Water Uptake and Diffusion in PHBV Tissue Engineering Scaffolds and Non-porous Thin Films

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. The water uptake and diffusion characteristics of tissue engineering (TE) scaffolds based on a natural biodegradable polymer, poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), were studied. The results obtained from porous scaffolds were compared with those obtained from non-porous thin films. The experiments were performed at 25, 37 and 50°C, respectively, using phosphate buffer saline (PBS) as the immersion medium for scaffold and thin films. The classical Fick’s diffusion theory was applied in the initial stage of immersion in PBS. Diffusion coefficients were calculated for both scaffolds and thin films. The activation energy for diffusion was also determined using the Arrhenius equation. Diffusion coefficients were much higher for porous scaffolds than for thin films and the activation energy for scaffolds was lower than that for thin films.

Keywords: water uptake, diffusion, PHBV, scaffold, thin film.

1. Introduction As the primary mechanism for polymer degradation in human bodies is hydrolysis, the study of water

uptake and diffusion characteristics of polymeric tissue engineering (TE) scaffolds is of high importance for their potential applications. The ingress of water into the struts of polymer-based porous scaffolds can have beneficial as well as adverse effects on the properties of scaffolds. Hydrolysis and micro crack formation take place in TE scaffolds due to exposure to water at 37°C. Breakdown of three-dimensional scaffolds can occur owing to excessive water uptake. Investigations have been conducted to study the water uptake characteristics of non-porous biomedical polymers and composites in fields such as dentistry [1, 2]. However, the water uptake and diffusion characteristics of TE scaffolds have been rarely assessed and reported. Therefore, in the current study, using poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) scaffolds intended for bone tissue engineering, an investigation was made into the water uptake and diffusion characteristics of TE scaffolds. For comparison, solvent-cast PHBV thin films were also used in the study.

The classical Fick’s diffusion theory was applied in the current study. According to Fick’s Second Law [3], for the initial stages of diffusion,

21

22 ⎟⎠⎞

⎜⎝⎛=

∞ lDt

MMt

(1) where Mt is the mass uptake at time t, M∞ is the mass uptake at equilibrium, 2l is the thickness of the specimen, D is the diffusion coefficient and t is the mass uptake time. A plot of Mt/M∞ against t½ should provide a straight line for the early stage of water uptake in the current study.

+ Corresponding authors Tel.: +852 2859 7903 (M.Wang); +6 07 5536496 (N.Sultana). Fax: +852 2858 5415 (M.Wang); +6 07 5536222 (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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The diffusion coefficient can be calculated from the slope of the initial straight line. The temperature dependence of the diffusion coefficient is given by the Arrhenius equation:

⎟⎠⎞

⎜⎝⎛−=

TRQDD 1lnln 0 (2)

where T is the absolute temperature (°K), Q is the activation energy for diffusion (J/mol), Do is a temperature-independent constant (pre-exponential constant) (m2/s), and R is the gas constant (8.314 J/mol-K).

2. Materials and Methods

2.1. Materials PHBV in the powder form was purchased from Tianan Biologic Material Ltd., Ningbo, China. PHBV

having 2.9 % of 3-hydroxyvalerate had an average molecular weight of 310,000 and a purity of 98.8%. It was used for producing scaffolds and thin films without further purification. All the chemicals such as chloroform, acetic acid and salts were analytical grade. Water, which was obtained from a Mili-Q water purification system, was ultra pure (<18 mΩ).

2.2. Experimental Methods PHBV scaffolds were fabricated using an emulsion freezing / freeze-drying technique [4, 5]. Typically,

scaffolds were made 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 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. Non-porous PHBV thin films were produced using the solvent casting method.

Test specimens for immersion in an aqueous solution were cut from PHBV scaffolds and thin films using a sharp razor blade and then weighed by an analytical balance. Scaffolds specimens had dimensions of 20mm×5mm×3mm and film specimens had dimensions of 20mm×5mm×0.5mm. The crystallinity of PHBV scaffolds and thin films were 56.9% and 59%, respectively, which were determined through DSC analysis previously. Phosphate buffered saline (PBS) was prepared by dissolving PBS tablets (Zymed laboratories Inc., USA) in ultra pure water. Test specimens were placed in 10 ml of PBS (pH7.4) in sealable vials, which were maintained at 25 (room temperature), 37 (body temperature) or 50°C in a shaking water bath. At regular intervals, test specimens were taken out of PBS, extensively rinsed with pure water, blotted dry on filter paper to remove excess water and weighed. After weighting, they were returned to PBS at respective temperatures for continuing the tests. The water uptake was calculated as

( ) 100/(%) ×−= ddw WWWeWateruptak (3) where Wd and Ww were specimen weights before and after immersion in PBS.

The morphologies of porous scaffolds and thin films before and after immersion in PBS were examined using scanning electron microscopy (SEM, Stereoscan 440, Cambridge, UK). SEM specimens were cut from test specimens 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.

3. Results and Discussion Fig. 1 displays the water uptake curve of PHBV thin films at room temperature in the form of Mt/ M∞ vs.

the square root of immersion time. The maximum water uptake at equilibrium was 1.4% (in reference to the original specimen weight). The diffusion coefficients were calculated from the initial linear region of this curve. In order to determine the activation energy for diffusion, tests were performed at three different temperatures, namely, 25, 37 and 50°C. The diffusion coefficients determined for these temperatures are listed in Table 1. The diffusion coefficient at 50°C is much higher than that at 25°C or 37°C. The Arrhenius

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0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.00340-30

-29

-28

-27

-26

-25

-24

-23

-22

ln D

1/T

plot was then constructed (Fig. 2). As the slope of the curve, the activation energy was calculated, which was 99.8± 21.9 KJ/mole with the regression coefficient r2 of 0.94.

Fig. 1 Water uptake curve of PHBV thin films at 25°C

Table 1 Diffusion coefficients of water in PHBV thin films

Test Temperature (°C) Diffusion coefficients (m2/s, ×10-13) 25 37 50

3.85 ± 1.54 11.30 ± 1.02 59.36 ± 12.00

Fig. 2 Arrhenius plot for PHBV thin films

Fig. 3 shows the water uptake curve of PHBV scaffolds at room temperature in the form of Mt/ M∞ vs. the square root of immersion time. Water diffusion coefficients were calculated from water uptake curves at 25, 37 and 50°C, respectively, and are given in Table 2. These values of water diffusion coefficient were at least two orders of magnitude higher than those for non-porous PHBV films. Fig. 4 is the Arrhenius plot for PHBV scaffolds. From the slope of the best fit linear line, the activation energy was obtained, which was 83.5±53 KJ/mole. This value is lower than that for PHBV films, indicating that water diffusion was easier in PHBV scaffolds.

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 00 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

S Q R T o f im m e r s io n t im e ( s e c )

∞MM t

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Fig. 3 Water uptake curve of PHBV scaffolds at 25°C

Table 2 Diffusion coefficients of water in PHBV scaffolds

Test Temperature (°C) Diffusion coefficients (m2/s, ×10-12) 25 37 50

51.75 ± 1.10 67.60 ± 3.20 165.00 ± 9.10

0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.00340

-34

-32

-30

-28

-26

-24

-22

ln D

1/T Fig. 4 Arrhenius plot for PHBV scaffolds

Fig. 5 exhibits the morphologies of a PHBV thin film and a PHBV scaffold after immersion in PBS. It

was observed that highly porous scaffolds contained many micropores. Water uptake increased significantly with increasing porosity of the scaffold. The scaffolds whose porosity was above 70% absorbed much larger amounts of water than non-porous PHBV films.

(a) (b)

0 200 400 600 800 1000 1200 14000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Square root of im m ersion tim e (sec)

∞MM t

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Fig. 5 SEM micrographs of (a) PHBV thin film and (b) PHBV scaffold

Water uptake by a material can occur by way of “absorbed water”, meaning the water absorbed from the media into the material, which depends mainly on the hydrophilicity of the material. Capillary water is the water that is “drawn in” through pores or capillaries of the material. The amount of water absorbed into a scaffold is related to the scaffold porosity and the amount of water available at the surface of the material. For this reason, a porous material can take up and store more water whereas its non-porous (dense) counterpart can store only a limited amount of water. It can be seen from the Mt/M∞ vs. square root of immersion time curves (Fig. 1 and Fig. 3) that the curves are almost linear for both PHBV thin films and scaffolds at the initial stage of immersion in PBS. As Fick’s Second Law fits for both thin films and scaffolds at the initial immersion period, it can be inferred that the initial water uptake is diffusion controlled. Similar phenomena were observed for non-porous polymeric composite materials for dental applications [1, 2]. The water diffusion coefficients are always higher for scaffolds than for thin films. The surface area to volume ratio of non-porous objects and porous scaffolds can affect the water diffusion coefficient.

4. Conclusions The water diffusion coefficients were higher for porous PHBV scaffolds than for non-porous thin PHBV

films. The porosity and hence surface to volume ratio of scaffolds had an effect on the water uptake of scaffolds. The activation energy for water diffusion in porous PHBV scaffolds was lower than that for non-porous PHBV films.

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] M. Braden, and R.L.Clarke. Water absorption characteristics of dental microfine composite filling materials: I.

Proprietary materials. Biomaterials, 1984, 5(6): 369-372.

[2] S. Deb, M. Braden, and W. Bonfield. Water absorption characteristics of modified hydroxyapatite bone cements. Biomaterials, 1995, 16(14): 1095-1100.

[3] J. Crank. The mathematics of diffusion. Oxford: Clarendon Press, 1979.

[4] N. Sultana, and M. Wang. Fabrication and characterization of polymer and composite scaffolds based on polyhydroxybutyrate and polyhydroxybutyrate-co-hydroxyvalerate. Key Engineering Materials, 2007, 334-335: 1229-1232.

[5] 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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