DESIGN OF MICROPOROUS SODIUM ALGINATE AND
IRON OXIDE-BASED SCAFFOLD FOR BONE TISSUE
REGENERATION
Submitted in partial fulfillment for the award of the degree of
Bachelor of Technology
in
Chemical Engineering
by
AISHWARYA M: 20BCM0049
PRATAKSHA GUPTA: 20BCM0072
PRABHAT RANJAN: 20BCM0151
Under the Guidance of
Dr. Dharmendra Kumar Bal
Associate Professor,
School of Chemical Engineering
VIT, Vellore 632014
May, 2024
3. OBJECTIVES AND SCHEDULE OF WORK
Our objectives revolved around creating an appropriate microfluidic device to bubble
nitrogen gas through the sodium alginate solution, thereby making a monodispersed scaffold.
We utilised Fusion 360 software to design the T-junction and employed ABS material for 3D
printing. A stable, red-coloured hydrogel was created with the assistance of a syringe pump and
subjected to freeze-drying to remove moisture. Subsequently, the lyophilized samples underwent
characterization using various methods, namely XRD, FTIR, and SEM. Drug release kinetics
were also performed for both scaffold types, with and without the drug.
Fig 1: Gantt Chart for the scheduling of the work
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4. MATERIALS AND METHODS
4.1 Design and fabrication of 3D printed T-junction
A fusion-360 model of the microfluidic channel (T-junction) was 3D printed using
Acrylonitrile Butadiene Styrene (ABS) using Fused Deposition Modelling (FDM) technology. The
diameter of both the channels is 1.7 mm. To minimise warping during cooling we set the infill
density to 20% in a grid geometry.
Fig 2: Cross-sectional view of T-junction Fig 3: Design made in Fusion-360
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4.2 Experimental Setup
18 gauge needles were used to establish the connections between the T-junction, syringe and
nitrogen gas supply. The cannula of the needles were sawed and filed to ensure a uniform cross
section and uniform flow, pressure through the T-junction. The needles were fixed at all three
openings of the channels at a depth of 1.8 cm from the opening, using M-seal.
Fig 4: Syringe Pump Fig 5: Connecting T-junction with 18-gauge needle.
Fig 6: Experimental Setup
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4.3 Preparation of Solutions
4.3.1 Solution I
15 mg of dry iron oxide nanoparticles were added to 94.485 grams of deionized water in a
250 ml beaker. Then, this mixture was put in a water bath sonicator for 30 minutes to prevent the
NPs from sticking to the surface of the beaker.
Fig 7: Solution A mixing in water bath sonicator
4.3.2 Solution II
3 grams of sodium alginate powder and 2 grams of Pluronic F127 surfactant were added to
the homogenized dispersion, solution I, in a 250 ml beaker. The mixture was then kept on a
magnetic stirrer at 300 rpm for one hour.
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4.3.3 Solution III
199.75 mg (200 ppm) of AAS solution was added to solution II and magnetically stirred for 10
minutes at 300 rmp.
4.3.4 Solution IV
3 grams of sodium alginate powder was added to 97 grams of DI water and kept for magnetic
stirring for one hour at 300 rpm.
4.3.5 Solution V
3 grams of sodium alginate and 2 grams of Pluronic F127 were added to 95 grams dI water and
magnetically stirred for one hour at 300 rpm.
Solutions II, III, IV were collected in centrifuge tubes and sent for FTIR, XRD analysis along with
the individual components in power form, i.e. SA , IONP, PF-127.
Fig 8: Preparation of Solution II Fig 9: Solutions (II, IV and V) collected in centrifugal tubes.
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4.3.6 Solution VI
The crosslinking solution was prepared by adding 4.41 grams (0.03M) of calcium chloride
powder to 100 grams of deionized water in a 250 ml beaker and mixing using a magnetic stirrer at
300 rpm for 10 minutes.
4.4 Preparation of Scaffold
Scaffold A is prepared using solution II, scaffold B (drug loaded) is prepared using solution
III. The method of extrusion and gelation for both scaffolds remains the same. The respective
solutions are loaded in a 20 mL syringe. This syringe is then attached to the 18 gauge needle
attached to the continuous channel of the T-junction. Nitrogen gas, discharged from a cylinder at 0.1
kg/cm is sent through the perpendicular end. The syringe pump is operated at 2 mL/min and the
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monodispersed liquid foam is extruded into a petri dish.
Fig 10: Preparation of scaffold using syringe pump
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Fig 11: Scaffold before gelation
The crosslinking agent (0.03M CaCl solution) was immediately added dropwise to the petri
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dish from the sides. It is left undisturbed for 30 minutes to create a solid foam (hydrogel).
Fig 12: Gelation method
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4.5 Lyophilization
The microporous scaffold was then sent for freeze drying in the TT facility, VIT University,
Vellore, India. However, before lyophilising them, the samples are kept in a deep freezer at a
temperature of -80℃ for 18 hours. Post that, they are freeze dried at -55℃ for 2 days to remove all
the moisture. The bone-dry scaffold is then crushed and given for characterization.
Fig 13: Deep Freezer Fig 14: Freeze Drier
Fig 15: Dried scaffolds
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both characteristics or semicrystalline nature. From Fig. 18, it can be observed that the material is
crystalline in nature [41]. The % crystallinity of the scaffold sample with Fe3O4 NPs was found to be
84.269%. Here B denotes Intensity(au).
Fig 18: X-ray diffraction pattern of the scaffold.
Fig 19: Identification of peaks at different angles using OriginPro
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5.4 SEM
Scanning Electron Microscopy (SEM) is a crucial tool for visualizing the morphology of
alginate-based scaffolds. The analyses were performed using EVO 18 Research Model: CARL
ZEISS.SEM is coupled with Image J analyzes software to measure the average pore size. In bone
tissue engineering, scaffolds serve as temporary structures that support cell growth and facilitate the
regeneration of damaged or missing bone tissue, pore size allows for effective nutrient exchange,
waste removal, and cellular infiltration throughout the scaffold, mimicking the natural extracellular
matrix of bone tissue. The average pore size varies from 530 nm to 750 nm.
Fig 20: SEM image of scaffold Fig 21: Image J analysis
5.5 Drug Release Kinetics
The amount of AAS drug absorbed by the scaffold can be calculated using formula:
Where Ww was the weight of the swollen scaffold and Wd was the weight of the dried scaffold.
The dried scaffold A was dipped in an AAS solution of 200 ppm for a period of 24 hours. The
measured Wd is 0.47 g and Ww is 0.9125g. % Drug absorbed is 94.15%.
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Fig 22: Two methods of preparing scaffold
(Image is taken from the paper: BTE drug delivery Pedro et. al 2015)
The cumulative concentration of drug released from scaffold A and scaffold B as a function
of time is shown in Fig. 23 and Fig. 24 respectively. The dried scaffold was gently dipped into the
brine solution following antibiotic absorption. To determine the concentration of antibiotics
released, the beakers were gently shaken, and samples were taken at regular intervals of 30 minutes.
To keep the overall volume consistent, an equal volume of brine solution was added each time a
sample was taken out of the beaker. An average error of 1% in the measured concentration is
anticipated due to the slight variation in the volume collected. The amount of light absorbed by the
samples at a wavelength of 244 nm was measured. A time period of 24 hours is required for scaffold
B to completely release the drug whereas a period of 10 hours is required for scaffold A to release
the AAS drug.
Fig 23: Cumulative % drug release in scaffold A
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Fig 24: Cumulative % drug release in scaffold B
Fig 25: % AAS drug in scaffold A
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Fig 26: % AAS drug in scaffold B
Fig 27: Comparison studies Scaffold B
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Fig 28: Comparison studies Scaffold A
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