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: saiful.zubairi08@imperial.ac.uk
INTRODUCTION Weight loss after surface treatment
No structural integrity problem.
No apparent detachable fraction.
The poor hydrophilic properties of PHA have hindered its extensive use for medical applications[1]. TABLE 3:
(a) rf-O2 plasma treatment PHB (4%, w/v) PHBV (4%, w/v) Weight loss of PHB and PHBV 4% (w/v)
Hence, it is imperative to improve the surface properties of PHA to render it suitable for tissue porous 3-D scaffolds after (a) rf-O2 plasma and
100 W, 10 min
engineering[2]. A possible and effective way is surface treatment. Tailoring surface properties of (b) NaOH surface treatment.
Weight loss (%) 1.64 ± 0.15 1.45 ± 0.25*
degradable polymer scaffolds is an essential requirement towards the development of biomimetic
support matrices. In this study, the PHA, particularly PHB and PHBV were fabricated into porous 3-D (b) NaOH treatment PHB (4%, w/v) PHBV (4%, w/v)
scaffolds with an improved thickness (greater than 4 mm). Later, they were treated with two types of 0.4 mol L-1 0.6 mol L-1 0.4 mol L-1 0.6 mol L-1
surface treatments to enhance the surface hydrophilicity and in turn, improving the cell-biomaterial Weight loss (%) 3.58 ± 0.48* 5.56 ± 0.42* 2.79 ± 0.75* 3.57 ± 0.87*
affinity. The PHB and PHBV foams were treated with NaOH and rf-oxygen plasma to modify their *p<0.05 as compared with PHB or PHBV treated with rf-O2 plasma (n = 4).
surface chemistry and hydrophilicity with the aim of increasing the cellular attachment of Chronic
Lymphocytic Leukaemia cell line (RL) as well as to identify which treatments suit best for the Structural analysis of polymeric porous 3-D scaffolds after surface treatment
biological surface coating.
0.4 M NaOH PHB 0.6 M NaOH PHB Rf-O2 plasma PHB FIGURE 4:
OBJECTIVE Scanning electron micrographs of PHB and PHBV
(4%, w/v) porous 3-D scaffolds subsequent to
alkaline and rf-oxygen plasma treatment. The
1. To analyze the morphology and surface properties of the modified polymeric 3-D scaffolds. treatment conditions for rf-oxygen plasma: 100 W,
2. To identify which surface treatments suit best for biological surface coating based on the RL cell 10 min. (a) 0.4M NaOH PHB; (b) 0.4M NaOH
line cellular response. PHBV; (c) 0.6M NaOH PHB; (d) 0.6M NaOH PHBV;
(e) PHB rf-oxygen plasma; (f) PHBV rf-oxygen
METHODOLOGY INNER SIDE
INNER SIDE
plasma.
0.4 M NaOH PHBV 0.6 M NaOH PHBV Rf-O2 plasma PHBV
PHBV (4%, w/v)
Solvent evaporation in fume
cupboard (Complied with ∼10
∼
∼ mm ∼10
∼
∼ mm
UK-SED, 2002: < 20 mg/m3) FIGURE 5:
∼
∼ 5 mm ξ = f(pH) for PHB (a) and PHBV (b) porous 3-D
INNER SIDE
INNER SIDE scaffolds before and after rf-oxygen plasma and
Polymer solution in
NaOH treatment. In Figure 2(a) and (b), the arrow
organic solvent Rectangular size of polymeric highlights the shift of the iep after NaOH treatment.
(c)
(a)
(e) porous 3-D scaffolds(> 4mm)
Porogen-DIW ζ-potential measurement of polymeric porous 3-D scaffolds
Polymer solution + Porogen leaching
(a) 3
20 20
(a) (b)
4 pH (103 M KCl) pH (103 M KCl)
2
1 0 0
5 Porous 3-D 0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11
scaffolds -20 -20
PHB PHBV
Zeta-Potential ζ [Mv]
(b)
Dried cast Polymer +
Zeta-Potential ζ [Mv]
Polymer + Solvent -40 -40
ζ
+ Porogen cast Porogen
ζ
ζplateau -60
-60
ζ plateau
-80 -80
Porogen (i.e., NaCl,
sucrose & etc.)
-100 -100
FIGURE 1: -120 -120
Schematic of the Solvent-Casting Particulate-Leaching (SCPL) process. The process comprises (1) mixing of polymer -140 -140
solution with porogen; (2) adding the polymer solution with porogen into a Petri-dish and then incubated in the Untreated PHB (4%, w/v) Treated PHB (4%, w/v) 0.4M NaOH 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
lyophilization flask to avoid development of etching surfaces; (3) evaporation of solvent for 48 h in the fume cupboard. Treated PHB (4%, w/v) 0.6M NaOH Treated PHB (4%, w/v) Oxygen-plas ma
Untreated PHB (4%, w/v) EtOH (2 hours ) Untreated PHBV (4%, w/v), EtOH (2 hours)
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 TABLE 4:
ζ-potential & water contact angle No significant changed were
deionized water for 48 h (changed twice/day) at 20 ± 1oC; (5) lyophilized porous 3-D scaffolds with the thickness ζ-potential results: iep and ζplateau values of the ζ = f(pH) for PHB and
measurement after sterilization process observed for both polymers.
greater than 4 mm; (6) A rectangular size of ∼10mm x ∼10mm x ∼5mm porous 3-D scaffolds is incised prior to the PHBV (4%, w/v) porous 3-D scaffolds before and after surface treatment. Similar ζ-potential profile & CA
surface treatments, in vitro degradation measurement, mechanical testing and cellular proliferation studies.
Polymer iep ζplateau (mV) with the untreated polymers
1A 1B O2 rf-plasma treatment*
PHB, untreated 3.8 -29 TABLE 5:
Alkaline treatment - NaOH* Porous
(0.2, 0.4, 0.6, 0.8, 1.0 mol L−1) 3-D scaffolds
(Optimum parameter: FIGURE 2: PHB, untreated, EtOH (2 h) 3.7 -29 Water contact angle (θH20) of untreated PHB and PHBV (4%, w/v) solvent-cast thin
100 W, 10 min) - Köse, et al. (2003) Schematic representation of the alkaline
Identify the ideal concentration PHB, NaOH 0.4M 3.7 -31 films pre- and post-sterilization (n = 4).
and rf-O2-plasma surface treatment and
PHB, NaOH 0.6M 2.7 -81 Surface physico-chemistry Polymeric porous 3-D scaffolds
2 physico-chemical characterization by
means of scanning electron microscopy PHB, 100W 10 min - -120 PHB (4%, w/v) PHBV (4%, w/v)
Water contact angle (θH2O) &
ζ-potential measurement (SEM), electrokinetic analyzer (EKA), PHBV, untreated 3.1 -37 Before sterilization process
NaOH 3 helium pycnometer and drop sessile PHBV, untreated, EtOH (2 h) 3.2 -36 Contact angle, θapparent (o) 66.80 ± 0.2 79.24 ± 0.4
Morphology of porous structure by using analyzer (DSA). Statistical analysis was PHBV, NaOH 0.4M 3.0 -53
scanning electron microscopy (SEM) After sterilization process
conducted by using the Students t-test and PHBV, NaOH 0.6M 2.7 -93
4 Contact angle, θapparent (o) 65.43 ± 0.3 78.11 ± 0.5
ANOVA Tukey’s test (SPSS version 17.0 PHBV, 100W 10 min - -128
In vitro cell-biomaterial interactions (2 weeks)
IBM co.)
5
In vitro degradation process in cell growth media
Identify the ideal treatment based
on the cellular proliferation study 110 (a) Ψ 9 (b)
*Ψ *Ψ
RESULTS FIGURE 6:
% Residual weight of porous 3-D scaffolds
100 * 8
* Ruptured
90
* *
Kinetics of the in vitro degradation process for PHB
7
80 * *Ψ Ψ *Ψ and PHBV (4%, w/v) porous 3-D scaffolds are
NaOH surface treatment of polymeric porous 3-D scaffolds *Ψ *Ψ *Ψ
* 6 *Ψ
70 measured via (a) mass and (b) pH. The mean
(f)
pH value
Ψ Ψ
FIGURE 3:
60 5 *Ψ Ψ
values obtained from 4 experiments ± standard
0.8 M 1.0 M 0.6 M 50
0.4 M 0.2 M
Morphology of polymeric porous PHB (4%, w/v) porous 3-D s caffold Ruptured 4 deviation (SD) for each time frame is shown below
3-D foams in a rectangular form
40
PHBV (4%, w/v) porous 3-D s caffold 3
PHB (4%, w/v) porous 3-D scaffold (n = 4). (Ψ) p<0.05 for the value compared to each
PHBV (4%, w/v) porous 3-D scaffold
(an approximate size of 10mm ×
30
of the polymers or control (pH analysis) and (*)
2 Cell growth media without scaffold
PHBV (4%, w/v) porous 3-D scaffolds PHB (4%, w/v) porous 3-D scaffolds 20
10mm × 5mm) after serial p<0.05 for the value change compared to the
Control + DIW 0.4 M 0.2 M 10 1
0.8 M 0.6 M 1.0 M
concentrations of NaOH surface previous value.
0
0
treatment. 0 7 14 21 28 35 42 49 56 63 70 0 7 14 21 28 35 42 49 56 63
Tim e (days ) Time (days )
Mechanical Properties TABLE 5:
TABLE 1: Mechanical properties of PHB and PHBV (4%, w/v) porous 3-D scaffolds. Cellular response of a CLL’s cell line (RL) on untreated and treated foams
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. Mechanical Properties
*
Polymers Compressive Ultimate compressive
1.4
Water contact angle of solvent-cast thin films post-surface treatment modulus (GPa) strength (MPa)[a]
NaOH treatment: Significantly PHB (4%, w/v) 0.0071 ± 0.72 1.97 ± 0.12 1.2
(a) Surface physico-chemistry PHB (4%, w/v) PHBV (4%, w/v) changed for 0.4 M & 0.6 M.
PHBV (4%, w/v) 0.0096 ± 0.18 1.83 ± 0.09
Absorbance (490 nm)
1
(rf-O2 plasma treatment) 100 W, 10 min[a] Plasma: Both polymers were [a] Samples are crushed, compacted and eventually ruptured into several fragments (n = 3).
Contact angle, θapparent (o) < 25[b][c] < 25[b][c] completely wet (<25o). 0.8
*
Colorimetric assay *
(b) Surface physico-chemistry PHB (4%, w/v) PHBV (4%, w/v) (MTS assay) 0.6
Seeding efficacy for all
(NaOH treatment) 0.4 mol L-1 0.6 mol L-1 0.4 mol L-1 0.6 mol L-1 untreated and treated foams
0.4
a) All scaffolds at day 14 displayed high in cell = 81.55 to 95.43%
Contact angle, θapparent (o) 65.88 ± 0.72 15.44 ± 0.33** < 25[b][c] < 25[b][c] proliferation as compared to day 7 (p<0.05). FIGURE 7:
0.2
**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 are
b) Data were obtained in 6 separate instances, each Cellular growth of a CLL cell line (RL) on PHB and
studied 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. in quadruplicates (n = 4). PHBV (4%, w/v) porous 3-D scaffolds without 0
Day 1 Day 7 Day 14
treatment (control) and with surface treatment
Type of PHAs porous 3-D scaffolds
(alkaline and rf-oxygen plasma).
TABLE 2:
CONCLUSIONS
PHB without treatment PHBV without treatment PHB 0.6M NaOH
Physical properties of PHB and PHBV (4%, w/v) porous 3-D scaffolds before and after surface treatment. PHBV 0.6M NaOH PHB plasma treatment PHBV plasma treatment
Physical properties of the polymeric foam pre- and post-surface treatment 1. No structural and sturdiness problems.
2. Altered the foams morphology by making more voids available for occupation by CLL cell line.
Polymeric foams (4%, w/v) BET surface area was found 3. Both polymers were incomparable in term of their compressive modulus (GPa) and ultimate compressive strength (MPa). They were much
Physical properties Before treatment to be significantly different better than other porous biodegradable polymers and composites.
PHB PHBV for both treatment. 4. CLL cell line was proliferated immensely on all treated & untreated polymeric foams after 14 days of incubation.
BET surface area, As, m2 g-1[a] 0.70 ± 0.02 0.82 ± 0.03 5. 0.6 M NaOH treatment is the best surface treatment for biological surface coating.
Geometrical bulk density, g cm-3 0.084 ± 0.15 0.072 ± 0.28*
Similar porosity + 6. CLL: No preferential on choosing which surface properties & material characteristics.
Skeletal density, g cm-3[b][c] 0.47 ± 0.52 0.92 ± 0.14* ↑ voids developed. 7. HIGH potential in developing an ex vivo 3-D mimicry of the human haematopoietic microenvironment model for the study of CLL.
Porosity, % 81.97 ± 1.22 92.17 ± 0.73*
Polymeric foams (4%, w/v)
Physical properties Alkaline treatment (0.6 M) rf-O2 plasma treatment REFERENCES
PHB PHBV PHB PHBV 1. S. F. Williams et al., International Journal of Biological Macromolecules, 1999, 25,111.
BET surface area, As, m2 g-1[a] 0.89 ± 0.02* 0.97 ± 0.03* 0.78 ± 0.03* 0.91 ± 0.01* 2. L. Jing et al., Journal of Biomedical Materials Research Part A, 2005, 75, 985.
Geometrical bulk density, g cm-3 0.084 ± 0.15 0.072 ± 0.28* 0.084 ± 0.15 0.072 ± 0.28* 3. G. T. Köse et al., Biomaterials 2003, 24, 1949.
Skeletal density, g cm-3[b][c] 0.47 ± 0.52 0.92 ± 0.14* 0.47 ± 0.52 0.92 ± 0.14* 4. G. T. Köse et al., Biomaterials 2003, 24, 4999.
Porosity, % 80.96 ± 0.21 91.05 ± 0.52 79.11 ± 0.87 91.74 ± 0.42 5. A. Atala et al., in Principles of Regenerative Medicine, Academic Press, 2007.
*(p<0.05) - Results are considered statistically significant (n = 4) as compared to prior treatment. Ψ(p<0.05) - Results are considered
statistically 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 higher
pore volume (the higher the amount of absorbate intruded), the lower the skeletal volume.
The authors would like to thank the Malaysian Higher Education and the Richard Thomas
Leukaemia Fund for providing financial support to this project. ACKNOWLEDGEMENTS