Tissue engineering (TE) is an interdisciplinary filed of science that aims
to meet the medical needs by recreating tissues and organs or improving
their original biological functions. It's based on cell biology, materials
science and engineering. Biomaterial-based scaffolds have the task to replace anatomical
and functional features, lost in injured or diseased tissues, and to
restore and maintain normal functions [1, 2]. Scaffolds can be obtained by
natural or synthetic materials. In particular, polycaprolac-tone (PCl) is
FDA-approved linear polyester, biodegradable, relatively cheap. Its properties
makes it fit for different biomedical application, such as medical implants,
medical devices, wound dressing materials, drug delivery systems, and
tissue engineering scaffolds (bone, cardiovascular, and liver
substitutes) [3-5]. Highly porous networks for cellular
support miming the natural extra-cellular matrix (ECM) can be
produced from PCl through different techniques like the electrospinning.
This technique allows to produce fibrillar structures characterized by
fiber diameters ranging from nanometers to microns . Electrospun
nanofibrous scaffolds show a high surface/volume ratio, tunable porosity, and flexibility
in order to suit over a wide variety of sizes and shapes [7-9].
Moreover, the biomaterial composition can be modulated in features and
functionality. In particular, in order to improve chemical-physical properties
and optimize cell growth, the scaffold can be prepared starting by blends
of PCl with a variety of natural polymers (chitosan, silk fibroin, collagen, elastin)
or synthetic polymers (PllA, PlGA, polystyrene) [7, 10]. For example, Fadaie
and colleagues prepared a bionanocomposite electrospun scaffold of PCl/chitosan
with improved mechanical properties, wettability and cell compatibility
. In addition, this blend scaffold, employed as vascular
engineered vascular graft, showed ECM deposition (especially elastin
and collagen) and, above all, endothelialisation . PCl/starch nanofibrillar
mat, prepared by elettrospinnig, was employed in haemostatic applications,
due to its ability to promote blood clotting formation in 156 s .
Different PCl/PlA blends have been assessed to optimize properties of electrospun
scaffold in term of morphology, in vitro degradation, mechanical behaviour
and cell compatibility .
account the advantages to use hybrid PCl scaffolds, this study aimed to assess
the resistance to bile and urine of an electrospun blend of PCl, α,β-poly(N-2-hydroxyethyl-D-l)-aspartamide
(PHEA) and polylactic acid (PlA) (named PHEA-PlA). Tubular PCl/PHEA-PlA
has been already employed to create arteriovenous fistulas (AFV) in pigs
[15, 16]. In this case, the material showed great elasticity, probably
thanks to regularity and dimensional uniformity of fibres, and good
mechanical features. A tubular PCl/PHEA-PlA scaffold was also tested
to create a biliary-digestive anastomosis, on rabbits. Three months
after implant, the fibrillar structure did not show any sign of lithic
digestion by bile and a new epithelial tissue appeared on scaffold surface,
suggesting potential reparative features of the material . The aim of
this work is to evaluate the biological stability of planar and tubular PCl/PHEA-PlA
matrices in different biological conditions (bile and urine).
Material and methods
in our experimental
procedures we utilized the PHEA-PlA+PCl scaffold in two female pigs to
assess its resistance to bile and urine.
(PHEA) was prepared and purified according to the reported procedures
. Spectroscopic data (FT-IR and 1H-NMR) were in
agreement with previous results . PHEA weight-average molecular weight
was determined by size exclusion chromatography and was equal to 38 kDa (MW/MN =
In the first step,
PlA N-hydroxysuccinimide (NHS) derivative was prepared and purified
[20-22]. In the second step, PHEA and PlA-NHS were solubilised using an
opportune amount of DEA as catalyst. The reaction between the hydroxyl
groups of PHEA and the activated carboxylic groups of PlA-NHS
was carried out for 24 hours . After work-up process, obtained
copolymer was characterized by 1H NMR, and the value of molar
derivatisation degree in PlA was in agreement with previous results.
Proton nuclear magnetic resonance (1H-NMR) was performed using
a Brucker AC-300 instrument of 300 MHz.
process, a programmable syringe pump (Aitecs PlUS SEP-21S) and a high voltage
power supply (Spellman CZE 1000 R) were used. Electrospinning process was
carried out horizontally with an accelerating voltage of 20-25 kV and a
constant polymeric solution rate of 1 ml/h obtained through a programmable
syringe pump. Scaffold with different shapes were obtained by
changing the rotative collector of the electrospinning apparatus. In
particular, we used a 2 cm2 planar scaffold and a tubular
scaffold with 6 mm diameter and 4 cm length. In both scaffolds the
thickness was 0.6 mm. All scaffolds obtained were washed several
time with bidistilled water, then dried under vacuum and sterilized.
experimental procedures were in compliance with the Animal Research: Reporting
of In Vivo Experiments guidelines. The study designed was initially
submitted for approval to the Animal Welfare Body (O.P.B.A.) of the
"A.Mirri" Institute (according to Italian legislative Decree No. 26
of 4 March 2014 transposing Directive 2010/63/EU on the protection of
animals used for scientific purposes) that expressed a favourable opinion.
We used two porcine
models, female of 20 kg and 4 months of age. During a procedure under general anaesthesia
(premedication: Zolazepam + Tileta-mine 6.3 mg/Kg + Xylazine 2.3 mg/Kg -
induction: Propofol 0.5 mg/Kg - Maintenance: Isoflurane + Pancuronium
0.07 mg/Kg) . In all animals a central venous catheter was placed to be
used also for post-operative blood tests . With the animal in a
prone position and the 4 limbs secured to the operating table, the region of
interest was shaved and the operating field was disinfected with
povidone iodine 10%. After the surgical procedure, all the pigs
received post-operative antibiotic treatment with oxytetracycline (20
mg/Kg a day for 3 days). In post-operative period, the animals were
monitored clinically and blood samples were drawn daily for the first
seven days and then once a week to control the inflammation and cholestasis
parameters. A follow-up ultrasound was performed at post-op day 7.
The sacrifice of the animals was scheduled at one month, except in the
case of a worsening of clinical conditions requiring a change of the scheduled deadlines.
1 (t1): resistance
test in vivo to bile
We assessed the
ability of our scaffold to replace a portion of the gallbladder wall. We already
tested the biocompatibility of the scaffold and its
resistance against bile in other surgical procedures in rat (17) and
rabbit (15). In this surgical procedure we want to simulate a bigger
damage as close as possible to real surgical injuries and to value how the
scaffold works. After a median longitudinal incision we accessed
the hepato-duodenal region and isolated the gallbladder. Once the gallbladder
was identified and isolated, we clamped the gallbladder fundus and made an
incision of about 2 cm2. We sutured a planar scaffold made
of PHEA-PlA+PCl on the gallbladder by means of interrupted 6-0 Prolene
2 (t2): resistance
test in vivo to urine
We assessed the
ability of our scaffold to resist against pig's urine. After a median longitudinal
incision we accessed the pelvic region and isolated the bladder. We
performed an approx. 5 cm incision on the bottom of the bladder. Inside
the bladder, about 1 cm from the section margin, we have sutured
the scaffold to the inner wall of the bladder with continuous suture in
non-absorbable material 5-0. After that we closed the surgical bladder
After the explant
of gallbladder and bladder, we evaluated scaffold morphology and cells
adhesion by electron microscopy (SEM) using a scanning electron microscope
(ESEM Quanta FEI) on sample cross section. After removal, all samples were
washed with phosphate buffer and were treated with a 4% (v/v) formaldehyde
solution. Samples were dehydrated with ethanol (30%, 50%, 70%, 90% and
pure ethanol), then were treated with hexamethyldisilazane and
freeze-dried. Histopathological analysis on tissue samples taken from areas of
contact with gallbladder mat were performed by the coloration
with hematoxylin-eosin. In particular, 5 |jm thick sections were cut and
were analysed using an optical digital microscope.
period, both the animals were monitored clinically and blood samples
were drawn daily for the first seven days and then once a week to
control the inflammation, cholestasis parameters in the first pig and kidney
function in the second one. According to our schedule, a follow-up ultrasound
was performed at post-op day 7. There wasn't leakage or peritonitis signs
in both pigs. After a month we performed cholecystectomy in the first
pig and a partial bladder resection in the second one.
In the first case
(t1), a microscopic histological examination of the grafted section was done to
assess if there were any response of the host and the degree of tissue
regeneration. After fixation and reduction, the samples were processed as
done routinely for paraffin embedding. Serial sections (5 μ m) of
each sample were stained with hematoxylin-eosin (H&E). An
immunohistochemical exam was done to search for specific endothelial
markers by using Ig anti-CD31. In both cases the samples were embedded in formalin,
treated with 30% sucrose, included in OCT (antibodies) and stored
at -80 °C. Part of the samples were cut into slices with a thickness of 8 mm in a cryostat for subsequent immunofluorescence and analysis
under confocal microscopy.
examination of tissue samples recovered from areas of contact with gallbladder mat
showed a mucosal tissue on the scaffold. In addition, a correct
stratification of epithelial cells was evident. The boundary line between
electrospun matrix and mucosal tissue formed was visible (fig. 1). Under
this transition zone, there was a monocyte infiltration with the presence
of giant cells and macrophages.
Fig. 1. Hystological microscopy on tissue sample after 30
days of implantation (20x)
obtained revealed an interpenetration of cells and extracellular matrix
components (fig. 2A). In addition, fibres appeared unchanged in term of
form and dimension and, above all, the absence of fusion among each other was
demonstrated (fig. 2B).
In the second case
(t2) a tubular PCl/PHEA-PlA scaffold was sutured into pig's bladder. After 30
days the scaffold was explanted and SEM analysis was conducted. Micrograph
acquired showed the optimal cell colonisation of electrospun matrix.
Fig. 2. SEM micrograph of PCl/PHEA-PlA scaffold after 30 days
of implantation in pig gallbladder. Scale bar corresponds to 20 μm for A and to
2 μm for C panel
structure of scaffold was maintained over time and the suture thread was
detectable after explant (fig. 3). In addition, the accumulation of
different type of insoluble inorganic salt (such as phosphate, oxalated or
uronic salts) appeared evident.
micrographs obtained confirmed that materials maintained in vivo unaltered
its fibrillary structures after the contact with bile and urine.
is already used to prepare drug carriers systems and as a starting materials
for different biomedical and pharmaceutical applications [22, 25-27].
PHEA-PLA derivative was extensively employed to design biocompatible scaffolds thank
its easy chemical processability and optimal biocompatibility toward
different cell lines [20, 22, 28]. In particular, nanofibrillar scaffolds
starting by PHEA-PLA can be obtained through electrospinning technique
how reported in a previous work . This polymeric derivative of PHEA
can be mixed with PCL to prepare different type of electrospun matrixes,
following a procedure reported elsewhere . Taking into account the
potential versatility of these scaffolds, the aim of this work was to study the
biological stability of blended PCL/PHEA-PLA matrixes in
vivo animal models.
The scaffold used
was characterized by a large surface area to volume ratio, superior mechanical performance
like stiffness and tensile strength compared with any other known form of the
material . Micrographs obtained in the two experimental trails confirmed
that materials maintained unaltered its fibrillary structures after the contact
with extracellular environment. Electrospun matrix seemed to mime
the native ECM structure promoting cells adhesion and the infiltration
of extracellular matrix components. These features were confirmed by histological
The opportune cell
infiltration and proliferation observed allowed to assess the biocompatibility
of material used. However, an inflammatory response of modest degree was
observed, which has a key role in the long-term resorption of the material
leading potentially to the migration of stem cells.
chronic inflammatory response can be a limit of this study because we observed
the scaffold ongoing only 30 days. This limit would be exceeded by
testing the material for a longer time frame. In addition, this type of
scaffold is easily functionalized with anti-inflammatory factors,
growth factors, antibiotics and anti-coagulants that can be
a key-factor in the future use of this device.
Fig. 3. SEM micrograph of PCL/PHEA-PLA tubular scaffold after 30 days of
implantation in pig bladder. Scale bar corresponds to 500 μm for A and to 20 μm for B panel
matrix showed a high biological versatility. Fibers maintained a
uniform size and shape over time. Data obtained allowed to suppose
that the three-dimensional architecture features promote the interaction
between cells and the regeneration of tissue. As a result, both mat
and tubular scaffold were colonized by cells and extracellular matrix elements.
these results confirmed the ability of PCL/PHEA-PLA scaffold to promote tissue
regeneration thanks to the integration with native component of tissues.
Further studies will be necessary to evaluate the long-term effects of the
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