1. Biological Systems Engineering Laboratory (BSEL)
“Development of ex-vivo three-dimensional model
of chronic lymphocytic leukaemia (CLL)”
SAIFUL IRWAN ZUBAIRI
SUPERVISOR: Dr. Sakis Mantalaris
CO-SUPERVISOR: Dr. Nicki Panoskaltsis

2. Outlines
PHAs
Chronic Lymphocytic Leukaemia (CLL)
An ideal scaffold?
Rationale, novelty, contribution & objectives
Experimental setup
Results
Future works
Conclusion
Biological Systems Engineering Laboratory (BSEL)

3. What are PHAs?
DEFINITION LOCATION
TISSUE
CLASSES ENGINEERING?
FACTORS TYPES OF PHAs
Biological Systems Engineering Laboratory (BSEL)

4. Molecular structure of PHB and PHBV
3 1
2
Source: http://biopol.free.fr
m = STRUCTURE BACKBONE = 1, 2, 3, etc. m = 1 is the most common
n = 100 - 30,000 monomers. 3-HB
R is a variable: Types of homo-polymers in the PHAs family.
m = 1, R = CH3, → 3-hydroxybutyrate (3-HB)
m = 1, R = C2H5, → 3-hydroxyvalerate (3-HV)
3-HB + 3-HV

5. The Role of PHAs in Tissue Engineering
2 1
Mimicking the abnormal
3-D BM niches
Williams et al. International Journal of Biological Macromolecules, (1999)
Biological Systems Engineering Laboratory (BSEL)

6. What is Chronic Lymphocytic Leukaemia?
FREQUENCY OF
DEFINITION OCCURENCES
PATHOGENESIS TREATMENT

7. An Ideal Scaffold for
the T.E.R.M.?
An ideal tissue engineering scaffold should fulfill a series of requirements which are:
The scaffold → inter-connecting pores → tissue integration &
vascularisation process.
Material → biocompatible → adverse responses.
Surface chemistry → cellular attachment, differentiation & proliferation.
Mechanical properties → intended site of implantation & handling.
Be easily fabricated into a variety of shapes & sizes.
Biological Systems Engineering Laboratory (BSEL) Tubes derived from PHOH film (left) and porous PHOH
(right) - Williams et al. (1999)

8. Rationale of doing this research?
Malaysia - 15 million tonnes - crude palm oil/year = 52% total world production
The process to extract oil - Fresh Fruit Bunch (FFB) - large amount of water -
sterilizing the fruits & oil clarification = discharge of organic + non-toxic
wastewater → Palm Oil Mill Effluent (POME).
POME = 95-96% water + 0.6-0.7% oil + 4-5% total solids.
To promote the usage of POME in producing PHAs via microbial fermentation
process as an ADDED VALUE MATERIALS for the T.E applications.
Novelty
Be able to fabricate porous 3-D scaffolds with an improved thickness of > 2 mm
from the commercially available PHB and PHBV materials

9. OBJECTIVES
1. The study of CLL - lack of appropriate ex vivo models - mimic the ABNORMAL
3-D niches.
2. To fabricate and optimize the suitable biomimetic scaffolds for culturing
leukaemic cells ex vivo → facilitate the study of CLL in its native 3-D niche.
3. No animal & clinical studies are conducted + Primary CLL are not wasted + Less
time consumed for choosing the right treatment.
Why PHB and PHBV are chosen for
3-
fabricating porous 3-D scaffolds?
The ONLY biodegradable polymers - slowly degraded by surface erosion - OTHER
biodegradable polymers (e.g. PLA, PLGA etc.) → rapid & bulk degradation →
suitable for long term leukaemic cell growth (8 weeks).

10. Porogen residual effect Vs. growth media
Experimental Setup Efficacy of SCPL
The solvent-casting and particulate-leaching (SCPL)
Polymer concentration vs. thickness
Polymer solution in Solvent evaporation
(Complied with UK-SED, Polymer concentration vs. time
organic solvent
2002: <20 mg/m3) Porogen-DIW
Polymer solution leaching
FABRICATION SP1
+ Porogen 3
4
2
1 Porous 3-D
scaffolds
Polymer + Polymer +
Solvent + Porogen cast
Porogen cast SP2
Porogen (i.e., NaCl, PHYSICO-CHEMICAL
sucrose etc.)
Principal physical analysis
Advantages: Simple → fairly reproducible method →
no sophisticated apparatus → controlled porosity &
interconnectivity.
Water contact angle
Disadvantages: Thickness limitations → structures
generally isotropic & angular → hazardous solvent →
lack of pores interconnectivity → limited mechanical Morphology of porous structure using SEM
properties → residual of porogen & solvent
Biological Systems Engineering Laboratory (BSEL)

11. Specific Objectives 1 (SP1)
“To fabricate a novel porous 3-D scaffolds with an improved thickness (more
than 2 mm) using the Solvent-Casting Particulate-Leaching (SCPL) technique”
Experimental works
(1) Polymer concentrations with respect to homogenization time
↓
(2) Polymer concentrations with respect to polymeric porous 3-D scaffolds
thickness
↓
(3) Efficacy of Solvent-Casting Particulate-Leaching (SCPL) via conductivity
(mS/cm) measurement
↓
(4) Effect of sodium chloride (Sigma-Aldrich) residual in polymeric porous 3-D
scaffolds on the cell growth media
Biological Systems Engineering Laboratory (BSEL)

12. “RESULTS:
SP1”
Biological Systems Engineering Laboratory (BSEL)

13. Polymer concentrations with respect to homogenization time
Biological Systems Engineering Laboratory (BSEL)

14. Polymer concentrations with respect to polymeric 3-D scaffolds thickness
The Best

15. Polymer concentrations with respect to polymer 3-D scaffolds thickness

16. Polymer concentrations with respect to polymer 3-D scaffolds thickness

17. Polymer concentrations with respect to polymer 3-D scaffolds thickness
PHB 4% (w/v) PHBV 4% (w/v)
INNER SIDE
INNER SIDE
PHBV 4% (w/v)
PHB 4% (w/v) ∼10 mm
∼10 mm
∼5 mm
INNER SIDE
INNER SIDE

18. Efficacy of Solvent-Casting Particulate-Leaching (SCPL) via
conductivity (mS/cm) measurement
(A) (B)
Source: http://www.4oakton.com
100
Salt solution Vs. Conductivity calibration curve
90
80
Conduc tiv ity (mS/c m)
70
60
50 y = 2.8475x + 8.5027
40 R2 = 0.9999
30
20
10
0
0 5 10 15 20 25 30 35
No lost of polymer mass
Efficiency: PHB > PHBV → throughout the SCPL process
Concentration of NaCl (mg/ml)
Hydrophilicity: PHB > PHBV
Biological Systems Engineering Laboratory (BSEL)

19. Effect of sodium chloride (Sigma-Aldrich) residual in
polymeric porous 3-D scaffolds on cell growth media
Conductivity of cell growth
media = 20.77 mS/cm @ 21 oC
κ
Conductivity (κ) of cell growth media as a function of time at
temperature of 21 oC. The polymeric porous 3-D scaffolds were
submerged in cell growth media (90% IMDM + 10% FBS + 1%
PS) and incubated at 37 oC, and 5% CO2 for 7 days.
http://www.joslinresearch.org/medianet/Reagent_Contents_main.asp
Biological Systems Engineering Laboratory (BSEL)

20. Specific Objectives 2 (SP2)
“To characterize the physico-chemical of polymeric porous 3-D scaffolds with
an improved thickness (> 2 mm)”
Analysis
(1) Analysis of porosity, surface area, PSD, void volume, bulk and skeletal
density & roughness
↓
(2) Observation of pores sizes and the pore distribution by using
scanning electron microscopy (SEM)
↓
(3) Water contact angle of polymeric porous 3-D scaffolds and the
corresponding thin films (T.I.P.S)
Biological Systems Engineering Laboratory (BSEL)

21. “RESULTS:
SP2”
Biological Systems Engineering Laboratory (BSEL)

22. Physical properties of polymeric porous 3-D scaffolds

23. Morphology of porous structure using scanning electron microscopy (SEM)
PHB 4% (w/v) PHB 4% (w/v) - Enlarged
PHBV 4% (w/v) PHBV 4% (w/v) - Enlarged

24. Water contact angle of polymeric porous 3-D scaffolds and thin films
T.I.P.S
S.C.P.L
Polymeric porous 3-D scaffolds are highly hydrophobic probably due to (1) surface
roughness; (2) air trapped inside the pore grooves; (3) contaminants of salt on the surfaces

25. “CONCLUSIONS”
Biological Systems Engineering Laboratory (BSEL)

26. Polymer concentration of 4% (w/v) → ideal concentration → thickness of
porous 3-D scaffolds → > 2 mm.
κ
The insignificant conductivity (κ) changes = insignificant amount of salt
trapped inside → to effect the cell growth media electrolytes balance →
CONSIDERED FREE FROM CONTAMINANTS & SAFE TO USED AS
SCAFFOLDS.
Highly hydrophobic → surface roughness + air trapped inside the pore
grooves + contaminants of salt on the surface.
High in hydrophobicity → EXPECTED → low degree of cell attachment &
proliferation.
Biological Systems Engineering Laboratory (BSEL)

27. “FUTURE WORKS”
Biological Systems Engineering Laboratory (BSEL)

28. Biological Systems Engineering Laboratory (BSEL)

29. “THANK YOU FOR
YOUR KIND
ATTENTION”
Biological Systems Engineering Laboratory (BSEL)

30. Pore interconnectivity analysis
3-D image analysis: X-ray micro- Mercury Intrusion Pycnometry (MIP)
computed tomography (XMT)
Fraction of non-pores solid material
Total porosity = Π = 1 - [0.076 g/ml/1.285 g/ml] = 1 - 0.0591 = 0.94 × 100% = 94%
(1) ρscaffolds = Gravimetry (but for the sake of an accuracy, result was taken from MIP = 0.076 g/ml)
(2) ρmaterial = PHB = 1.285 g/ml
π
The open porosity (π) [porosity accessible for mercury intrusion] = RESULT FROM THE MIP = 73%
The closed porosity (ϖ) [porosity not accessible to mercury] = Π - π = 94% - 73% = 21%
ϖ
So, we assumed that the DISTRIBUTION OF POROSITY INSIDE THE POROUS 3-D SCAFFOLDS
ARE AS FOLLOWS = out 94% total porosity = 73% open interconnected pores + 21% closed
pores + 6% non-pores solid material.