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Biological Systems Engineering Laboratory (BSEL)“Development of ex-vivo three-dimensional model    of chronic lymphocytic ...
Outlines PHAs Chronic Lymphocytic Leukaemia (CLL) An ideal scaffold? Rationale, novelty, contribution & objectives Experim...
What are PHAs? DEFINITION                                 LOCATION                                               TISSUECLA...
Molecular structure of PHB and PHBV                                3                1                                     ...
The Role of PHAs in Tissue Engineering                 2                                    1                             ...
What is Chronic Lymphocytic Leukaemia?                        FREQUENCY OF    DEFINITION           OCCURENCES   PATHOGENES...
An Ideal Scaffold for     the T.E.R.M.?An ideal tissue engineering scaffold should fulfill a series of requirements which ...
Rationale of doing this research?  Malaysia - 15 million tonnes - crude palm oil/year = 52% total world production  The pr...
OBJECTIVES1.     The study of CLL - lack of appropriate ex vivo models - mimic the ABNORMAL       3-D niches.2.     To fab...
Porogen residual effect Vs. growth media         Experimental Setup                                                       ...
Specific Objectives 1 (SP1) “To fabricate a novel porous 3-D scaffolds with an improved thickness (morethan 2 mm) using th...
“RESULTS:  SP1”Biological Systems Engineering Laboratory (BSEL)
Polymer concentrations with respect to homogenization time                  Biological Systems Engineering Laboratory (BSEL)
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
Polymer concentrations with respect to polymer 3-D scaffolds thickness            PHB 4% (w/v)                            ...
Efficacy of Solvent-Casting Particulate-Leaching (SCPL) via conductivity (mS/cm) measurement                 (A)          ...
Effect of sodium chloride (Sigma-Aldrich) residual in       polymeric porous 3-D scaffolds on cell growth media Conductiv...
Specific Objectives 2 (SP2)“To characterize the physico-chemical of polymeric porous 3-D scaffolds with                   ...
“RESULTS:  SP2”Biological Systems Engineering Laboratory (BSEL)
Physical properties of polymeric porous 3-D scaffolds
Morphology of porous structure using scanning electron microscopy (SEM) PHB 4% (w/v)                            PHB 4% (w/...
Water contact angle of polymeric porous 3-D scaffolds and thin films                                                      ...
“CONCLUSIONS”   Biological Systems Engineering Laboratory (BSEL)
Polymer concentration of 4% (w/v) → ideal concentration → thickness ofporous 3-D scaffolds → > 2 mm.                      ...
“FUTURE WORKS”   Biological Systems Engineering Laboratory (BSEL)
Biological Systems Engineering Laboratory (BSEL)
“THANK YOU FOR   YOUR KIND  ATTENTION”   Biological Systems Engineering Laboratory (BSEL)
Pore interconnectivity analysis     3-D image analysis: X-ray micro-                                 Mercury Intrusion Pyc...
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3rd Group Meeting VIVA M.Phil Transfer 2010 2nd Draft

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

  1. 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. 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. 3. What are PHAs? DEFINITION LOCATION TISSUECLASSES ENGINEERING?FACTORS TYPES OF PHAs Biological Systems Engineering Laboratory (BSEL)
  4. 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. 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. 6. What is Chronic Lymphocytic Leukaemia? FREQUENCY OF DEFINITION OCCURENCES PATHOGENESIS TREATMENT
  7. 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. 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. 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. 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. 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. 12. “RESULTS: SP1”Biological Systems Engineering Laboratory (BSEL)
  13. 13. Polymer concentrations with respect to homogenization time Biological Systems Engineering Laboratory (BSEL)
  14. 14. Polymer concentrations with respect to polymeric 3-D scaffolds thickness The Best
  15. 15. Polymer concentrations with respect to polymer 3-D scaffolds thickness
  16. 16. Polymer concentrations with respect to polymer 3-D scaffolds thickness
  17. 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. 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. 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. 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. 21. “RESULTS: SP2”Biological Systems Engineering Laboratory (BSEL)
  22. 22. Physical properties of polymeric porous 3-D scaffolds
  23. 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. 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. 25. “CONCLUSIONS” Biological Systems Engineering Laboratory (BSEL)
  26. 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. 27. “FUTURE WORKS” Biological Systems Engineering Laboratory (BSEL)
  28. 28. Biological Systems Engineering Laboratory (BSEL)
  29. 29. “THANK YOU FOR YOUR KIND ATTENTION” Biological Systems Engineering Laboratory (BSEL)
  30. 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.

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