Poly(3-hydroxybutyrate) (PHB) is a polyester which shows excellent biocompatibility and a PHB material is therefore considered suitable for many biomedical applications. A highly porous PHB material may be designed to facilitate the transport of small molecules and body fluids or serve as a biocompatible temporary barrier. In this study, PHB films with varying degree of porosity and pore interconnectivity were made by solvent casting using water-in-oil emulsion templates of varying composition. The morphology was characterized by SEM and the water permeability of the films was determined. The results show that an increased water content of the template emulsion resulted in a film with increased porosity. A fine tuning of the film morphology of the casted films was achieved by varying the salt content of the water phase of the template emulsion. The porosity of these films was roughly the same but the water permeability varied between
Poly(3-hydroxybutyrate) (PHB) is considered to be a polymer that has high potential as a biodegradable implant material. The polymer is a well examined thermoplastic polyester of microbial origin which shows excellent biocompatibility in contact with tissue and blood [
The fabrication of highly porous materials with interconnected pores is of great interest in the field of tissue engineering since it enables the passage of fluid and transport of small molecules such as nutrients, drugs, or bioactive molecules to soft tissue and cells. If the material is made macroporous then cells may interpenetrate via the pore structures. However, if the material is made microporous (pore sizes of a few micrometers and below) such a material has the potential to serve as a biocompatible temporary barrier preventing cellular ingrowth yet facilitating transport to adjacent soft tissue and cells. Moreover, by combining a porous and a dense backing layer, new permeability and degradation properties are acquired which opens up for additional applications such as directional transport of small bioactive molecules or side directed cell growth.
Currently there are several different techniques such as salt leaching [
Poly-[(R)-3-hydroxybutyrate], PHB, (natural origin), chloroform (purity 99.0–99.4%, stabilized with 1% ethanol), sorbitan monooleate (Span 80), and lithium sulphate monohydrate were purchased from Sigma-Aldrich, (Germany). Milli-Q water was used for the emulsion preparation. Ultima gold and tritiated water were purchased from PerkinElmer (USA). All chemicals were used as received.
Water-in-oil (w/o) template emulsions were prepared from a 7% (w/v) PHB solution and a water phase consisting of lithium sulphate salt and Milli-Q water. A range of template emulsions was produced by varying the amount of water (0–10% (v/v)) and lithium sulphate (0–14.3% (w/v)) of the emulsion. In a typical procedure making the emulsion, 560 mg PHB was dissolved in 8 mL of chloroform at 58°C, under vigorous stirring with a magnetic bar for 30–45 minutes until a clear solution was obtained. The polymer solution was cooled to room temperature before addition of 100
Films were prepared by solvent casting of PHB solutions into glass Petri dishes. Approximately 7 mL of the prepared emulsions or pure PHB solutions was poured into Petri dishes with an inner diameter of 7 cm. Tryouts were made to establish the optimal drying conditions and solvent evaporation rate that gave the best quality of the resulting film. Typically, the PHB solution was poured out and preconcentrated by solvent evaporation from the emulsion for two minutes with the lid open on the dish. Subsequently, the Petri dish was covered with a glass lid leaving an opening of about 1 mm between the dish and lid during film formation. The films were left to dry for about 36 hours at 22°C in a fume 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 sample.
The first layer was prepared from a solution of 2 mL of 3.5% (w/v) PHB dissolved in chloroform at 58°C, during vigorous stirring for 30–45 minutes until a clear solution was obtained. The PHB solution was casted in a Petri dish forming a first film layer by preconcentrating the polymer solution for two minutes without lid, closing the lid for two minutes, and finally the lid was removed for two more minutes generating a sticky semidry film. A second layer was casted on top of the first PHB layer in the Petri dish by adding 4 mL of an emulsion consisting of 10% (v/v) water, 7% (w/v) PHB, 2.9% (w/v) Li2SO4, and 1.5% (w/w) Span80. The emulsion was prepared according to the method described in Section
The dry films were cut into sections, repeatedly submerged into liquid nitrogen for a few minutes, and freeze-fractured to display the interior regions of the film. The film specimens were sputter-coated with gold and visualized by in-lens and horizontal detection mode using a field emission scanning electron microscope (SEM) (Leo Ultra 55 FEG SEM) at a magnification of 5–30 k at 3 kV.
The water permeability of the films was determined using a permeability chamber with a setup previously described [
Circular samples of 5 mm in diameter were punched and weighed and the thickness measured by a micrometer gauge (Mitutoyo, Japan). The apparent film density was determined from the weight and dimensions of each of the porous PHB films (
Template emulsions with adjustable amount of included water and lithium salt content were used for the fabrication of films with varying extent of porosity. Films were also made from a pure PHB solution to produce dense and nonporous PHB films. In addition, bilayer films were made by cocasting a dense PHB layer with a highly porous layer of PHB. The films were then characterized with respect to morphology, water permeability, and porosity.
All films were visually inspected after the casting and drying procedure. The film cast from a PHB solution was semitransparent with a smooth upper surface. The PHB films casted from emulsion templates appeared increasingly whiter and less transparent with increasing amount of included water in the template emulsion. The upper surfaces appear smooth for all the films produced from emulsion templates at an ocular inspection. SEM was used to visualize the film morphology and disclose the pore structures and distribution at the surface and cross sections of the films made from emulsion templates and pure PHB films. The SEM images of cross sections from films made with 3–10% of water in the emulsion template and pure PHB are shown in Figure
SEM images of PHB film cross sections made with (a) no emulsion template, (b) 3%, (c) 6%, (d) 8%, (e) 9%, and (f) 10% of water in the emulsion template. The lithium salt content of the water phase was 2.9% (w/v) for all template emulsions. The scale bar is 2
The SEM images show that the films made from emulsion templates including 3 and 6% water (Figures
SEM image of the surface of a PHB film made with 10% of water in the emulsion template taken with in-lens detection mode. The scale bar is 1
The results from the SEM experiments show that a high fraction of water in the template emulsion results in a highly porous PHB film. For the film with 10% of water in the emulsion template, the individual pores are connected through windows possibly forming a network of interconnected pores in the film. With interconnected pores, the passages and transport of water and other molecules are enabled through a film with the prospect to be used for delivery or barrier applications.
Formation of bilayer PHB films consisting of a porous and a dense backing layer was explored in order to obtain films with new degradation characteristics that could enable a directional transport of nutrients or drugs or provide additional mechanical strength to a porous film. A two-step method is in many cases the preferred choice since it offers a way of controlling the thickness and the morphology of this second layer in comparison to a spontaneously formed two-layered film. These film layers need to be well fused together with no gap or slip between the layers in order to give a mechanical support or directed drug delivery. The SEM image in Figure
SEM image taken by in-lens detection mode of a bilayer PHB film combining a dense PHB layer with a porous layer made with 10% of water in the emulsion template showing (a) the whole cross section (scale bar 10
There are many factors that affect the molecular diffusion of a permeant through a film, mainly the diffusion routes available in the polymer matrix but also the size and chemical properties of the permeant. A major factor determining the diffusion rate is the presence of pores or channels to pass through.
The permeability of a film is closely related to the diffusion of the solute or permeant. Fick’s first law of diffusion is
Exemplifying plot of the amount of radioactive water in CPM (counts per minute), having diffused across a single layer PHB film made from an emulsion template consisting of 10% of water and 2.9% (w/v) of Li2SO4.
For a porous PHB film, water will mainly pass through the available pores and channels, not penetrating the polymer matrix, due to the inherent crystallinity and hydrophobicity of PHB [
Plot of the water permeability of films casted from template emulsions with varying water content in the emulsion template. The permeability was normalized against the film thickness. Error bars indicate min/max deviation (
The permeability of double layered films was also determined. Figure
Water permeability of porous, dense (nonporous), and bilayer films casted from template emulsions with 10% (v/v) of water and 2.9% (w/v) of Li2SO4 in the emulsion template. The permeability was normalized against the film thickness. Error bars indicate min/max deviation (
In summary, these data show that the bilayer film has equally low permeability as a dense PHB film and in addition enables unidirectional drug release or new degradation characteristics.
It has previously been shown by us that including lithium sulphate in the water phase is a way of facilitating the translation of template emulsion droplets into spherical pores which are evenly dispersed in the resulting PHB film [
Plot of the water permeability for films casted from template emulsions with 10% of water and increasing Li2SO4 content in the water phase. The permeability was normalized against the film thickness. Error bars indicate min/max deviation (
The film made from 0% salt in the template emulsion has a very low permeability of approximately
The diverse water permeability of PHB films made from emulsion templates with varied salt content is most likely a result of differences in the film morphology and window formation between pores in film. The low permeable PHB films would reasonably then have a lower degree of porosity or interconnectivity between the pores than the high permeable PHB films. The SEM images of these films however are very similar, showing highly porous films with windows of approximately 0.2–0.5
The degree of porosity of films made from template emulsions with varying amount of water or lithium salt in the water phase was determined in order to see how well the degree of porosity correlates with the respective film permeability. The porosity (
The porosity of films casted from template emulsions with a variation in water volume and lithium salt content (
Water content | Li2SO4 content | Porosity |
---|---|---|
% (v/v) | % (w/v) | % (w/w) |
0 | 0 | 1 ± 0 |
5 | 2.9 | 16 ± 14 |
6 | 2.9 | 17 ± 14 |
8 | 2.9 | 39 ± 7 |
9 | 2.9 | 47 ± 6 |
10 | 2.9 | 52 ± 3 |
10 | 0 | 51 ± 3 |
10 | 1.4 | 51 ± 6 |
10 | 2.9 | 52 ± 3 |
10 | 8.7 | 57 ± 7 |
10 | 14.3 | 45 ± 3 |
The solvent casted pure PHB film made without emulsion template showed almost no porosity. The film porosity increases with the amount of water in the emulsion template. The porosities of films made from 5 and 6% (v/v) water in the template emulsion are about 16-17%. The quite large standard deviation for the 5 and 6% (v/v) films is probably an effect of the low viscosity of the sample which results in a less stabile template emulsion and variations in the template emulsion which is translated to the resulting film. The film morphology of these films is somewhat different than the other porous films made from emulsion templates. Films made from template emulsions with 8–10% (v/v) water showed an increase in degree of porosity from 47% up to 52% with increased concentration. However, films made from template emulsions of 10% (v/v) water show little effect of varied lithium salt content in the water phase on the degree of porosity (45–57%). A film made from a lithium salt deficient template emulsion results in a film with as high as 51% porosity. The results from porosity analysis show that the concentration of salt in the template emulsions has little or no effect on the porosity of the films. However it seems that there is a relationship between the amount of water in the emulsion template and the degree of porosity of the tested films.
Taken all data into account, the SEM images and the porosity analysis provide information about the film morphology and porosity. However it is essential to perform permeability analysis for these kinds of films to get an understanding of the pore interconnectivity.
In this study it was shown that it is possible to make highly porous PHB films with a tunable permeability by changing the composition of the emulsion template. The results show that the water permeability of the films can be varied by modifying the film morphology as a result of changing the lithium salt content of the template emulsion. The results further suggest that the water permeability of the films tested in this study is not directly correlated to the degree of porosity but to the extent of pore interconnectivity. Furthermore, we report on the formation of bilayer PHB films consisting of a porous and a dense backing layer which were well fused together with no visible gap between the layers. The permeability analysis showed that the bilayer film has equally low permeability as the dense PHB film. These porous PHB films of single and bilayer type are interesting in a future perspective for the use as temporary barriers or directional transport of nutrients or drugs. Possible future works would include the design and production of multilayered films with drugs or bioactive molecules incorporated in or between the different layers, investigate the degradation profile, and perform permeability studies using drugs or larger biomolecules.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank Anders Mårtensson at the Department of Chemical and Biological Engineering, Chalmers University of Technology, for his help with the SEM image analysis. This work was performed by financial support from the VINN Excellence Centre SuMo Biomaterials (Supermolecular Biomaterials-structure dynamics and properties) and from Chalmers Area of Advance, Materials Science, Chalmers University of Technology.