3d systems delivering vegf to promote angiogenesis for tissue engineering11
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7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 17
3D systems delivering VEGF to promote angiogenesis for tissue engineering
Anne des Rieux a Bernard Ucakar a Billy Paul Kaishusha Mupendwa a Didier Colau b Olivier Feron cPeter Carmeliet d Veacuteronique Preacuteat a
a Universiteacute catholique de Louvain Louvain Drug Research Institute Uniteacute de Pharmacie Galeacutenique 1200 Brussels Belgiumb Ludwig Institute for Cancer Research Brussels Branch 1200 Brussels Belgiumc Universiteacute catholique de Louvain Institut de recherche expeacuterimentale et clinique Pole of Pharmacology 1200 Brussels Belgiumd Vesalius Research Center (VRC) KU Leuven VIB 3000 Leuven Belgium
a b s t r a c ta r t i c l e i n f o
Article history
Received 15 July 2010
Accepted 28 November 2010
Available online 3 December 2010
Keywords
VEGF encapsulation
Hydrogel
Scaffolds
Angiogenesis
Tissue engineering
In most cases vascularization is the 1047297rst requirement to achieve tissue regeneration The delivery from
implants of angiogenic factors like VEGF has been widely investigated for establishing a vascular network
within the developing tissue In this report we investigated if encapsulation of VEGF in nanoparticles could
enhance angiogenesis in vivo as compared to free VEGF when incorporated into two different types of 3D
matrices Matrigeltrade hydrogels and PLGA scaffolds Negatively charged nanoparticles encapsulating VEGF
were obtained witha high ef 1047297ciency by complex formationwith dextran sulfate andcoacervationby chitosan
After 2 weeks encapsulation reduced VEGF release from hydrogels from 30 to 1 andincreased VEGF release
from scaffolds from 20 to 30 in comparison with free VEGF VEGF encapsulation consistently improved
angiogenesis in vivo with both type of 3D matrices up to 75- to 35-times more endothelial and red blood
cells were observed respectively into hydrogels and scaffolds Hence encapsulation in nanoparticles
enhanced VEGF ef 1047297ciency by protection and controlled release from 3D implants Encapsulation and
incorporation of VEGF into 3D implants that in addition to sustaining cell in 1047297ltration and organization will
stimulate blood vessel are a promising approach for tissue regeneration engineering
copy 2010 Elsevier BV All rights reserved
1 Introduction
Regardless of the tissue of interest implants developed for tissue
engineering must be biocompatible and biodegradable They should
serve as a three-dimensionaltemplate to provide structural support to
the newly formed tissue through an interpenetrating network of
pores (at least 100 μ m wide) to allow cell migration tissue in-growth
and vascularization [12] In addition further enhancing of the
functionality of these matrices by loading them with growth factors
acting on the surrounding tissues at therapeutic concentrations and
for an adequate period of time is highly desirable [3] The delivery
from implants of angiogenic factors like vascular endothelial growth
factor (VEGF) has been widely investigated to promote new blood
vessel formation [45] which is a basic requirement for establishing a
vascular network within the developing tissue [6] There are different
routes for VEGF administration that can be divided into local and
systemic delivery [7] Intravenous delivery of VEGF is problematic
The short circulation half-life extraneous interactions with multiple
binding sites and VEGF degradation are incompatible with the
sustained local concentrations of VEGF required for the development
of mature blood vessels [8] Incorporation of VEGF into poly(-lactic-
co-glycolic acid) (PLGA) scaffolds or into microspheres has shown the
potential to protect and locally deliver VEGF at a more constant rate
leading to site-speci1047297c angiogenesis [8] Still VEGF released from the
implant remains susceptible to degradation Current techniques to
encapsulate and protect VEGF often use harsh organic solvents and
submit proteins to mechanical stress resulting usually in low protein
loading [9] Huang et al [8] developed a method to ef 1047297ciently
encapsulate VEGF based on the VEGF heparin binding domain af 1047297nity
for dextran sulfate and subsequent coacervation by chitosan forming
nanoparticular polyelectrolyte complexes Complexation and encap-
sulation preserve VEGF activity provide a high loading ef 1047297ciency and
allow a controlled release making this formulation suitable for VEGF
incorporation into implants
A wide range of three-dimensional bioactive implants has been
developed with potential uses as delivery systems for therapeutic
drugs relevant for tissue repair processes [3] among which two main
types hydrogels and porous polymeric scaffolds Ideally hydrogels
should be injectable in the lesion for a non-destructive delivery
limiting then further damage Several macromolecules have been
used to form injectable hydrogels [10] but Matrigeltrade (composed of
structural proteins such as laminin collagen IV and heparan sulfate
proteoglycans [11]) has became the method of choice for many
studies involving in vivo testing of angiogenesis [1213] Depending
on the type of tissue the use of polymeric scaffolds may be
Journal of Controlled Release 150 (2011) 272ndash278
Corresponding author Universiteacute Catholique de Louvain Louvain Drug Research
Institute Uniteacute de Pharmacie Galeacutenique Avenue E Mounier 73-20 1200 Brussels
Belgium Tel +32 2 7647320 fax +32 2 7647398
E-mail address veroniquepreatuclouvainbe (V Preacuteat)
0168-3659$ ndash see front matter copy 2010 Elsevier BV All rights reserved
doi101016jjconrel201011028
Contents lists available at ScienceDirect
Journal of Controlled Release
j o u r n a l h o m e p a g e w w w e l s ev i e r c o m l o c a t e j c o n r e l
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 27
advantageous to offer a physical support to the regenerating cells
Previous studies have demonstrated that VEGF delivery from PLGA
scaffolds can achieve local concentrations of VEGF that induced blood
vessel growth and stimulated new tissue formation [14ndash17]
In this report we investigated if encapsulation of VEGF could
enhance angiogenesis in vivo when incorporated into two different
types of 3D matrices Matrigeltrade hydrogels and PLGA scaffolds VEGF
was encapsulated into nanoparticular polyelectrolyte complexes by1047297
rst binding to dextran sulfate and then by coacervation withchitosan VEGF incorporation ef 1047297ciency and release from implants
were characterized in vitro The ability of these implants to induce
angiogenesis in vivo was quanti1047297ed
2 Materials and methods
21 Materials
Buffered sorbitol-complex medium (BMSY-medium) contained
glycerol as the carbon source and consisted per liter of 200 mM
phosphate buffer pH 68 and 134 g of yeast nitrogen extract (YNB)
(Invitrogen Carlsbad USA) 10 g sorbitol (Sigma Saint Louis USA)
04 mg biotin (Sigma) and 100 mg zeocin (Invivogen Toulouse FR)
Buffered methanol-complex medium (BMMY) is similar to BMGY
except that the carbon source is 1 (vv) methanol (Merck
Darmstadt GE) and contains 1 (wv) casamino acids (Becton
Dickinson Bedford USA) Dextran sulfate (500 kDa) was purchased
from Fluka (Buchs CH) and chitosan (15 kDa) from Polysciences Inc
(Warrington USA) VEGF quanti1047297cation was performed by ELISA
using monoclonal anti-rat VEGF and biotinylated anti-rat VEGF (RampD
Systems Inc Minneapolis USA) as well as Piercereg high sensitivity
streptavidinndashhorseradish peroxidase and TMB substrate kit (Thermo-
Scienti1047297c Rockford USA) 7525 poly(lactide-co-glycolide) (PLGA RG
755 S) was kindly provided by Boehringer Ingelheim (Ingelheim GE)
Anti phospho-VEGFR2 (TYR1175) was from Cell Signaling (Beverly
USA) Zinc sulfate mannitol Triton X-100 bovine serum albumin
(BSA) and HUVEC (passage 1) were bought from Sigma used at
passage 6 and cultivated in an endothelial cell growth medium kit
(Sigma) Growth Factor Reduced (GFR)-Matrigeltrade was purchasedfrom Becton Dickinson
22 Recombinant Rat VEGF 164
221 Production
Pichia pastoris yeasts producing VEGF164 were kindly provided by
Prof Carmeliet (VesaliusResearch Center KU Leuven Belgium) Pichia
were inoculated in 50 ml of the BMSY-medium in 250 ml baf 1047298ed 1047298ask
24 h at 30 degC in an orbital shaker To induce production of VEGF164
yeasts were centrifuged (1500 g for 10 min) and resuspended at 1 U
OD (600 nm) in 250 ml of the BMMY-medium (2 L baf 1047298ed 1047298asks with
250 ml1047298ask) Induced cells were grown at 30 degC under orbital shaking
(150 rpm) Every 24 h 1 (vv) methanol was added After 2 days the
supernatant of the culture was collected after centrifugation (10000 g20 min) and 1047297ltered on 045 and 02 μ m 1047297lters
222 Puri 1047297cation
Recombinant VEGF164 was puri1047297ed from the cell supernatant by
af 1047297nity chromatography with heparin as ligand followed by size
exclusion chromatography Brie1047298y 500 ml of the culture supernatant
was 1047297rst concentrated to 10 ml by ultra1047297ltration (MWCO 10000) in
an Amicon chamber andthen diluted to 50 mlwith sodiumphosphate
buffer 50 mM pH 6 The sample was loaded on a 5 ml Hitrap Heparin
HP column (GE Healthcare Sweden) previously equilibrated with
sodium phosphate buffer 50 mM NaCl 200 mM pH 6 The column
waswashed with the same bufferadjusted to 500 mM NaCl VEGF was
eluted with 1 M NaCl Dimeric VEGF (MW~40000) was separated
from aggregated forms on a Superdex75 HR10300 column (GE
Healthcare) with PBS as an elution buffer Purity and integrity of VEGF
were checked by SDS-Page and concentration was evaluated by UV
spectrophotometry (calculated dimer molar extinction coef 1047297cient
e= 12115 Mminus1cmminus1) and ELISA The VEGF solution was 1047297lter-
sterilized and its concentration adjusted to 1 mgml The activity of
VEGF was assessed by detection of VEGF receptor 2 (VEGFR2)
phosphorylation of HUVEC cells by Western blot [18] It was
compared to a commercial VEGF (VEGF164 RampD Systems produced
in murine myeloma cell line NS0 derived) The activity of VEGF wasmeasured by its ability to phosphorylate the VEGF receptor 2 (R2) of
human umbilical vein endothelial cells (pre-screened HUVECs
ECACC S200-05n) The detailed protocol can be found in
Supplementary material 1
223 VEGF radioactive labeling and biotinylation
[3H] VEGF waspreparedby reductivealkylation of amino groupsas
previously described [19] The speci1047297c activity of [3H] VEGF was
obtained by measuring the total protein concentration by the
microBCA test (Pierce Thermo Scienti1047297c) according to the manufac-
turers instructions and the radioactivity by liquid scintillation The
speci1047297c activity of [3H] VEGF was 613middot10eminus4μ Ciμ g [125I] VEGF was
obtained by incubation of VEGF with sodium iodine [125I] carrier free
(1 mCi Perkin Elmer Waltham USA) and Iodination Beads (Pierce
Thermo Scienti1047297c) according to the manufacturers instructions [125I]
VEGF speci1047297c activity was 112 μ Ciμ g VEGF integrity after labeling
was checked by SDS-Page electrophoresis
Puri1047297ed recombinantrat VEGF164wasbiotinylated withthe EZ-Linkreg
Sulfo-N -hydroxysuccinimide ester of biotin (Thermo Scienti1047297c)
Unincorporated biotin was separated from biotinylated VEGF on a
HITRAP desalting column(GE Healthcare) with PBS as the elution buffer
Biotin incorporation was quantitated by 4prime-hydroxyazonenzene-2-
carboxylic acidavidin assay (Thermo Scienti1047297c) A mean ratio of 45
biotin per dimer of VEGF was obtained
23 Formation of VEGF nanoparticles
VEGF nanoparticles (VEGF NP) were prepared as described by
Huang et al [8] Brie1047298y 80 μ l of the VEGF solution (1 mgml in PBS)was added to 800 μ l of the dextran sulfate (DS) solution (1 wv in
water) under stirring (700 rpm) and were stirred for 30 min Next
16 ml of the chitosan (CS) solution (01 wv in acetic acid pH 6) was
added dropwise and stirred for 5 min Finally 80 μ l of the zinc sulfate
solution (1 M in water) was added and stirred for 30 min The
particles were either incubated in 5 mannitol for 30 min centri-
fuged resuspended in 5 mannitol and freeze-dried or centrifuged
and resuspended at the desired concentration in water The mean size
of nanoparticles with and without VEGF was determined by dynamic
light scattering and the zeta potential by phase analysis light
scattering using a NanoZS (Malvern UK) The in1047298uence of freeze-
drying on nanoparticle size and zeta potential was determined
Incorporation ef 1047297ciency was evaluated by incorporating [3H] VEGF
in the formulation Incorporation ef 1047297ciency was expressed as apercentage of thetotalamount of [3H] VEGF found in thenanoparticles
over the radioactivity in the supernatant and in the nanoparticles
24 Nanoparticle incorporation into 3D implants
241 Hydrogels
The free or encapsulated VEGF was mixed with 500 μ l of Growth
Factor Reduced (GFR)-Matrigeltrade and was kept on ice Matrigeltradesupplemented with PBS was used as a negative control Hydrogels
geli1047297ed at 37 degC forming a solid mass
242 Polymeric scaffolds
Polymeric scaffolds were fabricated using a gas foamingparticu-
late leaching process as previously described [20ndash
22] Brie1047298y free
273 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
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VEGF CSndashDS or CSndashDSndashVEGF nanoparticles were freeze-dried and
mixed 1047297rst with 36 mg of microspheres (PLGA 7525) and then with
100 mg of NaCl (250 μ mndash425 μ m diameter) Scaffolds composed of
PLGA microspheres and salt only were used as a negative control
Thenthe mixture was compressed ina 5 mmKBr die at 1500 psi using
a Carver press The PLGA scaffolds were then equilibrated with high
pressure CO2 gas (800 psi) for 16 h in a custom-made pressure vessel
[2324] The pressure was released over a period of 30 min To create
scaffold macroporosity scaffolds were leached 1047297
rst in 4 ml of sterilewater for 2 h and were then transferred in fresh sterile water for
30 min PLGA scaffolds were then dried and kept under dry and sterile
conditions [4]
The maximal amount of lyophilized nanoparticles that can be
incorporated in the PLGA scaffolds without disrupting their integrity
was evaluated by adding 3 to 20 mg of freeze-dried nanoparticles
prepared without VEGF (CSndashDS) to the microspheresalt mixture
(36 mg100 mg) Scaffold integrity was evaluated visually The
criteria were i) the general shape of the scaffold ii) it resistance to
manual manipulation (crumbling) iii) the shape of the pores
evaluated by an optical microscope and compared to scaffoldswithout
nanoparticles and iv) scaffold weight The scaffolds were considered
to be ldquonon disruptedrdquo when no difference was observed with control
scaffolds (without nanoparticles)
The VEGF loss during leaching was evaluated by incorporating
sup3H-VEGF (free or encapsulated) in the scaffolds and measuring
residual radioactivity in the scaffolds as well as in leaching super-
natants The loss during leaching was expressedas a percentage of the
total radioactivity found in the supernatants over the radioactivity in
the supernatants and in the scaffolds
25 Release of VEGF from the 3D matrix
Nanoparticles were formed either with [125I] VEGF and incorpo-
rated into PLGA scaffolds (1 μ g VEGFsample+1 μ l of [125I] VEGF
(0548 μ Ci)) or with biotinylated VEGF and incorporated into hydro-
gels (2 μ g of biotinylated VEGF500 μ l hydrogel) (n =3) Samples
were incubated in PBS at 37 degC After speci1047297c time periods PBS was
removed and replaced by fresh PBS Radioactivity of the supernatantthat contained the sample for that speci1047297c time period was measured
by gamma counter (Perkin Elmer) to quantify the amount of protein
that was released The cumulative release of VEGF from the samples
was calculated by dividing the cumulative radioactivity released into
the buffer by the total amount of radioactivity released over the
course of the study plus the radioactivity that remained inside the
sample at the end of the study The latter was analyzed by dissolving
the samples in 5 M NaOH to measure their radioactivity Biotinylated
VEGF was quanti1047297ed by ELISA The cumulative release of VEGF from
the hydrogels was calculated by dividing the cumulative VEGF
released into the buffer by the total amount of VEGF incorporated
inside the hydrogels
Activity of released VEGF was evaluated by detection of VEGF-R2
phosphorylation as described above
26 In 1047298uence of VEGF encapsulation on angiogenesis
Experimental protocols of animal studies were approved by the
Ethical Committee for Animal Care and Use of the Medicine Faculty of
the Universiteacute Catholique de Louvain
In1047298uence of NP of DS and CS alone on angiogenesis has been tested
by Huang et al [8] with no effect reported on HUVEC proliferation so
CSndashDS NP have not been tested here
1 μ and 2 μ g of free or encapsulated VEGF were incorporated in
hydrogels and 1 μ g in scaffolds Anesthetized BalbC female mice
received a subcutaneous injection of 05 ml of GFR-Matrigeltrade [1225]
PLGA scaffolds were implanted into the intraperitoneal fat pad of
NMRI male mice as previously described [26]
At designed time-points implants were retrieved and 1047297xed in 4
paraformaldehyde for 24 h before incubation in a 30 glucose solution
for 4 h and embedding in Tissue-Tek OCT compound (Sakura
Finetek Torrance CA) Sections were cut at 12 μ m thickness for
Matrigeltrade hydrogels and 14 μ m thickness for PLGA scaffolds using a
cryostat (Leica Microsystems Wetzlar GE) An antibody directed
against the murine endothelial cell surface marker (CD 31) was used
to determine the extent of endothelial cell colonization of the
implants After permeabilization (Triton X-100 01 (vv) in PBS)and blocking (5(wv) BSAin PBS) the primary antibody (rat anti-CD
31 (150 BD Biosciences San Jose USA)) was applied for 1 h at 37 degC
Secondaryantibody(Alexa Fluor 568 goat anti-rat(1500 Invitrogen))
was used to visualizethe antigen Lastly sections were incubated with
DAPI (Invitrogen) (50 ngml) for 5 min to allow visualization of cell
nuclei
Image acquisition was performed by a MIRAX microscope (Zeiss
Thornwood USA) allowing the acquisition of entire sections limiting
the information loss and experimenter in1047298uence by 1047297eld selection
In addition high quality pictures were obtained allowing high
magni1047297cation enlargements For each hydrogel condition 8 sections
at different levels within hydrogels were imaged For each PLGA
scaffold condition 24 sections were imaged 8 sections taken at one
extremity 8 in the middle and 8 at the other extremity of scaffolds
Quanti1047297cation of CD 31 and red blood cells was then performed on
whole sections using a script of Axiovision (Zeiss) The implant
size was normalized by delimiting the implant borders and by
quantifying the percentage of red pixels over the total amount of
analyzed pixels within these borders Results were expressed as
percentage of red pixels over the total amount of pixels within the
analyzed surface
27 Statistics
Statistical analyses were done using PRISM One-way and two-way
ANOVA with post-hoc Bonferronis multiple comparison test were
performed with a p-level of 005 Error bars represent the standard
error of the mean in all 1047297gures
3 Results
31 VEGF production
Pure 40 kDa VEGF164 was successfully produced Its activity was
compared as control to a commercial VEGF164 (RampD systems)
Our VEGF induced VEGFR2 phosphorylation of HUVEC cells at the
same concentration than the control (100 ngml) (Supplementary
material 2)
32 Nanoparticle characterization
To protect and stabilize VEGF it was complexed by dextran
sulfate (DS) and precipitated by coacervation with chitosan (CS) to
produce particular polyelectrolyte complexes encapsulating VEGF [8]
300 nm negatively charged nanoparticles were obtained (Table 1)
Encapsulation ef 1047297ciency was about 76 and the drug loading about
5 μ g VEGFmg of nanoparticles The size of CSndashDSndashVEGF nanoparticles
was slightly higher than CSndashDS nanoparticles and tends to increase
but notsigni1047297cantly after freeze-drying No loss of VEGF was detected
after freeze-drying
The nanoparticle size was stable in PBS for 4 h and then increased
to micrometers probably due to chitosan insolubility above pH 65
while the nanoparticle size was stable in water for at least 12 h
(data not shown)
274 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
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33 VEGF release from 3D implants
VEGF release would likely be in1047298uenced by its incorporation as
well as by the nature of the 3D matrix into which nanoparticles were
incorporated Release of free or encapsulated VEGF was evaluated
over time after incorporation into hydrogels (Matrigeltrade hydrogels)
and into PLGA scaffolds
A 10 to 15 burst release was observed for VEGF whatever the
matrices or the formulation (2 h of incubation) except for encapsu-
lated VEGF incorporated in hydrogel (03) followed by a sustained
release over time (Fig 1) When incorporated in scaffolds release of
free VEGF was lower than encapsulated VEGF (24 versus 36
respectively after 2 weeks) In hydrogels encapsulation signi1047297cantly
slowed down VEGF release Indeed 20-times more free VEGF was
released after 2 weeks than encapsulated VEGF (32 and 16 for free
and encapsulated VEGF respectively) Release of free VEGF from
hydrogels was similar to release of encapsulated VEGF from scaffolds
(2 weeks incubation time 31 of encapsulated VEGF and 32 of free
VEGF respectively) Concerning the activity of released VEGF Huang
et al [8] demonstrated a preserved bioactivity for released VEGF from
NP for 10 days by a HUVEC proliferation assay VEGF released fromPLGA bridges [27] and from hydrogels was still able to induce
phosphorylation of HUVEC VEGF-R2 over a period of 42 days [28] and
of 2 weeks (Supplementary material 3) respectively
34 Nanoparticle incorporation into hydrogels effect on angiogenesis
The angiogenesis potential of free VEGF versus encapsulated VEGF
incorporated into an injectable hydrogel was studied Matrigeltradewas
chosen as a model for tissue regeneration due to its injectability and
proven ability to support cell and blood vessel growth [122930]
Following injection Matrigeltrade solidi1047297es and permits subsequent
penetration by host cells that induce vascularization Growth factor
reduced Matrigeltrade
was used to avoid interferences with the activityof the incorporated VEGF Free or encapsulated VEGF and as control
PBS were mixed with Matrigeltrade before subcutaneous injection
In1047298uence of VEGF amount (1 μ g and 2 μ g) and of incubation times
(2 and 3 weeks) was evaluated The effect on angiogenesis was
determined by quanti1047297cation of endothelial and red blood cells inside
the Matrigeltrade hydrogels after implantation
Endothelial cells werevisualized by immunostainingwith an anti-CD
31 antibody and red blood cells were visible by auto1047298uoresence The
percentage of red pixel represents both endothelial and red blood cells
Entire sections of Matrigeltrade hydrogels were analyzed The bene1047297cial
effect of VEGF encapsulation on the stimulation of angiogenesis
was clearly demonstrated (Fig 2) Indeed 2 weeks after implantation
endothelial cells some of them organized as blood vessels were ob-
served within the samples containing the encapsulated VEGF (Fig 2e
and f) In addition red blood cells were present within these structures
indicating their perfusion and their functionality In contrast for the
hydrogels loaded with PBS or free VEGF endothelial cells were detected
only around the implants (Fig 2a to d)
Quantitative analyses of the hydrogels loaded with VEGF nano-
particles showed that these implants contained 58 to 74-fold more
endothelial and red blood cells in comparison to the hydrogels loaded
with PBS or free VEGF respectively (Fig 3) No signi1047297cant differences
Fig 1 In1047298uence of VEGF encapsulation on its release from 3D implants [125I] VEGF and
biotinylated VEGF were incorporated into 3D implants (hydrogels and PLGA scaffolds)
either free or encapsulated Samples were incubated in PBS at 37 degC Radioactivity or
biotinylated VEGF in the supernatants was measured over time (n=3 signi1047297cant
Pb
005)
Fig 2 In1047298uence of VEGF encapsulation on angiogenesis when incorporated into
hydrogels VEGF (1 μ g) free (c d) or encapsulated (e f) was mixedwith Matrigeltrade and
injected sc in mice (2 weeks) negative control (a b) consisted in PBS mixed with
Matrigeltrade Endothelial cells (red) were detected by immuno1047298uorescence (CD 31) and
red blood cells (red arrow) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Table 1
Properties of CSDSVEGF nanoparticles
Formulation Diameter
(nm)
PDI Zeta potential
(mV)
Encapsulation
ef 1047297ciency
()
a) Before freeze-drying
CSndashDSa 325+minus19 022 +minus003 minus26+minus06 NA
CSndashDSndashVEGFb 383+minus73 026 +minus006 minus26+minus2 765 +minus65
b) After freeze-drying CSndashDSa 319+minus17 020 +minus001 minus25+minus03 NA
CSndashDSndashVEGFb 457+minus24 029 +minus001 minus34+minus15 71 +minus144
NA Not Applicablea n=4b n=6
275 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
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were observed between 1 and 2 μ g of loaded VEGF with implantationduration of 2 and 3 weeks respectively The implants loaded with PBS
or free VEGF showed no signi1047297cant difference
35 Nanoparticle incorporation into PLGA scaffolds effect
on angiogenesis
The objective was to evaluate the effect of VEGF encapsulation on
angiogenesis when incorporated into polymeric scaffolds developed
for tissue engineering [421233132] Variable amounts of VEGF
nanoparticles were incorporated during scaffold processing and the
impact on scaffold integrity was determined The potential improve-
ment of encapsulated over free VEGF on VEGF loss during scaffold
leaching was estimated Then VEGF loaded scaffolds were implanted
in mice and angiogenesis was studiedFreeze-dried CSndashDS nanoparticles were mixed with PLGA micro-
spheres and NaCl crystals to form PLGA scaffolds Physical integrity
was estimated visually The physical integrity of scaffolds was
preserved up to 12 mg of incorporated nanoparticles per 36 mg
PLGA (n= 3) Incorporation of higher amounts induced scaffold
crumbling during the leaching phase To measure VEGF loss after
scaffold leaching lyophilized free or encapsulated [sup3H] VEGF was
used for the scaffold process VEGF loss during salt leaching was 25
for free VEGF and 44 for encapsulated VEGF (n=3) Taking these
ratios into account the amount of VEGF added to the scaffold process
was adapted to obtain 1 μ g of VEGF per implanted scaffold
VEGF loaded scaffolds were implanted in the fat pad of mice and
the in1047298uence on angiogenesis of encapsulated versus free VEGF was
studied 2 weeks after implantation Empty PLGA scaffolds were usedas control Sections of the implanted scaffolds were immunostained
Endothelial and red blood cells were identi1047297ed within and around the
scaffolds for all the conditions (Fig 4)
However as observed for hydrogels PLGA scaffolds containing
VEGF nanoparticles seemed to stimulate more ef 1047297ciently angiogenesis
(Fig 4e and f)
In order to determineif VEGF encapsulation signi1047297cantly increased
the amount of endothelial and red blood cells within and around the
scaffolds quanti1047297cation of the percentage of red pixels over the total
amount of analyzed pixels was performed Signi1047297cantly more
endothelial and red blood cells were detected with VEGF nanopar-
ticles incorporated into PLGA scaffolds (Fig 5) compared with free
VEGF or control although the difference was less marked than
observed for the Matrigeltrade
hydrogels (35-folds) This could be
explained by the fact that PLGA scaffolds alone seemed to support cell
in1047297
ltration and angiogenesis to a higher extent than Matrigeltrade
Nosigni1047297cant difference was observed between negative control and free
VEGF
4 Discussion
When designing systems for tissue engineering one of the 1047297rst
requirements is the colonization of the implant by blood vessels
bringing oxygen and nutriments to support tissue reconstruction
Fig 4 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in scaffolds
VEGF (1 μ g)free (c andd) or encapsulated(e andf) was incorporatedin PLGA scaffolds
and implanted in mouse fat pads (2 weeks) negative control (a and b) consisted in
empty scaffolds Endothelial cells (red) were detected by immuno1047298uorescence (CD 31)
and red blood cells (red) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Fig 5 In1047298uenceof VEGF encapsulation on angiogenesis when incorporated in scaffolds
quanti1047297cation of endothelial and red blood cells VEGF (1 μ g) free or encapsulated was
incorporated in PLGA scaffolds and implanted in mouse fat pads (2 weeks) negative
control consisted in empty scaffolds Endothelial cells were detected by immuno1047298u-
orescence (CD 31) and red bloodcells byauto1047298uorescence Pictures were acquired with
a MIRAX microscope 24sections by condition(n =3) were imagedin their entirety and
analyzed (Axiovision) 8 sections taken at one extremity 8 in the middle and 8 at the
other extremity of scaffolds (mean +minus
SEM Pb
005)
Fig 3 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in
hydrogels quanti1047297cation of endothelial and red blood cells VEGF (1 or 2 μ g) free or
encapsulated was incorporated in Matrigeltrade and injected sc in mice (2 or 3 weeks)
negative control consisted in PBS mixed with Matrigeltrade Endothelial cells were
detected by immuno1047298uorescence (CD 31) and red blood cells by auto1047298uorescence
Pictures were acquired with a MIRAX microscope 8 sections by condition (n=3) were
imaged in their entirety and analyzed (Axiovision) (mean +minusSEM Pb005)
276 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
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The major focus of this work was to determine if incorporation of
VEGFndashCSndashDS nanoparticles into twotypesof 3D implants (PLGA scaffold
or hydrogel) commonly used for tissue engineering (hydrogels and
polymeric scaffolds) improved angiogenesis within the implants and
thus tissue regeneration We investigated the effect of VEGF combined
release from nanoparticles and 3D matrices and the in1047298uence of the
matrix nature on angiogenesis The results indicate thatimplantation of
3D systems loaded with VEGF nanoparticles stimulated endothelial cell
colonization and blood vessel formation more ef 1047297
ciently than matricesloaded with free VEGF regardless of the VEGF dose implantation time
and implant type
The improvement probably resulted from i) VEGF protection by
complexation of the heparin binding site of VEGF with dextran sulfate
combined with ii) protection and controlled release brought by
complex stabilization by chitosan Indeed Huang et al [8] demon-
strated in vitro by ELISA and HUVEC proliferation test that VEGF
encapsulation by DS andCS protectedmost of VEGF activity forat least
10 days and that VEGF was slowly released from nanoparticles
We observed that encapsulation of VEGF in comparison with free
VEGF signi1047297cantly decreases burst release (03 versus 10 respec-
tively) from VEGF loaded Matrigeltrade hydrogels and slowed down
diffusionfrom the implant about 20-fold after 2 weeks It could be due
to higher size combined with electrostatic interactions due to the
negative surface charge of nanoparticles Encapsulated VEGF was
released from PLGA scaffolds slightly faster than free VEGF (24
versus 36 respectively after 2 weeks) Encapsulation did not affect
burst release but loss during leaching was taken into account for
scaffold preparation and the initial VEGF loading was adjusted to be
1 μ g of VEGF post-leaching for both conditions Indeed encapsulation
signi1047297cantly decreased VEGF loss during leaching (about 21) In that
perspective the burst release for free VEGF loaded scaffolds would be
approximately about 15 higher than for VEGF nanoparticle-loaded
scaffolds Finally compared to microspheres classically used to
protect and deliver proteins complexation by DS and coacervation
by CS achieved a higher loading and did not compromise implant
integrity Indeed approximately 5 μ g of VEGF was incorporated in
1 mg of CSndashDS nanoparticles versus approximately 50 ng per mg of
microspheres [9] This allows incorporation of a much higher amountof VEGF into implants compared to microspheres
Although encapsulated VEGF stimulated angiogenesis to a greater
extent than freeVEGF in both implant types differences between PLGA
scaffolds and Matrigeltrade hydrogels were observed The percentage of
endothelial and red blood cells quanti1047297ed in nanoparticle-loaded
scaffolds was 75 times higher compared to Matrigeltrade hydrogels but
the scaffold itself without VEGF stimulated angiogenesis at the same
level than hydrogels loaded with encapsulated VEGF This could be
explained by the factthat these PLGAscaffolds havebeen optimizedfor
tissue engineering [4] and thus promote cell in1047297ltration and
proliferation However encapsulation of VEGF did not affect release
from scaffolds as much as for hydrogels Hence the increase in
angiogenesis elicited by encapsulated versus free VEGF loaded into
Matrigeltradehydrogels wasabout 5 to 7-foldwhile it wasonlyabout 35-fold for scaffolds Considering these results it seems that it could be
interesting to combine thetwo systems by1047297lling PLGA scaffoldspores
with hydrogel both loaded with VEGF nanoparticles
5 Conclusion
Wehypothesized thatVEGFactivityin vivo would be enhanced by its
encapsulation in CSndashDS nanoparticles and further incorporation into
implants used in tissue engineering like injectable hydrogels and
polymeric scaffolds Angiogenesis the primary effect of VEGF was
clearly improved by encapsulation the extent of implant colonization
by endothelial cells and blood vessels was higher when both types of
implants were loaded with VEGF nanoparticles compared with free
VEGF(non-encapsulated) Nanoparticles composed of chitosan dextran
sulfate and VEGF were obtained and incorporated in implants without
impairingtheirstructuralpropertiesand allowing a higher VEGFloading
compared to VEGF classical techniques of encapsulation (up to 5 μ g of
VEGF per mg of nanoparticles) VEGF encapsulation guarantees VEGF
ef 1047297ciency by protection and sustained release from 3D implants
In the scope of the spinal cord regeneration systems to deliver
active VEGF are attractive since in addition to its angiogenic activity
VEGF has recently been shown to be a neurogenic factor [33ndash35]
Then encapsulation and incorporation of VEGF into 3D implants thatin addition to support cell in1047297ltration and organization will stimulate
blood vessel and axon growth are a promising approach to stimulate
tissue regeneration
Supplementary materialsrelated to this article canbe found online
at doi101016jjconrel201011028
Acknowledgments
The authors would like to thank Prof Carmeliet (Vesalius Research
Center KU Leuven Belgium) for the gift of VEGF producing Pichia
pastoris yeast The authors thank Sabine Cordi for her help with MIRAX
acquisition as well as Christophe Pierreux for the RGB quanti1047297cation
script SeacutebastienSartis thankedfor assistance with VEGF productionand
Laurenne Petit for the realization of VEGF activity tests This work wassupported by grants from the Belgian Fonds National de la Recherche
Scienti1047297que (FNRS Creacutedits aux chercheurs and FRSM 3461709) and
CTB (Belgium) Anne des Rieux is FNRS Post-Doctoral Researcher
References
[1] V Guarino F Causa L Ambrosio Bioactive scaffolds for bone and ligament tissueExpert Rev Med Devices 3 (2007) 405ndash418
[2] TM Freyman IV Yannas R Yokoo LJ Gibson Fibroblast contraction of acollagen-GAG matrix Biomaterials 21 (2001) 2883ndash2891
[3] V Mourino AR Boccaccini Bone tissue engineering therapeutics controlled drugdelivery in three-dimensional scaffolds J R Soc Interface 43 (2010) 209 ndash227
[4] CB Rives A des Rieux M Zelivyanskaya SR Stock WL Lowe Jr LD SheaLayered PLGscaffoldsfor invivo plasmid delivery Biomaterials3 (2009)394ndash401
[5] AH Zisch MP Lutolf JA Hubbell Biopolymeric delivery matrices for angiogenic
growth factors Cardiovasc Pathol 6 (2003) 295ndash310[6] ZS Patel AG Mikos Angiogenesis with biomaterial-based drug- and cell-
delivery systems J Biomater Sci Polym Ed 6 (2004) 701ndash726[7] BV Somayaji U Jariwala P Jayachandran K Vidyalakshmi RV Dudhani
Evaluation of antimicrobial ef 1047297cacy and release pattern of tetracycline andmetronidazole using a local delivery system J Periodontol 4 (1998) 409ndash413
[8] MHuang SN VitharanaLJ Peek T CoopC Berkland Polyelectrolyte complexesstabilize and controllably release vascular endothelial growth factor Biomacro-molecules 5 (2007) 1607ndash1614
[9] JS GolubYTKimCL Duvall RV BellamkondaD Gupta AS Lin DWeiss WRobertTaylor RE Guldberg Sustained VEGF delivery via PLGA nanoparticles promotesvascular growth Am J Physiol Heart Circ Physiol 6 (2010) H1959ndashH1965
[10] C He SW Kim DS Lee In situ gelling stimuli-sensitive block copolymerhydrogels for drug delivery J Control Release 3 (2008) 189ndash207
[11] HK Kleinman ML McGarvey JR Hassell VL Star FB Cannon GW LaurieGR Martin Basement membrane complexes with biological activity Biochemistry 2(1986) 312ndash318
[12] N Akhtar EB Dickerson R Auerbach The spongeMatrigel angiogenesis assayAngiogenesis 1ndash2 (2002) 75ndash80
[13] D Donovan NJ Brown ET Bishop CE Lewis Comparison of three in vitrohuman lsquoangiogenesisrsquo assays with capillaries formed in vivo Angiogenesis 2(2001) 113ndash121
[14] JM Kanczler PJ Ginty JJ Barry NM Clarke SM Howdle KM ShakesheffROOreffo Theeffectof mesenchymalpopulations and vascularendothelial growthfactor delivered from biodegradable polymer scaffolds on bone formationBiomaterials 12 (2008) 1892ndash1900
[15] FG Rocha CA Sundback NJ Krebs JK Leach DJ Mooney SW Ashley JPVacanti EE Whang The effect of sustained delivery of vascular endothelialgrowth factor on angiogenesis in tissue-engineered intestine Biomaterials 19(2008) 2884ndash2890
[16] JM Kanczler J Barry P Ginty SM Howdle KM Shakesheff RO OreffoSupercritical carbon dioxide generated vascular endothelial growth factorencapsulated poly(DL -lactic acid) scaffolds induce angiogenesis in vitro BiochemBiophys Res Commun 1 (2007) 135ndash141
[17] JM Kanczler PJ Ginty L White NM Clarke SM Howdle KM Shakesheff ROOreffo The effect of the delivery of vascular endothelial growth factor and bonemorphogenic protein-2 to osteoprogenitor cell populations on bone formation
Biomaterials 6 (2010) 1242ndash
1250
277 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 77
[18] TT Chen A Luque S Lee SM Anderson T Segura ML Iruela-Arispe Anchorageof VEGF to the extracellular matrix conveys differential signaling responses toendothelial cells J Cell Biol 4 (2010) 595ndash609
[19] GE Means RE Feeney Reductive alkylation of amino groups in proteinsBiochemistry 6 (1968) 2192ndash2201
[20] JH Jang LD Shea Controllable delivery of non-viral DNA from porous scaffolds J Control Release 1 (2003) 157ndash168
[21] LD Shea E Smiley J Bonadio DJ Mooney DNA delivery from polymer matricesfor tissue engineering Nat Biotechnol 6 (1999) 551ndash554
[22] DJ Mooney DF Baldwin NP Suh JP Vacanti R Langer Novel approach tofabricate porous sponges of poly(DL -lactic-co-glycolic acid) without the use of
organic solvents Biomaterials 14 (1996) 1417ndash
1422[23] JH Jang CB Rives LD Shea Plasmid delivery in vivo from porous tissue-engineering scaffolds transgene expression and cellular transfection Mol Ther 3(2005) 475ndash483
[24] MO Aviles CHLin M Zelivyanskaya JGGraham RMBoehlerPBMessersmithLD Shea The contribution of plasmid design and release to in vivo gene expressionfollowing delivery from cationic polymer modi1047297ed scaffolds Biomaterials 6 (2010)1140ndash1147
[25] P Sonveaux A Brouet X Havaux V Gregoire C Dessy JL Balligand O FeronIrradiation-induced angiogenesis through the up-regulation of the nitric oxidepathway implications for tumor radiotherapy Cancer Res 5 (2003) 1012 ndash1019
[26] H Blomeier X Zhang C Rives M Brissova E Hughes M Baker AC Powers DBKaufman LD Shea WL Lowe Jr Polymer scaffolds as synthetic microenviron-ments for extrahepatic islet transplantation Transplantation 4 (2006) 452ndash459
[27] Y Yang L De Laporte CB Rives JH Jang WC Lin KR Shull LD SheaNeurotrophin releasing single and multiple lumen nerve conduits J ControlRelease 3 (2005) 433ndash446
[28] L De Laporte A des Rieux ML Zelivyanska HM Tuinstra N De Clerck AAPostnov LD Shea VEGF and FGF-2 delivery from spinal cord bridges to enhanceangiogenesis following injury J Biomed Mater Res Part A (in press)
[29] ML Ponce Tube formationan in vitro matrigel angiogenesis assayMethods MolBiol (2009) 183ndash188
[30] A Albini R Benelli The chemoinvasion assay a method to assess tumor andendothelial cell invasion and its modulation Nat Protoc 3 (2007) 504ndash511
[31] MH Sheridan LD Shea MC Peters DJ Mooney Bioabsorbable polymer
scaffolds for tissue engineering capable of sustained growth factor delivery J Control Release 1ndash3 (2000) 91ndash102[32] JH Jang TL Houchin LD Shea Gene delivery from polymer scaffolds for tissue
engineering Expert Rev Med Devices 1 (2004) 127ndash138[33] P Carmeliet M Tessier-Lavigne Common mechanisms of nerve and blood vessel
wiring Nature 7048 (2005) 193ndash200[34] E Storkebaum D Lambrechts P Carmeliet VEGF once regarded as a speci1047297c
angiogenic factor now implicated in neuroprotection Bioessays 9 (2004) 943ndash954[35] E StorkebaumD LambrechtsM Dewerchin MPMoreno-Murciano S Appelmans
H Oh P Van Damme B Rutten WY Man M De Mol S Wyns D Manka KVermeulen L Van Den Bosch N Mertens C Schmitz W Robberecht EM ConwayD Collen L Moons P Carmeliet Treatment of motoneuron degeneration byintracerebroventricular delivery of VEGF in a rat model of ALS Nat Neurosci 1(2005) 85ndash92
278 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 27
advantageous to offer a physical support to the regenerating cells
Previous studies have demonstrated that VEGF delivery from PLGA
scaffolds can achieve local concentrations of VEGF that induced blood
vessel growth and stimulated new tissue formation [14ndash17]
In this report we investigated if encapsulation of VEGF could
enhance angiogenesis in vivo when incorporated into two different
types of 3D matrices Matrigeltrade hydrogels and PLGA scaffolds VEGF
was encapsulated into nanoparticular polyelectrolyte complexes by1047297
rst binding to dextran sulfate and then by coacervation withchitosan VEGF incorporation ef 1047297ciency and release from implants
were characterized in vitro The ability of these implants to induce
angiogenesis in vivo was quanti1047297ed
2 Materials and methods
21 Materials
Buffered sorbitol-complex medium (BMSY-medium) contained
glycerol as the carbon source and consisted per liter of 200 mM
phosphate buffer pH 68 and 134 g of yeast nitrogen extract (YNB)
(Invitrogen Carlsbad USA) 10 g sorbitol (Sigma Saint Louis USA)
04 mg biotin (Sigma) and 100 mg zeocin (Invivogen Toulouse FR)
Buffered methanol-complex medium (BMMY) is similar to BMGY
except that the carbon source is 1 (vv) methanol (Merck
Darmstadt GE) and contains 1 (wv) casamino acids (Becton
Dickinson Bedford USA) Dextran sulfate (500 kDa) was purchased
from Fluka (Buchs CH) and chitosan (15 kDa) from Polysciences Inc
(Warrington USA) VEGF quanti1047297cation was performed by ELISA
using monoclonal anti-rat VEGF and biotinylated anti-rat VEGF (RampD
Systems Inc Minneapolis USA) as well as Piercereg high sensitivity
streptavidinndashhorseradish peroxidase and TMB substrate kit (Thermo-
Scienti1047297c Rockford USA) 7525 poly(lactide-co-glycolide) (PLGA RG
755 S) was kindly provided by Boehringer Ingelheim (Ingelheim GE)
Anti phospho-VEGFR2 (TYR1175) was from Cell Signaling (Beverly
USA) Zinc sulfate mannitol Triton X-100 bovine serum albumin
(BSA) and HUVEC (passage 1) were bought from Sigma used at
passage 6 and cultivated in an endothelial cell growth medium kit
(Sigma) Growth Factor Reduced (GFR)-Matrigeltrade was purchasedfrom Becton Dickinson
22 Recombinant Rat VEGF 164
221 Production
Pichia pastoris yeasts producing VEGF164 were kindly provided by
Prof Carmeliet (VesaliusResearch Center KU Leuven Belgium) Pichia
were inoculated in 50 ml of the BMSY-medium in 250 ml baf 1047298ed 1047298ask
24 h at 30 degC in an orbital shaker To induce production of VEGF164
yeasts were centrifuged (1500 g for 10 min) and resuspended at 1 U
OD (600 nm) in 250 ml of the BMMY-medium (2 L baf 1047298ed 1047298asks with
250 ml1047298ask) Induced cells were grown at 30 degC under orbital shaking
(150 rpm) Every 24 h 1 (vv) methanol was added After 2 days the
supernatant of the culture was collected after centrifugation (10000 g20 min) and 1047297ltered on 045 and 02 μ m 1047297lters
222 Puri 1047297cation
Recombinant VEGF164 was puri1047297ed from the cell supernatant by
af 1047297nity chromatography with heparin as ligand followed by size
exclusion chromatography Brie1047298y 500 ml of the culture supernatant
was 1047297rst concentrated to 10 ml by ultra1047297ltration (MWCO 10000) in
an Amicon chamber andthen diluted to 50 mlwith sodiumphosphate
buffer 50 mM pH 6 The sample was loaded on a 5 ml Hitrap Heparin
HP column (GE Healthcare Sweden) previously equilibrated with
sodium phosphate buffer 50 mM NaCl 200 mM pH 6 The column
waswashed with the same bufferadjusted to 500 mM NaCl VEGF was
eluted with 1 M NaCl Dimeric VEGF (MW~40000) was separated
from aggregated forms on a Superdex75 HR10300 column (GE
Healthcare) with PBS as an elution buffer Purity and integrity of VEGF
were checked by SDS-Page and concentration was evaluated by UV
spectrophotometry (calculated dimer molar extinction coef 1047297cient
e= 12115 Mminus1cmminus1) and ELISA The VEGF solution was 1047297lter-
sterilized and its concentration adjusted to 1 mgml The activity of
VEGF was assessed by detection of VEGF receptor 2 (VEGFR2)
phosphorylation of HUVEC cells by Western blot [18] It was
compared to a commercial VEGF (VEGF164 RampD Systems produced
in murine myeloma cell line NS0 derived) The activity of VEGF wasmeasured by its ability to phosphorylate the VEGF receptor 2 (R2) of
human umbilical vein endothelial cells (pre-screened HUVECs
ECACC S200-05n) The detailed protocol can be found in
Supplementary material 1
223 VEGF radioactive labeling and biotinylation
[3H] VEGF waspreparedby reductivealkylation of amino groupsas
previously described [19] The speci1047297c activity of [3H] VEGF was
obtained by measuring the total protein concentration by the
microBCA test (Pierce Thermo Scienti1047297c) according to the manufac-
turers instructions and the radioactivity by liquid scintillation The
speci1047297c activity of [3H] VEGF was 613middot10eminus4μ Ciμ g [125I] VEGF was
obtained by incubation of VEGF with sodium iodine [125I] carrier free
(1 mCi Perkin Elmer Waltham USA) and Iodination Beads (Pierce
Thermo Scienti1047297c) according to the manufacturers instructions [125I]
VEGF speci1047297c activity was 112 μ Ciμ g VEGF integrity after labeling
was checked by SDS-Page electrophoresis
Puri1047297ed recombinantrat VEGF164wasbiotinylated withthe EZ-Linkreg
Sulfo-N -hydroxysuccinimide ester of biotin (Thermo Scienti1047297c)
Unincorporated biotin was separated from biotinylated VEGF on a
HITRAP desalting column(GE Healthcare) with PBS as the elution buffer
Biotin incorporation was quantitated by 4prime-hydroxyazonenzene-2-
carboxylic acidavidin assay (Thermo Scienti1047297c) A mean ratio of 45
biotin per dimer of VEGF was obtained
23 Formation of VEGF nanoparticles
VEGF nanoparticles (VEGF NP) were prepared as described by
Huang et al [8] Brie1047298y 80 μ l of the VEGF solution (1 mgml in PBS)was added to 800 μ l of the dextran sulfate (DS) solution (1 wv in
water) under stirring (700 rpm) and were stirred for 30 min Next
16 ml of the chitosan (CS) solution (01 wv in acetic acid pH 6) was
added dropwise and stirred for 5 min Finally 80 μ l of the zinc sulfate
solution (1 M in water) was added and stirred for 30 min The
particles were either incubated in 5 mannitol for 30 min centri-
fuged resuspended in 5 mannitol and freeze-dried or centrifuged
and resuspended at the desired concentration in water The mean size
of nanoparticles with and without VEGF was determined by dynamic
light scattering and the zeta potential by phase analysis light
scattering using a NanoZS (Malvern UK) The in1047298uence of freeze-
drying on nanoparticle size and zeta potential was determined
Incorporation ef 1047297ciency was evaluated by incorporating [3H] VEGF
in the formulation Incorporation ef 1047297ciency was expressed as apercentage of thetotalamount of [3H] VEGF found in thenanoparticles
over the radioactivity in the supernatant and in the nanoparticles
24 Nanoparticle incorporation into 3D implants
241 Hydrogels
The free or encapsulated VEGF was mixed with 500 μ l of Growth
Factor Reduced (GFR)-Matrigeltrade and was kept on ice Matrigeltradesupplemented with PBS was used as a negative control Hydrogels
geli1047297ed at 37 degC forming a solid mass
242 Polymeric scaffolds
Polymeric scaffolds were fabricated using a gas foamingparticu-
late leaching process as previously described [20ndash
22] Brie1047298y free
273 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 37
VEGF CSndashDS or CSndashDSndashVEGF nanoparticles were freeze-dried and
mixed 1047297rst with 36 mg of microspheres (PLGA 7525) and then with
100 mg of NaCl (250 μ mndash425 μ m diameter) Scaffolds composed of
PLGA microspheres and salt only were used as a negative control
Thenthe mixture was compressed ina 5 mmKBr die at 1500 psi using
a Carver press The PLGA scaffolds were then equilibrated with high
pressure CO2 gas (800 psi) for 16 h in a custom-made pressure vessel
[2324] The pressure was released over a period of 30 min To create
scaffold macroporosity scaffolds were leached 1047297
rst in 4 ml of sterilewater for 2 h and were then transferred in fresh sterile water for
30 min PLGA scaffolds were then dried and kept under dry and sterile
conditions [4]
The maximal amount of lyophilized nanoparticles that can be
incorporated in the PLGA scaffolds without disrupting their integrity
was evaluated by adding 3 to 20 mg of freeze-dried nanoparticles
prepared without VEGF (CSndashDS) to the microspheresalt mixture
(36 mg100 mg) Scaffold integrity was evaluated visually The
criteria were i) the general shape of the scaffold ii) it resistance to
manual manipulation (crumbling) iii) the shape of the pores
evaluated by an optical microscope and compared to scaffoldswithout
nanoparticles and iv) scaffold weight The scaffolds were considered
to be ldquonon disruptedrdquo when no difference was observed with control
scaffolds (without nanoparticles)
The VEGF loss during leaching was evaluated by incorporating
sup3H-VEGF (free or encapsulated) in the scaffolds and measuring
residual radioactivity in the scaffolds as well as in leaching super-
natants The loss during leaching was expressedas a percentage of the
total radioactivity found in the supernatants over the radioactivity in
the supernatants and in the scaffolds
25 Release of VEGF from the 3D matrix
Nanoparticles were formed either with [125I] VEGF and incorpo-
rated into PLGA scaffolds (1 μ g VEGFsample+1 μ l of [125I] VEGF
(0548 μ Ci)) or with biotinylated VEGF and incorporated into hydro-
gels (2 μ g of biotinylated VEGF500 μ l hydrogel) (n =3) Samples
were incubated in PBS at 37 degC After speci1047297c time periods PBS was
removed and replaced by fresh PBS Radioactivity of the supernatantthat contained the sample for that speci1047297c time period was measured
by gamma counter (Perkin Elmer) to quantify the amount of protein
that was released The cumulative release of VEGF from the samples
was calculated by dividing the cumulative radioactivity released into
the buffer by the total amount of radioactivity released over the
course of the study plus the radioactivity that remained inside the
sample at the end of the study The latter was analyzed by dissolving
the samples in 5 M NaOH to measure their radioactivity Biotinylated
VEGF was quanti1047297ed by ELISA The cumulative release of VEGF from
the hydrogels was calculated by dividing the cumulative VEGF
released into the buffer by the total amount of VEGF incorporated
inside the hydrogels
Activity of released VEGF was evaluated by detection of VEGF-R2
phosphorylation as described above
26 In 1047298uence of VEGF encapsulation on angiogenesis
Experimental protocols of animal studies were approved by the
Ethical Committee for Animal Care and Use of the Medicine Faculty of
the Universiteacute Catholique de Louvain
In1047298uence of NP of DS and CS alone on angiogenesis has been tested
by Huang et al [8] with no effect reported on HUVEC proliferation so
CSndashDS NP have not been tested here
1 μ and 2 μ g of free or encapsulated VEGF were incorporated in
hydrogels and 1 μ g in scaffolds Anesthetized BalbC female mice
received a subcutaneous injection of 05 ml of GFR-Matrigeltrade [1225]
PLGA scaffolds were implanted into the intraperitoneal fat pad of
NMRI male mice as previously described [26]
At designed time-points implants were retrieved and 1047297xed in 4
paraformaldehyde for 24 h before incubation in a 30 glucose solution
for 4 h and embedding in Tissue-Tek OCT compound (Sakura
Finetek Torrance CA) Sections were cut at 12 μ m thickness for
Matrigeltrade hydrogels and 14 μ m thickness for PLGA scaffolds using a
cryostat (Leica Microsystems Wetzlar GE) An antibody directed
against the murine endothelial cell surface marker (CD 31) was used
to determine the extent of endothelial cell colonization of the
implants After permeabilization (Triton X-100 01 (vv) in PBS)and blocking (5(wv) BSAin PBS) the primary antibody (rat anti-CD
31 (150 BD Biosciences San Jose USA)) was applied for 1 h at 37 degC
Secondaryantibody(Alexa Fluor 568 goat anti-rat(1500 Invitrogen))
was used to visualizethe antigen Lastly sections were incubated with
DAPI (Invitrogen) (50 ngml) for 5 min to allow visualization of cell
nuclei
Image acquisition was performed by a MIRAX microscope (Zeiss
Thornwood USA) allowing the acquisition of entire sections limiting
the information loss and experimenter in1047298uence by 1047297eld selection
In addition high quality pictures were obtained allowing high
magni1047297cation enlargements For each hydrogel condition 8 sections
at different levels within hydrogels were imaged For each PLGA
scaffold condition 24 sections were imaged 8 sections taken at one
extremity 8 in the middle and 8 at the other extremity of scaffolds
Quanti1047297cation of CD 31 and red blood cells was then performed on
whole sections using a script of Axiovision (Zeiss) The implant
size was normalized by delimiting the implant borders and by
quantifying the percentage of red pixels over the total amount of
analyzed pixels within these borders Results were expressed as
percentage of red pixels over the total amount of pixels within the
analyzed surface
27 Statistics
Statistical analyses were done using PRISM One-way and two-way
ANOVA with post-hoc Bonferronis multiple comparison test were
performed with a p-level of 005 Error bars represent the standard
error of the mean in all 1047297gures
3 Results
31 VEGF production
Pure 40 kDa VEGF164 was successfully produced Its activity was
compared as control to a commercial VEGF164 (RampD systems)
Our VEGF induced VEGFR2 phosphorylation of HUVEC cells at the
same concentration than the control (100 ngml) (Supplementary
material 2)
32 Nanoparticle characterization
To protect and stabilize VEGF it was complexed by dextran
sulfate (DS) and precipitated by coacervation with chitosan (CS) to
produce particular polyelectrolyte complexes encapsulating VEGF [8]
300 nm negatively charged nanoparticles were obtained (Table 1)
Encapsulation ef 1047297ciency was about 76 and the drug loading about
5 μ g VEGFmg of nanoparticles The size of CSndashDSndashVEGF nanoparticles
was slightly higher than CSndashDS nanoparticles and tends to increase
but notsigni1047297cantly after freeze-drying No loss of VEGF was detected
after freeze-drying
The nanoparticle size was stable in PBS for 4 h and then increased
to micrometers probably due to chitosan insolubility above pH 65
while the nanoparticle size was stable in water for at least 12 h
(data not shown)
274 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 47
33 VEGF release from 3D implants
VEGF release would likely be in1047298uenced by its incorporation as
well as by the nature of the 3D matrix into which nanoparticles were
incorporated Release of free or encapsulated VEGF was evaluated
over time after incorporation into hydrogels (Matrigeltrade hydrogels)
and into PLGA scaffolds
A 10 to 15 burst release was observed for VEGF whatever the
matrices or the formulation (2 h of incubation) except for encapsu-
lated VEGF incorporated in hydrogel (03) followed by a sustained
release over time (Fig 1) When incorporated in scaffolds release of
free VEGF was lower than encapsulated VEGF (24 versus 36
respectively after 2 weeks) In hydrogels encapsulation signi1047297cantly
slowed down VEGF release Indeed 20-times more free VEGF was
released after 2 weeks than encapsulated VEGF (32 and 16 for free
and encapsulated VEGF respectively) Release of free VEGF from
hydrogels was similar to release of encapsulated VEGF from scaffolds
(2 weeks incubation time 31 of encapsulated VEGF and 32 of free
VEGF respectively) Concerning the activity of released VEGF Huang
et al [8] demonstrated a preserved bioactivity for released VEGF from
NP for 10 days by a HUVEC proliferation assay VEGF released fromPLGA bridges [27] and from hydrogels was still able to induce
phosphorylation of HUVEC VEGF-R2 over a period of 42 days [28] and
of 2 weeks (Supplementary material 3) respectively
34 Nanoparticle incorporation into hydrogels effect on angiogenesis
The angiogenesis potential of free VEGF versus encapsulated VEGF
incorporated into an injectable hydrogel was studied Matrigeltradewas
chosen as a model for tissue regeneration due to its injectability and
proven ability to support cell and blood vessel growth [122930]
Following injection Matrigeltrade solidi1047297es and permits subsequent
penetration by host cells that induce vascularization Growth factor
reduced Matrigeltrade
was used to avoid interferences with the activityof the incorporated VEGF Free or encapsulated VEGF and as control
PBS were mixed with Matrigeltrade before subcutaneous injection
In1047298uence of VEGF amount (1 μ g and 2 μ g) and of incubation times
(2 and 3 weeks) was evaluated The effect on angiogenesis was
determined by quanti1047297cation of endothelial and red blood cells inside
the Matrigeltrade hydrogels after implantation
Endothelial cells werevisualized by immunostainingwith an anti-CD
31 antibody and red blood cells were visible by auto1047298uoresence The
percentage of red pixel represents both endothelial and red blood cells
Entire sections of Matrigeltrade hydrogels were analyzed The bene1047297cial
effect of VEGF encapsulation on the stimulation of angiogenesis
was clearly demonstrated (Fig 2) Indeed 2 weeks after implantation
endothelial cells some of them organized as blood vessels were ob-
served within the samples containing the encapsulated VEGF (Fig 2e
and f) In addition red blood cells were present within these structures
indicating their perfusion and their functionality In contrast for the
hydrogels loaded with PBS or free VEGF endothelial cells were detected
only around the implants (Fig 2a to d)
Quantitative analyses of the hydrogels loaded with VEGF nano-
particles showed that these implants contained 58 to 74-fold more
endothelial and red blood cells in comparison to the hydrogels loaded
with PBS or free VEGF respectively (Fig 3) No signi1047297cant differences
Fig 1 In1047298uence of VEGF encapsulation on its release from 3D implants [125I] VEGF and
biotinylated VEGF were incorporated into 3D implants (hydrogels and PLGA scaffolds)
either free or encapsulated Samples were incubated in PBS at 37 degC Radioactivity or
biotinylated VEGF in the supernatants was measured over time (n=3 signi1047297cant
Pb
005)
Fig 2 In1047298uence of VEGF encapsulation on angiogenesis when incorporated into
hydrogels VEGF (1 μ g) free (c d) or encapsulated (e f) was mixedwith Matrigeltrade and
injected sc in mice (2 weeks) negative control (a b) consisted in PBS mixed with
Matrigeltrade Endothelial cells (red) were detected by immuno1047298uorescence (CD 31) and
red blood cells (red arrow) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Table 1
Properties of CSDSVEGF nanoparticles
Formulation Diameter
(nm)
PDI Zeta potential
(mV)
Encapsulation
ef 1047297ciency
()
a) Before freeze-drying
CSndashDSa 325+minus19 022 +minus003 minus26+minus06 NA
CSndashDSndashVEGFb 383+minus73 026 +minus006 minus26+minus2 765 +minus65
b) After freeze-drying CSndashDSa 319+minus17 020 +minus001 minus25+minus03 NA
CSndashDSndashVEGFb 457+minus24 029 +minus001 minus34+minus15 71 +minus144
NA Not Applicablea n=4b n=6
275 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 57
were observed between 1 and 2 μ g of loaded VEGF with implantationduration of 2 and 3 weeks respectively The implants loaded with PBS
or free VEGF showed no signi1047297cant difference
35 Nanoparticle incorporation into PLGA scaffolds effect
on angiogenesis
The objective was to evaluate the effect of VEGF encapsulation on
angiogenesis when incorporated into polymeric scaffolds developed
for tissue engineering [421233132] Variable amounts of VEGF
nanoparticles were incorporated during scaffold processing and the
impact on scaffold integrity was determined The potential improve-
ment of encapsulated over free VEGF on VEGF loss during scaffold
leaching was estimated Then VEGF loaded scaffolds were implanted
in mice and angiogenesis was studiedFreeze-dried CSndashDS nanoparticles were mixed with PLGA micro-
spheres and NaCl crystals to form PLGA scaffolds Physical integrity
was estimated visually The physical integrity of scaffolds was
preserved up to 12 mg of incorporated nanoparticles per 36 mg
PLGA (n= 3) Incorporation of higher amounts induced scaffold
crumbling during the leaching phase To measure VEGF loss after
scaffold leaching lyophilized free or encapsulated [sup3H] VEGF was
used for the scaffold process VEGF loss during salt leaching was 25
for free VEGF and 44 for encapsulated VEGF (n=3) Taking these
ratios into account the amount of VEGF added to the scaffold process
was adapted to obtain 1 μ g of VEGF per implanted scaffold
VEGF loaded scaffolds were implanted in the fat pad of mice and
the in1047298uence on angiogenesis of encapsulated versus free VEGF was
studied 2 weeks after implantation Empty PLGA scaffolds were usedas control Sections of the implanted scaffolds were immunostained
Endothelial and red blood cells were identi1047297ed within and around the
scaffolds for all the conditions (Fig 4)
However as observed for hydrogels PLGA scaffolds containing
VEGF nanoparticles seemed to stimulate more ef 1047297ciently angiogenesis
(Fig 4e and f)
In order to determineif VEGF encapsulation signi1047297cantly increased
the amount of endothelial and red blood cells within and around the
scaffolds quanti1047297cation of the percentage of red pixels over the total
amount of analyzed pixels was performed Signi1047297cantly more
endothelial and red blood cells were detected with VEGF nanopar-
ticles incorporated into PLGA scaffolds (Fig 5) compared with free
VEGF or control although the difference was less marked than
observed for the Matrigeltrade
hydrogels (35-folds) This could be
explained by the fact that PLGA scaffolds alone seemed to support cell
in1047297
ltration and angiogenesis to a higher extent than Matrigeltrade
Nosigni1047297cant difference was observed between negative control and free
VEGF
4 Discussion
When designing systems for tissue engineering one of the 1047297rst
requirements is the colonization of the implant by blood vessels
bringing oxygen and nutriments to support tissue reconstruction
Fig 4 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in scaffolds
VEGF (1 μ g)free (c andd) or encapsulated(e andf) was incorporatedin PLGA scaffolds
and implanted in mouse fat pads (2 weeks) negative control (a and b) consisted in
empty scaffolds Endothelial cells (red) were detected by immuno1047298uorescence (CD 31)
and red blood cells (red) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Fig 5 In1047298uenceof VEGF encapsulation on angiogenesis when incorporated in scaffolds
quanti1047297cation of endothelial and red blood cells VEGF (1 μ g) free or encapsulated was
incorporated in PLGA scaffolds and implanted in mouse fat pads (2 weeks) negative
control consisted in empty scaffolds Endothelial cells were detected by immuno1047298u-
orescence (CD 31) and red bloodcells byauto1047298uorescence Pictures were acquired with
a MIRAX microscope 24sections by condition(n =3) were imagedin their entirety and
analyzed (Axiovision) 8 sections taken at one extremity 8 in the middle and 8 at the
other extremity of scaffolds (mean +minus
SEM Pb
005)
Fig 3 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in
hydrogels quanti1047297cation of endothelial and red blood cells VEGF (1 or 2 μ g) free or
encapsulated was incorporated in Matrigeltrade and injected sc in mice (2 or 3 weeks)
negative control consisted in PBS mixed with Matrigeltrade Endothelial cells were
detected by immuno1047298uorescence (CD 31) and red blood cells by auto1047298uorescence
Pictures were acquired with a MIRAX microscope 8 sections by condition (n=3) were
imaged in their entirety and analyzed (Axiovision) (mean +minusSEM Pb005)
276 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 67
The major focus of this work was to determine if incorporation of
VEGFndashCSndashDS nanoparticles into twotypesof 3D implants (PLGA scaffold
or hydrogel) commonly used for tissue engineering (hydrogels and
polymeric scaffolds) improved angiogenesis within the implants and
thus tissue regeneration We investigated the effect of VEGF combined
release from nanoparticles and 3D matrices and the in1047298uence of the
matrix nature on angiogenesis The results indicate thatimplantation of
3D systems loaded with VEGF nanoparticles stimulated endothelial cell
colonization and blood vessel formation more ef 1047297
ciently than matricesloaded with free VEGF regardless of the VEGF dose implantation time
and implant type
The improvement probably resulted from i) VEGF protection by
complexation of the heparin binding site of VEGF with dextran sulfate
combined with ii) protection and controlled release brought by
complex stabilization by chitosan Indeed Huang et al [8] demon-
strated in vitro by ELISA and HUVEC proliferation test that VEGF
encapsulation by DS andCS protectedmost of VEGF activity forat least
10 days and that VEGF was slowly released from nanoparticles
We observed that encapsulation of VEGF in comparison with free
VEGF signi1047297cantly decreases burst release (03 versus 10 respec-
tively) from VEGF loaded Matrigeltrade hydrogels and slowed down
diffusionfrom the implant about 20-fold after 2 weeks It could be due
to higher size combined with electrostatic interactions due to the
negative surface charge of nanoparticles Encapsulated VEGF was
released from PLGA scaffolds slightly faster than free VEGF (24
versus 36 respectively after 2 weeks) Encapsulation did not affect
burst release but loss during leaching was taken into account for
scaffold preparation and the initial VEGF loading was adjusted to be
1 μ g of VEGF post-leaching for both conditions Indeed encapsulation
signi1047297cantly decreased VEGF loss during leaching (about 21) In that
perspective the burst release for free VEGF loaded scaffolds would be
approximately about 15 higher than for VEGF nanoparticle-loaded
scaffolds Finally compared to microspheres classically used to
protect and deliver proteins complexation by DS and coacervation
by CS achieved a higher loading and did not compromise implant
integrity Indeed approximately 5 μ g of VEGF was incorporated in
1 mg of CSndashDS nanoparticles versus approximately 50 ng per mg of
microspheres [9] This allows incorporation of a much higher amountof VEGF into implants compared to microspheres
Although encapsulated VEGF stimulated angiogenesis to a greater
extent than freeVEGF in both implant types differences between PLGA
scaffolds and Matrigeltrade hydrogels were observed The percentage of
endothelial and red blood cells quanti1047297ed in nanoparticle-loaded
scaffolds was 75 times higher compared to Matrigeltrade hydrogels but
the scaffold itself without VEGF stimulated angiogenesis at the same
level than hydrogels loaded with encapsulated VEGF This could be
explained by the factthat these PLGAscaffolds havebeen optimizedfor
tissue engineering [4] and thus promote cell in1047297ltration and
proliferation However encapsulation of VEGF did not affect release
from scaffolds as much as for hydrogels Hence the increase in
angiogenesis elicited by encapsulated versus free VEGF loaded into
Matrigeltradehydrogels wasabout 5 to 7-foldwhile it wasonlyabout 35-fold for scaffolds Considering these results it seems that it could be
interesting to combine thetwo systems by1047297lling PLGA scaffoldspores
with hydrogel both loaded with VEGF nanoparticles
5 Conclusion
Wehypothesized thatVEGFactivityin vivo would be enhanced by its
encapsulation in CSndashDS nanoparticles and further incorporation into
implants used in tissue engineering like injectable hydrogels and
polymeric scaffolds Angiogenesis the primary effect of VEGF was
clearly improved by encapsulation the extent of implant colonization
by endothelial cells and blood vessels was higher when both types of
implants were loaded with VEGF nanoparticles compared with free
VEGF(non-encapsulated) Nanoparticles composed of chitosan dextran
sulfate and VEGF were obtained and incorporated in implants without
impairingtheirstructuralpropertiesand allowing a higher VEGFloading
compared to VEGF classical techniques of encapsulation (up to 5 μ g of
VEGF per mg of nanoparticles) VEGF encapsulation guarantees VEGF
ef 1047297ciency by protection and sustained release from 3D implants
In the scope of the spinal cord regeneration systems to deliver
active VEGF are attractive since in addition to its angiogenic activity
VEGF has recently been shown to be a neurogenic factor [33ndash35]
Then encapsulation and incorporation of VEGF into 3D implants thatin addition to support cell in1047297ltration and organization will stimulate
blood vessel and axon growth are a promising approach to stimulate
tissue regeneration
Supplementary materialsrelated to this article canbe found online
at doi101016jjconrel201011028
Acknowledgments
The authors would like to thank Prof Carmeliet (Vesalius Research
Center KU Leuven Belgium) for the gift of VEGF producing Pichia
pastoris yeast The authors thank Sabine Cordi for her help with MIRAX
acquisition as well as Christophe Pierreux for the RGB quanti1047297cation
script SeacutebastienSartis thankedfor assistance with VEGF productionand
Laurenne Petit for the realization of VEGF activity tests This work wassupported by grants from the Belgian Fonds National de la Recherche
Scienti1047297que (FNRS Creacutedits aux chercheurs and FRSM 3461709) and
CTB (Belgium) Anne des Rieux is FNRS Post-Doctoral Researcher
References
[1] V Guarino F Causa L Ambrosio Bioactive scaffolds for bone and ligament tissueExpert Rev Med Devices 3 (2007) 405ndash418
[2] TM Freyman IV Yannas R Yokoo LJ Gibson Fibroblast contraction of acollagen-GAG matrix Biomaterials 21 (2001) 2883ndash2891
[3] V Mourino AR Boccaccini Bone tissue engineering therapeutics controlled drugdelivery in three-dimensional scaffolds J R Soc Interface 43 (2010) 209 ndash227
[4] CB Rives A des Rieux M Zelivyanskaya SR Stock WL Lowe Jr LD SheaLayered PLGscaffoldsfor invivo plasmid delivery Biomaterials3 (2009)394ndash401
[5] AH Zisch MP Lutolf JA Hubbell Biopolymeric delivery matrices for angiogenic
growth factors Cardiovasc Pathol 6 (2003) 295ndash310[6] ZS Patel AG Mikos Angiogenesis with biomaterial-based drug- and cell-
delivery systems J Biomater Sci Polym Ed 6 (2004) 701ndash726[7] BV Somayaji U Jariwala P Jayachandran K Vidyalakshmi RV Dudhani
Evaluation of antimicrobial ef 1047297cacy and release pattern of tetracycline andmetronidazole using a local delivery system J Periodontol 4 (1998) 409ndash413
[8] MHuang SN VitharanaLJ Peek T CoopC Berkland Polyelectrolyte complexesstabilize and controllably release vascular endothelial growth factor Biomacro-molecules 5 (2007) 1607ndash1614
[9] JS GolubYTKimCL Duvall RV BellamkondaD Gupta AS Lin DWeiss WRobertTaylor RE Guldberg Sustained VEGF delivery via PLGA nanoparticles promotesvascular growth Am J Physiol Heart Circ Physiol 6 (2010) H1959ndashH1965
[10] C He SW Kim DS Lee In situ gelling stimuli-sensitive block copolymerhydrogels for drug delivery J Control Release 3 (2008) 189ndash207
[11] HK Kleinman ML McGarvey JR Hassell VL Star FB Cannon GW LaurieGR Martin Basement membrane complexes with biological activity Biochemistry 2(1986) 312ndash318
[12] N Akhtar EB Dickerson R Auerbach The spongeMatrigel angiogenesis assayAngiogenesis 1ndash2 (2002) 75ndash80
[13] D Donovan NJ Brown ET Bishop CE Lewis Comparison of three in vitrohuman lsquoangiogenesisrsquo assays with capillaries formed in vivo Angiogenesis 2(2001) 113ndash121
[14] JM Kanczler PJ Ginty JJ Barry NM Clarke SM Howdle KM ShakesheffROOreffo Theeffectof mesenchymalpopulations and vascularendothelial growthfactor delivered from biodegradable polymer scaffolds on bone formationBiomaterials 12 (2008) 1892ndash1900
[15] FG Rocha CA Sundback NJ Krebs JK Leach DJ Mooney SW Ashley JPVacanti EE Whang The effect of sustained delivery of vascular endothelialgrowth factor on angiogenesis in tissue-engineered intestine Biomaterials 19(2008) 2884ndash2890
[16] JM Kanczler J Barry P Ginty SM Howdle KM Shakesheff RO OreffoSupercritical carbon dioxide generated vascular endothelial growth factorencapsulated poly(DL -lactic acid) scaffolds induce angiogenesis in vitro BiochemBiophys Res Commun 1 (2007) 135ndash141
[17] JM Kanczler PJ Ginty L White NM Clarke SM Howdle KM Shakesheff ROOreffo The effect of the delivery of vascular endothelial growth factor and bonemorphogenic protein-2 to osteoprogenitor cell populations on bone formation
Biomaterials 6 (2010) 1242ndash
1250
277 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 77
[18] TT Chen A Luque S Lee SM Anderson T Segura ML Iruela-Arispe Anchorageof VEGF to the extracellular matrix conveys differential signaling responses toendothelial cells J Cell Biol 4 (2010) 595ndash609
[19] GE Means RE Feeney Reductive alkylation of amino groups in proteinsBiochemistry 6 (1968) 2192ndash2201
[20] JH Jang LD Shea Controllable delivery of non-viral DNA from porous scaffolds J Control Release 1 (2003) 157ndash168
[21] LD Shea E Smiley J Bonadio DJ Mooney DNA delivery from polymer matricesfor tissue engineering Nat Biotechnol 6 (1999) 551ndash554
[22] DJ Mooney DF Baldwin NP Suh JP Vacanti R Langer Novel approach tofabricate porous sponges of poly(DL -lactic-co-glycolic acid) without the use of
organic solvents Biomaterials 14 (1996) 1417ndash
1422[23] JH Jang CB Rives LD Shea Plasmid delivery in vivo from porous tissue-engineering scaffolds transgene expression and cellular transfection Mol Ther 3(2005) 475ndash483
[24] MO Aviles CHLin M Zelivyanskaya JGGraham RMBoehlerPBMessersmithLD Shea The contribution of plasmid design and release to in vivo gene expressionfollowing delivery from cationic polymer modi1047297ed scaffolds Biomaterials 6 (2010)1140ndash1147
[25] P Sonveaux A Brouet X Havaux V Gregoire C Dessy JL Balligand O FeronIrradiation-induced angiogenesis through the up-regulation of the nitric oxidepathway implications for tumor radiotherapy Cancer Res 5 (2003) 1012 ndash1019
[26] H Blomeier X Zhang C Rives M Brissova E Hughes M Baker AC Powers DBKaufman LD Shea WL Lowe Jr Polymer scaffolds as synthetic microenviron-ments for extrahepatic islet transplantation Transplantation 4 (2006) 452ndash459
[27] Y Yang L De Laporte CB Rives JH Jang WC Lin KR Shull LD SheaNeurotrophin releasing single and multiple lumen nerve conduits J ControlRelease 3 (2005) 433ndash446
[28] L De Laporte A des Rieux ML Zelivyanska HM Tuinstra N De Clerck AAPostnov LD Shea VEGF and FGF-2 delivery from spinal cord bridges to enhanceangiogenesis following injury J Biomed Mater Res Part A (in press)
[29] ML Ponce Tube formationan in vitro matrigel angiogenesis assayMethods MolBiol (2009) 183ndash188
[30] A Albini R Benelli The chemoinvasion assay a method to assess tumor andendothelial cell invasion and its modulation Nat Protoc 3 (2007) 504ndash511
[31] MH Sheridan LD Shea MC Peters DJ Mooney Bioabsorbable polymer
scaffolds for tissue engineering capable of sustained growth factor delivery J Control Release 1ndash3 (2000) 91ndash102[32] JH Jang TL Houchin LD Shea Gene delivery from polymer scaffolds for tissue
engineering Expert Rev Med Devices 1 (2004) 127ndash138[33] P Carmeliet M Tessier-Lavigne Common mechanisms of nerve and blood vessel
wiring Nature 7048 (2005) 193ndash200[34] E Storkebaum D Lambrechts P Carmeliet VEGF once regarded as a speci1047297c
angiogenic factor now implicated in neuroprotection Bioessays 9 (2004) 943ndash954[35] E StorkebaumD LambrechtsM Dewerchin MPMoreno-Murciano S Appelmans
H Oh P Van Damme B Rutten WY Man M De Mol S Wyns D Manka KVermeulen L Van Den Bosch N Mertens C Schmitz W Robberecht EM ConwayD Collen L Moons P Carmeliet Treatment of motoneuron degeneration byintracerebroventricular delivery of VEGF in a rat model of ALS Nat Neurosci 1(2005) 85ndash92
278 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 37
VEGF CSndashDS or CSndashDSndashVEGF nanoparticles were freeze-dried and
mixed 1047297rst with 36 mg of microspheres (PLGA 7525) and then with
100 mg of NaCl (250 μ mndash425 μ m diameter) Scaffolds composed of
PLGA microspheres and salt only were used as a negative control
Thenthe mixture was compressed ina 5 mmKBr die at 1500 psi using
a Carver press The PLGA scaffolds were then equilibrated with high
pressure CO2 gas (800 psi) for 16 h in a custom-made pressure vessel
[2324] The pressure was released over a period of 30 min To create
scaffold macroporosity scaffolds were leached 1047297
rst in 4 ml of sterilewater for 2 h and were then transferred in fresh sterile water for
30 min PLGA scaffolds were then dried and kept under dry and sterile
conditions [4]
The maximal amount of lyophilized nanoparticles that can be
incorporated in the PLGA scaffolds without disrupting their integrity
was evaluated by adding 3 to 20 mg of freeze-dried nanoparticles
prepared without VEGF (CSndashDS) to the microspheresalt mixture
(36 mg100 mg) Scaffold integrity was evaluated visually The
criteria were i) the general shape of the scaffold ii) it resistance to
manual manipulation (crumbling) iii) the shape of the pores
evaluated by an optical microscope and compared to scaffoldswithout
nanoparticles and iv) scaffold weight The scaffolds were considered
to be ldquonon disruptedrdquo when no difference was observed with control
scaffolds (without nanoparticles)
The VEGF loss during leaching was evaluated by incorporating
sup3H-VEGF (free or encapsulated) in the scaffolds and measuring
residual radioactivity in the scaffolds as well as in leaching super-
natants The loss during leaching was expressedas a percentage of the
total radioactivity found in the supernatants over the radioactivity in
the supernatants and in the scaffolds
25 Release of VEGF from the 3D matrix
Nanoparticles were formed either with [125I] VEGF and incorpo-
rated into PLGA scaffolds (1 μ g VEGFsample+1 μ l of [125I] VEGF
(0548 μ Ci)) or with biotinylated VEGF and incorporated into hydro-
gels (2 μ g of biotinylated VEGF500 μ l hydrogel) (n =3) Samples
were incubated in PBS at 37 degC After speci1047297c time periods PBS was
removed and replaced by fresh PBS Radioactivity of the supernatantthat contained the sample for that speci1047297c time period was measured
by gamma counter (Perkin Elmer) to quantify the amount of protein
that was released The cumulative release of VEGF from the samples
was calculated by dividing the cumulative radioactivity released into
the buffer by the total amount of radioactivity released over the
course of the study plus the radioactivity that remained inside the
sample at the end of the study The latter was analyzed by dissolving
the samples in 5 M NaOH to measure their radioactivity Biotinylated
VEGF was quanti1047297ed by ELISA The cumulative release of VEGF from
the hydrogels was calculated by dividing the cumulative VEGF
released into the buffer by the total amount of VEGF incorporated
inside the hydrogels
Activity of released VEGF was evaluated by detection of VEGF-R2
phosphorylation as described above
26 In 1047298uence of VEGF encapsulation on angiogenesis
Experimental protocols of animal studies were approved by the
Ethical Committee for Animal Care and Use of the Medicine Faculty of
the Universiteacute Catholique de Louvain
In1047298uence of NP of DS and CS alone on angiogenesis has been tested
by Huang et al [8] with no effect reported on HUVEC proliferation so
CSndashDS NP have not been tested here
1 μ and 2 μ g of free or encapsulated VEGF were incorporated in
hydrogels and 1 μ g in scaffolds Anesthetized BalbC female mice
received a subcutaneous injection of 05 ml of GFR-Matrigeltrade [1225]
PLGA scaffolds were implanted into the intraperitoneal fat pad of
NMRI male mice as previously described [26]
At designed time-points implants were retrieved and 1047297xed in 4
paraformaldehyde for 24 h before incubation in a 30 glucose solution
for 4 h and embedding in Tissue-Tek OCT compound (Sakura
Finetek Torrance CA) Sections were cut at 12 μ m thickness for
Matrigeltrade hydrogels and 14 μ m thickness for PLGA scaffolds using a
cryostat (Leica Microsystems Wetzlar GE) An antibody directed
against the murine endothelial cell surface marker (CD 31) was used
to determine the extent of endothelial cell colonization of the
implants After permeabilization (Triton X-100 01 (vv) in PBS)and blocking (5(wv) BSAin PBS) the primary antibody (rat anti-CD
31 (150 BD Biosciences San Jose USA)) was applied for 1 h at 37 degC
Secondaryantibody(Alexa Fluor 568 goat anti-rat(1500 Invitrogen))
was used to visualizethe antigen Lastly sections were incubated with
DAPI (Invitrogen) (50 ngml) for 5 min to allow visualization of cell
nuclei
Image acquisition was performed by a MIRAX microscope (Zeiss
Thornwood USA) allowing the acquisition of entire sections limiting
the information loss and experimenter in1047298uence by 1047297eld selection
In addition high quality pictures were obtained allowing high
magni1047297cation enlargements For each hydrogel condition 8 sections
at different levels within hydrogels were imaged For each PLGA
scaffold condition 24 sections were imaged 8 sections taken at one
extremity 8 in the middle and 8 at the other extremity of scaffolds
Quanti1047297cation of CD 31 and red blood cells was then performed on
whole sections using a script of Axiovision (Zeiss) The implant
size was normalized by delimiting the implant borders and by
quantifying the percentage of red pixels over the total amount of
analyzed pixels within these borders Results were expressed as
percentage of red pixels over the total amount of pixels within the
analyzed surface
27 Statistics
Statistical analyses were done using PRISM One-way and two-way
ANOVA with post-hoc Bonferronis multiple comparison test were
performed with a p-level of 005 Error bars represent the standard
error of the mean in all 1047297gures
3 Results
31 VEGF production
Pure 40 kDa VEGF164 was successfully produced Its activity was
compared as control to a commercial VEGF164 (RampD systems)
Our VEGF induced VEGFR2 phosphorylation of HUVEC cells at the
same concentration than the control (100 ngml) (Supplementary
material 2)
32 Nanoparticle characterization
To protect and stabilize VEGF it was complexed by dextran
sulfate (DS) and precipitated by coacervation with chitosan (CS) to
produce particular polyelectrolyte complexes encapsulating VEGF [8]
300 nm negatively charged nanoparticles were obtained (Table 1)
Encapsulation ef 1047297ciency was about 76 and the drug loading about
5 μ g VEGFmg of nanoparticles The size of CSndashDSndashVEGF nanoparticles
was slightly higher than CSndashDS nanoparticles and tends to increase
but notsigni1047297cantly after freeze-drying No loss of VEGF was detected
after freeze-drying
The nanoparticle size was stable in PBS for 4 h and then increased
to micrometers probably due to chitosan insolubility above pH 65
while the nanoparticle size was stable in water for at least 12 h
(data not shown)
274 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 47
33 VEGF release from 3D implants
VEGF release would likely be in1047298uenced by its incorporation as
well as by the nature of the 3D matrix into which nanoparticles were
incorporated Release of free or encapsulated VEGF was evaluated
over time after incorporation into hydrogels (Matrigeltrade hydrogels)
and into PLGA scaffolds
A 10 to 15 burst release was observed for VEGF whatever the
matrices or the formulation (2 h of incubation) except for encapsu-
lated VEGF incorporated in hydrogel (03) followed by a sustained
release over time (Fig 1) When incorporated in scaffolds release of
free VEGF was lower than encapsulated VEGF (24 versus 36
respectively after 2 weeks) In hydrogels encapsulation signi1047297cantly
slowed down VEGF release Indeed 20-times more free VEGF was
released after 2 weeks than encapsulated VEGF (32 and 16 for free
and encapsulated VEGF respectively) Release of free VEGF from
hydrogels was similar to release of encapsulated VEGF from scaffolds
(2 weeks incubation time 31 of encapsulated VEGF and 32 of free
VEGF respectively) Concerning the activity of released VEGF Huang
et al [8] demonstrated a preserved bioactivity for released VEGF from
NP for 10 days by a HUVEC proliferation assay VEGF released fromPLGA bridges [27] and from hydrogels was still able to induce
phosphorylation of HUVEC VEGF-R2 over a period of 42 days [28] and
of 2 weeks (Supplementary material 3) respectively
34 Nanoparticle incorporation into hydrogels effect on angiogenesis
The angiogenesis potential of free VEGF versus encapsulated VEGF
incorporated into an injectable hydrogel was studied Matrigeltradewas
chosen as a model for tissue regeneration due to its injectability and
proven ability to support cell and blood vessel growth [122930]
Following injection Matrigeltrade solidi1047297es and permits subsequent
penetration by host cells that induce vascularization Growth factor
reduced Matrigeltrade
was used to avoid interferences with the activityof the incorporated VEGF Free or encapsulated VEGF and as control
PBS were mixed with Matrigeltrade before subcutaneous injection
In1047298uence of VEGF amount (1 μ g and 2 μ g) and of incubation times
(2 and 3 weeks) was evaluated The effect on angiogenesis was
determined by quanti1047297cation of endothelial and red blood cells inside
the Matrigeltrade hydrogels after implantation
Endothelial cells werevisualized by immunostainingwith an anti-CD
31 antibody and red blood cells were visible by auto1047298uoresence The
percentage of red pixel represents both endothelial and red blood cells
Entire sections of Matrigeltrade hydrogels were analyzed The bene1047297cial
effect of VEGF encapsulation on the stimulation of angiogenesis
was clearly demonstrated (Fig 2) Indeed 2 weeks after implantation
endothelial cells some of them organized as blood vessels were ob-
served within the samples containing the encapsulated VEGF (Fig 2e
and f) In addition red blood cells were present within these structures
indicating their perfusion and their functionality In contrast for the
hydrogels loaded with PBS or free VEGF endothelial cells were detected
only around the implants (Fig 2a to d)
Quantitative analyses of the hydrogels loaded with VEGF nano-
particles showed that these implants contained 58 to 74-fold more
endothelial and red blood cells in comparison to the hydrogels loaded
with PBS or free VEGF respectively (Fig 3) No signi1047297cant differences
Fig 1 In1047298uence of VEGF encapsulation on its release from 3D implants [125I] VEGF and
biotinylated VEGF were incorporated into 3D implants (hydrogels and PLGA scaffolds)
either free or encapsulated Samples were incubated in PBS at 37 degC Radioactivity or
biotinylated VEGF in the supernatants was measured over time (n=3 signi1047297cant
Pb
005)
Fig 2 In1047298uence of VEGF encapsulation on angiogenesis when incorporated into
hydrogels VEGF (1 μ g) free (c d) or encapsulated (e f) was mixedwith Matrigeltrade and
injected sc in mice (2 weeks) negative control (a b) consisted in PBS mixed with
Matrigeltrade Endothelial cells (red) were detected by immuno1047298uorescence (CD 31) and
red blood cells (red arrow) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Table 1
Properties of CSDSVEGF nanoparticles
Formulation Diameter
(nm)
PDI Zeta potential
(mV)
Encapsulation
ef 1047297ciency
()
a) Before freeze-drying
CSndashDSa 325+minus19 022 +minus003 minus26+minus06 NA
CSndashDSndashVEGFb 383+minus73 026 +minus006 minus26+minus2 765 +minus65
b) After freeze-drying CSndashDSa 319+minus17 020 +minus001 minus25+minus03 NA
CSndashDSndashVEGFb 457+minus24 029 +minus001 minus34+minus15 71 +minus144
NA Not Applicablea n=4b n=6
275 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 57
were observed between 1 and 2 μ g of loaded VEGF with implantationduration of 2 and 3 weeks respectively The implants loaded with PBS
or free VEGF showed no signi1047297cant difference
35 Nanoparticle incorporation into PLGA scaffolds effect
on angiogenesis
The objective was to evaluate the effect of VEGF encapsulation on
angiogenesis when incorporated into polymeric scaffolds developed
for tissue engineering [421233132] Variable amounts of VEGF
nanoparticles were incorporated during scaffold processing and the
impact on scaffold integrity was determined The potential improve-
ment of encapsulated over free VEGF on VEGF loss during scaffold
leaching was estimated Then VEGF loaded scaffolds were implanted
in mice and angiogenesis was studiedFreeze-dried CSndashDS nanoparticles were mixed with PLGA micro-
spheres and NaCl crystals to form PLGA scaffolds Physical integrity
was estimated visually The physical integrity of scaffolds was
preserved up to 12 mg of incorporated nanoparticles per 36 mg
PLGA (n= 3) Incorporation of higher amounts induced scaffold
crumbling during the leaching phase To measure VEGF loss after
scaffold leaching lyophilized free or encapsulated [sup3H] VEGF was
used for the scaffold process VEGF loss during salt leaching was 25
for free VEGF and 44 for encapsulated VEGF (n=3) Taking these
ratios into account the amount of VEGF added to the scaffold process
was adapted to obtain 1 μ g of VEGF per implanted scaffold
VEGF loaded scaffolds were implanted in the fat pad of mice and
the in1047298uence on angiogenesis of encapsulated versus free VEGF was
studied 2 weeks after implantation Empty PLGA scaffolds were usedas control Sections of the implanted scaffolds were immunostained
Endothelial and red blood cells were identi1047297ed within and around the
scaffolds for all the conditions (Fig 4)
However as observed for hydrogels PLGA scaffolds containing
VEGF nanoparticles seemed to stimulate more ef 1047297ciently angiogenesis
(Fig 4e and f)
In order to determineif VEGF encapsulation signi1047297cantly increased
the amount of endothelial and red blood cells within and around the
scaffolds quanti1047297cation of the percentage of red pixels over the total
amount of analyzed pixels was performed Signi1047297cantly more
endothelial and red blood cells were detected with VEGF nanopar-
ticles incorporated into PLGA scaffolds (Fig 5) compared with free
VEGF or control although the difference was less marked than
observed for the Matrigeltrade
hydrogels (35-folds) This could be
explained by the fact that PLGA scaffolds alone seemed to support cell
in1047297
ltration and angiogenesis to a higher extent than Matrigeltrade
Nosigni1047297cant difference was observed between negative control and free
VEGF
4 Discussion
When designing systems for tissue engineering one of the 1047297rst
requirements is the colonization of the implant by blood vessels
bringing oxygen and nutriments to support tissue reconstruction
Fig 4 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in scaffolds
VEGF (1 μ g)free (c andd) or encapsulated(e andf) was incorporatedin PLGA scaffolds
and implanted in mouse fat pads (2 weeks) negative control (a and b) consisted in
empty scaffolds Endothelial cells (red) were detected by immuno1047298uorescence (CD 31)
and red blood cells (red) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Fig 5 In1047298uenceof VEGF encapsulation on angiogenesis when incorporated in scaffolds
quanti1047297cation of endothelial and red blood cells VEGF (1 μ g) free or encapsulated was
incorporated in PLGA scaffolds and implanted in mouse fat pads (2 weeks) negative
control consisted in empty scaffolds Endothelial cells were detected by immuno1047298u-
orescence (CD 31) and red bloodcells byauto1047298uorescence Pictures were acquired with
a MIRAX microscope 24sections by condition(n =3) were imagedin their entirety and
analyzed (Axiovision) 8 sections taken at one extremity 8 in the middle and 8 at the
other extremity of scaffolds (mean +minus
SEM Pb
005)
Fig 3 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in
hydrogels quanti1047297cation of endothelial and red blood cells VEGF (1 or 2 μ g) free or
encapsulated was incorporated in Matrigeltrade and injected sc in mice (2 or 3 weeks)
negative control consisted in PBS mixed with Matrigeltrade Endothelial cells were
detected by immuno1047298uorescence (CD 31) and red blood cells by auto1047298uorescence
Pictures were acquired with a MIRAX microscope 8 sections by condition (n=3) were
imaged in their entirety and analyzed (Axiovision) (mean +minusSEM Pb005)
276 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 67
The major focus of this work was to determine if incorporation of
VEGFndashCSndashDS nanoparticles into twotypesof 3D implants (PLGA scaffold
or hydrogel) commonly used for tissue engineering (hydrogels and
polymeric scaffolds) improved angiogenesis within the implants and
thus tissue regeneration We investigated the effect of VEGF combined
release from nanoparticles and 3D matrices and the in1047298uence of the
matrix nature on angiogenesis The results indicate thatimplantation of
3D systems loaded with VEGF nanoparticles stimulated endothelial cell
colonization and blood vessel formation more ef 1047297
ciently than matricesloaded with free VEGF regardless of the VEGF dose implantation time
and implant type
The improvement probably resulted from i) VEGF protection by
complexation of the heparin binding site of VEGF with dextran sulfate
combined with ii) protection and controlled release brought by
complex stabilization by chitosan Indeed Huang et al [8] demon-
strated in vitro by ELISA and HUVEC proliferation test that VEGF
encapsulation by DS andCS protectedmost of VEGF activity forat least
10 days and that VEGF was slowly released from nanoparticles
We observed that encapsulation of VEGF in comparison with free
VEGF signi1047297cantly decreases burst release (03 versus 10 respec-
tively) from VEGF loaded Matrigeltrade hydrogels and slowed down
diffusionfrom the implant about 20-fold after 2 weeks It could be due
to higher size combined with electrostatic interactions due to the
negative surface charge of nanoparticles Encapsulated VEGF was
released from PLGA scaffolds slightly faster than free VEGF (24
versus 36 respectively after 2 weeks) Encapsulation did not affect
burst release but loss during leaching was taken into account for
scaffold preparation and the initial VEGF loading was adjusted to be
1 μ g of VEGF post-leaching for both conditions Indeed encapsulation
signi1047297cantly decreased VEGF loss during leaching (about 21) In that
perspective the burst release for free VEGF loaded scaffolds would be
approximately about 15 higher than for VEGF nanoparticle-loaded
scaffolds Finally compared to microspheres classically used to
protect and deliver proteins complexation by DS and coacervation
by CS achieved a higher loading and did not compromise implant
integrity Indeed approximately 5 μ g of VEGF was incorporated in
1 mg of CSndashDS nanoparticles versus approximately 50 ng per mg of
microspheres [9] This allows incorporation of a much higher amountof VEGF into implants compared to microspheres
Although encapsulated VEGF stimulated angiogenesis to a greater
extent than freeVEGF in both implant types differences between PLGA
scaffolds and Matrigeltrade hydrogels were observed The percentage of
endothelial and red blood cells quanti1047297ed in nanoparticle-loaded
scaffolds was 75 times higher compared to Matrigeltrade hydrogels but
the scaffold itself without VEGF stimulated angiogenesis at the same
level than hydrogels loaded with encapsulated VEGF This could be
explained by the factthat these PLGAscaffolds havebeen optimizedfor
tissue engineering [4] and thus promote cell in1047297ltration and
proliferation However encapsulation of VEGF did not affect release
from scaffolds as much as for hydrogels Hence the increase in
angiogenesis elicited by encapsulated versus free VEGF loaded into
Matrigeltradehydrogels wasabout 5 to 7-foldwhile it wasonlyabout 35-fold for scaffolds Considering these results it seems that it could be
interesting to combine thetwo systems by1047297lling PLGA scaffoldspores
with hydrogel both loaded with VEGF nanoparticles
5 Conclusion
Wehypothesized thatVEGFactivityin vivo would be enhanced by its
encapsulation in CSndashDS nanoparticles and further incorporation into
implants used in tissue engineering like injectable hydrogels and
polymeric scaffolds Angiogenesis the primary effect of VEGF was
clearly improved by encapsulation the extent of implant colonization
by endothelial cells and blood vessels was higher when both types of
implants were loaded with VEGF nanoparticles compared with free
VEGF(non-encapsulated) Nanoparticles composed of chitosan dextran
sulfate and VEGF were obtained and incorporated in implants without
impairingtheirstructuralpropertiesand allowing a higher VEGFloading
compared to VEGF classical techniques of encapsulation (up to 5 μ g of
VEGF per mg of nanoparticles) VEGF encapsulation guarantees VEGF
ef 1047297ciency by protection and sustained release from 3D implants
In the scope of the spinal cord regeneration systems to deliver
active VEGF are attractive since in addition to its angiogenic activity
VEGF has recently been shown to be a neurogenic factor [33ndash35]
Then encapsulation and incorporation of VEGF into 3D implants thatin addition to support cell in1047297ltration and organization will stimulate
blood vessel and axon growth are a promising approach to stimulate
tissue regeneration
Supplementary materialsrelated to this article canbe found online
at doi101016jjconrel201011028
Acknowledgments
The authors would like to thank Prof Carmeliet (Vesalius Research
Center KU Leuven Belgium) for the gift of VEGF producing Pichia
pastoris yeast The authors thank Sabine Cordi for her help with MIRAX
acquisition as well as Christophe Pierreux for the RGB quanti1047297cation
script SeacutebastienSartis thankedfor assistance with VEGF productionand
Laurenne Petit for the realization of VEGF activity tests This work wassupported by grants from the Belgian Fonds National de la Recherche
Scienti1047297que (FNRS Creacutedits aux chercheurs and FRSM 3461709) and
CTB (Belgium) Anne des Rieux is FNRS Post-Doctoral Researcher
References
[1] V Guarino F Causa L Ambrosio Bioactive scaffolds for bone and ligament tissueExpert Rev Med Devices 3 (2007) 405ndash418
[2] TM Freyman IV Yannas R Yokoo LJ Gibson Fibroblast contraction of acollagen-GAG matrix Biomaterials 21 (2001) 2883ndash2891
[3] V Mourino AR Boccaccini Bone tissue engineering therapeutics controlled drugdelivery in three-dimensional scaffolds J R Soc Interface 43 (2010) 209 ndash227
[4] CB Rives A des Rieux M Zelivyanskaya SR Stock WL Lowe Jr LD SheaLayered PLGscaffoldsfor invivo plasmid delivery Biomaterials3 (2009)394ndash401
[5] AH Zisch MP Lutolf JA Hubbell Biopolymeric delivery matrices for angiogenic
growth factors Cardiovasc Pathol 6 (2003) 295ndash310[6] ZS Patel AG Mikos Angiogenesis with biomaterial-based drug- and cell-
delivery systems J Biomater Sci Polym Ed 6 (2004) 701ndash726[7] BV Somayaji U Jariwala P Jayachandran K Vidyalakshmi RV Dudhani
Evaluation of antimicrobial ef 1047297cacy and release pattern of tetracycline andmetronidazole using a local delivery system J Periodontol 4 (1998) 409ndash413
[8] MHuang SN VitharanaLJ Peek T CoopC Berkland Polyelectrolyte complexesstabilize and controllably release vascular endothelial growth factor Biomacro-molecules 5 (2007) 1607ndash1614
[9] JS GolubYTKimCL Duvall RV BellamkondaD Gupta AS Lin DWeiss WRobertTaylor RE Guldberg Sustained VEGF delivery via PLGA nanoparticles promotesvascular growth Am J Physiol Heart Circ Physiol 6 (2010) H1959ndashH1965
[10] C He SW Kim DS Lee In situ gelling stimuli-sensitive block copolymerhydrogels for drug delivery J Control Release 3 (2008) 189ndash207
[11] HK Kleinman ML McGarvey JR Hassell VL Star FB Cannon GW LaurieGR Martin Basement membrane complexes with biological activity Biochemistry 2(1986) 312ndash318
[12] N Akhtar EB Dickerson R Auerbach The spongeMatrigel angiogenesis assayAngiogenesis 1ndash2 (2002) 75ndash80
[13] D Donovan NJ Brown ET Bishop CE Lewis Comparison of three in vitrohuman lsquoangiogenesisrsquo assays with capillaries formed in vivo Angiogenesis 2(2001) 113ndash121
[14] JM Kanczler PJ Ginty JJ Barry NM Clarke SM Howdle KM ShakesheffROOreffo Theeffectof mesenchymalpopulations and vascularendothelial growthfactor delivered from biodegradable polymer scaffolds on bone formationBiomaterials 12 (2008) 1892ndash1900
[15] FG Rocha CA Sundback NJ Krebs JK Leach DJ Mooney SW Ashley JPVacanti EE Whang The effect of sustained delivery of vascular endothelialgrowth factor on angiogenesis in tissue-engineered intestine Biomaterials 19(2008) 2884ndash2890
[16] JM Kanczler J Barry P Ginty SM Howdle KM Shakesheff RO OreffoSupercritical carbon dioxide generated vascular endothelial growth factorencapsulated poly(DL -lactic acid) scaffolds induce angiogenesis in vitro BiochemBiophys Res Commun 1 (2007) 135ndash141
[17] JM Kanczler PJ Ginty L White NM Clarke SM Howdle KM Shakesheff ROOreffo The effect of the delivery of vascular endothelial growth factor and bonemorphogenic protein-2 to osteoprogenitor cell populations on bone formation
Biomaterials 6 (2010) 1242ndash
1250
277 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 77
[18] TT Chen A Luque S Lee SM Anderson T Segura ML Iruela-Arispe Anchorageof VEGF to the extracellular matrix conveys differential signaling responses toendothelial cells J Cell Biol 4 (2010) 595ndash609
[19] GE Means RE Feeney Reductive alkylation of amino groups in proteinsBiochemistry 6 (1968) 2192ndash2201
[20] JH Jang LD Shea Controllable delivery of non-viral DNA from porous scaffolds J Control Release 1 (2003) 157ndash168
[21] LD Shea E Smiley J Bonadio DJ Mooney DNA delivery from polymer matricesfor tissue engineering Nat Biotechnol 6 (1999) 551ndash554
[22] DJ Mooney DF Baldwin NP Suh JP Vacanti R Langer Novel approach tofabricate porous sponges of poly(DL -lactic-co-glycolic acid) without the use of
organic solvents Biomaterials 14 (1996) 1417ndash
1422[23] JH Jang CB Rives LD Shea Plasmid delivery in vivo from porous tissue-engineering scaffolds transgene expression and cellular transfection Mol Ther 3(2005) 475ndash483
[24] MO Aviles CHLin M Zelivyanskaya JGGraham RMBoehlerPBMessersmithLD Shea The contribution of plasmid design and release to in vivo gene expressionfollowing delivery from cationic polymer modi1047297ed scaffolds Biomaterials 6 (2010)1140ndash1147
[25] P Sonveaux A Brouet X Havaux V Gregoire C Dessy JL Balligand O FeronIrradiation-induced angiogenesis through the up-regulation of the nitric oxidepathway implications for tumor radiotherapy Cancer Res 5 (2003) 1012 ndash1019
[26] H Blomeier X Zhang C Rives M Brissova E Hughes M Baker AC Powers DBKaufman LD Shea WL Lowe Jr Polymer scaffolds as synthetic microenviron-ments for extrahepatic islet transplantation Transplantation 4 (2006) 452ndash459
[27] Y Yang L De Laporte CB Rives JH Jang WC Lin KR Shull LD SheaNeurotrophin releasing single and multiple lumen nerve conduits J ControlRelease 3 (2005) 433ndash446
[28] L De Laporte A des Rieux ML Zelivyanska HM Tuinstra N De Clerck AAPostnov LD Shea VEGF and FGF-2 delivery from spinal cord bridges to enhanceangiogenesis following injury J Biomed Mater Res Part A (in press)
[29] ML Ponce Tube formationan in vitro matrigel angiogenesis assayMethods MolBiol (2009) 183ndash188
[30] A Albini R Benelli The chemoinvasion assay a method to assess tumor andendothelial cell invasion and its modulation Nat Protoc 3 (2007) 504ndash511
[31] MH Sheridan LD Shea MC Peters DJ Mooney Bioabsorbable polymer
scaffolds for tissue engineering capable of sustained growth factor delivery J Control Release 1ndash3 (2000) 91ndash102[32] JH Jang TL Houchin LD Shea Gene delivery from polymer scaffolds for tissue
engineering Expert Rev Med Devices 1 (2004) 127ndash138[33] P Carmeliet M Tessier-Lavigne Common mechanisms of nerve and blood vessel
wiring Nature 7048 (2005) 193ndash200[34] E Storkebaum D Lambrechts P Carmeliet VEGF once regarded as a speci1047297c
angiogenic factor now implicated in neuroprotection Bioessays 9 (2004) 943ndash954[35] E StorkebaumD LambrechtsM Dewerchin MPMoreno-Murciano S Appelmans
H Oh P Van Damme B Rutten WY Man M De Mol S Wyns D Manka KVermeulen L Van Den Bosch N Mertens C Schmitz W Robberecht EM ConwayD Collen L Moons P Carmeliet Treatment of motoneuron degeneration byintracerebroventricular delivery of VEGF in a rat model of ALS Nat Neurosci 1(2005) 85ndash92
278 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 47
33 VEGF release from 3D implants
VEGF release would likely be in1047298uenced by its incorporation as
well as by the nature of the 3D matrix into which nanoparticles were
incorporated Release of free or encapsulated VEGF was evaluated
over time after incorporation into hydrogels (Matrigeltrade hydrogels)
and into PLGA scaffolds
A 10 to 15 burst release was observed for VEGF whatever the
matrices or the formulation (2 h of incubation) except for encapsu-
lated VEGF incorporated in hydrogel (03) followed by a sustained
release over time (Fig 1) When incorporated in scaffolds release of
free VEGF was lower than encapsulated VEGF (24 versus 36
respectively after 2 weeks) In hydrogels encapsulation signi1047297cantly
slowed down VEGF release Indeed 20-times more free VEGF was
released after 2 weeks than encapsulated VEGF (32 and 16 for free
and encapsulated VEGF respectively) Release of free VEGF from
hydrogels was similar to release of encapsulated VEGF from scaffolds
(2 weeks incubation time 31 of encapsulated VEGF and 32 of free
VEGF respectively) Concerning the activity of released VEGF Huang
et al [8] demonstrated a preserved bioactivity for released VEGF from
NP for 10 days by a HUVEC proliferation assay VEGF released fromPLGA bridges [27] and from hydrogels was still able to induce
phosphorylation of HUVEC VEGF-R2 over a period of 42 days [28] and
of 2 weeks (Supplementary material 3) respectively
34 Nanoparticle incorporation into hydrogels effect on angiogenesis
The angiogenesis potential of free VEGF versus encapsulated VEGF
incorporated into an injectable hydrogel was studied Matrigeltradewas
chosen as a model for tissue regeneration due to its injectability and
proven ability to support cell and blood vessel growth [122930]
Following injection Matrigeltrade solidi1047297es and permits subsequent
penetration by host cells that induce vascularization Growth factor
reduced Matrigeltrade
was used to avoid interferences with the activityof the incorporated VEGF Free or encapsulated VEGF and as control
PBS were mixed with Matrigeltrade before subcutaneous injection
In1047298uence of VEGF amount (1 μ g and 2 μ g) and of incubation times
(2 and 3 weeks) was evaluated The effect on angiogenesis was
determined by quanti1047297cation of endothelial and red blood cells inside
the Matrigeltrade hydrogels after implantation
Endothelial cells werevisualized by immunostainingwith an anti-CD
31 antibody and red blood cells were visible by auto1047298uoresence The
percentage of red pixel represents both endothelial and red blood cells
Entire sections of Matrigeltrade hydrogels were analyzed The bene1047297cial
effect of VEGF encapsulation on the stimulation of angiogenesis
was clearly demonstrated (Fig 2) Indeed 2 weeks after implantation
endothelial cells some of them organized as blood vessels were ob-
served within the samples containing the encapsulated VEGF (Fig 2e
and f) In addition red blood cells were present within these structures
indicating their perfusion and their functionality In contrast for the
hydrogels loaded with PBS or free VEGF endothelial cells were detected
only around the implants (Fig 2a to d)
Quantitative analyses of the hydrogels loaded with VEGF nano-
particles showed that these implants contained 58 to 74-fold more
endothelial and red blood cells in comparison to the hydrogels loaded
with PBS or free VEGF respectively (Fig 3) No signi1047297cant differences
Fig 1 In1047298uence of VEGF encapsulation on its release from 3D implants [125I] VEGF and
biotinylated VEGF were incorporated into 3D implants (hydrogels and PLGA scaffolds)
either free or encapsulated Samples were incubated in PBS at 37 degC Radioactivity or
biotinylated VEGF in the supernatants was measured over time (n=3 signi1047297cant
Pb
005)
Fig 2 In1047298uence of VEGF encapsulation on angiogenesis when incorporated into
hydrogels VEGF (1 μ g) free (c d) or encapsulated (e f) was mixedwith Matrigeltrade and
injected sc in mice (2 weeks) negative control (a b) consisted in PBS mixed with
Matrigeltrade Endothelial cells (red) were detected by immuno1047298uorescence (CD 31) and
red blood cells (red arrow) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Table 1
Properties of CSDSVEGF nanoparticles
Formulation Diameter
(nm)
PDI Zeta potential
(mV)
Encapsulation
ef 1047297ciency
()
a) Before freeze-drying
CSndashDSa 325+minus19 022 +minus003 minus26+minus06 NA
CSndashDSndashVEGFb 383+minus73 026 +minus006 minus26+minus2 765 +minus65
b) After freeze-drying CSndashDSa 319+minus17 020 +minus001 minus25+minus03 NA
CSndashDSndashVEGFb 457+minus24 029 +minus001 minus34+minus15 71 +minus144
NA Not Applicablea n=4b n=6
275 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 57
were observed between 1 and 2 μ g of loaded VEGF with implantationduration of 2 and 3 weeks respectively The implants loaded with PBS
or free VEGF showed no signi1047297cant difference
35 Nanoparticle incorporation into PLGA scaffolds effect
on angiogenesis
The objective was to evaluate the effect of VEGF encapsulation on
angiogenesis when incorporated into polymeric scaffolds developed
for tissue engineering [421233132] Variable amounts of VEGF
nanoparticles were incorporated during scaffold processing and the
impact on scaffold integrity was determined The potential improve-
ment of encapsulated over free VEGF on VEGF loss during scaffold
leaching was estimated Then VEGF loaded scaffolds were implanted
in mice and angiogenesis was studiedFreeze-dried CSndashDS nanoparticles were mixed with PLGA micro-
spheres and NaCl crystals to form PLGA scaffolds Physical integrity
was estimated visually The physical integrity of scaffolds was
preserved up to 12 mg of incorporated nanoparticles per 36 mg
PLGA (n= 3) Incorporation of higher amounts induced scaffold
crumbling during the leaching phase To measure VEGF loss after
scaffold leaching lyophilized free or encapsulated [sup3H] VEGF was
used for the scaffold process VEGF loss during salt leaching was 25
for free VEGF and 44 for encapsulated VEGF (n=3) Taking these
ratios into account the amount of VEGF added to the scaffold process
was adapted to obtain 1 μ g of VEGF per implanted scaffold
VEGF loaded scaffolds were implanted in the fat pad of mice and
the in1047298uence on angiogenesis of encapsulated versus free VEGF was
studied 2 weeks after implantation Empty PLGA scaffolds were usedas control Sections of the implanted scaffolds were immunostained
Endothelial and red blood cells were identi1047297ed within and around the
scaffolds for all the conditions (Fig 4)
However as observed for hydrogels PLGA scaffolds containing
VEGF nanoparticles seemed to stimulate more ef 1047297ciently angiogenesis
(Fig 4e and f)
In order to determineif VEGF encapsulation signi1047297cantly increased
the amount of endothelial and red blood cells within and around the
scaffolds quanti1047297cation of the percentage of red pixels over the total
amount of analyzed pixels was performed Signi1047297cantly more
endothelial and red blood cells were detected with VEGF nanopar-
ticles incorporated into PLGA scaffolds (Fig 5) compared with free
VEGF or control although the difference was less marked than
observed for the Matrigeltrade
hydrogels (35-folds) This could be
explained by the fact that PLGA scaffolds alone seemed to support cell
in1047297
ltration and angiogenesis to a higher extent than Matrigeltrade
Nosigni1047297cant difference was observed between negative control and free
VEGF
4 Discussion
When designing systems for tissue engineering one of the 1047297rst
requirements is the colonization of the implant by blood vessels
bringing oxygen and nutriments to support tissue reconstruction
Fig 4 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in scaffolds
VEGF (1 μ g)free (c andd) or encapsulated(e andf) was incorporatedin PLGA scaffolds
and implanted in mouse fat pads (2 weeks) negative control (a and b) consisted in
empty scaffolds Endothelial cells (red) were detected by immuno1047298uorescence (CD 31)
and red blood cells (red) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Fig 5 In1047298uenceof VEGF encapsulation on angiogenesis when incorporated in scaffolds
quanti1047297cation of endothelial and red blood cells VEGF (1 μ g) free or encapsulated was
incorporated in PLGA scaffolds and implanted in mouse fat pads (2 weeks) negative
control consisted in empty scaffolds Endothelial cells were detected by immuno1047298u-
orescence (CD 31) and red bloodcells byauto1047298uorescence Pictures were acquired with
a MIRAX microscope 24sections by condition(n =3) were imagedin their entirety and
analyzed (Axiovision) 8 sections taken at one extremity 8 in the middle and 8 at the
other extremity of scaffolds (mean +minus
SEM Pb
005)
Fig 3 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in
hydrogels quanti1047297cation of endothelial and red blood cells VEGF (1 or 2 μ g) free or
encapsulated was incorporated in Matrigeltrade and injected sc in mice (2 or 3 weeks)
negative control consisted in PBS mixed with Matrigeltrade Endothelial cells were
detected by immuno1047298uorescence (CD 31) and red blood cells by auto1047298uorescence
Pictures were acquired with a MIRAX microscope 8 sections by condition (n=3) were
imaged in their entirety and analyzed (Axiovision) (mean +minusSEM Pb005)
276 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 67
The major focus of this work was to determine if incorporation of
VEGFndashCSndashDS nanoparticles into twotypesof 3D implants (PLGA scaffold
or hydrogel) commonly used for tissue engineering (hydrogels and
polymeric scaffolds) improved angiogenesis within the implants and
thus tissue regeneration We investigated the effect of VEGF combined
release from nanoparticles and 3D matrices and the in1047298uence of the
matrix nature on angiogenesis The results indicate thatimplantation of
3D systems loaded with VEGF nanoparticles stimulated endothelial cell
colonization and blood vessel formation more ef 1047297
ciently than matricesloaded with free VEGF regardless of the VEGF dose implantation time
and implant type
The improvement probably resulted from i) VEGF protection by
complexation of the heparin binding site of VEGF with dextran sulfate
combined with ii) protection and controlled release brought by
complex stabilization by chitosan Indeed Huang et al [8] demon-
strated in vitro by ELISA and HUVEC proliferation test that VEGF
encapsulation by DS andCS protectedmost of VEGF activity forat least
10 days and that VEGF was slowly released from nanoparticles
We observed that encapsulation of VEGF in comparison with free
VEGF signi1047297cantly decreases burst release (03 versus 10 respec-
tively) from VEGF loaded Matrigeltrade hydrogels and slowed down
diffusionfrom the implant about 20-fold after 2 weeks It could be due
to higher size combined with electrostatic interactions due to the
negative surface charge of nanoparticles Encapsulated VEGF was
released from PLGA scaffolds slightly faster than free VEGF (24
versus 36 respectively after 2 weeks) Encapsulation did not affect
burst release but loss during leaching was taken into account for
scaffold preparation and the initial VEGF loading was adjusted to be
1 μ g of VEGF post-leaching for both conditions Indeed encapsulation
signi1047297cantly decreased VEGF loss during leaching (about 21) In that
perspective the burst release for free VEGF loaded scaffolds would be
approximately about 15 higher than for VEGF nanoparticle-loaded
scaffolds Finally compared to microspheres classically used to
protect and deliver proteins complexation by DS and coacervation
by CS achieved a higher loading and did not compromise implant
integrity Indeed approximately 5 μ g of VEGF was incorporated in
1 mg of CSndashDS nanoparticles versus approximately 50 ng per mg of
microspheres [9] This allows incorporation of a much higher amountof VEGF into implants compared to microspheres
Although encapsulated VEGF stimulated angiogenesis to a greater
extent than freeVEGF in both implant types differences between PLGA
scaffolds and Matrigeltrade hydrogels were observed The percentage of
endothelial and red blood cells quanti1047297ed in nanoparticle-loaded
scaffolds was 75 times higher compared to Matrigeltrade hydrogels but
the scaffold itself without VEGF stimulated angiogenesis at the same
level than hydrogels loaded with encapsulated VEGF This could be
explained by the factthat these PLGAscaffolds havebeen optimizedfor
tissue engineering [4] and thus promote cell in1047297ltration and
proliferation However encapsulation of VEGF did not affect release
from scaffolds as much as for hydrogels Hence the increase in
angiogenesis elicited by encapsulated versus free VEGF loaded into
Matrigeltradehydrogels wasabout 5 to 7-foldwhile it wasonlyabout 35-fold for scaffolds Considering these results it seems that it could be
interesting to combine thetwo systems by1047297lling PLGA scaffoldspores
with hydrogel both loaded with VEGF nanoparticles
5 Conclusion
Wehypothesized thatVEGFactivityin vivo would be enhanced by its
encapsulation in CSndashDS nanoparticles and further incorporation into
implants used in tissue engineering like injectable hydrogels and
polymeric scaffolds Angiogenesis the primary effect of VEGF was
clearly improved by encapsulation the extent of implant colonization
by endothelial cells and blood vessels was higher when both types of
implants were loaded with VEGF nanoparticles compared with free
VEGF(non-encapsulated) Nanoparticles composed of chitosan dextran
sulfate and VEGF were obtained and incorporated in implants without
impairingtheirstructuralpropertiesand allowing a higher VEGFloading
compared to VEGF classical techniques of encapsulation (up to 5 μ g of
VEGF per mg of nanoparticles) VEGF encapsulation guarantees VEGF
ef 1047297ciency by protection and sustained release from 3D implants
In the scope of the spinal cord regeneration systems to deliver
active VEGF are attractive since in addition to its angiogenic activity
VEGF has recently been shown to be a neurogenic factor [33ndash35]
Then encapsulation and incorporation of VEGF into 3D implants thatin addition to support cell in1047297ltration and organization will stimulate
blood vessel and axon growth are a promising approach to stimulate
tissue regeneration
Supplementary materialsrelated to this article canbe found online
at doi101016jjconrel201011028
Acknowledgments
The authors would like to thank Prof Carmeliet (Vesalius Research
Center KU Leuven Belgium) for the gift of VEGF producing Pichia
pastoris yeast The authors thank Sabine Cordi for her help with MIRAX
acquisition as well as Christophe Pierreux for the RGB quanti1047297cation
script SeacutebastienSartis thankedfor assistance with VEGF productionand
Laurenne Petit for the realization of VEGF activity tests This work wassupported by grants from the Belgian Fonds National de la Recherche
Scienti1047297que (FNRS Creacutedits aux chercheurs and FRSM 3461709) and
CTB (Belgium) Anne des Rieux is FNRS Post-Doctoral Researcher
References
[1] V Guarino F Causa L Ambrosio Bioactive scaffolds for bone and ligament tissueExpert Rev Med Devices 3 (2007) 405ndash418
[2] TM Freyman IV Yannas R Yokoo LJ Gibson Fibroblast contraction of acollagen-GAG matrix Biomaterials 21 (2001) 2883ndash2891
[3] V Mourino AR Boccaccini Bone tissue engineering therapeutics controlled drugdelivery in three-dimensional scaffolds J R Soc Interface 43 (2010) 209 ndash227
[4] CB Rives A des Rieux M Zelivyanskaya SR Stock WL Lowe Jr LD SheaLayered PLGscaffoldsfor invivo plasmid delivery Biomaterials3 (2009)394ndash401
[5] AH Zisch MP Lutolf JA Hubbell Biopolymeric delivery matrices for angiogenic
growth factors Cardiovasc Pathol 6 (2003) 295ndash310[6] ZS Patel AG Mikos Angiogenesis with biomaterial-based drug- and cell-
delivery systems J Biomater Sci Polym Ed 6 (2004) 701ndash726[7] BV Somayaji U Jariwala P Jayachandran K Vidyalakshmi RV Dudhani
Evaluation of antimicrobial ef 1047297cacy and release pattern of tetracycline andmetronidazole using a local delivery system J Periodontol 4 (1998) 409ndash413
[8] MHuang SN VitharanaLJ Peek T CoopC Berkland Polyelectrolyte complexesstabilize and controllably release vascular endothelial growth factor Biomacro-molecules 5 (2007) 1607ndash1614
[9] JS GolubYTKimCL Duvall RV BellamkondaD Gupta AS Lin DWeiss WRobertTaylor RE Guldberg Sustained VEGF delivery via PLGA nanoparticles promotesvascular growth Am J Physiol Heart Circ Physiol 6 (2010) H1959ndashH1965
[10] C He SW Kim DS Lee In situ gelling stimuli-sensitive block copolymerhydrogels for drug delivery J Control Release 3 (2008) 189ndash207
[11] HK Kleinman ML McGarvey JR Hassell VL Star FB Cannon GW LaurieGR Martin Basement membrane complexes with biological activity Biochemistry 2(1986) 312ndash318
[12] N Akhtar EB Dickerson R Auerbach The spongeMatrigel angiogenesis assayAngiogenesis 1ndash2 (2002) 75ndash80
[13] D Donovan NJ Brown ET Bishop CE Lewis Comparison of three in vitrohuman lsquoangiogenesisrsquo assays with capillaries formed in vivo Angiogenesis 2(2001) 113ndash121
[14] JM Kanczler PJ Ginty JJ Barry NM Clarke SM Howdle KM ShakesheffROOreffo Theeffectof mesenchymalpopulations and vascularendothelial growthfactor delivered from biodegradable polymer scaffolds on bone formationBiomaterials 12 (2008) 1892ndash1900
[15] FG Rocha CA Sundback NJ Krebs JK Leach DJ Mooney SW Ashley JPVacanti EE Whang The effect of sustained delivery of vascular endothelialgrowth factor on angiogenesis in tissue-engineered intestine Biomaterials 19(2008) 2884ndash2890
[16] JM Kanczler J Barry P Ginty SM Howdle KM Shakesheff RO OreffoSupercritical carbon dioxide generated vascular endothelial growth factorencapsulated poly(DL -lactic acid) scaffolds induce angiogenesis in vitro BiochemBiophys Res Commun 1 (2007) 135ndash141
[17] JM Kanczler PJ Ginty L White NM Clarke SM Howdle KM Shakesheff ROOreffo The effect of the delivery of vascular endothelial growth factor and bonemorphogenic protein-2 to osteoprogenitor cell populations on bone formation
Biomaterials 6 (2010) 1242ndash
1250
277 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 77
[18] TT Chen A Luque S Lee SM Anderson T Segura ML Iruela-Arispe Anchorageof VEGF to the extracellular matrix conveys differential signaling responses toendothelial cells J Cell Biol 4 (2010) 595ndash609
[19] GE Means RE Feeney Reductive alkylation of amino groups in proteinsBiochemistry 6 (1968) 2192ndash2201
[20] JH Jang LD Shea Controllable delivery of non-viral DNA from porous scaffolds J Control Release 1 (2003) 157ndash168
[21] LD Shea E Smiley J Bonadio DJ Mooney DNA delivery from polymer matricesfor tissue engineering Nat Biotechnol 6 (1999) 551ndash554
[22] DJ Mooney DF Baldwin NP Suh JP Vacanti R Langer Novel approach tofabricate porous sponges of poly(DL -lactic-co-glycolic acid) without the use of
organic solvents Biomaterials 14 (1996) 1417ndash
1422[23] JH Jang CB Rives LD Shea Plasmid delivery in vivo from porous tissue-engineering scaffolds transgene expression and cellular transfection Mol Ther 3(2005) 475ndash483
[24] MO Aviles CHLin M Zelivyanskaya JGGraham RMBoehlerPBMessersmithLD Shea The contribution of plasmid design and release to in vivo gene expressionfollowing delivery from cationic polymer modi1047297ed scaffolds Biomaterials 6 (2010)1140ndash1147
[25] P Sonveaux A Brouet X Havaux V Gregoire C Dessy JL Balligand O FeronIrradiation-induced angiogenesis through the up-regulation of the nitric oxidepathway implications for tumor radiotherapy Cancer Res 5 (2003) 1012 ndash1019
[26] H Blomeier X Zhang C Rives M Brissova E Hughes M Baker AC Powers DBKaufman LD Shea WL Lowe Jr Polymer scaffolds as synthetic microenviron-ments for extrahepatic islet transplantation Transplantation 4 (2006) 452ndash459
[27] Y Yang L De Laporte CB Rives JH Jang WC Lin KR Shull LD SheaNeurotrophin releasing single and multiple lumen nerve conduits J ControlRelease 3 (2005) 433ndash446
[28] L De Laporte A des Rieux ML Zelivyanska HM Tuinstra N De Clerck AAPostnov LD Shea VEGF and FGF-2 delivery from spinal cord bridges to enhanceangiogenesis following injury J Biomed Mater Res Part A (in press)
[29] ML Ponce Tube formationan in vitro matrigel angiogenesis assayMethods MolBiol (2009) 183ndash188
[30] A Albini R Benelli The chemoinvasion assay a method to assess tumor andendothelial cell invasion and its modulation Nat Protoc 3 (2007) 504ndash511
[31] MH Sheridan LD Shea MC Peters DJ Mooney Bioabsorbable polymer
scaffolds for tissue engineering capable of sustained growth factor delivery J Control Release 1ndash3 (2000) 91ndash102[32] JH Jang TL Houchin LD Shea Gene delivery from polymer scaffolds for tissue
engineering Expert Rev Med Devices 1 (2004) 127ndash138[33] P Carmeliet M Tessier-Lavigne Common mechanisms of nerve and blood vessel
wiring Nature 7048 (2005) 193ndash200[34] E Storkebaum D Lambrechts P Carmeliet VEGF once regarded as a speci1047297c
angiogenic factor now implicated in neuroprotection Bioessays 9 (2004) 943ndash954[35] E StorkebaumD LambrechtsM Dewerchin MPMoreno-Murciano S Appelmans
H Oh P Van Damme B Rutten WY Man M De Mol S Wyns D Manka KVermeulen L Van Den Bosch N Mertens C Schmitz W Robberecht EM ConwayD Collen L Moons P Carmeliet Treatment of motoneuron degeneration byintracerebroventricular delivery of VEGF in a rat model of ALS Nat Neurosci 1(2005) 85ndash92
278 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 57
were observed between 1 and 2 μ g of loaded VEGF with implantationduration of 2 and 3 weeks respectively The implants loaded with PBS
or free VEGF showed no signi1047297cant difference
35 Nanoparticle incorporation into PLGA scaffolds effect
on angiogenesis
The objective was to evaluate the effect of VEGF encapsulation on
angiogenesis when incorporated into polymeric scaffolds developed
for tissue engineering [421233132] Variable amounts of VEGF
nanoparticles were incorporated during scaffold processing and the
impact on scaffold integrity was determined The potential improve-
ment of encapsulated over free VEGF on VEGF loss during scaffold
leaching was estimated Then VEGF loaded scaffolds were implanted
in mice and angiogenesis was studiedFreeze-dried CSndashDS nanoparticles were mixed with PLGA micro-
spheres and NaCl crystals to form PLGA scaffolds Physical integrity
was estimated visually The physical integrity of scaffolds was
preserved up to 12 mg of incorporated nanoparticles per 36 mg
PLGA (n= 3) Incorporation of higher amounts induced scaffold
crumbling during the leaching phase To measure VEGF loss after
scaffold leaching lyophilized free or encapsulated [sup3H] VEGF was
used for the scaffold process VEGF loss during salt leaching was 25
for free VEGF and 44 for encapsulated VEGF (n=3) Taking these
ratios into account the amount of VEGF added to the scaffold process
was adapted to obtain 1 μ g of VEGF per implanted scaffold
VEGF loaded scaffolds were implanted in the fat pad of mice and
the in1047298uence on angiogenesis of encapsulated versus free VEGF was
studied 2 weeks after implantation Empty PLGA scaffolds were usedas control Sections of the implanted scaffolds were immunostained
Endothelial and red blood cells were identi1047297ed within and around the
scaffolds for all the conditions (Fig 4)
However as observed for hydrogels PLGA scaffolds containing
VEGF nanoparticles seemed to stimulate more ef 1047297ciently angiogenesis
(Fig 4e and f)
In order to determineif VEGF encapsulation signi1047297cantly increased
the amount of endothelial and red blood cells within and around the
scaffolds quanti1047297cation of the percentage of red pixels over the total
amount of analyzed pixels was performed Signi1047297cantly more
endothelial and red blood cells were detected with VEGF nanopar-
ticles incorporated into PLGA scaffolds (Fig 5) compared with free
VEGF or control although the difference was less marked than
observed for the Matrigeltrade
hydrogels (35-folds) This could be
explained by the fact that PLGA scaffolds alone seemed to support cell
in1047297
ltration and angiogenesis to a higher extent than Matrigeltrade
Nosigni1047297cant difference was observed between negative control and free
VEGF
4 Discussion
When designing systems for tissue engineering one of the 1047297rst
requirements is the colonization of the implant by blood vessels
bringing oxygen and nutriments to support tissue reconstruction
Fig 4 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in scaffolds
VEGF (1 μ g)free (c andd) or encapsulated(e andf) was incorporatedin PLGA scaffolds
and implanted in mouse fat pads (2 weeks) negative control (a and b) consisted in
empty scaffolds Endothelial cells (red) were detected by immuno1047298uorescence (CD 31)
and red blood cells (red) by auto1047298uorescence Nuclei (blue) were labeled with DAPI
Pictures were acquired with a MIRAX microscope
Fig 5 In1047298uenceof VEGF encapsulation on angiogenesis when incorporated in scaffolds
quanti1047297cation of endothelial and red blood cells VEGF (1 μ g) free or encapsulated was
incorporated in PLGA scaffolds and implanted in mouse fat pads (2 weeks) negative
control consisted in empty scaffolds Endothelial cells were detected by immuno1047298u-
orescence (CD 31) and red bloodcells byauto1047298uorescence Pictures were acquired with
a MIRAX microscope 24sections by condition(n =3) were imagedin their entirety and
analyzed (Axiovision) 8 sections taken at one extremity 8 in the middle and 8 at the
other extremity of scaffolds (mean +minus
SEM Pb
005)
Fig 3 In1047298uence of VEGF encapsulation on angiogenesis when incorporated in
hydrogels quanti1047297cation of endothelial and red blood cells VEGF (1 or 2 μ g) free or
encapsulated was incorporated in Matrigeltrade and injected sc in mice (2 or 3 weeks)
negative control consisted in PBS mixed with Matrigeltrade Endothelial cells were
detected by immuno1047298uorescence (CD 31) and red blood cells by auto1047298uorescence
Pictures were acquired with a MIRAX microscope 8 sections by condition (n=3) were
imaged in their entirety and analyzed (Axiovision) (mean +minusSEM Pb005)
276 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 67
The major focus of this work was to determine if incorporation of
VEGFndashCSndashDS nanoparticles into twotypesof 3D implants (PLGA scaffold
or hydrogel) commonly used for tissue engineering (hydrogels and
polymeric scaffolds) improved angiogenesis within the implants and
thus tissue regeneration We investigated the effect of VEGF combined
release from nanoparticles and 3D matrices and the in1047298uence of the
matrix nature on angiogenesis The results indicate thatimplantation of
3D systems loaded with VEGF nanoparticles stimulated endothelial cell
colonization and blood vessel formation more ef 1047297
ciently than matricesloaded with free VEGF regardless of the VEGF dose implantation time
and implant type
The improvement probably resulted from i) VEGF protection by
complexation of the heparin binding site of VEGF with dextran sulfate
combined with ii) protection and controlled release brought by
complex stabilization by chitosan Indeed Huang et al [8] demon-
strated in vitro by ELISA and HUVEC proliferation test that VEGF
encapsulation by DS andCS protectedmost of VEGF activity forat least
10 days and that VEGF was slowly released from nanoparticles
We observed that encapsulation of VEGF in comparison with free
VEGF signi1047297cantly decreases burst release (03 versus 10 respec-
tively) from VEGF loaded Matrigeltrade hydrogels and slowed down
diffusionfrom the implant about 20-fold after 2 weeks It could be due
to higher size combined with electrostatic interactions due to the
negative surface charge of nanoparticles Encapsulated VEGF was
released from PLGA scaffolds slightly faster than free VEGF (24
versus 36 respectively after 2 weeks) Encapsulation did not affect
burst release but loss during leaching was taken into account for
scaffold preparation and the initial VEGF loading was adjusted to be
1 μ g of VEGF post-leaching for both conditions Indeed encapsulation
signi1047297cantly decreased VEGF loss during leaching (about 21) In that
perspective the burst release for free VEGF loaded scaffolds would be
approximately about 15 higher than for VEGF nanoparticle-loaded
scaffolds Finally compared to microspheres classically used to
protect and deliver proteins complexation by DS and coacervation
by CS achieved a higher loading and did not compromise implant
integrity Indeed approximately 5 μ g of VEGF was incorporated in
1 mg of CSndashDS nanoparticles versus approximately 50 ng per mg of
microspheres [9] This allows incorporation of a much higher amountof VEGF into implants compared to microspheres
Although encapsulated VEGF stimulated angiogenesis to a greater
extent than freeVEGF in both implant types differences between PLGA
scaffolds and Matrigeltrade hydrogels were observed The percentage of
endothelial and red blood cells quanti1047297ed in nanoparticle-loaded
scaffolds was 75 times higher compared to Matrigeltrade hydrogels but
the scaffold itself without VEGF stimulated angiogenesis at the same
level than hydrogels loaded with encapsulated VEGF This could be
explained by the factthat these PLGAscaffolds havebeen optimizedfor
tissue engineering [4] and thus promote cell in1047297ltration and
proliferation However encapsulation of VEGF did not affect release
from scaffolds as much as for hydrogels Hence the increase in
angiogenesis elicited by encapsulated versus free VEGF loaded into
Matrigeltradehydrogels wasabout 5 to 7-foldwhile it wasonlyabout 35-fold for scaffolds Considering these results it seems that it could be
interesting to combine thetwo systems by1047297lling PLGA scaffoldspores
with hydrogel both loaded with VEGF nanoparticles
5 Conclusion
Wehypothesized thatVEGFactivityin vivo would be enhanced by its
encapsulation in CSndashDS nanoparticles and further incorporation into
implants used in tissue engineering like injectable hydrogels and
polymeric scaffolds Angiogenesis the primary effect of VEGF was
clearly improved by encapsulation the extent of implant colonization
by endothelial cells and blood vessels was higher when both types of
implants were loaded with VEGF nanoparticles compared with free
VEGF(non-encapsulated) Nanoparticles composed of chitosan dextran
sulfate and VEGF were obtained and incorporated in implants without
impairingtheirstructuralpropertiesand allowing a higher VEGFloading
compared to VEGF classical techniques of encapsulation (up to 5 μ g of
VEGF per mg of nanoparticles) VEGF encapsulation guarantees VEGF
ef 1047297ciency by protection and sustained release from 3D implants
In the scope of the spinal cord regeneration systems to deliver
active VEGF are attractive since in addition to its angiogenic activity
VEGF has recently been shown to be a neurogenic factor [33ndash35]
Then encapsulation and incorporation of VEGF into 3D implants thatin addition to support cell in1047297ltration and organization will stimulate
blood vessel and axon growth are a promising approach to stimulate
tissue regeneration
Supplementary materialsrelated to this article canbe found online
at doi101016jjconrel201011028
Acknowledgments
The authors would like to thank Prof Carmeliet (Vesalius Research
Center KU Leuven Belgium) for the gift of VEGF producing Pichia
pastoris yeast The authors thank Sabine Cordi for her help with MIRAX
acquisition as well as Christophe Pierreux for the RGB quanti1047297cation
script SeacutebastienSartis thankedfor assistance with VEGF productionand
Laurenne Petit for the realization of VEGF activity tests This work wassupported by grants from the Belgian Fonds National de la Recherche
Scienti1047297que (FNRS Creacutedits aux chercheurs and FRSM 3461709) and
CTB (Belgium) Anne des Rieux is FNRS Post-Doctoral Researcher
References
[1] V Guarino F Causa L Ambrosio Bioactive scaffolds for bone and ligament tissueExpert Rev Med Devices 3 (2007) 405ndash418
[2] TM Freyman IV Yannas R Yokoo LJ Gibson Fibroblast contraction of acollagen-GAG matrix Biomaterials 21 (2001) 2883ndash2891
[3] V Mourino AR Boccaccini Bone tissue engineering therapeutics controlled drugdelivery in three-dimensional scaffolds J R Soc Interface 43 (2010) 209 ndash227
[4] CB Rives A des Rieux M Zelivyanskaya SR Stock WL Lowe Jr LD SheaLayered PLGscaffoldsfor invivo plasmid delivery Biomaterials3 (2009)394ndash401
[5] AH Zisch MP Lutolf JA Hubbell Biopolymeric delivery matrices for angiogenic
growth factors Cardiovasc Pathol 6 (2003) 295ndash310[6] ZS Patel AG Mikos Angiogenesis with biomaterial-based drug- and cell-
delivery systems J Biomater Sci Polym Ed 6 (2004) 701ndash726[7] BV Somayaji U Jariwala P Jayachandran K Vidyalakshmi RV Dudhani
Evaluation of antimicrobial ef 1047297cacy and release pattern of tetracycline andmetronidazole using a local delivery system J Periodontol 4 (1998) 409ndash413
[8] MHuang SN VitharanaLJ Peek T CoopC Berkland Polyelectrolyte complexesstabilize and controllably release vascular endothelial growth factor Biomacro-molecules 5 (2007) 1607ndash1614
[9] JS GolubYTKimCL Duvall RV BellamkondaD Gupta AS Lin DWeiss WRobertTaylor RE Guldberg Sustained VEGF delivery via PLGA nanoparticles promotesvascular growth Am J Physiol Heart Circ Physiol 6 (2010) H1959ndashH1965
[10] C He SW Kim DS Lee In situ gelling stimuli-sensitive block copolymerhydrogels for drug delivery J Control Release 3 (2008) 189ndash207
[11] HK Kleinman ML McGarvey JR Hassell VL Star FB Cannon GW LaurieGR Martin Basement membrane complexes with biological activity Biochemistry 2(1986) 312ndash318
[12] N Akhtar EB Dickerson R Auerbach The spongeMatrigel angiogenesis assayAngiogenesis 1ndash2 (2002) 75ndash80
[13] D Donovan NJ Brown ET Bishop CE Lewis Comparison of three in vitrohuman lsquoangiogenesisrsquo assays with capillaries formed in vivo Angiogenesis 2(2001) 113ndash121
[14] JM Kanczler PJ Ginty JJ Barry NM Clarke SM Howdle KM ShakesheffROOreffo Theeffectof mesenchymalpopulations and vascularendothelial growthfactor delivered from biodegradable polymer scaffolds on bone formationBiomaterials 12 (2008) 1892ndash1900
[15] FG Rocha CA Sundback NJ Krebs JK Leach DJ Mooney SW Ashley JPVacanti EE Whang The effect of sustained delivery of vascular endothelialgrowth factor on angiogenesis in tissue-engineered intestine Biomaterials 19(2008) 2884ndash2890
[16] JM Kanczler J Barry P Ginty SM Howdle KM Shakesheff RO OreffoSupercritical carbon dioxide generated vascular endothelial growth factorencapsulated poly(DL -lactic acid) scaffolds induce angiogenesis in vitro BiochemBiophys Res Commun 1 (2007) 135ndash141
[17] JM Kanczler PJ Ginty L White NM Clarke SM Howdle KM Shakesheff ROOreffo The effect of the delivery of vascular endothelial growth factor and bonemorphogenic protein-2 to osteoprogenitor cell populations on bone formation
Biomaterials 6 (2010) 1242ndash
1250
277 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 77
[18] TT Chen A Luque S Lee SM Anderson T Segura ML Iruela-Arispe Anchorageof VEGF to the extracellular matrix conveys differential signaling responses toendothelial cells J Cell Biol 4 (2010) 595ndash609
[19] GE Means RE Feeney Reductive alkylation of amino groups in proteinsBiochemistry 6 (1968) 2192ndash2201
[20] JH Jang LD Shea Controllable delivery of non-viral DNA from porous scaffolds J Control Release 1 (2003) 157ndash168
[21] LD Shea E Smiley J Bonadio DJ Mooney DNA delivery from polymer matricesfor tissue engineering Nat Biotechnol 6 (1999) 551ndash554
[22] DJ Mooney DF Baldwin NP Suh JP Vacanti R Langer Novel approach tofabricate porous sponges of poly(DL -lactic-co-glycolic acid) without the use of
organic solvents Biomaterials 14 (1996) 1417ndash
1422[23] JH Jang CB Rives LD Shea Plasmid delivery in vivo from porous tissue-engineering scaffolds transgene expression and cellular transfection Mol Ther 3(2005) 475ndash483
[24] MO Aviles CHLin M Zelivyanskaya JGGraham RMBoehlerPBMessersmithLD Shea The contribution of plasmid design and release to in vivo gene expressionfollowing delivery from cationic polymer modi1047297ed scaffolds Biomaterials 6 (2010)1140ndash1147
[25] P Sonveaux A Brouet X Havaux V Gregoire C Dessy JL Balligand O FeronIrradiation-induced angiogenesis through the up-regulation of the nitric oxidepathway implications for tumor radiotherapy Cancer Res 5 (2003) 1012 ndash1019
[26] H Blomeier X Zhang C Rives M Brissova E Hughes M Baker AC Powers DBKaufman LD Shea WL Lowe Jr Polymer scaffolds as synthetic microenviron-ments for extrahepatic islet transplantation Transplantation 4 (2006) 452ndash459
[27] Y Yang L De Laporte CB Rives JH Jang WC Lin KR Shull LD SheaNeurotrophin releasing single and multiple lumen nerve conduits J ControlRelease 3 (2005) 433ndash446
[28] L De Laporte A des Rieux ML Zelivyanska HM Tuinstra N De Clerck AAPostnov LD Shea VEGF and FGF-2 delivery from spinal cord bridges to enhanceangiogenesis following injury J Biomed Mater Res Part A (in press)
[29] ML Ponce Tube formationan in vitro matrigel angiogenesis assayMethods MolBiol (2009) 183ndash188
[30] A Albini R Benelli The chemoinvasion assay a method to assess tumor andendothelial cell invasion and its modulation Nat Protoc 3 (2007) 504ndash511
[31] MH Sheridan LD Shea MC Peters DJ Mooney Bioabsorbable polymer
scaffolds for tissue engineering capable of sustained growth factor delivery J Control Release 1ndash3 (2000) 91ndash102[32] JH Jang TL Houchin LD Shea Gene delivery from polymer scaffolds for tissue
engineering Expert Rev Med Devices 1 (2004) 127ndash138[33] P Carmeliet M Tessier-Lavigne Common mechanisms of nerve and blood vessel
wiring Nature 7048 (2005) 193ndash200[34] E Storkebaum D Lambrechts P Carmeliet VEGF once regarded as a speci1047297c
angiogenic factor now implicated in neuroprotection Bioessays 9 (2004) 943ndash954[35] E StorkebaumD LambrechtsM Dewerchin MPMoreno-Murciano S Appelmans
H Oh P Van Damme B Rutten WY Man M De Mol S Wyns D Manka KVermeulen L Van Den Bosch N Mertens C Schmitz W Robberecht EM ConwayD Collen L Moons P Carmeliet Treatment of motoneuron degeneration byintracerebroventricular delivery of VEGF in a rat model of ALS Nat Neurosci 1(2005) 85ndash92
278 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 67
The major focus of this work was to determine if incorporation of
VEGFndashCSndashDS nanoparticles into twotypesof 3D implants (PLGA scaffold
or hydrogel) commonly used for tissue engineering (hydrogels and
polymeric scaffolds) improved angiogenesis within the implants and
thus tissue regeneration We investigated the effect of VEGF combined
release from nanoparticles and 3D matrices and the in1047298uence of the
matrix nature on angiogenesis The results indicate thatimplantation of
3D systems loaded with VEGF nanoparticles stimulated endothelial cell
colonization and blood vessel formation more ef 1047297
ciently than matricesloaded with free VEGF regardless of the VEGF dose implantation time
and implant type
The improvement probably resulted from i) VEGF protection by
complexation of the heparin binding site of VEGF with dextran sulfate
combined with ii) protection and controlled release brought by
complex stabilization by chitosan Indeed Huang et al [8] demon-
strated in vitro by ELISA and HUVEC proliferation test that VEGF
encapsulation by DS andCS protectedmost of VEGF activity forat least
10 days and that VEGF was slowly released from nanoparticles
We observed that encapsulation of VEGF in comparison with free
VEGF signi1047297cantly decreases burst release (03 versus 10 respec-
tively) from VEGF loaded Matrigeltrade hydrogels and slowed down
diffusionfrom the implant about 20-fold after 2 weeks It could be due
to higher size combined with electrostatic interactions due to the
negative surface charge of nanoparticles Encapsulated VEGF was
released from PLGA scaffolds slightly faster than free VEGF (24
versus 36 respectively after 2 weeks) Encapsulation did not affect
burst release but loss during leaching was taken into account for
scaffold preparation and the initial VEGF loading was adjusted to be
1 μ g of VEGF post-leaching for both conditions Indeed encapsulation
signi1047297cantly decreased VEGF loss during leaching (about 21) In that
perspective the burst release for free VEGF loaded scaffolds would be
approximately about 15 higher than for VEGF nanoparticle-loaded
scaffolds Finally compared to microspheres classically used to
protect and deliver proteins complexation by DS and coacervation
by CS achieved a higher loading and did not compromise implant
integrity Indeed approximately 5 μ g of VEGF was incorporated in
1 mg of CSndashDS nanoparticles versus approximately 50 ng per mg of
microspheres [9] This allows incorporation of a much higher amountof VEGF into implants compared to microspheres
Although encapsulated VEGF stimulated angiogenesis to a greater
extent than freeVEGF in both implant types differences between PLGA
scaffolds and Matrigeltrade hydrogels were observed The percentage of
endothelial and red blood cells quanti1047297ed in nanoparticle-loaded
scaffolds was 75 times higher compared to Matrigeltrade hydrogels but
the scaffold itself without VEGF stimulated angiogenesis at the same
level than hydrogels loaded with encapsulated VEGF This could be
explained by the factthat these PLGAscaffolds havebeen optimizedfor
tissue engineering [4] and thus promote cell in1047297ltration and
proliferation However encapsulation of VEGF did not affect release
from scaffolds as much as for hydrogels Hence the increase in
angiogenesis elicited by encapsulated versus free VEGF loaded into
Matrigeltradehydrogels wasabout 5 to 7-foldwhile it wasonlyabout 35-fold for scaffolds Considering these results it seems that it could be
interesting to combine thetwo systems by1047297lling PLGA scaffoldspores
with hydrogel both loaded with VEGF nanoparticles
5 Conclusion
Wehypothesized thatVEGFactivityin vivo would be enhanced by its
encapsulation in CSndashDS nanoparticles and further incorporation into
implants used in tissue engineering like injectable hydrogels and
polymeric scaffolds Angiogenesis the primary effect of VEGF was
clearly improved by encapsulation the extent of implant colonization
by endothelial cells and blood vessels was higher when both types of
implants were loaded with VEGF nanoparticles compared with free
VEGF(non-encapsulated) Nanoparticles composed of chitosan dextran
sulfate and VEGF were obtained and incorporated in implants without
impairingtheirstructuralpropertiesand allowing a higher VEGFloading
compared to VEGF classical techniques of encapsulation (up to 5 μ g of
VEGF per mg of nanoparticles) VEGF encapsulation guarantees VEGF
ef 1047297ciency by protection and sustained release from 3D implants
In the scope of the spinal cord regeneration systems to deliver
active VEGF are attractive since in addition to its angiogenic activity
VEGF has recently been shown to be a neurogenic factor [33ndash35]
Then encapsulation and incorporation of VEGF into 3D implants thatin addition to support cell in1047297ltration and organization will stimulate
blood vessel and axon growth are a promising approach to stimulate
tissue regeneration
Supplementary materialsrelated to this article canbe found online
at doi101016jjconrel201011028
Acknowledgments
The authors would like to thank Prof Carmeliet (Vesalius Research
Center KU Leuven Belgium) for the gift of VEGF producing Pichia
pastoris yeast The authors thank Sabine Cordi for her help with MIRAX
acquisition as well as Christophe Pierreux for the RGB quanti1047297cation
script SeacutebastienSartis thankedfor assistance with VEGF productionand
Laurenne Petit for the realization of VEGF activity tests This work wassupported by grants from the Belgian Fonds National de la Recherche
Scienti1047297que (FNRS Creacutedits aux chercheurs and FRSM 3461709) and
CTB (Belgium) Anne des Rieux is FNRS Post-Doctoral Researcher
References
[1] V Guarino F Causa L Ambrosio Bioactive scaffolds for bone and ligament tissueExpert Rev Med Devices 3 (2007) 405ndash418
[2] TM Freyman IV Yannas R Yokoo LJ Gibson Fibroblast contraction of acollagen-GAG matrix Biomaterials 21 (2001) 2883ndash2891
[3] V Mourino AR Boccaccini Bone tissue engineering therapeutics controlled drugdelivery in three-dimensional scaffolds J R Soc Interface 43 (2010) 209 ndash227
[4] CB Rives A des Rieux M Zelivyanskaya SR Stock WL Lowe Jr LD SheaLayered PLGscaffoldsfor invivo plasmid delivery Biomaterials3 (2009)394ndash401
[5] AH Zisch MP Lutolf JA Hubbell Biopolymeric delivery matrices for angiogenic
growth factors Cardiovasc Pathol 6 (2003) 295ndash310[6] ZS Patel AG Mikos Angiogenesis with biomaterial-based drug- and cell-
delivery systems J Biomater Sci Polym Ed 6 (2004) 701ndash726[7] BV Somayaji U Jariwala P Jayachandran K Vidyalakshmi RV Dudhani
Evaluation of antimicrobial ef 1047297cacy and release pattern of tetracycline andmetronidazole using a local delivery system J Periodontol 4 (1998) 409ndash413
[8] MHuang SN VitharanaLJ Peek T CoopC Berkland Polyelectrolyte complexesstabilize and controllably release vascular endothelial growth factor Biomacro-molecules 5 (2007) 1607ndash1614
[9] JS GolubYTKimCL Duvall RV BellamkondaD Gupta AS Lin DWeiss WRobertTaylor RE Guldberg Sustained VEGF delivery via PLGA nanoparticles promotesvascular growth Am J Physiol Heart Circ Physiol 6 (2010) H1959ndashH1965
[10] C He SW Kim DS Lee In situ gelling stimuli-sensitive block copolymerhydrogels for drug delivery J Control Release 3 (2008) 189ndash207
[11] HK Kleinman ML McGarvey JR Hassell VL Star FB Cannon GW LaurieGR Martin Basement membrane complexes with biological activity Biochemistry 2(1986) 312ndash318
[12] N Akhtar EB Dickerson R Auerbach The spongeMatrigel angiogenesis assayAngiogenesis 1ndash2 (2002) 75ndash80
[13] D Donovan NJ Brown ET Bishop CE Lewis Comparison of three in vitrohuman lsquoangiogenesisrsquo assays with capillaries formed in vivo Angiogenesis 2(2001) 113ndash121
[14] JM Kanczler PJ Ginty JJ Barry NM Clarke SM Howdle KM ShakesheffROOreffo Theeffectof mesenchymalpopulations and vascularendothelial growthfactor delivered from biodegradable polymer scaffolds on bone formationBiomaterials 12 (2008) 1892ndash1900
[15] FG Rocha CA Sundback NJ Krebs JK Leach DJ Mooney SW Ashley JPVacanti EE Whang The effect of sustained delivery of vascular endothelialgrowth factor on angiogenesis in tissue-engineered intestine Biomaterials 19(2008) 2884ndash2890
[16] JM Kanczler J Barry P Ginty SM Howdle KM Shakesheff RO OreffoSupercritical carbon dioxide generated vascular endothelial growth factorencapsulated poly(DL -lactic acid) scaffolds induce angiogenesis in vitro BiochemBiophys Res Commun 1 (2007) 135ndash141
[17] JM Kanczler PJ Ginty L White NM Clarke SM Howdle KM Shakesheff ROOreffo The effect of the delivery of vascular endothelial growth factor and bonemorphogenic protein-2 to osteoprogenitor cell populations on bone formation
Biomaterials 6 (2010) 1242ndash
1250
277 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 77
[18] TT Chen A Luque S Lee SM Anderson T Segura ML Iruela-Arispe Anchorageof VEGF to the extracellular matrix conveys differential signaling responses toendothelial cells J Cell Biol 4 (2010) 595ndash609
[19] GE Means RE Feeney Reductive alkylation of amino groups in proteinsBiochemistry 6 (1968) 2192ndash2201
[20] JH Jang LD Shea Controllable delivery of non-viral DNA from porous scaffolds J Control Release 1 (2003) 157ndash168
[21] LD Shea E Smiley J Bonadio DJ Mooney DNA delivery from polymer matricesfor tissue engineering Nat Biotechnol 6 (1999) 551ndash554
[22] DJ Mooney DF Baldwin NP Suh JP Vacanti R Langer Novel approach tofabricate porous sponges of poly(DL -lactic-co-glycolic acid) without the use of
organic solvents Biomaterials 14 (1996) 1417ndash
1422[23] JH Jang CB Rives LD Shea Plasmid delivery in vivo from porous tissue-engineering scaffolds transgene expression and cellular transfection Mol Ther 3(2005) 475ndash483
[24] MO Aviles CHLin M Zelivyanskaya JGGraham RMBoehlerPBMessersmithLD Shea The contribution of plasmid design and release to in vivo gene expressionfollowing delivery from cationic polymer modi1047297ed scaffolds Biomaterials 6 (2010)1140ndash1147
[25] P Sonveaux A Brouet X Havaux V Gregoire C Dessy JL Balligand O FeronIrradiation-induced angiogenesis through the up-regulation of the nitric oxidepathway implications for tumor radiotherapy Cancer Res 5 (2003) 1012 ndash1019
[26] H Blomeier X Zhang C Rives M Brissova E Hughes M Baker AC Powers DBKaufman LD Shea WL Lowe Jr Polymer scaffolds as synthetic microenviron-ments for extrahepatic islet transplantation Transplantation 4 (2006) 452ndash459
[27] Y Yang L De Laporte CB Rives JH Jang WC Lin KR Shull LD SheaNeurotrophin releasing single and multiple lumen nerve conduits J ControlRelease 3 (2005) 433ndash446
[28] L De Laporte A des Rieux ML Zelivyanska HM Tuinstra N De Clerck AAPostnov LD Shea VEGF and FGF-2 delivery from spinal cord bridges to enhanceangiogenesis following injury J Biomed Mater Res Part A (in press)
[29] ML Ponce Tube formationan in vitro matrigel angiogenesis assayMethods MolBiol (2009) 183ndash188
[30] A Albini R Benelli The chemoinvasion assay a method to assess tumor andendothelial cell invasion and its modulation Nat Protoc 3 (2007) 504ndash511
[31] MH Sheridan LD Shea MC Peters DJ Mooney Bioabsorbable polymer
scaffolds for tissue engineering capable of sustained growth factor delivery J Control Release 1ndash3 (2000) 91ndash102[32] JH Jang TL Houchin LD Shea Gene delivery from polymer scaffolds for tissue
engineering Expert Rev Med Devices 1 (2004) 127ndash138[33] P Carmeliet M Tessier-Lavigne Common mechanisms of nerve and blood vessel
wiring Nature 7048 (2005) 193ndash200[34] E Storkebaum D Lambrechts P Carmeliet VEGF once regarded as a speci1047297c
angiogenic factor now implicated in neuroprotection Bioessays 9 (2004) 943ndash954[35] E StorkebaumD LambrechtsM Dewerchin MPMoreno-Murciano S Appelmans
H Oh P Van Damme B Rutten WY Man M De Mol S Wyns D Manka KVermeulen L Van Den Bosch N Mertens C Schmitz W Robberecht EM ConwayD Collen L Moons P Carmeliet Treatment of motoneuron degeneration byintracerebroventricular delivery of VEGF in a rat model of ALS Nat Neurosci 1(2005) 85ndash92
278 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11
httpslidepdfcomreaderfull3d-systems-delivering-vegf-to-promote-angiogenesis-for-tissue-engineering11 77
[18] TT Chen A Luque S Lee SM Anderson T Segura ML Iruela-Arispe Anchorageof VEGF to the extracellular matrix conveys differential signaling responses toendothelial cells J Cell Biol 4 (2010) 595ndash609
[19] GE Means RE Feeney Reductive alkylation of amino groups in proteinsBiochemistry 6 (1968) 2192ndash2201
[20] JH Jang LD Shea Controllable delivery of non-viral DNA from porous scaffolds J Control Release 1 (2003) 157ndash168
[21] LD Shea E Smiley J Bonadio DJ Mooney DNA delivery from polymer matricesfor tissue engineering Nat Biotechnol 6 (1999) 551ndash554
[22] DJ Mooney DF Baldwin NP Suh JP Vacanti R Langer Novel approach tofabricate porous sponges of poly(DL -lactic-co-glycolic acid) without the use of
organic solvents Biomaterials 14 (1996) 1417ndash
1422[23] JH Jang CB Rives LD Shea Plasmid delivery in vivo from porous tissue-engineering scaffolds transgene expression and cellular transfection Mol Ther 3(2005) 475ndash483
[24] MO Aviles CHLin M Zelivyanskaya JGGraham RMBoehlerPBMessersmithLD Shea The contribution of plasmid design and release to in vivo gene expressionfollowing delivery from cationic polymer modi1047297ed scaffolds Biomaterials 6 (2010)1140ndash1147
[25] P Sonveaux A Brouet X Havaux V Gregoire C Dessy JL Balligand O FeronIrradiation-induced angiogenesis through the up-regulation of the nitric oxidepathway implications for tumor radiotherapy Cancer Res 5 (2003) 1012 ndash1019
[26] H Blomeier X Zhang C Rives M Brissova E Hughes M Baker AC Powers DBKaufman LD Shea WL Lowe Jr Polymer scaffolds as synthetic microenviron-ments for extrahepatic islet transplantation Transplantation 4 (2006) 452ndash459
[27] Y Yang L De Laporte CB Rives JH Jang WC Lin KR Shull LD SheaNeurotrophin releasing single and multiple lumen nerve conduits J ControlRelease 3 (2005) 433ndash446
[28] L De Laporte A des Rieux ML Zelivyanska HM Tuinstra N De Clerck AAPostnov LD Shea VEGF and FGF-2 delivery from spinal cord bridges to enhanceangiogenesis following injury J Biomed Mater Res Part A (in press)
[29] ML Ponce Tube formationan in vitro matrigel angiogenesis assayMethods MolBiol (2009) 183ndash188
[30] A Albini R Benelli The chemoinvasion assay a method to assess tumor andendothelial cell invasion and its modulation Nat Protoc 3 (2007) 504ndash511
[31] MH Sheridan LD Shea MC Peters DJ Mooney Bioabsorbable polymer
scaffolds for tissue engineering capable of sustained growth factor delivery J Control Release 1ndash3 (2000) 91ndash102[32] JH Jang TL Houchin LD Shea Gene delivery from polymer scaffolds for tissue
engineering Expert Rev Med Devices 1 (2004) 127ndash138[33] P Carmeliet M Tessier-Lavigne Common mechanisms of nerve and blood vessel
wiring Nature 7048 (2005) 193ndash200[34] E Storkebaum D Lambrechts P Carmeliet VEGF once regarded as a speci1047297c
angiogenic factor now implicated in neuroprotection Bioessays 9 (2004) 943ndash954[35] E StorkebaumD LambrechtsM Dewerchin MPMoreno-Murciano S Appelmans
H Oh P Van Damme B Rutten WY Man M De Mol S Wyns D Manka KVermeulen L Van Den Bosch N Mertens C Schmitz W Robberecht EM ConwayD Collen L Moons P Carmeliet Treatment of motoneuron degeneration byintracerebroventricular delivery of VEGF in a rat model of ALS Nat Neurosci 1(2005) 85ndash92
278 A des Rieux et al Journal of Controlled Release 150 (2011) 272ndash 278
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