Fabrication of 3-D Scaffolds from poly(3-hydroxybutyric acid) (PHB) and poly(3-hydroxybutyric acid-co-3-hydroxyvalerate) (PHBV) for Leukaemia Tissue Engineering Applications

Saiful Zubairi1, Alexander Bismarck1, Apostolis Koutinas2, Nicki Panoskaltsis3 and Athanasios Mantalaris1

1Department of Chemical Engineering, Imperial College London, 2Department of Food Science and Technology, Agricultural University of Athens, and 3Department of

Haematology, Northwick Park & St. Mark’s campus, Imperial College London. For additional information please contact: saiful.zubairi08@imperial.ac.uk

INTRODUCTION

Over the past 30 years, polyhydroxy acids (PHA), particularly poly-3-hydroxybutyrate (PHB) andcopolymers of 3-hydroxybutyrate with 3-hydroxyvalerate (PHBV) have been demonstrated to besuitable for tissue engineering applications. Specifically, these polymers have been used as a woundhealing matrix and also as a wrap-around implant. However, to our knowledge, scaffolds from PHB withthickness greater than 1 mm have not been produced yet. In this work, PHB and PHBV porous 3-Dscaffolds with an improved thickness greater than 4 mm were fabricated and evaluated.

Fig. 5. Morphology of the polymeric

(a) (b)

(c) (d)

PHB 5% (w/v) PHBV 5% (w/v)

Polymer concentrations with respect to polymeric 3-D scaffolds thickness

(a)

(b) (d)

(c)

PHBV (4%, w/v)PHB (4%, w/v)

FIGURE 5Fig. 4. Morphology of scaffolds at different

polymer concentrations (a) Aerial view of

PHB (5%, w/v), (b) Aerial view of PHBV

(5%, w/v), (c) Aerial view of PHB (1%,

w/v), (d) Aerial view of PHBV and PHB

(3%, w/v).

FIGURE 4

PhD Symposium 2011, Department of Chemical Engineering, Imperial College London

92.59

99.67

82.20

99.97

0

10

20

30

40

50

60

70

80

90

100

110

120

Salt-leaching process Lyophilization process

Type of polyhydroxyalkanoates (PHAs) porous 3-D scaffolds

% E

ffic

acy

PHB (4%, w/v) PHBV (4%, w/v)

METHODOLOGY

Different concentrations of PHB and PHBV ranging from 1% to 5% (w/v) were prepared in chloroform.Porous 3-D scaffold were fabricated using the Solvent-Casting Particulate-Leaching (SCPL) method.The efficacy of the SCPL method was determined using ion conductivity measurement and gravimetricanalysis (to determine any potential of polymer weight loss during the salt-leaching process). The saltremnants left inside the scaffolds were measured using ion conductivity as an ultimate validation priorto physico-chemical analysis and cell proliferation studies. Analysis of statistical significance wasperformed using one-way analysis of variance (ANOVA) test and Students t-test with a significancelevel of p<0.05.

Fig. 5. Morphology of the polymeric

porous 3-D scaffolds in a rectangular

shape with an approximate size of 10 ×

10 × 5 mm3: (a) Aerial view of PHB (4%,

w/v), (b) Side view of PHB (4%, w/v), (c)

Aerial view of PHBV (4%, w/v), (d) Side

view of PHBV (4%, w/v).

(c) (d)

PHB 3% (w/v) PHBV 3% (w/v)

PHB 1% (w/v)

Fig. 1. Schematic of the Solvent-Casting

Particulate-Leaching (SCPL) process. The

process comprises of (1) mixing of polymer

solution with porogen; (2) adding the polymer

solution with porogen into a Petri-dish and then

20.5

20.55

20.6

20.65

20.7

20.75

20.8

20.85

0 1 2 3 4 5 6 7

Con

du

ctiv

ity (

mS

/cm

)

Time (days)

PHB (4%, w/v) porous 3-D scaffolds

PHBV (4%, w/v) porous 3-D scaffolds

Control: Cell growth media without a scaffold

Conductivity of cell growth media = 20.77 mS/cm @ 20 ±±±±1 oC

Polymer solution in Solvent evaporation in fume

Polymer concentration vs. thickness

Efficacy of Salt Removal

Porogen residual effect Vs. growth media

Efficacy of salt removal measured via ion conductivity and gravimetric analysis Effect of salt remnants in polymeric 3-D scaffolds on cell growth media

(b) (d)

PHBV (4%, w/v)

∼∼∼∼10 mm ∼∼∼∼10 mm

∼∼∼∼ 5 mm

PHB (4%, w/v)

INNER SIDE

INNER SIDE INNER SIDE

INNER SIDE

**

NOVELTY

Ability to fabricate porous 3-D scaffolds with an improved thickness greater than 4 mm from PHB andPHBV without the presence of etching surfaces and structural instability.

NS

FIGURE 1

FIGURE 7

FIGURE 6

RESULTS

Fig. 7. Conductivity (κ) of cell

growth media in the presence

of scaffolds as a function of

time at 20 ± 1 oC (n = 3).

Fig. 6. Efficacy of (A) salt-leaching process and

(B) salt removal after lyophilization process via

gravimetric analysis for PHB and PHBV (4%,

ACKNOWLEDGEMENTS

The authors would like to thank the Malaysian Higher Education andthe Richard Thomas Leukemia Fund for providing financial support tothis project.

Polymer

concentration

General observation Thickness (mm)

PHB PHBV

1% (w/v) Completely dissolved, homogenous solution appeared < 1.0 < 1.0

2% (w/v) Completely dissolved, homogenous solution appeared < 1.0 < 1.0

3% (w/v) Completely dissolved, homogenous solution appeared 1.80 ± 0.79 1.60 ± 0.79*

4% (w/v) Completely dissolved, homogenous solution appeared 5.25 ± 0.36 4.40 ± 0.52*

solution with porogen into a Petri-dish and then

incubate it in lyophilization flask to avoid

development of etching surfaces; (3)

evaporation of solvent for 48 hrs in fume

cupboard; (4) leaching out porogen from dried

cast polymer + porogen using 10 liters of

deionized water for 48 hrs (changed twice/day)

at 20 ± 1oC; (5) lyophilized porous 3-D

scaffolds with the thickness greater than 4 mm.

Cut into 10 sections

Porous 3-D

scaffolds

Replication (n = 10)

Average thickness

Randomly selected

of 5 sections

Polymer solution in

organic solvent

Porogen (i.e., NaCl,

sucrose etc.)

Polymer solution

+ Porogen

Solvent evaporation in fume

cupboard (Complied with UK-

SED, 2002: <20 mg/m3)

Dried cast

Polymer + Porogen

Porous 3-D

scaffolds

Porogen-DIW

leaching

12

34

Polymer +

Solvent +

Porogen cast

Polymer concentration vs. time

FABRICATION

5

Polymer concentrations with respect to polymeric 3-D scaffolds thickness

CONCLUSIONS

1. Polymer concentration of 4% (w/v) was considered an optimal concentration to produce an ideal porous 3-Dscaffolds with thickness greater than 4 mm without the presence of etching surfaces and structural instability.

2. High efficacies of salt-leaching process for both polymeric 3-D porous scaffolds were observed (99%) with noloss of polymer weight throughout the process.

3. The small amount of salt left inside the porous 3-D scaffolds might not be producing any adverse effect to the cellgrowth due to the electrolytes imbalance from the hypertonic media solution (excessive amount of salt in the cellgrowth media) - Insignificant changes of ion conductivity for both polymers as compared to the control.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Time of complete homogenization (mins)

Po

lym

ers

co

nce

ntr

atio

n, %

(w

/v)

Poly(3-hydroxybutyric acid): PHB

Poly(3-hydroxybutyric acid-co-hydroxyvalerate): PHBV

(A) Inhomogeneous polymer solutions contain glutinous semi-solid residual

*

*

*

* *

*

*

*

Ψ

Ψ

Ψ

Ψ

Ψ

Polymer concentrations with respect to homogenization time

No lost of polymer mass

throughout the SCPL process

Efficiency: PHB > PHBV →→→→

Hydrophilicity: PHB > PHBV

Fig. 3. Kinetics of PHB and PHBV homogenization

process with respect to different concentration, % (w/v).

(A): Inhomogeneous polymer solutions were occurred

with the appearances of glutinous polymer materials at

the bottom of the SCHOTT Duran bottle. The mean

values obtained from 10 experiments ± SEM are shown

(n = 10). *Significant difference with p<0.05 for the value

changed as compared to the previous value. (Ψ) p<0.05

for solubility rate of PHB vs. PHBV.

FIGURE 3FIGURE 2

gravimetric analysis for PHB and PHBV (4%,

w/v). *Significant difference with p<0.05 between

the samples were highlighted by lines (n = 10).

Fig. 2. Thickness of scaffolds at

different polymer concentrations.

Measurement was done using Digital

Vernier Caliper (accuracy ± 0.01 mm).

*Significant difference with p<0.05 as

compared to PHB (n = 10).