1-s2.0-s1959031810001144-main

8/13/2019 1-s2.0-S1959031810001144-main

http://slidepdf.com/reader/full/1-s20-s1959031810001144-main 1/7

IRBM 32 (2011) 214–220

Original article

Adhesion and proliferation of human bladder RT112 cellson functionalized polyesters

Adhésion et prolifération de cellules humaines du type RT112 sur des polyesters fonctionnalisés

E. Renard , G. Vergnol , V. Langlois∗

UMR 7182, institut de chimie et des matériaux de Paris Est (ICMPE), université Paris Est, 2 à 8, rue Henri-Dunant, 94320 Thiais, France

Received 22 June 2010; received in revised form 2 December 2010; accepted 5 December 2010

Available online 12 January 2011

Abstract

Attachment and growth of human bladder carcinoma RT112 cells were investigated in vitro on biopolyesters films in a view to use

them as scaffold in tissue engineering. Effect of the chemical structure of different bacterial polyesters on cell adhesion and proliferation

was studied. Four poly(3-hydroxyalkanoate)s (PHAs): poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), poly(3-hydroxyoctanoate)

(PHO), poly(3-hydroxyoctanoate-co-3-hydroxy-9-carboxydecanoate) (PHOD-COOH) and a graft copolymer poly(3-hydroxyoctanoate-co-3-

hydroxy-9-carboxydecanoate-g-polyethyleneglycol) (PHOD-g-PEG) have been used. PHB-HV and PHO are natural hydrophobic PHAs

obtained by bioconversion. PHOD-COOH and PHOD-g-PEG were prepared by chemical modification from unsaturated PHAs to modify the

hydrophilic–hydrophobic balance. Measurements of cell adhesion have been carried out in the presence of collagen IV or fetal calf serum. Best

results for attachment wereobtained with PHOD-COOH, whatever the experimental conditions. The hydrophobic surface of PHO filmsinduces also

a good adhesion density of RT112 cells. PHOD-g-PEG has a contrasted behavior, due to the presence of polyethylene glycol grafts. Proliferation of

human bladder carcinoma RT112 cells has been observed on the same polymers. PHOD-COOH does not improve the cell proliferation and seems

not to be a favorable support. This preliminary study showed that PHO has a good potential to induce regeneration of a functional bladder wall.

© 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Poly(3-hydroxyalkanoate)s; Human bladder RT112 cell; Adhesion; Proliferation; Polyesters

Résumé

L’adhésion et la prolifération de cellules humaines de la vessie (RT112) ont été étudiées in vitro sur quatre biopolyesters de structure chimique

différente, sous forme de films, en vue d’une application en ingénierietissulaire. Les polyesters étudiés sont des poly(3-hydroxyalcanoate)s (PHAs) :

le poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), le poly(3-hydroxyoctanoate) (PHO), le poly(3-hydroxyoctanoate-co-3-hydroxy-9-

carboxydecanoate) (PHOD-COOH) et le poly(3-hydroxyoctanoate-co-3-hydroxy-9-carboxydecanoate-g-polyéthylèneglycol) (PHOD-g-PEG). Les

deux premiers polymères (PHB-HV et PHO) sont naturels et hydrophobes, les deux autres sont obtenus par modification chimique d’un polyester

précurseur contenant des insaturations en groupements pendants pour améliorer le caractère hydrophile de ces polymères. Les mesures d’adhésion

et de prolifération ont été réalisées en présence de collagène IV ou de sérum fœtal de veau. Les meilleurs résultats d’adhésion concernent le

PHOD-COOH, quelque soit le prétraitement des films, alors que le caractère cytotoxique de ce support apparaissant à plus long terme n’en fait

pas un bon candidat pour la prolifération des cellules RT112. Cette étude préliminaire a permis de sélectionner le PHO comme matériau potentiel

pour induire une reconstruction tissulaire de la vessie après chirurgie.© 2010 Elsevier Masson SAS. Tous droits réservés.

Mots clés : Poly(3-hydroxyalcanoate)s ; Cellules urothéliales RT112 ; Adhésion ; Prolifération ; Polyesters

∗ Corresponding author.

E-mail address: [email protected] (V. Langlois).

1. Introduction

In recent years, biodegradable polymers have attracted scien-

tific and technological interest for medical and pharmacological

applications [1–3]. Tissue engineering has been a great deal of

1959-0318/$ – see front matter © 2010 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.irbm.2010.12.001

http://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.irbm.2010.12.001

mailto:[email protected]



mailto:[email protected]


8/13/2019 1-s2.0-S1959031810001144-main


E. Renard et al. / IRBM 32 (2011) 214–220 215

attention, and becomes one of the major fields in biotechnol-

ogy, because of its potential as a new method in the treatment

of damaged or lost human tissue and organs. The concept of

tissue engineering is widely based on a cell–polymer system

in which a polymeric material function for a certain time as

a substrate or scaffold to promote tissue formation. In tissue

engineering, scaffolds play an important role by serving as

substrates for bone regeneration [4,5], and temporary three-

dimensional supports to form new tissue or organ [6]. Both

natural extracellular materials and synthetic biodegradable poly-

mers have been used to fabricatescaffolds for tissueengineering.

One of the materials for this application which has attracted

most interest is poly(lactic acid) (PLA) and derivatives because

it degrades to natural metabolites, can be easily processed,

and its mechanical properties and degradation properties can

be adjusted to meet particular needs [7–9]. The second large

family of polyesters concerns the poly(3-hydroxyalkanoate)s

(PHAs). These microbial polyesters constitute a class of poly-

mers produced by many bacteria as intracellular carbon and

energy storage polymers [10–12]. Some PHAs exhibit thermaland mechanical properties similar to those of traditional ther-

moplastics such as polyethylene and polypropylene, as well

as offering a wide range of good biocompatibility. PHAs are

also inherently biodegradable. Advances of PHAs research have

brought these natural polymers to the attention of tissue engi-

neers as potential materials for medical applications [13–21].

To promote tissue formation, the biomaterial should provide a

highly biocompatible substrate to enable cell adhesion, migra-

tion, proliferation and differentiation. In vitro tests have shown

that PHAs are biocompatible to various cell lines including

fibroblasts, chondrocytes, osteoblasts and mesenchymal stem

cells. The interaction between cells and biomaterial aimed totissue engineering is not only influenced by surface topogra-

phy [22] but also by surface physicochemical properties such

as surface free energy, electric charges and chemical structure

of polymers which affect cell attachment and cellular behavior

[23,24]. These characteristicshaveto be considered in thechoice

of a material suitable for biomedical device. They govern cel-

lular adhesion, subsequent cellular viability and performances.

We have developed a series of experiments to reveal the use

of poly(3-hydroxyalkanoate)s in the bladder cancer research

domain in cooperation with the CHU Henri-Mondor (France,

Pr D. Chopin). On one hand, new copolymers have been used

for the encapsulation and release of antitumor drug doxorubicin

[25,26] and, on the other hand, interactions between differentPHAs and human bladder carcinoma RT112 cells have been

evaluated as models for tissue engineering from the adhesion

and proliferation viewpoint. The purpose of this research is to

select a biocompatible polymer aimed at a partial reconstruc-

tion of the bladder after surgery for invasive bladder cancer.

In a first stage, it was therefore necessary to consider the

in vitro ability of urothelial cells to adhere and proliferate

on different poly(3-hydroxyalkanoate)s surfaces. Human blad-

der carcinoma RT112 cell line has been chosen as model due

to its fast growth. In this paper, we present results concern-

ing interaction between RT112 cells and bacterial polyesters

films. Four polymers have been tested. Two of them are directly

prepared by biotechnology (PHO andPHB-HV). An uncommon

biopolyester (noted PHOU), containing unsaturated pendant

groups has been subjected to chemical modification in order to

introduce reactive and hydrophilic groups. The polymer con-

taining carboxylate end groups in the side chains is noted

PHOD-COOH. The fourth polymer studied is obtained by graft-

ing PEG onto the side chains of PHOD-COOH, and named

PHOD-g-PEG.

2. Materials and methods

Different polyesters were chosen to study cellular adhesion.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) is a

copolymer made of 78% of 3-hydroxybutyrate units and

22% of 3-hydroxyvalerate units provided by ICI (Billing-

ham, UK). Poly(3-hydroxyoctanoate) (PHO) was synthesized

using Pseudomonas sp. Gpo1. Poly(3-hydroxyoctanoate-co-3-

hydroxyundecenoate)(PHOU) with75% of 3-hydroxyoctanoate

units and 25% of 3-hydroxyundecenoate units was produced

with the same strain and grown on a mixture of octanoate

and undecenoic acid. Bacterial polyesters were extracted from

lyophilized cells with chloroform and then purified by precipi-

tation in ethanol.

PHOU was oxidized according to the method described by

Kurth et al. [27]. The formed polymer contains carboxylic acid

functions (PHOD-COOH) in side chains.

In a further reaction, poly(ethyleneglycol) (PEG) was grafted

onto PHOD-COOH [28,29]. 257 mg of PHOD-COOH were dis-

solved in 12.8 mL of anhydrous methylene chloride. 101.6 mg

of N,N’-dicyclohexylcarbodiimide (DCC, 1.2 equiv. per COOH)

and 11.7 mg of 4-diméthylaminopyridine (DMAP, 0.24 equiv.)

were added to the solution. After dissolving, 172.4 mg of monomethylated PEG (1.2 equiv., Mn = 350 g.mol−1) were

introduced to the mixture. This reaction was led at room temper-

ature for 4 hours. The resulting solution was filtered to eliminate

formed dicyclohexylurea (DCU). The excess of PEG was fil-

tered out by dialysis in aqueous solution. PHOD-g-PEG was

freeze-dried. According to these experimental conditions, the

final polymer contained 75% of 3-hydroxyoctanoate units, 11%

of 3-hydroxy-9-carboxydecanoate units and 14% of esterified

units, as determined by NMR spectroscopy.

2.1. Film preparation

The different polymers were dissolved in chloroform and

poured onto glass microscope slides by the solvent cast method.

Films were dried overnight and then dried in vacuum to remove

the residual solvent. Each film fixed to its glass support or glass

microscope slidesas referencewere puton each well of a twelve-

well culture plates. The plates were placed in the laminar flow

cabinet under UV-C germicidal lamp for 15 hours to sterilize the

shell and contents. Some of them were pre-treated by depositing

proteins: collagen IV or fetal calf serum. 1 mL of a solution of

collagen IV with a concentration of 12.5g.mL−1 or 1 mL of a

fetalcalfserumwasdepositedonfilmsurfaceandplacedat37 ◦C

for 1 hour. After incubation the supernatant was eliminated.

8/13/2019 1-s2.0-S1959031810001144-main


216 E. Renard et al. / IRBM 32 (2011) 214–220

2.2. Size exclusion chromatography (SEC)

The molar masses were determined by using the size exclu-

sion chromatography (SEC), a refractive index detector and

three columns in series PL gel (Polymer Laboratories, 10 000,

1000 and 500 A, five microns). The mobile phase was chlo-

roform, with an eluent flow rate of 1 mL.min−1. The sample

concentration was 10 mg.mL−1 and the injection volume was

50L. A calibration curve was generated with polystyrene stan-

dards of low polymolecularity purchased from Polysciences

(M g.mol−1): 2656000; 841700; 320000; 148000; 59500;

28 500 ; 10 850 ; 2930 and 580.

2.3. Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) measurements were

conducted on Mettler Toledo 822 apparatus. DSC was used

to study the glass-transition temperature (Tg) and the crystal-

lization behavior of native and modified PHAs film. Samples

were heated from 25 to 190 ◦C (first run) with a heating rateof 20 ◦C.min−1. Melting points (Tm) were determined after the

first heating run from the maximum of the endothermic peak

and Hm was calculated from the area of the endothermic peak

after the first run. Then, the sample was cooled to −45 ◦C and

then heated up again to 190 ◦C at a heating rate of 20 ◦C.min−1

(second run). The glass-transition temperature (Tg) was taken

as the midpoint of the transition, in the second heating run.

2.4. Water contact angle

The water contact angle of polymer films wasmeasured on an

apparatus Krüss G10 in air using the sessile droplet method. Thevolume of deionized water (MilliQ water) droplet was 20L.

For each sample, at least five different spots were measured and

the data were averaged.

2.5. Cell adhesion and growth

1 mL of human bladder carcinoma RT112 cells suspension

containing 300 000 cells and RPMI culture medium supple-

mented with 10% fetal calf serum, 0.05% streptomycin and

0.05% penicillin was put on each well. Then the plates were

placed at 37 ◦C. The medium was changed every day. At differ-

ent times, adhesive RT112 cells were counted by a colorimetricMTT test as described by Zund et al. [30]. Briefly a solution of

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

(MTT) 1 mg.mL−1 was added in each well containing cells fixed

on polymer after removing medium of culture with non-attached

cells. Plates were placed at 37 ◦C for 1 hour. The aqueous

solution of tetrazolium salt was converted by mitochondrial suc-

cinate deshydrogenase into formazan suspension. The intensity

of this suspension was proportionalto thequantity of living cells.

By adding isopropanol, cells burst and release the colored solu-

tion whose absorption was measured by UV spectrophotometer

(= 550nm). Cell adhesion was observed at 4 hours and cell

growth for 3 days. Experiments were repeated three times.

3. Results and discussion

3.1. Poly(3-hydroxyalkanoate)s, PHAs

All natural biopolyesters are hydrophobic due to the

presence of alkyl pendant groups in the side chains.

Poly(3-hydroxybutyrate) (PHB) and poly(3-hydoxybutyrate-co-

3-hydroxyvalerate) (PHB-HV) are the main representatives of the PHAs. PHB is a highly crystalline and brittle polymer. PHB-

HV copolymers have a lower melting point and higher flexibility

than the homopolymer PHB. The amount of HV monomer

units in the copolymer strongly influences the properties of

these polymers, as crystallinity, melting point and crystalliza-

tion rate. In this study the PHB-HV composition was 78% of HB

and 22% of HV (Fig. 1). Characteristics of these polymers are

shown in Table 1. Poly(3-hydroxyoctanoate) is a medium chain

length polyhydroxyalkanoate with 85% of 3-hydroxyoctanoate,

as monomer units and 15% of 3-hydroxyhexanoate. It can

be considered as an elastomer-thermoplastic due to the pres-

ence of long pendant chains compared with those of PHB-HV.PHOU is a particular bacterial polyester. Only a few bac-

teria are able to produce unsaturated polyhydroxyalkenoates

and often with a low yield. Pseudomonas sp. Gpo1 has been

largely investigated thanks to its high ability to grow on

sodium octanoate/10-undecenoic acid mixtures and on lonely

products. The major result is the incorporation of unsatu-

rated pendant groups in the polymers. The proportion of

these unsaturated monomer units in the bacterial polyester

is directly related to the nutrient composition. It is therefore

possible to prepare copolyesters poly(3-hydroxyoctanoate-co-

3-hydroxyundecenoate) (PHO100-xUx) with 0 to 100% of

3-hydroxyundecenoate. To achieve specific properties, the

unsaturated units can be converted to other functional groups.

Carboxylic groups are of greatest importance to bind bioactive

molecules, hydrolysable or hydrophilic oligomers or target-

ing proteins. Moreover, the presence of this functional group

changes the hydrophobic/hydrophilic balance and promotes

the hydrolysis of corresponding polymers [31,32]. PHOU was

totally oxidized in PHOD-COOH using KMnO4 and 18-crown-

6-ether as phase transfer anddissociating agent as determined by1H and 13C NMR. PHOD-COOH was purified by dialysis. This

long dialysis period can lead to chain scission due to hydrol-

ysis of ester bonds as shown by a decrease of the molar mass

(Table 1). Nevertheless, molarmass remains enough highto have

good mechanical properties to make films. The synthesisof graftPHAs was carried out by direct esterification of carboxylic lat-

eral end groups with hydroxyl groups of poly(ethylene glycol)

oligomers (350 g.mol-1) (PEG). PEG was selected because it

could increase the hydrophilicity of polymers and plays a role

in the cell adhesion. Monohydroxylated PEG has been used in

order to avoid crosslinking, which occurs with dihydroxylated

PEG. As explained in the experimental part, grafting of PEG

is not quantitative; all side COOH groups are not accessible to

esterification reaction; long pendant hydrophobic alkyl groups

can avoid the approach of PEG oligomers from reactive groups,

due to their size. The average molar mass of the modified PHAs

is given in Table 1. After PEG-grafting, a long dialysis period

8/13/2019 1-s2.0-S1959031810001144-main



CH3

O CH

(CH2)4

CH2 C O CH

(CH2)2

CH2 C

O O

CH3

1585

PHOCH3

O CH

CH3

CH2 C O CH

CH2

CH2 C

O O

2278

PHB-HV

O CH

(CH2)4

CH2 C O CH

(CH2)6

COOH

CH2 C

CH3

OO2575

PHOD-COOH

O CH

(CH2)4

CH2 C O CH

(CH2)6

COOH

CH2 C

CH3

OO

O CH

(CH2)6

C

CH2 C

O

O

O CH2 CH2 OCH3

1175 14

7

PHOD-g-PEG

O CH

(CH2)4

CH2 C O CH

(CH2)6

CH

CH2 C

CH3

CH2

OO

2575

PHOU

Fig. 1. Different PHAs used.

(7 days) is needed for the removal of no grafted PEG. The pH of

the dialysis solution is not controlled and a little decrease of the

average molar mass of PHOD-g-PEG was observed, because of

some degradation, as explained in a previous study [29].

The hydrophobic/hydrophilic balance plays a major role in

the attachment of proteins and cells to biomaterials, beside other

interactions likeelectrostatic charges,dipole interactions, hydro-

gen bonding. To examine the wettability of PHAs the water

contact angle of the films surface were measured in air using the

sessile droplet method. The PHO water contact angle (104◦) is

significantly higher than PHB-HV (79◦) (Table 2), due to long

pendant alkyl groups in the PHO macromolecular chains, which

give a more hydrophobic material than PHB-HV. The presence

of hydrophilic groups such as COOH (PHOD-COOH) and PEG

(PHOD-g-PEG) do not modify the wettability compared with

PHO. These results could be explained by the presence at the

film surface of a few quantities of carboxylic groups and PEG

graft oligomers, which does not decrease significantly the mate-

rials hydrophobicity. Indeed the mostprobable explanation is the

internalization of the polar groups during the film preparationprocess in chloroform as apolar solvent [33].

Table 1

Characteristics of polyesters used.

Mn (g.mol−1) Ip Tg (◦C) Tm (◦C) Hm (J/g)

PHOU 74 000 2.2 −39 41 6

PHO 101 000 1.9 −39 57 26

PHOD-g-PEG 34 000 2.2 −55 – –

PHOD-COOH 49 000 2.1 −20 – –

PHB-HV 184 000 2.0 +1 160 59

Films have been prepared and sterilized under UV-C ger-

micidal lamp in the laminar flow cabinet. This method of

sterilization does not significantly affect the average molar mass

of the polyesters studied. These materials were used just as

they were or after a pretreatment with a collagen IV or fetal

calf serum deposit. Type IV collagen is an ubiquitous compo-

nent in basement membranes and provides the major structural

support for this matrix. When the collagen IV meshwork is

assembled, it provides a scaffold for the assembly of other base-

ment membrane components through interactions with laminin,

entactin/nidogen and heparan sulfate proteoglycan. Collagen IV

is useful as a substrate for growth of epithelial, endothelial,

muscle and nerve cells. It plays a role in the regulation of cell

growth, differentiation and adhesion, as well as tissue formation.

Fetal calf serum is commonly used to promote cell growth and

differentiation by containing a group of promoting factors.

3.2. Adhesion

The mechanism of interaction between cells and substrate isvery complex and surface properties have to be taken in account.

Results of cell adhesion are displayed on Fig. 2. Firstly results

show that human bladder carcinoma RT112 cells can adhere to

PHAs films. In absence of protein, adhesion of cells seems to

be linked to the hydrophilic–hydrophobic balance in the case of

the no modified polyesters. Themost hydrophilic support (glass)

leads to the best cell adhesion and the most hydrophobic (PHO)

leads to the worst attachment as observed by other authors [34].

RT112 cell attachment is better on PHOD-COOH and PHOD-

g-PEG films than on PHO and PHB-HV films. The ability of

cells to adhere to a biomaterial scaffold is linked not only to the

surface properties as hydrophilic–hydrophobic balance but also

8/13/2019 1-s2.0-S1959031810001144-main



Table 2

Water contact angle of polyesters used.

Polymers Glass PHO PHOD-g-PEG PHOD-COOH PHB-HV

Water contact angle (◦) 51± 2 104±2 102±3 95±3 79±2

to theirintrinsic properties as cristallinity. In presence of proteins

(collagen IV or fetal calf serum), cell attachment is favored on

PHOD-COOH. Proteins form a layer at the surface of the film.

Collagen IV and bovine serum albumin (main component of

fetal calf serum) adhere preferentially on hydrophobic surface

[35]. By adding proteins the support surface is changed and the

adsorption of proteins from surrounding medium has to be taken

into account, forming a layer and contributing to adhesion. The

cell attachment ability can be linked to the type of protein at

the surface of the scaffold and to its ability to interact with the

material surface. This improvement is moderate for PHOD-g-

PEG film because PEG is known to inhibit adsorption of serum

proteins [36].

3.3. Proliferation

After three days of incubation (72 hours) without added pro-

teins, PHO support appears as the most interesting material

(Figs. 3 and 4). Even though hydrophilicity is higher in the case

of PHB-HV compared with PHO, it does not lead to a better

cell growth. The hydrophilic–hydrophobic balance is of major

importance for cell-support interaction but the thermal proper-

ties and porosity of films must also be taken into account. By

solvent castmethod, evaporation of solvent leads to porous films.

But this porosity depends on the capacity of polymer chains to

reorganize. The more the polymer is amorphous, the more thechains can move easily. PHO is more amorphous than PHB-

HV (Table 1). The obtained PHO films should be less porous

than PHB-HV films. This could explain that there is no signifi-

cant difference of cell growth on PHB-HV films and PHO films,

despite a difference of wettability. In presence of type IV col-

lagen or fetal calf serum, cell growth on glass is not affected.

Fig. 2. Cell adhesion on PHAs films after 4 hours of incubation at 37◦

C.

On the contrary, cell growth is increased on no modified PHAs

films. Forming a layer at the surface of the polymer, protein

support interactions have to be taken in account. Collagen and

bovine serum albumin (major component of fetal calf serum)

are known to better adhere on hydrophobic surface [35]. The

better adsorption of proteins on PHO and PHB-HV films leads

to a better effect of the presence of proteins on cell growth.

In the case of modified PHAs (Fig. 4) and without any pro-

teins, the results show a worse cell growth on PHOD-COOH

and PHOD-g-PEG films than on PHO. This difference could

Fig. 3. Cell proliferation on native PHAs films without any protein coating (A),

with collagen IV coating (B) and with fetal calf serum coating (C).

8/13/2019 1-s2.0-S1959031810001144-main



0

1

2

3

4

5 A

B

C

24h 48h 72h

N u m b e r o f c e l l s ( 1 0

5 )

PHO

PHOD-COOH

PHOD-g-PEG

0

1

2

3

4

5

24h 48h 72h


5 )

PH O

PHOD-COOH

PHOD-g-PEG

0

1

2

3

4

5

24h 48h 72h


5 )

PHO

PHOD-COOH

PHOD-g-PEG

Fig. 4. Cell proliferation on modified PHAs films without any protein coating

(A), with collagen IV coating (B) and with fetal calf serum coating (C).

be attributed to the cristallinity of the polymer. By the presence

of the PEG grafts and the COOH side chains, PHOD-g-PEG

and PHOD-COOH are amorphous compared to PHO (Table 1).

Cristallinity plays a role on cell attachment and growth. RT112

cells seem to better grow on semi-crystalline support like PHO.

As expected when type IV collagen is added to the culture

medium the cell growth is better whatever the modified poly-

mer support. This protein favors cell attachment, growth and

differentiation. Results obtained for PHOD-g-PEG could becompared to those obtained for PHO. According to the serum

proteins adsorption inhibition effect of PEG, cell proliferation

is better on PHO than on PHOD-g-PEG for the first 24 hours.

Surprisingly cell growth on PHOD-g-PEG increases for the fol-

lowing hours up to cell viability obtained for PHO. In regard

to the hydrophilic–hydrophobic balance, these polymers show a

similar wettability but PEG graft could lead to a different topog-

raphyand roughness of the surface thatcouldchange interactions

between surface and collagen.

According to the water contact angle measurements PHO

is more hydrophobic than PHOD-COOH. The better interac-

tion of collagen with hydrophobic support [35] leads to a better

cell growth on PHO than PHOD-COOH. A cytotoxic charac-

ter of PHOD-COOH appears after 72 hours of incubation with

a decrease of cell viability. This cytotoxicity could be favored

by a change of chemical structure of the surface. By contact of

PHOD-COOH film with water, a partial reorganization of the

polymeric material takes place after 72 hours of incubation, and

internalized carboxylic groups move to the surface, as explained

in a previous study [35]. The support becomes more hydrophilic

and the ability of collagen to adhere to the surface decreases.

On one hand this reorganization of the surface could change

surface free energy leading to a delamination of the type IV col-

lagen layer with a part of viable cells. And on the other hand

carboxylic functions appearing at the surface have a cytotoxic

character on cell growth by the presence of negative charges, as

already reported [24].

By adding fetal calf serum Human bladder carcinoma RT112

cell growth is inhibited on PHOD-g-PEG films. This phe-

nomenon is explained by the serum protein adsorption resistant

character of PEG [36]. Human bladder carcinoma RT112 cell

growth on PHOD-g-PEG depends on the adsorbed proteins. Onthe contrary, results show a better cell growth when proteins

of fetal calf serum form a layer on the PHOD-COOH films.

After 72 hours of incubation a decrease of cell viability can be

explained by a cytotoxic effect of PHOD-COOH. As described

previously a partial reorganization of the polymeric material

during the 72 hours of incubation can lead to the presence of

carboxylic groups at the surface and an increased hydrophilic

surface [33]. As seen earlier bovine serum albumin interacts

preferentially with hydrophobic surfaces. As for collagen the

change of surface hydrophobia probably leads to a delaminat-

ing of the proteic layer on which cells adhere leading to less

viable cells.In the proliferation, it is also necessary to taken in account

proteins that come in between material and cells.

4. Conclusions

This in vitro study of adhesion and proliferation of human

bladder carcinoma RT112 cells on different natural and artifi-

cial biopolyesters displays that many structural and chemical

factors contribute to these phenomena, as in the case of other

biodegradablepolymers. The hydrophilic–hydrophobicbalance,

cristallinity and topography of the surface have to be taken

into account. Moreover the type, conformation and amountof proteins adhered to the surface affect also the cellular

adhesion and proliferation. Among the tested PHAs, poly(3-

hydroxyoctanoate) appears as the most suitable PHA. This

semi-crystalline and hydrophobic polyester seems to be a good

candidate for a supplementary study in the research of a bio-

compatible biomaterial aimed at a partial reconstruction of the

bladder after surgery for invasive bladder cancer.

Conflict interest statement

None.

8/13/2019 1-s2.0-S1959031810001144-main



Acknowledgements

The authors are grateful to the professors Philippe Guérin,

Richard Bourbouze and Dominique Chopin, for their helpful

discussions and their confidence in this project.

References

[1] Nair LS, Laurencin CT. Biodegradable polymers as biomaterials. Prog

Polym Sci 2007;32:762–98.

[2] Gunatillake PA, Adhikari R. Biodegradable synthetic polymers for tissue

engineering. Eur Cell Mater 2003;5:1–16.

[3] Steinbüchel A, Marchessault RH. Biopolymers for medical and pharma-

ceutical applications. Wiley-VCH; 2005.

[4] Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the

art and future trends. Macromol Biosci 2004;4:743–65.

[5] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Bio-

materials 2000;21:2529–43.

[6] Schmidt CE, Baier JM. Acellular vascular tissues: natural biomate-

rials for tissue repair and tissue engineering. Biomaterials 2000;21:

2215–31.

[7] Brannon-Peppas L, Vert M, Polylactic. polyglycolic acids as drug deliv-

ery carriers. In: Wise DL, editor. Handbook of pharmaceutical controlled

release technology. New York: Marcel Dekker, Inc.; 2000. p. 99–130.

[8] Yang A, Yang L, Liu W, Li Z, Xu H, Yang X. Tumor necrosis factor alpha

blockingpeptide loaded PEG-PLGA nanoparticles:preparationand in vitro

evaluation. Int J Pharm 2007;331:123–32.

[9] Langlois V, Renard E, Vergnol G, Guerin P, Loirand G, Haroun F, et al.

Elaboration of biodegradable systemsfor electrograftingontoendovascular

metallic stents. Biomed Eng Res 2010;31:111–4.

[10] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role,

and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev

1990;54:450–72.

[11] Lee SY. Bacterial polyhydroxyalkonoates. Biotechnol Bioeng

1996;49:1–14.

[12] Steinbüchel A, Füchtenbusch B. Bacterial and other biological systems for

polyester production. Trends Biotechnol 1998;16:419–27.

[13] Marois Y, Zhang Ze, Vert M, Xiaoyan D, Lenz R, Guidoin R. Effect of

sterilization on the physical and structural characteristics of polyhydroxy-

octanoate. J Biomater Sci 1999;10(4):469–82 [polymer edition].

[14] Hu YJ, Wei X, Zhao W, Liu YS, Guo-Qiang Chen. Biocompatibility of

poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate)

with bone marrow mesenchymal stem cells. Acta Biomater

2009;5(4):1115–25.

[15] DengY, LinXS, Zheng Z, DengJG, ChenJC, Ma H. Poly(hydroxybutyrate-

co-hydroxyhexanoate) promoted production of extracellular matrix

of articular cartilage chondrocytes in vitro. Biomaterials 2003;24:

4273–81.

[16] Wang YW, Wu Q, Chen GQ. Attachment, proliferation and differentiation

of osteoblasts on random biopolyester poly(3-hydroxybutyrate-co-3-

hydroxyhexanoate) scaffolds. Biomaterials 2004;25:669–75.

[17] Wang L, Wang Z, Shen C, You M, Xiao J, Chen G. Differen-

tiation of human bone marrow mesenchymal stem cells grown in

terpolyesters of 3-hydroxyalkanoates scaffolds into nerve cells. Bioma-

terials 2010;31(7):1691–8.

[18] Yang M, Zhu S, Chen Y, Chang Z, Chen GQ, Gong Y. Studies on

bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-

hydroxyhexanoate). Biomaterials 2004;25:1365–73.

[19] Ji Y, Li XT, Chen GQ. Interactions between a poly(3-hydroxybutyrate-

co-3-hydroxyvalerate-co-3-hydroxyhexanoate) terpolyester and human

keratinocytes. Biomaterials 2008;29:3807–14.

[20] Williams SF, Martin DP, Horowitz DM, Peoples OP. PHA applications:

addressing the price performance issue I. Tissue engineering. Int J Biol

Macromol 1999;25:111–21.[21] Köse GT, Ber S, Korkusuz F, Hasirci V. Poly(3-hydroxybutyric acid-co-3-

hydroxyvaleric acid) based tissue engineering matrices. J Mater Sci Mater

Med 2003;14:121–6.

[22] Kunsler TP, Drobek T, Schuler M, Spencer ND. Systematic study of

osteoblast and fibroblast response to roughness by means of surface-

morphology gradients. Biomaterials 2007;28:2175–82.

[23] Anselme K. Osteoblast adhesion on biomaterials. Biomaterials

2000;21(7):667–81.

[24] Lee LH, Lee JW, Khang GH, Be H. Interaction of cells on chargeable

functional group gradient surfaces. Biomaterials 1997;18(4):351–8.

[25] Timbart L. Thesis. Université Paris-12–Val-de-Marne; 2005.

[26] Timbart L, Renard E, Tessier M, Langlois V. Monohydroxylated poly(3-

hydroxyoctanoate) oligomers and its functionalized derivatives used as

macroinitiators in the synthesis of degradable diblock copolyesters.

Biomacromolecules 2007;8(4):1255–65.

[27] Kurth N, Renard E, Brachet F, Robic D, Guerin P, Bourbouze R.

Polyhydroxyoctanoate containing pendant carboxilic groups for the prepa-

ration of nanoparticles aimed at drug transport and release. Polymer

2002;42:1095–101.

[28] Renard E, Ternat C, Langlois V, Guérin Ph. Synthesis of graft bacterial

polyesters for nanoparticlespreparation. Macromol Biosci 2003;3:248–52.

[29] Domenek S, Langlois V, Renard E. Bacterial polyesters grafted

polyethylenglycol: behavior in aqueous medium. Polym Deg Stab

2007;92:1384–92.

[30] Zund G, Ye Q, Hoerstrup SP, Schoeberlein A, Schmid AC, Grunenfelder

J, et al. Tissue engineering in cardiovascular surgery: MTT, a rapid and

reliable quantitative method to assess the optimal human cell seeding on

polymeric meshes. Eur J Cardiothorac Surg 1999;15:519–24.

[31] Renard E, Walls M, Guérin Ph, Langlois V. Hydrolytic degradation of

blends of polyhydroxyalkanoates and functionalized polyhydroxyalka-

noates. Polym Deg Stab 2004;85:2779.

[32] Renard E, Langlois V, Guérin P. Chemical modifications of bacterial

polyesters: from stability to controlled degradation of resulting polymers.

Corros Eng Sci Technol 2007;42(4):300–11.

[33] Renard E, Timbart L, Vergnol G, Langlois V. Role of carboxyl pendant

groups of medium chain length poly(3-hydroxyalkanoate)s in biomedical

temporary applications. J Appl Polym Sci 2010;117:1888–96.

[34] Wei J, Yoshinari M, Takemoto S, Hattori M, Kawada E, Liu B, et al.

Adhesion of mouse fibroblasts on hexamethyldisiloxane surfaces with

wide range of wettability. J Biomed Mater Res Part B Appl Biomater

2007;81B:66–75.

[35] PeiqingYing,Yong Yu, GangJin. ZulaiTaoCompetitive protein adsorption

studied with atomic force microscopy and imaging ellipsometry. Colloids

Surf B Biointerfaces 2003;32:1–10.

[36] Malmsten M, Emoto K, Van Alstine JM. Effect of chain density on inhibi-

tionof proteinadsorptionby poly(ethyleneglycol) basedcoatings.J Colloid

Interface Sci 1998;202:507–17.

1-s2.0-s1959031810001144-main

Documents