Accepted Manuscript

Development of Hybrid Scaffold with Biomimetic 3D

Architecture for Bone Regeneration

Priya Vashisth, Jayesh R Bellare

PII: S1549-9634(18)30074-1
DOI: doi:10.1016/j.nano.2018.03.011
Reference: NANO 1783
To appear in:
Received date: 13 November 2017
Revised date: 16 March 2018
Accepted date: 29 March 2018

Please cite this article as: Priya Vashisth, Jayesh R Bellare , Development of Hybrid
Scaffold with Biomimetic 3D Architecture for Bone Regeneration. The address for the
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appropriate. Nano(2018), doi:10.1016/j.nano.2018.03.011

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 ACCEPTED MANUSCRIPT

Title Page
Development of Hybrid Scaffold with Biomimetic 3D Architecture for Bone
Regeneration
Priya Vashisth (PhD)a & Jayesh R Bellare (PhD)*a,b
a
Wadhwani Research Center for Bioengineering, Indian Institute of Technology Mumbai, Maharashtra,
400076, India
b
Department of Chemical Engineering, Indian Institute of Technology Mumbai, Maharashtra, 400076,

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India

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First Author: Dr. Priya Vashisth

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Post Doctoral Fellow
Wadhwani Research Centre for Bioengineering

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Indian Institute of Technology Mumbai, Mumbai,
Maharashtra, India, Pin code-400076
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Email: p.vashisth@iitb.ac.in
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*Corresponding Author: Professor Jayesh Ramesh Bellare

Department of Chemical Engineering
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Indian Institute of Technology Mumbai, Mumbai

Maharashtra, India, Pin code-400076
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Email: jb@iitb.ac.in
Phone: +91-(22)-25767207, Fax: +91-(22)-2572-6895
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Word count for abstract: 150

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Word count for manuscript: 4928

Number of references: 53
Number of figures: 8
Number of tables: 0

We wish to confirm that there are no known conflicts of interest associated with this
publication. This study is financially supported by Wadhwani Research center for
Bioengineering, Indian Institute of Technology Bombay.

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Abstract
In the present study, a biomimetic three-dimensional hybrid scaffold has been designed

considering the bone natural architecture with favorable interconnected porous structure,

nano-microscale features and mechanical strength. The chief components of the hybrid

scaffold are core-sheath nanofibers and hydrogel, suitably arranged to create a bone like

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microenvironment. Specifically, the core-sheath nanofibers were coiled tightly into a ring

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to mimic the osteon, and reinforced in a hydrogel matrix. Morphological analysis using

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SEM and 4D-X-ray microscopy revealed that the hybrid scaffold consists of coiled rings

of nanofibers in highly porous hydrogel matrix showing structural similarity to osteons.

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The reinforcement of electrospun nanofibers in hydrogel influenced the mechanical
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properties of scaffold. The potential application of the biomimetic hybrid scaffold, and

the role of its specific architecture, was subsequently investigated in vitro using a human
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osteosarcoma fibroblast cell line. Furthermore, DNA quantification, alkaline-phosphatase

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and alizarin assay validated the potential of fabricated scaffold for bone tissue-

regeneration.
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Keywords: Biomimetic, nanofibers, hydrogel, bone regeneration

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Background
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Bone is a diversified tissue of the vertebrate skeletal system, which possesses a unique

combination of nanoscale to microscale features with precise mechanical strength. It

displays a tremendous capacity of self-regeneration and shows continuous remodeling

throughout human life, which can be seen during the injury repair and skeletal

development.1 However, there are many common painful bone conditions (such as bone

trauma, infection, skeletal abnormalities, avascular necrosis and osteoporosis), which are

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beyond the normal potential of self-healing and cannot heal on its own. Due to inability

of bone self-regeneration in some unavoidable circumstances, the demand for functional

bone grafts is steeply increasing.2,3 Presently, autografts serve as a gold standard method

to heal the bone damage. However, it is associated with donor site morbidity and is

therefore not recommended for large segmental bone damage. Allografts, another bone

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substitute is often associated with certain ethical concerns and possibilities of disease

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transmission.4 The limitations associated with conventional therapies have led to the

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research driven towards the tissue-engineering approaches, which include reconstruction

of biodegradable and biocompatible orthopedic scaffolds/graft with better filler properties

and mechanical strength.5–7

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Nanomaterials, especially electrospun nanofibers have received a remarkable attention

from research scientists for bone tissue-regeneration.8 The nano-topography of scaffolds

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can significantly influence the cell fate by providing a unique niche mimicking the bone
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extracellular matrix (ECM).6,8 Several natural, synthetic and composite biomaterials have

been exploited for synthesis of nanofibrous scaffolds.3,9–14 Though electrospun nanofibers

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can support the osteoinduction of bone cells and can provide enough mechanical support
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in small bone defects, it is tough to use nanofibrous matrix for large segmental bone. It is

difficult to create clinically relevant 3D-constructs using electrospinning and therefore,

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despite being a good therapy candidate, nanofibrous scaffolds fail to support the

expansion of cells at rapid rate to meet the clinical requirement.15,16

Hydrogels are another class of tissue engineered scaffolds that have received much

attention for bone regeneration.4,17–20 The structural and compositional features of

hydrogels are significantly similar to native bone ECM. The microscale pores and high

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porosity of hydrogels allows cell penetration and migration, which in turn promotes

vascularization process throughout the scaffold. Globally, numerous hydrogels including

injectable hydrogels have been explored for bone tissue engineering.4,21 However, it has

been reported that these hydrogels having inappropriate mechanical properties cannot be

used for load bearing bone defects. Therefore, to address this clinical complication, it is

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important to design a three dimensional scaffold which consist of nanoscale as well as

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microscale features all in one.

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In this study, taking clues from natural bone matrix, we have synthesized a biomimetic

hybrid-scaffold consisting both micro and nanoscale features. To mimic the nano-

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hierarchy of bone, core-sheath nanofibers of gelatin/polycaprolactone (gelatin/PCL)
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embedded with hydroxyapatite nanocrystals were fabricated into non-woven sheets using

a versatile electrospinning approach. Gelatin, a derivative of collagen, is a primary

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component of bone ECM.8,22 PCL was used to enhance the overall mechanical strength of
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nanofibers whereas HA nanocrystals were used due to their well-known capability to

promote mineralization.14 The coaxial nanofibers were then reinforced in the form of
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tightly coiled spiral rings in hydrogel matrix to mimic the osteon morphology of cortical
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bone. Reinforcement of electrospun PCL/Gelatin/HA nanofibers in the hydrogel matrix is

expected to provide 3D-bone architecture to the osteocytes as well as improve

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mechanical properties to the scaffold for bone tissue regeneration.

Methods

Materials

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Gelatin type A, extracted from porcine skin, was procured from Rama Industries, India.

Poly ε-caprolactone (Mw-90,000), gellan gum (Gelzan; Mw-1000), trifluroethanol (TFE)

were purchased from Himedia Labs, India. Hydroxyapatite (HA, 50-60nm nanorods)

powder was provided by Plasma Biotal Ltd., UK. Dulbecco's modified eagle medium

(DMEM) was procured from Gibco, Invitrogen, USA. Fetal bovine serum (FBS),

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penicillin-streptomycin antibiotics, trypsin-EDTA, thiazolyl blue tetrazolium bromide

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(MTT), Triton X-100, paraformaldehyde (PFA), dimethyl sulfoxide (DMSO),

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glutaraldehyde and other used chemical unless notified were procured from Sigma

Aldrich, India.

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Fabrication of Gelatin/PCL/HA nanofibers

Coaxial electrospinning technique was used to fabricate nanofibers with unique core–
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sheath structures.23 For preparation of core-sheath nanofibers 10wt% gelatin and 7wt%
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PCL solutions were prepared separately in TFE. Nanofibers were electrospun using 18

kV of applied DC voltage with 12 cm of work distance. The solution flow rate of 0.4
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ml/h was optimized for gelatin solution (comprising the sheath layer) whereas 0.7 ml/h
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for PCL solution (comprising the inner core). Aluminum sheet wrapped grounded

collector was used to collect the nanofibers.

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Preparation of crosslinked gellan and gellan/HA hydrogel

Gellan was dissolved in milliQ water to prepare 2wt% aqueous solution under constant

stirring at 90oC. After complete dissolution of gellan, the transparent solution was

allowed to cool down to 50oC. At this maintained temperature, the CaCl2 (ionic

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crosslinking agent) was added to gellan solution keeping the final concentration of CaCl2

at 0.1wt%. The solution was then stirred for additional half an hour. Additionally, for

preparation of gellan/HA hydrogel, 10wt% HA powder was added into the above-

described solution. The resultant solutions were poured into molds and kept in vacuum

desiccator for overnight to remove any air space. The molded hydrogels were first frozen

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at -20oC for approximately 2 h and then transferred to -80oC for 24 h following by

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lyophilization to obtain crosslinked gellan and gellan/HA hydrogel scaffold.

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Reinforcement of nanofibers in crosslinked gellan/HA hydrogel

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To obtain hybrid scaffold, the core-sheath nanofibrous sheets (0.05mm thick) were
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tightly coiled around a thin cylindrical wire to form a spiral-cylindrical column. Varying

the deposition time of nanofibers, the thickness of nanofibrous spiral rings or the coil
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diameter can be controlled. This would allow the spirals to be in diameter range from
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200-1000μm,which is analogous to native osteons dimensions.

The nanofibrous spiral rings were then reinforced in crosslinked gellan/HA hydrogel at
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about 50oC (as below this temperature gellan/HA solution starts to gel, which inhibits the
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proper integration of hydrogel with coaxial nanofibers). Figure 1 diagrammatically

represents the scaffold fabrication process. The molded hybrid scaffolds were vacuum
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filtrated to avoid any air space between nanofibrous sheath and hydrogel matrix. The

freezing and drying conditions were same as that for crosslinked gellan hydrogel.

Morphological analysis

The morphological properties of different scaffolds were analyzed using scanning

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electron microscopy (SEM, FEI, Quanta 200 (D 7548) equipped with Energy dispersive

X-ray spectroscopy (EDS). All samples were coated with a thin platinum layer for 120

seconds. The coaxial structure of nanofibers and presence of HA nanoparticles on sheath

were shown using transmission electron microscopy (TEM, JEM 2100 ultra HRTEM,

Jeol, Japan) at 200 kV accelerating voltage.

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Porosity and Pore size distribution

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The average porosity of scaffolds was measured using four-dimensional X-ray

microscopy (Xradia Versa 520, Zeiss). For analysis, true spatial resolution X-ray slices

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were obtained and analyzed using Fiji Image J software.
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Mechanical Analysis
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The compressive moduli of scaffolds (10×10 mm) was measured in dry as well as in
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hydrated conditions using a Universal Testing Machine with a crosshead speed of 1

mm/min until 70% deformation of sample. For analysis in dry state, the scaffolds were
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completely dried using lyophilization process. For hydrated condition, the scaffolds were
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soaked in 100% ethanol for 1h before compression testing. Linear region of the obtained

curve was used to determine the Young's modulus.

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Analysis of swelling properties

Swelling property of scaffolds was assessed by immersing the pre-weighed lyophilized

scaffolds in double distilled water (DDW) at room temperature for 24 h. After incubation,

excess water was removed from the scaffolds using tissue paper and the wet weight was

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determined. Subsequently, the scaffolds were dried in an oven and the dry weight was

calculated. Finally, The percentage of swelling was calculated using following equation:

𝑊𝑤 − 𝑊𝑑
𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 (%) = × 100
𝑊𝑑

In this equation Ww represents wet weight of scaffold and Wd represents dry weight of

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scaffold.

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Mineralization potential of scaffolds in simulated body fluid (SBF)

The mineralization potential of the scaffolds was examined by observing the

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hydroxyapatite formation onto the scaffold’s surfaces using ESEM, EDS and X-ray

diffraction (XRD) analysis. For analysis, the disk shaped scaffold (5×5 mm) were soaked
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in 30 ml of SBF at 36.5°C. At scheduled time points (7 and 14 days), these scaffolds
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were retrieved, washed thrice with distilled water, dried and observed using ESEM and
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XRD techniques.
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Cell line and culture conditions

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The biocompatibility assay was carried out using human osteosarcoma fibroblast cell line

(MG63, procured from NCCS Pune, India) which was maintained in DMEM media
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supplemented with 10% FBS, 100 U/ml Penicillin, 100 mg/ml streptomycin. The cultures

were maintained in a humidified growth chamber with 5% CO2 at 37°C. For assay, the

different scaffolds (gellan/HA hydrogel, core-sheath nanofibers and hybrid scaffold) were

first sterilized with 70% ethanol and then with ultraviolet radiation for 30 min each.

Afterwards, the scaffolds were dried overnight and pre-conditioning was performed by

incubating the scaffolds in cell culture media for 4 h. To avoid the floating of cells

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around the scaffold, excess culture medium was removed before the cell seeding. Prior to

the experiment, the cells were counted using a hemocytometer and seeded at a

concentration of 1× 104 cells/ml per well in 24-well tissue culture plate. Culture media

was replenished after every two days till the incubation period of 14 days.

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Assessment of cell adhesion, viability and proliferation on fabricated scaffolds

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For evaluating the adhesion and morphology of cells on different scaffolds (hydrogel,

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core-sheath nanofibers and hybrid scaffold), at pre-determined incubation points (day 7

and 14), media was discarded and the cell-scaffold constructs were rinsed twice with

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PBS. Subsequently the cells on scaffolds were fixed with 2.5% glutaraldehyde at 4°C for
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4 h and then dehydrated through gradient concentrations of ethanol (25%, 50%, 75%,

95% and 100%). All samples were completely dried in a desiccator, sputter-coated with a
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thin layer of platinum and observed under the SEM.

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The cell viability in presence of scaffolds was examined using MTT assay for a period of

14 days. After particular incubation points, media was discarded and 200μl of MTT (5
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mg/ml) was added into each well. Then the plates were incubated for 4 h at 37°C in
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humid atmosphere containing 5% CO2. After 4 h, 1800μl of DMSO was added to

dissolve the MTT formazan crystals.24 Subsequently, 100μl of the dissolved formazan
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solution of each test sample was transferred to individual wells of 96-well plate and the

absorbance was determined using ELISA plate reader (Spectra max M2e) at 570 nm.

Cell proliferation on different scaffolds was studied by DNA quantification assay. The

total DNA content from the cells seeded on different test scaffolds was isolated using Tri

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reagent (Sigma Aldrich) and subsequently was measured using NanoDrop1000,

spectrophotometer (Thermofisher, USA) at 260 nm.

Confocal microscopy

For confocal analysis, after day 7 and 14, the cells seeded on the scaffolds were fixed

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with 4% paraformaldehyde for 6 h. The fixed cells were first stained with fluorescein

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isothiocyanate- phalloidin (FITC-ph) for 6 h at 4°C and then with 4′,6-diamidino-2-

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phenylindole (DAPI) for 5 min. FITC-ph stains actin filaments of cells and gives green

florescence while DAPI stains the cell nuclei and fluoresce blue. For analysis, the cells

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were observed under the laser scanning confocal microscope (Carl Zeiss Meditec AG,
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Jena, Germany) and processed using Zen software (Zeiss).
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Alkaline phosphatase (ALP) activity

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For measuring the ALP activity of MG63 cells, cells from scaffolds were harvested using

a cell lysis buffer containing 0.1% Triton X-100 at predetermined time points (3, 7 and
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14 days). The harvested cell lysates were then centrifuged at 2500g for 10 min and the
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supernatant was taken to measure ALP activity using ALP assay kit (Sensolyte,

Anaspec). Briefly, 50 μl of para-nitro phenyl phosphate (PNPP) solution was mixed with
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50μl of cell lysate suspension and kept at 37°C for 60 min. After 1h of incubation, the

enzymatic reaction was stopped by adding 100μl of 1N NaOH. The production of p-

nitrophenol was observed by measuring the absorbance of the solution at 405 nm using

ELISA plate reader. Finally, the ALP activity was determined using a standard curve.

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BCA Protein Assay Kit with bovine serum albumin (BSA) was used to calculate the total

protein content.

Alizarin red staining (ARS) assay

Alizarin red assay was carried out to quantify cell-mineralization. For analysis, the MG63

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cells grown on scaffolds for 3, 7 and 14 days were fixed with 2.5% glutaraldehyde for 4 h

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and then thoroughly washed with PBS. After washing, 2mM ARS working solution (pH

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4.1–4.3) was added to the cell-scaffolds construct and incubated at room temperature for

30 min. Extra dye was rinsed off with thorough DDW washing. For quantification of

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mineral particles formed by the cells, the supernatant was extracted using 10% acetic
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acid. Subsequently, 500 μl of the above supernatant was neutralized with equal volume of

10% ammonium hydroxide and absorbance was measured at 405 nm using a micro-plate
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reader.
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Statistical analysis
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The statistical analysis of all data set was performed using the OriginLab software.
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Numerical data are presented as the mean ± standard deviation. For the statistical

significant comparisons, one-way analysis of variance (ANOVA) post hoc Tukey's test
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was applied. p ≤ 0.05(*) and p ≤ 0.005(**) were used to represent the statistically

significant differences.

Results

Fabrication of three dimensional biomimetic hybrid scaffold

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Figure 1 displays a schematic representation of a combinational technique to fabricate a

biomimetic hybrid scaffold with unique well-defined nano architecture and precise

surface micro-texture. To achieve biomimetic hybrid scaffold, spiral-cylinders of core-

sheath nanofibers with high mechanical strength were reinforced within a highly porous

gellan/HA hydrogel matrix.

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Morphology of gellan/HA hydrogel, core-sheath nanofibers and hybrid scaffold

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Figure 2 a–f shows the surface and cross-sectional SEM images of core-sheath

nanofibers, gellan hydrogel and biomimetic hybrid scaffold consisting reinforced spiral

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rings. The SEM micrograph of nanofibers (Figure 2a & b) represented smooth surface of
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random nanofibers and non-homogenous distribution of HA particles. TEM structure of

nanofibers as observed in Figure 2c & d confirmed the core-sheath structure of

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nanofibers comprising PCL in core and gelatin/HA on outer sheath surface.

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SEM micrographs of gellan hydrogel displayed an ample distribution of the

interconnected pores, with an average pore diameter of 350 ± 30μm as shown in Figure
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2e. SEM morphology of hybrid scaffold (Figure 2f) revealed the biomimetic features of
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3D hybrid scaffold and osteon-like impressions of coiled nanofibers in micro-porous

hydrogel matrix.
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Morphology and porosity assessment

4D X-ray microscopic images reveal highly interconnected porous structure of gellan

hydrogel (Figure 3A(a)) and distribution of nanofibrous spiral coils in the porous

hydrogel matrix (Figure 3A(b-d)). The scaffolds showed combination of large and small

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pores, which is required for cells to migrate, infiltrate as well as for nutrient and waste

transfer. The porosity of gellan hydrogel and hybrid scaffold evaluated using 4D X-ray

microscopy, was noted to be approximately 89.50 ± 9.34 and 66 ± 6.77%, respectively.

Though, the total porosity get reduced for hybrid scaffold after incorporation of tightly

coiled nanofibrous-rings, but it was enough to promote cell infiltration in the scaffold to

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provide necessary condition for bone-regeneration.

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Mechanical properties

Biomedical materials for bone tissue engineering application should exhibit suitable

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mechanical properties to provide support at defect site until the regeneration of new bone
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ECM.25,26 Figure 3B & C shows the compressive strength of scaffolds in dry and wet

conditions, respectively. As expected, the gellan hydrogel without any modification

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showed least moduli in both states as compared to other modified hydrogels. The calcium
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chloride crosslinked gellan hydrogel and gellan/HA hydrogel possessed significantly

better compressive strength but was not appropriate for load bearing bone defects. The
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compressive moduli of 3D-hybrid scaffold significantly improved as compared to gellan

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hydrogel alone with a Young’s modulus 13.9 MPa in dry and 9.1 MPa in wet state.
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Swelling properties

The swelling ratio and change in volume of scaffold is a direct measure of its water

reabsorption potential.27 The tissue engineering scaffolds should be sufficiently

hydrophilic so that it can wet well. A wet surface of the correct chemical nature can

support cell responses such as adhesion, proliferation, migration and differentiation. In

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addition, the scaffolds should permit transfer of water born nutrients and bio-chemical

through itself to reach internal cells, which in turn supports the cell growth within the

scaffold.28–30

For evaluating the swelling potential of different formulations, the samples were first

lyophilized and placed in PBS until the swelling equilibrium was reached. All samples

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achieved their swelling equilibrium within 10 h. Figure 4a illustrate the swelling ability

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of different formulations. It was noticed that, non-crosslinked and crosslinked gellan

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hydrogel had shown almost similar swelling profiles. The water uptake and change in

volume for HA incorporated gellan hydrogel and the hybrid scaffold was found to be

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lower than the gellan hydrogel. The nanofibers showed the least water absorption in this
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time duration, which could be attributed to their hydrophobic PCL core.
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Degradation studies
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The degradation profiles of scaffolds provide useful information about its degradation

kinetics in vivo.31,32 The weight loss data for different formulations after immersion in
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aqueous medium is summarized in Figure 4b. It was observed that the weight loss was
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highest for CaCl2 crosslinked gellan hydrogels. On the contrary, least weight loss was

noted for core-sheath nanofibers as compared to other formulations during the entire
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immersion period. The weight loss was significantly reduced with incorporation of HA

and reinforced nanofibrous rings into the gellan hydrogel matrix. After 28 days analysis,

the total weight loss recorded for hybrid scaffold was approximately 27% whereas for

crosslinked gellan hydrogel, it was approximately 50%.

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In vitro mineralization ability of scaffolds

The mineralization ability of scaffold evaluated in SBF has become a recognized

approach to validate the potential of such fabricated scaffolds for in vivo bone formation

application.26,33 The SEM images showing the presence of mineralized particles onto the

surfaces of scaffolds can be seen in Figure 5. The surfaces of fabricated scaffolds have

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shown the signs of mineralization. However, the maximum mineralization was noticed on

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the surface of gellan/HA scaffold and hybrid scaffold as compared to core-sheath

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nanofibers. In addition, the elemental composition of calcium and phosphate was

analyzed using EDS (inset of Figure 5: Panel A), which was found to be comparable with

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the stoichiometric ratio of HA crystal in bone. Moreover, to observe the composition of
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the mineralized particles onto the surfaces of scaffolds, XRD analysis was carried out.

Figure 5, Panel C shows the XRD spectra of different scaffolds. The distinguishable
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diffraction peaks at 31.9°, 39.9° and 49.8° attributed to HA were noticed in XRD spectra
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for all scaffold. However, the crosslinked gellan/HA hydrogel and hybrid scaffolds were

observed to exhibit strongest X-ray diffraction peak for HA. For comparative analysis,
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the SEM micrographs of scaffolds prior to immersion in SBF have been provided in
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supplementary file with the supporting EDS and XRD data (supplementary figure 1).
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Cytocompatibility assay

The cell adhesion behavior of MG63 cells on different scaffolds is represented in Figure

6. The developed biomimetic hybrid scaffold allowed the improved cell adhesion along

with the cell infiltration and migration of MG63 cells within the 3D matrix. SEM analysis

only enabled the visualization of cells on the surface of scaffold. Therefore, to observe

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the cells within the 3D planes of scaffold, the cell-seeded scaffolds at different incubation

points were observed using confocal microscopy. The z-stack confocal images taken after

day 7 and 14 (Figure 7a), MTT assay (Figure 7b) and DNA quantification assay (Figure

7c) confirmed the abundant cell adhesion, migration and proliferation on hybrid scaffold

comparable to hydrogel. The data obtained, showed that the developed 3D biomimetic

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hybrid scaffold, exhibiting specific physical cues can be used for improved bone cell

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growth. However, these are the preliminary results, and thus further examination is

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required to investigate the role of fabricated scaffold on cell differentiation. Nonetheless,

significant augmentation in growth of bone cells in the 3D biomimetic environment

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provided by the hybrid scaffold demonstrated its significant potential in the bone tissue-
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engineering field.
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ALP and ARS assay

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The ALP activity for different formulations at different incubation points (3, 7 and 14) is

represented in Figure 8a. No significant difference in ALP activity was noticed in case of
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cells grown without scaffold, at all time points. Though, a significant difference was
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observed in ALP activity from day 3 to 7 for cells grown on different scaffolds as

compared to control cells. In contrary, the variation in ALP activity between day 7 and 14
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was not significant, which can be attributed to the fact that the ALP activity is an early

stage marker of differentiation.

Alizarin red is a marker of bio-mineralization activity and indicates newly deposited

mineralized matrix produced by the cells. Figure 8b shows the ARS activity by the

different formulations. The cells grown on only polystyrene surface without any scaffold

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(control group) have shown low level of mineralization at all time points as compare to

gellan/HA hydrogels and biomimetic hybrid scaffold.

Discussion

When comparing native bone with an ideal scaffold, two broad set of parameters need to

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be addressed. The first is the qualitative nature of micro-structural morphology and

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hierarchical organization. Native bone has very specific fundamental micro-structural

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features, most important of which is osteon. Osteon is the functional unit of bone, which

is responsible to provide structural support and niche for cells.34 Osteons are several

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millimeters long coarse cylindrical structures with diameter of around 200μm that
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consists of concentric layers called lamellae with a spot in the center.35 The ideal scaffold

should mimic and promote the formation of osteons, which can provide channels for
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mass-transfer. The second set of parameters includes quantitatively measurable

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mechanical properties and chemical parameters such as bone mineral density and Ca-P

ratio.
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In this study, we have demonstrated one key requirement of an ideal bone scaffold i.e. the
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presence of osteon like structure, which have been missing in most of the reported

scaffolds so far. Therefore, herein, we have developed a three dimensional biomimetic-

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hybrid scaffold encompassing a hierarchical organization and properties similar to native

bone.

The SEM micrograph of core-sheath nanofibers represented smooth surface morphology

and non-homogenous distribution of HA particles on sheath layer (Figure 2a & b). The

nanofibers containing PCL in core and gelatin/HA at outer-sheath were designed in such

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a way that the core PCL can provide high mechanical strength to the scaffolds whereas

gelatin/HA at outer surface can lead to better attachment/adhesion of cells (Figure 6).36,37

In addition, hydrophilic gelatin at surface was found to merge well with surrounding

hydrophilic gellan hydrogel matrix. The hydrogel matrix was synthesized using gellan

and HA. Gellan is a naturally biodegradable, biocompatible and hydrophilic polymer.

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These inherent properties of gellan make it a potential candidate for tissue engineering

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applications.38,39 HA nanoparticles were also incorporated in the hydrogel matrix with

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gellan. As reported earlier, incorporation of HA particles in a hydrogel matrix can initiate

rapid mineralization and osteogenesis process.40 The gellan/HA hydrogel were

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characterized to have pore diameter between 300-350μm (Figure 2e). The micro-porous
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architecture with well-defined interconnected pores of the fabricated biomimetic scaffold

can support the migration, adhesion and proliferation of bone cells (Figure 6 & 7), which
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subsequently can encourage the in-growth of cells within the scaffold to promote the
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bone formation within the scaffold as well as around the scaffold.

By coiling nanofibrous sheets into a spiral-rings and reinforcing them in a hydrogel

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matrix, we tried to mimic the native osteons. The dimensions and thickness of nanofibers
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can be controlled to better mimic the osteon structure. Figure 2f depicted the fabricated

osteon-like structure i.e. several millimeters long with varying diameter from 200μm to
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1000μm. The deep orientation of nanofibrous spiral rings, displayed in Figure 3A (c &

d), mimics the compact osteon and the surrounding hydrogel matrix mimics the highly

porous trabecular bone. This architecture of synthesized scaffold is advantageous to

direct the alignment of bone cells throughout the scaffold.

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Additionally, bone scaffolds should have high porosity with interconnected pores and

comparable mechanical properties to act as a temporary skeleton until the neo-ECM

regeneration.41,42 However, high porosity and pore interconnectivity can significantly

reduce its mechanical properties. So it’s a challenge to fabricate scaffold with high

porosity while maintaining appropriate mechanical properties.26 In present study, along

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with mimicking the architectural features of bone, the enforcement of nanofibrous coils

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and HA particles in the hydrogel matrix enhanced the overall compressive modulus of

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scaffold as compared to gellan hydrogel alone. The Young’s modulus of the hybrid

scaffold was found relatively less compared to native bone. However, our findings are in

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line with the earlier reports which suggested that an ideal scaffold should not exhibit
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similar mechanical properties as the host tissue and be able to degrade with time at a

controlled bone-resorption rate.28,43–48

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The swelling assay showed a variation in the water absorption capacity by the scaffolds,
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which follow a similar trend to scaffold’s porosity. It might be explained that the gellan

and gellan/HA hydrogel consist of high gellan concentration and thus higher porosity,
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which offered more space to retain water. Yielding an increase in water retaining
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efficiency can help in entrapment of cells and nutrients in the scaffolds.18,49

Biodegradability is another crucial factor for bone regeneration scaffolds. An ideal bone
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scaffold should display a suitable biodegradability so that it can serve as a temporary

substitute until the neo-tissue regeneration, and then degrade naturally.

The degradation behavior of the scaffolds could vary based on the applications. For e.g.

stability for about nine months or more is required for scaffolds in spinal fusion and three

to six months is required for scaffolds for cranio-maxillofacial applications. Furthermore,

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It is reported that scaffold fabricated using natural polymers, having multi-scale porosity

and related biodegradation can have a potential for bone tissue engineering.43 In line with

the earlier reports, the fabricated hybrid scaffold herein possess an improved stability and

approximately 27% degradation over a month i.e. the scaffold can withstand eventually

with the new bone formation for 3-5 months.

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Data obtained from in vitro mineralization study suggested that, the mineralization was

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significantly greater onto the surface of gellan/HA hydrogel and hybrid scaffold as

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compared to gellan hydrogel and core-sheath nanofibers. This concluded that the

presence of HA particles on scaffold surface can significantly influence the in vivo

mineralization process (i.e. bone formation).

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Furthermore, in vitro cell culture assay supported our hypothesis that the 3D biomimetic

scaffold fabricated with precise nano-microscale features can stimulate the cell
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behavior.50 The vertical distribution of pores and pore size (about 350μm) enabled
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osteocytes to grow and migrate in different planes of three-dimensional scaffold, which is

a critical step in bone regeneration. As shown in Figure 6, Cells were observed to adhere
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at different locations on the scaffolds initially which then migrated and proliferated on
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both the horizontal and vertical surface of gellan/HA hydrogel and hybrid scaffold.

Whereas on nanofibers, the migration and proliferation of cells was restricted to 2D plane
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only. The migration and proliferation of MG63 cells on 3D-hybrid scaffold resulted in the

formation of distinct cell colonies that expanded vertically throughout the scaffold.51,52 Z-

stack imaging confirmed the migration of cells deep down the layers of hybrid-scaffold

due to interconnected pores. However, in nanofibers the migration and infiltration of cells

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deep inside the scaffold was lower as compared to 3D-hydrogel and hybrid scaffold,

which could be attributed to the presence of larger pores in hydrogel and hybrid matrix.

The bone regeneration potential of fabricated biomimetic hybrid scaffold was further

characterized in terms of alkaline phosphatase and bio-mineralization activity using ALP

and alizarin red staining assay, respectively.53 Alizarin red is a marker of bio

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mineralization activity that stains only newly deposited mineralized matrix produced by

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the bone cells. ALP and ARS activity of hybrid scaffold was found to be comparable with

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hydrogel and significantly higher than the control and core-sheath nanofibers at all

incubation time points. The results obtained from ALP and ARS studies are in line with

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the in vitro mineralization study. Based on the collective data obtained, we can conclude
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that the reported scaffolds herein demonstrated a key requirement of an ideal bone

scaffold i.e. osteon like structure, which have been missing in the reported bone scaffolds
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so far. Furthermore, reinforcement of nanofibrous rings into the hydrogel matrix

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improved the overall mechanical properties of hydrogel, surface bio-mineralization and

cellular responses.
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Acknowledgement

We are thankful to Central facilities and Centre for Research in Nanotechnology and
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Science (CRNTS), IIT Bombay for providing characterization facility. Authors also

acknowledge Dr. Mayur Temgire for TEM analysis.

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Figure Legends

Figure 1: (a) Microstructure of bone ECM and (b) fabrication process of biomimetic

three-dimensional (3D) hybrid scaffold.

Figure 2: SEM micrographs of (a) PCL-gelatin core-sheath nanofibers (b) PCL-gelatin

core-sheath nanofibers embedded with HA (c) TEM micrograph for PCL-gelatin core-

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sheath nanofibers (d) TEM micrograph for PCL-gelatin/HA core-sheath nanofibers

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(e) Gellan/HA hydrogel and (d) biomimetic hybrid scaffold. Arrows shows the presence

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of HA particles in the core-sheath nanofibers.

Figure 3: (A) 4D X-Ray microscopic image of (a) gellan hydrogel (b) biomimetic hybrid

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scaffold and (c and d) cross-sectional view of biomimetic hybrid scaffold (B) & (C)
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Stress–strain curves for (a) gellan hydrogel (b) CaCl2 crosslinked gellan hydrogel (c)

CaCl2 crosslinked gellan/HA hydrogel and (d) biomimetic hybrid scaffolds in dry and wet
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state, respectively.
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Figure 4: (a) percent swelling of different formulations and (b) in vitro degradation

profiles for different formulations at pre-decided time in PBS.

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Figure 5: (Panel A) SEM micrographs and respective EDS spectra of different fabricated
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scaffold (crosslinked gellan/HA hydrogel, core-sheath nanofibers and biomimetic hybrid

scaffold) soaked in SBF solution after day 7 (Panel B) respective SEM images at high
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magnification and (Panel C) respective XRD spectra.

Figure 6: SEM micrographs of cellular adhesion on fabricated scaffolds at day 7 and 14.

Figure 7: (a) The study of cell morphology grown on 3D matrices at day 7 and 14 using

confocal laser scanning microscopy (b) MTT assay and (c) DNA quantification assay.

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Figure 8: (a) ALP and (b) Alizarin red mineralization activity of MG63 cells on

fabricated scaffolds after 3, 7 and 14 days of culture.

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Graphical Abstract

In this work, a biomimetic three-dimensional hybrid scaffold has been designed by

considering the architectural and compositional features of natural bone, which contain
collagen nano-fibrils alongwith interconnected micro porous matrix. To mimic the nano-
hierarchy of bone, tightly coiled spiral rings of core-sheath nanofibers were reinforced
within a gellan/HA hydrogel matrix, which gives an osteon like arrangement of core-
sheath nanofibers, in a highly porous gel matrix. Along with mimicking the bone
structure (cortical and trabecular), the reinforcement of nanofibrous spiral coils in the
hydrogel matrix enhanced the overall mechanical strength of scaffold, which is a pre-

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requisite for bone tissue regeneration.

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Graphics Abstract
Figure 1
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