# PLGA Microspheres Incorporated Gelatin Scaffold: Microspheres Modulate Scaffold Properties

Authors: Indranil Banerjee; Debasish Mishra; Tapas K. Maiti
Journal: International Journal of Biomaterials
 (2009)

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## Abstract

Freeze drying is one of the popular methods of fabrication for poly(lactide-co-glycolide) (PLGA)  microspheres incorporated polymer scaffolds. However, the consequence of  microspheres incorporation on physical and biological properties of scaffold has not been studied yet. In this study, attempt has been made to characterize the effect of PLGA microsphere incorporation on the physical properties of freeze-dried gelatin scaffold and its influence on cytocompatibility. Scaffolds loaded with varying amount of PLGA microspheres (10%, 1%, 0.1% w/w) were subjected to microarchitecture analysis, swelling, porosity, mechanical properties, biodegradation, cell adhesion, and cell proliferation studies. Results revealed that an increase in percentage loading of microspheres reduced the pore size and uniformity of the pore structure. Moreover, loading of PLGA microspheres up to 1% w/w significantly increased porosity, swelling, and mechanical properties of the scaffold but variations were not proportional for 10% w/w loading. Results also showed that PLGA microspheres have no significant effect on cell adhesion but influenced the growth kinetics.


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## Body

## 1. Introduction

Freeze drying is  one of the popular methods of  scaffold
formation in tissue engineering. This is a method of choice especially for the
preparation of natural polymeric scaffold like gelatin, chitosan, collagen, and
so forth
[1–3]. Freeze-drying
method involves the formation of ice crystals inside polymer solution during
freezing, and those ice crystals act as porogens during lyophilization that
results a porous three-dimensional polymer scaffold. In case of freeze drying,
any physical or chemical factors that can change the pattern of ice crystal
formation and  distribution (e.g., freezing
temperature [4]) can change scaffold
microarchitecture.Recently,
effort has been made to fabricate a smart controlled release tissue engineering
matrix by incorporating therapeutics loaded microspheres into polymer solution
followed by freeze drying [5]. Objective of such efforts is the efficient and
controlled delivery of therapeutic molecules during tissue remodeling and
regeneration
[6–10]. Among all
these microsphere-based controlled release systems, PLGA microspheres
have got an edge over the
others because of its biocompatibility and known efficiency to deliver a number
of growth factors, proteins, or drugs in a time dependent manner both in vitro
and in vivo
[11–13]. A couple of
literatures have clearly stated that therapeutics molecule loaded PLGA
microsphere embedded scaffold can be fabricated by freeze drying, and those
scaffolds can act as a controlled release engineered matrix. In all these
efforts, the main aim was to achieve the desired therapeutic benefit but
variation in physical properties of the scaffold with PLGA microspheres
incorporation was not properly characterized
[14–16]. This
overlooked phenomenon may be crucial for the stability and performance of PLGA
microspheres loaded freeze-dried natural polymeric scaffold. PLGA is
hydrophobic as compared to many natural biopolymers like gelatin, alginate
[17–19]; therefore,
presence of PLGA microspheres inside the hydrophilic polymers at the time of
freezing can alter the size and distribution of ice crystals formed and such
alteration can change the microarchitecture of the freeze-dried scaffold.
Furthermore, microspheres may cause changes in the mechanical properties of the
scaffold [20]. In this present work, an attempt has been taken to characterize
the effect of PLGA microspheres incorporation on physical properties of
freeze-dried scaffold and its impact on the performance of the cells cultured
on the scaffold. For this purpose, PLGA (65:35) microspheres loaded gelatin
scaffold made by freeze drying was chosen as model. Gelatin is widely used in
tissue engineering as scaffold materials because of its biocompatibility, low
immunogenicity, and biodegradability [21]. Influence of PLGA microsphere
incorporation on the physical properties of the freeze-dried gelatin scaffold
was studied by analyzing the changes in scaffold microarchitecture, porosity,
swelling, mechanical strength and biodegradation with varying amount of PLGA
microspheres loading. Impact of PLGA microsphere incorporation in the above
mentioned scaffolds on cellular performance was further characterized by
studying the adhesion and growth kinetics of murine fibroblast L929 cells on
these scaffolds.


## 2. Materials and Methods

Gelatin, glutaraldehyde, poly(DL-lactide-co-glycolide)
PLGA (65:35) were  purchased from
Sigma-Aldrich (St. Louis, MO, USA). DMEM was from GibcoBRL (Grand Island, NY, USA). FBS was
obtained from Hyclone (South Logan, UT, USA). Murine L929 cells were obtained from NCCS Pune, India.
Other chemicals used were purchased from local
vendors.


### 2.1. Preparation of PLGA and Gelatin Film

A 5% (w/v) PLGA (65:35) solution in dichloromethane was
prepared and cast into Petri dishes followed by solvent evaporation at room
temperature. The film formed was vacuum dried for 48 hours and kept in
desiccators until further use. In case of preparation of gelatin film, 3%
gelatin solution was crosslinked with glutaraldehyde (0.05%) and cast on Petri
dish for film formation.


### 2.2. Contact Angle Measurement of PLGA and Gelatin Film

To
keep an account of hydrophobicity of the two polymers used in this model, the
advancing contact angles of three replicates of PLGA (65:35) and gelatin
(3% w/v, glutaraldehyde crosslinked) films were determined using a dynamic
contact angle meter and tensiometer (model: D CAT, 11 DataPhysics). Briefly,
each sample was attached to a microbalance and immersed into the wetting medium
(deionised water). The wetting force at the solid/liquid/vapor interface was
automatically recorded via an electrobalance as function of both time and
immersion depth; this was converted into the advancing
contact angle.


### 2.3. Preparation of PLGA Microspheres

PLGA
microspheres were prepared using emulsion-solvent evaporation method. In brief,
200 mg of PLGA (65:35) was dissolved in 4 mL methylene chloride. The solution was
mixed with 20 mL aqueous solution of 1% poly vinyl alcohol (PVA) and sonicated
using an ultraprobe adopting regular pattern of ultrasonic vibration for 30
seconds followed by a pause of 30 seconds thrice (at 15 watt) in an ice bath.
The resulting emulsion was stirred for 3 hours at room temperature followed by
methylene chloride evaporation. The microsphere prepared in this way was collected
by centrifugation at 10000 g. Then, it was washed thrice with PBS to remove
excess PVA and finally lyophilized to get
powder.


### 2.4. Characterization of Microsphere

Lyophilized powdered
microspheres were examined by a scanning electron microscope (model: JEOL
JSM-5800). Prior to observation, samples were
sputter coated with gold, and the imaging was conducted
at an accelerating voltage of 10 kV. At least 100 particles were examined to
get average diameter and particle size
distribution.


### 2.5. Preparation of PLGA Microspheres Incorporated Gelatin Scaffolds

Aqueous
suspension of PLGA microspheres 0.1% (w/v) was mixed proportionally to 3%
gelatin solution under constant stirring to
prepare three different blends of
PLGA and gelatin (10%, 1%, 0.1% PLGA microspheres
with respect  to gelatin weight). 2.5 mL of each suspension was cast in Petri
dish (60 mm diameter) in presence of 0.05% glutaraldehyde. All the resulting
suspensions were allowed to crosslink for 12 hours at room temperature. The
crosslinked hydrogels were frozen
at−20°C for 12 hours followed by 24 hours
lyophilization.


### 2.6. Study of Scaffold Morphology

Scaffold
morphology was analyzed using phase contrast microscope (Olympus CK X 41) and
scanning electron microscope for elucidation of influence of PLGA incorporation
on scaffold microarchitecture. Prior to observation through scanning electron
microscope scaffolds were sputter coated with gold and analyzed at an
accelerating voltage of 20 kV. The objective of the study was to characterize
distribution of microspheres inside the scaffold and the effect of microsphere
incorporation on pore size of the scaffold. For each analysis, at least 50
pores were examined.


### 2.7. Study of the Water Uptake Ability (Swelling Test)

Effect of microsphere incorporation on water absorption capacity was determined by
swelling the scaffolds in water at room temperature. A known weight of scaffold
material was placed in water and after 24 hours incubation, its wet weight was
determined. The percentage water absorption (Wsw) of the scaffold was
calculated from the expression (1)Wsw=[(W24h−W0)W0]×100, where W24h represents the wet weight
of scaffold after 24 hours of incubation, and W0 is the initial weight of the scaffolds. The values
were expressed as mean ± SD (n=3).


### 2.8. Porosity Analysis

Variation
in porosity of the scaffold due to microspheres loading was determined using a
mercury intrusion porosimeter (Poremaster, Quantachrome). In brief, scaffolds
were degassed under vacuum and placed inside the penetrometer. Analysis was
done at low pressure using mercury keeping 10-second equilibration time at each
pressure step.


### 2.9. Study of Mechanical Properties

Mechanical
properties of the scaffolds were tested using a universal testing machine (Hounsfield
H25kS, Surrey, England). 30 × 10 × 1 mm scaffold pieces were subjected for tensile
strength measurement at dry condition using a cross-head speed 1 mm/min. Tensile
strength and percentage elongation at break were recorded. Data were analyzed
using Q.MAT 3.1 software. The values were expressed
as mean ± SD (n=3).


### 2.10. Biodegradation Study

To
study the effect of  microspheres loading on in vitro biodegradation, scaffolds
were incubated in PBS (pH 7.4) for 10 days at 37°C. The biodegradation was calculated
in terms of percentage (%) weight loss using the formula (2)[(W0−Wn)W0×100], where
Wn is the dry
weight of scaffold after “n” days incubation in PBS, and W0 is its initial weight. The values were expressed as
the mean ± SD (n=3).


### 2.11. Cell Adhesion Study

Cell
adhesion study on the scaffolds was performed using mouse fibroblast L929
cells. In brief, murine L929 cells were cultured in DMEM containing 10% FBS in
a 5% CO2 incubator at 37°C. At confluence, cells were harvested from the flask by
trypsinization, and  5×104 cells/cm2 were seeded on each
scaffold of 1 cm × 1 cm × 0.1 cm dimension. Cells were allowed to adhere on the
scaffold at 37°C for 4 hours. Cell adhesion on the scaffold was assessed by MTT
method [22]. The values were expressed as
mean ± SD (n=3).


### 2.12. Cell Proliferation Study

Mouse
fibroblast L929 cells were cultured in DMEM containing 10% FBS in a 5% CO2 incubator at 37°C. At
confluence, cells were harvested from the flask by trypsinization, and  4×105cells/cm2 were seeded on each
type of scaffolds of 1 cm × 1 cm ×
0.1 cm dimension. Scaffolds seeded with cells
were kept days in 5% CO2 incubator at 37°C, and cells were allowed to grow up to 7 days. Media was
replaced in each alternative day. After definite intervals, scaffolds were
taken out and cell growth was estimated using MTT assay. All the experiments
were done in triplicate.


### 2.13. Study of Cell Morphology

To
check the influence of incorporated PLGA microspheres on cell growth and cell
morphology, cell-seeded scaffold (after 3 days of initial seeding) was taken
and subjected for analysis using scanning electron microscopy. Cells were fixed
with 2.5% glutaraldehyde for 4 hours at room temperature, and it was then
serially dehydrated using alcohol, sputter coated with gold, and then examined
by a scanning electron microscope
(model: JEOL JSM-5800).


### 2.14. Statistical Analysis

Experiments
were run in triplicate for each sample. All data were expressed as mean ±
standard deviation (SD) forn=3. Student’s t-test analysis was done to
assess the statistical significance of
the data  sets.


## 2.1. Preparation of PLGA and Gelatin Film

A 5% (w/v) PLGA (65:35) solution in dichloromethane was
prepared and cast into Petri dishes followed by solvent evaporation at room
temperature. The film formed was vacuum dried for 48 hours and kept in
desiccators until further use. In case of preparation of gelatin film, 3%
gelatin solution was crosslinked with glutaraldehyde (0.05%) and cast on Petri
dish for film formation.


## 2.2. Contact Angle Measurement of PLGA and Gelatin Film

To
keep an account of hydrophobicity of the two polymers used in this model, the
advancing contact angles of three replicates of PLGA (65:35) and gelatin
(3% w/v, glutaraldehyde crosslinked) films were determined using a dynamic
contact angle meter and tensiometer (model: D CAT, 11 DataPhysics). Briefly,
each sample was attached to a microbalance and immersed into the wetting medium
(deionised water). The wetting force at the solid/liquid/vapor interface was
automatically recorded via an electrobalance as function of both time and
immersion depth; this was converted into the advancing
contact angle.


## 2.3. Preparation of PLGA Microspheres

PLGA
microspheres were prepared using emulsion-solvent evaporation method. In brief,
200 mg of PLGA (65:35) was dissolved in 4 mL methylene chloride. The solution was
mixed with 20 mL aqueous solution of 1% poly vinyl alcohol (PVA) and sonicated
using an ultraprobe adopting regular pattern of ultrasonic vibration for 30
seconds followed by a pause of 30 seconds thrice (at 15 watt) in an ice bath.
The resulting emulsion was stirred for 3 hours at room temperature followed by
methylene chloride evaporation. The microsphere prepared in this way was collected
by centrifugation at 10000 g. Then, it was washed thrice with PBS to remove
excess PVA and finally lyophilized to get
powder.


## 2.4. Characterization of Microsphere

Lyophilized powdered
microspheres were examined by a scanning electron microscope (model: JEOL
JSM-5800). Prior to observation, samples were
sputter coated with gold, and the imaging was conducted
at an accelerating voltage of 10 kV. At least 100 particles were examined to
get average diameter and particle size
distribution.


## 2.5. Preparation of PLGA Microspheres Incorporated Gelatin Scaffolds

Aqueous
suspension of PLGA microspheres 0.1% (w/v) was mixed proportionally to 3%
gelatin solution under constant stirring to
prepare three different blends of
PLGA and gelatin (10%, 1%, 0.1% PLGA microspheres
with respect  to gelatin weight). 2.5 mL of each suspension was cast in Petri
dish (60 mm diameter) in presence of 0.05% glutaraldehyde. All the resulting
suspensions were allowed to crosslink for 12 hours at room temperature. The
crosslinked hydrogels were frozen
at−20°C for 12 hours followed by 24 hours
lyophilization.


## 2.6. Study of Scaffold Morphology

Scaffold
morphology was analyzed using phase contrast microscope (Olympus CK X 41) and
scanning electron microscope for elucidation of influence of PLGA incorporation
on scaffold microarchitecture. Prior to observation through scanning electron
microscope scaffolds were sputter coated with gold and analyzed at an
accelerating voltage of 20 kV. The objective of the study was to characterize
distribution of microspheres inside the scaffold and the effect of microsphere
incorporation on pore size of the scaffold. For each analysis, at least 50
pores were examined.


## 2.7. Study of the Water Uptake Ability (Swelling Test)

Effect of microsphere incorporation on water absorption capacity was determined by
swelling the scaffolds in water at room temperature. A known weight of scaffold
material was placed in water and after 24 hours incubation, its wet weight was
determined. The percentage water absorption (Wsw) of the scaffold was
calculated from the expression (1)Wsw=[(W24h−W0)W0]×100, where W24h represents the wet weight
of scaffold after 24 hours of incubation, and W0 is the initial weight of the scaffolds. The values
were expressed as mean ± SD (n=3).


## 2.8. Porosity Analysis

Variation
in porosity of the scaffold due to microspheres loading was determined using a
mercury intrusion porosimeter (Poremaster, Quantachrome). In brief, scaffolds
were degassed under vacuum and placed inside the penetrometer. Analysis was
done at low pressure using mercury keeping 10-second equilibration time at each
pressure step.


## 2.9. Study of Mechanical Properties

Mechanical
properties of the scaffolds were tested using a universal testing machine (Hounsfield
H25kS, Surrey, England). 30 × 10 × 1 mm scaffold pieces were subjected for tensile
strength measurement at dry condition using a cross-head speed 1 mm/min. Tensile
strength and percentage elongation at break were recorded. Data were analyzed
using Q.MAT 3.1 software. The values were expressed
as mean ± SD (n=3).


## 2.10. Biodegradation Study

To
study the effect of  microspheres loading on in vitro biodegradation, scaffolds
were incubated in PBS (pH 7.4) for 10 days at 37°C. The biodegradation was calculated
in terms of percentage (%) weight loss using the formula (2)[(W0−Wn)W0×100], where
Wn is the dry
weight of scaffold after “n” days incubation in PBS, and W0 is its initial weight. The values were expressed as
the mean ± SD (n=3).


## 2.11. Cell Adhesion Study

Cell
adhesion study on the scaffolds was performed using mouse fibroblast L929
cells. In brief, murine L929 cells were cultured in DMEM containing 10% FBS in
a 5% CO2 incubator at 37°C. At confluence, cells were harvested from the flask by
trypsinization, and  5×104 cells/cm2 were seeded on each
scaffold of 1 cm × 1 cm × 0.1 cm dimension. Cells were allowed to adhere on the
scaffold at 37°C for 4 hours. Cell adhesion on the scaffold was assessed by MTT
method [22]. The values were expressed as
mean ± SD (n=3).


## 2.12. Cell Proliferation Study

Mouse
fibroblast L929 cells were cultured in DMEM containing 10% FBS in a 5% CO2 incubator at 37°C. At
confluence, cells were harvested from the flask by trypsinization, and  4×105cells/cm2 were seeded on each
type of scaffolds of 1 cm × 1 cm ×
0.1 cm dimension. Scaffolds seeded with cells
were kept days in 5% CO2 incubator at 37°C, and cells were allowed to grow up to 7 days. Media was
replaced in each alternative day. After definite intervals, scaffolds were
taken out and cell growth was estimated using MTT assay. All the experiments
were done in triplicate.


## 2.13. Study of Cell Morphology

To
check the influence of incorporated PLGA microspheres on cell growth and cell
morphology, cell-seeded scaffold (after 3 days of initial seeding) was taken
and subjected for analysis using scanning electron microscopy. Cells were fixed
with 2.5% glutaraldehyde for 4 hours at room temperature, and it was then
serially dehydrated using alcohol, sputter coated with gold, and then examined
by a scanning electron microscope
(model: JEOL JSM-5800).


## 2.14. Statistical Analysis

Experiments
were run in triplicate for each sample. All data were expressed as mean ±
standard deviation (SD) forn=3. Student’s t-test analysis was done to
assess the statistical significance of
the data  sets.


## 3. Results

### 3.1. Contact Angle Measurement of PLGA and Gelatin Film

Contact
angle is a measure of hydrophobicity of a material. The higher the contact
angle is, the higher the hydrophobicity of the material is. Here, the
advancing contact angle measured for gelatin film was55.2±1.3° and that of
PLGA film was 71.2±0.4°. This result confirms the significant difference in
the hydrophobicity of two used polymers.


### 3.2. Characterization of PLGA Microsphere

The
PLGA microspheres were prepared by emulsion—solvent
evaporation method. The microspheres were spherical in shape and had a smooth
surface as judged by SEM
(see Figure1).
Size distribution of microsphere was
found in the range of 1–15 μm (96% of
total population among which 53%
were in the range of
2–5 μm (data not shown)).Figure 1
Scanning electron
micrograph of PLGA microsphere formed by
emulsion-solvent evaporation method.


### 3.3. Microarchitecture of PLGA Microsphere Loaded Gelatin Scaffold

It
is evident from the phase contrast and SEM micrographs
(see Figures2 and
3)
that PLGA microspheres were uniformly distributed through out the gelatin
matrix irrespective of the amount of microsphere added. Incorporation of PLGA
microspheres during fabrication of gelatin scaffold by freeze—drying has
significant effect on the overall microarchitecture of the scaffold. Effect of
microsphere doping on scaffold pore size was summarized in Table 1. Among
these four different scaffolds, control gelatin scaffold has regular pore
structure (average pore diameter 160 μm). Loading of 0.1% microspheres reduced
the average pore size to 110 μm. In case of 1% w/w PLGA microsphere loaded
scaffold, there were two set of pores (average diameter 150–120 μm which is
60% of total abundance and average diameter 50–30 μm which is 40%
of total abundance). 10% w/w PLGA microsphere loaded scaffold has no regular
pore structure (pore diameter varied
from 30–150 μm).Table 1
Effect of microsphere doping on scaffold
microarchitecture. For each
scaffold, 50 pores were analyzed to get the
average pore size.
%age of PLGA microsphere
doping (w/w) in gelatinAverage pore size (μm)0%1600.1%1101%[150–120], [50–30]10%150–30Phase contrast
micrograph of PLGA microsphere incorporated gelatin scaffolds. (a) Pure gelatin
scaffold, (b) 0.1% w/w PLGA microsphere
incorporated scaffold, (c) 1% w/w PLGA
microsphere incorporated scaffold, and (d) 10% w/w PLGA microsphere incorporated
scaffold. Photographs were taken at 40X.
(a)(b)(c)(d)Scanning electron micrograph of microspheres incorporated gelatin scaffolds.
(a) Pure gelatin scaffold, (b) 0.1% w/w PLGA microsphere incorporated scaffold, (c) 1% w/w
PLGA microsphere incorporated scaffold, and (d) 10% w/w PLGA microsphere
incorporated scaffold.
(a)(b)(c)(d)


### 3.4. Swelling Property

The
water uptake ability of the scaffolds was in the range of 1400–2100% (see
Figure4). It was expected that incorporation of hydrophobic PLGA microsphere would
reduce the water uptake in a dose dependent manner. However, no such trend was
observed. Result showed that 1% w/w PLGA microspheres loaded scaffold has the highest
swelling properties (2144%), and 10% PLGA microsphere loaded scaffold has shown
the least swelling of (1435.33%).Figure 4
Effect of PLGA microsphere incorporation on the swelling property of gelatin
scaffold. Values are mean ± S.D. (n=3). P<.005*, compared to gelatin
scaffold having no PLGA microsphere.


### 3.5. Porosity

All
the scaffolds had porosity in the range of 25 to 42 percent (see
Figure5).
Porosity of 10% PLGA microsphere loaded scaffold (26.13%) is significantly less
than that of control (31.83%), where 0.1% and 1% w/w PLGA scaffold has porosity
greater than control (41.5% and 38.53%, resp.).Figure 5
Effect of PLGA
microsphere incorporation on scaffold porosity. Values are mean ± S.D. (n=3). P<.05*, compared to gelatin scaffold having no
PLGA microsphere.


### 3.6. Mechanical Properties of the Scaffold

PLGA microspheres loaded
scaffolds were tested for tensile properties in dry condition. Tensile  strength
(see Figure6) of 0.1% and 1% w/w PLGA microsphere loaded scaffolds
(0.448 MPa
and 0.406 MPa, resp.)  was
significantly higher than that of control
(0.228 MPa) and among all, 0.1% has
the highest tensile strength. Although tensile strength of 10% w/w PLGA
microsphere loaded is higher (0.280 MPa) than that of control, the value was not
significant. From the data, it was evident that there was no clear relation
between the weight percentages of PLGA microspheres doping and tensile strength
of the scaffold. Trend observed in percentage elongation at break is almost
similar to the previous one, that is, percentage elongation at break for 0.1%
and 1% w/w PLGA microsphere loaded scaffolds is significantly higher than that
of control (see Figure 7). Percentage elongation at break for 0.1% and 1% w/w
PLGA microspheres doped scaffold is 2.20 and 2.49 fold greater than that of
control, where it is 0.96 fold less for 10% w/w PLGA doped scaffold.Figure 6
Effect of PLGA microsphere incorporation on the tensile strength of gelatin
scaffold. Values are mean ± S.D. (n=3). P<.05*, compared to gelatin
scaffold having no PLGA microsphere.Figure 7
Effect of PLGA microsphere incorporation on percentage elongation at break
of gelatin scaffold. Values are mean ± S.D. (n=3). P<.05*, compared
to gelatin scaffold having no PLGA microsphere.


### 3.7. Biodegradation

Biodegradation
study showed that incorporation of microspheres in the scaffold up to a certain
extent (up to 1% w/w) has no significant effect on early phase of degradation (see
Figure8). The extent of biodegradation after 48 hours was within 18-19% for
all three scaffolds except 10% w/w PLGA microsphere loaded scaffold (25.6%
biodegradation). However, with the progress of time, a variation in extent of
biodegradation was observed. Result revealed that the rate and total extent of
biodegradation were
higher for scaffolds having higher PLGA microsphere content. This may be
because of the time dependant degradation of PLGA itself by means of hydrolysis
[23] which rendered the scaffold microenvironment acidic. Under these circumstances
chemical intervention become predominant over the physical influence of the
hydrophobic microsphere on scaffold degradation; therefore scaffolds having
higher PLGA content showed higher degradation.Figure 8
Effect of PLGA microsphere incorporation on biodegradation of gelatin
scaffold. Degradation was studied for 10 days by incubating the scaffolds in
PBS (pH-7.4) at 37°C. Values are mean ± S.D. (n=3). (A1) Gelatin scaffold, (A2)
0.1% w/w PLGA microsphere loaded scaffold, (A3) 1% w/w PLGA microsphere loaded
scaffold, and (A4) 10% w/w PLGA microsphere loaded
scaffold *.


### 3.8. Study of Cell Adhesion

Cell
adhesion study showed that incorporation has no significant effect on cell
adhesion (see Figure9). All the scaffolds showed comparable cell adhesion
properties. This might be because the total number of microspheres present in
the upper surface is not sufficient to exert any significant effect on cell
adhesion.Figure 9
Effect of PLGA microsphere
incorporation on cell adhesion after 4 hours of cell seeding. Values are mean
± S.D. (n=3).


### 3.9. Study of Cell Proliferation

Cell
proliferation study (see Figure10) showed that there was not much variation in
cell proliferation for the first three days of culture. Growth of the cells on
all three types scaffolds loaded with varying amount of PLGA microspheres was
similar to that of control. Highest cell proliferation was achieved at day 5
for all type of scaffolds; however at day 5, the extent of cell proliferation
on 0.1% and 1% w/w PLGA microspheres loaded scaffolds was significantly higher than that of
control; on the other hand cell proliferation on 10% w/w PLGA microsphere
incorporated scaffold was less than not only to that of 0.1% and 1% w/w PLGA
microspheres loaded scaffold but even significantly less than the control. The
same trend was observed up to day 7 but extent of proliferation was decreased
compared to day 5 for all set of scaffolds.Figure 10
Effect of PLGA microsphere incorporation on murine L929 cells proliferation
on gelatin scaffold. Proliferation was studied for 7 days. Values are mean
± S.D. (n=3). (A1) Gelatin scaffold, (A2)
0.1% w/w PLGA microsphere loaded
scaffold, (A3) 1% w/w PLGA microsphere loaded scaffold, and (A4) 10% w/w PLGA
microsphere loaded scaffold.  P<.05*, compared to gelatin scaffold
having no PLGA microsphere.


### 3.10. Study of Cell Morphology

From
the scanning electron micrographs of cells cultured on microspheres loaded
scaffold (see Figure11), it was evident that microspheres  have no adverse
effect on cell growth. However, a stricken observation was that cells cultured
on 10% w/w PLGA microsphere loaded scaffold has an elongated structure compared
to the cells cultured on other scaffolds.Scanning
electron micrograph of mouse L929 cells cultured on PLGA microsphere
incorporated gelatin scaffold after 3 days of initial seeding. (a) Gelatin scaffold,
(b) 0.1% w/w PLGA microsphere loaded scaffold, (c) 1% w/w PLGA microsphere
loaded scaffold, and (d) 10% w/w PLGA microsphere
loaded scaffold.P<.05*,
compared to gelatin scaffold having no PLGA
microsphere.
(a)(b)(c)(d)


## 3.1. Contact Angle Measurement of PLGA and Gelatin Film

Contact
angle is a measure of hydrophobicity of a material. The higher the contact
angle is, the higher the hydrophobicity of the material is. Here, the
advancing contact angle measured for gelatin film was55.2±1.3° and that of
PLGA film was 71.2±0.4°. This result confirms the significant difference in
the hydrophobicity of two used polymers.


## 3.2. Characterization of PLGA Microsphere

The
PLGA microspheres were prepared by emulsion—solvent
evaporation method. The microspheres were spherical in shape and had a smooth
surface as judged by SEM
(see Figure1).
Size distribution of microsphere was
found in the range of 1–15 μm (96% of
total population among which 53%
were in the range of
2–5 μm (data not shown)).Figure 1
Scanning electron
micrograph of PLGA microsphere formed by
emulsion-solvent evaporation method.


## 3.3. Microarchitecture of PLGA Microsphere Loaded Gelatin Scaffold

It
is evident from the phase contrast and SEM micrographs
(see Figures2 and
3)
that PLGA microspheres were uniformly distributed through out the gelatin
matrix irrespective of the amount of microsphere added. Incorporation of PLGA
microspheres during fabrication of gelatin scaffold by freeze—drying has
significant effect on the overall microarchitecture of the scaffold. Effect of
microsphere doping on scaffold pore size was summarized in Table 1. Among
these four different scaffolds, control gelatin scaffold has regular pore
structure (average pore diameter 160 μm). Loading of 0.1% microspheres reduced
the average pore size to 110 μm. In case of 1% w/w PLGA microsphere loaded
scaffold, there were two set of pores (average diameter 150–120 μm which is
60% of total abundance and average diameter 50–30 μm which is 40%
of total abundance). 10% w/w PLGA microsphere loaded scaffold has no regular
pore structure (pore diameter varied
from 30–150 μm).Table 1
Effect of microsphere doping on scaffold
microarchitecture. For each
scaffold, 50 pores were analyzed to get the
average pore size.
%age of PLGA microsphere
doping (w/w) in gelatinAverage pore size (μm)0%1600.1%1101%[150–120], [50–30]10%150–30Phase contrast
micrograph of PLGA microsphere incorporated gelatin scaffolds. (a) Pure gelatin
scaffold, (b) 0.1% w/w PLGA microsphere
incorporated scaffold, (c) 1% w/w PLGA
microsphere incorporated scaffold, and (d) 10% w/w PLGA microsphere incorporated
scaffold. Photographs were taken at 40X.
(a)(b)(c)(d)Scanning electron micrograph of microspheres incorporated gelatin scaffolds.
(a) Pure gelatin scaffold, (b) 0.1% w/w PLGA microsphere incorporated scaffold, (c) 1% w/w
PLGA microsphere incorporated scaffold, and (d) 10% w/w PLGA microsphere
incorporated scaffold.
(a)(b)(c)(d)


## 3.4. Swelling Property

The
water uptake ability of the scaffolds was in the range of 1400–2100% (see
Figure4). It was expected that incorporation of hydrophobic PLGA microsphere would
reduce the water uptake in a dose dependent manner. However, no such trend was
observed. Result showed that 1% w/w PLGA microspheres loaded scaffold has the highest
swelling properties (2144%), and 10% PLGA microsphere loaded scaffold has shown
the least swelling of (1435.33%).Figure 4
Effect of PLGA microsphere incorporation on the swelling property of gelatin
scaffold. Values are mean ± S.D. (n=3). P<.005*, compared to gelatin
scaffold having no PLGA microsphere.


## 3.5. Porosity

All
the scaffolds had porosity in the range of 25 to 42 percent (see
Figure5).
Porosity of 10% PLGA microsphere loaded scaffold (26.13%) is significantly less
than that of control (31.83%), where 0.1% and 1% w/w PLGA scaffold has porosity
greater than control (41.5% and 38.53%, resp.).Figure 5
Effect of PLGA
microsphere incorporation on scaffold porosity. Values are mean ± S.D. (n=3). P<.05*, compared to gelatin scaffold having no
PLGA microsphere.


## 3.6. Mechanical Properties of the Scaffold

PLGA microspheres loaded
scaffolds were tested for tensile properties in dry condition. Tensile  strength
(see Figure6) of 0.1% and 1% w/w PLGA microsphere loaded scaffolds
(0.448 MPa
and 0.406 MPa, resp.)  was
significantly higher than that of control
(0.228 MPa) and among all, 0.1% has
the highest tensile strength. Although tensile strength of 10% w/w PLGA
microsphere loaded is higher (0.280 MPa) than that of control, the value was not
significant. From the data, it was evident that there was no clear relation
between the weight percentages of PLGA microspheres doping and tensile strength
of the scaffold. Trend observed in percentage elongation at break is almost
similar to the previous one, that is, percentage elongation at break for 0.1%
and 1% w/w PLGA microsphere loaded scaffolds is significantly higher than that
of control (see Figure 7). Percentage elongation at break for 0.1% and 1% w/w
PLGA microspheres doped scaffold is 2.20 and 2.49 fold greater than that of
control, where it is 0.96 fold less for 10% w/w PLGA doped scaffold.Figure 6
Effect of PLGA microsphere incorporation on the tensile strength of gelatin
scaffold. Values are mean ± S.D. (n=3). P<.05*, compared to gelatin
scaffold having no PLGA microsphere.Figure 7
Effect of PLGA microsphere incorporation on percentage elongation at break
of gelatin scaffold. Values are mean ± S.D. (n=3). P<.05*, compared
to gelatin scaffold having no PLGA microsphere.


## 3.7. Biodegradation

Biodegradation
study showed that incorporation of microspheres in the scaffold up to a certain
extent (up to 1% w/w) has no significant effect on early phase of degradation (see
Figure8). The extent of biodegradation after 48 hours was within 18-19% for
all three scaffolds except 10% w/w PLGA microsphere loaded scaffold (25.6%
biodegradation). However, with the progress of time, a variation in extent of
biodegradation was observed. Result revealed that the rate and total extent of
biodegradation were
higher for scaffolds having higher PLGA microsphere content. This may be
because of the time dependant degradation of PLGA itself by means of hydrolysis
[23] which rendered the scaffold microenvironment acidic. Under these circumstances
chemical intervention become predominant over the physical influence of the
hydrophobic microsphere on scaffold degradation; therefore scaffolds having
higher PLGA content showed higher degradation.Figure 8
Effect of PLGA microsphere incorporation on biodegradation of gelatin
scaffold. Degradation was studied for 10 days by incubating the scaffolds in
PBS (pH-7.4) at 37°C. Values are mean ± S.D. (n=3). (A1) Gelatin scaffold, (A2)
0.1% w/w PLGA microsphere loaded scaffold, (A3) 1% w/w PLGA microsphere loaded
scaffold, and (A4) 10% w/w PLGA microsphere loaded
scaffold *.


## 3.8. Study of Cell Adhesion

Cell
adhesion study showed that incorporation has no significant effect on cell
adhesion (see Figure9). All the scaffolds showed comparable cell adhesion
properties. This might be because the total number of microspheres present in
the upper surface is not sufficient to exert any significant effect on cell
adhesion.Figure 9
Effect of PLGA microsphere
incorporation on cell adhesion after 4 hours of cell seeding. Values are mean
± S.D. (n=3).


## 3.9. Study of Cell Proliferation

Cell
proliferation study (see Figure10) showed that there was not much variation in
cell proliferation for the first three days of culture. Growth of the cells on
all three types scaffolds loaded with varying amount of PLGA microspheres was
similar to that of control. Highest cell proliferation was achieved at day 5
for all type of scaffolds; however at day 5, the extent of cell proliferation
on 0.1% and 1% w/w PLGA microspheres loaded scaffolds was significantly higher than that of
control; on the other hand cell proliferation on 10% w/w PLGA microsphere
incorporated scaffold was less than not only to that of 0.1% and 1% w/w PLGA
microspheres loaded scaffold but even significantly less than the control. The
same trend was observed up to day 7 but extent of proliferation was decreased
compared to day 5 for all set of scaffolds.Figure 10
Effect of PLGA microsphere incorporation on murine L929 cells proliferation
on gelatin scaffold. Proliferation was studied for 7 days. Values are mean
± S.D. (n=3). (A1) Gelatin scaffold, (A2)
0.1% w/w PLGA microsphere loaded
scaffold, (A3) 1% w/w PLGA microsphere loaded scaffold, and (A4) 10% w/w PLGA
microsphere loaded scaffold.  P<.05*, compared to gelatin scaffold
having no PLGA microsphere.


## 3.10. Study of Cell Morphology

From
the scanning electron micrographs of cells cultured on microspheres loaded
scaffold (see Figure11), it was evident that microspheres  have no adverse
effect on cell growth. However, a stricken observation was that cells cultured
on 10% w/w PLGA microsphere loaded scaffold has an elongated structure compared
to the cells cultured on other scaffolds.Scanning
electron micrograph of mouse L929 cells cultured on PLGA microsphere
incorporated gelatin scaffold after 3 days of initial seeding. (a) Gelatin scaffold,
(b) 0.1% w/w PLGA microsphere loaded scaffold, (c) 1% w/w PLGA microsphere
loaded scaffold, and (d) 10% w/w PLGA microsphere
loaded scaffold.P<.05*,
compared to gelatin scaffold having no PLGA
microsphere.
(a)(b)(c)(d)


## 4. Discussion

Water
molecules interacting with the hydrophobic surfaces do not form hydrogen bond
with the surface. Instead, it forms a highly connected self-assembled structure using its own
hydrogen bonding [24]. This exceptional behavior of water molecule on
hydrophobic surfaces evoked the thought that presence of hydrophobic
microspheres in natural hydrophilic polymer solution can alter the size and
distribution of ice crystals formed at the time of freezing and thus can change
the whole microarchitecture of the scaffold made by freeze drying. To verify
this concept, we have taken PLGA microspheres incorporated gelatin scaffold as
a model system. Prior to the fabrication of the model, the contact angle of
PLGA and gelatin film was measured to confirm the difference in the
hydrophobicity of two polymers used in the model. Result ensured that PLGA
(65:35) is much hydrophobic than the gelatin used. During the fabrication of
the model, care was taken to keep the size of the microspheres considerably
lower (at least 5 times lower than the minimum pore size of the scaffold) than
the pore sizes of the scaffold; otherwise, microspheres may block the pore
structure. Microarchitecture analysis indicated that presence of PLGA
microsphere during freeze drying actually reduced the pore size of scaffold.
However in case of 1% w/w PLGA microsphere loaded scaffold, there were two sets of pores (150–120 μm and 50–30 μm). A probable
explanation of such heterogeneity in pore size is the fusion of two or three
adjacent pores by the rupture of common pore walls. This might be due to the
steric effect of the microspheres embedded or because of strong water repelling
force exerted by the hydrophobic microspheres. This assumption was strongly
supported as 10% w/w PLGA microsphere loaded scaffold has no regular structure
or pore size which varies from 30–150 μm.
Experimental result (see Figure 4) revealed that variations in swelling
property of the scaffolds are
not proportional with the amount of microspheres incorporation. Extent of
swelling of 0.1% and 1% w/w PLGA microsphere loaded scaffolds was even higher than that
of control (scaffold having no microsphere). However, 10% w/w PLGA microspheres
incorporated scaffold has less swelling than control. This was contrary to our
expectation that presence of greater amount of hydrophobic materials inside the scaffolds reduces the water up take
to a greater extent. Probably incorporation of PLGA microspheres up to a
certain extent in the scaffold enhanced its porosity that allows it to restore
greater amount of water. Aforementioned assumption was supported by the trend
observed in the variation in porosity (see Figure 5). Porosity data showed that
porosity of 1% and 0.1% w/w microspheres loaded scaffolds was higher than that of
control. This increased porosity might be because of higher interconnectivity
of the 0.1% and 1% w/w PLGA microspheres incorporated scaffold as evident from
the SEM micrographs (see Figure 3). Tensile strength of 0.1% and 1% w/w
microsphere incorporated scaffold (see Figure 6) implied that presence of PLGA
microspheres within the scaffold could enhance its mechanical strength. The
same trend was observed in case of percentage elongation at break of those
scaffolds. Higher values of percentage elongation at break in case of 0.1% and
1% w/w PLGA microsphere incorporated scaffold with respect to control and
10% w/w PLGA microsphere incorporated scaffold indicated the greater mechanical
flexibility of 0.1% and 1% w/w PLGA microsphere incorporated scaffold. It was
expected that presence of hydrophobic microspheres can contribute to the
hydration of the scaffold thus can affect the initial phase of biodegradation
but no significant difference in the rate of biodegradation was observed for
first 48 hours in case of 0.1% and 1% w/w microspheres loaded scaffolds (see
Figure 8). Although 10%
PLGA microspheres incorporated scaffold had lower
swelling properties, its high degradation rates indicate predominance of chemical factors over the
extent of hydration. Cell adhesion study was aiming to characterize the
influence of the presence of hydrophobic microspheres in the gelatin scaffold
on cell adhesion. PLGA itself is a moderate substrate for cell adhesion
[25],
and it was expected that presence of hydrophobic PLGA microspheres inside the
scaffold may play a critical role in early phase of cell adhesion because that
phase of cell adhesion is primarily governed by simple electrostatic
interaction between cells and substrate but result showed that all scaffolds
have comparable cell adhesion property (see Figure 9). A probable explanation of this observation could
be the presence of gelatin which is well known for its excellent cell adhesion
property. Analysis of growth kinetics of L929 cells on PLGA microspheres
incorporated scaffold revealed that microspheres have no direct influence on
cell proliferation at the early phase of culture (up to day 3) but with time
the effect become pronounced as evident from day 5 proliferation data. This
may be because of the higher interconnectivity and porosity of 0.1 and 1% w/w
PLGA microspheres loaded scaffold which leads to efficient diffusion of toxic
products (produced by the cells) from the scaffold to the media rendering the
scaffold microenvironment more hospitable to
the cultured cells.


## 5. Conclusion

This effort has brought
forward the consequence of PLGA microsphere incorporation during freeze drying
of gelatin scaffold. Study has revealed that loading of hydrophobic PLGA
microspheres in relatively hydrophilic gelatin scaffold not only change the
scaffold microenvironment but also modulate other physical properties of the
scaffold which influence the cell growth kinetics. The model gives an insight
to the happening taken place inside the hydrophilic scaffold microenvironment
due to the presence of microscale hydrophobic moieties at the time of scaffold
fabrication by freeze-drying method. This study may find its application in
designing a better-controlled release matrix for improved tissue engineering.
Further investigation is required to find out the threshold difference in
hydrophobicity of two polymers that may give rise to this kind of variation and effect of size
variation of polymer spheres on scaffold’s
properties.



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*Source: 143659-2009-03-30.xml*