2nd bsel group meeting presentation

“Development of ex-vivo three-dimensional model

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

Outlines

Introduction

An ideal scaffold?

Aims & objectives

Experimental setupExperimental setup

Results

Future works

Conclusion


Introduction Introduction Introduction 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) →→→→ →→→→

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).


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)


The Potential Use of PHAs in MedicineThe Potential Use of PHAs in MedicineThe Potential Use of PHAs in MedicineThe Potential Use of PHAs in Medicine

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)

TO DATETO DATETO DATETO DATE


An Ideal Scaffold for

the T.E.R.M.?

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

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.

Tubes derived from PHOH film (left) and porous PHOH

(right) - Williams et al. (1999)


“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

Aim 1Aim 1Aim 1Aim 1

Objectives Objectives Objectives Objectives

(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


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

with an improved thickness (more than 2 mm)”

(1) Analysis of porosity/surface area/PSD/void volume/roughness

↓↓↓↓

Aim 2Aim 2Aim 2Aim 2

Objectives Objectives Objectives Objectives

↓↓↓↓

(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


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

A

The solvent-casting and particulate-leaching (SCPL)

Porogen (i.e., NaCl,

sucrose etc.)

Polymer +

Porogen cast

Contact angle and surface free energy

PHYSICO-CHEMICAL

Porosity analysisRoughness analysis

Pores size and interconnectivity using SEM

Polymer +

Solvent +

Porogen cast B

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 and solvent


“RESULTS:

PART A” PART A”


Polymer concentrations with respect to homogenization time


Polymer concentrations with respect to polymeric 3-D scaffolds thickness

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)

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

90

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

“Mass balance of sodium chloride were calculated

after the leaching and lyophilization process”

y = 2.8475x + 8.5027

R2 = 0.9999

0

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25 30 35

Concentration of NaCl (mg/ml)

Co

nd

uc

tivity

(m

S/c

m)


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.


“RESULTS:

PART B” PART B”


Physical properties of polymeric porous 3-D scaffolds

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

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

“CONCLUSIONS”“CONCLUSIONS”


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


“FUTURE WORKS”“FUTURE WORKS”


“THANK YOU FOR

YOUR KIND

ATTENTION”ATTENTION”


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 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

OMIT SLIDESOMIT SLIDES

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)]


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.

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).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.


2nd bsel group meeting presentation

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