1

Effect of Different Growth Factors(VEGF and

PDGF) and Different Scaffolds(PGA and Collagen)
on the Growth of Skeletal Muscle Tissue
Inseong Joe and Kevin Suzuki

AbstractSkeletal muscle injuries are one of the most common

injuries caused by sports. In this experiment, we attempted to
generate skeletal muscle tissue in vitro, from bovine bone marrow
stem cells by supplementing them with different growth factors
(VEGF and PDGF) and using 2 different scaffolds: PGA and
Collagen. This paper focuses on how the different conditions
of growth factors and scaffolds affect the engineering of skeletal
muscle tissue. A collagen assay was done for biochemical analysis
and measure the amount of collagen deposition on PGA scaffolds,
and Hematoxylin and Eosin (H&E) staining was done for
histology to see whether the different growth factors and scaffolds
affected the rate of growth of the muscle tissue and the accuracy
of physical structure. Results show that while for all conditions
and scaffolds, muscle like characteristics appear by week 4; there
was no identifiable difference in muscle tissue growth between
the three different growth conditions. However, PGA scaffold
samples differentiated into muscle tissue at a faster rate and
showed more similarity in qualitative structure, suggesting that
PGA scaffolds may be better suited for engineering muscle tissue.

I. I NTRODUCTION
In the human body, there are three different types of muscle
tissues: skeletal, cardiac and smooth. Skeletal muscle tissue
is the most abundant tissue in vertebrates. It is composed of
individual muscle fibers that have branches of blood vessels
following the connective tissue of the muscle [1]. Its main
function is coordinating the movement of bones. Without
skeletal muscle tissue we would be unable to move.
Sports injuries are the most common method of skeletal
muscle injuries, which account for up to 55% of all muscle
injuries [2]. There are two different types of injuries that can
occur: direct trauma (lacerations, strains, and contusions) and
indirect injuries: (ischemia and neurological disfunctions) [2].
In our experiment, we hope expedite the process of skeletal
muscle regeneration and focus on the direct trauma injuries
because they are more common injuries that can be addressed
with tissue engineering.
The current methods of sports injuries treatment include
the RICE protocol (rest, ice, compression and elevation) [2],
local application of heat and passive motion exercises, and
drug therapies which include nonsteroidal anti-inflammatory
drugs (NSAIDs) and intramuscular corticosteroids [2].
Unfortunately, current therapy does not completely restore
the skeletal muscle tissue back to preinjury status due to
the large variation in injury severity and affected muscle
group, which make it hard to find suitable treatments [9].

Fig. 1: leg showing severe skeletal muscle injury after sports

In order to increase the chances of finding a successful

treatment, it is vital to understand the cellular processes in
healing skeletal muscle injuries. Fortunately, newer biological
therapies including cell therapy, tissue engineering and the
use of growth factors have been studied to increase the rate
of the healing process in skeletal muscle tissue.
The healing process of injured skeletal muscle involves
bioactive molecules [2], which includes proinflammatory
cytokines, transforming growth factor-beta (TGF-B),
superfamily members and angiogenic factors [2]. Thus
in order to increase the rate of regeneration of injured
skeletal muscles, growth factors and cytokines have been
experimented with as a potential therapeutic treatment [2].
The signaling molecules activate the myogenic precursor
cells, which accelerates the regeneration of injured muscle
tissue.
The two growth factors that we experimented with: vascular
endothelial growth factor (VEGF) and platelet derived growth
factor (PDGF) have been shown to be effective in cell

therapy. VEGF is a signal protein produced by cells that

stimulates angiogenesis and vasculogenesis. VEGF is part
of the system that restores oxygen supply to tissues when
blood circulation is insufficient. VEGFs normal function is
to promote endothelial cells mitogenesis and migration and
stimulate myoblast migration [2]. We decided to use VEGF
because it creates new blood vessels after injury and helps
create new muscle after exercise.
PDGF is a protein that regulates cell growth and
division and plays a significant role in angiogenesis, blood
vessel formation, and the growth of blood vessels from
existing blood vessel tissue. PDGF also promotes the
mitogenesis of mesenchymal cells and fibroblasts, induces
proliferation of satellite cells, and inhibits the end stages of
myoblast differentiation [2]. As the tissues begin to grow,
neoangiogenesis plays a large role in the healing process
of muscle tissue. The new blood vessels that form provide
a supply of oxygen, growth factor and blood stem cell to
increase the regeneration of tissue.
Skeletal muscle tissue engineering can be done in vitro
and in vivo [2]. In vitro tissue engineering is the process
of expanding and seeding stem cells onto a 3D structured
scaffold to produce a cell-biomaterial construct [2]. Skeletal
muscle regeneration is related to the biomaterial used to
scaffold the stem cells. The main purpose of the scaffolds is
to simulate the anatomical and biomechanical properties of
the injured tissue.
The two types of scaffolds we used were polylacticcoglycolic acid (PGA) and collagen scaffolds. PGA
scaffolds are synthetic and biodegradable scaffolds that
show appropriate rigidity and connection for muscle tissue
engineering [2]. PGA scaffolds also have high and predictable
bioabsorbability and tensile strength, which make them
a preferable scaffold type [16]. Collagen scaffolds with
parallel oritented pores have been used to reproduce the
three-dimensional organization of skeletal muscle [2].
We used collagen as a scaffold because it is the most common
and abundant protein in the human body and is also the
primary constituent of the extracellular matrix of most tissues
[8].
In our experiment, we aim to identify how different growth
factors and scaffolds effect the cell growth and differentiation.
We expect to see a realtively higher and faster rate of
differentiation with cells treated with PDGF compared to
cells treated with VEGF. All cells with growth factors
are expected to grow better than controls. PGA scaffolds
are hypothesized to maintain a better structure of the
differentiated cells than collagen scaffolds, thus having more
similar characteristics of an actual muscle tissue.

II. M ATERIALS AND M ETHODS

Cell Isolation and Culture
Bone marrow Mesenchymal Stem Cells (MSCs) were
isolated from a bovine calf femur. The end of the femur was
removed and bone marrow was collected. To break up the
bone marrow, expansion media [10] (DMEM supplemented

Fig. 2: An image of a synthetic and degradable PGA scaffold

Fig. 3: An image of the natural and degradable collagen

scaffolds that we made

with 10% FBS, 1% penicillin-streptomycin-glutamate, 5

mM HEPES, 10ng/mL PDGF-BB, 50ug/mL vitamin C,
3ng/mLCuSO4 , and 50ug/mL of proline, alanine, and
glycine) was added and bone marrow was passed through
25 guage needles and pipetted repeatedly. After washing,
cells were resuspended in expansion media and plated on
tissue culture plates at an approximate density of 1x106 per
PGA scaffold and 0.5x106 cells/mL collagen [1]. Media was
replaced three times a week and cells were passed when about

80% confluent. When passing cells, PBS without Ca/Mg was

used to wash and trypsin was used to detach cells. Cells were
replated by a 1:10 ratio. Cells were cultured for 3 weeks prior
to seeding on scaffolds. 3 replicates of each condition were
made (3x PDGF/3x VEGF/ 3x control) * 2 types of scaffolds
(collagen/PGA) = (27 collagen scaffolds, 27 PGA scaffolds),
leading to a total of 54 wells
Preparation of Scaffolds
Polyglycolic acid (PGA) disc scaffolds (5 mm diameter,
2 mm high, Synthecon, Houston, TX) were washed with
ethanol and pre-wetted in culture medium in 6 well plates,
and incubated in a humidified 37 CCO2 incubator.
3mg/mL concentration collagen scaffolds were made in
a 48 well plate with 200L gels per well. 0.5x106 cells/mL
were added to the wells and incubated in a humidified
37 CCO2 incubator for 30 minutes. Collagen scaffolds were
made right before starting the experiment.
Cell Seeding
Cells that were expanded in expansion media for 3 weeks
were drip seeded on PGA scaffolds. 1x106 cells were seeded
per PGA scaffold. Collagen scaffolds were made according
to the description above and the referenced protocol [11].
The experiment was set up so that each scaffold of each
condition (50 ng/mL VEGF (50 g, Prospec, East Burnswick,
NJ), 5 ng/mL PDGF (10 g, Prospec, East Burnswick,
NJ), and no growth factor) had 3 replicates and samples
were fixed at 1, 2, and 4 weeks within multiple six well plates.

referenced protocol [11].

Histology
The other half of each sample harvested was put in a
histology cassette and fixed in 10% buffered formalin for
at least 5 days. After fixation, all samples were dehydrated
and embedded in paraffin. Then, after the samples were
sectioned and fixed onto slides, H&E staining was performed.
After staining, samples were imaged on a microscope at 10x
magnification using the uEye camera.

III. R ESULTS
A. Extracellular Matrix Content
To determine the amount of collagen per mg construct, a
hydroxyproline collagen assay was performed on PGA scaffold samples. The standard curve made with hydroxyproline
was overall linear up to the 4th sample (Fig 5). After the 4th
sample, the line started to curve (Fig 5). Thus, the linear line
upto the 4th sample was used to calculate collagen deposition.
The weights of PGA scaffolds were also used for calculation.
Average weights for each condition for each time period is
shown in figure 6. There is some consistency in the weights
across time for control samples, but high varience in VEGF
and PDGF samples. Mixed results were identified from the
assay. The data shows that the amount of collagen decreased
when using the PDGF substantially from week one to week
four (Fig 7). During week one, there was 301.6 g collagen
and in week two, there was a slight decrease to 263.3 g.
However, by week four, data showed that there was only
66.5 g of collagen, indicating that the amount of collagen
decreased by a large amount. Figure 8 displays the total
collagen weight vs the weight of the entire tissue sample.
From this graph, we are able to see that the ratio of collagen to
total tissue sample decreases over time for the PDGF sample,
whereas this relationship increases over time for the VEGF
and control samples.

Fig. 4: Experimental Setup

Experimental setup layout for this experiment. 3 replicates of
well for each week.
Cell Feeding
Cells were fed every 2-3 days and the entire media was
replaced with the appropriate media. Both PGA and collagen
scaffolds were seen to decrease in size over the course of four
weeks. Unfortunately, 3 wells were lost when the aspirating
tip hit the scaffold, rendering it useless.
Biochemical Analysis
A collagen assay was done on PGA scaffolds to analyze
the amount of collagen deposited. Samples harvested at
each time period were cut in half and one half was stored
in -80 C. After thawing, and digestion in papain, [12] and
hydrolyzation, collagen assay was done according to the

Fig. 5: Hydroxyproline Standard Curve

Standard Curve made for hydroxyproline while doing hydroxyproline collagen assay.

Fig. 6: Average weights in grams of each sample

Average weights of PGA scaffolds are graphed for each week
and each condition

stains the extracellular matrix and fibroblasts, whereas the

Hematoxylin stains nuclei. Fibroblast development can be
seen in newly cultivated tissue samples as well as the cell
nuclei. Figure 9, shows that the samples harvested after the
first week are still forming and still in fragments. Figures
9(a) and 9(c) show mainly long rectangular figures which are
PGA fibers and they are sparse with minimal tissue growth.
From samples harvested after two weeks, muscle tissues
have started to form and figures 9(d) and 9(f) show a large
increase in tissue growth, displayed by the striations towards
the center of the sample. There is a decrease in the excess
PGA fibers, which are the smaller cell groupings in the center
as seen in figure 9. These two images also display uniformity
throughout the cell, displaying its proper growth. The samples
harvested after week four show that the muscle tissue is still
structurally stable and many different fascicles of similar size
can be seen. All of these figures show myofibers of relatively
uniform size along with nuclei which are present. Figure
9(i) looks very very close to figure 10(i), the umbilical cord,
because it has similar textures and similar striations and
validates our muscle tissues proper growth.

Fig. 7: collagen deposition in g

Average amount of collagen in each sample is graphed for
each week and each condition.

Fig. 8: collagen deposition in mg collagen per mg sample

tissue
Amount of collagen per mg of tissue sample is shown for each
week and each condition.

B. Histology
PGA Scaffolds
A Hematoxylin and Eosin (H&E) stain was also performed
on the PGA scaffolds. H&E stain was chosen because Eosin

Fig. 9: H&E staining on Collagen Scaffolds shown at 10x

magnification.
Top row shows three samples that were harvested after one
week, each treated with (a) Control, (b) VEGF, (c) PDGF.
Second row shows three samples harvested after two weeks,
each treated with (a) Control, (b) VEGF, (c) PDGF. Third row
shows three samples harvested after four weeks, each treated
with (a) Control, (b) VEGF, (c) PDGF
Collagen Scaffolds
The HE stain was also performed on collagen scaffolds.
All three conditions control, with VEGF, and with PDGF
of samples from week 1 had fewer cells compared to the
later weeks and looked much less dense. Figures 10(a), 10(b),
and 10(c) show clearly that the samples have a small amount
of cells mixed in collagen. Samples that were harvested
after two weeks(Fig 10(d),10(e),10(f)) looked denser than the
previous three. This may be due to compaction of collagen.

However, the samples do seem to have started to form more

of a muscle like feature comparing it to the previous week.
The week 4 sample shown in figure 10(g) looks similar to
the muscle sample (fig 10(h)) and it can be seen that the
two have the matching texture and appearance. By week 4,
qualitatively, cells have developed into more of a muscle tissue
like structure.

Fig. 10: H&E staining on Collagen Scaffolds shown at 10x

magnification
Top row shows three samples that were harvested after one
week, each treated with (a) Control, (b) VEGF, (c) PDGF.
Second row shows three samples harvested after two weeks,
each treated with (a) Control, (b) VEGF, (c) PDGF. (g) shows
a sample after four weeks treated with VEGF. (h) shows a
muscle sample collectd from bovine calf femur. (i) shows an
umbelical cord sample.

IV. D ISCUSSION
In this experiment we aimed to grow skeletal muscle tissue
from bone marrow stem cells (BMSC) and evaluate the
effects of different growth factors (VEGF and PDGF) and
scaffold types (PGA and Collagen) on the cells growth and
differentiation. Though the experiments were preliminary, the
results from H&E staining and collagen assay suggest the
production of some muscle tissue. However, from our results,
there seemed to be no significant effect of the different growth
factors on the cells differentiation. This was inconsistent with
our initial hypothesis and most research that has been done on
the role of growth factors [1].
One reason why this may be the case is that we had
based the growth factor concentration off of S. Levenberg
et. al. (2005) [1], while the experiment were fundamentally
different from our experiment. Although they were working
with endothelial cells, we were working with mesenchymal
stem cells. In their paper, it made sense that they would
supplement with a higher concentration of VEGF and a lower
concentration of PDGF in order to grow more vascularized

endothelial cells. However, for our results, in order to see see

more clear and significant data, we would have needed a higher
concentration of PDGF because the data on the PDGF yielded
unexpected results within the collagen assay. M. Kamimura et
al. (2004) has used 10 ng/mL in engineering muscle tissue
which is 5 ng more concentrated than what we had used [17].
Additionally, the PDGF may have been in bad condition due
to its alliquoted concentration. The protocol[13] stated that it
should be frozen in a concentration of 100g/L. However, we
had placed it in a concentration of 25g/L. Although this may
be a minor issue, it could have contributed to the malfunction
of PDGF.
From figure 7, it is clear that the PGA scaffold with PDGF
had less collagen deposition after four weeks. Our reason for
this dropoff is due to the fact that the collagen buildup on the
scaffolds were becoming more concentrated and thus getting
smaller and smaller as it contracted as the weeks progressed.
Because the PGA scaffold was so small, obtaining accurate
results is challenging. The ratio of collagen to total tissue mass
also decreases over time for the PGA scaffold with PDGF as
seen in Figure 8. However both decreases may be attributed
to the diminishing size of the scaffold and not necessarily the
growth factor.
Collagen assay results showed fairly consistent results for
control and VEGF treated samples. We saw that both show
stable collagen deposition. However, due to our week four
scaffolds being so small, we cannot accurately verify that this
collagen deposition data is completely valid. Also, the collagen
assay results have fairly low reliability and validity because the
hydroxyproline standard curve is not very linear. To make the
curve as linear as we could, we only used the values up to the
4th sample on the standard curve (Fig 5), leading to invalid
results. Another error that was made was that when performing
the papain digestion, we did not add the proper buffer and
instead directly added the papain stock solution which could
have affected the inconsistent numbers. In addition, the mass
values were also questionable as the samples were very small
and thus were hard to weigh (Fig 6).
The two different scaffolds that we used have very distinct
characteristics. PGA scaffolds are synthetic and biodegradable
scaffolds, while collagen scaffolds are also biodegradable but
are natural scaffolds. Both scaffolds are fairly highly porous
scaffolds, but comapred to collagen, PGA scaffolds are more
porous [18]. PGA scaffolds have comparably large fibers and
starts out much stiffer than collagen. Collagen is used in tissue
engineering as a scaffold because because of its abundance,
biocompatability and natural ubiquity within the human body
[7]. However, a downside of using collagen scaffolds is the
lack of mechanical stability [8].PGA scaffolds have more
chemical stability, but as they are not natural, there may be
different effects that must be taken into account when growing
tissue.
Our results show that in the 4 week timespan, although both
PGA and collagen scaffold samples have developed into somewhat muscle-like structures, PGA scaffold samples seemed
to have differentiated faster than collagen scaffold samples.
While muscle like characteristics are shown in week 4 for
collagen scaffolds, this muscle like characteristic is starting

to show from week 2 for PGA scaffolds. The downside with

collagen scaffolds is that they have poor chemical stability and
do not maintain shape very well. Thus, it was expected for the
collagen scaffold to show a sufficient amount deterioration in
comparison to the PGA scaffold. However, in our experiment,
PGA scaffolds also showed contraction over the course of
time. No paper has reported this observation, which questions
some of our aspects of our experiment. Additionally, in order
to have more chemically stable collagen scaffolds, the final
collagen concentration can be increased or the initial size of
the collagen scaffolds can be increased.
If we were to redo this experiment, there will be multiple
changes that we would make. The first change would be the
addition of one more scaffold and one more growth factor. We
would add a polylactic-co-glycolic acid (PLGA) scaffold. We
would use a PLGA scaffodl because it has been shown to display biocompatibility, a tailored biodegredation rate, potential
to modify surface properties to provide better interaction with
biological materials, good mechanical properties, especially
high tensile strength and high Youngs modulus relative to
collagen, and excellent processability [14]. Thus it is more
rigid than collagen and can potentially lead to less and more
controlled degredation over time.
We would also use transforming growth factor (TGF-)
because this growth factor stimulates mesenchymal cell proliferation, regulates endothelial cell activity and angiogenesis,
promotes the proliferation of fibroblasts and the biosynthesis
of the extracellular matrix, including collagen, and inhibits
satellite cell proliferation and differation [2]. However, it is
important to manage the concentration of TGF- used because
an overexpression can lead to inhibition of myoblast differentiation and muscle fiber regeneration [15]. As we mentioned,
we would increase the PDGF concentration because it showed
substantially lower results in comparison to the other two
conditions. We could also create more samples to ensure that
we have more accurate data.
We also would run more tests and do different histological
tests. Although we had an actin staining protocol that would
have given us different results, we were unable to pursue that
due to a lack of time after the microtone machine broke. We
could also use a higher density, bigger collagen scaffold with
lower cell density to determine whether the growth factors will
have a larger impact on muscle growth.
V. C ONCLUSION
From this experiment, we were able to successively generate
a muscle-like substance from bone marrow mesenchymal stem
cells. However, we were unable to decisively conclude which
growth factor and scaffold combination yield the best results
in generation of skeletal muscle tissue. Our initial hypothesis:
PDGF on a PGA scaffold will grow the quickest, seems to not
have been true because from the histology samples, VEGF on
a PGA scaffold appears to be more structureally stable. Based
on the collagen assay, the PDGF sample seems to yield the
lowest amoount of collagen deposition after four weeks, but
the validity of this fact can not be verified by this experiment.
From our histological data, it appears as if PGA scaffolds

show generation of muscle tissue by week 2, whereas the

collagen scaffolds display generation of muscle tissue by week
4. Even though our results are inconclusive, we believe that
tissue engineering can be a viable solution in increasing the
rate of muscle regeneration and can help millions of athletes
around the world who suffer skeletal muscle injuries due to
the fact that there is still a sign of muscle tissue growing on
both scaffolds, under all three growth conditions.
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