1. Biological Systems Engineering Laboratory (BSEL)
“Development of ex-vivo three-dimensional model
of chronic lymphocytic leukemia (CLL)”
SAIFUL IRWAN ZUBAIRI
SUPERVISOR: Dr. Sakis Mantalaris
CO-SUPERVISOR: Dr. Nicki Panoskaltsis
2. Outlines
Introduction
An ideal scaffold?
Aims & objectives
Experimental setup
Results
Future works
Conclusion
Biological Systems Engineering Laboratory (BSEL)
3. Introduction
Polyhydroxyalkanoates (PHAs) → a family of biopolyesters → bacteria →
intracellular carbon & energy-storage compounds.
Tissue engineering materials → GOOD → physical properties, biodegradability &
biocompatibility.
Poly(3-hydroxybutyrate) (PHB) & poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
(PHBV) → biomaterials → in vitro & in vivo studies
> 150 types → PHAs → various monomers
Types of bacterium & growth conditions → chemical composition → PHAs & Mw
→ 2×105 to 3×106 Da.
× ×
3 classes → (sclPHA, C3 - C5) → (mclPHA, C6 - C14) → (lclPHA, >C14).
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
Williams et al. International Journal of Biological Macromolecules, (1999)
Biological Systems Engineering Laboratory (BSEL)
6. The Potential Use of PHAs in Medicine
TO DATE
Zinn et al. Advanced Drug Delivery Reviews, 2001
The approval of TephaFLEX Absorbable suture by FDA which derived from a type of PHA named
poly-4-hydroxybutyrate (P-4HB) for the use in the surgical applications (Dai et al. 2009)
Biological Systems Engineering Laboratory (BSEL)
7. An Ideal Scaffold for
the T.E.R.M.?
An ideal scaffold should possess the following characteristics to bring about the
desired biological response (Liu, W. & Y. Cao, 2007):
The scaffold → inter-connecting pores → tissue integration &
vascularisation process.
Material → biodegradability/bio-resorbability.
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. Aim 1
“To fabricate a novel porous 3-D scaffolds with an improved thickness (more
than 2 mm) using the Solvent-Casting Particulate-Leaching (SCPL) technique”
Objectives
(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)
9. Aim 2
“To characterize the physico-chemical of polymeric porous 3-D scaffolds
with an improved thickness (more than 2 mm)”
Objectives
(1) Analysis of porosity/surface area/PSD/void volume/roughness
↓
(2) Analysis of pores size and interconnectivity using
scanning electron microscopy (SEM)
↓
(3) Contact angle and surface free energy of dry PHB and PHBV
porous 3-D scaffolds
Biological Systems Engineering Laboratory (BSEL)
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 A
+ Porogen 3
4
2
1 Porous 3-D
scaffolds
Polymer + Polymer +
Solvent + Porogen cast
Porogen cast B
Porogen (i.e., NaCl, PHYSICO-CHEMICAL
sucrose etc.)
Porosity analysis
Advantages: Simple → fairly reproducible method → Roughness analysis
no sophisticated apparatus → controlled porosity &
interconnectivity.
Contact angle and surface free energy
Disadvantages: Thickness limitations → structures
generally isotropic & angular → hazardous solvent →
lack of pores interconnectivity → limited mechanical
properties → residual of porogen and solvent
Pores size and interconnectivity using SEM
Biological Systems Engineering Laboratory (BSEL)
16. Polymer concentrations with respect to polymer 3-D scaffolds thickness
PHB 4% (w/v) PHBV 4% (w/v)
PHBV 4% (w/v)
PHB 4% (w/v) ∼10 mm
∼10 mm
∼5 mm
17. Efficacy of Solvent-Casting Particulate-Leaching (SCPL) via
conductivity (mS/cm) measurement
100 Source: http://www.4oakton.com
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
Concentration of NaCl (mg/ml)
“Mass balance of sodium chloride were calculated
after the leaching and lyophilization process”
Biological Systems Engineering Laboratory (BSEL)
18. Effect of sodium chloride (Sigma-Aldrich) residual
in polymeric porous 3-D scaffolds on cell growth
media
The effect of sodium chloride residual inside PHB and PHBV porous 3-D scaffolds on the cell growth media
measured by pH changes. The polymeric porous 3-D scaffolds were submerged in the cell growth media (90%
IMDM+10% FBS+1% PS) and incubated at 37 oC, and 5% CO2 (n = 3) for 7 days. NS indicates no significant
differences as compared to control.
Biological Systems Engineering Laboratory (BSEL)
21. Pores size and interconnectivity analysis using scanning electron microscopy (SEM)
PHB 4% (w/v) PHB 4% (w/v) - enlarged
PHBV 4% (w/v) PHBV 4% (w/v) - enlarged
22. Wettability and surface energy of polymeric porous 3-D scaffolds
(a, b) Schematic of a simple derivation of Young’s equation using surface tension vectors for a
liquid on a solid substances (ideal solid surfaces). (c) Wenzel’s model of non-ideal solid surfaces
23. “CONCLUSIONS”
Biological Systems Engineering Laboratory (BSEL)
24. Polymer concentration of 4% (w/v) for PHB and PHBV → ideal concentration →
thickness of porous 3-D scaffolds → more than 2 mm.
The insignificant → pH values → cell growth media Vs. control → insignificant
amount of porogen residual remained.
No contaminants/residual → No effect on the in vitro cell proliferation studies.
Both polymeric porous 3-D scaffolds → highly hydrophobic materials.
Lack of pores interconnectivity and highly hydrophobicity of the surfaces
→ EXPECTED → low degree of cell attachment and proliferation.
Modifying its surface chemistries → polymer surface becomes chemically more
homogeneous (smoothing effect) → physically more pores interconnectivity were
created → functionalization with oxygen-containing groups into hydrophilic surfaces
→ allow better cell attachment and proliferation
Biological Systems Engineering Laboratory (BSEL)
25. “FUTURE WORKS”
Biological Systems Engineering Laboratory (BSEL)
27. “THANK YOU FOR
YOUR KIND
ATTENTION”
Biological Systems Engineering Laboratory (BSEL)
28. Question:
1. Why the thickness of 5-mm? ANS: (1) Previous studies show that the thickness of 5-
mm was the optimum level for the cell-depth penetration to be occurred - Problem could
occurred if >5-mm e.g.: no nutrient, oxygen and waste could be transported across the
scaffolds – this could trigger apostosis (programmed cell death) due to the starvation. (2)
Since our aim to mimic the BM micro-environment for transplanting HSC into the leukemia
BM, we’re aiming to mimic the thickness as similar to the human BM. Thickness of human
BM in reticular (resembling a net in form; netlike) connective tissue area which consist of
a complex sinusoidal system (arterial vascular system) + hematopoietic cells + stroma
(non-hemato).
2. Novelty of your research? – Can fabricate 3-D scaffolds with an improved thickness of
more than 2 mm – Up the extent of our knowledge - none of the studies produce 3-D
scaffolds with thickness than 2 mm with this particular type of biopolyesters – most of
them are at the µm size.
3. Why PHB and PHBV? Why not PU, PP and others? – This polymers can be
synthesized – waste/renewable sources – to become as added value product – for the
application of leukemia treatment
30. Weight fraction and ratio of materials and chemicals
in fabricating polymeric porous 3-D scaffolds of
Solvent-Casting Particulate-Leaching (SCPL)
As the salt weight fraction increased from 60% to 90% (w/w), the porosities increased
gradually from 0.69 to 0.90 and porosities are homogenous with interconnected pores -
[Lu et al. (2000) & Mikos (1994)]
Biological Systems Engineering Laboratory (BSEL)
31. World’s Manufacturer of PHB & PHBV - May 2010
Gurieff, N. and P. Lant, Comparative life cycle
assessment and financial analysis of mixed culture
polyhydroxyalkanoate production. Bioresource
Technology, 2007. 98(17): p. 3393-3403.
32. In vitro degradation studies for PHB and PHBV porous 3-D scaffolds - PBS & cell
growth media.
↓
Mechanical testing (compressive moduli): Untreated & immersion porous 3-D
scaffolds with cell growth media (4 wks & 8 wks)
↓
Surface treatment via O2 rf-plasma or alkaline treatment Vs. cellular proliferation
studies (2 weeks).
↓
Surface modification via immersion freeze-dried coating with 2 types of BM main
proteins (collagen type I & fibronectin) Vs. cellular proliferation
studies (2 weeks).
↓
Modeling the abnormal hematopoietic 3-D culture system for short- and long-term
of 4 and 8 weeks respectively.
Biological Systems Engineering Laboratory (BSEL)
