Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating

doi:10.4236/jbnb.2012.34043

Paper Menu >>

Journal Menu >>

Journal of Biomaterials and Nanobiotechnology, 2012, 3, 431-439

http://dx.doi.org/10.4236/jbnb.2012.34043 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)

431

Preparation of Porous Poly(3-Hydroxybutyrate) Films by

Water-Droplet Templating

Anna Bergstrand1*, Helene Andersson2, Johanna Cramby1, Kristin Sott2, A n et t e Lars s o n1

1Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden; 2Structure and Mate-

rial Design, The Swedish Institute for Food and Biotechnology, Göteborg, Sweden.

Email: *[email protected]

Received July 5th, 2012; revised August 24th, 2012; accepted September 3rd, 2012

ABSTRACT

Porous resorbable implants are of great interest since they may deliver bioactives or drugs, facilitate the transport of

body fluids or degradation products and provide a favorable environment for cell attachment and growth. In this work

we report on a method using concentrated emulsions to template interconnected solid foam materials and to produce

highly porous poly(3-hydroxybutyrate) (PHB) materials. Porous PHB films were cast made from water-in-oil template

emulsions including Span 80 and lithium sulphate. The films were characterized by SEM-EDX and DMA. The water

uptake of the films was recorded in order to determine the fraction water available pores. The results show that the ad-

dition of lithium sulphate allows a fine tuning of the film morphology with respect to porosity and interconnected po-

rous structure. The film porosity was determined to 51% ± 3%, 52% ± 3% and 45% ± 3% for the films made with 0%,

2.9% and 14.3% lithium sulphate in the template emulsion, respectively. The fraction water available pores was sig-

nificantly lower, 11% ±3%, 38% ±12% and 48% ± 7% for films with 0%, 2.9% and 14.3% litium sulphate respectively.

Differences in fraction water available pores and total porosity for the films reflects the film morphology and differ-

ences in pore interconnection.

Keywords: Biodegradable; Emulsion Templating; Porous; Implant

1. Introduction

Biodegradable polymers have received much attention in

the past decades due to their potential as implant materi-

als for controlled drug release or tissue engineering. The

implants act as a drug delivery device or as a temporary

support for transplantation of cells and tissue. The bio-

degradable polymer implant is being integrated and in

some cases resorbed in the body upon hydrolytic and

enzymatic degradation and there is no need for surgical

removal as it is for some non-degradable implant materi-

als. Depending of the function of the material, the biode-

gradable implant may be dense and non-porous or porous

with a low-density. Dense materials may be used for

nerve guides and vascular stents and porous materials for

injectable micro particles and scaffolds for tissue engi-

neering. Porous materials can be produced by salt par-

ticulate leaching [1-3], phase separation [4] or micro

structuring techniques [5,6]. The pore size and degree of

porosity varies, but generally these techniques result in

materials with macro porous structures of sizes from 10 -

500 μm and they include several preparation steps which

make them labor intensive and time-consuming.

A simple and straight forward method is to use emul-

sions in order to template porous structures in materials.

The emulsions may be a water-in-oil (w/o) emulsion type,

having water droplets as the internal dispersed phase and

oil as the external and continuous phase or an oil-in-wa-

ter (o/w) emulsion (with oil as the dispersed droplet

phase and water as the continuous phase). Concentrated

emulsions also referred to as high internal phase emul-

sions (HIPE) can be used to create highly porous materi-

als by emulsion templating. The HIPE has typically an

internal phase volume of more than 74%. Commonly, the

external non-droplet phase is converted into a solid

polymer and the water droplets template porous struc-

tures during solvent evaporation. This produces a highly

porous and permeable material, so called solid foam. The

spherical cavities created by the water droplets are com-

monly defined as “voids” or “cells”. In this work we will

use the term void. Under favorable conditions, small in-

terconnected pores are formed between adjacent emul-

sion droplets. These interconnecting pores between each

void and its neighbors are referred to as “windows”.

Only a few reports have been made on the production

of highly porous biodegradable polymer materials by

*Corresponding author.

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating

432

emulsion templating. They may be prepared by cross-

linking of monomers in the continuous phase of a HIPE

(forming a so called polyHIPE) [7,8] or simply solidifi-

cation of the continuous phase of an emulsion by solvent

evaporation forming a solid foam. In the present study,

we report on w/o emulsion templating in combination

with solvent casting to fabricate highly porous and per-

meable poly(3-hydroxybutyrate) (PHB) foams. PHB is a

naturally occurring polyester. When implanted in the

body it is degraded into the monomer 3-hydroxybutyric

acid, a normal mammalian metabolite [9,10]. The poly-

mer is highly crystalline and hydrophobic in nature and

degrades in a very slow rate compared to most biode-

gradable polyesters. PHB is developed as an implant ma-

terial for the regeneration and redirection of nerve cells

[11,12] and as a biodegradable gastrointestinal patch [13].

A highly porous and permeable PHB material with an

interconnected pore structure could potentially be used as

a resorbable implant platform or scaffold, resistant to

mechanical deformation for a prolonged time in a wet

environment. The polymer platform offer possibilities to

achieve controlled release of drugs. An interconnection

between pores enables the transport of acidic degradation

products out from the interior region. It could also enable

the flow of nutrients and oxygen through the material

which could be essential for cellular adhesion and growth.

To the authors’ knowledge, a porous PHB material

created by emulsion templating has only been reported in

one previous study [14]. In that study a microemulsion

was used to create highly porous PHB materials. The

usage of microemulsions to produce porous materials has

advantages, but an aspect to take into account is that mi-

croemulsions usually contain large amounts of emulsifier.

An alternative approach to make porous PHB material is

to use a regular emulsion to create water-droplet tem-

plates. In the present study we use a low amount of

emulsifier to create a w/o emulsion. The solvent is effi-

ciently driven off to increase the portion internal phase to

form a concentrated emulsion and bring the emulsion

droplets sufficiently close to create windows and voids

during solidification a process very much equivalent to

pore formation utilizing HIPE templates (See Figure 1).

Lithium sulphate is used to stabilize the emulsion and to

tune the emulsion droplet sizes and the resulting film

morphology.

2. Materials and Methods

2.1. Materials

Poly-[(R)-3-hydroxybutyric acid] (PHB), Chloroform (pu-

rity 99.0% - 99.4%, stabilized with 1% ethanol), sorbitan

monooleate (Span 80) and lithium sulphate monohydrate

were purchased from Sigma-Aldrich, (Germany) and used

as received.

2.2. Preparation of PHB Template Emulsions

and PHB Solution

A w/o template emulsion was prepared from a 7% (w/v)

PHB solution. The PHB is dissolved in the organic phase

and constitutes the continuous phase. Water is dispersed

in the oil phase to produce an emulsion with a water-

to-oil ratio of roughly 1/10. In a typical procedure mak-

ing the emulsion, 560 mg PHB was dissolved in 8 ml of

chloroform at 58˚C, under vigorous stirring for 30 - 45

minutes until a clear solution was obtained. The lithium

sulphate concentration in the water phase was 0%, 2.9%

and 14.3% (w/w), based on the polymer weight. The poly-

mer solution was cooled to room temperature before ad-

dition of 100 µl of 1.5% (w/v) Span80 in chloroform fol-

lowed by stirring for a few minutes. Aqueous solutions

of 2.9 % and 14.3% (w/v) Li2SO4 in MilliQ water were

prepared. Pure MilliQ water was used as the water phase

for 0% Li2SO4 emulsion. 800 µl of the aqueous solution

with or without lithium sulphate was slowly injected to

the PHB solution with a Hamilton syringe during homo-

genization with a disperser (IKA DI18, Brazil) for 5 min

at 22000 rpm, on iced water. For pure PHB films a 7%

(w/v) PHB solution was prepared by dissolving 560 mg

PHB in 8 ml chloroform at 58˚C, during vigorous stirring

for at least 30 min, without homogenization.

2.3. Visualization of Template Emulsions

The droplet sizes of the emulsions were visualized with a

Solvent casting of water-in-oil emulsion Formation of high water phase emulsion

and fixation of water droplets

Solid foam formation by evaporation of

solvent remains

Figure 1. Schematic picture of the preparation of porous PHB material by combining emulsion templating and solvent cast-

ng. i

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating 433

light microscope (Nikon, Microphot-FXA) equipped with

a Plan 40 × 0.7 differential interference contrast, DIC, oil

objective. The images were acquired using a 8 bit RGB

color camera (Altran 20, Soft imaging system).

2.4. Casting of Films from Template Emulsions

and Pure PHB Solutions

Films were prepared by solvent casting of PHB solutions

into glass petri dishes. About 7 ml of the prepared emul-

sions or pure PHB solutions were poured into petri dishes

with a diameter of 6 cm. Tryouts were made to establish

the optimal drying conditions and solvent evaporation

rate that rendered the best quality of the resulting film.

The solvent evaporation rate during film formation was

monitored and determined by gravimetric analysis. Typi-

cally, the petri dish was covered with a glass lid leaving

an opening of about 1 mm between the dish and lid dur-

ing film formation to achieve the best film quality. The

resulting films were left to dry for about 36 hours at 22˚C

in a foiume cupboard and subsequently in a vacuum oven,

at 40˚C for 24 hours, in order to evaporate the remains of

chloroform. The film thickness films was measured by a

micrometer gauge (Mitutoyo, Japan) and determined from

an average of the thickness at five positions of each sam-

ple. The range of thickness means was 145 - 175 µm.

2.5. Film Morphology and Elemental Analysis

The dry films were cut into sections, repeatedly sub-

merged into liquid nitrogen for a few minutes and freeze

fractured. The films were sputter-coated with gold and

visualized by in-line and horizontal detection mode using

a field emission scanning electron microscope (SEM),

(Leo Ultra 55 FEG SEM), at a magnification of 5000 at 3

kV. The location of sulphur in the films, originating from

lithium sulfate in water phase of the template emulsion,

was established by elemental analysis using energy dis-

persive x-ray (EDX), (Inca Oxford Instruments), analysis

on selected areas on the film surface.

2.6. Water Uptake and Porosity in Films

The water uptake of the films was determined gravimet-

rically. The experiments were carried out on triplicate

samples (1.7 × 1.7 cm) from 4 different types of film

preparations. The film samples were submerged in phos-

phate buffered saline (PBS) buffer, pH 7.4 at 37˚C during

magnetic stirring. The film samples were removed at pre-

determined time points, excess PBS was carefully wiped

off with a Kleenex, and finally the samples were weighed

and afterwards re-submerged in PBS. In order to deter-

mine the apparent porosity, circular samples of Ø 5 mm

were punched, weighed and the thickness measured by a

micrometer gauge (Mitutoyo, Japan). The apparent den-

sity was calculated from the weight and dimensions for

each of the porous PHB films (n = 3).

2.7. Dynamic Mechanical Analysis on Films

Submerged in Water

The mechanical properties of PHB films prior to and

after submersion in water were measured using a Rheo-

metrics RSAII (Rheometrics Scientific, Piscataway, USA)

dynamic mechanical analysis (DMA) apparatus equipped

with an in-house designed submersion cell [15]. Film

strips were prepared to a width of 4 mm using a razor-

edged punch. The thickness was determined as the aver-

age of three measurements and the effective initial sam-

ple length in the DMA was approximately 22 mm. The

films were mounted in the DMA apparatus and a small

static force was applied to ensure proper stretching of the

sample. After 7 min from start, 40 ml of PBS solution

adjusted to a temperature of 22˚C was added. Measure-

ments were performed in a strain-controlled stretching

mode with a static force. The amplitude of the dynamic

strain was set to 0.03% and the static force was set to

exceed the amplitude of the harmonic dynamic force by

20%. The complex modulus (E*) and the loss factor (tanδ)

were calculated from the force response and the dimen-

sions of the sample as recorded during the measurement.

Equilibrium tanδ and E* values were taken as the average

of the plateau values prior to and after PBS buffer addi-

tion.

3. Results and Discussion

3.1. Template Emulsion Morphology and

Stability

As the film porous structure is derived from the template

emulsion the emulsion preparation conditions, the droplet

size, and droplet distance will strongly influence the

morphology of the final film material. The template

emulsions were studied by light microscopy and pictures

were taken about 15 minutes after emulsion preparation.

Figure 2 shows representative light microscopy pictures

of template emulsions prepared with: (a) no Li2SO4 and

(b) 2.9% Li2SO4 in the water phase. The pictures show

that the water droplets are manifold and that there is a

great variation in droplet size. As clearly can be seen, the

template emulsion including litium sulphate (Figure 2(b))

mainly contain droplets sizes of 0.5 - 3 µm. A few larger

droplet species can occasionally be observed but the

main population is in the mentioned size range. The size

distribution is reasonably narrow and the droplets are

located quite tight in the initial emulsion. The emulsion

without Li2SO4 (Figure 2(a)) in the water phase displays

sizes between 0.5 and 7 µm and the emulsion shows

greater polydispersity in droplet size compared to all

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating

434

(a) (b)

Figure 2. Light microscope pictures of template emulsions prepared with: (a) No Li2SO4 and (b) 2.9% Li2SO4 in the water

phase. The pictures were taken about 15 minutes after emulsion preparation. The scale bar is 10 µm.

emulsions with litium sulphate included in the water

droplets.

The emulsion droplet size is an important parameter

which governs the kinetic stability of an emulsion. It is

well established that an emulsion containing uniform and

small droplets is the most favorable one in terms of sta-

bility. The small droplet sizes reduce the tendency of the

emulsion to undergo coalescence and/or Ostwald ripen-

ing. Ostwald ripening is a process where larger emulsion

droplets grow on the expense of smaller species, due to

diffusion of internal phase molecules through the con-

tinuous phase. The outcome of Ostwald ripening is pro-

gressive coarsening of the emulsion, coalescence events

and eventually emulsions break-down. Emulsions con-

taining litium sulphate all display smaller droplet sizes

and a narrow size distribution which is favorable in terms

of emulsion stability. The emulsion stability test (results

not shown) demonstrations that the emulsion stability

was greatly influenced by the Li2SO4 content. It was

found that emulsions including litium sulphate are stabile

for 24 hours or longer. The Li2SO4 depleted emulsions

have separated after 30 minutes which is observed as a

creaming layer atop in the vial. The litium sulphate

clearly has a stabilizing effect on the emulsion by gov-

erning the droplet sizes. There are examples in literature

of the stabilizing effect of electrolytes in the water phase

of w/o emulsions [16,17]. Increased electrolyte concen-

tration decreases the tendency for Ostwald ripening and

increase the emulsion stability. The addition of electro-

lytes to the water phase decreases the transport of inter-

nal phase into the external phase, which results in slower

Oswald ripening.

3.2. Morphology of PHB Films

The PHB films were produced from solvent casting of

PHB solution and w/o template emulsions and then cha-

racterized by SEM. The result is displayed in Figure 3.

The pictures (a)-(c) show the cross sections and surfaces

(d)-(f) of films cast from emulsions with varying Li2SO4

content. The films made from PHB solutions are non-

porous and dense (see Figures 3(g) and (h)). The mor-

phology of the films produced from the emulsion tem-

plates varies in appearance. The interior of the film lack-

ing Li2SO4 in the water phase of the template emulsion

show non-spherical, slightly elongated voids whose sizes

ranging from about 1 - 6 µm. The polymer walls are dif-

fering in thickness and a few small windows of about 0.5

µm are visible. The upper film surface is rough (see

Figure 3(d)) with only a few small pores of 0.5 µm. The

appearance of the upper surface of film (Figure 3(d))

made from the emulsion template without Li2SO4 sug-

gests that there has been a phase separation during the

film casting procedure resulting in skin formation on the

upper surface.

Cross sections of the films made from emulsion tem-

plates with 2.9% and 14.3 % Li2SO4 in the water phase

show a more uniform void size of approximately 1 - 2

µm, with several interconnecting windows of about 0.5

µm. The polymer walls are thin and of reasonably even

thickness. The upper film surfaces show some variation

in film morphology but mainly display pores of ap-

proximately the same sizes and there is no significant

skin formation on the film. The results show that the wa-

ter droplet sizes of the emulsion templates (sizes 0.5 - 3

µm) is mostly translated into the resulting films and that

a solid foam material with neighboring voids is formed.

The SEM pictures also show the presence of intercon-

nected windows between adjacent voids. The intercon-

nected windows are created in an analogue manner to the

solidification of foam materials from HIPE, where the

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating 435

(a) (b) (c)

(d) (e) (f)

(g) (h)

Figure 3. SEM micrographs of surfaces and freeze fractured cross sections of non-porous PHB films and porous PHB films

made from w/o emulsion templates with varying amount of Li2SO4 in the water phase. The pictures show the cross sections

(a)-(c) and upper surface (d)-(f) of films with 0% Li2SO4, 2.9% Li2SO4 and 14.3% Li2SO4 (from left to the right) and the up-

per surface (g) and cross section (h) of a PHB film.

high concentration of inner phase and tight packing of

emulsion droplets gives rise to window formation. Here

the highly volatile organic solvent of the template emul-

sion is rapidly driven off during film casting rendering

the emulsion into a highly concentrated emulsion. This

leads to an increase in solution viscosity and a fixation of

the water droplets in the polymer matrix during film

casting. The formation of a highly concentrated emulsion

enables the formation of windows between adjacent wa-

ter droplets during the film casting.

The solvent evaporation and film formation must be

made in a controlled manner. A too slow evaporation rate

may induce phase separation of the emulsion during the

film casting whereas a too quick evaporation on the other

hand leads to a curly and deformed film. It is therefore

necessary to monitor and optimize the drying process in

order to avoid phase separation. Several try-outs were

made in order to produce films of required quality. The

mass loss during film drying shown in Figure 4 is pre-

sented to demonstrate the range of conditions that were

tested to achieve different solvent evaporation rates and

to produce films of different qualities. The evaporation

rate was too quick for films cast with half open lid and

too slow for films cast with totally closed lid resulting in

films of bad qualities. The films cast in a fume hood

with a gap of approximately 1 mm between the petri

dish and a glass lid, in order to make a solvent saturated

microclimate and to control the evaporation rate, were

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating

436

Figure 4. Solvent evaporation rate of the PHB film casting.

found to be of good quality. At 120 minutes, 50% of the

solvent has evaporated and a highly concentrated emul-

sion is present. During these conditions, the droplets

sizes of the template emulsion were translated into the

casted film during film casting and films were produced

in a reproducible manner. Considering the different film

morphologies, it is apparent that Li2SO4 is a vital com-

ponent of the emulsion template in order to obtain films

with controlled structures and uniform sizes. Also the

film casting procedure with the solvent evaporation is a

key step in order to produce reproducible films of desired

quality.

3.4. Location of Li2SO4 in the Porous PHB Films

as Studied by SEM-EDX

SEM-EDX was made in order to establish the location of

Li2SO4 after the film formation. SEM-EDX were made

on two areas of equal size, taken on the flat surface and

deep in a pore of the upper surface of porous PHB films

(See Figure 5). The upper EDX spectrum show the pres-

ence of sulphur peaks in the interior of the pore. No sul-

phur peak is visible when EDX analysis was made on the

flat upper surface (see the lower EDX spectra in Figure

5). From these results it can be concluded that Li2SO4 is

located mainly inside the pores, probably lining the pore

wall. Since no sulphur peak was recorded from the area

on the flat surface, then there is probably no litium sul-

phate residing in the polymer matrix. The Li2SO4 salt

precipitation which is lining the pore walls might influ-

ence the material properties of the porous film.

3.5. Mechanical Properties of PHB Films before

and after Water Exposure

Dynamic mechanical analysis, DMA, was used to exam-

ine if the mechanical properties of the porous material

were retained after water exposure. The complex mo-

dulus, , is considered as a measure of rigidity and

mirrors the ability of a material to resist deformation

when it is exposed to stress. The deformation of a mate-

rial may be a combination of an elastic and a viscous

portion. The viscous and the elastic portion are described

by the loss modulus and storage modulus



E







respectively. As the loss factor, tan



, is the ratio be-

tween E



 and E



an ideal elastic solid would have no

viscous contribution and tan



would be zero. The

complex modulus and tan



before and after water ex-

posure were determined for the different films, see Table

1. The percent change after water exposure of the com-

plex modulus





E was calculated according to:

*100





 (1)

where and are the plateau values for the com-

plex modulus of the dry and water exposed films, respec-

tively. Similarily, the percent change of the loss factor





tan



 after water exposure was determined from the

plateau values of the loss factor from the dry, tan d



, and

wet film, tan w



, using the following equation:

tan tan

tan 100

tan







 (2)

In the dry state the non-porous PHB film had an

of 1.9 GPa, which is considerable larger than the porous

films with values of 0.7 - 1.2 GPa. This decrease in

is quite reasonable since porous films normally have

a lower mechanical strength compared to the corre-

sponding dense material [18]. The DMA measurements

on the porous PHB films in the dry condition show that

the increased with increasing litium sulphate con-

tent in the template emulsion (Table 1). This is probably

an effect of the spatial location of litium sulphate, lining

the interior of the pores as indicated in Figure 5, rein-

forcing the structure and thereby increasing the value of

for these materials. However, since litium sulphate

is a water soluble additive it is readily washed out upon

water contact leading to a decrease in between 12%

and 35% after water exposure, which can be seen in Ta -

ble 1.

The values of the loss factor, tan



, in Table 1 are

very low, indicating that both the porous and non-porous

PHB materials behave as an elastic solid material in the

dry state. Submerging the films in water could theoreti-

cally have a plasticizing effect on the material due to

water penetration into the material and this should then

increase the loss factor. The non-porous PHB film is

more or less unaffected by water exposure in comparison

to the emulsion prepared films which have tan





(percent change after exposure to water) values all above

40% (Table 1). From this one could argue that water acts

as a plasticizer for the emulsion prepared films, but not

for the pure PHB film. However, the loss factor is still

very low and the increase in tan



could be due to that

the dissolved additives migrates to the water phase,

hich results in a somewhat more flexible material. w

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating

437

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Full Scale 105 cts Cursor: 3.490 (1 cts) keV

Electron Image 1

10 μm

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Full Scale 276 cts Cursor: 1.603 (11 cts) keV

Electron Image 1

10 μm

Figure 5. SEM-EDX on two areas of equal size taken on the flat surface and in a pore of the porous PHB films. The upper

spectrum shows sulphur peaks w hich originates from the counter ion of lithium. Please note that the scale on the lower graph

is enlarged compared to the upper graph.

Table 1. The complex modulus, *

, and loss factor, , of the dry non-porous PHB films and dry porous PHB films

made from emulsion templates, and percent change after water exposure determined according to Equations (1) and (2). Val-

ues within parentheses indicate min/max deviation from the average of (n = 2) measurements.

tan d

E tand



E tan





(GPa) (%) (%)

PHB 1.9 (0.096) 0.036 (0.0010) 3.59 (0.1) 1.8 (0.7)

PHB emulsion 0% Li2SO4 0.7 (0.008) 0.024 (0.0002) –12.1 (0.7) 41.7 (2.6)

PHB emulsion 2.86 % Li2SO4 0.8 (0.004) 0.023 (0.0003) –16.5 (0.3) 58.2 (1.7)

PHB emulsion 14.3 % Li2SO4 1.2 (0.006) 0.029 (0.0006) –35.3 (0.8) 72.2 (1.3)



  (3)

3.6. Water Uptake and Film Porosity

The water uptake of a porous polymer material may ei-

ther take place in the pore structures only, as penetration

of water in the polymer matrix or as a combination of

both, depending on the time scale, the hydrophilic nature

of the polymer material and the pore interconnectivity.

The porosity was determined to 52% ± 3% and 45% ±

3% for the films with 2.9% and 14.3% litium sulphate in

the template emulsion, respectively. The film made from

template emulsion without the litium sulphate showed to

have 51% ± 3% porosity.

The total film porosity (Φ), see Equation (3), was de-

termined by calculating the apparent density of the film





and comparing it to the true density





of PHB

(1.22 g·cm–3) [19]. The a



was derived from the weight

and dimensions of the porous PHB films.

The degree of water uptake is an important factor since

it reflects the wettability of the film interior but more

important it reflects the void interconnectivity of the

porous material. Figure 6 shows a plot of the water

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating

438

100

0 204060801001

Water absorption (%)

Time (min)

PHB 0 % Li2.86 % Li14.3 % Li

Figure 6. Plot of the water absorption capacity of PHB films

made either by w/o emulsion te mplates with varying amount

of Li2SO4 in the water phase, or PHB only.

absorption capacity of porous and non-porous PHB films

during 120 minutes. The water uptake is calculated as

follows:

100





(4)

where A is absorbed water amount, o

W is the initial

weight of the film and Ww is the weight of the wetted film.

Initially there is fast water absorption for the porous

PHB films made from emulsion templates. At 120 min-

utes the curves level out to reach a value of 15%, 65%

and 70% water absorption for the films made from emul-

sion templates with 0%, 2.9% and 14.3% Li2SO4. The

water uptake of the non-porous PHB film is on a minor

level, as expected. The higher the amount of Li2SO4 in

the template emulsion, the higher the water uptake and

the rate of water intrusion into the PHB film. The ini-

tially fast water absorption of films made from template

emulsions including litium sulphate may be a result from

capillary forces arising from the greater extent of small

voids and connections between the voids compared to

films made from lithium deficient template emulsions. It

could also be due to the presence of Li2SO4 which is lin-

ing the interior of the pore walls rendering the material

hydrophilic in character, hence increasing the wettability

of the otherwise hydrophobic polymer.

Considering the hydrophobic nature of PHB it is rea-

sonable to believe that the water uptake of the porous

PHB films is to a major extent dependent on the pore

structure, the number of voids and interconnecting win-

dows. The water uptake of the films can be used to cal-

culate the portion of water available pores for each film

type by simply correcting the film weights for the density

difference between water and the polymer. Films pro-

duced from template emulsions including litium sulphate

have comparable volume fractions of water available

pores, 38% ± 12% and 48% ± 7% for films with 2.9%

and 14.3% litium sulphate respectively. The morphology

of the films also appear very similar as observed from

SEM images (Figure 3). However, the film made from

lithium deficient template emulsions show a substantially

lower fraction of water available pores of 11% ± 3% only.

The total porosity was considerably higher, 51% ± 3%.

This difference probably reflects the film morphology

(See Figure 3) that shows irregular and thick polymer

walls between the voids, leading to less interconnection

between the voids. If there are pores which are not inter-

connected there may be areas which are not reached by

the water intrusion. The total porosity is more or less the

same for all film types but the fraction water available

pores differs. It is significantly less for the film made

from litium sulphate deficient emulsion template which

indicates lower pore interconnectivity due to different

film morphology.

4. Conclusion

Highly porous biodegradable PHB films were prepared

by combining basic w/o emulsion assembly with solvent

casting to achieve concentrated emulsions suitable for

templating interconnected solid foam materials. The

films have a tunable morphology with a resulting porous

and interconnected structure. Differences in fraction wa-

ter available pores and total porosity for the films made

from emulsion templates reflects the film morphology

and differences in interconnection between the voids.

5. Acknowledgements

We sincerely thank Anders Mårtensson at the Depart-

ment of Chemical and Biological Engineering for help

with the SEM micrographs. Financial support was kindly

provided by the Vinn Excellence Centre SuMo Biomate-

rials (Supermolecular Biomaterials—structure dynamics

and properties). Additional funding from Chalmers Area

of Advance—Materials Science is gratefully acknow-

ledged.

REFERENCES

[1] L. Lu, S. J. Peter, M. D. Lyman, H. L. Lai, S. M. Leite, J.

A. Tamada, S. Uyama, J. P. Vacanti, R. Langer and A. G.

Mikos, “In Vitro and in Vivo Degradation of Porous

Poly(dl-lactic-co-glycolic Acid) Foams,” Biomaterials,

Vol. 21, No. 18, 2000, pp. 1837-1845.

doi:10.1016/S0142-9612(00)00047-8

[2] A. G. Mikos, G. Sarakinos, S. M. Leite, J. P. Vacanti and

R. Langer, “Laminated Three-Dimensional Biodegradable

Foams for Use in Tissue Engineering,” Biomaterials, Vol.

14, No. 5, 1993, pp. 323-330.

doi:10.1016/0142-9612(93)90049-8

[3] C. Vaquette, C. Frochot, R. Rahouadj and X. Wang, “An

Preparation of Porous Poly(3-Hydroxybutyrate) Films by Water-Droplet Templating

439

Innovative Method to Obtain Porous Poly(L-lactic Acid)

Scaffolds with Highly Spherical and Interconnected

Pores,” Journal of Biomedical Materials Research, Part

B, Vol. 86B, No. 1, 2008, pp. 9-17.

doi:10.1002/jbm.b.30982

[4] H. Lo, M. S. Ponticiello and K. W. Leong, “Fabrication

of Controlled Release Biodegradable Foams by Phase

Separation,” Tissue Engineering, Vol. 1, No. 1, 1995, pp.

15-28. doi:10.1089/ten.1995.1.15

[5] A. C. R. Grayson, I. S. Choi, B. M. Tyler, P. P. Wang, H.

B rem, M. J. Cima and R. Langer, “Multi-Pulse Drug De-

livery from a Resorbable Polymeric Microchip Device,”

Nature Materials, Vol. 2, No. 11, 2003, pp. 767-772.

doi:10.1038/nmat998

[6] W. H. Ryu, M. Vyakarnam, R. S. Greco, F. B. Prinz and

R. J. Fasching, “Fabrication of Multi-Layered Biode-

gradable Drug Delivery Device Based on Micro-Struc-

turing of PLGA Polymers,” Biomedical Microdevices,

Vol. 9, No. 6, 2007, pp. 845-853.

doi:10.1007/s10544-007-9097-8

[7] E. M. Christenson, W. Soofi, J. L. Holm, N. R. Cameron

and A. G. Mikos, “Biodegradable Fumarate-Based Poly-

High Internal Phase Emulsions (HIPE) as Tissue Engi-

neering Scaffolds,” Biomacromolecules, Vol. 8, No. 12,

2007, pp. 3806-3814. doi:10.1021/bm7007235

[8] Y. Lumelsky and M. S. Silverstein, “Biodegradable Po-

rous Polymers through Emulsion Templating,” Macro-

molecules, Vol. 42, No. 5, 2009, pp. 1627-1633.

doi:10.1021/ma802461m

[9] R. N. Reusch, A. W. Sparrow and J. Gardiner, “Transport

of Poly-β-Hydroxybutyrate in Human Plasma,” Biochi-

mica et Biophysica Acta, Vol. 1123, No. 1, 1992, pp. 33-

40.

[10] T. Saito, K. Tomita, K. Juni and K. Ooba, “In Vivo and in

Vitro degradation of Poly(3-Hydroxybutyrate) in Rat,”

Biomaterials, Vol. 12, No. 3, 1991, pp. 309-312.

doi:10.1016/0142-9612(91)90039-D

[11] M. Aberg, C. Ljungberg, E. Edin, H. Millqvist, E. Nordh,

A. Theorin, G. Terenghi and M. Wiberg, “Clinical Eva-

luation of a Resorbable Wrap-Around Implant as an Al-

ternative to Nerve Repair: A Prospective, Assessor-

Blinded, Randomised Clinical Study of Sensory, Motor

and Functional Recovery after Peripheral Nerve Repair,”

Journal of Plastic, Reconstructive and Aesthetic Surgery,

Vol. 62, No. 11, 2009, pp. 1503-1509.

doi:10.1016/j.bjps.2008.06.041

[12] P. N. Mohanna, R. C. Young, M. Wiberg and G. Terenghi,

“A Composite Poly-Hydroxybutyrate-Glial Growth Fac-

tor Conduit for Long Nerve Gap Repairs,” Journal of

Anatomy, Vol. 203, No. 6, 2003, pp. 553-565.

doi:10.1046/j.1469-7580.2003.00243.x

[13] T. Freier, C. Kunze, C. Nischan, S. Kramer, K. Sternberg,

M. Sass, U. T. Hopt and K.-P. Schmitz, “In Vitro and in

Vivo Degradation Studies for Development of a Biode-

gradable Patch Based on Poly(3-Hydroxybutyrate),” Bio-

materials, Vol. 23, No. 13, 2002, pp. 2649-2657.

doi:10.1016/S0142-9612(01)00405-7

[14] Z. Cai, “Biocompatibility and Biodegradation of Novel

PHB Porous Substrates with Controlled Multi-Pore Size

by Emulsion Templates Method,” Journal of Materials

Science: Materials in Medicine, Vol. 17, No. 12, 2006, pp.

1297-1303. doi:10.1007/s10856-006-0604-x

[15] S. Edrud, M. Petersson and M. Stading, “DMA Analysis

of Biopolymer Film Swelling,” Annual Transactions of

the Nordic Rheology Soceity, Vol. 11, 2003, pp. 155-156.

[16] N. R. Cameron, “High Internal Phase Emulsion Templat-

ing as a Route to Well-Defined Porous Polymers,” Poly-

mer, Vol. 46, No. 5, 2005, pp. 1439-1449.

doi:10.1016/j.polymer.2004.11.097

[17] M. Y. Koroleva and E. V. Yurtov, “Effect of Ionic

Strength of Dispersed Phase on Ostwald Ripening in Wa-

ter-in-Oil Emulsions,” Colloid Journal, Vol. 65, No. 1,

2003, pp. 40-43. doi:10.1023/A:1022362807131

[18] S. Ji, Q. Gu and B. Xia, “Porosity Dependence of Me-

chanical Properties of Solid Materials,” Journal of Mate-

rials Science, Vol. 41, No. 6, 2006, pp. 1757-1768.

doi:10.1007/s10853-006-2871-9

[19] C. Pedros-Alio, J. Mas and R. Guerrero, “The Influence

of Poly-β-Hydroxybutyrate Accumulation on Cell Vol-

ume and Buoyant Density in Alcaligenes Eutrophus,”

From Archives of Microbiology, Vol. 143, No. 2, 1985,

pp. 178-184. doi:10.1007/BF00411044