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2nd BSEL Group Meeting Presentation


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2nd BSEL Group Meeting Presentation

  1. 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. 2. Outlines Introduction An ideal scaffold? Aims & objectives Experimental setup Results Future works Conclusion Biological Systems Engineering Laboratory (BSEL)
  3. 3. IntroductionPolyhydroxyalkanoates (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 monomersTypes 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. 4. Molecular structure of PHB and PHBV 3 1 2Source: 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 Williams et al. International Journal of Biological Macromolecules, (1999) Biological Systems Engineering Laboratory (BSEL)
  6. 6. The Potential Use of PHAs in Medicine TO DATEZinn et al. Advanced Drug Delivery Reviews, 2001The approval of TephaFLEX Absorbable suture by FDA which derived from a type of PHA namedpoly-4-hydroxybutyrate (P-4HB) for the use in the surgical applications (Dai et al. 2009) Biological Systems Engineering Laboratory (BSEL)
  7. 7. An Ideal Scaffold for the T.E.R.M.?An ideal scaffold should possess the following characteristics to bring about thedesired 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. 8. Aim 1 “To fabricate a novel porous 3-D scaffolds with an improved thickness (morethan 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. 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. 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 analysisAdvantages: Simple → fairly reproducible method → Roughness analysisno sophisticated apparatus → controlled porosity &interconnectivity. Contact angle and surface free energyDisadvantages: Thickness limitations → structuresgenerally isotropic & angular → hazardous solvent →lack of pores interconnectivity → limited mechanicalproperties → residual of porogen and solvent Pores size and interconnectivity using SEM Biological Systems Engineering Laboratory (BSEL)
  11. 11. “RESULTS: PART A”Biological Systems Engineering Laboratory (BSEL)
  12. 12. Polymer concentrations with respect to homogenization time Biological Systems Engineering Laboratory (BSEL)
  13. 13. Polymer concentrations with respect to polymeric 3-D scaffolds thickness
  14. 14. Polymer concentrations with respect to polymer 3-D scaffolds thickness
  15. 15. Polymer concentrations with respect to polymer 3-D scaffolds thickness
  16. 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. 17. Efficacy of Solvent-Casting Particulate-Leaching (SCPL) viaconductivity (mS/cm) measurement 100 Source: 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 calculatedafter the leaching and lyophilization process” Biological Systems Engineering Laboratory (BSEL)
  18. 18. Effect of sodium chloride (Sigma-Aldrich) residualin polymeric porous 3-D scaffolds on cell growthmedia 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)
  19. 19. “RESULTS: PART B”Biological Systems Engineering Laboratory (BSEL)
  20. 20. Physical properties of polymeric porous 3-D scaffolds
  21. 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. 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. 23. “CONCLUSIONS” Biological Systems Engineering Laboratory (BSEL)
  24. 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 → insignificantamount 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 morehomogeneous (smoothing effect) → physically more pores interconnectivity werecreated → functionalization with oxygen-containing groups into hydrophilic surfaces→ allow better cell attachment and proliferation Biological Systems Engineering Laboratory (BSEL)
  25. 25. “FUTURE WORKS” Biological Systems Engineering Laboratory (BSEL)
  26. 26. Biological Systems Engineering Laboratory (BSEL)
  27. 27. “THANK YOU FOR YOUR KIND ATTENTION” Biological Systems Engineering Laboratory (BSEL)
  28. 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
  29. 29. OMIT SLIDES
  30. 30. Weight fraction and ratio of materials and chemicalsin fabricating polymeric porous 3-D scaffolds ofSolvent-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. 31. World’s Manufacturer of PHB & PHBV - May 2010Gurieff, N. and P. Lant, Comparative life cycleassessment and financial analysis of mixed culturepolyhydroxyalkanoate production. BioresourceTechnology, 2007. 98(17): p. 3393-3403.
  32. 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)