Characterization of polycaprolactone based electrospun scaffold towards in vitro human trabecular meshwork model

Abstract

Glaucoma is the world´s second leading cause of irreversible blindness according to the World Health Organization. Age, race, family history of glaucoma, myopia and elevated intraocular pressure (IOP) are the most important risk factors to develop this pathology. The human trabecular meshwork (HTM), which is responsible for the regulation of the IOP by draining the aqueous humor (AH), is usually dysregulated during Glaucoma development. A decrease of draining capacity would render an increase of the IOP, which causes the stiffening of the trabecular meshwork (TM). However, the biophysical mechanisms by which these pathologic changes contribute to IOP elevation remain unclear. Moreover, the lack of appropriate in vitro TM models makes it challenging to understand the specifics of outflow physiology as well as to derive new anti-glaucoma therapies. For this reason, we propose a TM-emulating model based on nanofibrous composite scaffold fabricated by means of electrospinning technologies. In this work, randomly oriented polycaprolactone (PCL) nanofibers were used as scaffolds of the TM. Scanning electron microscopy and mechanical testing were performed in order to quantify the microstructure porosity and stiffness of both the scaffolds and the native tissue. Permeability studies were also undertaken by passing out a specific flow rate through the electrospun mesh, which simulates the AH. Our results show cellular viability of human trabecular meshwork cells onto the scaffolds. Finally, this in vitro platform will allow further research on the effects of TM rigidity on regulation of the IOP and glaucoma progression. In all likelihood, this research will pave the way for the development of new treatment strategies for this pathology.

Full text

Characterization of polycaprolactone based electrospun scaffold
towards in vitro human trabecular meshwork model
M. Bikuña Izagirre1, E. Carnero González2, L. Extramiana Esquisabel2, J. Aldazabal1, J. Moreno
Montañés2, J. Paredes Puente1
1 Departamento de Ingeniería Biomédica, Universidad de Navarra TECNUN, San Sebastián, España,
mbikunai,jaldazabal,jparedes@tecnun.es
2 Departamento de Oftalmología, Clínica Universidad de Navarra, Pamplona, España, ecarnero,lextramiana@unav.es
Abstract
Glaucoma is the world´s second leading cause of irreversible
blindness according to the World Health Organization. Age, race,
family history of glaucoma, myopia and elevated intraocular
pressure (IOP) are the most important risk factors to develop this
pathology. The human trabecular meshwork (HTM), which is
responsible for the regulation of the IOP by draining the aqueous
humor (AH), is usually dysregulated during Glaucoma
development. A decrease of draining capacity would render an
increase of the IOP, which causes the stiffening of the trabecular
meshwork (TM). However, the biophysical mechanisms by which
these pathologic changes contribute to IOP elevation remain
unclear. Moreover, the lack of appropriate in vitro TM models
makes it challenging to understand the specifics of outflow
physiology as well as to derive new anti-glaucoma therapies. For
this reason, we propose a TM-emulating model based on
nanofibrous composite scaffold fabricated by means of
electrospinning technologies. In this work, randomly oriented
polycaprolactone (PCL) nanofibers were used as scaffolds of the
TM. Scanning electron microscopy and mechanical testing were
performed in order to quantify the microstructure porosity and
stiffness of both the scaffolds and the native tissue. Permeability
studies were also undertaken by passing out a specific flow rate
through the electrospun mesh, which simulates the AH. Our
results show cellular viability of human trabecular meshwork
cells onto the scaffolds. Finally, this in vitro platform will allow
further research on the effects of TM rigidity on regulation of the
IOP and glaucoma progression. In all likelihood, this research
will pave the way for the development of new treatment strategies
for this pathology.
1. Introduction
Glaucoma is an optic neuropathy that is characterized by
the progressive functional degeneration of the optic nerve.
It is a major public health concern, as it represents the
second leading cause of irreversible blindness in the
world[1]. There are several types of glaucoma, out of
which primary open angle glaucoma (POAG) is the most
extended one. A recent review estimated the global number
of diagnosed POAG cases in 2013 to be around 44 million,
while also foreshadowing an increase to 53 million by 2020
due to population aging[2]. Some uncertainty regarding the
actual number of people with POAG remains, since as it is
an asymptomatic disease, the number of affected
individuals could be substantially larger than the number
of confirmed cases[1,3]. The American Academy of
Opthalmology recommends lowering the intraocular
pressure (IOP) of the eye toward specific values that are
believed to lower the progression of the disease, avoiding
in this manner, the major retinal damage[3] for which
glaucoma is notorious. IOP is generated by aqueous humor
(AH) circulation system in the anterior eye segment. AH
passes from the posterior chamber, through the pupil, into
the anterior chamber, and then exits the eye via two
pathways: (1) the conventional or trabecular outflow
pathways, or (2) the unconventional or uveoscleral outflow
pathway[4]. The conventional outflow pathway is more
relevant, as it performs 80-90% of the AH drainage and
provides resistance against the flow of AH, which
inadvertently generates the IOP[4]. The human trabecular
meshwork (TM) is composed of a connective tissue that
contains a core of collagenous and elastic fibers, which are
covered by flat cells that rest on basal lamina. These beams
attach to one another in several layers and form a porous
filter-like structure, which essentially divide the TM into
three main regions: the uveoscleral meshwork, the
corneoscleral meshwork and the juxtacanalicular tissue,
localized directly adjacent to the inner wall endothelium of
the Schlemm canal [4]. Changes in HTM structure and
functionality are likely to affect tissue rigidity and
biomechanical properties, modifying its resistance to AH
flow and augmenting the probability of developing
glaucoma.
Despite the pressing issue that this malady poses,
Glaucoma research is severely limited by the use of animal
models that do not translate well to human forms of the
disease. Due to the wide anatomical inter-species variation
in the TM and aqueous humour outflow pathway, using an
animal in vivo model of the TM represents a substantial
problem. Although advances in glaucoma research have
been facilitated by in vitro 2D culture models, they are not
ideal to gain a good understanding of the AH outflow
process and TM physiology[5]. The uniqueness of this
tissue, along with the lack of good animal models, makes
the study of Glaucoma a challenging and complex task.
In consequence, developing a 3D artificial TM has become
a justified endeavor whose necessity is reaching
unprecedented heights. Until now, very few bioengineered
in vitro HTM models have been developed. Torrejon et al.
fabricated a SU-8 based scaffold, coated with 1% gelatin
and with pores of 12 µm, which turned out to be the
optimum combination for cell adhesion and growth[6].
More recently, a collagen and sulfate chondroitin
tridimensional matrix has been developed. The authors of
this work observed that the TM porcine cells were able to
proliferate while maintaining their functionality[7].
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ISBN: 978-84-09-25491-0, pags. 389- 392

Building upon current knowledge of HTM morphology
and physiology, in this study we fabricate an electrospun
based porous membrane of polycaprolactone (PCL) to
generate an in vitro HTM model. The PCL is a
biocompatible and biodegradable polyester that allows cell
adhesion and growth, as well as over time progressive
degradation and elasticity to accommodate contractile
functions[8]. The electrospinning technology allows the
fabrication of porous microstructures to support HTM cells
and to simulate similar outflow characteristics to in vivo
TM. The fiber sizes as well as the stiffness were analyzed
by scanning electron microscopy (SEM) and traction trials,
respectively, with the goal of ensuring the native
mechanical properties. These properties will allow the
physiological proliferation of human trabecular meshwork
cell (HTMC) on PCL fibers.
Following this, human trabecular meshwork cells (HTMC)
were seeded onto PCL scaffolds to study the expression of
their markers and characterize the secretion of extracellular
matrix proteins. Subsequently, the permeability was
studied introducing the bioengineered scaffold in a
watertight pressure camera as part of a flow and pressure-
controlled system, specifically designed to mimic the AH
outflow. These studies established that higher permeability
rates lead to lower scaffold stiffness because of lower
pressure values.
2. Materials and Method
2.1. Materials & reagents
PCL pellets were purchased from Sigma-Aldrich (St.
Louis, MO). Chloroform and methanol were purchased
from Fisher Scientific. Dulbecco´s modified eagle medium
(DMEM) was obtained from Gibco. The HTMC were
obtained from donors.
2.2. Nanofibrous scaffold fabrication
Firstly, PCL (10 wt. %) has been dissolved in the
chloroform/methanol solvent mixture with a volume rate
of 4:1, v/v and by agitating the mixture with a magnetic
stirrer at 600rpm overnight at room temperature (21 ±1 °C).
Similarly, PCL (20 wt. %) was dissolved in a
chloroform/methanol (3:1, v/v) solvent mixture by
agitating the mixture with a magnetic stirrer at 600 rpm
overnight at room temperature. For the electrospinning
process, all the solutions were placed in a 1 mL plastic
syringe exiting the solution with a speed of 1 mL/h through
a 20G metallic needle. For PCL (10 wt. %) a voltage of
10kV was applied, with a distance of 15cm between the
needle and the collector. For the higher concentration
polymer, the voltage was raised to 15kV. The nanofibers
were collected on a 9x9 cm2 aluminum foils.
2.3. Characterization of nanofibrous scaffolds
The morphology of the composite nanofibrous scaffolds
was studied with SEM. The fiber diameters were calculated
based on SEM images by using image analysis software
(ImageJ, NIH, USA).
The elasticity studies were carried out using the Zwik Roell
(Ulm, Germany) traction instrument. All the samples were
6x1cm large, with a grip separation of 10mm and a test
speed of 10mm/min.
2.4. Perfusion studies
The constructed circuit is equipped with a programmable
syringe pump (Chemyx® Fusion 100, Stafford, USA) and
two pressure sensors (Elveflow®, Paris, France) as shown
in Figure 1. The system follows a simple working
principle. A control program designed using the graphical
programming environment LabView (National
Instruments® Austin, Texas) is used to monitor and ensure
the correct functioning of the apparatus. The interface
portrays the real time values of the pressure sensors and
generates a scenario-representative graph. The nanofibrous
scaffolds were secured inside the camera. This system
allows the control of the flow and the measurement of the
transmembrane pressure, enabling the study of the
permeability characteristics of our HTM model. In our
experiment, we first introduced the different electropsun
meshes inside the camera and perfused them with a flow
rate of 20 µL/min for 20 min. On forward steps, HTMC
seeded on PCL scaffolds will be introduced in the camera
with the same flow rate to observe pressure differences. For
future lines, the effect of pressure on these cells will be
studied and different drugs will be perfused to observe the
cell´s response.
Figure 1. Complete scheme of the perfusion system with the syringe pump, the PC and the pressure sensor with the reader.The camera is
located inside the incubator. In the right-hand side of the figure, the inner structure of the camera can be seen. (A) Represents the way
the liquid follows, entering from the upper part to exit from the bottommost hole. (B) The lowest piece of the camera, which contains the
PCL mesh with the HTMC
A
B
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XXXVIII Congreso Anual de la Sociedad Española de Ingeniería Biomédica. 25 – 27 Nov, 2020

2.5. HTMC culture
The primary HTMC were obtained from donor eyes
(University of Navarra) or corneal ring donors (Barcelona
tissue bank). HTMC isolated from the trabecular
meshwork, were firstly cultured in a 6 well plate with 0.1%
gelatin coating and DMEM, 20% FBS and 1% P/S. Fresh
culture medium was supplied every 48h. Cells were
maintained at 37°C in a humidified atmosphere with 5%
carbon dioxide until 80–90% confluence was achieved at
which point the cells were trypsinized using 0.25%
Trypsin/0.5mM EDTA and subcultured. HTMC were
subcultured on gelatin-coated 75 cm2 cell culture flasks.
All studies were conducted using cells before the 5th
passage.
2.6. Culture of HTM cells on PCL scaffolds
PCL 10% and PCL 20% scaffolds were sterilized under a
UV hood. The electrospun meshes were suspended on
cellular crowns (Figure 2) for 12 well plate (Sigma Aldrich,
St. Louis, MO) and coated with gelatin 0.1 % to promote
HTM cell attachment by soaking them in sterile solution
for 30 min. HTM cells were seeded on the scaffolds in a
density of 1x104 cells/cm2 and cultured with 10 % FBS.
Cell growth was monitored every 48h for 15 days, to
finally treat them with 300nM of dexamethasone for 5 days
to observe changes on F-actin distribution and to measure
MYOC expression levels to corroborate the good
proliferation of the cells over the nanofibrous scaffold.
2.7. Inmunocytochemistry
Cells were fixed and stained for characteristic F-actin
cytoskeleton and the nucleus. After 15 days in culture, the
cells grown on the scaffolds were fixed with 4%
paraformaldehyde, permeabilized with 0.2 % Triton X-
100, and blocked with 5 % goat serum in PBS. HTM cells
were subsequently incubated with Phalloidin ifluor 594
reagent (Abcam, Netherlands) to visualize the F-actin and
stained with DAPI to reveal cell nuclei.
3. Results and Discussion
3.1. Mechanical characterization of PCL
nanofibrous scaffolds
The 10 wt. % and 20wt. % PCL scaffolds were prepared by
means of electrospinning methods and mechanically
characterized. The fiber structure was observed under SEM
as shown in Figure 3. The PCL 10% scaffolds had a mean
fiber diameter of 1.85±0.21µm (mean ± sd) and mean
porous area of 143,141±181,043 µm2. For the PCL 20%
scaffolds the mean fiber diameter value was 2.97 µm with
a standard deviation of 0.38 µm, and the mean porous area
was 366,501±507,185 µm2. Pore dimensions among these
sizes have shown successful results in cellular migration
through scaffolds[9], with inadequate proliferation when
pores are too small.
With regard to the traction studies, PCL 10% meshes show
a considerably lower stiffness than the PCL 20% ones
(Figure 4), the former type mesh having a Young Modulus
of 3.9MPa versus the 20MPa Young Modulus of the latter
type. Some studies showed the Young Modulus of healthy
HTM states to be around 4kPa, whereas for the
glaucomatous one it rises up to 80kPa [10]. Even despite
the evident stiffening of the tissue due to the disease, the
PCL scaffolds remain distant from those values.
3.2. Permeability studies
Permeability studies were carried out in order to study the
outflow resistance of the nanofibrous scaffolds. Thus, PCL
10% and PCL 20% meshes were introduced in the chamber
and were exposed to different flow rates (10, 20 and 40
µL/min). The observed outcome is shown in Figure 5
which portrays how the pressure values increase as the
flow also grows. Pressure measurements at different flow
rates allow for the calculation of the outflow facility of the
bioengineered HTM. The slope of the transmembrane
pressure (P) versus flow rate (F) was 0.304 mBar/ µL/min
for the PCL 10% and 0.209 mBar/µL/min for the PCL
20%, and the outflow facility (ΔF/ ΔP), calculated by
taking the inverse of the slope, was 3.289 µL/min/mBar
and 4.784 µL/min/mBar, respectively. These results
concur with what has been shown in the literature, where
Figure 2. Cell crown with PCL 10% mesh where HTMC
are seeded
C
Figure 3. Fiber analysis under SEM. (A) PCL 10%
fibers (B) Fiber diameter histogram showing the
prevalence of a diameter value of 1.85 µm. (C) PCL
20% fibers. (D) Fiber diameter histogram showing
common sizes between 2.59 and 3.35 µm.
A
B
D
Figure 4. Strain stress graphic comparing the both PCL
scaffolds
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XXXVIII Congreso Anual de la Sociedad Española de Ingeniería Biomédica. 25 – 27 Nov, 2020

the conventional AH drainage resistance moves around
3.99-5.33mBar/ µL/min [11]. Therefore, PCL 10% meshes
show higher permeability values in consequence of lower
values in pressure.
3.3. HYM cell proliferation over PCL 10%
mesh
The cellular viability and proliferation was evaluated over
PCL 10% scaffolds (Figure 6). There was not any
significant difference between dexamethasone treated and
non-treated cultures. Both cultures show a successful
cellular proliferation obtaining 30-100ng/µL of RNA,
together with MYOC expression, concluding a good
cytoskeletal functioning.
4. Conclusion
In this study, we confirm the feasibility of using an
electrospinning based PCL nanofibrous scaffold to
construct an in vitro HTM model for the purpose of
studying the HTM outflow physiology and its response to
biological agents. Despite the fact that PCL 10% meshes
displayed a good mechanical and cellular response, we
would like to study the PCL scaffolds with different natural
polymers such as collagen, gelatin or elastin to study their
mechanical characteristics and cellular viability.
Moreover, our HTM perfusion platform and nanofibrous
scaffold are ready for perfusion studies with HTM cells on
the scaffolds to bring them under different drugs in order
to study outflow facility and the cellular response.
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Figure 5. Pressure values depending on the flow rate for
PCL 10% and PCL 20%
A
B
Figure 6. PCL 10% cultures stained with DAPI for the nucleus
and in red for F-actin. (A) Culture without dexamethasone (B)
culture with dexamethasone. Scale bar: 100µm
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