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Biomaterials 23 (2002) 13911397

Effect of surface treatment on the biocompatibility of microbialpolyhydroxyalkanoates

Xianshuang Yang, Kai Zhao, Guo-Qiang Chen*

Laboratory of Microbiology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

Received 9 January 2001; accepted 26 July 2001

Abstract

The biocompatibility of microbial polyesters polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate)

(PHBHHx) were evaluated in vitro. The mouse fibroblast cell line L929 was inoculated on films made of PHB, PHBHHx and theirblends, polylactic acid (PLA) as control. It was found that the growth of the cells L929 was poor on PHB and PLA films. The viable

cell number ranged from 8.8102 to 1.8104/cm2 only. Cell growth on the films made by blending PHB and PHBHHx showed a

dramatic improvement. The viable cell number observed increased from 9.7102 to 1.9105 on a series of PHB/PHBHHx blended

film in ratios of 0.9/0.1 : 0/1, respectively, indicating a much better biocompatibility in the blends contributed by PHBHHx.

Biocompatibility was also strongly improved when these polymers were treated with lipases and NaOH, respectively. However, the

effects of treatment were weakened when PHBHHx content increased in the blends. It was found that lipase treatment had more

increased biocompatibility than NaOH. After the treatment biocompatibility of PHB was approximately the same as PLA, while

PHBHHx and its dominant blends showed improved biocompatibility compared to PLA. r 2002 Elsevier Science Ltd. All rights

reserved.

Keywords: Poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx); Polyhydroxybutyrate (PHB); Biocompatibility

1. Introduction

Polyhydroxyalkanoates (PHA) is a family of biopo-

lyesters synthesized by many bacteria [1,2]. Over 90

different types of PHA consisting of various monomers

have been reported and the number is still increasing [2].

Some PHA behave similarly to conventional plastics

such as polyethylene and polypropylene, while others

are elastomeric. All natural PHA normally possess

biodegradability [2]. Therefore, many research and

industrial efforts have been made to turn PHA into

biodegradable plastics [35]. However, the high produc-

tion cost of PHA has prohibited its application as

plastics. Several studies were conducted using poly-

hydroxybutyrate (PHB), the most common member of

the PHA family, as biomaterial for in vitro and in vivo

studies. Results showed various degrees of biocompat-

ibility and biodegradability [6,7]. The application of

PHA as biomaterial will add values to this novel

biopolymer [8].

Due to its biodegradability and biocompatibility,

PHA may well serve as materials for tissue engineer-

ing [8]. PHA, including fragile PHB, a flexible co-

polymer consisting of 3-hydroxybutyrate and 3-hydro-

xyvalerate (PHBV), and an elastomeric copolymer

consisting of 3-hydroxyoctanoate and 3-hydroxy-

hexanoate (PHOH) may have promising applications

as tissue scaffolds and cardiovascular tissue [8]. How-

ever, some studies showed that PHB and PHBV have

induced prolonged acute inflammatory responses when

implanted in vivo [9]. These phenomena may

be caused by the polymer surfaces that may not be

biocompatible with the in vivo environments.

In addition, PHB and PHBV have poor mechanical

properties, which may limit their applications in

situations that require high tensile strength. Therefore,

PHA with better mechanical properties, such as

copolymers of 3-hydroxybutyrate and 3-hydroxyhex-

anoate or PHBHHx [10], may be more suitable for the

tissue engineering application, provided PHBHHx is

biocompatible.

*Corresponding author. Tel: +86-10-6278-3844; fax: +86-10-6278-

8784.

E-mail address: [email protected] (G.-Q. Chen).

0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 2 6 0 - 5


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It is, therefore, desirable to modify the material

surface to suit the intended application, often with-

out altering other properties of the scaffold, such as

its mechanical strength or thermal properties [8,11].

Effective surface modifications include changes in

chemical group functionality, surface charge, hydro-

philicity, hydrophobicity, and wettability [8,11,12].Modification of a polymer surface can be achieved by

means of various chemical or physical processes includ-

ing plasmaion beam treatment, electric discharge,

surface grafting, chemical reaction, vapor deposition

of metals and flame treatment [8].

In this study, we intended to compare the in vitro

biocompatibility of PHB, PHBHHx their blends and

polylactic acid (PLA) before and after the surface

treatment with lipases and NaOH, respectively. This is

the first study concerning biocompatibility of the

copolyester PHBHHx and its comparison with PLA

and PHB.

2. Experimental section

2.1. Materials

2.1.1. Preparation of pure PHA polymers

PHBHHx and PHB were provided by Procter &

Gamble Co., Cincinnati, USA and Jiangmen Center

for Biotech Development, Guangdong, China, respec-

tively.

1 g PHBHHx (Mw 1,000,000 Da) was dissolved in

100 ml acetone and refluxed at 601

C for 1h. ThePHBHHx acetone solution was centrifuged to remove

non-soluble particles (5000g, 20 min). The supernatant

was added with methanol to obtain PHBHHx precipi-

tates in high purity.

PHB (Mw 300,000 Da) in powder form was already in

99.99% purity and it was not subjected to further

purification.

PLA (Mw 350,000 Da) was provided by the Institute

of Macromolecules, Nankai University, Tianjin, China.

2.1.2. Polymer film casting

Totally, 1 g of PHB and PHBHHx in a series of

weight ratios (1 : 0, 0.9 : 0.1, 0.75 : 0.25, 0.5 : 0.5,

0.25 : 0.75, 0.1 : 0.9, 0 : 1), respectively, was dissolved in

100 ml chloroform. Each 100 ml chloroform PHA

solution was evenly distributed into 20 petri dishes

(diameter 60 mm). The dishes were maintained at room

temperature to allow the complete evaporation of

chloroform. The evaporation of solvent resulted in the

formation of PHA films in the petri dishes. Vacuumdrying was further applied to completely remove any

possible solvent remaining in the films as the solvent is

toxic to cells and may influence the results. Before

inoculation, the films were sterilized under ultraviolet

radiation overnight.

PLA was cast into film as described above. The PHA

and PLA films were then subjected to the following

treatments.

2.1.3. Agents used to treat the polymer films

To increase the biocompatibility of the polyesters

used in this study, lipases and NaOH were used to break

the ester-linkage of the polymers, respectively. Thehydrolyzation process will generate more hydroxyl

groups that lead to increased hydrophilicity, thus

improving the capacity for the cells to adhere to the

polymer surfaces.

Lipolase 100T, Lipolase Ultra 50T and Lipoprime

50T were donated by Novo Nordisk China (Beijing,

China). The optimal working conditions for the lipases

are listed in Table 1. Each polymer film in the Petri

dishes was immersed in 10 ml of one of the above lipase

solutions (0.1 g/l) under 301C and pH 7.0 for 24 h. 1n

NaOH was also used here to treat the PHA films at 601C

for 1 h.

2.2. Methods

2.2.1. Cell line and cell culture

The mouse fibroblast cell line L929 was purchased

from the Institute of Virology Academia Sinica, Beijing.

The cell line was cultured in 50 ml polystyrene

flasks incubated in a CO2 incubator (Sanyo, MCO-15

A, Japan) supplied with 5% CO2 at 371C. The culture

medium was Dulbeccos modified eagle medium

(DMEM) supplemented with 10% fetal calf serum

and 1% penicillinstreptomycin solution. After

the cell line grew to completely cover the 50 ml cell

Table 1

Activities of the three lipases used in this studya

Lipase type Activity determination

Substrate Temperature (1C) pH Lipolytic activity

Lipolase 100T Tributyrin 30 7.0 100 KLU/g

Lipolase Ultra50T Tributyrin 30 7.0 50 KULU/g

LipoPrime 50T Tributyrin 30 7.0 50 KLU(P)/g

aKLU and KULU are Novo Nordisk lipase activity units.

X. Yang et al. / Biomaterials 23 (2002) 139113971392


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culture flask, 5 ml of DMEM containing 0.01%

trypsinase was added to the culture flask to digest the

interconnected cells.

2.2.2. Growth of the cell line on polymer films

The mouse fibroblasts cell line L929 was treated with

trypsinase to create a single cell suspension beforecultivation on the films. Cell number was determined by

direct counting via a haemacytometer. Effects of lipases

and NaOH treatment on cell growth were carried out by

direct cultivation of 0.2 ml suspended mouse fibroblast

cell line L929 (5 104/ml) on the treated polymer films

immersing in 5 ml DMEM containing 10% fetal bovine

serum.

The above seeded films were then inoculated in the

CO2 incubator (5% CO2) for 68h. The medium was

then removed and the films washed with phosphate

buffer solution (PBS, pH 7.4) to completely remove

serum. 1 ml 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetra-

zolium bromide (MTT) and 5 ml serum-free DMEMwere added to each film and the cells were allowed to

grow for another 4 h. Cell morphological examinations

of every dish were performed daily under an inverted

phase contrast microscope (JNOEC XD-101, Nanjin,

China).

2.2.3. [3-(4,5-dimethylthiazol-2-yl)-diphenyl-

tetrazolium bromide] MTT assay

Biocompatibility of untreated or treated polymers was

evaluated by observing the number of mouse fibroblast

cell lines L929 grown on the above polymer films using

the MTT assay [1316].MTT assay determines the viable cell number and is

based on the mitochondrial conversion of the tetra-

zolium bromide salt. MTT assay was employed in this

study to quantitatively assess the viable cell numbers of

L929 attached and grown on polymer film surfaces. As

stated above, the original medium was replaced by 1 ml

MTT PBS solution containing 5 mg MTT in 1 ml PBS,

and 5 ml serum-free DMEM medium. The films in new

MTT containing medium were incubated at 371C for an

additional 4 h for MTT formazan formation. At the end

of the culturing period, the medium and MTT were

removed and rinsed twice with PBS (pH 7.4). 10 ml

dimethyl sulfoxide (DMSO) was added to each film to

dissolve formazan. Formazan was completely dissolved

after 30 min. 200ml of the above formazan solution was

taken from each inoculum and added to one well of the

96-well plate. Six parallel samples were prepared. The

absorbencies of the samples were measured using a

Microplate Reader (Bio-RAD Benchmark, USA) set at

wavelength 550 nm; DMSO was used as blank. To

ensure that the polymers themselves did not contribute

to the absorbance, polymer films alone were assayed as

background controls. Six parallel replicates were mea-

sured for each film. MTT assay was performed on a

direct count of L929 cells consisting of the following

numbers: 1106, 5105 2.5 105, 1.25 105,6.25 104 and 3.1 104. The absorbance values were

plotted against the counted cell numbers, and thus a

standard calibration curve was established (Fig. 1).

Viable cells grown on a polymer film were determined

based on their MTT.

2.2.4. Scanning electron microscopy (SEM)

The polymer films prepared as in Section 2.1.2,

including the untreated and treated samples, were

coated with gold and observed under scanning electron

microscope (Chinese Academy of Science, KYKY-2800,

Beijing, China).

2.2.5. Statistical analysis

Results were expressed as mean7standard error of

the mean. Comparisons between groups were performed

by one-way ANOVA test. Statistical significance was set

at Po0:05: Data were processed with Microcal origin

6.0 software (Microcal, USA). Linear regression analy-

sis was utilized to evaluate the correction using

Microsoft Excel software.

3. Results

Growth of the cells L929 was poor on untreated PHB

and PLA films; viable cell number ranged only from

8.8102 cells/cm2 on untreated PHB film and to

1.8104 cells/cm2 on untreated PLA film (Fig. 2ad).

Viable cells on the untreated PHBHHx film were

1.9105/cm2, approximately 216 times more than that

on the PHB film and 11 times more than on PLA film.

Films made by blending PHB and PHBHHx showed a

dramatic improvement, and the viable cell number

increased from 9.7102 to 1.3 105 on blended films

of PHB/PHBHHx in ratios of 0.9/0.1 and 0.1/0.9,

Fig. 1. Relationship between MTT absorbance at 550nm and L 929

cell density It is a linear correlation between L 929 cell number directly

counted and the MTT absorbance: Y 0:0407 2:54 106x; r2

0:993:

X. Yang et al. / Biomaterials 23 (2002) 13911397 1393


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Fig. 2. Effect of lipase and NaOH treatment on the viability of cells grown on PHB, PHBHHx, blend polymers of PHB and PHBHHx and PLA (a)

Lipolase 100T; (b) Lipolase Ultra 50T; (c) Lipoprime 50T; (d) 1 n NaOH. Statistical analysis performed by ANOVA showed P values in all data are

smaller than 0.05.



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respectively. The cells grew better in the presence of

more PHBHHx in the blends. The number of viable cells

increased dramatically when films of PHB, PHBHHx,

their blends and PLA were treated with three different

lipases and 1n NaOH, respectively. Lipase treatment

increased the viable cell number on the PHB film from

107 to 201 times compared to the untreated PHB film.Viable cells on the PLA film also increased from

1.8 104 to 8.5104 and 2.5105 cells/cm2 after the

lipase treatment, a 4.7- and 14-fold increase, depending

on the type of lipase used (Fig. 3). NaOH treatment on

PHB film also indicated an increase of 25 times on the

viable cell number compared with the untreated PHB

film.

As the PHBHHx content increased in the blends,

the effects of lipase and NaOH treatment were

significantly weakened. The lipase treatment on a

blended film consisting of PHB/PHBHHx with a ratio

of 0.90.1 resulted in at least a 103-fold in-crease in

viable cells. While the same treatment showed much less

effect on a blend containing PHB/PHBHHx in a ratio of

0.10.9, a maximum 0.6-fold increase in viable cells was

observed. The lipase treatment on PHBHHx alone

showed mixed effects, lipase Lipoprime 50T even

decreased viable cell number on the film, Lipolase

100T and Lipolase Ultra 50T improved the growth of

viable cells on PHBHHx slightly (Fig. 3). The treated

and untreated PLA films showed a similar behavior

affecting viable cell growth compared to a blend

consisting of 50% PHB and 50% PHBHHx, although

a little bit weaker than the blend.

NaOH treatment appeared less effective in improv-

ing the growth of viable cells on all PHA films. Cell

number increased maximally only 13-fold after 1n

NaOH treatment compared with 201-fold observed

after lipase treatment. PLA is soluble in 1 n NaOH, no

comparison was made. The effect of NaOH on blend

polymers of PHB and PHBHHx behaved similarly tolipase treatment, albeit weaker than lipase treatment

(Fig. 2d).

SEM showed that the surface of the PHB film had

many pores with sizes ranging from 1 to 5 mm. The lipase

and NaOH treatment processes reduced the pore sizes to

less than 1 mm on the surface of PHB films. No apparent

morphological change was observed on the surfaces of

PHBHHx films (Picture not shown). It seems that the

surface morphologies, together with the hydrophilicity

increase were the factors behind enhanced cell growth

on the polymer surfaces.

4. Discussion

As a new member of the PHA family, PHBHHx

still has many properties that may be interesting to a

wide range of applications. PHBHHx has much better

mechanical properties over PHB and PHBV [10].

However, no study was done so far concerning

this promising material for applications such as tissue

engineering material. The results of the present

study have shown that PHBHHx can well support

cell growth (Fig. 2). Viable cell growth on untreated

Fig. 3. Comparison of effects of lipase and NaOH treatments on the biocompatibility of PHB, PHBHHx, their blends and PLA. Statistical analysis

performed by ANOVA showed that P values in all data are smaller than 0.05.



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PHBHHx was better than that on PHB and PLA films,

indicating a promising property of PHBHHx for use as

biomaterial. This study may provide first-hand data to

compare the biocompatibility of PLA, PHB and

PHBHHx.

The surface properties of a biomaterial, especially

hydrophilicity, influence cell adhesion to the materials[1720]. In general, the higher the hydrophilicity

of a material surface, the stronger the cells attached

to the material. In this study, lipases and NaOH

were employed to improve the hydrophilicity of

the polymer films. The improved hydrophilicity of the

films allowed cells in its suspension to easily attach

on the polymer films compared to that on the untreated

ones. This is why we observed better cell growth on

the treated PHB and PLA. Lipase is an enzyme

specifically acting to break the ester bonds, and the

lipase treatment probably produced many hydroxyl

groups resulting from the hydrolyzation of the ester

bonds of PHB, PHBHHx and PLA, thus contributing tothe improved hydrophilicity of the polymer films.

NaOH should act similarly to lipase; however, NaOH,

which is strongly alkaline, may also destroy the fine

polymer structure and reduce the ability of the cells to

attach to the treated films. This may be the reason why

we observed weaker growth on NaOH treated films on

lipase-treated films.

SEM examination revealed significant surface mor-

phological change in the PHB film surface after lipase

treatment NaOH treatment also changed the PHB film

surface, although to a lesser extent (photos not shown).

PHB film treated with lipase had a reduced pore size,cells grew better on it compared to the untreated larger

porous film and the NaOH treated film, which had

reduced pore sizes compared to the untreated film. The

morphological changes could be attributed to the action

of lipases and/or NaOH, which hydrolyze the rough and

larger polymer film surface, and slowly smooth the

polymer surfaces. Therefore, a reduced pore size and a

surface smoothing phenomenon were observed as the

result. Besides the surface morphological changes,

hydrophilicity increase is expected to play an important

role for enhanced cell growth on the polymer films.

Interestingly, the PHBHHx film showed little change

on its surface after the lipase treatment, corresponding

to an insignificant cell growth change before and after

surface treatment. It is clearly observable that the

increasing content of PHBHHx contributed to the

increasing growth of viable cells in the blends consisting

of PHB and PHBHHx in treated or untreated polymer

films.

This study has demonstrated that PHBHHx, a

microbially synthesized polyester with good biodegrad-

ability and biocompatibility, as well as strong mechan-

ical properties, may be a very promising biomaterial for

tissue engineering applications.

Acknowledgements

Tsinghua University 985 Project and the Natural

Science Foundation of China, Grant No. 20074020,

supported this study.

References

[1] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic

role, and industrial uses of bacterial polyhydroxyalkanoates.

Microbiol Rev 1990;54:45072.

[2] Steinb.uchel A. Polyhydroxyalkanoic acids. In: Byrom D, editor.

Biomaterials: novel materials from biological sources. New York:

Stockton, 1991. p. 124213.

[3] Chen GQ, Koenig KH, Lafferty RM. Production of poly D-(-)-3-

hydroxybutyrate and poly D-(-)-3-hydroxybalerate by strains of

Alcaligenes latus. Antonie van Leeuwenhoek 1991;60:616.

[4] Byrom D. Production of poly b-hydroxybutyrate and poly b-

hydroxyvalerate copolymers. FEMS Microbiol Rev

1992;103:24750.

[5] Chen GQ, Page WJ. Production of poly b-hydrobutyrate by

Azotobacter vinelandii UWD in a two-stage fermentation process.

Biotechnol Technol 1997;11:34750.

[6] Doyle C, Tanner ET, Bonfield W. In vitro and in vivo evaluation

of polyhydroxybutyrate and of polyhydroxbutyrate reinforced

with hydroxyapatite. Biomaterials 1991;12:8417.

[7] Knowles JC, Hastings GW, Ohta H, Niwa S, Boeree N.

Development of a degradable composite for orthopedic use: in

vivo biomechanical and histological evaluation of two bioactive

degradable composites based on the polyhydroxybutyrate poly-

mer. Biomaterials 1992;13:4916.

[8] Williams SF, Martin DP, Horowitz DM, Peoples OP. PHA

applications: addressing the price performance issue I. Tissue

engineering. Int J Biol Macromol 1999;25:11121.

[9] Akhtar S. Physiochemical properties of bacterial P (HB-HV)polyesters and their uses in drug delivery. PhD Thesis, University

of Bath, 1990.

[10] Doi Y, Kitamura S, Abe H. Microbial synthesis and character-

ization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

Macromolecules 1995;28:48228.

[11] Liu JH, Jen HL, Chung YC. Surface modification of polyethylene

membranes using phosphorylcholine derivatives and their platelet

compatibility. J Appl Polym Sci 1999;74:294754.

[12] Sacristan J, Reinecke H, Mijangos C. Surface modification of

PVC films in solvent-non-solvent mixtures. Polymer 2000;41:

557782.

[13] Zund G, Ye Q, Hoerstrup S P, Shoeberlein A, Schmid AC,

Grunenfelder J, Vogt P, Turina M. Tissue engineering in

cardiovascular surgery: MTT, a rapid and reliable quantitative

method to assess the optimal human cell seeding on polymericmeshes. Eur J Cardio-thoracic Surg 1999;15:51924.

[14] Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A. In vitro

evaluation of cell/biomaterial interaction by MTT assay. Bioma-

terials 1993;14(5):35964.

[15] Marois Y, Guidoin R, Roy R, Vidovsky T, Jakubiec B, Sigot-

Luzard M, Braybrook J, Mehri Y, Laroche G, King M. Selecting

valid in vitro biocompatibility tests that predict the in vivo healing

response of synthetic vascular prostheses. Biomaterials 1996;

17(19):183542.

[16] Sgouras D, Duncan R. Methods for the evaluation of biocompat-

ibility of soluble synthetic polymers which have potential for

biomedical use: 1-use of the tetrazolium-based colorimetric assay

(MTT) as a preliminary screen for evaluation of in vitro

cytotoxicity. J Mater Sci: Mater Med 1990;1:618.



7/7

[17] Zhao J, Geuskens G. Surface modification of polymers VI.

Thermal and radiochemical grafting of acrylamide on polyrthy-

lene and polystyrene. Eur Polym J 1999;35:211523.

[18] Furukawa T, Matsusue Y, Yasunaga T, Shikinami Y, Okuno M,

Nakamura T. Biodegradation behavior of ultra-high-strength

hydroxyapatite/poly(L-lactide) composite rods for internal fixa-

tion of bone fractures. Biomaterials 2000;21:88998.

[19] Chanvel-Lesrat DJ, Pellen-Mussi P, Auroy P, Bonnaure-Mallet

M. Evaluation of the in vitro biocompatibility of various

elastomers. Biomaterials 1999;20:2919.

[20] Qing TW, Yang ZM, Cai SX, Xu SR, Wu ZZ. Interaction of cell

adhesion to materials in tissue engineering. Chin Reparative

Reconstruct Surg 1999;13(1):317.


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