esb 2011 - dublin (ireland) 01 sept 2011
TRANSCRIPT
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).