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Dual-Acting Biofunctionalised Scaffolds For Applications in Regenerative Medicine
Dual-Acting Biofunctionalised Scaffolds For Applications in Regenerative Medicine
DOI 10.1007/s10856-017-5849-z
Received: 22 November 2016 / Accepted: 6 January 2017 / Published online: 20 January 2017
Springer Science+Business Media New York 2017
Abstract Off the shelf scaffolds for replacing ultra-small endothelialisation were assessed on explants. Biofunctio-
diameter vascular grafts are valuable for reconstruction of nalised scaffolds were also grafted subcutaneously and
diseased or damaged vessels. The limitations for such grafts intraperitoneally to evaluate integration, inammation and
include optimal handling with ready availability of varied angiogenesis reactions. The potential wider applications of
lengths of grafts, graft patency with the ability to replace the this dual acting scaffold were evaluated for its interactions
function of active cellular mechanisms and adequate with human dermal broblasts as well as bronchial epithe-
mechanical properties to maintain physicochemical func- lial cells. Physicochemical property evaluation of the
tion. We used a well-established, solvent casting method for functionalised grafts has claried the mechanical strength
potential tissue replacement scaffold fabrication with and permeability. This study conrmed the microsurgical
incorporated bioactive molecules, which we have pre- suturability of tubular grafts and graft patency of functio-
viously explored to confer haemocompatibility. These nalized scaffolds. The study demonstrated the potential of a
grafts were tested in-vivo within the abdominal aorta of 10 dual acting biofunctionalised scaffolds use for a wide range
Wistar rats and the patency was clinically and echo- of tissue engineering applications where micro-porous, yet
graphically evaluated. Haemocompatibility and impermeable scaffolds are needed.
bioactive molecules that encourage vessel formation and a 250 ml reaction ask, equipped with mechanical stirrer and
degree of scaffold porosity are signicant for efcacious graft nitrogen inlet, and heated to 90 C.
function. Whilst porosity is a signicant feature in tissue 4,4-Methylenebis(phenyl isocyanate), (MDI), was added
engineering [79], the process of tubular structure construc- to the polyol and then reacted, under nitrogen, at 7585 C
tion, requires the graft exterior to be impermeable so that the for 90 min to form a pre-polymer. Dimethylacetamide was
luminal content is contained and provides a channel of added slowly to the pre-polymer to form a solution; the
delivery for the relevant content. Therefore, direct fabrication solution was cooled to 40 C. Chain extension of the pre-
of a porous yet impermeable scaffold is useful. This study polymer was carried out by the drop wise addition of
demonstrates a single-step fabrication method of obtaining ethylenediamine and isobutylamine in dimethylacetamide to
porous, non-permeable scaffold through elution of bioactive, form a solution of polycarbonate urea-urethane in dime-
yet biologically favourable molecules. thylacetamide. After completion of the chain extension 1-
A previous study has considered the above properties butanol in dimethylacetamide was added to the polymer
in vitro where a poly(carbonate-urea) urethane (PCU), was solution.
functionalised with L-Arginine Methyl Ester (L-AME) [10]. 90, 180 and 270 mg of L-arginine methyl ester dihy-
L-AME is an analogue of L-Arginine (L-Arg), which is an drochloride (L-AME) (Sigma-Aldrich) were dissolved in
endogenous substrate of nitric oxide synthase and thus plays a 500 l of deionised water and vortexed to produce a clear
signicant role in the cardiovascular [11], central nervous solution. The solution was added drop by drop to 5 g of
system [12], respiratory system [13], as well as in wound 18% PCU with 2 ml of dimethylacetamide (DMAC), fol-
healing. This study aimed to clarify the in vivo biocompat- lowed by vortexing and gentle heating in a water bath to
ibility of the preliminary in vitro results and the angiogenesis produce a clear solution.
potential.
Considering the 3Rs (replacement, reduction, renement) 2.2 Tubular scaffold fabrication
linked to in vivo studies, we have opted for a rat model as a
small animal model to test material properties [14]. Despite Five millimeter diameter stainless steel mandrels (Metal
their suitability for the assessment of micro-vascular con- Mania online retailer) were attached to a exible lament,
duits, rat models are generally recognised to have a robust (to adopt a vertical balance during solvent evaporation)
vascular system that can resist many features associated with when suspended to hang freely in an oven (Fig. 1). The
test-graft failure. Therefore, in this study as a compromise of mandrels were dipped into 50 ml centrifuge tube containing
observing 3Rs and obtaining meaningful results, we intro- PCU with 180 mg of L-AME (L-AME-PCU) and a control
duced a graft mismatch at the site of anastomosis in order to with PCU only. The coated mandrels were placed in an
induce local turbulence in blood ow, [15] with the aim of oven set at 65 C for 3 h. They were then inverted and re-
comparing and evaluating the better performing scaffold in dipped into the centrifuge tube to ensure that the polymer
the presence of adverse vascular events. Therefore, the aim was evenly distributed. This process was repeated to pro-
of the aortic vessel anastomosis was to evaluate the scaffold duce scaffolds with 6, 8 and 10 coats. Tubes were then
that can better resist thrombosis inducing factors, indepen- immersed overnight in propanol-lled test tubes before
dent of the scaffold mechanical properties in vivo. separating from the mandrels.
This scaffold in previous studies has shown positive
interactions with human dermal broblasts, (HDFs) and 2.3 Scaffold fabrication for in vitro tests
endothelial progenitor cells (EPC) in vitro [10] and here we
demonstrate its interactions with epithelial cells, with the PCU and L-AME-PCU polymer solution were poured on
intention to evaluate the practical application and suitability 10 cm-diameter circular glass plates and left in an air-
to apply this functionalised material in medical devices and circulating oven at 65 C for at least 12 h to produce a cast
tissue replacements. Figure 1 presents a summary of the polymer. Thirteen millimeter diameter disks were cut from
experimental plan aimed to address the main aims of this the casted polymer and sterilised in 70% ethanol for 1 h, and
article. left under UV light for 1 h, followed by three rounds of
washing in phosphate-buffered saline (PBS) of 5 min each.
Sterilised disks were incubated in respective cell culture
2 Materials and methods medium for at least 3 h prior to cell seeding.
Scaffolds were generated from 18% PCU synthesised as Scaffolds were immersed in 70% ethanol solution overnight
below. Polycarbonate polyol, 2000 mwt was placed in a (Sigma) and ethanol was evaporated in a biosafety cabinet
J Mater Sci: Mater Med (2017) 28:32 Page 3 of 12 32
under ultra violet light to ensure sterility of the scaffolds. 2% PFA and 1.5% GTA) and critical point dried before
Scaffolds that have been removed from the mandrel were being processed for analysis.
immersed in deionized water-lled 50 ml centrifuge tubes
for 48 h. The tubes were placed over a roller and was
changed with fresh water every 24 h. 2.6 Permeability testing
2.5 Scanning electron microscope (SEM) Test scaffolds completely covered the 1 cm2 cut-out in the
acrylic holding sheet as part of the main permeability testing
Samples were sputter-coated with 5 nm of gold- device. (Supplementary Fig. 1AF) The holding sheets
nanoparticles using a plasma sputter-coater (Quorum were tightened to create a water-tight chamber. Water
Q15ORS) and samples were imaged using JEOL JSM- pressure in the device was increased and measured by
7401F eld emission scanning electron microscope or Zeiss C9557 Pressure Meter (Comark). The volume of water
EVO HD15 microscope. Test samples for SEM with cells that escaped from the main device through the scaffold was
were xed (in 300 L 0.1 M sodium cacodylate buffer with collected by the beaker.
32 Page 4 of 12 J Mater Sci: Mater Med (2017) 28:32
2.7 Burst pressure testing the proximal and distal aortic segments was rinsed to pre-
vent thrombi formation (Fig. 3a and b). Rounded 10/0
A dynamic model was assembled to test PCU and L-AME- Ethilon was used to perform end-to-end anastomosis
PCU tubes using a water bath, pressure monitor, connection between the vascular scaffolds and ends of the aorta. The
tubes. The test grafts were introduced with a solution with a lumen was rinsed again to ush the air bubbles and the
blue coloured dye (Supplementary Fig. 1GI) anastomosis was checked under saline. The clamp was
The 6, 8, and 10 coated tubes were subjected to burst released distally, and then proximally. Anastomosis was
pressure measurements. Each tube was connected to the checked 3 min after unclamping to check for leakage. The
infusion pump on one side and to the pressure monitor on peritoneum and muscular layer were closed with 4/0 Mer-
the other. Blue died water was infused at a rate of 0.2 ml/ suture. No anticoagulants were introduced in order to
min and the rate was subsequently increased up to 50 ml/ avoid a bias and reduce confounding. The rats were
min. The pressure inside the tube was recorded simulta- immediately warmed and placed in cages. Their weights
neously using the pressure monitor. were monitored post-surgery.
Test polymers were cut into dumbbell shape samples Prosthetic patency was evaluated by evaluating the ow
(Fig. 2b and c, Supplementary Fig 2) according to ISO 37 proximal to the implanted scaffold. The rats that survived
Type 3. Instron 5565 gripped two ends of the dumbbell at more than a day were brought for Doppler ultrasound
20 mm, and scaffolds were pulled to their breaking point. evaluation. Rats were anesthetised with 4% isourane in air
Tensile strengths were subsequently measured by Bluehill for induction and maintained using 2.5% isourane. They
software. were positioned in supine position and their vitals mon-
itored. The abdomen was shaved and then depilated using
2.9 In vivo study depilatory cream. We used VEVO2100 ultrasound ima-
ging system (Visualsonics, Toronto, CA), with a MS250
Tubular scaffolds were excised into 10 mm. 10 Wister rats probe at 18 MHz, to visual blood ow proximal and distal
were included in this study as was considered optimal to to the anastomotic sites at 21, and 120 days after the sur-
obtain the experimental clarication, whilst observing gery. Pulsed Doppler measurements were performed
replacement, reduction, renement (3Rs) within the allo- upstream and downstream to the implanted scaffold when
cated research funds for this study. Five male rats were possible.
implanted with PCU scaffolds (control), and 4 female and 1
male rats with bio-functionalised L-AME-PCU scaffolds. 2.11 Histology
(Fig. 3, Supplementary Fig 3) This study was approved by
the Regional Committee of Ethics on Animal Experiments Amongst the ve control rats: four died within 1 day fol-
(Paris Descartes University) (registration number lowing the procedure. one control rat died on day 4 after
CEEA34.15-021). grafting (this rat was sacriced due to display of pain and
The rats were anesthetized with intramuscular injection hind limb immobility). Two test rats were sacriced on day
of 10 ml of ketamine (50 mg/ml) and 1.5 ml of chlorpro- 120 as one of test rats died during surgical procedure, and
mazine (5 mg/ml). Then, they were placed in supine posi- one during transport between labs for analysis (but the
tion with their heads at the left of the surgeon. Their four explanted graft demonstrated a patent graft). All samples of
limbs were attached to the table with adhesive elastic bands of PCU and L-AME-PCU explant scaffold and rat tissues
(Elastoplast). were xed on with 4% paraformaldehyde then embedded in
A 7 cm median xipho-pubic skin incision was made. parafn and stained with hematoxylin-eosin. PCU and L-
Followed by dissection of muscular and peritoneal layers to AME-PCU scaffolds were also implanted intraperitoneal
expose the aorta and the vena cava, and the intestines were and subcutaneously , which were xed with 4% paraf-
mobilised to the left. A further dissection was performed ormaldehyde and embedded in parafn and stained with
under a microscope (Carl Zeiss) to free the subrenal hematoxylin-eosin to evaluate the inammatory response.
branches of the abdominal aorta. The collaterals between The histology sample sectioning was met with difcul-
the renal arteries and the iliac bifurcation were cauterised ties in obtaining full and complete sections of the scaffold
(Supplementary Fig 3). attached to live tissues. Due to this damage to the samples
The aorta was clamped with Gilbert double clamp 5 mm during processing, no quantication analysis could be per-
proximal to the iliac bifurcation and distal to the renal formed. The interface between the scaffold and tissues were
vessels, and 5 mm of the aorta was excised. The lumen of not embedded, thereby making comparison between the
J Mater Sci: Mater Med (2017) 28:32 Page 5 of 12 32
Fig. 2 a Mandrels of 1.5 mm (second from left), scaffold on mandrel uorescence microscopy. Scaffold in dumbbell shape before elution
(third from left), 6 coats (fourth from right), 8 coats (third from right), (b) after elution demonstrating greater compliance with expanded and
10 coats (second from right) Mandrels with varying diameters can be elongated scaffold (c). SEM images of g luminal surface of L-AME-
used to fabricate grafts with required diameters. d 6 coats of L-AME- PCU graft immersed in propanol. h External surface of L-AME-PCU
PCU under uorescence microscopy. e 8 coats of L-AME-PCU under graft immersed in propanol. i Luminal surface of L-AME-PCU graft
uorescence microscopy. f 10 coats of L-AME-PCU under immersed in water
samples difcult. A modied protocol, which was used to medium in each well was made up to 1 ml and changed
study the interface between bones and implants could be every two days.
implemented here to strengthen the samples analysis [16].
2.13 Fluorescence microscopy
2.12 Cell cultures The disk was removed on day and xed in 500 l of 4%
formaldehyde in PBS (Gibco) for 1 h at room temperature.
Human bronchial epithelial cells (HBEC) (Caltag Medsys- Formaldehyde was aspirated and disks were then washed
tems) were cultured in human bronchial epithelial cell with PBS twice. Disks were protein-blocked in 500 l 1%
medium with 1% antibiotic (50 l/ml streptomycin, 50 l/ml bovine serum albumin (BSA) with 0.25% triton in PBS for
penicillin) and supplements (Caltag Medsystems). HBEC 45 min at room temperature and washed in PBS twice.
were seeded onto sterilised 13 mm disks of scaffold at a Disks were then submerged in 200 l of 0.5% phalloidin
density of 5*105 cells per well on 48-well plates. The cell (Sigma) in PBS for 1 h, followed by 100 l of DAPI
32 Page 6 of 12 J Mater Sci: Mater Med (2017) 28:32
(Sigma) for 15 min, at room temperature and in the dark. microscopy (Fig. 2df) were used to measure the average
Stained cells were visualised using EVOS FL Cell Ima- scaffold wall thickness as 330.25 20.57 m for six coats,
ging System (Life Technologies). 348.70 14.82 m for eight coats, 497.28 49.23 m for
10 coats (Table 1). The scaffold wall thickness increased
with increasing number of coats. Mandrels with a range of
2.14 Statistical analysis diameters can be used to manufacture these grafts with
ease.
DAgostino & Pearson omnibus normality tests were con- SEM images of luminal and external surface of L-AME-
ducted. P < 0.05 was considered statically signicant, and PCU graft immersed in propanol (Fig. 2g and h) demon-
the null hypothesis that the data is normally distributed is strated that L-AME did not elute from the scaffold in the
rejected. Ordinary ANOVA test was conducted for nor- presence of propanol while it was eluted when immersed in
mally distributed data with Bonferroni post hoc test. water (Fig. 2i). This proved to be a signicant nding as
Kruskal-Walli test was conducted for data that is not nor- immersion of the polymer coated mandrels in water is
mally distributed with Dunns post hoc test. required to allow the grafts formed to be detached away
from the mandrels, but substitution in propanol facilitated
this whilst retaining L-AME within the graft. Luminal
3 Results surface of L-AME-PCU scaffolds had regular, honeycomb
structure. The fabrication resulted in a transparent structure
3.1 Macro and micro structure of tubular scaffolds for the control and an opaque surface for the functionalised
scaffold.
Mandrel with diameters of 1.5 mm (second from left L-AME concentrations ranging up to (3.75, 7.5, 15, 30
Fig. 2a) were used, for in vivo studies. The cross-sections and 60 mg) were tested and found to generate smaller pores.
of 6, 8 and 10 coated tubular scaffold under uorescence (results not included) However, there was no signicant
J Mater Sci: Mater Med (2017) 28:32 Page 7 of 12 32
0.191 0.0183
Table 1 The average tube thickness measured using ImageJ for tubes
27.198 3.563
with 6, 8 and 10 coats (n = 9)
374.3 6.372
Number of coats 6 8 10
10 coats
Average tube 330.25 20.57 348.70 14.82 497.28 49.23
thickness (m)
0.167 0.0163
25.177 3.619
314.3 6.197
difference between biomechanical properties measured by
Table 2 Tensile strength of control scaffold, L-AME-PCU non-porous scaffold, and L-AME-PCU porous scaffold (where L-AME was eluted in water)
microscopy (results not included).
0.1548 0.00121
22.193 3.219
3.2 Permeability and burst pressure
298.2 5.785
The scaffold was impermeable to de-ionised water at pres-
6 coats
sures of 200 mmHg when it was tested by the permeability
testing device as a at scaffold (Supplementary Fig. 1AF).
The 6, 8 and 10 coated L-AME-PCU tubes resisted burst
0.229 0.0286
47.062 25.662
pressures of 450 mmHg, 700 mmHg and 1028 mmHg
520.67 152.7
respectively. Distension of the tubes was noted, and there
was an increase in their diameters. 2 0.25 cm for the six
coats tube, 1.5 0.15 cm for the eight coats tube and 1 10 coats
0.1 cm for the ten coats tube (Supplementary Fig. 1, Sup-
plementary video).
0.193 0.0211
39.536 19.261
509.51 150.6
L-AME-P (non-Porous/before elution)
679.3 59.400
Fig. 4 Photographs of representative images of explants of the test adventitia (TA), the blood vessels (BV) and some red blood cells
scaffolds, which were placed in the rat model in situ a1 and b1 and a (RBC). Ultrasound of the test rat 21 days after the implantation in
longitudinal section of the scaffold a2 and b2; Histological sections transversal (c1) and sagittal (c2) view; (c3) Pulsed Wave Doppler
illustrating the interface between the scaffold (indicated in dotted line), blood ow velocity measurement distal to bionfunctionalised L-AME-
with b3, b4, b5, b6, b7 and b8 L-AME, and the aorta, stained with PCU scaffold in abdominal aorta. (c4) Pulsed Wave Doppler blood
hematoxylin-eosin, showing, at different magnication (4; 10; ow velocity measurement proximal to biofunctionalised L-AME-
20; 40), the implant (IM), the endothelium of the tunica intima PCU scaffold in abdominal aorta. (c5) Pulsed Wave Doppler blood
(Endo), the tunica intima (TI), the tunica media (TM), the tunica ow velocity measurement in distal abdominal aorta, t
Fig. 5 Photographs of the inammatory studies of the PCU-scaffold with a2,a3,a4,a5,a6,a7,c2,c3 L-AME, and the aorta, stained with
implanted intraperitoneally a1 and b1 and subcutaneously c1 and d1 in hematoxylin-eosin, showing, at different magnication (4; 10;
the same rat; Histological sections showing the interface between the 20; 40), the implant (IM), the blood vessels (BV) and some red
scaffold (indicated in dotted line), without b2,b3,d2, d3, and d4 and blood cells (RBC)
demonstrated in Supplementary Fig. 4 showing the difference poor cell adhesion (Supplementary Fig 5A). The test sam-
in diameter between rat aorta and PCU implant. ples with L-AME functionalised scaffolds, had bronchial
epithelial cells with healthy cobblestone morphology and
sheet-like appearance, which demonstrated healthy adhe-
3.6 Human bronchial epithelial cells (HBEC) sion and proliferation. (Supplementary Fig. 5BD) SEM of
interactions with functionalised scaffold bronchial epithelial cells, cultured on L-AME-PCU at
scaffolds, demonstrated microivili-like protrusions on cell
Human bronchial epithelial cells were seeded on PCU and membranes. There were healthy HBEC cell morphology
L-AME-PCU scaffolds, where the latter had L-AME eluted. and cell-cell interaction, as well as complete covering of the
On day 7, on the control scaffolds, epithelial cells appear to scaffold surface indicating good cell adhesion and pro-
be round with a small nucleus and cytoplasm. They dis- liferation after seeding (Supplementary Fig. 5E, F).
played abnormal bronchial epithelial cell morphology and
32 Page 10 of 12 J Mater Sci: Mater Med (2017) 28:32
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