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

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

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

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)

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

0

2

4

6

8

10

12

14

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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110

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