3rd group meeting viva m.phil transfer 2010 2nd draft

“Development of ex-vivo three-dimensional model

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

OutlinesPHAs

Chronic Lymphocytic Leukaemia (CLL)

An ideal scaffold?

Rationale, novelty, contribution & objectives Rationale, novelty, contribution & objectives

Experimental setup

Results

Future works

Conclusion


What are PHAs? What are PHAs? What are PHAs? What are PHAs?

DEFINITION LOCATION

CLASSESTISSUE


CLASSES

TYPES OF PHAsFACTORS

TISSUE

ENGINEERING?

Molecular structure of PHB and PHBV

31

2

Source: http://biopol.free.fr

m = STRUCTURE BACKBONE = 1, 2, 3, etc. m = 1 is the most common

n = 100 - 30,000 monomers.

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

3-HB

The Role of PHAs in Tissue EngineeringThe Role of PHAs in Tissue EngineeringThe Role of PHAs in Tissue EngineeringThe Role of PHAs in Tissue Engineering

12

Williams et al. International Journal of Biological Macromolecules, (1999)


Mimicking the abnormal

3-D BM niches

What is Chronic Lymphocytic Leukaemia?What is Chronic Lymphocytic Leukaemia?What is Chronic Lymphocytic Leukaemia?What is Chronic Lymphocytic Leukaemia?

DEFINITIONFREQUENCY OF

OCCURENCES

PATHOGENESIS TREATMENT

An Ideal Scaffold for

the T.E.R.M.?

The scaffold →→→→ inter-connecting pores →→→→ tissue integration &

vascularisation process.

An ideal tissue engineering scaffold should fulfill a series of requirements which are:

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.

Tubes derived from PHOH film (left) and porous PHOH

(right) - Williams et al. (1999)


Rationale of doing this research? Rationale of doing this research? Rationale of doing this research? 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.

NoveltyNoveltyNoveltyNovelty

Be able to fabricate porous 3-D scaffolds with an improved thickness of > 2 mm

from the commercially available PHB and PHBV materials

� 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.

OBJECTIVES OBJECTIVES OBJECTIVES 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 Why PHB and PHBV are chosen for Why PHB and PHBV are chosen for Why PHB and PHBV are chosen for

fabricating porous 3fabricating porous 3fabricating porous 3fabricating porous 3----D scaffolds? D scaffolds? D scaffolds? 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).

Experimental Setup

Polymer solution in

organic solvent

Polymer solution

+ Porogen

Solvent evaporation

(Complied with UK-SED,

2002: <20 mg/m3)

Polymer +

Porous 3-D

scaffolds

Porogen-DIW

leaching

Polymer concentration vs. time

Polymer concentration vs. thickness

FABRICATION

Efficacy of SCPL

Porogen residual effect Vs. growth media

12

34

SP1

The solvent-casting and particulate-leaching (SCPL)

Porogen (i.e., NaCl,

sucrose etc.)

Polymer +

Porogen cast

Water contact angle

PHYSICO-CHEMICAL

Principal physical analysis

Morphology of porous structure using SEM

Polymer +

Solvent +

Porogen cast SP2

Advantages: Simple →→→→ fairly reproducible method →→→→no sophisticated apparatus →→→→ controlled porosity & interconnectivity.

Disadvantages: Thickness limitations →→→→ structures generally isotropic & angular →→→→ hazardous solvent →→→→lack of pores interconnectivity →→→→ limited mechanical properties →→→→ residual of porogen & solvent


“To fabricate a novel porous 3-D scaffolds with an improved thickness (more than 2 mm) using the Solvent-Casting Particulate-Leaching (SCPL) technique”

(1) Polymer concentrations with respect to homogenization time

↓↓↓↓(2) Polymer concentrations with respect to polymeric porous 3-D scaffolds

Specific Objectives 1 (SP1)Specific Objectives 1 (SP1)Specific Objectives 1 (SP1)Specific Objectives 1 (SP1)

Experimental works Experimental works Experimental works Experimental works

(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


“RESULTS:

SP1” SP1”


Polymer concentrations with respect to homogenization time


Polymer concentrations with respect to polymeric 3-D scaffolds thickness

The BestThe Best

Polymer concentrations with respect to polymer 3-D scaffolds thickness

Polymer concentrations with respect to polymer 3-D scaffolds thickness

PHBV 4% (w/v)PHB 4% (w/v)

∼∼∼∼10 mm∼∼∼∼10 mm

∼∼∼∼5 mm

PHBV 4% (w/v)

PHB 4% (w/v)

INNER SIDEINNER SIDE

INNER SIDEINNER SIDE

Efficacy of Solvent-Casting Particulate-Leaching (SCPL) via conductivity (mS/cm) measurement

Source: http://www.4oakton.com

(B)(A)

y = 2.8475x + 8.5027

R2 = 0.9999

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35

Concentration of NaCl (mg/ml)C

on

du

ctiv

ity (

mS

/cm

)

Source: http://www.4oakton.com


Salt solution Vs. Conductivity calibration curve

Efficiency: PHB > PHBV →→→→Hydrophilicity: PHB > PHBV

No lost of polymer mass

throughout the SCPL process

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


Conductivity (κκκκ) 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

“To characterize the physico-chemical of polymeric porous 3-D scaffolds with

an improved thickness (> 2 mm)”

(1) Analysis of porosity, surface area, PSD, void volume, bulk and skeletal

density & roughness

Specific Objectives 2 (SP2)Specific Objectives 2 (SP2)Specific Objectives 2 (SP2)Specific Objectives 2 (SP2)

Analysis Analysis Analysis Analysis

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)


“RESULTS:

SP2” SP2”


Physical properties of polymeric porous 3-D scaffolds

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

Water contact angle of polymeric porous 3-D scaffolds and thin films

T.I.P.S

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

S.C.P.LT.I.P.S

“CONCLUSIONS”“CONCLUSIONS”


Polymer concentration of 4% (w/v) →→→→ ideal concentration →→→→ thickness of porous 3-D scaffolds →→→→ > 2 mm.

The insignificant conductivity (κκκκ) 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.


“FUTURE WORKS”“FUTURE WORKS”


“THANK YOU FOR

YOUR KIND

ATTENTION”ATTENTION”


Pore interconnectivity analysis

3-D image analysis: X-ray micro-

computed tomography (XMT)

Mercury Intrusion Pycnometry (MIP)

Total porosity = ΠΠΠΠ = 1 - [0.076 g/ml/1.285 g/ml] = 1 - 0.0591 = 0.94 ×××× 100% = 94%

(1) ρρρρscaffolds = Gravimetry (but for the sake of an accuracy, result was taken from MIP = 0.076 g/ml)

ρρρρ

Fraction of non-pores solid material

(2) ρρρρmaterial = PHB = 1.285 g/ml

The open porosity (ππππ) [porosity accessible for mercury intrusion] = RESULT FROM THE MIP = 73%

The closed porosity (ϖϖϖϖ) [porosity not accessible to mercury] = ΠΠΠΠ - ππππ = 94% - 73% = 21%

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.

3rd group meeting viva m.phil transfer 2010 2nd draft

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