Polymeric Porous 3-D Scaffolds


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  • (Pfeffer, 1992; Salehizadeh and Van Loosdrecht, 2004) – References quoted from Koutinas et al., 2006 (Anshuman et al., 2006) - PHB FROM FOOD WASTE AS A CARBON SOURCES FILES – NO. 3 – END NOTE
  • I’ve identified 3 main objectives in my MPhil research project. I’ve found a few journals that used renewable feedstocks as their nutrient supplement – but most of them are converted the feedstocks by using fungal fermentation process before being used as a primary source of nutrient media for the bacterial fermentation process – They want to break down more to simplify the nutrient
  • ( Lowenberg et al ., 1999) – Quoted from Teresa’s thesis references
  • (Lee, 1999; Lee, 1996) - q uoted from this website - http://2008.igem.org/Team:Utah_State/Project
  • And now, lets look at the properties of this type of microb which is R. eutrophus a.k.a
  • (Verlinden, 2007) - quoted from this website - http://2008.igem.org/Team:Utah_State/Project
  • Why are we going to use PHB instead of using other natural polyesters? ( Deng et al., 2002) - Literature MS WORD (Lee et al., 1998; Van et al., 1985) – Teresa’s thesis references
  • KH 2 PO 4 - Potassium dihydrogen phosphate
  • Microbial biosynthesis of PHB starts with the condensation of two molecules of acetyl- CoA to give acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA. This latter compound is then used as a monomer to polymerize PHB
  • Polymeric Porous 3-D Scaffolds

    1. 1. “ Development of an ex vivo 3-D model of chronic lymphocytic leukemia (CLL)” SAIFUL IRWAN ZUBAIRI SUPERVISOR: Dr. Sakis Mantalaris CO-SUPERVISOR: Dr. Nicki Panoskaltsis
    2. 2. Brief research introduction: <ul><li>No one could ever wonder the complex renewable feedstocks - e.g.: tapioca hydrolysates, whey, food scraps from our daily consumption and pulp fiber sludge - ground breaking materials - T.E.R.M (Koutinas et al., 2006). </li></ul><ul><li>Recently, scientist has discovered a few natural polymers - complex renewable sources - produced - aliphatic group (e.g.: PHA and PHB) - used effectively - support niches structure known as scaffolds. </li></ul><ul><li>Natural scaffolds - deliver self renewal hematopoietic stem cells (HSCs) - transplanting - the scaffolds + HSCs into the leukemic patient’s bone marrow - adverse effects. </li></ul>
    3. 3. Continue: <ul><li>The main interest - natural polymers - polyhydroxybutyrate (PHB). </li></ul><ul><li>Poly  -hydroxybutyric acid (PHB) is the most extensively studied PHA - produced in nature in the presence of excess carbon by bacteria as storage granules - provide food and energy - during its limited food sources (Pfeffer, 1992; Salehizadeh and Van Loosdrecht, 2004). </li></ul><ul><li>PHB has properties similar to petroleum derived synthetic plastics - polypropylene (PP) - completely biodegradable in the environment (Anshuman et al., 2006). </li></ul><ul><li>Negative gram bacterial ( Ralstonia eutropha a.k.a Alcaligenes euthropus NCIMB 11599) - produce - polyhydroxybutyrate (PHB) - via batch and fed-batch fermentation process. </li></ul>
    4. 4. Aim and hypothesis 1 (MPhil level): <ul><li>(1) To synthesize natural polyesters (PHB) from the pure commercial nutrient media (high in carbon and nitrogen sources) by using batch and fed-batch bacterial fermentation process. </li></ul><ul><li>It is hypothesized that by using the pure commercial nutrient media - the high yield of PHB (mg) can be obtained - purity up to 60% - regardless - batch or fed-batch fermentation process - an excellent benchmark/standard as if the REAL renewable feedstocks are used as a nutrient supplement. </li></ul>
    5. 5. Aim and hypothesis 2 (MPhil level): <ul><li>(2) To fabricate scaffolds from the naturally synthesized PHB (bacterial fermentation) and commercialized natural origin PHB (Sigma-Aldrich, 99%) and compared it with the non-biodegradable polyurethane (PU) from the previous studies. </li></ul><ul><li>It is hypothesized that by using a 3-D culture system that resembled the highly porous structure of the bone marrow - a good depiction of the original leukemic bone marrow architecture can be reproduced . </li></ul><ul><li>Leukemic cell culture could be maintained with its original genetic features - independent from cytokine addition - HOPEFULLY - natural CLL growth could be studied in its natural condition ex vivo . </li></ul>
    6. 6. Aim and hypothesis 3 (MPhil level): <ul><li>(3) To evaluate the efficacy of all cell seeded scaffolds by checking its cell seeding efficiency and long term proliferation. </li></ul><ul><li>This investigation is based on the hypothesis that 3-D growth design for CLL proliferation will imitate the in vivo marrow environment more closely than 2-D culture - resulting in an enhanced in vitro culture without the need to add exogenous cytokines. </li></ul><ul><li>Two natural and one synthetic based polymeric scaffolds are selected and tested as suitable materials for CLL seeding and growth. </li></ul><ul><li>These scaffolds will have interrelated pores to allow cell penetration and multiply - to mimic the bone marrow structure. </li></ul>
    7. 7. What is leukemia in relation with BM? <ul><li>Bone marrow - complex 3-D tissue - hematopoiesis is regulated by the intercellular microenvironments niches. </li></ul><ul><li>The dysregulation of this niche, either in structure or function - can contribute in the pathogenesis (step-by-step development of disease) of a certain disease - e.g.: Leukemia ( Lowenberg et al ., 1999). </li></ul><ul><li>Leukemia = a cancer of the blood or bone marrow - is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). </li></ul><ul><li>The latest statistics - incurable blood tumors which are known as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) - the main common blood tumors - kill approximately 1.4 millions peoples in United States for the past 20 years ( Leukemia & Lymphoma Society, 2008). </li></ul>
    8. 8. Continue: <ul><li>Leukemia - clinically and pathologically subdivided into several large groups. </li></ul><ul><li>The first division is between its acute and chronic forms. </li></ul><ul><li>To be more specific, the diseases are subdivided according to which kind of blood cell is affected. </li></ul><ul><li>In lymphoblastic or lymphocytic leukemias - the cancerous change takes place in a type of marrow cell that normally goes on to - lymphocytes, which are infection-fighting immune system cells. </li></ul><ul><li>In myeloid or myelogenous leukemias - the cancerous change takes place in a type of marrow cell that normally goes on to - red blood cells, some other types of white cells, and platelets. </li></ul>
    9. 9. Combining these two classifications provides a total of four main categories: <ul><li>Definition in relation with rate of the cell growth: </li></ul><ul><li>Acute Leukemia = an overgrowth of very immature blood cells - progressing quickly - this is a life threatening situation - low in mature blood cells - anemia, infection & bleeding to death. </li></ul><ul><li>Chronic Leukemia = an overgrowth of mature blood cells – progressing slowly </li></ul>
    10. 10. What is chronic lymphocytic leukemia (CLL)? <ul><li>Chronic lymphocytic leukemia - a type of leukemia or cancer of the white blood cells (lymphocytes). </li></ul><ul><li>CLL affects a particular lymphocyte - e.g.: the B cell - originates in the bone marrow, develops in the lymph nodes, and normally fights infection. </li></ul><ul><li>In CLL, the DNA of a B cell is damaged - it can't fight infection - it grows out of control and crowds out in the healthy blood cells that can fight infection. </li></ul><ul><li>CLL is a disease of ADULTS . </li></ul><ul><li>Most people newly diagnosed with CLL are over the age 50 (>75%) - majority are men. </li></ul><ul><li>In the United States during 2007 - it is estimated there are 15,340 new cases diagnosed and 4,500 deaths, but because of prolonged survival - due to medicine breakthrough - many more people are living with CLL. </li></ul><ul><li>Early CLL is not treated. </li></ul><ul><li>Late CLL is treated with chemotherapy and monoclonal antibodies . </li></ul><ul><li>Survival varies from 5 years to more than 25 years. </li></ul>
    11. 11. What is Polyhydroxylbutyrate (PHB)? <ul><li>Polyhydroxybutyrate (PHB) belongs to and is the most common member of a broader class of polyesters - polyhydroxyalkanoates (PHA). </li></ul><ul><li>It is an intracellular accumulated by a wide range of microorganisms - carbon and energy reserve material in response to an environmental stress - e.g.: nutrient limitation. </li></ul><ul><li>Among all microbes - Ralstonia eutrophus a.k.a Alcaligenes eutrophus - produced - the highest % dry weight of biomass - 96 % (w/w) - purity of PHB - up to 60 % (w/w). </li></ul><ul><li>Over 80 different PHAs have been classified - each varies in mechanical properties. </li></ul><ul><li>The difference within the polymers depends on the R side chain . </li></ul><ul><li>In particular, PHB possesses material properties comparable to petrochemically-derived plastics (Lee, 1999; Lee, 1996). </li></ul><ul><li>In addition to physical and mechanical similarities with conventional plastics like polypropylene and polyethylene, its biocompatibility properties with human tissues make it appealing for the use in biomedical applications . </li></ul>
    12. 12. Ralstonia eutrophus a.k.a Alcaligenes eutrophus <ul><li>-ve gram bacteria. </li></ul><ul><li>Non-spore forming bacillus . </li></ul><ul><li>Growth at T = 30 o C (optimal) - environment that contain toxic heavy metal (mM). </li></ul><ul><li>Synthesize PHB - as a way to store LIPIDS - excess carbon is present but limited in nitrogen and phosphate. </li></ul>
    13. 13. Metabolic Pathway for PHB accumulation <ul><li>Consists of three major steps which is involved in 3 types of enzymes (Verlinden, 2007): </li></ul><ul><ul><ul><li>1. 3-ketothiolase - produces acetoacetyl-CoA by joining two molecules of acetyl-CoA </li></ul></ul></ul><ul><ul><ul><li>2. Acetoacetyl-CoA reductase - promotes the reduction of acetoacetyl-CoA by NADH to 3-hydroxybutyryl-CoA. </li></ul></ul></ul><ul><ul><ul><li>3. PHA synthase - polymerizes the 3-hydroxybutyryl-CoA to form PHB. </li></ul></ul></ul><ul><li>Acetyl-coenzyme-A (acetyl-CoA) is a PHB precursor that is naturally produced by these bacteria. </li></ul>R-chain side PHB - consist - combination of 20,000 monomers
    14. 14. Why PHB but not other classes of polyester (e.g.: PHA, PHV, PHH & etc.)? <ul><li>Most of the polyesters (aliphatic & aromatic) are water soluble and moisture sensitive - hydrolysis process (degradation induced by water). </li></ul><ul><li>But for PHB - the only polyester - H 2 O insoluble. </li></ul><ul><li>**Although the cells adhere & grow better on hydrophilic surface scaffolds (Lee et al., 1998; Van et al., 1985) - PHB can be treated to be slightly hydrophilic by using the lipases and NaOH treatment ( Deng et al., 2002)** </li></ul><ul><li>Relatively resistant to hydrolytic degradation. </li></ul><ul><li>Non-toxic materials. </li></ul><ul><li>Shows good O 2 permeability. </li></ul><ul><li>Soluble in chloroform and other chlorinated hydrocarbons (e.g.: methyl chloride, dichloromethane). </li></ul>
    15. 15. Research Approach (MPhil Level)
    16. 16. (1) Acquiring the polyhydroxybutryrate (PHB) - Bacterial batch and fed-batch fermentation process: <ul><li>The batch and fed-batch fermentation process are conducted in triplicate on 2 different occasions/treatment (N = 6). </li></ul><ul><li>There are 4 dependant variables that needs to be observed which are as follows: (a) total glucose consumed (mg); (b) microbial biomass concentration (mg/ml); (c) purified total dry weight (mg); and (d) yield of PHB (mg/mg). </li></ul><ul><li>The results are expressed as mean  standard deviation (mean  SD). </li></ul><ul><li>Significantly different results (between the treatments) are evaluated by using one-way analysis of variance (ANOVA) with a level of p<0.05 or p<0.01 depending on the measurement that considered significant . </li></ul>
    17. 17. Continue: <ul><li>A level of p<0.05 or p<0.01 is considered as significant data (significantly different with the other treatments). </li></ul><ul><li>The fermentation media are formulated by mixing the pure glucose (30 mg/ml) + protein hydrolysates (30 mg/ml) + yeast extracts (5 - 10 mg/ml) + sterile tap water (to dissolve all nutrients). </li></ul><ul><li>The nutrient is supplemented with mineral - which contained 1.0 mg/ml KH 2 PO 4 + 0.005 mg/ml CaCl 2 + 0.1 ml trace element solution. </li></ul><ul><li>The fed-batch fermentation process : runs up to 70 - 100 hrs ( Kim et al., 1994) - the amount of pure glucose consumed (mg) & microbial biomass concentration (mg/ml) - measure - interval time of 10 hrs. </li></ul><ul><li>The batch fermentations process : the amount of pure glucose consumed (30 mg/ml) - observed - 1 hr interval despite the fact that the batch process usually end up earlier than the fed-batch process. </li></ul><ul><li>The estimated time is between 35 - 50 hrs (Koutinas et al., 2006) - the time could be vary depending on the kinetics of the fermentation process. </li></ul>
    18. 18. (2) Production of scaffolds: Fabrication and characterization <ul><li>3 types of scaffolds are fabricated in the lab by using T.I.P.S. </li></ul><ul><li>1 scaffold: naturally synthesized PHB - R. eutropha fermentation process. </li></ul><ul><li>2 other scaffolds: (a) commercialized natural origin PHB (Sigma-Aldrich); (b) polyurethane (PU) from the previous studies. </li></ul><ul><li>Polyurethane (PU) - is chosen - can significantly mimic the in vivo micro environment of BM - allowing cells to proliferate profoundly. </li></ul><ul><li>Both are meant for comparison only. </li></ul><ul><li>The experiments - triplicate for each of the scaffolds. </li></ul><ul><li>The total numbers of experiment: 9 (N = 9). </li></ul><ul><li>4 dependant variables need to be observed which are as follows: (a) porosity (%); (b) mass of the approximate 5x5x5 mm 3 cubes (g); (c) scaffold volume (V s : ml); (d) pores volume (V p : ml). </li></ul><ul><li>Dependant variables for (a), (c) and (d) - Pycnometer </li></ul><ul><li>The results are expressed as mean  standard deviation (mean  SD). </li></ul>
    19. 19. Type of polymeric materials which are used for the fabrication of scaffolds and comparison purposes:
    20. 20. (3) Product testing (scaffolds + cells): Evaluating the cell proliferation <ul><li>4 cell line cultures for each of 3 different scaffolds are evaluating on 12 different occasions (4 cell lines  3 different scaffolds), in triplicate on each occasions. </li></ul><ul><li>The total numbers of treatment are 36 (N = 36). </li></ul><ul><li>The scaffolds are evaluated in terms of seeding efficiency (%) - which is measured 24 h after seeding - and cellular proliferation using the MTS assays at different time intervals of: 48 hr, 1, 2, 3, and 4 weeks. </li></ul><ul><li>Once the best scaffolds suitable for CLL expansion is identified - long-term culture tests are performed on the selected ones (up to 8 weeks). </li></ul><ul><li>Finally, the results obtained are compared with PU - a conclusion of the most suitable scaffold can be identified. </li></ul><ul><li>The results data are expressed as mean  standard deviation (mean  SD). </li></ul><ul><li>Significantly different of the results (between the treatments) are evaluated by using one-way analysis of variance (ANOVA) with a level of p<0.05 or p<0.01 depending on the measure considered significant . </li></ul><ul><li>A level of p<0.05 or p<0.01 is considered as significant data (significantly different with the other treatments). </li></ul><ul><li>Data are analyzed by linear regression using SPSS version 17.0 (SPSS Inc. Ca). </li></ul>
    21. 21. Cell seeding and proliferating observation of 4 different types of human’s CLL cell lines into 3 different types of polymeric materials
    23. 23. Metabolic Pathway: Continue