SASTRA CeNTAB

Fabrication of Nanofibrous scaffolds by electrospinning

An ideal tissue scaffold should possess sufficient porosity, surface area and surface chemistry to permit
cell adhesion, growth, migration and differentiation as well as an appropriate degradation rate to match the
rate of regeneration of natural tissue. A nanofibrous polymeric material offers an effective scaffold due to
their nano dimensions and capability to orient cell which is the prime requisite for tissue engineering.
Though many methods have been attempted to make nanofibers such as drawing, phase separation, solvent
evaporation etc, electrospinning offers an attractive method to fabricate nanofibers owing to its ease in
controlling nanofibers dimensions.

PRINCIPLE:

The electrospinning process is achieved by applying a high voltage source to polymer solution, causing
charges to buildup in a droplet of solution, to form a conical deformation known as Taylor cone. At a
certain threshold, a polymer jet is ejected from the cone deformation and travels to the region of reduced
electrical potential (a grounded plate). During the passage to the collector, the jet experiences
destabilization forces due to repulsion between positively charged particles (electrostatic repulsion) as the
volatile solvent evaporates. As a result, the jet splits into multiple smaller fibers which are then collected
on the grounded metallic collector.

EXPERIMENTAL PARAMETERS INFLUENCING FIBER MORPHOLOGY

The major parameters influencing fiber morphology are:

1. Polymer concentration

2. Flow rate of polymer solution

3. Needle gauge of the syringe

4. Tip to target distance

5. Applied voltage

Optimization of these parameters is essential to obtain defect free fiber morphology. Polymer
concentration affects the solution viscosity. If the solution has low viscosity then fiber formation does not
occur due to the increased strain and leads to formation of discontinuous fibers. At high viscosities,
polymer solution might not be able to come out of the needle. Electrospinning requires low surface tension
to spin the polymers into fibers. This is achieved by the choice of appropriate solvent.

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SASTRA CeNTAB

High voltage creates necessary electrostatic force to overcome the solution surface tension. Higher the
applied voltage, higher is the columbic repulsive force present within the polymer jet thus promoting a
reduction in fiber diameter.

The tip to target distance also influences fiber morphology. As the distance is increased a reduction in fiber
dimension is expected as more time is available for evaporation of solvent and formation of greater
repulsive forces, leading to finer splitting of the jet.

Low flow rates of polymer solution are generally preferred as sufficient time is provided for the formation
of a Taylor cone at the needle tip. Small diameter needles will lead to formation of finer particles. Fine
tuning of these parameters will lead to the formation of defect free fibers.

Figure: A simplified schematic representation of the electrospinning process

A polymer solution held in a syringe (A) is fed to a metal needle (B). A high voltage
supply (C) is connected to the needle, producing a fine jet of polymer solution (D). This
dries out in transit, resulting in fine fibers which are collected on a grounded collector
(E).

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SASTRA CeNTAB

Expt No: 01
Date:

INFLUENCE OF POLYMER CONCENTRATION ON FIBER MORPHOLOGY

AIM:

To study the influence of polymer concentration on fiber morphology during electrospinning.

MATERIALS:

PLGA (50:50)(Poly L-lactide co-glycolic acid) (Lakeshore Biomaterials, USA) was dissolved in
Chloroform (Merck, India) by constant stirring until clear solution of different w/v concentration of 7 %,
10 %, 12% and 15 %were obtained. The operating parameters for various trials are tabulated below.

Operating parameters Value

Applied voltage 20 kV
Flow rate 0.003 mL/min
Polymer concentration 7%, 10%, 12% and 15% (w/v)
Tip to target distance 10 cm
Solvent Chloroform
Needle gauge size 24 G
PROCEDURE:

3 mL of clear transparent polymer solution was taken in a syringe with the gauge size of 24 G and fitted in
the syringe pump (KD Scientific, USA). The flow rate was set at 0.003 mL/min and the positive of the
high voltage (Zeonics, India) was connected to the tip of the syringe needle. An aluminum foil was stuck
on metal collector which was kept 12 cm away from the tip of the needle and was grounded. The voltage
was gradually increased from 0 to 20 kV and the formation of Taylor cone at the tip of the needle was
observed. The voltage was turned off after five minutes and the aluminum foil was removed. The opaque
deposit on the aluminum foil was analyzed using field emission scanning electron microscope (FESEM,
JEOL 6701 F, Japan).

A 0.5× 0.5 cm portion of the opaque area on the aluminum foil was cut and placed on a brass stub using
double sided carbon tape. A thin coat of platinum was sputtered at a current of 20 mA for 40 seconds. The
sputter coated samples were then introduced into the specimen chamber of the electron microscope through
the exchange chamber. An accelerating voltage of 3 kV was used to image the samples. All samples were
imaged using upper secondary electron detector.

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RESULTS:

The scanning electron micrographs of various samples are shown below:

Figure: Influence of PLGA polymer concentration on morphology of electro spun fibers.

A: 7% of PLGA, B: 10% of PLGA, C: 12% of PLGA, D: 15% of PLGA.
Flow rate: 0.003mL/min, Tip-target distance: 10cm, Applied voltage: 20 kV, Solvent: chloroform,
Needle guage size: 24 G

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INFERENCE:

From the SEM images, following inferences can be made:

As the polymeric concentration increases, fibers start forming. As the polymer concentration increases
fibers start forming. At 5% polymer concentration there were only beads and no fibers form. Formations of
fibers were seen from 7% polymer concentration. Only at 10% polymer concentration beads free fibers
were obtained but they showed uneven distribution non-uniform fiber diameter. The range of fiber
diameter is 0.487 to 0.814 µm.

REPORT:

PLGA fibers were electro spun at different polymer concentrations. Completely defect free fibers were not
obtained. Best results could be observed at a polymer concentration between 7 to 10%.

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Expt No: 02
Date:

INFLUENCE OF TIP TO TARGET DISTANCE ON FIBER MORPHOLOGY

AIM:

To study the influence of tip to target distance on fiber morphology during electrospinning.

MATERIALS:

PLGA (50:50) (Poly L- lactide co-glycolic acid) (Lakeshore Biomaterials, USA) was dissolved in
Chloroform (Merck, India) by constant stirring until clear solution of various tip to target distances (10 cm,
12 cm, 14 cm, 16 cm, and 18 cm) were obtained. The operating parameters for various trials are tabulated
below.

Operating parameters Value

Applied voltage 20 kV
Flow rate 0.003 mL/min
Polymer concentration 10 %
Tip to target distance 10 cm,12 cm,14 cm,16 cm and 18 cm
solvent Chloroform
Needle gauge size 24 G

PROCEDURE:

3 mL of clear transparent polymer solution was taken in a syringe with the gauge size of 24 G and fitted in
the syringe pump (KD Scientific, USA). The flow rate was set at 0.003 mL/min and the positive of the
high voltage (Zeonics, India) was connected to the tip of the syringe needle. An aluminum foil was stuck
on metal collector which was kept at various distances(10 cm, 12 cm, 14 cm, 16 cm, 18 cm) away from the
tip of the needle and was grounded. The voltage was gradually increased from 0 to 20 kV and the
formation of Taylor cone at the tip of the syringe was observed. The voltage was turned off after five
minutes and the aluminum foil was removed. The opaque deposit on the aluminum foil was analyzed using
field emission scanning electron microscope (FESEM, JEOL 6701 F, Japan).

A 0.5× 0.5 cm portion of the opaque area on the aluminum foil was cut and placed on a brass stub using
double sided carbon tape. A thin coat of platinum was sputtered at a current of 20 mA for 40 seconds. The
sputter coated samples were then introduced into the specimen chamber of the electron microscope through
the exchange chamber. An accelerating voltage of 3 kV was used to image the samples. All samples were
imaged using upper secondary electron detector.

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RESULTS:

The scanning electron micrographs of various samples are shown below:

A B

D C

F
F

E
E F

Figure: Influence of tip to target distances on fiber morphology of electro spun fibers
A: 8 cm, B: 10 cm, C: 12 cm, D: 14 cm, E: 16 cm and F: 18 cm. Flow rate: 0.003 mL/min, Concentration:
10 %, Applied voltage: 20 kV, Needle gauge size: 24 G, Solvent: chloroform.

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INFERENCE:

From the scanning electron micrographs, the following inference can be made.

The SEM image shows the presence of large number of defects at all tip-target distances. Almost all the
showed the presence of large size spindles like beads with thick fibers (diameter in micron range). Among
the various tip to target distances, it appears that at a distance of 18cm, we find more no. of continuous
fibers with comparatively less no. of spherical beads. The dimension of fibers formed with a tip- target
distance of 18cm was between 0.13 to 0.179 µm.

REPORT:

PLGA fibers were electro spun at different tip-target distances. Completely defect free fibers were not
obtained. Best results could be observed at a tip-target distance of 18cm.

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Expt No: 03
Date:

INFLUENCE OF FLOW RATE ON FIBER MORPHOLOGY

AIM:

To study the influence of flow rate on fiber morphology during electrospinning.

MATERIALS:

PLGA (50:50) (Poly L-lactide co-glycolic acid) (Lakeshore Biomaterials, USA) was dissolved in
Chloroform (Merck, India) by constant stirring until clear solution of different flow rate 0.001 mL/min,
0.003 mL/min, 0.005 mL/min, 0.007 mL/min, 0.009 mL/min and 0.011 mL/min were obtained. The
operating parameters for various trials are tabulated below.

Operating parameters Value

Applied voltage 20 kV
Flow rate 0.001mL/min,0.003mL/min,0.005mL/min,0.007
mL/min, and 0.009ml/min
Polymer concentration 10 %
Tip to target distance 10 cm
Solvent Chloroform
Needle gauge size 24 G

PROCEDURE:

3 mL of clear transparent polymer solution was taken in a syringe with the gauge size of 24 G and fitted in
the syringe pump (KD Scientific, USA). The flow rate was set at specified rate(0.001 mL/min, 0.003
ml/min, 0.005 mL/min, 0.007 mL/min and 0.009 mL/min) and the positive of the high voltage (Zeonics,
India) was connected to the tip of the syringe needle. An aluminum foil was stuck on metal collector which
was kept 10 cm away from the tip of the needle and was grounded. The voltage was gradually increased
from 0 to 20 kV and the formation of Taylor cone at the tip of the needle was observed. The voltage was
turned off after five minutes and the aluminum foil was removed. The opaque deposit on the aluminum foil
was analyzed using field emission scanning electron microscope (FESEM, JEOL 6701 F, Japan).

A 0.5× 0.5 cm portion of the opaque area on the aluminum foil was cut and placed on a brass stub using
double sided carbon tape. A thin coat of platinum was sputtered at a current of 20 mA for 40 seconds. The
sputter coated samples were then introduced into the specimen chamber of the electron microscope through
the exchange chamber. An accelerating voltage of 3 kV was used to image the samples. All samples were
imaged using upper secondary electron detector.

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RESULTS:

The scanning electron micrographs of various samples are shown below:

Figure: Influence of flow rate on morphology of electro spun fibers

A: 0.001mL/min, B: 0.003mL/min, C: 0.005mL/min, D: 0.007mL/min, E: 0.009mL/min. Tip-target
distance: 10 cm, Concentration: 10%, Applied voltage: 20kV, Solvent: chloroform. Needle gauge size:

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INFERENCE:

From the scanning electron micrographs, the following inference can be made.

The SEM image shows the presence of large number of defects at all defined flow rates (0.001mL/min to
0.009 mL/min). But increased values of flow rates showed the formation of large number of beads with
increasing size. At the lower flow rate (0.003mL/min) fibers were formed. Fine tuning of the flow rate give
defect free fibers in nano range.

REPORT:

PLGA fibers were electro spun at different flow rates. Completely defect free fibers were not obtained.
Best results could be observed at the flow rate of 0.003 mL/min.

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Expt No: 04
Date:

INFLUENCE OF HIGH VOLTAGE ON FIBER MORPHOLOGY

AIM:

To study the influence of high voltage on fiber morphology during electrospinning

MATERIALS:

PLGA (50:50) (Poly L-lactide co-glycolic acid) (Lakeshore Biomaterials, USA) was dissolved in
Chloroform (Merck, India) by constant stirring until clear solution of different voltage ranges from 10 kV,
12 kV, 14 kV, 16 kV, 18 kV and 20 kV were obtained. The operating parameters for various trials are
tabulated below.

Operating parameters Value

Applied voltage 10 kV,12 kV,14 kV,16 kV,18 kV and 20 kV
Flow rate 0.003 mL/min
Polymer concentration 10 %
Tip to target distance 10 cm
Solvent Chloroform
Needle gauge size 24 G

PROCEDURE:

3 mL of clear transparent polymer solution was taken in a syringe with the gauge size of 24 G and fitted in
the syringe pump (KD Scientific, USA). The flow rate was set at 0.003 mL/min and the positive terminal
of the high voltage (Zeonics, India) was connected to the tip of the syringe needle. An aluminum foil was
stuck on metal collector which was kept 10 cm away from the tip of the needle and was grounded. The
voltages were set at specified rates (10 kV, 12 kV, 14 kV, 16 kV, 18 kV and 20 kV) and the formation of
Taylor cone at the tip of the needle was observed. The voltage was turned off after five minutes and the
aluminum foil was removed. The opaque deposit on the aluminum foil was analyzed using field emission
scanning electron microscope (FESEM, JEOL 6701 F, Japan).

A 0.5× 0.5 cm portion of the opaque area on the aluminum foil was cut and placed on as brass stub using
double sided carbon tape. A thin coat of platinum was sputtered at a current of 20 mA for 40 seconds. The
sputter coated samples were then introduced into the specimen chamber of the electron microscope through
the exchange chamber. An accelerating voltage of 3 kV was used to image the samples. All samples were
imaged using upper secondary electron detector.

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RESULTS:
The scanning electron micrographs of various samples are shown below:

Figure: Influence of voltage on fiber morphology of electro spun fibers

A: 10 kV, B: 12 kV, C: 14 kV, D: 16 kV, E: 18 Kv, F: 20 kV. Tip to target distance: 12 cm,
Concentration: 10%, Flow rate: 0.003 mL/min, Needle gauge size: 24 G, Solvent: chloroform

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SASTRA CeNTAB

INFERENCE:

From the SEM images, following inferences can be made:

They show the presence of large no. of defects at all defined voltages. There are beads in almost all the
micrographs from 10 kV to 20 kV.

At 20 kV, the size and number of beads have reduced dramatically. Fine timing of other parameters such
as flow rate, concentration etc. will provide defect free fibers of 0.19 µm. Very low voltage is insufficient
to cause splitting of jet into fibers.

REPORT:

PLGA fibers were electro spun at different voltages. Completely defect free fibers were not obtained and
best results were observed at 20 kV.

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Expt No: 05
Date:

INFLUENCE OF NEEDLE GAUGE SIZE ON FIBER MORPHOLOGY

AIM:

To study the influence of needle gauge size on fiber morphology during electrospinning.

MATERIALS:

PLGA (50:50) (Poly L- lactide co-glycolic acid) (Lakeshore Biomaterials, USA) was dissolved in
Chloroform (Merck, India) by constant stirring until clear solution of different needle gauge size ranges
from 18 G,20 G,22 G,23 G and 26 G were obtained. The operating parameters for various trials are
tabulated below.

Operating parameters Value

Applied voltage 20 kV
Flow rate 0.003 mL/min
Polymer concentration 10 %
Tip to target distance 10 cm
Solvent Chloroform
Needle gauge size 18 G, 20 G, 22 G, 23 G and 26 G

PROCEDURE:

3 mL of clear transparent polymer solution was taken in a syringe with different gauge sizes (18 G, 20 G,
22 G, 23 G and 26 G) and fitted in the syringe pump (KD Scientific, USA). The flow rate was set at 0.003
mL/min and the positive of the high voltage (Zeonics, India) was connected to the tip of the syringe
needle. An aluminum foil was stuck on metal collector which was kept 10 cm away from the tip of the
needle and was grounded. The voltage was gradually increased from 0 to 12 kV and the formation of
Taylor cone at the tip of the syringe was observed. The voltage was turned off after five minutes and the
aluminum foil was removed. The opaque deposit on the aluminum foil was analyzed using field emission
scanning electron microscope (FESEM, JEOL 6701 F, Japan).

A 0.5× 0.5 cm portion of the opaque area on the aluminum foil was cut and placed on a brass stub using
double sided carbon tape. A thin coat of platinum was sputtered at a current of 20 mA for 40 seconds. The
sputter coated samples were then introduced into the specimen chamber of the electron microscope through
the exchange chamber. An accelerating voltage of 3 kV was used to image the samples. All samples were
imaged using upper secondary electron detector.

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RESULTS:

The scanning electron micrographs of various samples are shown below:

Figure: Influence of voltage on morphology of electro spun fibers

A: 18G, B: 20G, C: 22G, D: 23G, E: 26G
Tip-target distance: 10 cm, Concentration: 10%, Flow rate: 0.003mL/min,
Solvent: chloroform Needle gauge size: 18 G, 20 G, 22 G,23 G and 26 G

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INFERENCE:

From the scanning electron micrographs, the following inference can be made.

Among the various needle gauge sizes, 18 G has the largest diameter and the fibers deposited are thick and
very sparse.20 G fibers have more beads than fibers and the fibers are thick as well.22 G fibers have
comparatively less number of defects and uniform fibers but large no. of defects. At 26 G, fibers are thin
and uniformly distributed.

REPORT:

PLGA fibers were electro spun at different needle gauge sizes. Completely defect free fibers were not
obtained. Best results could be observed at needle gauge size of 24 G.

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Expt No: 06
Date:

FABRICATION OF POLYMERIC MICROSPHERES BY SINGLE EMULSION

TECHNIQUE USING SDS AS SURFACTANT

AIM:

To fabricate polymeric microspheres using single emulsion technique with SDS as surfactant.

PRINCIPLE:

Sintered scaffolds are widely used in tissue engineering because the pores allow the diffusion of nutrients
from the media which is essential for cell survival. Polymeric microspheres can be used for making
sintered scaffolds. Microspheres were cast in a mold and then heated to high temperature which results in
formation of sintered scaffolds with highly inter connected pores. Polymers when dissolved in a suitable
organic solvent containing surfactant will result in formation of microspheres.

MATERIALS:

10 % PLGA (50:50) (Lakeshore Biomaterials, USA) was dissolved in Chloroform (Merck, India) and an
aqueous solution 1 % (w/v) of SDS (Sodium Dodecyl Sulphate) (Sigma chemicals, India) was used as
surfactant.

PROCEDURE:

5 mL of 10 % w/v solution of PLGA polymer solution was prepared by dissolving the 0.5 g of polymer in
chloroform. Then this polymer solution was added to the 300 mL of aqueous phase containing 1 % w/v
SDS under constant stirring using a mechanical stirrer. The emulsion was stirred continuously at 600 rpm
and 1200 rpm for 1 and 3 hrs each. The resultant product was collected by filtration.

The microspheres were then air-dried and characterized using Scanning Electron Microscopy (FESEM,
JEOL 6701F, Japan). A small quantity of sample was placed in brass stub using carbon paste. The sample
was then sputter coated with a thin layer of platinum at 20 mA current for40 seconds. The sputtered sample
was then introduced into the specimen chamber of electron microscope through the exchange chamber. An
accelerating voltage of 3kV was used to image the samples. All the images were taken using upper
secondary electron detector.

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RESULTS:

The scanning electron micrographs of various samples are shown below.

Figure: Fabrication of polymeric microspheres.

A: 600 rpm, 1hr; B: 600 rpm, 3hr; C: 1200 rpm, 1hr; D: 1200 rpm, 3hr;
Surfactant-SDS (Sodium Dodecyl Sulfate) (Sigma Chemicals, USA).

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SASTRA CeNTAB

INFERENCE:

From SEM images, the following inferences can be made.

At 600 rpm for 1 hour and 3 hours, the range of 60 µm – 130 µm diameters of spheres can be observed. At
1200 rpm, the time factor does not influence the dimension of microsphere. The range of diameter
observed was 20 µm – 80 µm. Hence for the same surfactant (SDS) the optimization of time and rpm was
done and it was found that time factor does not play a key role while increase in rpm gives better
dimension of microspheres.

REPORT:

By comparing the SEM micrograph results obtained from both the surfactants we report that the time
factor is not a major constraint and CTAB proved to be a better surfactant than SDS.

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Expt No: 07
Date:

FABRICATION OF POLYMERIC MICROSPHERES BY SINGLE EMULSION

TECHNIQUE USING CTAB AS SURFACTANT

AIM:

To fabricate polymeric microspheres using single emulsion technique using CTAB as surfactant

PRINCIPLE:

Sintered scaffolds are widely used in tissue engineering because the pores allow the diffusion of nutrients
from the media which is essential for cell survival. Polymeric microspheres can be used for making
sintered scaffolds. Microspheres were cast in a mold and then heated to high temperature which results in
formation of sintered scaffolds with highly inter connected pores. Polymers when dissolved in a suitable
organic solvent containing surfactant will result in formation of microspheres.

MATERIALS:

10 % PLGA (50:50) (Lakeshore Biomaterials, USA) was dissolves in Chloroform (Merck, India) and an
aqueous solution 1 % (w/v) of Cetyl Tri-methyl ammonium used as surfactant.

PROCEDURE:

5 mL of 10 % w/v solution of PLGA polymer solution was prepared by dissolving the 0.5 g of polymer in
chloroform. Then this polymer solution was added to the 200 mL of aqueous phase containing 1 %w/v
CTAB under constant stirring using a high torque mechanical stirrer. The emulsion was stirred
continuously at 600 rpm and 1200 rpm for 1 and 3 hrs each. The resultant product was collected by
filtration.

The resultant product was collected by filtration. The microspheres were then air-dried and characterized
using Scanning Electron Microscopy (FESEM, JEOL 6701F, Japan). A small quantity of sample was
placed in brass stub using carbon paste. The sample was then sputter coated with a thin layer of platinum at
20 mA current for 40 seconds. The sputtered sample was then introduced into the specimen chamber of
electron microscope through the exchange chamber. An accelerating voltage of 3 kV was used to image
the samples. All the images were taken using upper secondary electron detector.

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OBSERVATION:

Fine particles were observed to be deposited at the bottom of the beaker.

RESULTS:

The scanning electron micrographs of various samples are shown below.

Figure: Fabrication of polymeric microspheres.

A: 600 rpm, 1hr, B: 1200 rpm, 3hr, C: 600 rpm, 1hr, D: 1200 rpm, 3hr. Surfactant-CTAB (Cetyl
Trimethyl Ammonium Bromide) (Sigma Chemicals, USA).

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INFERENCE

From SEM images, the following inferences can be made.

On par with SDS, CTAB gives better and finer microspheres. At 600 rpm for 1 hour, the range of 50 µm –
80 µm microspheres were observed and when they were prolonged till 3 hrs, there was a reduction in
microsphere dimension (40-70µm) at 1200 rpm the dimension further reduced to 30-50 mm and the
number of microspheres with smaller dimensions was less at one hour stirring and it gradually increased at
3 hr mechanical stirring.

REPORT:

Polymeric microspheres were formed by using CTAB as surfactant at different rpm and time. Completely
defect free spheres were not obtained. Best results were obtained at 1200 rpm (when kept for 3hrs).
Overall, CTAB was able to form more uniform microspheres than SDS.

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Expt No: 8(a)
Date:

FABRICATION OF POROUS SCAFFOLD BY PARTICULATE LEACHING

USING SODIUM CHLORIDE AS POROGEN

AIM:

To fabricate porous scaffold by particulate leaching using sodium chloride as porogen

MATERIALS:

1 g of Poly (L-Lactide-Co-Glycolic acid) (Lakeshore Biomaterials, USA) was dissolved in 10 ml of

chloroform by constant stirring until a clear transparent solution was obtained. NaCl crystals of average
size 32 and 64 µ were used as porogen with polymer: porogen ratio of 1:5, 1:10 respectively.

PRINCIPLE:

Porosity decides the success of tissue engineering scaffold. Particulate leaching is one of the simplest
methods that are used for the fabrication of porous scaffold. The pore size and porosity of the scaffold can
be controlled by the porogen size and ratio of the polymer and porogen. Interconnected pores are preferred
for tissue engineering applications. Polymer was dissolved in organic solvent and the appropriate quantity
of water soluble porogen (salt, sucrose) is dispersed in the polymer solution. The polymer solution was
cast into thin film. After the organic solvent was evaporated, the scaffold is placed in warm water which
will causes the porogen to dissolve leaving behind pores in the polymer matrix.

PROCEDURE:

10 %( w/v) solution of PLGA in chloroform was prepared. NaCl crystals were added to the polymer
solution with Polymer: porogen ratio of 1:5, 1:10 respectively. The mixture was stirred well to obtain a
homogenous suspension. The sample was poured into a clean Petri dish lined with non-sticky polymer film
(Bytac) without any air bubbles. The solvent was allowed to evaporate by air drying. The dried polymer
film was placed in 250 ml of water at room temperature. The salt was allowed to leach for two days and
the water was replaced for every 24 hrs. After 2 days the polymer scaffold was rinsed well with double-
distilled water and allowed to air-dry. The dried scaffold was then subjected to scanning electron
microscopic analysis.

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RESULTS:

The scanning electron micrographs of the various samples are shown below.

Figure: SEM images for PLGA porous scaffold after leaching.

A & B- polymer: porogen ratio of 1:5 with 32 & 64 µ size respectively and C & D- polymer:
porogen ratio of 1:10 with 32 & 64 µ size respectively. Porogen- NaCl crystals

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SASTRA CeNTAB

INFERENCE:

From the scanning electron micrographs, the following inferences can be made:

In all the SEM images irregular pores of non-uniform diameter can be seen. For NaCl crystals of 32 µ
size, average diameter of the pores is lesser than that formed by NaCl crystals of 64 µ size. The number of
pores increases as NaCl: polymer is increased from 1:5 to 1:10. For higher amount of NaCl, the salt forms
a continuous clustered distribution within PLGA. This leads to better formation of water filled pores within
the polymer and hence better access of water to interiorly placed NaCl crystals. When comparing to
sodium citrate crystals, pore formation is better for NaCl crystals owing to higher solubility in water and
hence better leaching.

REPORT:

Porous scaffolds of PLGA using NaCl crystals as porogen were obtained for crystal sizes of 32 and 64 µ
respectively for polymer: porogen ratios of 1:5 and 1:10. Completely defect-free scaffolds were not
obtained. Pores obtained were irregular, and of non-uniform size and distribution. Best results were
obtained for NaCl of size 32/64 microns, 1:10 polymer: porogen ratio.

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SASTRA CeNTAB

Expt No: 8(b)

Date:

FABRICATION OF POROUS SCAFFOLD BY PARTICULATE LEACHING

USING SODIUM CITRATE AS POROGEN

AIM:

To fabricate porous scaffold by particulate leaching using sodium citrate as porogen.

MATERIALS:

1 g of Poly (L-Lactide-Co-Glycolic acid) (Lakeshore Biomaterials, USA) was dissolved in 10 ml of

chloroform (Merck, India) by constant stirring until a clear transparent solution was obtained. Sodium
citrate crystals of average size 32 and 64 microns were used as porogen with polymer: porogen ratio of 1:5
and 1:10 respectively.

PRINCIPLE:

Porosity decides the success of tissue engineering scaffold. Particulate leaching is one of the simplest
methods that are used for the fabrication of porous scaffold. The pore size and porosity of the scaffold can
be controlled by the porogen size and ratio of the polymer and porogen. Interconnected pores are preferred
for tissue engineering applications. Polymer was dissolved in organic solvent and the appropriate quantity
of water soluble porogen (salt, sucrose,) is dispersed in the polymer solution. The polymer solution was
cast into thin film. After the organic solvent was evaporated, the scaffold is placed in warm water which
will causes the porogen to dissolve leaving behind pores in the polymer matrix.

PROCEDURE:

10 % (w/v) solution of PLGA in chloroform was prepared. Sodium citrate crystals were added to the
polymer solution with Polymer: porogen as 1:5 and 1:10 respectively. The mixture was stirred well to
obtain a homogenous suspension. The sample was poured into a clean Petri dish lined with non-sticky
polymer film (Bytac) without any air bubbles. The solvent was allowed to evaporate by air drying. The
dried polymer film was placed in 250 ml of water at room temperature. The salt was allowed to leach for
two days and the water was replaced for every 24 hrs. After 2 days the polymer scaffold was rinsed well
with double-distilled water and allowed to air-dry. The dried scaffold was then subjected to scanning
electron microscopic analysis.

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SASTRA CeNTAB

RESULTS:

The scanning electron micrographs of the various samples are shown below.

Figure: SEM images of PLGA porous scaffold after leaching.

A & B-polymer: porogen ratio of 1:5 with 32 & 64 µ size respectively and C & D-polymer: porogen
ratio of 1:10 with 32 & 64 µ size respectively. Porogen- Sodium Citrate.

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SASTRA CeNTAB

INFERENCE:

From the scanning electron micrographs, the following inferences can be made:

In all the SEM images irregular pores of non-uniform diameter can be seen. For Sodium citrate crystals of
32 µ size, average diameter of the pores is lesser than that formed by Sodium citrate crystals of 64 microns
size. The number of pores increases as Sodium citrate: polymer is increased from 1:5 to 1:10. For higher
amount of Sodium citrate; the salt forms a continuous clustered distribution within PLGA. This leads to
better formation of water filled pores within the polymer and hence better access of water to interiorly
placed Sodium citrate crystals. When comparing to sodium citrate crystals, pore formation is better for
NaCl crystals owing to higher solubility in water and hence better leaching.

REPORT:

Porous scaffolds of PLGA using Sodium citrate crystals as porogen were obtained for crystal sizes of 32
and 64 microns respectively for polymer:porogen ratios of 1:5 and 1:10. Completely defect-free scaffolds
were not obtained. Pores obtained were irregular, and of non-uniform size and distribution. Best results
were obtained for Sodium citrate crystals of 32/64 µ with 1:10 polymer: porogen ratio.

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