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3rd Group Meeting VIVA M.Phil Transfer 2010 2nd Draft

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  • 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 TISSUECLASSES ENGINEERING?FACTORS TYPES OF PHAs Biological Systems Engineering Laboratory (BSEL)
  • 4. Molecular structure of PHB and PHBV 3 1 2Source: 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. NoveltyBe able to fabricate porous 3-D scaffolds with an improved thickness of > 2 mmfrom the commercially available PHB and PHBV materials
  • 9. OBJECTIVES1. 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 analysisAdvantages: Simple → fairly reproducible method →no sophisticated apparatus → controlled porosity &interconnectivity. Water contact angleDisadvantages: Thickness limitations → structuresgenerally isotropic & angular → hazardous solvent →lack of pores interconnectivity → limited mechanical Morphology of porous structure using SEMproperties → 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 (morethan 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 massEfficiency: 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 ofporous 3-D scaffolds → > 2 mm. κThe insignificant conductivity (κ) changes = insignificant amount of salttrapped inside → to effect the cell growth media electrolytes balance →CONSIDERED FREE FROM CONTAMINANTS & SAFE TO USED ASSCAFFOLDS.Highly hydrophobic → surface roughness + air trapped inside the poregrooves + 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 materialTotal 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 πThe 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% ϖSo, we assumed that the DISTRIBUTION OF POROSITY INSIDE THE POROUS 3-D SCAFFOLDSARE AS FOLLOWS = out 94% total porosity = 73% open interconnected pores + 21% closedpores + 6% non-pores solid material.