 550 nm using spectrophotometer.
3.9. Evaluation of cellular growth and expression of collagen type I
The osteoblast activity was evaluated through immunocytochemis-
try against expression of collagen type I. The cell-scaffold construct cul-
tured for two weeks were collected and fixed with 4%
paraformaldehyde solution for 15 min, washed twice with PBS and
then incubated in 3% bovine serum albumin (BSA) solution for 30 min.
Thereafter, samples were washed with PBS and incubated with anti-
collagen type I antibody for 30 min. Then, the samples were washed
twice with PBS and incubated with FITC-conjugated goat anti mouse an-
tibody for 15min. After antibody incubation samples were washed and
then incubated with TRITC-phalloidin for 15 min. Finally, samples were
incubated with DAPI for 5 min, washed with PBS and then images were
collected using confocal microscopy (Carl Zeiss).
3B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
3.10. Evaluation of in-vivo bone tissue regeneration potential
Developed composite scaffold was evaluated for its biocompati-
bility and bone tissue regeneration potential through artificially cre-
ating non-union segmental bone defect (10–12 mm) in ulna of rabbit
left forearm (The study was approved by the Ethical Committee of
the Department of Orthopedics, IMS BHU, Varanasi, India). Rabbits
were anaesthetized using standard protocol, hairs were shaved and
skin was disinfected before the surgery. In the left forearm ulna of
test rabbit a non-union segmental bone defect (10–12 mm) was cre-
ated and sterilized cylindrical composite scaffold (10–12 mm) was
implanted (Fig. 1). However, in case of control rabbit defect was
not implanted with any scaffold. After surgery, the wound was
closed properly by suture and the forearm was fixed with bandage.
All the operated rabbits were followed up for 12 week and investi-
gated through X-ray imaging. Thereafter, the rabbits were sacrificed
to obtain the specimens of the regenerated bone from the test and
control rabbits. The obtained specimens were then fixed with 10%
formalin buffer solution and embedded in paraffin block. The ob-
tained gross sections were deparaffinized and stained with hema-
toxylin & eosin (H&E) and Masson's Trichrome stain for the
histopathological evaluation.
3.11. Statistical analysis
Each experiment was performed in triplicate and data were pre-
sented as mean ± standard deviation. Statistical significance wasevalu-
ated by one way ANOVA using Graph Pad Prism software. Pvalues b0.05
were considered as significant.
4. Result and discussion
4.1. Nano-bioactive glass characterization
FESEM (Fig. 2A, a) and TEM (Fig. 2A, b) images of nBG shows syn-
thesis of fine nano-sized bioglass particles (~100 nm). However, par-
ticle size analysis (Fig. 2A, d) shows presence of heterogeneous
bioglass particles due to aggregation of nBG under aqueous condi-
tion. Also, EDS analysis (Fig. 2A, c) of nBG shows presence of Si
(58.18%), Ca (34.86%) and P (6.96%) as a major constituent. Further-
more, SAED image (Fig. 2A, b) confirms formation amorphous nBG,
which was reported to be desired property of bioglass.
The in-vitro apatite forming abilities of bioglass was evaluated
using SBF. FESEM images (Fig. 2B, a) of nBG soaked in SBF for a
week exhibited apatite formation over the surface. Deposited apatite
was observed to be appeared as aggregated beads on string like
structure. Also, X-ray diffractogram (Fig. 2B, b) of pure nano-
bioactive glass and bioactive glass after soaking in SBF confirms
broad peak around 30° with absence of any specific peak signifies
amorphouspropertiesofaspreparedpurenBGandappearance
peak specific to apatite after soaking in SBF. The characteristic XRD
peak associated with hydroxyapatite deposited over nBG after
soaking in SBF was observed to be present at around 26° (002),
31.8° (211), 34° (202), 39.5° (310), 50° (213) and 53° (004) [31].
Moreover, EDS analysis (Fig. 2B, c) of nBG treated with SBF shows
Si (31.93%), Ca (42.50%) and P (25.57%) indicates dissolution of
bioglass followed by gradual release of Si ion and deposition of apa-
tite with Ca/P ratio of 1.66, which is closely similar to the Ca/P ratio of
1.67 associated with hydroxyapatite. Thus, synthesized bioglass was
amorphous in nature and facilitates apatite formation under physio-
logical condition.
4.2. Composite scaffold fabrication and physical evaluation
In the present study, nBG incorporated composite scaffold was
fabricated through PEC followed by phase separation and replace-
ment of separated water with gelatin solution (Fig. 3). Chondroitin
sulfate was slowly added and dissolved in chitosan solution with
pH b3. As chondroitin sulfate was added a milky white color solution
generated through electrostatic interaction mediated PEC between
cationic chitosan and anionic chondroitin sulfate. The resulting poly-
electrolyte solution shows an increase in pH (~5). Also, the resulting
CH/CS solution shows presence of suspended polyelectrolyte com-
plex. Moreover, as the nBG was added in prepared CH/CS solution
Fig. 1. Schematic representation of the surgical procedure adopted for non-union segmental bone defect creation and scaffold implantation into the defect area in a rabbit model.
4B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
Fig. 2. (A) FESEM (a), TEM (b), EDS (c) and DLS histogram (d) images of synthesized nano-bioactive glass (nBG). (B) FESEM (a) images of nBG after SBF treatment, X-ray diffractogram before/after SBF treatment (b) and EDS (c) of nBG after SBF
treatment.
5B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
the pH of solution was obser ved to be abruptly rises (pH 6.5–6.7) due
to surrounding alkalization as a result of faster reactivity of nBG and
resulted in generation of surface silanol group over the nBG. As the
pH of solution rises, positively charged amino group of CH react vig-
orously with negatively charged group present in the medium such
as sulfonic and carboxylate groups of CS and silanol group (Si-OH)
of nBG through ionic bonding, amide group form hydrogen bonding
with silanol group and covalent bond through esterification between
silanol group of nBG and hydroxyl group of chitosan. [32,33]. Thus,
enhanced hydrogen bonding, ionic bonding and covalent interaction
between CH, CS and nBG resulted in phase separation. Also, the ob-
tained polyelectrolyte complexes were micron sized spherical
shape and nBG were encapsulated within the developed PECs
microparticles. Moreover, bioactivity of bioglass depends on specific
surface area or the contact surface between the material and physio-
logical fluid. Thus, higher the specific surface area higher will be its
bioactivity. Therefore, as the particle size reduced from micron
scale to nano scale, the surface to the volume ratio increases and
thus enhanced the bioactivity of the scaffold. Moreover, nano-
bioglass also provide higher surface area for optimal reactivity with
polyelectrolyte complex and thus allows nano-bioglass encapsula-
tion in developed polyelectrolyte complex. Thereby, the optimum
size of nano-bioglass to fabricate stable scaffold was observed to be
≤100 nm. Furthermore, the transparent solvent phase replaced
with equal volume of gelatin solution. The separated PECs were uni-
formly distributed in gelatin solution followed by freeze drying of
Fig. 3. Schematic representation of composite scaffold fabrication through polyelectrolyte complexation and phase separation mechanism followed by freeze drying method.
6B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
Fig. 4. (A) FESEM images of developed porous scaffold. (B) Pore size (a) and porosity (b), of the scaffolds. (C) Stress vs strain curve (a) and compressive strength (b) of the scaffolds.
7B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
obtained solution. Also, gelatin play important role as binder and
might be significantly enhance the strength and stability of compos-
ite scaffold.
4.3. Scaffold morphology, porosity, and mechanical properties evaluation
Fig. 4A shows FESEM images of the scaffolds depict porous architec-
ture of homogeneous multicomponent system. Pore size (Fig. 4B, a) of
CH/CS/G scaffold was observed to be uniform and in the range of
399 ± 138 μm. However, the pore size of the CH/CS/G/4nBG, CH/CS/G/
8nBG, and CH/CS/G/12nBG scaffolds were observed to be decreases as
the nBG content increases due to the enhanced microparticles forma-
tion and measured to be in the range of 348 ± 92 μm, 265 ± 74 μm
and 196 ± 59 μm respectively. Furthermore, it has been reported that
pore size in range of 100–300 μm of ceramic based scaffold facilitate
bone tissue formation [34]. Thus, the developed composite scaffold ex-
hibit pores with sizes in range of 100–300 μm and thereby suitable to
initiate bone tissue regeneration.
The developed CH/CS/G, CH/CS/G/4nBG, CH/CS/G/8nBG, and CH/CS/
G/12nBG scaffolds shows 77.1 ± 4.9%, 75 ± 8.0%, 69 ± 2.6%, and 62.6 ±
4.9% porosity respectively (Fig. 4B, b). Thus, as the nBG content in-
creased, porosity of the scaffolds decreased gradually. Higher porosity
of scaffold favors cells migration and bone in-growth, however it
might further compromise with mechanical and structural stability of
scaffold. Thus, porosity of scaffold needs to be optimized in reference
to the strength of the scaffolds. Furthermore, CH/CS/nano-SiO
2
based
scaffold has been reported to be suitable for bone tissue engineering ap-
plications and possess pore size in range of 150–200 μm and porosity
around 65% [19]. Also, Chitosan/Hydroxyapatite based scaffold was
also reported with porosity around 50% and observed to support cell
growth and infiltration [35]. Thus, scaffolds with porosity ≥60% might
be suitable for bone tissue regeneration. The compressive strength
(Fig. 4C) of developed CH/CS/G, CH/CS/G/4nBG, CH/CS/G/8nBG, and
CH/CS/G/12nBG scaffolds were measured to be 0.077 ± 0.009, 0.19 ±
0.01, 0.29 ± 0.06, 0.62 ± 0.18 (MPa) respectively. Recently, chitosan
based microparticle incorporated porous scaffold containing calcium
phosphate as inorganic constituent has been reported with compressive
strength in the range of 0.2–0.4 MPa in hydrated condition [36]. Fur-
thermore, chitosan/Chondroitin sulfate/Hyaluronic acid/Nano-
hydroxyapatite based composite was also reported with compressive
strength around 0.12 MPa in hydrated condition [37]. Thereby, the de-
veloped nBG incorporated composite scaffold (CH/CS/G/nBG) through
polyelectrolyte complexation followed by phase separation and re-
placement of separated water with gelatin, leads to enhancement of
compressive strength of chitosan based scaffold in hydrated condition.
Thus, the developed composite scaffolds possess superior mechanical
strength in hydrated condition and might be a suitable biomaterial for
bone tissue regeneration.
4.4. Analysis of structural, functional, and swelling properties of the
scaffolds
Chitosan is a crystalline macromolecule with characteristic XRD
peak around 10.50°, 15° and 20.50°, whereas gelatin exhibit peak
around 20° and CS is a macromolecule without crystallization [38,39].
Thereby, the XRD diffractogram (Fig. 5A) of CH/CS/G shows broad
semi-crystalline peak around 2θ= 21°. However, the amorphous prop-
erty of the composite scaffold was observed to be enhances as the amor-
phous nBG content was increases in the scaffold and thus the
broadening of peak around 21° was observed. Also, usually the fabri-
cated composite scaffold lacks any significant peak as nBG is amorphous
in nature. Furthermore, scaffolds were also characterized for the func-
tional properties using FT-IR. The obtained FT-IR spectra (Fig. 5B) of
the scaffolds shows broad peak around 1640 cm
−1
due to C_O
stretching (amide I) of gelatin, peak around 1550 cm
−1
due to N\\H
(amide II) bending and the peak around 1407 cm
−1
as well as
1507 cm
−1
indicates presence of\\COO
−
and\\NH
3
+
respectively sug-
gesting\\NH
3
−
COO
+
complex formation dueto PEC [40,41]. The charac-
teristic peak of \\CONH
2
and \\NH
3
+
group at 1646 cm
−1
and
1560 cm
−1
respectively associated withCH/CS was observed to be over-
lapping with the peak associated with gelatin [41]. Also, the broad peak
associated with \\NH
4
+
SO
4
−
due to polyelectrolyte complexation has
been observed in the range of 1150–1300 cm
−1
[41,42]. Furthermore,
the FT-IR spectra of composite scaffold shows presence of new vibration
band around 467 cm
−1
and a shoulder at 1200 cm
−1
is associated with
Si-O-Si bending mode [43]. FT-IR peak around 870 cm
−1
indicates Si\\O
stretch, which is attributed to the silicon bond of phosphorous rich
phase [44]. Whereas the vibration band at around 1030–1070 cm
−1
in-
dicates stretching vibration of phosphate group [43]. Also, the band at
around 1030–1070 cm
−1
, was observed to be broadened as the nBG
content was increased and possibly due to the Si\\O\\Cbonding
through esterification between silanol group of nBG and hydroxyl
group of chitosan. Thereby, the XRD and FT-IR analysis confirms the
generation of polyelectrolyte complexation mediated nBG incorporated
composite scaffolds.
Swelling behavior of scaffold regulates the surface area/volume ratio
under physiological condition and facilitates cell attachment, migration
and allows availability of required culture media/nutrient for cellular
growth [45]. Developed CH/CS/G based scaffold shows swelling ratio
(Fig. 5C) in the range of 5.3–6.2. However, the polyelectrolyte complex-
ation of CH and CS was observed to regulate swelling behavior of CH/CS
based scaffold and thus allows better control over structural integrity
and mechanical properties [41]. Furthermore, it has been observed ear-
lier that incorporation of bioceramics in composite scaffold reduces
swelling behavior of polymeric scaffold and thereby enhances scaffold
stability under physiological condition [45]. Similarly, the developed
CH/CS/G/4nBG, CH/CS/G/8nBG and CH/CS/G/12nBG composite scaffolds
were observed to show lower swelling ratio in comparison of CH/CS/G
in the range of 2.1–2.5, 2.2–3.0 and 2.6–3.1 respectively. However, the
swelling ratio was observed to be increases in a controlled manner
without much affecting the structural stability and mechanical proper-
ties of scaffolds with increases in nBG content. Hence, the developed
scaffold might be suitable for cell infusion and attachment under phys-
iological condition for neobone tissue regeneration.
4.5. In-vitro biomineralization potential of the scaffold
FESEM images (Fig. 6A) of scaffolds soaked in SBF for 7 days shows
formation of apatite layer over the scaffolds. Scaffold without nBG
(CH/CS/G) shows limited extent of apatite grains deposition over the
surface after SBF treatment. Whereas, nanobioglass based composite
scaffolds show deposition of homogenous grains of hydroxyapatite.
However, CH/CS/G/12nBG shows higher apatite deposition potential
than CH/CS/G and lower apatite deposition in comparison to CH/CS/G/
4nBG and CH/CS/G/8nBG. This might be due to enhanced nBG encapsu-
lation under superior polyelectrolyte complexation of CH and CS upon
incorporation of 12 w/v of nBG and thus exhibit controlled apatite depo-
sition overthe surfaces of the CH/CS/12nBG scaffold. Furthermore, it has
been observed that the developed CH/CS/G/12nBG composite scaffold
shows better control over the surface reactivity under physiological
condition and thereby the pH (Fig. 6B) of the surrounding medium
was rises to an optimum level (~7.8). Also, pH of the matrix around
7.75 was observed to be optimal for osteoblast cell growth [46].
Whereas, higher surface reactivity potential of CH/CS/G/4nBG leads to
the deposition of higher level of apatite and thereby thepH of surround-
ing medium was also observed to be rises to ~9, whereas CH/CS/G/8nBG
shows reduction in pH rise and was recorded to be around 8.0–8.5.
Thereby, a better control on pH increment of surrounding medium
was observed by increasing the nBG content and this might be attrib-
uted due to the enhanced polyelectrolyte complexation of biopolymers
with increasing nBG content. Thereby, microparticle encapsulated nBG
controls the surface reactivity of nBG through preventing direct contact
8B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
with surrounding medium and also control the exchange of ionic com-
ponent across the surface of microparticle. Also, it has been previously
observed that abrupt or uncontrolled mineralization of scaffold is detri-
mental for cell growth and shows poor osteogenic potential in compar-
ison to matrix with controlled mineralization potential [47]. Thus, CH/
CS/G/12nBG shows better control over rise in pH (7.78) and leads to
the controlled apatite deposition under physiological condition. The
XRD diffractogram (Fig. 6C) of CH/CS/G shows absence of any character-
istic peak associated with deposited apatite might be due to very low
level of mineralization. Whereas, CH/CS/G/4nBG and CH/CS/G/8nBG
shows prominent broad peak at around 26° (002), 31.9° (211), 46.8°
(222) and 53° (004) indicating deposition of apatite phase according
to the standard card (JCPDS file No. 09-0432). Moreover, peak at 29°
shows formation CaCO
3
as calcite due to faster dissolution of nBG and
release of calcium ions. Thus, nBG leads to the deposition of poorly crys-
talline hydroxyapatite similar to the apatite formation during bone tis-
sue remodeling or regeneration. Furthermore, CH/CS/G/12nBG shows
lower peak intensity associated with apatite deposition, which might
be due to controlled surface reactivity and exchange of ions. Moreover,
FT-IR spectra (Fig. 6D) shows peak at around 1000–1100 cm
−1
and
976 cm
−1
indicates P\\O stretching band for phosphate group of the de-
posited apatite over the scaffold [48,49]. Also, peak at about 604 cm
−1
and 568 cm
−1
are associated with O-P-O bending and stretching
mode of PO
4
−3
group associated with deposited hydroxyapatite
[50,51]. FT-IR peak at 800 cm
−1
shows formation of Si-OH silanols
group during initial phase of interfacial reaction [44]. Moreover, peak
at around 1400–1450 cm
−1
indicate deposition of carbonated apatite
similar to natural bone tissue inorganic apatite layer. Furthermore, FT-
IR analysis also shows lower peak intensity associated with apatite
deposition over CH/CS/G/12nBG as compared to other composite scaf-
folds. Thereby, FESEM, XRD and FT-IR shows controlled mineralization
potential of the developed CH/CS/G12nBG composite scaffold under
physiological condition.
4.6. Cell viability, metabolic activity and growth over the scaffold
Developed CH/CS/G, CH/CS/G/12nBG and the control (CH/CS) scaf-
folds were further evaluated for cells supportive properties through
live/dead assay, MTT assay and SEM analysis. Live/dead assay (Fig. 7A)
shows that cells were viable over all the scaffolds after culturing for
24 h. However, MTT assay (Fig. 7A, d) shows that cells over CH/CS/G/
12nBG are insignificantly less metabolically active in comparison to
control and CH/CS/G after 24 h of culture duration. This might be due
to the variation in pH of the surrounding medium. Moreover, on the
day 3 cells were more metabolically active over composite scaffold
and subsequently show significantly higher metabolic activity by day
7 in comparison to control and CH/CS/G respectively. Also, encapsula-
tion of nBG within the polyelectrolyte complex limits the direct contact
of nBG with surrounding medium and thus decreases the faster
bioreactivity of nBG. Thereby, limits the pH dependent cytotoxicity
through limiting the vigorous rise in pH of the surrounding medium
and thus allows MG63 pre-osteoblast cells to grow over the scaffold
surfaces.
Furthermore, SEM (Fig. 7B) and confocal (Fig. 7C) images of cells cul-
tured over the scaffolds show that all the scaffold favors cell growth and
distribution irrespective of the compositions. SEM images of cells over
control and CH/CS/G shows spherical and elongated fibroblast like
shape over the culture period. However, cell over CH/CS/G was
Fig. 5. XRD (A), FT-IR (B) and swelling ratio (C) of the developed porous composite scaffolds.
9B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
observed to possess higher tendency for spreadingas compared of con-
trol, whichmight be dueto the presence of cell binding RGD motif asso-
ciated with gelatin. However, the cells were often spread and were
polygonal in shape over the composite scaffold by day 14. Thus,
composite scaffold favors more spreading and differentiation towards
osteoblast like cells as the roughsurface topography due to the incorpo-
ration of nBG in composite scaffold provides more target spot for cell at-
tachment. As under physiological condition bioglass enhances apatite
Fig. 6. FESEM (A), pH variation (B), XRD (C) and FT-IR (D) of scaffold after soaking in SBF solution.
10 B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
Fig. 7. (A) Live/Dead and MTT assay to evaluate viability and metabolic activity of cells over the scaffold. (B) SEM and (C) confocal images of cultured cells over the scaffold after culturing for day 7 and 14.
11B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
Fig. 8. (A) ALP activity (a) and Alizarin red S assay (b) of scaffold after culturing for 2 weeks. (B) Inverted phase contrast microscopic images of Alizarin red S stained cultured scaffold.
(C) Immunocytochemistry for collagen type I expression by cells cultured over the scaffold for day 7 and 14.
12 B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
deposition over the scaffold and thereby enhances roughness of the
scaffold with time. Also, it has been reported earlier that Si and Ca
ions released through bioglass dissolution, play vital role in stimulating
synthesis of bone specific protein by MG63 cells, enhances expression
for various growth factors (IGF I or IGF II), favor cell attachment, prolif-
eration and differentiation over composite scaffold [52–54]. Moreover,
SEM images also confirm stability of scaffold architecture during in-
vitro cells growth over the scaffold throughout the culture duration.
Thus, developed scaffold maintain its structural integrity and allows
cells to grow and proliferate over the scaffold to facilitate generation
of tissue engineered construct.
4.7. Evaluation of in-vitro ALP activity, biomineralization and collagen I
expression
Based on cellular compatibility, cell adhesion and morphological be-
havior of cells, the designed scaffolds were characterized for ALP activ-
ity, biomineralization activity and expression of collagen type I to
evaluate the bone tissue regeneration potential. ALP activity is the
early marker for the osteoblast activity and thereby evaluated to inves-
tigate the osteogenic potential of the scaffold. Fig. 8A (a), shows that by
day 7 the ALP activity of cells over the scaffolds differs insignificantly.
Moreover, ALP activity of cells over CH/CS/G/12nBG and CH/CS/G scaf-
folds was observed to be higher than control. This might be due to the
controlled bioreactivity of encapsulated nBG. However by day 14, the
ALP activity was observed to be significantly enhanced over both the
scaffolds as compared of control. Moreover, cells over CH/CS/G/12nBG
show significantly higher ALP activity in comparison to CH/CS/G and
control. This might be due to the development of osteogenic inductive
microenvironment rich in inorganic ions (Si & Ca ions) and deposition
of apatite layer through dissolution of nBG [55,56]. Thus, developed
composite material triggers osteogenic differentiation of pre-
osteoblast cells. Alizarin red S dye was used, which efficiently stain
only newly deposited mineralized matrix by bone cells over the scaffold
surfaces [57]. Furthermore, alizarin red S assay (Fig. 8A, b) shows that
cells over CH/CS/G/12nBG deposited significantly higher level of miner-
alized matrix in comparison of CH/CS/G and control after 14 days of cul-
ture duration. Also, phase contrast images (Fig. 8B) shows deposition of
reddish mineral nodule by cultured cells. Deposition of mineralized ma-
trix was observed to be higher over the composite scaffold and also en-
hanced with culture duration. Also, thedeposited mineralized matrix by
cultured cells over the scaffold was also evaluated for the collagen type I
through immunocytochemistry (Fig. 8C). The obtained result shows
that expression of collagen type I was higher over the composite scaf-
fold on both day 7 and day 14. However, the expression was very low
or negligible over the CH/CS/G and control on day 7 and shows late ex-
pression by day 14. The enhanced expression of collagen type I over the
CH/CS/G/nBG composite scaffold might be due to the release of silicic
acid and soluble silicon through dissolution of encapsulated nBG,
which play a vital role in upregulation of prolyl hydroxylase mediated
collagen type I expression and cross-linking of collagen and proteogly-
cans at the inorganic-organic interface while bone tissue formation
[58,59]. Thereby, the developed scaffold provide cytocompatible cell
supportive surface for cell attachment, growth, biomineralization and
deposition of collagen rich mineralized matrix for bone tissue
regeneration.
4.8. Histological and radiographic examination
The obtained result shows that all the rabbits with implants were
survived and no sign of any adverse inflammatory reaction was noticed.
Fig. 9A, (a) clearly shows no bone formation in control defect and open-
ing of both the end of ulna segmental defects was observed to be closed
in order to prevent the loss of marrow. Although, X-ray radiograph
(Fig. 9B, a) of the control shows existence of unhealednon-union critical
sized bone defect after 12 week of post-implantation. However, defect
implanted with scaffold (Fig. 9A, b) shows formation callus bridging
both the end of ulna at the non-union gap and also the regenerated re-
gion was rigid and cannot be removed forcibly. Also, X-ray radiograph
(Fig. 9B, b) shows formation of calcified new bone at the site of defect.
Fig. 9. (A) Gross photographs of the control sample (a) without scaffold and test sample (b) implanted with scaffold after 12 weeks of post-implantation. (B) Radiographs of the bone
defect created in control (a) and test (b) sample after 12 weeks of post-implantation.
13B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
The non-union gap was observed to be dominated by regenerated neo
calcified bone tissue at both the end of defect margin, which allows
bridging at critical sized bone defect of ulna.
Moreover, H&E images (Fig. 10A) clearly shows migration of bone
cells within the implanted scaffold. Thereby, the developed scaffold ex-
hibit suitable osteoconductivity and porosity for cells migration and dis-
tribution. Also, regenerated new bone tissue exhibits nearly similar
bone tissue morphology as normal bone of radius. The total regenerated
bone area was measured to be nearly 64.9 ± 9% of the total implanted
scaffold at the defectsite after 12 week of implantation. Also, scaffold re-
maining at defect site was observed to be populated with bonecells and
degraded in accordance with the regeneration of bone tissue. Thus, in-
vivo study confirms stability of scaffold architecture to facilitate regen-
eration of bone tissue during 12 week of implantation. Furthermore, re-
generated bone also possesses regenerated blood vessels with varying
diameter. Thus, the developed composite scaffold provides a suitable
Fig. 10. (A) H&E and (B) Masson's Trichrome stained tissue sections after 12 week of post-implantation. RB –regenerated bone, S –scaffold, BV –blood vessel, BM –bone marrow.
14 B.N. Singh et al. / International Journal of Biological Macromolecules 153 (2020) 1–16
template to facilitate osteogenesis as well as neovascularization of the
regenerated bone tissue. Moreover, Masson's Trichrome staining
(Fig. 10B) also shows deposition of collagen across the regenerated
bone tissue. However, the degree of collagen deposition across regener-
ated bone was observed to be lower than natural bone tissue of radius
and expected to enhance with maturation or complete regeneration of
the defected bone tissue. The deposition of collagen indicates
osteoinductive property of the developed scaffold and was observed
to be suitable biomaterial with superior ability to allow migration of
bone cells, interact with surrounding bone tissue to facilitate vasculari-
zation and finally allows restoration of non-union segmental bone de-
fect through bone tissue regeneration.
5. Conclusion
In the present study, nano-bioactive glass encapsulated microparticle
loaded composite scaffold was fabricated and characterized for various
physico-chemical properties. Developed novel porous scaffold shows con-
trolled rise in pH, maintain structural stability under physiological condi-
tion and provide micro/nano architecture for tissue engineering
application. Also, scaffold provides suitable wet strength and controlled ap-
atite deposition potential for bone tissue engineering. In-vitro cell study re-
veals cytocompatibility, cell supportive properties and biomineralization
potential of the composite scaffold. Furthermore, in-vivo animal study re-
veals biocompatibility and absence any adverse inflammatory reaction as-
sociated with the developed composite scaffold. Also, the developed
bioresorbable scaffold provides superior structural framework,
osteoconductivity and osteoinductivity to restore artificially created non-
union segmental defect. Moreover, the composite scaffold favors neovas-
cularization for efficient regeneration of better quality of bone to restore
the non-union defects. Thus, the developed composite scaffold might be
a suitable biomaterial for bone tissue engineering applications.
CRediT authorship contribution statement
Bhisham Narayan Singh:Conceptualization, Methodology, Writing
- original draft, Writing - review & editing.Vivek Veeresh:Methodology,
Conceptualization.Sarada Prasanna Mallick:Writing - original draft,
Writing - review & editing.Shivam Sinha:Supervision, Resources.Amit
Rastogi:Supervision, Methodology.Pradeep Srivastava:Supervision,
Resources, Writing - original draft, Writing - review & editing.
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgement
This study was supported by the Central Instrument Facility Center
(CIFC), IIT (BHU), Animal House (BHU) and Department of Pathology,
IMS BHU. The authors also thankful to IIT BHU for providing PDF insti-
tute fellowship and other infrastructural facility to the first author to
carry this work.
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