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Porous Three-Dimensional Scaffolds of Poly(3-Hydroxybutyric Acid) (PHB) and Poly(3-Hydroxybutyric-co-3-Hydroxyvaleric Acid) (PHBV) With
An Improved Thickness As Cell Growth Supporting Materials
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
Polymer solution in organic solvent
Polymer solution + Porogen
Solvent evaporation in fume cupboard (Complied with
UK-SED, 2002: < 20 mg/m3)
Dried cast Polymer +
Porous 3-D scaffolds
Porogen-DIW leaching
12
34
Polymer + Solvent
Polymer Concentration Vs. Thickness
Polymer Concentration Vs. Time
FABRICATION
Efficacy of Salt Removal
Effect of Porogen Residual On Cell Growth Media
5
(a)
(b)
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 be
suitable 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 PHBwith thickness greater than 1 mm have not been produced yet. In this work, PHB and PHBV porous
3-D scaffolds with an improved thickness greater than 4 mm were fabricated and evaluated in terms oftheir physico-chemical characterization and cellular response on the Acute Myeloid Leukaemia cell line
(HL-60) for 14 days.
20.5
20.55
20.6
20.65
20.7
20.75
20.8
20.85
0 1 2 3 4 5 6 7
Cond
uctiv
ity (
mS
/cm
)
PHB (4%, w/v) porous 3-D scaffolds
PHBV (4%, w/v) porous 3-D scaffolds
Control: Cell growth media without a scaffold
NS
92.59
99.67 99.97
82.20
0
10
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60
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120
Salt-leaching process Lyophilization process
Type of polyhydroxyalkanoates (PHAs) porous 3-D scaffolds
% E
ffic
ac
y
PHB (4%, w/v) PHBV (4%, w/v)
**
No lost of polymer mass throughout the SCPL process
Efficiency: PHB > PHBV →→→→Hydrophilicity: PHB > PHBV
FIGURE 6: Conductivity (κ) of cell growth media in the presence of
scaffolds as a function of time at 20 ± 1 oC (n = 3).
FIGURE 5: Efficacy of (A) salt-leaching process and (B) salt
removal after lyophilization process via gravimetric analysis for
PHB and PHBV (4%, w/v). *Significant difference with p<0.05
between the samples were highlighted by lines (n = 10).NOVELTY
METHODOLOGY
Efficacy of salt removal measured via ion conductivity and gravimetric analysis
(a) (c)(b) (d)Through direction
Through direction
Effect of salt remnants in polymeric 3-D scaffolds on cell growth media
To promote the usage of POME in producing PHA via
microbial fermentation process as an ADDED VALUE
MATERIALS for Tissue Engineering applications.
1. To fabricate and optimize the suitable biomimetic scaffolds for culturing
leukaemic cells ex vivo.
2. Study of CLL - lack of appropriate ex vivo models - mimic the ABNORMAL 3-D
BM niches → To facilitate the study of CLL in its native 3-D niches.
Rationale of this research Objectives
Ability to fabricate porous 3-D scaffolds with an improved thickness greater than 4 mm from PHB andPHBV without the presence of the etching surfaces and structural instability.
(a)(c)(e)
Porogen (i.e., NaCl, sucrose & etc.)
Dried cast Polymer + Porogen
Polymer + Solvent + Porogen cast
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 andgravimetric analysis (to determine any potential of polymer weight loss during the salt-leachingprocess). The salt remnants left inside the scaffolds were measured using ion conductivity as an
ultimate validation prior to the physico-chemical characterization and cellular proliferation studies onthe Acute Myeloid Leukaemia cell line (HL-60). Analysis of statistical significance was performed using
one-way analysis of variance (ANOVA) test and Students t-test with a significance level of p<0.05.
Time (days)
Conductivity of cell growth media = 20.77 mS/cm @ 20 ±±±±1 oC
Replication (n = 10)
0
0.5
1
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2
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5.5
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Time of complete homogenization (mins)
Poly
mers
concentra
tio
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
FIGURE 3:Kinetics of PHB and PHBV homogenization
process with respect to differentconcentration, % (w/v). (A): Inhomogeneouspolymer solutions were occurred with the
appearances of glutinous polymer materialsat the bottom of the SCHOTT Duranbottle. The mean values obtained from 10
experiments ± SEM are shown (n = 10).*Significant difference with p<0.05 for thevalue changed as compared to the previous
value. (Ψ) p<0.05 for solubility rate of PHBvs. PHBV.
FIGURE 2:
Thickness of scaffolds at different polymer
concentrations. Measurement was done
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 thickness greater than 4 mm;(6) A rectangular size of ∼10 mm x ∼10 mm x ∼5 mm porous 3-D scaffolds is incised prior to the physico-chemical
characterization, in vitro degradation measurement and cellular proliferation studies.
RESULTS
Polymer concentrations with respect to polymeric 3-D scaffolds thickness Structural properties Polymeric porous 3-D scaffolds
PHB (4%, w/v) PHBV (4%, w/v)
Scaffold thickness, mm 5.25 ± 0.36 4.40 ± 0.52**
Pore size distribution
(diameter: µm)
Micro-pores 10 - 100µm +
Macro-pores 100 - 350µm
Micro-pores 10 - 100µm +
Macro-pores 100 - 350µm
Physical properties
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*
Surface physico-chemistry
Solvent-cast thin film[a][b]
PHB
(4%, w/v)
PHBV
(4%, w/v)
Contact angle, θapparent (deg.) 66.80 ± 0.2 79.24 ± 0.4*
Surface free energy, mN m-1 (γs) 54.13 ± 0.3 46.93 ± 0.2*
Work of adhesive, mN m-1 (WSL)[c] +109.42 ± 0.2 +97.41 ± 0.3*
Spreading coefficient (SH20/scaffolds)[d] -36.38 ± 0.3 -48.39 ± 0.2*
Physico-chemical characterization
(a) (c)
(b) (d)
Through direction Through direction
Through direction Through direction
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16
0 50 100 150 200 250 300 350 400
PHBV (4%, w/v) porous 3-D scaffold
PHB (4%, w/v) porous 3-D scaffold
-dV
/d(log
D)/
cm
3/g
Pore Diameter, D/µm
FIGURE 8: Pore size distribution (PSD)
of PHBV and PHB (4%, w/v) porous 3-
D scaffolds determined using mercury
intrusion porosimetry (MIP).
TABLE 1: Physical properties of PHB and PHBV
(4%, w/v) porous 3-D scaffolds. The table
summarizes the principal physical properties of two
polymeric porous 3-D scaffolds prior to the in vitro
cell proliferation studies.
**(p<0.01) - Results are considered statistically significant (n = 10) ascompared with PHB. *(p<0.05) - Results are considered statisticallysignificant (n = 4) as compared with PHB. [a] BET surface area (m2 g-1) =Total skeletal surface area (m2)/skeletal mass (g). [b] ρs is the skeletal densityof the crushed scaffolds, which is determined from helium pycnometry. [c]The higher pore volume (the higher the amount of absorbate intruded), thelower the skeletal volume.
TABLE 2: Wetting and wettability of water on
PHB and PHBV solvent-cast thin films surfaces.
[a] Equilibrium contact angle on solvent-cast thin films on polypropylene sheet (n = 10). [b]Contact angle of polypropylene (PP) sheet without PHB and PHBV coating = 92.43 ± 0.3 o. [c](+) or (-) work of adhesive: A non-spontaneous or spontaneous process respectively. [d] (+) or (-)spreading coefficient: Water will spread or not spread over the surface respectively. *p<0.05 ascompared with PHB. #p<0.05 as compared with apparent contact angle.
Polymer[a][b][d] Melting
temperature
Heat of fusion
at melting (∆Hm)
Crystalinity (%)[c] TABLE 3: Melting temperature, heat of fusion at melting point
and crystallinity of PHB and PHBV.
FIGURE 7: Scanning electron micrograph
of: (a) PHBV (4%, w/v) porous 3-D
scaffolds vertical cross-section (35x); (b)
PHB (4%, w/v) porous 3-D scaffolds
vertical cross-section 35x). The enlarged
views of (a) and (b) are shown in (c) and
(d) respectively (100x). Particles size:
212-850 µm.
FIGURE 5: Morphology of the polymeric porous3-D scaffolds in a rectangular shape with an
approximate size of 10 × 10 × 5 mm3: (a) Aerialview 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).
The authors would like to thank the Malaysian Higher Education (MOHE), National University of Malaysia
(UKM) and the Richard Thomas Leukemia Fund for providing financial support to this project.
Cut into 10 sections
Porous 3-D
scaffolds
Average thickness
Randomly selected of 5 sections
1. Polymer concentration of 4% (w/v) was considered an optimal concentration to produce an ideal porous 3-D scaffolds with athickness 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%, w/w) with no loss of
polymer weight throughout the process.3. The small amount of salt left inside the porous 3-D scaffolds might not give any adverse effect to the cell growth due to the
electrolytes imbalance from the hypertonic media solution (excessive amount of salt in the cell growth media).4. High in surface hydrophobicity → surface roughness + air trapped inside the pore grooves + contaminants of the salt on the
pore surface.
5. High in surface hydrophobicity → EXPECTED → low degree of cell attachment & proliferation (14 days of cellular response onthe AML cell line (HL-60)).
(a)
(b) (d)
(c)
PHB (4%, w/v)
PHBV (4%, w/v)PHB (4%, w/v)
PHBV (4%, w/v)
∼∼∼∼10 mm ∼∼∼∼10 mm
∼∼∼∼ 5 mmINNER SIDE
INNER SIDE INNER SIDE
INNER SIDE
concentrations. Measurement was done
using Digital Vernier Caliper (accuracy ±
0.01 mm). *Significant difference with p<0.05
as compared to PHB (n = 10).
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*
ACKNOWLEDGEMENTS
temperature at melting (∆Hm)
PHB 179.9 oC 104,974 J kg-1 71.9
PHBV (12% PHV) 152.1 oC 72,708 J kg-1 49.8 [a] Thermal analysis data are provided by the Sigma-Aldrich. [b] Thermal analyses of PHB andPHBV are done using a model DSC-7 differential scanning calorimeter (Perkin Elmer, USA) undera nitrogen atmosphere, at a heating rate of 10 oC min-1. [c] Crystallinity is determined using thefollowing heat of fusion values for 100 % crystalline materials: ∆H
0, PHB =146,000 J kg-1. The ∆H0
for PHBV is assumed to be the same as that for PHB.[106][d] The degree of crystallinity, H* (%); ofthe polymer could thus be estimated by using the following equation: H* (%) = ∆H
m/∆H0 × 100 %.
FIGURE 9: Kinetics of in vitro degradation process for PHB and PHBV (4%, w/v)
porous 3-D scaffolds are measured via mass analysis. The polymeric porous 3-D
scaffolds are submerged in phosphate buffered saline (PBS) and incubated at 37 oC.
Samples are periodically removed and dried under vacuum prior to analysis. (*)
p<0.05 for percent decreased from the previous value (n = 6). ΨSignificant difference
with p<0.01 between each polymers were highlighted by line (n = 6).
Incubation time
(days)
Type of PHAs
(4%, w/v)
Cell number[a]
1 PHB 207,657 ± 76,869
PHBV 136,182 ± 41,574
7 PHB 170,714 ± 105,416
PHBV 165,793 ± 133,283
14 PHB 195,000 ± 69,114
PHBV* 346,428 ± 32,732**
Cell Proliferation Assay of Acute Myeloid Leukaemia Cell
Line (HL-60) on Polymeric Porous 3-D Scaffolds
TABLE 4: Change of cell numbers on PHB and PHBV porous 3-D
scaffolds with time (by MTS assay).
[a] Initial seeding 370,000 cells per sample.**p<0.01 relative to day 1.*p<0.05 relative to PHB at day 14.
(a) (b) (c) (d)
PHB 3% (w/v) PHBV 3% (w/v)PHB 5% (w/v) PHBV 5% (w/v) PHB 1% (w/v)
FIGURE 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).
0
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0 7 14 21 28 35 42 49 56 63 70 77 84
Time (days)
% R
esid
ua
l w
eig
ht o
f p
oro
us 3
-D s
ca
ffo
lds
PHB (4%, w/v) porous 3-D scaffold
PHBV (4%, w/v) porous 3-D scaffold
Ψ
*
* *
**
*
CONCLUSIONS
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