ESB 2011 - Dublin (Ireland) 01 sept 2011

Page 1

Surface Treatment of Poly(3-Hydroxybutyric Acid) (PHB) and Poly(3-Hydroxybutyric-co-3-Hydroxyvaleric Acid) (PHBV) Porous 3-D Scaffolds With An Improved Thickness

To Enhance Cell-Biomaterial Adhesion and Interactions

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: [email protected]

The poor hydrophilic properties of PHA have hindered its extensive use for medical applications[1].Hence, it is imperative to improve the surface properties of PHA to render it suitable for tissue

engineering[2]. A possible and effective way is surface treatment. Tailoring surface properties ofdegradable polymer scaffolds is an essential requirement towards the development of biomimeticsupport matrices. In this study, the PHA, particularly PHB and PHBV were fabricated into porous 3-D

scaffolds with an improved thickness (greater than 4 mm). Later, they were treated with two types ofsurface treatments to enhance the surface hydrophilicity and in turn, improving the cell-biomaterial

affinity. The PHB and PHBV foams were treated with NaOH and rf-oxygen plasma to modify theirsurface chemistry and hydrophilicity with the aim of increasing the cellular attachment of ChronicLymphocytic Leukaemia cell line (RL) as well as to identify which treatments suit best for the

biological surface coating.

INTRODUCTION

METHODOLOGYSolvent evaporation in fume

cupboard (Complied with UK-SED, 2002: < 20 mg/m3)

PHBV (4%, w/v)

∼∼∼∼10 mm ∼∼∼∼10 mm

∼∼∼∼ 5 mm

INNER SIDE

INNER SIDE

INNER SIDEINNER SIDE

OBJECTIVE

1. To analyze the morphology and surface properties of the modified polymeric 3-D scaffolds.2. To identify which surface treatments suit best for biological surface coating based on the RL cell

line cellular response.

Structural analysis of polymeric porous 3-D scaffolds after surface treatment

0.4 M NaOH PHB 0.6 M NaOH PHB

0.4 M NaOH PHBV 0.6 M NaOH PHBV

Rf-O2 plasma PHB

Rf-O2 plasma PHBV

Weight loss after surface treatmentNo structural integrity problem.

No apparent detachable fraction.

(a) rf-O2 plasma treatment PHB (4%, w/v) PHBV (4%, w/v)

100 W, 10 min

Weight loss (%) 1.64 ± 0.15 1.45 ± 0.25*

(b) NaOH treatment PHB (4%, w/v) PHBV (4%, w/v)

0.4 mol L-1 0.6 mol L-1 0.4 mol L-1 0.6 mol L-1

Weight loss (%) 3.58 ± 0.48* 5.56 ± 0.42* 2.79 ± 0.75* 3.57 ± 0.87*

TABLE 3:

Weight loss of PHB and PHBV 4% (w/v)

porous 3-D scaffolds after (a) rf-O2 plasma and

(b) NaOH surface treatment.

*p<0.05 as compared with PHB or PHBV treated with rf-O2 plasma (n = 4).

FIGURE 4:

Scanning electron micrographs of PHB and PHBV

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

alkaline and rf-oxygen plasma treatment. The

treatment conditions for rf-oxygen plasma: 100 W,

10 min. (a) 0.4M NaOH PHB; (b) 0.4M NaOH

PHBV; (c) 0.6M NaOH PHB; (d) 0.6M NaOH PHBV;

(e) PHB rf-oxygen plasma; (f) PHBV rf-oxygen

plasma.

FIGURE 5:

ξ = f(pH) for PHB (a) and PHBV (b) porous 3-D

scaffolds before and after rf-oxygen plasma and

(a)(c)(e)

Morphology of porous structure by using scanning electron microscopy (SEM)

Porous3-D scaffolds

O2 rf-plasma treatment*(Optimum parameter:

100 W, 10 min) - Köse, et al. (2003)

Alkaline treatment - NaOH*(0.2, 0.4, 0.6, 0.8, 1.0 mol L−1)

Identify the ideal concentration

Water contact angle (θH2O) &

ζ-potential measurement

1A 1B

2

3

In vitro cell-biomaterial interactions (2 weeks)

Identify the ideal treatment based on the cellular proliferation study

4

5

NaOH

Polymer solution in organic solvent

Porogen (i.e., NaCl, sucrose & etc.)

Polymer solution + Porogen

Dried cast Polymer + Porogen

Porous 3-D scaffolds

Porogen-DIW leaching

12

3

4

Polymer + Solvent + Porogen cast

Rectangular size of polymeric porous 3-D scaffolds(> 4mm)

5

(a)

(b)

INNER SIDE

FIGURE 1:

Schematic of the Solvent-Casting Particulate-Leaching (SCPL) process. The process comprises (1) mixing of polymer

solution with porogen; (2) adding the polymer solution with porogen into a Petri-dish and then incubated in the

lyophilization flask to avoid development of etching surfaces; (3) evaporation of solvent for 48 h in the fume cupboard.

The solvent evaporation is complied with the United Kingdom Solvent Emission Directive (SED), 2002 for Halogenated

VOCs: <20 mg/m3 (<≅ 12 kg of CHCl3); (4) leaching out porogen from dried cast polymer + porogen by using 10 liters of

deionized water for 48 h (changed twice/day) at 20 ± 1oC; (5) lyophilized porous 3-D scaffolds with the thicknessgreater than 4 mm; (6) A rectangular size of ∼10mm x ∼10mm x ∼5mm porous 3-D scaffolds is incised prior to the

surface treatments, in vitro degradation measurement, mechanical testing and cellular proliferation studies.

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

0.8 M

0.8 M

1.0 M

1.0 M

0.6 M

0.6 MControl + DIW 0.4 M

0.4 M

0.2 M

0.2 M

ζ-potential measurement of polymeric porous 3-D scaffolds

ζ-potential & water contact angle measurement after sterilization process

No significant changed were

observed for both polymers.

Similar ζ-potential profile & CA

with the untreated polymers

-140

-120

-100

-80

-60

-40

-20

0

20

0 1 2 3 4 5 6 7 8 9 10 11

Untreated PHBV (4%, w/v) Treated PHBV (4%, w/v) 0.4M NaOH

Treated PHBV (4%, w/v) 0.6M NaOH Treated PHBV (4%, w/v) Oxygen-plasma

Untreated PHBV (4%, w/v), EtOH (2 hours)

pH (103 M KCl)

Zet

a-P

ote

ntia

l ζζ ζζ[M

v]

(b)

PHBV

ζζζζplateauplateauplateauplateau

-140

-120

-100

-80

-60

-40

-20

0

20

0 1 2 3 4 5 6 7 8 9 10 11

Untreated PHB (4%, w/v) Treated PHB (4%, w/v) 0.4M NaOH

Treated PHB (4%, w/v) 0.6M NaOH Treated PHB (4%, w/v) Oxygen-plasma

Untreated PHB (4%, w/v) EtOH (2 hours)

pH (103 M KCl)

Zet

a-P

ote

ntia

l ζζ ζζ[M

v]

(a)

PHB

ζplateau

RESULTS

FIGURE 2:

Schematic representation of the alkaline

and rf-O2-plasma surface treatment and

physico-chemical characterization by

means of scanning electron microscopy

(SEM), electrokinetic analyzer (EKA),

helium pycnometer and drop sessile

analyzer (DSA). Statistical analysis was

conducted by using the Students t-test and

ANOVA Tukey’s test (SPSS version 17.0

IBM co.)

NaOH treatment. In Figure 2(a) and (b), the arrow

highlights the shift of the iep after NaOH treatment.

Polymer iep ζplateau (mV)

PHB, untreated 3.8 -29

PHB, untreated, EtOH (2 h) 3.7 -29

PHB, NaOH 0.4M 3.7 -31

PHB, NaOH 0.6M 2.7 -81

PHB, 100W 10 min - -120

PHBV, untreated 3.1 -37

PHBV, untreated, EtOH (2 h) 3.2 -36

PHBV, NaOH 0.4M 3.0 -53

PHBV, NaOH 0.6M 2.7 -93

PHBV, 100W 10 min - -128

TABLE 4:

ζ-potential results: iep and ζplateau values of the ζ = f(pH) for PHB and

PHBV (4%, w/v) porous 3-D scaffolds before and after surface treatment.

Surface physico-chemistry Polymeric porous 3-D scaffolds

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

Before sterilization process

Contact angle, θapparent (o) 66.80 ± 0.2 79.24 ± 0.4

After sterilization process

Contact angle, θapparent (o) 65.43 ± 0.3 78.11 ± 0.5

TABLE 5:

Water contact angle (θH20) of untreated PHB and PHBV (4%, w/v) solvent-cast thin

films pre- and post-sterilization (n = 4).

0

1

2

3

4

5

6

7

8

9

pH

va

lue

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

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

Cell growth media without scaffold

(b)

*

*

*

*

*

*

*

*

* *

Ψ

Ψ

Ψ

Ψ

Ψ

Ψ

ΨΨ

Ψ

Ψ

Ψ

Ψ

Ψ(f)

0

10

20

30

40

50

60

70

80

90

100

110

% R

esid

ua

l w

eig

ht o

f p

oro

us

3-D

sca

ffo

lds

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

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

Ψ

Ruptured

(a)

*

**

*

*

Ruptured

FIGURE 6:

Kinetics of the in vitro degradation process for PHB

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

measured via (a) mass and (b) pH. The mean

values obtained from 4 experiments ± standard

deviation (SD) for each time frame is shown below

(n = 4). (Ψ) p<0.05 for the value compared to each

of the polymers or control (pH analysis) and (*)

p<0.05 for the value change compared to the

previous value.

FIGURE 3:

Morphology of polymeric porous

3-D foams in a rectangular form

(an approximate size of 10mm ×10mm × 5mm) after serial

concentrations of NaOH surface

treatment.

NaOH surface treatment of polymeric porous 3-D scaffolds

In vitro degradation process in cell growth media

1. S. F. Williams et al., International Journal of Biological Macromolecules, 1999, 25,111.2. L. Jing et al., Journal of Biomedical Materials Research Part A, 2005, 75, 985.

3. G. T. Köse et al., Biomaterials 2003, 24, 1949.4. G. T. Köse et al., Biomaterials 2003, 24, 4999.5. A. Atala et al., in Principles of Regenerative Medicine, Academic Press, 2007.

The authors would like to thank the Malaysian Higher Education and the Richard ThomasLeukaemia Fund for providing financial support to this project.

Water contact angle of solvent-cast thin films post-surface treatmentNaOH treatment: Significantly

changed for 0.4 M & 0.6 M.

Plasma: Both polymers were

completely wet (<25o).

Physical properties of the polymeric foam pre- and post-surface treatment

BET surface area was found

to be significantly different

for both treatment.

Similar porosity +

↑ voids developed.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Day 1 Day 7 Day 14

Type of PHAs porous 3-D scaffolds

Ab

sorb

an

ce

(4

90 n

m)

PHB without treatment PHBV without treatment PHB 0.6M NaOH

PHBV 0.6M NaOH PHB plasma treatment PHBV plasma treatment

*

Seeding efficacy for all untreated and treated foams

= 81.55 to 95.43%

Cellular response of a CLL’s cell line (RL) on untreated and treated foams

Colorimetric assay

(MTS assay)

a) All scaffolds at day 14 displayed high in cell

proliferation as compared to day 7 (p<0.05).b) Data were obtained in 6 separate instances, each

in quadruplicates (n = 4).

CONCLUSIONS

REFERENCES

ACKNOWLEDGEMENTS

1. No structural and sturdiness problems.2. Altered the foams morphology by making more voids available for occupation by CLL cell line.3. Both polymers were incomparable in term of their compressive modulus (GPa) and ultimate compressive strength (MPa). They were much

better than other porous biodegradable polymers and composites.

4. CLL cell line was proliferated immensely on all treated & untreated polymeric foams after 14 days of incubation.5. 0.6 M NaOH treatment is the best surface treatment for biological surface coating.

6. CLL: No preferential on choosing which surface properties & material characteristics.7. HIGH potential in developing an ex vivo 3-D mimicry of the human haematopoietic microenvironment model for the study of CLL.

(a) Surface physico-chemistry

(rf-O2 plasma treatment)

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

100 W, 10 min[a]

Contact angle, θapparent (o) < 25[b][c] < 25[b][c]

(b) Surface physico-chemistry

(NaOH treatment)

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

0.4 mol L-1 0.6 mol L-1 0.4 mol L-1 0.6 mol L-1

Contact angle, θapparent (o) 65.88 ± 0.72 15.44 ± 0.33** < 25[b][c] < 25[b][c]**p<0.01 as compared to 0.4 mol L-1 NaOH and untreated PHB (66.80 ± 0.2o) (n = 10). [a] Optimized operational parameters arestudied by Köse et al.[3, 4] [b] The surface is completely wet by re-distilled water droplet (n = 10). Contact angle of fully wetting <25o.[5][c] Thin films of PHB and PHBV are fabricated on the polypropylene (PP) sheet and then treated with both treatments.

TABLE 1:

Water contact angle (θH2O) of PHB and PHBV 4% (w/v) solvent-cast thin

films after (a) rf-O2 plasma and (b) NaOH surface treatment.

Physical properties

Polymeric foams (4%, w/v)

Before treatment

PHB PHBV

BET surface area, As, m2 g-1[a] 0.70 ± 0.02 0.82 ± 0.03

Geometrical bulk density, g cm-3 0.084 ± 0.15 0.072 ± 0.28*

Skeletal density, g cm-3[b][c] 0.47 ± 0.52 0.92 ± 0.14*

Porosity, % 81.97 ± 1.22 92.17 ± 0.73*

Physical properties

Polymeric foams (4%, w/v)

Alkaline treatment (0.6 M) rf-O2 plasma treatment

PHB PHBV PHB PHBV

BET surface area, As, m2 g-1[a] 0.89 ± 0.02* 0.97 ± 0.03* 0.78 ± 0.03* 0.91 ± 0.01*

Geometrical bulk density, g cm-3 0.084 ± 0.15 0.072 ± 0.28* 0.084 ± 0.15 0.072 ± 0.28*

Skeletal density, g cm-3[b][c] 0.47 ± 0.52 0.92 ± 0.14* 0.47 ± 0.52 0.92 ± 0.14*

Porosity, % 80.96 ± 0.21 91.05 ± 0.52 79.11 ± 0.87 91.74 ± 0.42

TABLE 2:

Physical properties of PHB and PHBV (4%, w/v) porous 3-D scaffolds before and after surface treatment.

*(p<0.05) - Results are considered statistically significant (n = 4) as compared to prior treatment. Ψ(p<0.05) - Results are consideredstatistically significant (n = 4) as compared to rf-O2 plasma treatment. [a] BET surface area (m2 g-1) = Total surface area in all direction(m2)/skeletal mass (g). [b] ρs is the skeletal density of the crushed scaffolds, which is determined from helium pycnometry. [c] The higherpore volume (the higher the amount of absorbate intruded), the lower the skeletal volume.

0

0 7 14 21 28 35 42 49 56 63

Time (days)

0

0 7 14 21 28 35 42 49 56 63 70

Time (days)

treatment.

Mechanical Properties

Polymers

Mechanical Properties

Compressive

modulus (GPa)

Ultimate compressive

strength (MPa)[a]

PHB (4%, w/v) 0.0071 ± 0.72 1.97 ± 0.12

PHBV (4%, w/v) 0.0096 ± 0.18 1.83 ± 0.09

TABLE 5:

Mechanical properties of PHB and PHBV (4%, w/v) porous 3-D scaffolds.

[a] Samples are crushed, compacted and eventually ruptured into several fragments (n = 3).

**

FIGURE 7:

Cellular growth of a CLL cell line (RL) on PHB and

PHBV (4%, w/v) porous 3-D scaffolds without

treatment (control) and with surface treatment

(alkaline and rf-oxygen plasma).