PHBV Tissue Engineering Scaffolds Fabricated via Emulsion Freezing / Freeze-drying: Effects of Processing Parameters

Naznin Sultana 1 , 2 and Min Wang 1 1 Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong

2 Medical Implant Technology Group (Mediteg), Department of Biomechanics and Biomedical Materials, FKBSK, Universiti Teknologi Malaysia, Johor, Malaysia

Abstract. Biodegradable polymers have been widely used for scaffolds in tissue engineering which aims to form completely natural tissues without leaving permanently synthetic element(s) in the human body. Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) is a natural, biocompatible and biodegradable polymer suitable for tissue engineering applications. This paper reports the fabrication and characterization of three-dimensional, highly porous PHBV scaffolds produced through the emulsion freezing/freeze-drying technique. PHBV emulsions having 2.5%, 5%, 7.5%, 10%, 12.5% (w/v) PHBV concentrations were used. Freezing and freeze-drying of polymer/solvent/water phase emulsions produced hard and tough scaffolds with interconnected pores having good handling quality. The porosity of scaffolds changed from 85% to 71% when the PHBV concentration in emulsions increased from 2.5% to 12.5%. Compressive mechanical properties of scaffolds were increased with increasing PHBV concentration. The scaffolds had pore sizes ranging from several microns to a few hundred microns.

Keywords: scaffolds, tissue engineering, emulsion freezing / freeze-drying, PHBV

1. Introduction Tissue engineering (TE) emerged two decades ago as a new approach for the repair of diseased or

traumatized human body tissues. With many advantages over conventional treatment methods [1], tissue engineering holds great promises for millions of patients around the world. In scaffold-based tissue engineering, scaffolds play several important roles and the properties of a scaffold can determine the success or failure of a TE strategy. Therefore, scaffold materials and scaffold fabrication techniques must be selected carefully. Biodegradable polymers, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and their copolymer poly(lactic acid-co-glycolic acid) (PLGA), are commonly used as scaffold materials [1] and a few other polymers such as poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) are also made into TE scaffolds. There are some basic requirements for polymeric scaffolds. Scaffold materials should be easily made into the desired porous structure. The degradation product(s) of scaffolds should be non-toxic and easily taken up or excreted via metabolic pathways. Scaffolds should have a controlled porous architecture, allowing for cell migration, attachment and growth and vascularisation, leading to tissue regeneration. Scaffolds should be mechanically strong so as to maintain their structural integrity during culture. The mechanical properties of polymers at large deformations are thus important for selecting particular polymers for TE applications.

Poly(hydroxybutyrate) (PHB) and its copolymer, which are formed by microorganisms via fermentation [2], PHBV are natural biodegradable polymers. PHBV possesses good biocompatibility and degrades in vivo into d-3-hydroxybutyric acid which is a normal constituent of human blood. It has therefore been

+ Corresponding authors. Tel.: +852 2859 7903 (M.Wang); +6 07 55 36496 (N.Sultana). Fax: +852 2858 5415 (M.Wang); +6 07 55 36222 (N.Sultana). E-mail address: memwang@hku.hk (M.Wang); naznin@utm.my (N.Sultana).

2011 International Conference on Biomedical Engineering and Technology IPCBEE vol.11 (2011) © (2011) IACSIT Press, Singapore

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investigated for tissue engineering applications [3, 4]. There are many techniques for scaffold fabrication, including salt leaching, electrospinning, gas foaming, phase separation and rapid prototyping. The emulsion freezing / freeze-drying technique, due to its usefulness to create high-porosity scaffolds and control the pore size, has also been used for constructing TE scaffolds [5, 6]. In the current investigation, the effects of processing parameters of this scaffold fabrication technique on the structure and properties of PHBV scaffolds were studied.

2. Materials and Methods

2.1. Materials PHBV (Tianan Biologic Material Ltd., Ningbo, China) in the powder form was used. It contained 2.9 %

of 3-hydroxyvalerate, had an average molecular weight of 310,000 and was 98.8% pure. All chemicals used in this investigation, such as chloroform, acetic acid and salt, were analytical grade. Water, which was obtained from a Mili-Q water purification system, was ultra pure (<18 mΩ).

2.2. Fabrication of Scaffolds For scaffold fabrication, PHBV powder was weighed accurately and poured into a centrifuge

tube. Then an accurately measured amount of chloroform was added to the tube to make a solution with a desired PHBV concentration at 2.5% - 10% (w/v). To obtain a homogeneous PHBV solution, the mixture was kept at 50°C in a water bath and mixed thoroughly. After obtaining the polymer solution, the water phase (aqueous acetic acid solution or ultra pure water) was added to make an emulsion. A small amount of salts (NaCl) were optionally added and the emulsion was homogenized using a homogenizer (Ultra –Turrax, T-25; IKA-WERKE) at different speeds.

PHBV scaffolds were made via emulsion freezing / freeze-drying [5, 6]. Typically, scaffolds were produced through the following steps: 10 ml of PHBV emulsion were poured into a beaker (30 ml). The beaker containing the emulsion was rapidly transferred into a deep freezer at a preset temperature to quickly solidify the emulsion. The solidified emulsion was maintained at that temperature overnight. The frozen emulsion was then placed into a freeze-drying vessel (LABCONCO-Freeze dry system, USA) at a preset temperature of -10°C. The samples were freeze-dried for at least 46 hrs to remove the solvent and the water phase completely and polymer scaffolds were subsequently obtained. The scaffolds were stored in a vacuum dessiccator at room temperature for storage and for further removal of any residual solvent.

2.3. Characterization of Scaffolds The viscosity of emulsions was measured using a viscometer (Brookfield, USA). The density and

porosity of PHBV scaffolds were measured using the liquid displacement method. Ethanol, which is a non-solvent of polymers, was used as the displacement liquid as it penetrated easily into the pores and at the same time no shrinkage or swelling was caused.

The porous structures of PHBV scaffolds were studied through scanning electron microscopy (SEM; Stereoscan 440, Cambridge, UK). SEM specimens were cut from scaffold samples using a sharp razor blade after they had been frozen at -35°C for 24 hrs. They were then coated with a thin layer of gold prior to SEM examination. The pore diameters were calculated using SEM micrographs. From each scaffold sample, cubic specimens of 5×5×5mm3 in size were obtained for mechanical testing. Compressive mechanical properties of PHBV scaffolds were determined at room temperature using an Instron mechanical tester (Instron 5848, USA) with a 100 N load cell and at a crosshead speed of 0.5 mm/min. The compressive modulus was calculated from the initial linear region of stress-strain curves.

3. Results and Discussion Fig. 1a shows two scaffold samples produced through emulsion freezing / freeze-drying. All scaffolds

were relatively large in size and homogeneous in appearance. They could be handled easily and normally did not contain voids (viz., macropores with sizes greater than 1 mm). The scaffolds fabricated had a nonporous thin skin layer. Fig. 1b is an SEM micrograph, revealing the porous structure of the interior of a scaffold

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sample. PHBV scaffolds, when produced using appropriate parameters, had pores which were highly interconnected and exhibited a large pore size distribution.

Fig. 2 displays some poor-quality scaffold samples that could be produced when non-optimized processing parameters were used for scaffold fabrication. If an inappropriate solvent such as dioxane was used to make PHBV polymer solutions, scaffolds with broken structures were commonly formed (Fig. 2a). If the solvent removal speed was too high for high concentration PHBV solutions, nonporous structures would be formed (Fig. 2b).

(a) (b) Fig. 1 Good-quality PHBV scaffolds: (a) general appearance, (b) porous structure inside a PHBV scaffold

(a) (b) Fig. 2 Poor-quality PHBV scaffolds: (a) broken structure, (b) non-porous structure

Table 1 Effects of process variables on the scaffold handling quality

Process variables Scaffold handling quality

Increase in polymer concentration (from 2.5% to 12.5% w/v)

Type of water phase Acetic acid Ultra pure water

Increase in water phase to solvent ratio Increase in emulsion viscosity Use of emulsion stabilizer and porogen [NaCl] Increase in homogenizing speed

+

+ - - + + +

+: handling quality improved; -: handling quality deteriorated.

The processing parameters were found to have significant influence on the quality of PHBV scaffolds. Table 1 summarizes the effects of process variables on the scaffold handling quality. The handling quality here is defined as the capability of scaffold specimens to be handled for characterization and evaluation (including manual handling during specimen preparation). The polymer concentration in emulsions on scaffold porosity and handling quality was evaluated. Scaffolds produced from emulsions at the 2.5% (w/v)

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PHBV concentration were weak structures and were considered to be structurally inadequate. Emulsions having a PHBV concentration higher than 12.5% (w/v) possessed high viscosity, which ultimately prevented adequate emulsion homogenization, and hence scaffolds produced from these emulsions exhibited poor porous structures. Scaffolds produced from emulsions at PHBV concentrations of 5%, 7.5%, 10% or 12.5% (w/v) had better porous structures and handling quality. Higher homogenization speeds were found to provide better homogenized emulsions, resulting in better quality scaffolds.

The emulsion viscosity increased rapidly with the increase in PHBV concentration when the PHBV concentration was above 7.5% (w/v), forming porous structures with pore sizes over three hundred microns and reasonable pore interconnectivity. The water phase to solvent volume ratio of emulsions had direct influence on emulsion stability and also resultant scaffold size. The acetic acid to solvent ratio of 1:1 or ultra pure water to solvent ratio of 1:2 was found to be appropriate for scaffold fabrication. These ratios produced the thickest and most stable emulsions to scaffold manufacture, which were used for making the majority of scaffolds in this study. If the ratio increased from the optimized values, poor-quality scaffolds were formed. Fig. 3 shows porous structures of PHBV scaffolds made from emulsions of different water phase to solvent ratios.

(a) (b)

Fig. 3 PHBV scaffolds fabricated from emulsions with different water phase to solvent ratios: (a) 1:1, (b) 2:1

(a) (b) Fig. 4 PHBV scaffolds fabricated from emulsions with different PHBV concentrations: (a) 5%, (b) 10%

PHBV scaffolds with high porosity were fabricated using emulsions of different PHBV concentrations. At the same quenching temperature, with increasing PHBV concentration in emulsions, the scaffold density increased whereas the scaffold porosity decreased. When the PHBV concentration increased from 2.5% to 12.5% (w/v), the scaffold density increased from 0.0540 g/cm3 to 0.2926 g/cm3 and the scaffold porosity dropped from 85% to 71%.

A series of PHBV scaffolds were fabricated from PHBV/chloroform/acetic acid emulsions with the PHBV concentration ranging from 2.5% to 12.5% (w/v). Scaffolds made from 5% PHBV emulsions showed low scaffold interconnectivity (Fig. 4a). Scaffolds produced from 10% PHBV emulsions were hard and tough and had desirable pore morphologies (Fig. 4b). As the polymer concentration increased, the pore walls

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became thicker and the total porosity decreased. The pore structure became more uniform, with pore sizes ranging from 60-70 to 300-600 microns.

Compressive properties of PHBV scaffolds increased with increasing PHBV concentration (Fig. 5). In the 2.5%-7.5% strain range, scaffolds from 7.5% PHBV emulsions had a compressive modulus of 1.4±0.61 MPa whereas scaffolds from 12.5% PHBV emulsions had a compressive modulus of 6.41±1.03 MPa.

Fig. 5 Compressive properties of scaffolds produced from emulsions with different PHBV concentrations:

(a) 7.5%, (b) 10%, (c) 12.5% (w/v)

4. Conclusions The emulsion freezing / freeze-drying technique could be employed to fabricate high porosity PHBV

tissue engineering scaffolds. Highly organized three-dimensional porous structures could be obtained if processing parameters were carefully selected and used. It was possible to fabricate interconnected porous structures with pore sizes ranging from several microns to a few hundred microns with porosities above 70%. The water phase to solvent ratio for emulsions and the polymer concentration in emulsions were dominant factors for the quality and properties of resultant scaffolds.

5. Acknowledgements N. Sultana thanks The University of Hong Kong (HKU) for providing her with a research studentship.

This work was supported by a GRF grant (HKU 7182/05E) from the Research Grants Council of Hong Kong. Assistance provided by technical staff in the Dept. of Mechanical Engineering, HKU, is acknowledged. N. Sultana acknowledges the financial support provided by UTM research grant (Vote: RJ 13000077364D002) and RMC to attend the conference.

6. References [1] B.D. Ratner, A.S. Hoffman, F.J. Schoen, and J.E. Lemons. Biomaterials Science: An introduction to materials in

medicine. 2nd edition, Academic Press, San Diego, 2004.

[2] P.A. Holmes. Developments in crystalline polymers. In D.C. Basset (Ed.). Elsevier Applied Science., 1982

[3] B.Duan, M.Wang, W.Y.Zhou, W.-L.Cheung, Z.Y.Li, and W.W.Lu. Three-dimensional Nanocomposite Scaffolds Fabricated via Selective Laser Sintering for Bone Tissue Engineering. Acta Biomaterialia, 6 (2010), 4495–4505

[4] H.-W.Tong, M.Wang, Z.-Y.Li, and W.W.Lu. Electrospinning, Characterization and In Vitro Biological Evaluation of Nanocomposite Fibers Containing Carbonated Hydroxyapatite Nanoparticles. Biomedical Materials, 5 (2010), 054111, 13.

[5] N. Sultana, and M. Wang. Fabrication of HA/PHBV Composite Scaffolds through the Emulsion Freezing / Freeze-drying Process and Characterisation of the Scaffolds. Journal of Materials Science: Materials in Medicine, 19 (2008), 2555-2561

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[6] N. Sultana, and M. Wang. PHBV/PLLA-based Composite Scaffolds Containing Nano-sized Hydroxyapatite Particles for Bone Tissue Engineering. Journal of Experimental Nanoscience. 2008, 3(2), 121-132.

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