The response of rat cerebellar granule neurons (rCGNs) to various polyhydroxyalkanoate (PHA) films

The response of rat cerebellar granule neurons (rCGNs) to

various polyhydroxyalkanoate (PHA) films

Bo-Yi Yua,b, Chi-Ruei Chenb, Yi-Ming Suna,c,d,*, Tai-Horng Youngb

aDepartment of Chemical Engineering and Materials Science, Yuan Ze University, Chung-Li, Taoyuan,Taiwan 320, Republic of China

Tel. +886-3-4638800 ext. 2558; Fax +886-3-4559373; email: [email protected] of Medical Engineering, National Taiwan University, Taipei, Taiwan 100, Republic of China

cGraduate School of Biotechnology and Bioengineering, Yuan Ze University, Chung-Li, Taoyuan, Taiwan 320,Republic of China

dR&D Center for Membrane Technology, Chung Yuan University, Chung-Li, Taoyuan, Taiwan 320,Republic of China

Received 30 June 2008; revised 04 January 2009; accepted 09 February 2009

Abstract

The aim of this study is to control the behavior of rat cerebellar granule neurons (rCGNs) by adjusting the

surface characteristics of polyhydroxyalkanoate (PHA) films which were created by using compression-mold-

ing, solvent-casting, and electrospinning methods. The compression-molded PHA membranes were dense and

flat substrates, the cast ones showed higher roughness than the compression-molded ones, and the electrospun

membranes were fibrous substrates. RCGNs could aggregate into three-dimensional (3-D) spheroid and develop

many synapses on the compression-molded and solvent-cast membranes, and they aggregated into two-dimen-

sional (2-D) flat sheet on the electrospun film in contrast. The viability of rCGNs on the electrospun membranes

was higher than that on the other PHA films because the nutrients and metabolizes could easily transport through

the highly fibrous structure of the electrospun films. RCGNs did not respond to the environmental stimuli

created by the surface characteristics of the compression-molded and solvent-cast films, while they showed

obvious difference to the specific fibrous characteristics of electrospun film in terms of morphology and via-

bility.

Keywords: Cell-substrate interaction; Electrospun fibrous film

Desalination 246 (2009) 266–273

*Corresponding author.

Presented at the conference Engineering with Membranes 2008; Membrane Processes: Development, Monitoring andModelling – From the Nano to the Macro Scale – (EWM 2008), May 25–28, 2008, Vale do Lobo, Algarve, Portugal.

0011-9164/09/$– See front matter © 2009 Elsevier B.V. All rights reserved.

doi: 10.1016/j.desal.0000.00.000

B.-Y. Yu et al. / Desalination 246 (2009) 266–273 267

1. Introduction

Polyhydroxyalkanoates (PHAs) are a class of

polyesters produced by microorganisms as intra-

cellular carbon and energy storage polymers under

unbalanced growth conditions. More than 150

kinds of PHAs consisting of various co-monomers

have been reported, but only a few of them have

been considered for commercial production, such

as poly(3-hydroxybutyrate) (PHB), poly(3-

hydroxybutyrate-co-3-hydroxyvalerate) (PHBV),

and poly(3-hydroxybutyrate-co-3- hydroxyhexa-

noate) (PHBHHx). In general, PHAs are native

polymers whose thermal and mechanical proper-

ties can be adjusted from thermoplastic to elas-

tomeric by using various compositions of their

copolymeric components. The advantageous char-

acteristics of PHAs are biocompatible, biodegrad-

able, and nontoxic [1–5]. Recently, PHAs have

been applied widely in regenerative medicine and

tissue engineering [6–7].

Cerebellar granule neurons (CGNs) constitute

the largest homogeneous neuronal population in

mammalian brain. The postnatal neurogenesis of

cerebellar granule neurons suggested that cere-

bella explanted from neonatal rats could be

advantageously used as an easy source of primary

neurons to be grown in vitro. Since the culture of

CGNs was established, the cells have become one

of the most important in vitro model to study all

the aspects of developmental, functional, and

pathological neurobiology in a rather homoge-

neous population of neurons [8]. Furthermore,

CGNs were highly sensitive to the stimulus from

the environment, so there could be a model cell to

detect the different surface characteristics of bio-

materials [9].

Electrospinning is a technology with some his-

tory as the first patent on this area was issued in

the 1930s [10]. This technique has recently been

applied to produce polymeric fibrous scaffolds for

cell culture and tissue engineering. Meshes of ran-

dom and orientated fibers with an average diam-

eter ranging from 100 nm to over 1 μm have been

prepared through the controlling of various

processing parameters. Previous studies showed

that cells on these fibrous meshes could have bet-

ter performances of proliferation, differentiation,

and metabolism than that on other films [10–15].

The micro-environment provided by the

fibrous PHA mesh was superior to that by com-

pression-molded and solvent-cast PHA films for

the proliferation of human mesenchymal stem

cells (hMSCs) in our previous study [16]. The

compression-molded PHA membranes were

dense and flat substrates, the cast ones showed

higher roughness than the compression-molded

ones and contained some dents, and the electro-

spun membranes were fibrous nonwoven sub-

strates. More detailed information about the

surface characteristics of PHA films has been dis-

cussed in our previous study [16–17], and the

behaviors of rCGNs cultured on those films are

presented here. In this study, PHAs are used as

the substrate materials. The behaviors of rCGNs

are modulated by adjusting the surface character-

istics of PHA films. It is intended to demonstrate

that the micro-environment provided by various

PHA films is also suitable for the proliferation of

rCGNs.

2. Materials and methods

2.1. Materials

Poly(3-hydroxybutyrate-co-5 mol%-3-

hydroxyvalerate) (PHBV5) and poly(3-hydroxy-

butyrate-co-12 mol%-3-hydroxyvalerate)

(PHBV12) were purchased from Aldrich, Inc.

(USA). Poly(3-hydroxybutyrate-co-8.3 mol%-3-

hydroxyhexanoate) was kindly provided by the

Procter & Gamble Co. (West Chester, Ohio,

USA). The mol% indicates the molar content of

hydroxyvalerate (HV) or hydroxyhexanoate

(HHx), respectively, in those copolymers.

2.2. Membrane preparation

The compression-molded PHBV5, PHBV12,

and PHBHHx membranes were prepared by a

268 B.-Y. Yu et al. / Desalination 246 (2009) 266–273

compression molding machine (Gotech GT-

7014). The polymer samples were placed

between two aluminum plates, heated without

any pressure at 170°C for 5 min, and then pressed

under a pressure of 9.8 MPa at 170°C for 15 min.

The melted samples were cooled to 120°C in a

period of 30 min and then cooled to ambient tem-

perature in another 10 min (step cooling). The

cast PHA films were prepared as follows. The

casting solution was prepared by dissolving 10

wt% PHBV12 (or 5 wt% for PHBHHx) in chlo-

roform (CHCl3, Mallinckrodt), and then was

poured onto a glass plate. A film-casting knife

(Braive) was pulled over the solution with con-

trolled clearance to adjust the thickness. After the

solvent evaporated in the air for 24 h, a cast PHB-

HHx film was obtained. The solvent evaporation

procedures were carried out at two temperatures,

18 and 32°C, in order to obtain different topo-

graphic morphology or surface roughness on the

cast PHA films. The apparent thickness of com-

pression-molded and solvent-cast films ranged

from 50 to 60 μm. The films were washed with

deionized water at least three times before further

applications or treatments.

Fibrous PHA membranes were prepared via

electrospinning. The polymer was dissolved in an

organic solvent mixture of chloroform (CHCl3,

Mallinckrodt) and N,N-dimethylformamide

(DMF, Tedia). The polymer solution was loaded

in a syringe capped with 21 gauge metal needle.

An electric field was created by a power supply at

12 kV between the needle and the rectangular

stainless steel receiver at the distance of 25 cm.

The polymer solution was drawn from the needle

under an accurate controlled syringe pump and

then sprayed onto the receiver by combined

forces of gravity and electrostatic charge.

2.3. Primary rCGNs culture

Cerebellar granule neurons were obtained

from 7-day-old Wistar rats according to Levi et

al . [18] with slight modification. Briefly, neurons

were dissociated from freshly dissected cerebella

by mechanical disruption in the presence of

trypsin and DNase. A variety of the PHA films

were placed in 24-well tissue culture plates

(Orange). The films were cut into 13 mm-diame-

ter discs and were tightly wedged into the culture

wells. In order to avoid film floating in the pres-

ence of medium, Teflon O-rings were used to fix

the films on the bottom of the wells. Subse-

quently, neurons were added to the culture wells

at a density of 1 × 106 cells/well in basal Eagle

medium (BME, Gibco) supplemented with 10%

fetal calf serum, 25 mM KCl, penicillin G (100

IU/ml) and streptomycin (100 mg/mL). Cultures

were maintained at 37°C in a 95% humidified

atmosphere with 5% CO2

in air. Cytosine arabi-

noside (10 mM) was added to the culture medium

to prevent replication of non-neuronal cells 1 day

after plating [18]. The samples were cultured up

to 21 days, and there was no medium change dur-

ing the process of this study. The cell behaviors

were observed by using phase contrast

microscopy every day.

2.4. Scanning electron microscopy

Various PHA films with cells were washed by

PBS and subsequently fixed in 4% glutaraldehyde

before dehydration with increasing concentrations

of ethanol, and finally they were treated with a

critical point drying (CPD) process in order to fur-

ther extract water. Dehydrated films with or with-

out cells were mounted on aluminum studs,

sputter-coated with gold-palladium, and examined

under scanning electron microscopy (Joel, JSM-

5600) at an accelerating voltage of 15 kV.

2.5. MTT assay

The viability of rCGNs cells was determined

using MTT assays after 7, 14, and 21 days of cul-

turing. A MTT assay, which assesses the rate of

mitochondrial reduction of MTT, was used to

measure the viability or the relative proliferation

B.-Y. Yu et al. / Desalination 246 (2009) 266–273 269

activity of cells. The MTT assay kit (KPL) was

used as the procedures suggested by the supplier.

Each test was quadruplicated by using an ELISA

reader (Thermo Lab systems).

3. Results and discussions

The surface characteristics of various PHA

films via compression-molding (thermal-press-

ing), solvent-casting, and electrospinning meth-

ods could obviously affect the behaviors of

rCGNs in cell morphology and proliferation. The

morphology of rCGNs on compression-molded

PHBV12 film after being seeded for 6 days is

shown in Fig. 1. Not only could rCGNs develop

many synapses, but they also established synapsis

network structure. During the same time, rCGNs

could aggregate and form 3-D cell clusters (Fig.

1b). In addition, the glia cells also adhered to the

surface and built some kinds of synapses network

structure (Fig. 1c). It was noted that rCGNs could

(a)

(b)

(c)

Fig. 1. The morphology of rCGNs on the compression-

molded PHBV12 membranes: (a) ×150, (b) and (c)

×1000 after being cultured for 6 days.

(a)

(b)

Fig. 2. The morphology of rCGNs on the compression-

molded PHBHHX membranes: (a) ×150 and (b) ×1000

after being cultured for 6 days.

270 B.-Y. Yu et al. / Desalination 246 (2009) 266–273

not be completely isolated by the current method

of rCGNs purification, but the existence of glia

cells could benefit the construction of neural net-

work. It indicated that the micro-environment of

compression-molded PHBV12 film was suitable

for rCGNs to proliferate and differentiate mor-

phologically. The behaviors of rCGNs on the

compression-molded PHBHHx film are shown in

Fig 2. RCGNs showed the same behaviors as that

on the PHBV12 film (Fig. 1). There were com-

plicated synapsis networks among the 3-D cell

clusters and glia cells (Fig. 2b). The same cell

behaviors were also found on the compression-

molded PHBV5 film and the solvent-cast

PHBV12 films.

The morphology of rCGNs on various PHA

membranes after being seeded for 11 days is

presented in Fig. 3. It was obvious that the length of

the neurites reduced and the neuronal network

structure demolished with time for rCGNs on the

cast PHBV12 films prepared at 18°C under

unchanged medium condition (Fig. 3a). RCGNs on

the compression-molded PHBV5, PHBHHx film,

and the solvent-cast PHBHHx film (prepared at

18°C) also showed the same behaviors as that on

the solvent-cast PHBV12 film. Those results

demonstrated that rCGNs could not recognize the

difference of chemical composition among various

compression-molded and solvent-cast PHA films.

The evaporation rate of the solvent, which

depends on the casting temperature, has an obvi-

ous effect on the surface morphology of films.

The surface roughness of the cast films increased

with an increase of temperature. Namely, there

(a) (c)

(b) (d)

Fig. 3. The morphology of rCGNs (a) on cast PHBV12 prepared at 18°C, (b) cast PHBV12 membrane prepared at 32°C,

(c) and (d) electrospun PHBHHx membrane after being seeded for 11 days.

B.-Y. Yu et al. / Desalination 246 (2009) 266–273 271

was a porous structure on the surface of the sol-

vent-cast film at 32°C (Fig. 3b), and the pores

were significantly larger than that (if there was

any) on the solvent-cast film at 18°C (Fig. 3a).

The films with the different degree of surface

roughness or morphologic topography were pre-

pared in order to discuss the efforts of that on the

behaviors of rCGNs. RCGNs first piled up on

each other and built a 3-D cell cluster on the com-

pression-molded PHBV12 film (Fig. 1b), and the

behaviors of rCGNs on the solvent-cast PHBV12

film also showed the same as that on the com-

pression-molded PHBV12 film. Even the surface

roughness of the cast PHBV12 film was obvi-

ously higher than that of compression-molded

PHBV12 film (Fig. 3a and b). The results illus-

trated that the surface roughness or morphologic

topography didn’t have significant effect on the

aggregation and the formation of 3-D cell clusters

for rCGNs. However, rCGNs displayed a good

spread and developed many synapses on the elec-

trospun PHBHHx film after being cultured for 11

days (Fig. 3c). This cellular behavior was signif-

icantly different from that on the compression-

molded or solvent-cast PHBHHx film. This result

indicated that the interconnected space between

fibers in the interior of the electrospun film still

had influence on the morphology of rCGNs,

although rCGNs could only adhere to the surface

and could not migrate into the interior of this film

with time. Even the cell size of rCGNs, after

being cultured for 11 days, was smaller than 10

μm, rCGNs on the electrospun film could not fall

in the space between the electrospun fibers,

which was larger than 10 μm (Fig. 3(c)).

The results of MTT assay of rCGNs on various

PHA films under unchanged medium are shown

in Fig. 4. RCGNs on the electrospun PHBHHx

film presented much higher viability than those on

the other PHA films and TCPS (as reference) on

the 7th day. Because the medium was not replaced,

the nutrients in the medium would decrease and

the metabolites from rCGNs would accumulate

with time, and then the environment became

MTT assay

BV5 BV12 BV12C HX HX-ES TCPS

O.D

. val

ue 5

70 n

m

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.457 days14 days21 days

Fig. 4. MTT assay results of rCGNs cultured on the com-

pression-molded PHBV5 (BV5), PHBV12 (BV12), and

PHBHHx (HX), solvent-cast PHBV12 (BV12C), elec-

trospun PHBHHx (HX-ES), and TCPS films after being

cultured for 7, 14, and 21days, respectively.

unsuitable for rCGNs to survive. On the 14th day,

the viabilities of rCGNs on most PHA films and

TCPS were lower that on the 7th day, however, the

viability of rCGNs on the electrospun film was

kept about the same. Even on the 21st days, the

viability of rCGNs on the electrospun film was

significantly higher than that on other PHA films.

Moreover, rCGNs on other PHA films would

aggregate and detach from the surface of film and

then suspend in the medium (Fig. 5), but this phe-

nomenon was never found on the electrospun

film. Those results suggested that the electrospun

PHBHHx film provided a suitable micro-environ-

ment for rCGNs to have a better viability in long-

term culture. Although the films with various

surface properties (chemical composition, surface

roughness, texture, and hydrophilic nature) were

prepared, rCGNs were not sensitive to those dif-

ferences and presented similar behaviors except

that on the electrospun film.

It was of interest to discuss the effect of surface

morphology of films on the behaviors of rCGNs.

Now, we focused on the results from PHBHHx

films with various surface roughness or topo-

graphic morphology, and discussed the effect of

inner fibrous structure of electrospun films on the

behaviors of rCGNs. RCGNs on the solvent-cast

272 B.-Y. Yu et al. / Desalination 246 (2009) 266–273

film and compression-molded film had similar

morphology and viability, although the surface

characteristics of those films were significantly dif-

ferent. Even though the surfaces of both solvent-

cast films and electrospun films were porous,

rCGNs on those films showed obviously different

behaviors in morphology and viability. Those

results demonstrated that the fibrous characteristics

(including surface and bulk) of electrospun film

played an important role on the cellular behaviors

in terms of adhesion, morphology, and viability.

Although rCGNs only adhered to the surface and

could not migrate into the interior of the electrospun

film, the interconnected space between fibers of

electrospun film still had influence on the cell

behaviors. We speculated that the interconnected

space facilitated the transport of nutrients and meta-

bolic products. In other words, the specific fibrous

structure of electrospun film could induce rCGNs

to perform better spread and higher viability.

4. Conclusion

The PHA films with various surface character-

istics (surface roughness and topographic mor-

phology) were prepared by compression-molding,

solvent-casting, and electrospinning methods.

RCGNs could form 3-D cell clusters and build

synapsis network on various PHA films. It

demonstrated that PHAs were suitable materials

for rCGNs, although rCGNs were not sensitive to

the change of the surface properties of films

except that on the electrospun film. Furthermore,

the viability of rCGNs on the electrospun PHB-

HHx film was higher than that on the compres-

sion- molded and solvent-cast PHBHHx films and

TCPS, and it demonstrated that the specific

fibrous characteristics of electrospun film was

suitable for rCGNs to perform better viability in

long-term culture. Because the interconnected

space between fibers of electrospun film can ben-

efit the transport of nutrients and metabolic prod-

ucts, the inner fibrous structure still hade influence

on the behaviors of rCGNs in terms of cell mor-

phology and viability.

Acknowledgments

This work was supported by the National Sci-

ence Council of the Republic of China through

the grant of NSC95-2218-E-155-001. The

authors would like to thank Dr. Isao Noda of the

Procter & Gamble Co. for his kindness in provid-

ing PHBHHx.

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