Response to Reviewers:
Reviewer 1:
Overall Comment:
The paper is well written and logically ordered. The authors need to focus on choosing
either fiber diameter or hypoxia and delve deeper into its dependence on endothelial
cell behavior. Please refer to the attachment for the reviewer comments for detailed
comments.
Response:
The authors would like to thank you for taking the time in reviewing the manuscript.
While we agree that choosing a focus is important, we believe that all the conditions
we have studied are important to the study. Our conclusion is that the major finding
of this study is that fibre diameter has the biggest effect on endothelial cell behaviour.
We have therefore used the majority of the discussion to delve deeper into fibre diameter
and put less of a focus on the other factors we studied. We have altered the structure
and focus our Conclusions to ensure this comes across as our major finding/focus.
Edits made to Conclusions section:
In this study, three different aspects of the scaffold and culture environment were
assessed to evaluate which had the biggest impact on HUVEC performance. It was noted
that the incorporation of ECM into the fibres had minimal effects on HUVEC gene expression
when cultured in both a hypoxic and normoxic environment, but it did have the effect
of increasing cell deposits on the scaffold. Furthermore, the hypoxic culture had
a limited effect on the HUVECs when cultured on both the PCL and the ECM scaffolds.
On the other hand, fibre diameter had the greatest influence on cell performance.
The largest fibre diameter led to increased HUVEC infiltration and significantly increased
cell viability and expression of CD31 compared to the three other fibre diameters
(significance was only noted between the extra-large fibre and medium fibre at 6 days
of culture).
Therefore, the present study suggests that fibre diameter plays the biggest role in
the modulation of cell proliferation and angiogenic gene response.
Comment 1:
The author states in the introduction that ‘fibre diameter on endothelial cells has
not yet been studied’. However, there has been ample research done on the effects
of fiber diameter on endothelial cell behavior.
Some of the references are
Rüder, Constantin, Tilman Sauter, Karl Kratz, Tobias Haase, Jan Peter, Friedrich
Jung, Andreas Lendlein, and Dietlind Zohlnhöfer. "Influence of fibre diameter and
orientation of electrospun copolyetheresterurethanes on smooth muscle and endothelial
cell behaviour." Clinical hemorheology and microcirculation 55, no. 4 (2013): 513-522.
Ko, Young-Gwang, Ju Hee Park, Jae Baek Lee, Hwan Hee Oh, Won Ho Park, Donghwan Cho,
and Oh Hyeong Kwon. "Growth behavior of endothelial cells according to electrospun
poly (D, L-lactic-co-glycolic acid) fiber diameter as a tissue engineering scaffold."
Tissue engineering and regenerative medicine 13, no. 4 (2016): 343-351.
The authors need to cite appropriate references and remove claims that are not justified.
Response:
Thank you very much for comment and for finding those references. All references will
be included and any unjustified statement will be removed. Statements will also be
amended to emphasise that this is the first time (to the best of our knowledge) that
the effect of fibre diameter using polycaprolactone will be assessed with endothelial
cells.
Edits made to Introduction section:
For example, it has been shown with human kidney primary epithelial cells and chondrocytes
that fibre diameter and fibre orientation have dramatic effects on cell morphology
and the expression of key genes [27,28]. While there has been some work looking at
fibre diameter and endothelial cells, to the best of our knowledge, the present study
is the first that looks at the effect of fibre diameter in electrospun PCL scaffolds
on endothelial cells [29,30]. Furthermore, recent work by our group has shown that
incorporating native vascular ECMs into the fibre had positive effects on HUVEC proliferation
and gene expression [9,31].
Comment 2:
The authors need to check the paper for typographical errors and grammatical mistakes.
Eg. Superscript -1 in the sentence ‘Coherent anti-stokes Raman scattering (CARS) imaging
at 2911cm-1 was used to image the PCL scaffold fibres, whilst simultaneously exciting
two photon fluorescence (TPEF) from Phalloidin and DAPI stained cells’.
Response:
Thank you for the comment and picking up on the mistake. All mistakes like this will
be changed in the text and the paper will be thoroughly proofread.
Comment 3:
The authors need to clarify on how the diameters of fibers were measured. How many
FESEM images for each sample were used to obtain the statistical significance with
regard to fiber diameter? The difference between the 3um and 4um fibers are quite
close. Is it possible that the diameters might have not been statistically significant
if other images of the same sample were used?
Response:
Thank you for the comment regarding more detail on how the fibres were measured. Fibre
diameters were measured using a minimum of 50 individual fibres from four different
scaffolds. Due to fibre morphology being fairly uniform throughout the scaffold, a
small standard deviation was observed. Furthermore due to the high number of measurements
taken and the small standard deviation, the differences noted between the 3um and
4um were significantly different. Information will be added to the methods section
to clarify how fibre diameter is measured.
Edits made to Methods section:
Fibre and pore properties
Scanning electron images were analysed using ImageJ software (NIH). Briefly, SEM images
of the scaffolds analysed using the DiameterJ plugin for fibre diameter and pore width
and the OrientationJ plugin for fibre orientation [33]. Fibre diameters were measured
on a minimum for 50 different fibres on four different scaffolds. Variance in fibre
diameters along individual fibres was measured using 5 diameter measurements per fibre
along a total of 5 fibres.
Comment 4:
The authors need to elaborate on the reproducibility of the electrospinning results.
Environmental factors play a major role in the diameter and the subsequent fiber morphology.
Response:
Thank you for the comment. You are absolutely correct that environmental factors do
play a major role. Our system is not environmentally controlled and work has shown
that temperature and humidity both affect electrospinning, so this will be added as
a limitation into the discussion [1]. We electrospun all the scaffolds in the same
week, which would limit the overall effect that the environment has between each scaffold.
While these electrospinning parameters would not produce identical scaffolds when
reproduced, they would lead to similar scaffolds with differing fibre diameters.
Edits made to Discussion section:
This study is not without its limitations. Firstly, we were unable to differentiate
between whether fibre size or pore size was having the major effect. One issue with
electrospinning is that these two morphological characteristics are somewhat interconnected,
with a study by Pham et al. showing a linear correlation between the two characteristics
[59]. A further limitation is that we were unable to study the full spectrum of fibre
diameters. We were only able to look at a limited number of scaffold morphologies
and our apparatus only allowed for fibre diameters of up to approximately 5μm. Furthermore,
the system used in this study does not have environmental controls (humidity and temperature),
therefore some minor differences in fibre morphology would be expected when repeated.
However, the problem of environmental control was kept to a minimum by electrospinning
all scaffolds in the same week. In addition, while fibre morphology might change due
to environmental factors, the electrospinning parameters used in this study will still
create scaffolds with a range of fibre diameters. Moreover, further analysis on how
the scaffolds perform when cultured under flow/shear would more accurately mimic the
native environment. While these limitations merit further study in an attempt to gain
an overarching image of exactly how scaffold morphology/composition is affecting the
seeded endothelial cells; we have shown that through altering the scaffold’s fibre
diameter that we can influence cellular performance.
Comment 5:
The authors have performed a one-way ANOVA. Why did the authors choose ANOVA and not
regression-based analysis?
Response:
Thank you very much for the comment. One-way ANOVA with tukey post hoc was used as
this is the most commonly used method for determining statistical significance between
means of three or more independent groups. This method of measuring significance has
been widely used in similar biological analyses [2–8]. (reference list can be found
at the end of this document)
Comment 6:
The mechanical properties and stiffness of the fibers also changes along with diameter.
Why have the authors chosen to just represent that fiber diameter is the sole factor
leading to changes in expression? The authors need to think about performing AFM if
possible to get a better understanding about the scaffold properties.
Response:
Thank you for the comment. While the mechanical properties and stiffness of the fibres
are changing between the scaffolds, the differences noted are small on the grand scheme
of stiffnesses that have been tested in vitro. You are correct that the assumption
cannot be made, therefore a comment will be added to the limitations to discuss this
point. With regards to performing AFM, this would give us a better understanding of
the scaffold’s topographical properties, however this equipment is not available to
us. A discussion point will be added to discuss how AFM could be utilised in future
studies.
Edits made to Discussion section:
One issue with electrospinning is that these two morphological characteristics are
somewhat interconnected, with a study by Pham et al. showing a linear correlation
between the two characteristics [59]. The mechanical properties of the scaffolds also
changed between each scaffold, meaning that the scaffold’s mechanical properties might
play a part in the differences in cellular performance noted. A further limitation
is that we were unable to study the full spectrum of fibre diameters. We were only
able to look at a limited number of scaffold morphologies and our apparatus only allowed
for fibre diameters of up to approximately 5μm.
More analysis is required in order to get a deeper understanding of the scaffold’s
topographical properties and how these might affect seeded endothelial cells. One
method would be to use technologies such as atomic force microscopy (AFM), which have
previously been used to study the interaction of cells with their scaffold/substrate
[60.61]. Moreover, further analysis on how the scaffolds perform when cultured under
flow/shear would more accurately mimic the native environment. While these limitations
merit further study in an attempt to gain an overarching image of exactly how scaffold
morphology/composition is affecting the seeded endothelial cells; we have shown that
through altering the scaffold’s fibre diameter that we can influence cellular performance.
Comment 7:
The thickness of the fabricated scaffolds needs to be mentioned in the main paper.
Differences in cell infiltration can only be compared when the thickness of the scaffolds
are similar. The variable thickness could also be a factor in expression of genes.
The author needs to further investigate and elaborate on the different variables.
Response:
Thank you for the comments. The thickness of the scaffold will be added to the paper.
While we agree that thickness can have an impact on infiltration, we have only noted
infiltrations that are in the top 25-80um of the scaffold. These infiltration depths
will be the same whether the scaffold is 300um or 3000um thick. All the scaffolds
used in this study were much thicker than the deepest noted infiltration, therefore
thickness should not have had an effect on infiltration. The same applies for gene
expression whereby the cells will not be affected by any excess thickness. However,
thickness would play a major role in nutrient delivery, especially when cells were
found deeper in the scaffold, which would cascade down into gene expression. However,
due to infiltration depths being less than 100um, this should not have an effect on
in vitro cultured cells.
Edits made to Results section:
Table 4: Mechanical and physical properties of all four scaffolds.
Fibre diameter (µm) Pore diameter (μm) Variance in fibre diameter along fibre (%)
Scaffold thickness (µm) Porosity (%) Ultimate tensile strength (MPa) Failure strain
(%) Contact angle at 0.2s (°) Stiffness (MPa)
0-5% 5-10%
S 1.64 ± 0.18 8.4 ± 3.7 1.23 140 ± 9.4 91.0 ± 1.6 1.08 ± 0.17 843 ± 143 110.8 ± 7.7
4.10 ± 0.33 2.17 ± 0.25
M 2.95 ± 0.16 14.7 ± 4.8 0.83 336 ± 36.9 89.6 ± 2.4 1.50 ± 0.09 1202 ± 62 128.2 ±
4.0 6.50 ± 0.72 2.99 ± 0.46
L 3.37 ± 0.27 16.4 ± 6.7 2.28 549 ± 33.8 83.9 ± 1.1 1.55 ± 0.16 1071 ± 21 129.1 ±
2.5 5.80 ± 0.36 3.16 ± 0.48
XL 4.83 ± 0.49 23.3 ± 9.0 2.83 598 ± 59.6 83.3 ± 0.9 1.26 ± 0.06 855 ± 120 131.5 ±
1.1 5.77 ± 0.39 3.66 ± 0.28
Edits made to Discussion section:
Furthermore, the infiltration depth of cells could have an impact on the noted gene
expression. It has been previously documented that oxygen transfer to deeper parts
of a scaffold can be limited depending on the scaffold’s architecture [62]. While
this is unlikely in the present study due to the highly porous nature of the scaffolds,
it is worth considering in future experiments. Moreover, further analysis on how the
scaffolds perform when cultured under flow/shear would more accurately mimic the native
environment. While these limitations merit further study in an attempt to gain an
overarching image of exactly how scaffold morphology/composition is affecting the
seeded endothelial cells; we have shown that through altering the scaffold’s fibre
diameter that we can influence cellular performance.
Comment 8:
There are no appropriate controls for the cell titer blue viability assays. A positive
and negative control needs to be represented. A standard tissue culture is an ideal
positive control.
Response:
Negative controls were run alongside and they are used to remove the background fluorescence
from the data. All data has been represented with this background fluorescence removed.
Any value above 0 can be counted as excess fluorescence over the background. A positive
control of cells grown on tissue culture plastic can be added, however, this study
is looking at comparisons between scaffolds and not making comparisons with a standard
culture on tissue culture plastic. We have added a supplementary file showing a comparison
between cells cultured on tissue culture plastic and those cultured on scaffolds.
Edits made to Methods section:
CellTiter-Blue® cell viability assay
The assay was performed as per manufacturer’s instructions (Promega) and as described
in previous studies[9]. Measurements were taken after 3.5 h at Ex: 525 nm and Em:
580-640 nm. For each condition group, n=4. All data has been represented with the
background fluorescence removed (negative control). A comparison of cells cultured
on scaffolds to cells cultured on tissue culture plastic can be found in S1 Fig.
Edits made to Supplementary section:
Caption: Cell viability of HUVECs grown on tissue culture plastic (positive control)
compared to HUVECs grown on a small fibred PCL scaffold after 4 days of culture. (N
= 4).
Comment 9:
Controls need to be established for the gene expression studies.
Response:
Thank you very much for the comment. HUVECs were cultured on tissue culture plastic
up until 70% confluence. These cells were then run for all the genes and are used
in the ΔΔCt method. All values in the gene expression graphs were normalized to the
values found for HUVECs on tissue culture plastic. Therefore, the positive control
is represented as 1 on each graph and all other values are relative to this positive
control. Negative controls were run to ensure no contamination but were not included
in the graph as they did not show up values when analysed.
Edits made to Methods section:
Reverse transcription polymerase chain reaction (RT-PCR)
RNA was extracted from the cell seeded scaffolds using a Tri-Reagent (Invitrogen,
Thermofisher) method and purified using Qiagen’s RNeasy spin column system. Real-time
polymerase chain reaction was performed using a LightCycler® 480 Instrument II (Roche
Life Science) and Sensifast™ SYBR® High-ROX system (Bioline). Forward and reverse
sequences were designed online. Relative quantification of RT-PCR results was carried
out using the 2^(-∆∆ct) method [41]. Gene expression levels were expressed relative
to GAPDH (housekeeping gene) and normalised to 70% confluent HUVECs on tissue culture
plastic (positive control).
Comment 10:
Surface characterization of the fibers need to be performed in order to demonstrate
that the surface properties are similar across all scaffold morphologies.
Response:
Thank you for the comment. While we agree that more advanced scaffold analysis would
be helpful in comparing the scaffolds, however, this is not in the scope of this submitted
paper. The highest resolution images our SEM was capable of taking showed no difference
between the different fibres. While SEM images don’t show the nanotopography of the
fibres, they do show that all fibres are uniform in size (as seen with the small standard
deviation) and they also show that the fibres all appear smooth in structure and no
melting of fibres is occurring.
We have performed some analysis on the SEM images to show the variability in fibre
diameter along each individual fibre.
Edits made to Methods section:
Fibre and pore properties
Scanning electron images were analysed using ImageJ software (NIH). Briefly, SEM images
of the scaffolds analysed using the DiameterJ plugin for fibre diameter and pore width
and the OrientationJ plugin for fibre orientation [33]. Fibre diameters were measured
on a minimum for 50 different fibres on four different scaffolds. Variance in fibre
diameters along individual fibres was measured using 5 diameter measurements per fibre
along a total of 5 fibres.
Edits made to Results section:
Table 4: Mechanical and physical properties of all four scaffolds.
Fibre diameter (µm) Pore diameter (μm) Variance in fibre diameter along fibre (%)
Scaffold thickness (µm) Porosity (%) Ultimate tensile strength (MPa) Failure strain
(%) Contact angle at 0.2s (°) Stiffness (MPa)
0-5% 5-10%
S 1.64 ± 0.18 8.4 ± 3.7 1.23 140 ± 9.4 91.0 ± 1.6 1.08 ± 0.17 843 ± 143 110.8 ± 7.7
4.10 ± 0.33 2.17 ± 0.25
M 2.95 ± 0.16 14.7 ± 4.8 0.83 336 ± 36.9 89.6 ± 2.4 1.50 ± 0.09 1202 ± 62 128.2 ±
4.0 6.50 ± 0.72 2.99 ± 0.46
L 3.37 ± 0.27 16.4 ± 6.7 2.28 549 ± 33.8 83.9 ± 1.1 1.55 ± 0.16 1071 ± 21 129.1 ±
2.5 5.80 ± 0.36 3.16 ± 0.48
XL 4.83 ± 0.49 23.3 ± 9.0 2.83 598 ± 59.6 83.3 ± 0.9 1.26 ± 0.06 855 ± 120 131.5 ±
1.1 5.77 ± 0.39 3.66 ± 0.28
Reviewer 2:
Overall Comment:
The research topic on electrospun fiber scaffold for tissue engineering is promising.
However, the experimental design of this work will produce very limited new information
in the field. The motivation of this research is not clear.
Response:
The authors would like to thank you for taking the time in reviewing the manuscript.
We agree that the motivation may not be that clear. We have rewritten some of the
introduction to help clarify the motivation of this paper.
Edits made to Introduction section:
While these ECM scaffolds have been looked at in normoxic culture, they have not yet
been studied in hypoxic cultures that more accurately mimic the native oxygen content
found in most native tissue types. Therefore, there is further motivation in not only
studying fibre diameter but also looking at how hypoxic culture affects blended PCL/ECM
electrospun scaffolds. By looking at these different compositional and morphological
aspects of the electrospun scaffold, the aim is to pin-point which one has the biggest
effect on seeded endothelial cells to help guide future scaffold design.
Comment 1:
Why did authors tried to compare the ECs gene expression under hypoxic normoxia or
hypoxia condition?
Response:
Thank you very much for the comment. The reason we have compared normal incubator
conditions (normoxia) to hypoxic conditions is that hypoxia is a truer representation
of the in vivo conditions. Most in vitro experiments take place in 16% oxygen which
is higher than the oxygen content found in most parts of the body. For example, most
healthy tissues in the body have oxygen contents ranging between 3% and 7.4%, and
most tumours have oxygen contents ranging between 0.3% and 4.2% [9].
Comment 2:
What is the biological mechanism for increase in viability and cell proliferation
on the large diameter fiber scaffold?
Response:
Thank you for the comment. We will try and discuss this further in the discussion.
While we don’t know the exact answer to this, one theory is that the changes in viability/gene
expression are being driven by the integrin binding sites which have previously been
shown to drive gene expression in endothelial cells [10–13].
Edits made to Discussion section:
In addition, there was an upregulation of CD31 in the extra-large scaffold compared
to the three other scaffolds. CD31 is an angiogenic gene that is heavily involved
in endothelial cell-cell association that is required for their reorganization into
tubular networks [24]. A significant relative upregulation in CD31 was noted in the
extra-large scaffold after 6 days compared to the medium scaffold. Relative increases
were also noted between the extra-large scaffold and the three other morphologies
at both 6 and 12 days, albeit without significance. No trend in data could be noted
between the three other morphologies suggesting that the jump from the large fibre
to extra-large fibre (3.37µm to 4.83µm) ultimately led to this increase in CD31 expression.
While this study was not able to pin point exactly why changing fibre diameter led
to alterations in gene expression, it did gives insights into potential mechanisms.
For example, Figure 3 shows how the HUVECs are binding to each scaffold, with fewer
binding sites present on the largest fibre. A plethora of work has shown how integrin-mediated
adhesion in endothelial cells can lead to angiogenesis and other changes in gene expression
[56-59]. Therefore, with the final aim of creating an environment most suited for
endothelial cells to proliferate into a healthy endothelium, the results suggest that
the extra-large scaffold fibre is the best candidate.
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