1

Polyelectrolyte capsules as growth factor releasing centers in tissue engineering

scaffolds

De Cock L.J.1, De Wever O.

2, Van Vlierberghe S.

3, Vanderleyden E.

3, Dubruel P.

3, De Vos F.

4, Vervaet C.

1, Remon

J.P.1

and De Geest B.G.1,*

1 Laboratory of Pharmaceutical Technology, Department of Pharmaceutics, Ghent University, Harelbekestraat 72, 9000

Ghent, Belgium

2 Laboratory of Experimental Cancer Research, Department of Radiotherapy and Nuclear Medicine, Ghent University

Hospital, Belgium

3 Polymer Chemistry and Biomaterials Research Group, Ghent University, Krijgslaan 281, 9000 Ghent, Belgium

4 Laboratory of Radiopharmacy, Department of Pharmaceutical analysis, Ghent University, Harelbekestraat 72, 9000 Ghent,

Belgium

* Corresponding author: Dr. Bruno De Geest

Br.DeGeest@UGent.be

ABSTRACT

Spatial and temporal controlled delivery of growth factors which contribute to efficient repair upon

tissue injury or failure remains a major challenge in tissue engineering. Here we evaluate polyelectrolyte

multilayer microcapsules as growth factor carriers in order to allow spatial and temporal control over growth

factor release. Transforming growth factor-β1 (TGF-β1) was used as model growth factor and bio-specific

engineering of the microcapsules allowed to bind relative high amount of TGF-β1. Subsequently we investigated

the interactions between TGF-β1 and respectively microcapsules and gelatin based tissue engineering scaffolds.

By monitoring TGF-β1 induced transdifferentiation of fibroblasts into myofibroblasts we were able to

demonstrate that TGF-β1 encapsulation into polyelectrolyte multilayer microcapsules and subsequent

incorporation into gelatin based hydrogels did not alter the bioactivity of the growth factor.

2

1. INTRODUCTION

The field of regenerative medicine faces the challenge to treat damaged or diseased tissue in an efficient

and minimally invasive way. Tissue regeneration is a complex biological process requiring concerted action of

cell proliferation, differentiation and migration in addition to the generation of extracellular matrix components.

This process can be efficiently induced by appropriately combining cells, growth factors and scaffolds. The

scaffold which supports cell adhesion should degrade as soon as sufficient tissue regeneration has occurred and

this regeneration is enhanced by stimulating cells with growth factors.1 Growth factors are polypeptides which

transmit signals to control cellular activities such as proliferation, differentiation, migration, adhesion and

protein expression.2 Under normal circumstances, growth factors are produced by endogenous cells however, in

case of tissue repair, they must be provided exogenously.1 Optimal tissue regeneration requires the endogenous

profile of growth factors secreted during natural tissue regeneration to be closely mimicked.3

Delivery of growth factors to the site of tissue regeneration is challenging since these proteins have

short half-lives, high molecular weight and slow tissue penetration. Moreover, bolus injection should often be

avoided due to potential toxicity or undesirable side effects in the tissue surrounding the injection site. Growth

factors can be incorporated into polymeric scaffolds to stabilize the growth factor and to control their release

kinetics.4-6

Another promising strategy to enhance in vivo efficacy of growth factors is the use of growth factor

loaded drug delivery systems which release their payload in a controlled fashion. In this perspective, several

studies have focused on the encapsulation of growth factors into microsphere carriers based on PLGA7,8

,

gelatin9-11

, alginate12

, micelles13

, silk7, calcium carbonate

14 or polyelectrolytes

15,16. Incorporation of these

microcarriers into synthetic matrices 17-20

might be performed during scaffold preparation by mixing the

microcarriers with a building component of the synthetic scaffold 21,22

or after scaffold preparation through e.g.

microcapsule seeding 23

or injection 24

.

Fig. 1. Schematic overview of the design of growth factor binding polyelectrolyte multilayer microcapsules. (A) Synthesis of

heparin (orange) loaded CaCO3 microparticles by co-precipitating CaCl2 and Na2CO3 in the presence of heparin. (B) Layer-

by-layer coating of CaCO3 microparticles with heparin (polyanion; green) and poly-L-arginine (polycation; brown). (C) Hollow

microcapsules are obtained after dissolving the CaCO3 core templates with EDTA. (D) Growth factors (purple) are

postloaded into the capsules in slightly acidic buffer at low ionic strength.

3

In this paper we evaluate polyelectrolyte multilayer microcapsules25

as growth factor releasing centers

in tissue engineering scaffolds. These capsules are fabricated by layer-by-layer (LbL) coating of a sacrificial

template with alternately charged polyelectrolytes using electrostatic interaction as driving force, followed by

the decomposition of this template yielding hollow capsules.26-28

These capsules can be either preloaded or

postloaded with bio-active molecules.29

A preloading approach involves the therapeutic molecules of interest to

be embedded within the sacrificial template and subsequently to remain encapsulated within the hollow

capsules formed by LbL coating and decomposition of the core templates. This is an interesting strategy for the

encapsulation of rather high doses of larger proteins such as vaccine antigens.30,31

However, low dosed

expensive growth factors with mostly a molecular weight below 40 kDa, will be prone to premature release

upon dissolution of the core templates.29

Therefore, in this paper we introduce a postloading approach by

e gi eeri g the apsules i su h a way that they a t as growth fa tor i di g i ro-spo ges . This approa h is

schematically shown in Fig. 1 and comprises in a first step the synthesis of porous calcium carbonate (CaCO3)

microparticles filled with heparin in their pores by co-precipitating CaCl2 and Na2CO3 in the presence of heparin.

Subsequently, these microparticles are coated with 2 bilayers of again heparin as polyanion and poly-L-arginine

as polycation, followed by decomposition of the CaCO3 core in an aqueous EDTA solution. In this way, hollow

capsules are obtained with heparin both as membrane component as well as being suspended in their hollow

void. Heparin is a highly sulfated (thus bearing a net negative charge at physiological conditions)

glycosaminoglycan and has a high affinity for several growth factors.32-34

Therefore we hypothesize that

engineering the microcapsules with a high content of heparin will enhance their growth factor binding capacity.

Postloading with growth factor is then performed by incubating the microcapsules with growth factor in slightly

acidic buffer at low ionic strength to maximize the electrostatic interaction between growth factor (being more

protonated and thus bearing a higher net positive charge at lower pH) and heparin-engineered microcapsules.

Release of growth factor from the microcapsules is then expected to occur upon incubation in physiological

conditions through ionic competition and concentration gradient.

As model growth factor in this paper we encapsulate transforming growth factor-β1 (TGF-β1), a growth

factor known to induce transdifferentiation of fibroblasts into myofibroblasts, into heparin engineered

microcapsules. We demonstrate that TGF-β1 can be loaded and released without affecting its biological activity

and further demonstrate that these capsules can be incorporated within a gelatin tissue engineering cryogel

scaffold without affecting the properties of this scaffold.

4

2. MATERIALS AND METHODS

2.1. Materials

Calcium chloride (CaCl2), sodium carbonate (Na2CO3), ethylenediaminetetraacetic acid (EDTA),

fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), Hoechst 33258, potassium persulfate (KPS),

N,N,N ,N -tetramethylethylenediamine (TEMED), p-nitrophenyl phosphate (pNPP), poly-L-arginine (Mw > 70

kDa), anti-α-SMA (clone 1A4), anti-tubulin (clone B-5-1-2) and anti-N-cadherin were purchased from Sigma-

Aldrich. Anti-tenascin-C (anti-TNC; clone BC-8) was provided by L. Zardi. Anti-fibronectin was obtained from

Abcam. Heparin was purchased from Diosynth Biotechnology. Pronase was purchased from Roche. Human

dermal fibroblasts and fibroblast growth medium were obtained from Promocell. LysoTracker Red, CellTracker

Red and the FITC-conjugated secondary antibody were purchased from Invitrogen. TGF- 1 was purchased from

R&D systems. The DC protein assay was obtained from Biorad. Hybond membranes were purchased from

Amersham. Methacrylated gelatin (degree of substitution of 60 %) was synthesized according to Van Den Bulcke

et al.35

Glass bottom dishes were purchased from Mattek. Irgacure 2959 was obtained from Ciba. Tissue Tek was

purchased from Sakura. FlexiPERM rings were purchased from Vivascience.

2.2. Fabrication and degradation of polyelectrolyte capsules

Calcium carbonate (CaCO3) microparticles doped with heparin were fabricated by adding 625 µl CaCl2

and 625 µl Na2CO3 (both 1M in deionized water) to 5 ml of an aqueous (deionized water) 0.2 mg/ml heparin

solution (1 mg/ml; 0.5 M NaCl). The heparin doped CaCO3 microparticles were centrifuged and subsequently

incubated for 5 min. in a heparin solution. After two centrifugation and washing steps with deionized water to

remove non-adsorbed polyelectrolytes, the microparticles were incubated for 5 min. with poly-L-arginine (1

mg/ml; 0.5 M NaCl). This cycle was repeated twice to deposit 2 heparin/poly-L-arginine bilayers followed by

dissolving the CaCO3 core templates in an aqueous EDTA (0.2 M) solution, yielding hollow microcapsules.

Fluorescent labeling of the microcapsules was performed using FITC or RITC labeled poly-L-arginine.36

Microcapsule degradability was assessed by suspending the microcapsules in a pronase solution (0.5

mg/ml in Tris buffer containing 0.5% sodium lauryl sulfate) followed by monitoring the turbidity of the

suspension using an automated turbidity reader (Bioscreen-C). Microcapsules suspended in water were used as

control. All experiments were run in triplicate.

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2.3. Microscopy

Fluorescent labeled samples were visualized on a Nikon EZ-C1 confocal microscope. Scanning electron

microscopy images of dry samples being sputtered with gold were recorded with a Quanta 200 FEG FEI scanning

electron microscope operating at an acceleration voltage of 5 kV.

2.4. Interaction between capsules and fibroblasts

Normal human dermal fibroblasts were cultured in fibroblast growth medium containing bFGF (1 ng/ml),

insulin (5 µg/ml) and antibiotics (penicillin and streptomycin). Cells were grown in a humidified atmosphere

containing 5 % CO2.

Fibroblasts were exposed to green fluorescently labeled microcapsules for 12 hours and subsequently

visualized by confocal microscopy. Lysosomal vesicels were stained red fluorescent using LysoTracker Red

(1/5000 Invitrogen). Cell nuclei were stained blue fluorescent with Hoechst.

Cytotoxicitiy of the microcapsules was assessed according to De Koker et al.37

Human dermal fibroblasts

were seeded in a 96-well plate at a density of 2500 cells per well and incubated for 6 hours with several

amounts of microcapsules (with and without dissolution of the CaCO3 core templates). After an additional

cultivation period of 48 hours, cell viability was evaluated using a p-nitrophenyl phosphate (pNPP) cell viability

assay as previously described.36

All experiments were run in triplicate.

2.5. Encapsulation and release of TGF-β1

Hollow microcapsules were postloaded with TGF-β1 by 1 h incubation in 1 µg/ml TGF-β1 acetate buffer

(pH 5; 50 mM; 0.1 % Tween 20). After the incubation period, the growth factor loaded microcapsules were

washed twice with acetate buffer and subsequently suspended in phosphate buffered saline (PBS; pH 7.4; 150

mm NaCl; 0.1 % Tween 20) for further experiments.

To quantify encapsulation and release, TGF-β1 was radioactive labeled with 131

I by incubating 10 μg TGF-

β1 with 131

I in a vial coated with 70 μg iodogen in PBS containing 0.1 % Tween 20 during 20 min. at room

temperature. Subsequently, residual 131

I was separated from 131

I-TGF-β1 on a PD-10 column by size exclusion

chromatography using acetate buffer (pH 5; 50 mM; 0.1 % Tween 20) as eluent. The concentration of the

radioactive growth factor was calculated via the percentage of nonspecific adsorption of TGF-β1 on the PD-10

column and the original amount of growth factor, and the concentrated 131

I-TGF-β1 solution was further diluted

in acetate buffer (pH 5; 50 mM; 0.1 % Tween 20). 131

I-TGF-β1 was postloaded into the microcpasules as

6

described above and subsequently suspended in PBS. Encapsulation and release at several time points was

quantified by measuring the radioactivity in supernatant of centrifuged samples by a Packard cobra automated

γ-counter. All experiments were run in triplicate.

2.6. Biological activity of released TGF-β1

2.6.1. Immunocytochemistry

Fibroblasts were treated with cell medium, cell medium containing TGF-β1 (10 ng/ml), empty

microcapsules and TGF-β1 loaded microcapsules during 7 days. After treatment, the cells were fixed and

permeabilized using ice-cold methanol. Nonspecific binding was blocked by incubating the cells with Tris-

buffered saline (TBS) containing 5 % bovine serum albumin (BSA) for 30 minutes at room temperature. Alpha-

smooth muscle actin (α-SMA) was stained with a primary antibody (1/100; clone 1A4) followed by a FITC-

conjugated secondary antibody (1/1000). The cell nuclei were counterstained with Hoechst (1/5000).

2.6.2. Western blotting

Fibroblasts were treated with cell medium, cell medium containing TGF-β1 (10 ng/ml), empty capsules

and TGF-β1 loaded microcapsules during 7 days. Cells were lysed using Laemmli buffer and protein

concentrations were determined using a Lowry-based assay (DC protein assay). Samples were heated at 100°C

followed by loading on a 8 % SDS polyacrylamide gel. After transferring the separated proteins to a Hybond

membrane, nonspecific binding was blocked using PBS containing 5 % (w/v) nonfat milk powder and 0.5 % (w/v)

Tween 20 during 1 hour at room temperature. The separated proteins were then incubated with primary

antibodies against α-SMA (1/1000; clone 1A4), tubulin (1/4000; clone B-5-1-2), N-cadherin (1/1000), TNC

(1/1000; clone BC-8) and fibronectin (1/1000) during 1 hour at room temperature. After extensive washing in

PBS containing 5 % (w/v) nonfat milk powder and 0.5 % (w/v) Tween 20, membranes were incubated with HRP-

conjugated secondary antibodies. The intensity of the bands on the membrane was analyzed using Image J

software.

2.6.3. Collagen contraction assay

Fibroblast treated with cell medium, cell medium containing TGF-β1 (10 ng/ml), empty capsules and

TGF-β1 loaded microcapsules during 7 days were incorporated in a collagen gel. This was performed by

suspending the pretreated cells in a mixture of collagen type I, CMF-HBSS, MEM, NaHCO3 (0.25 M) and NaOH. A

final concentration of 250 000 cells/ml was obtained. 200 µl of the suspension was pipetted in a 24-well plate

7

followed by a 1 hour incubation period in a humidified atmosphere containing 5 % CO2 to allow gelification. The

fabricated gels were released and contraction of the gel was evaluated by measuring the diameter of the gel

after 24 hours. The procentual contraction was calculated according to the formula: [(diameter t0 – mean

diameter t24)/diameter t0)] were t0 represents the time point of collagen gel release and t24 represents the time

point 24 hours after collagen release.

2.6.4. Biological activity of TGF-β1 released from polyelectrolyte microcapsules incorporated within a gelatin

hydrogel film

TGF-β1 loaded microcapsules were suspended in 500 µl of a 10 % (w/v) methacrylated gelatin solution.

After adding 18 µl KPS (50 mg/ml) and 10 µl TEMED (neutralized with 4N HCl) to initiate radical polymerization

of the methacrylate moieties, the suspension was pipetted in a glass bottom dish. After 2 h reaction the

obtained hydrogel film was extensively rinsed with PBS to remove KPS and TEMED. Subsequently, fibroblasts

were seeded on the hydrogel film and after 7 days culturing, immunocytochemistry was performed to visualize

-SMA expression. Therefore, the cells were fixed and permeabilized using PBS containing 0.1 % Triton x-100.

Nonspecific binding was blocked by incubating the cells with Tris-buffered saline (TBS) containing 5 % bovine

BSA for 30 min. at roo te perature. α-SMA was stained with a primary antibody (1/100; clone 1A4) followed

by counterstaining with a FITC-conjugated secondary antibody (1/1000). Cell nuclei were stained with Hoechst

(1/5000).

2.7. Surface plasmon resonance (SPR)

Interaction between TGF-β1 and gelatin was evaluated using surface plasmon resonance (SPR; Biacore-

X). Gold sensor chips were spin coated with a 5 % (w/v) gelatin solution containing Irgacure 2959 (2 mol %) as

photoinitiator and exposed to UV light (276 nm; 10 mW/cm2; 5 min.) to crosslink the methacrylate moieties.

38

After inserting the chip in the SPR instrument, 50 µl TGF- 1 solution (Tris buffer; pH 7.4; 0.1 % Tween 20) was

injected (5 µl/min flow rate) and the surface plasmon resonance signal was monitored. Three concentrations

(i.e. 0.5, 1 and 2 µg/ml TGF-β1) were evaluated.

2.8. Incorporation of polyelectrolyte capsules in gelatin cryogels

1 g methacrylated gelatin was dissolved in 10 ml deionized water at 40 °C followed by the addition of

microcapsules and Irgacure 2959 (2 mol %) as photoinitiator. The mixture was injected into the mold of a cryo-

unit and was allowed to gel for 1 h at room temperature. Subsequently, crosslinking of the methacrylate

moieties was performed by exposing the hydrogel to UV light (276 nm; 10 mW/cm2) for 2 hours and pores were

8

formed using a freezing step (cryo-unit). Dry porous gelatin scaffolds were obtained through lyophilization of the

frozen hydrogels. Cryosections of Tissue Tek embedded cryogels were cut at 10 µm thickness.

The cryogels were mechanically tested using a TA 500 Texture Analyser equipped with a 10 N load cell.

An indentation of 2 mm was performed on a hydrated cryogel sample having a size of 1 mm x 1 mm x 0.7 mm.

Stiffness was calculated as slope of the load extension curve. For each data point, three samples were

measured.

2.9. Population of the synthetic scaffold with fibroblasts

Cell seeding was performed according to the method described by Dubruel et al.39

Briefly, hydrated

cryogels having a size of 15 mm x 4 mm x 3 mm were seeded with 160 000 cells using the drop-seeding method.

Seeded hydrogels were fixed under flexiPERM rings in a 6-well plate to prevent them from floating in the cell

medium. After 24 hours incubation, the flexiPERM rings were removed and the hydrogels were flipped. Cells

were stained red fluorescent using CellTracker Red (1/5000) while the gelatin was stained green fluorescent

through electrostatic adsorption of FITC-labeled poly-L-arginine.

9

3. RESULTS AND DISCUSSION

3.1. Synthesis and characterization of (hep/pARG)2 capsules

As stated in the introduction and schematically represented in Fig. 1, we aim to engineer polyelectrolyte

microcapsules in such a way that they exhibit an enhanced affinity to bind growth factors. This is done by

initially co-precipitating heparin into calcium carbonate microparticles followed by the deposition of 2

polyelectrolyte bilayers composed of heparin and poly-L-arginine (further on denoted as (hep/pARG)2) and

subsequent dissolution of the calcium carbonate core templates in an aqueous EDTA solution. Fig. 2 shows

scanning electron microscopy (SEM) images of the calcium carbonate microparticles (A) before and (B) after LbL

coating. The hollow capsules obtained after EDTA treatment are visualized by SEM in Fig. 2C and confocal

microscopy in Fig. 2D. Note that the collapsed structure in Fig. 2C is due to the drying of the sample and a proof

of their hollow structure. The capsules are fairly monodisperse and have mean diameter of 3.7 µm as measured

by laser diffraction.

Fig. 2 Scanning electron microscopy images of (A) CaCO3 microparticles and (B) polyelectrolyte microparticles composed

of 2 bilayers heparin and poly-L-arginine before and (C) after dissolution of the CaCO3 cores. (D) Confocal microscopy image

of hollow (hep/pARG)2 microcapsules which are red fluorescent due to the use of RITC-labeled heparin in the LbL film.

To be incorporated into tissue engineering scaffolds that should degrade over time during tissue

regeneration, capsule degradability and biocompatibility is undoubtly an important issue. Capsule degradability

was investigated by incubating (hep/pARG)2 microcapsules in a pronase, a non-specific protease mixture,

solution while monitoring the turbidity of the suspension. Fig. 3A shows a decrease in turbidity during the first 6

hours of incubation followed by a constant turbidity which might be caused by reduction of the enzyme activity.

As tissue engineering scaffolds should degrade over weeks to months, with a continuous renewal of extracellular

proteases to which the capsule will be exposed, it is reasonable to conclude from the short term (i.e. 15 h) data

shown in Fig. 3A that progressive capsule degradation is likely to occur.

10

Fig. 3 (A) Turbidity measurements of hollow (hep/pARG)2 microcapsules suspended in water or a pronase solution. (B)

Confocal microscopy image of dermal fibroblasts incubated with green fluorescent hollow (hep/pARG)2 microcapsules.

Lysosomes and cell nuclei were stained respectively red (LysoTracker Red) and blue (Hoechst) fluorescent. Internalization of

microcapsules by cells is observed as a yellow signal due to co-localization of green (capsules) and red (lysosomes)

fluorescence (arrow). (C) Cell viability of dermal fibroblasts treated with different amounts of (hep/pARG)2 microcapsules

before and after dissolution of CaCO3 cores.

In a next series of experiments we assessed the interaction between (hep/pARG)2 microcapsules and

living cells in vitro. For this purpose, human dermal fibroblasts were selected as a model cell type. First we

incubated these fibroblasts during 12 h with (hep/pARG)2 microcapsules that were labeled green fluorescent

using FITC-labeled poly-L-arginine and investigated by confocal microscopy whether these capsules had an

11

influence on cell morphology and whether they were phagocyted. We did not observe an effect of the capsules

on cell spreading and morphology, indicating that these capsules are well tolerated by living cells. Furthermore,

by counterstaining the cellular lysosomes red fluorescent with LysoTracker Red 40-42

we only sparsely observed

co-localization of green and red fluorescence (yielding a yellow/orange signal), indicating that capsule

internalization by primary fibroblasts is unlike to occur (Fig. 3B). The observation that these capsules were well

tolerated was further confirmed by quantifying cell viability according to the method described by De Koker et

al.37

Fibroblasts were incubated for 6 hours with (hep/pARG)2 microcapsules (with and without the CaCO3 cores

being dissolved) followed by an additional cultivation period of 48 hours in cell medium followed by measuring

cell viability. As shown in Fig. 3C, hollow microcapsules did not affect cell viability up to an amount of 1000

microcapsules per cell. This result is in agreement with earlier published data showing the relatively inert

character of hollow polyelectrolyte microcapsules on cell viability.37

In contrast, incubation of fibroblasts with

microcapsules that still contained their CaCO3 core, did have a negative influence on cell viability starting from

250 microcapsules per cell or more. The higher mass of these CaCO3 containing microcapsules, thus exerting a

higher mechanical pressure onto the cells is assumed to be responsible for this.

3.2. Encapsulation, realease and biological activity of TGF-β1

Postloading polyelectrolyte multilayer microcapsules with proteins results mostly in protein

accumulation within the capsule membrane.29

Therefore we engineered our capsules in such a way that they are

strongly anionic by virtue of heparin both in their shell as well as in their hollow void. TGF-β1 abundantly

possesses basic residues (the isoelectric point of TGF-β1 is 9.5) which bind through electrostatic interaction with

heparin.32

Furthermore, we loaded the capsules in acetate buffer at pH 5 to enhance the electrostatic

i tera tio etwee the hepari s a io i sulfate groups a d the a i o groups of TGF-β1 which should be

extensively protonated at pH 5. We also kept the ionic strength of the loading buffer low, i.e. 50 mM, to

minimize charge shielding by the buffer ions. Radioactive labeling of TGF-β1 and subsequent determination of

radioactivity within the capsules after loading and washing allowed us to measure an amount of 59 ± 3 ng TGF-

β1 per 106 capsules. Related to the amount of TGF-β1 in the postloading solution, this is an encapsulation

efficiency of 35 ± 2 %. When these TGF-β1 loaded capsules are incubated in phosphate buffered saline at pH 7.4,

thus mimicking physiological conditions, an initial burst release of TGF-β1 is observed followed by a sustained

release over several days (Fig. 4).

12

0 20 40 60 80 100 120 140 160 1800

10

20

30

TG

F-

1 r

ele

ase

[%

]

Time [hours]

Fig. 4 Cummulative release profile of radioactive labeled TGF-β1 from (hep/pARG)2 microcapsules.

TGF-β1 is a homodimer of two 12.5 kDa peptides joined by a sulphydryl bond forming a 25 kDa molecule

and is involved in wound healing by affecting cellular processes as growth inhibition, differentiation,

angiogenesis and deposition of extracellular matrix components.43-45

After injury, blood platelets release a TGF-

β1 bolus to recruit inflammatory cells to the site of injury. Moreover, TGF-β1 stimulates transdifferentiation of

fibroblasts into myofibroblasts and stimulates (myo)fibroblasts to produce collagen and fibronectin in addition

to contract the newly formed connective tissue to bring together the edges of the wound in order to reduce the

wound size.46-51

Transdifferentiation of fibroblasts into myofibroblasts is expressed by an upregulation of several

proteins including tenascin (TNC), fibronectin, N-cadherin (N-cadh) and alpha-s ooth us le a ti (α-SMA). To

evaluate whether TGF-β1 retained its biological activity after being loaded and released from the microcapsules,

we exposed fibroblasts for 7 days to TGF-β1 loaded microcapsules. Subsequently, cell lysates were analyzed by

western blotting to determine several differentiation and functional markers of myofibroblasts such as tenascin,

fibronectin, N-cadherin and α-SMA. Tubulin was used as control to verify that equal amounts of proteins were

loaded in each lane as tubulin should exhibit the same relative intensity for each sample. The western blots in

Fig. 5A show increased expression of tenascin, fibronectin, N-cadherin and α-SMA after fibroblast treatment

with TGF-β1 both in solution (10 ng/ml) as well as released from the microcapsules, compared to fibroblasts

treated with empty capsules or cell medium without growth factor. Immunocytochemistry revealed that α-SMA

was organized in stress fibers (Fig. 5B1-5B2) after fibroblast treatment with both native TGF-β1 (10 ng/ml) as

well as TGF-β1 that was released from the microcapsules. Since contractile activity of (myo)fibroblasts is

enhanced when de novo expressed alpha-s ooth us le a ti (α-SMA) is incorporated in stress fibers,

fu tio ality of α-SMA incorporated in stress fibers was evaluated by incorporating TGF-β1 treated fibroblasts in

13

a 3D collagen gel. Contraction of the collagen matrix was evaluated 24 hours after fabrication of the collagen gel

(Fig. 5C-5D1-5D2). Fibroblasts exposed to TGF-β1 in solution (10 ng/ml) or to TGF-β1 released from microcapsules

showed enhanced contraction of the collagen gel compared to fibroblasts exposed to cell medium without

growth factor or blanco microcapsules. Taken together, these results clearly demonstrate that TGF-β1 loaded

and released from (hep/pARG)2 polyelectrolyte microcapsules is still bioactive and can stimulate fibroblast

transdifferentiation into myofibroblasts as potent as a native TGF-β1 solution.

Fig. 5 Fibroblast transdifferentiation into myofibroblasts by exposure to cell medium without growth factor, TGF-β1

solution (10 ng/ml), blanco microcapsules and TGF-β1 loaded microcapsules for 7 days. (A) Western blot analysis of

cellysates. Co fo al i ros opy i ages of α-SMA and nuclei staining of fibroblasts exposed to (B1) TGF-β1 loaded

14

microcapsules and (B2) empty microcapsules as control. α-SMA was stai ed gree fluores e t with α-SMA antibody and

FITC-labeled secondary antibody while the nuclei were stained blue fluorescent with Hoechst. (C) Contraction of collagen

gels incorporating fibroblasts exposed to cell medium without growth factor, TGF-β1 solution (10 ng/ml), empty

microcapsules and TGF-β1 loaded microcapsules for 7 days. Digital images of collagen gels incorporating fibroblasts exposed

during 7 days to (D1) TGF-β1 loaded microcapsules and (D2) blanco microcapsules after a 24 h incubation period.

3.3. Incorporation and interaction of TGF-β1 in tissue engineering scaffolds

The final aim of this paper is to incorporate growth factor loaded microcapsules into tissue engineering

scaffolds and to evaluate their interaction with these scaffolds. Dubruel et al. previously demonstrated the

potential of gelatin based cryogels as tissue engineering scaffolds. Gelatin is a natural polymer derived from

collagen and allows excellent cellular adhesion and growth. To obtain gels which are stable at physiological

temperature, gelatin was grafted with methacrylate moieties which can be crosslinked by radical

polymerization.35

Cryogel scaffolds are 3D structures with interconnected macropores (size of 100 µm and more)

which are formed by a cryogenic treatment (i.e. controlled cooling cyclus) of an aqueous gelatin solution which

induces phase separation of the solution into ice crystals, which will form the pores after freeze-drying, and a

gelatin rich phase which is then further stabilized by crosslinking the methacrylate moieties.39,52

Here we used

these gelatin cryogels as a model scaffold to incorporate TGF-β1 loaded hollow polyelectrolyte microcapsules. In

order to assess whether TGF-β1 released from the microcapsules would bind to gelatin and whether this would

preserve its bioactivity we first investigated the interaction between TGF-β1 and planar gelatin films. Surface

plasmon resonance (SPR) was used to specifically investigate the adsorption behavior of TGF-β1 onto gelatin.

Therefore, gold sensor chips were spin coated with methacrylated gelatin followed by UV irradiation, in the

presence of Irgacure 2959 as photoinitiator, to form a stable crosslinked hydrogel film. Subsequently, the chips

were placed in the flow cell of the SPR apparatus and TGF-β1 solutions were injected. At the start of the injection

phase (t = 500 s) TGF-β1 bound to the gelatin film which resulted in an increase of the response units and, as

shown in Fig. 6A, this binding was concentration dependent. The driving force for adsorption is electrostatic

interaction since TGF-β1 (IEP = 9.5) and gelatin (IEP = 5) are oppositely charged at physiological conditions. At the

end of the injection phase (t = 1100 s) loosely bound TGF-β1 dissociates from the gelatin film resulting in a

limited decrease of the response units. At t = 1700 s an extra washing step was performed however this did not

evoke a further decrease of the response units (data not shown) demonstrating that TGF-β1 was stably adsorbed

onto the gelatin.

To investigate whether TGF-β1 retains its biological activity upon encapsulation in (hep/pARG)2

microcapsules and subsequent incorporation within gelatin hydrogels we incorporated TGF-β1 loaded

microcapsules in a gelatin hydrogel film by mixing the TGF-β1 loaded microcapsules with a gelatin solution. After

15

adding the agents to crosslink the gelati s pe di g etha rylates, the mixture was quickly transferred into a

glass bottom dish to allow the formation of a stable hydrogel film. Next, fibroblasts were cultured for 7 days

onto this gelatin film followed by immunocytochemical staining of -SMA expression. As shown in Fig. 6B,

fibroblasts cultured on a gelatin film containing TGF-β1 loaded microcapsules did express -SMA which was

organized in stress fibers. By contrast, -SMA expression was absent in fibroblasts cultured on a gelatin film

containing empty microcapsules. This demonstrates that upon encapsulation in polyelectrolyte multilayer

capsules and subsequent incorporation into gelatin hydrogels, TGF-β1 is released from the microcapsules and

can reach cells cultured on the surface of the gelatin hydrogels while retaining its biological activity.

Fig. 6 (A) Surface plasmon resonance sensorgram showing the interaction between TGF-β1 and gelatin. (B) -SMA

expression by fibroblasts cultured on a gelatin film containing TGF-β1 loaded microcapsules. α-SMA was stained green

fluorescent with a primary antibody against α-SMA and FITC-labeled secondary antibody while the nuclei were stained blue

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fluorescent with Hoechst. (C) Scanning electron microscopy image of a gelatin cryogel. (D) Confocal microscopy image of a

microtomed section of a cryogel (labeled green fluorescent through adsorption of FITC-labeled pARG) loaded with red

fluorescence (due to RITC-labeled poly-L-arginine) microcapsules. (E) Overview and (F) zoomed confocal microscopy image

of a cryogel (green fluorescent) populated with fibroblasts (red fluorescent). The green fluorescence is due to adsorption of

FITC-labeled pARG to the gelatin. The red fluorescence is due to staining of the fibroblasts with CellTracker Red.

In a next series of experiments we incorporated the microcapsules in a 3D gelatin cryogel by mixing the

microcapsules with methacrylated gelatin in solution, followed by a freezing and thawing cycle to induce

porosity through phase separation of ice crystals (i.e. the pores) and a gelatin rich phase. UV irradiation of the

matrix resulted in crosslinking of the methacrylate moieties to covalently crosslink the cryogel. Fig. 6C shows a

scanning electron microscopy image of a cryogel, clearly demonstrating its 3D structure. Confocal microscopy

image (Fig. 6D) of a microtomed section shows that the microcapsules (labeled red fluorescent) are

homogeneously distributed throughout the cryogel (green fluorescent). To assess whether the incorporation of

microcapsules had an impact on the mechanical properties of the cryogels the stiffness of the scaffolds without

and with different amount of capsules (2.8*107, 7*10

7 and 14*10

7) was measured. These measurements

indicated that, independent on the amount of incorporated capsules, the stiffness remained around 0.3 N/mm.

This demonstrates that one is able to incorporate high amounts of microcapsules within the scaffold without

affecting its mechanical properties. This is beneficial for the design of scaffolds with specific mechanical

properties which are independent on the presence of growth factor releasing centers.

Finally we aimed to assess whether gelatin cryogels containing microcapsules could still be populated

with living cells in vitro. Therefore, dermal fibroblasts were drop-seeded on cryogels. This drop-seeding

procedure is a static cell seeding method involving the deposition of a drop of cells suspended in culture

medium on top of the cryogels as described by Dubruel et al. After 7 days culturing, confocal microscopy was

used to visualize the distribution of the cells throughout the gelatin cryogels. For this purpose the cells were

stained red fluorescent with CellTracker Red and the cryogels were stained green fluorescent through

electrostatic adsorption of FITC-labeled poly-L-arginine. As shown in Fig. 6E and 6F complete and dense cellular

population within the cryogels was observed.

4. CONCLUSIONS

In this paper, we evaluated the potential of hollow (hep/pARG)2 microcapsules as growth factor

releasing centers for tissue engineering applications. First we optimized the microcapsule design and the loading

conditions to optimally bind TGF- 1 which was used as model growth factor in this work. Incubation of these

capsules in physiological buffer induced an initial burst release followed by a sustained release of TGF- 1,

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without affecting its biological activity. Subsequently we demonstrated the incorporation of these growth factor

loaded microcapsules within a gelatin cryogel tissue engineering scaffold which did not alter the cryogel

morphology, the mechanical properties and the bioactivity of released TGF- 1.

5. ACKNOWLEDGEMENTS

LJDC wishes to express her gratitude to the Institute for the Promotion of Innovation by Science and

Technology in Flanders (IWT-Flanders) for their financial support. BGDG acknowledges the FWO-Flanders for a

postdoctoral scholarship.

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