3d systems delivering vegf to promote angiogenesis for tissue engineering11

7232019 3D systems delivering VEGF to promote angiogenesis for tissue engineering11

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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



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



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)




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


NA Not Applicablea n=4b n=6

275 A des Rieux e t al Journal of Controlled Release 150 (2011) 272ndash 278



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


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)




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




[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




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















conditions [4]































































nuclei















analyzed surface

27 Statistics





3 Results

31 VEGF production





material 2)










after freeze-drying




(data not shown)




























































Pb

005)







Table 1



(nm)

PDI Zeta potential

(mV)

Encapsulation

ef 1047297ciency

()













on angiogenesis


























(Fig 4e and f)











in1047297



VEGF

4 Discussion


















SEM Pb

005)





















and implant type











































5 Conclusion



















tissue regeneration



Acknowledgments









References




















1250






































conditions [4]































































nuclei















analyzed surface

27 Statistics





3 Results

31 VEGF production





material 2)










after freeze-drying




(data not shown)




























































Pb

005)







Table 1



(nm)

PDI Zeta potential

(mV)

Encapsulation

ef 1047297ciency

()













on angiogenesis


























(Fig 4e and f)











in1047297



VEGF

4 Discussion


















SEM Pb

005)





















and implant type











































5 Conclusion



















tissue regeneration



Acknowledgments









References




















1250



















































































Pb

005)







Table 1



(nm)

PDI Zeta potential

(mV)

Encapsulation

ef 1047297ciency

()













on angiogenesis


























(Fig 4e and f)











in1047297



VEGF

4 Discussion


















SEM Pb

005)





















and implant type











































5 Conclusion



















tissue regeneration



Acknowledgments









References




















1250






























on angiogenesis


























(Fig 4e and f)











in1047297



VEGF

4 Discussion


















SEM Pb

005)





















and implant type











































5 Conclusion



















tissue regeneration



Acknowledgments









References




















1250





































and implant type











































5 Conclusion



















tissue regeneration



Acknowledgments









References




















1250

























3d systems delivering vegf to promote angiogenesis for tissue engineering11

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