Hydrophobic and hydrophilic substituted polyphosphazenes as scaffolds for liver cell growth

HYDROPHOBIC AND HYDROPHILIC SUBSTITUTED POLYPHOSPHAZENES AS SCAFFOLDS FOR LIVER CELL GROWTH KO CHOON YING NATIONAL UNIVERSITY OF SINGAPORE 2004 HYDROPHOBIC AND HYDROPHILIC SUBSTITUTED POLYPHOSPHAZENES AS SCAFFOLDS FOR LIVER CELL GROWTH KO CHOON YING (B. Eng. (Hons.), UTM) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS I would like to express my heartfelt thanks to my supervisor, Dr. Tong Yen Wah who do science with pure, unselfish and honest passion. With his support and advice in guiding me throughout my research, I have been gained fruitfully both academically and in terms of character building. Without his help, this work would not be possible. I would also like to thank Dr Cao Tong and his research students, Liu Hua, Judy Saw Tzuen Yih, Zou Xiao Hui and Shi Zheng for their invaluable advice and helpful assistance on cell culture works and cell cytotoxicity testing. I would also like to thank Chaw Su Thwin, Hidenori Nishioka, Jeremy D. Lease and Cui Weiyi for many occasions. Without them, my research life could be much lonelier. A special thanks also goes to Dr Fan Wan Yip and Ms Liu Shaoqiong who help me to carry out the NMR testing. I am also indebted to all lab officers who have helped me either in the acquisition of chemicals, apparatus and the operations of various equipment, especially Ms. Lee Chai Keng and Mr. Boey Kok Hong. Lastly, I would like to thank my family and friends for their continuous support. i TABLE OF CONTENTS ACKNOWLEDGEMENTS.......................................................................................... i TABLE OF CONTENTS............................................................................................. ii SUMMARY................................................................................................................ iv NOMENCLATURE ................................................................................................... vi LIST OF FIGURES ..................................................................................................viii LIST OF TABLES...................................................................................................... xi CHAPTER 1 INTRODUCTION ................................................................................. 1 1.1 General Background ....................................................................................... 1 1.2 Objective and Scopes...................................................................................... 2 CHAPTER 2 LITERATURE REVIEW ...................................................................... 4 2.1 Tissue Engineering.......................................................................................... 4 2.1.1 Introduction................................................................................................. 4 2.1.2 Methods of tissue engineering .................................................................... 5 2.1.3 History of tissue engineering ...................................................................... 6 2.1.4 The needs of tissue engineering.................................................................. 7 2.1.5 Growth of tissue engineering ...................................................................... 9 2.1.6 Current limitations and issues................................................................... 10 2.2 Liver and cells............................................................................................... 11 2.2.1 The liver .................................................................................................... 11 2.2.2 Liver tissue engineering............................................................................ 12 2.2.3 Liver cells.................................................................................................. 13 2.2.4 Cells seeding and cell behaviors ............................................................... 14 2.2.5 Hepatocytes............................................................................................... 16 2.3 Polymers ....................................................................................................... 17 2.3.1. Synthetic and natural polymers................................................................. 17 2.3.2 Material and cell interaction ..................................................................... 20 2.3.3 Polymer Scaffold ...................................................................................... 21 2.4 Polyphosphazenes ......................................................................................... 29 2.4.1 Introduction................................................................................................... 29 2.4.2 History........................................................................................................... 30 2.4.3 Applications .................................................................................................. 30 2.4.4 Polyphosphazenes in tissue engineering................................................... 31 2.4.5 Methods of synthesis................................................................................. 33 CHAPTER 3 MATERIALS & METHODS .............................................................. 39 3.1 Polymer Synthesis............................................................................................... 39 3.1.1 Synthesis of poly(dichlorophosphazene) .................................................. 40 3.1.2 Poly [bis(glycinato ethyl ester)phosphazene] [PGP] ................................ 41 3.1.3 Poly[bis(p-methylphenoxy)phosphazene] PMPP ..................................... 42 3.1.4 Poly [(ethyl glycinato)(methylphenoxy)phosphazenes PPHOS ............... 43 ii 3.1.5 Preparation of polymer films for cell growth studies ............................... 44 3.2 Measurements ............................................................................................... 45 3.2.1 Water contact angle film and SEM sample preparation ........................... 46 3.3 Cell culture................................................................................................... 46 3.3.1 Cell Culture Hep3B................................................................................... 46 3.3.2 MTT assays............................................................................................... 48 3.3.3 Albumin Determination by ELISA........................................................... 49 3.3.4 Cytochrome P450...................................................................................... 52 3.4 In Vitro Cytotoxicity Testing........................................................................ 53 3.4.1 Interaction between the substituted polyphosphazenes and L929 ............ 53 3.4.2 Protocols for in vitro cytotoxicity testing ................................................. 53 3.4.3 Phase contrast microscopy and SEM........................................................... 54 CHAPTER 4 RESULTS & DISCUSSIONS ............................................................. 56 4.1 Polymer Characterization.............................................................................. 56 4.2 In Vitro Cytotoxicity Testing........................................................................ 74 4.2.1 In vitro cytotoxicity testing based on extraction....................................... 75 4.3 Interaction between polyphosphazenes and Hep3B ..................................... 80 4.3.1 Cell proliferation....................................................................................... 80 4.3.2 Hep3B function-albumin synthesis........................................................... 82 4.3.3 Hep3B function- Cytochrome P450.......................................................... 84 4.3.4 Morphology of the Scaffold and the Hep3B Cells.................................... 85 4.4. Discussion .......................................................................................................... 91 CHAPTER 5 CONCLUSIONS & SUGGESTIONS................................................. 95 5.1 Conclusions................................................................................................... 95 5.2 Suggestions ................................................................................................... 97 References.................................................................................................................. 99 iii SUMMARY Biodegradable polymers have become increasingly useful as a part of a system that treats, augments, or replaces any tissue, organ, or function of the body. Among the biodegradable polymers, PGA (Poly(glycolic acid)), PLA (Poly(lactic acid)) and PLGA (Poly(lactide-co-glycolide)) are popularly used polymers in liver tissue engineering because of their good compatibility. However, these polymers have their limitations such as not having functional groups that can be easily modified. In comparison, polyphosphazenes are a broad class of inorganic polymers with the general formula of [NPR]2, possess easily modified inorganic or organic side groups. Hence, the versatility of this polymer is high. The different chemical side groups can control the chemical and physical properties of the polymer. Therefore, this bioerodible polymer has shown a promise for use in tissue engineering. In this research, (PGP), poly[bis(ethyl-glycinato)phosphazene] Poly[bis(methylphenoxy)phosphazene] (PMPP) and different copolymer compositions of the poly [(ethyl glycinato)(methylphenoxy)phosphazenes] (PPHOS) were synthesized from ring opening polymerization of poly(dichlorophosphazene) and side group reactions. These polymers were characterized by using elemental analyzer, FTIR (Fourier transforms infrared), 1H NMR( Nuclear magnetic resonance) , 31 P NMR, DSC (Differential scanning calorimeter), GPC (Gel permeation chromatographic), SEM (Scanning electron microscopy), EDX (Energy dispersive X-ray), XRD (X Ray diffraction), TGA (Termogravimetric analysis) and dynamic water contact angle. The polymers glass transition temperatures were found to be in the range of –18 to 0oC, and molecular weights were found in the order of 104 g/mol. GPC results also were showed low polydispersity (1-1.5). An increase in the content of the methyl phenoxy group resulted in an increase in glass transition temperature and water contact angle. SEM iv images of the polymers casted as films showed unusual morphologies. In vitro cytotoxicity testing was done by using L929 cells to examine the biocompatibility of these polymers. The polymer films also were seeded with Hep3B, a cancerous liver cell line, to evaluate the effects of the different chemical functional groups and surface wettability on cell growth. Cell proliferation was examined by using MTT assays and two function markers, the secretion of albumin and ethoxyresorufin 0-dealkylase (EROD) activity were also investigated. Results revealed that different side group of the polyphosphazenes influenced the thermal properties, morphology and crystallinity. In addition, hepatocytes attachment, morphology, and growth are dependent on the wettability of the substrate. The final results showed that moderate surface wettability (60-70o) is the major factor promoting high levels of cell attachment but not cell functionality. The morphology of the cells was found to be dependent on surface composition which has a direct influence on cell functionality and proliferation. Overall, our research has led us to understand how the side groups of polyphosphazenes affect the properties of polymer and the consequences of these findings on liver cells proliferation and functionality. v NOMENCLATURE AFM Atomic force microscopy ATCC American tissue culture centre AVONA One-way analysis of variance CaH2 Calcium hydride DMEM Dulbecco’s modified eagle medium DMSO Dimethyl sulfoxide DSC Differential scanning calorimeter ECM Extracellular matrix EDX Energy dispersive X-ray analysis ELISA Enzyme linked immunosorbent assay EROD Ethoxyresorufin O-dealkylse FBS Fetal bovine serum FDA Food and Drug Administration FTIR Fourier transforms infrared GPC Gel permeation chromatographic HBSS Hank’s balanced salt solution H3PO4 Phosphoric acid HAHs Planar halogenated aromatic hydrocarbons HDPE High density polyethylene MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) NMR Nuclear magnetic resonance NSF National Science Foundation PAHs Polycyclic aromatic hydrocarbons PLA Poly(lactic acid) vi PGA Poly(glycolic acid) PLGA Poly(lactide-co-glycolide) PCBs Polychlorinated biphenyls PBS Phosphate buffered saline PGP Poly[bis(glycinato ethyl ester)phosphazene] PMPP Poly[bis(p-methylphenoxy)phosphazene] PPHOS Poly[(ethyl glycinato)(methylphenoxy)phosphazene] SEM Scanning electron microscopy TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TBS Tris Buffered Saline TEA Triethlyamine TGA Termogravimetric analysis THF Tetrahydrofuran TMB 3,3’,5,5’-Tetramethyl-Benzidine WTEC World Technology Evaluation Center XRD X Ray diffraction ZDEC Zinc diethyldithiocarbamate vii LIST OF FIGURES Figure 2.1: Approximate sequence of development of tissue engineering since 1970.........7 Figure 2.2: Current limitations and issues of tissue engineering ........................................11 Figure 2.3: Cellular arrangement within the liver lobules ..................................................12 Figure 2.4: Fundamental steps in liver tissue engineering..................................................13 Figure 2.5: Hepatocytes ......................................................................................................16 Figure 2.6: Classification of biomaterials………………………………………...........……18 Figure 2.7: Factors controlling tissue reaction to implantation materials...........................20 Figure 2.8: Scaffold [12].....................................................................................................21 Figure 2.9: Designs of the scaffold .....................................................................................26 Figure 2.10: Polymer structure of PGA and PLA...............................................................27 Figure 2.11: Polymer structure of PLGA............................................................................28 Figure 2.12: [NPR]2 structure .............................................................................................29 Figure 2.13: [NPCl]2 structure ............................................................................................29 Figure 2.14: Conversion of hexachlorocyclotriphosphazene to polydichlorophazene .......34 Figure 2.15: Methods of Macromolecular substitutions .....................................................36 Figure 2.16: PCl5 initiated polymerization of trichloro(trimethylsilyl)phosphoranimine...37 Figure 3.1: Thermal polymerization of poly(dichlorophosphazene)…………..……………40 Figure 3.2: Conversion of poly[(dichloro)phosphazene] into PGP ....................................41 Figure 3.3: Conversion of poly(dichlorophosphazene) into PMPP ....................................42 Figure 3.4: PPHOS polymer structure ................................................................................43 Figure 3.5: Measurements of polymer characterization .....................................................45 Figure 4.1: FTIR spectrum of the monomer (hexachlorocyclotriphosphazene) and poly(dichlorophosphazene)……………………………………..............................57 Figure 4.2: 1H NMR spectrum of PGP ...............................................................................59 Figure 4.3: 1H NMR spectrum of PMPP ............................................................................59 viii Figure 4.4: 1H NMR spectrum of PPHOS2 ........................................................................60 Figure 4.5: SEM of substituted polyphosphazene films on microscope slide (a) PGP, (b) PPHOS1, (c) PPHOS2, (d) PPHOS3, (e) PMPP.........................................64 Figure 4.6: XRD diagram of the five different polyphosphazenes polymer films .............66 Figure 4.7: EDX spectrum for PPHOS2 .............................................................................67 Figure 4.8: SEM of (a) PPHOS1, (b) PPHOS2 and (c) PPHOS3 with application of EDX ..................................................................................................................67 Figure 4.9: Schematic representation of the structure of a PPHOS copolymer. The thicker lines represent the methyl phenoxy-rich domain and the thinner lines the ethyl glycinato-rich group domain. ....................................................69 Figure 4.10: AFM images of PPHOS1 ...............................................................................70 Figure 4.11: AFM image of PPHOS2.................................................................................70 Figure 4.12: The TGA scans of the polymers.....................................................................71 Figure 4.13: Water contact angles for substituted polyphosphazenes. Values are the mean ±standard deviations (n=6)...................................................................73 Figure 4.14: Three approaches for cytotoxicity testing ......................................................74 Figure 4.15: L929 cell proliferations at 1,2,3,6 and 9 days after seeding on PGP, PPHOS2 and PMPP thin film. .......................................................................75 Figure 4.16: MTT results of pH dependent effect on L929 after 24h and 48h...................76 Figure 4.17: Light microscope of cell after exposure to (a) negative control (b) PGP (c) PPHOS2 (d) PMPP (e) positive control.........................................................77 Figure 4.18: MTT results of exclude pH dependent effect on L929 after 24h and 48h .....78 Figure 4.19: Light micrograph of cell after exposure to (a) negative control (b) PGP.......79 Figure 4.20: MTT assays of Hep3B cells measured as optical density (OD) function of culture time on polymers.*Statistically significant differences in data (calculated using one-way ANOVA with p0.05). 3.4 In Vitro Cytotoxicity Testing L929 cell lines (ATCC, USA) were used for in vitro cytotoxicity testing since they are recommended by the standard institutions. The cells were maintained in continuous culture in MEM supplemented with 1.5g/L of sodium bicarbonate, 10% FBS, 1mM sodium pyruvate, 0.1mM non-essential amino acids and 2mM L-glutamine (Sigma, Germany) at 37oC in an air atmosphere containing 5% CO2 and 95% relative humidity. Besides of the polyphosphazenes samples, the evaluation was also carried out by using reference materials of polyurethane film containing 0.1% ZDEC as the positive control and HDPE as the negative control (Hatano Research Institute, Japan). The reference materials were chosen according to the ISO 10993 standard [107-108]. 3.4.1 Interaction between the substituted polyphosphazenes and L929 Before the in vitro cytotoxicity testing was carried out, the interactions between the substituted polyphosphazenes of the cells were investigated. PGP, PPHOS2 and PMPP were coated as a film on 60mm Petri dish. Later, 1x104/cm2 of L929 was plated onto coated Petri dish and incubated for 1 to 9 days. 3.4.2 Protocols for in vitro cytotoxicity testing The protocols for in vitro cytotoxicity testing were based on the ISO 10993 standard [94-96]. Methods to evaluate the cytotoxicity levels are the assessments of cell damage by morphological via phase contrast microscope and measurement of cell metabolism 53 Chapter 3 Material & Methods by using MTT assays. The study was carried out to evaluate the effects of pH on cell growth and also to exclude the pH effect whereby the solution of the polymers were adjusted into pH 7.4 by adding medium. Polymer samples were stored in serum supplemented tissue culture medium (0.01g/ml) for 48h at 37oC. The polymer extracts are collected and filtered by using 0.22µm filter. To exclude cytotoxic effects due to changes in pH of the polymer solutions, the pH of one of the polymer extraction were adjusted to 7.4. L929 cells were plated onto 96well microplates at a density of 3 x 104 cells/ well. After 24 h or until the cells have reached 80% confluence, culture medium was washed twice with PBS to ensure the complete removal of serum protein. Then it was replaced by 100µL of the polymers extraction. At various period (after 24h and 48h), medium with the polymer extracts were aspirated and replaced by 200µL DMEM without serum to minimize the chance of aggregate formation between the charged sites of proteins and polymer before the MTT assays. 20µL sterile filtered stock solution in PBS (5mg/ml) pH 7.4 were added to each well with a final concentration of 5mg MTT/ml. After 4 hours incubation, unreacted dye and media was removed by aspiration, and the insoluble formazan crystals were dissolved in 200µL/ well DMSO to lyse the cell and to dissolve the dye by vigorously shaking for at least 15 min. The samples were measured spectrophotometrically in an ELISA reader at a wavelength of 570nm (test) and 620nm (reference). 3.4.3 Phase contrast microscopy and SEM Cell- seeded scaffolds will be studied under phase contrast light microscope (IX 70, Olympus, Germany) and SEM microscope (Jeol JSM-5600LV, Japan). After removing 54 Chapter 3 Material & Methods the supernatant in cell culture dish, 2.5% Glutaraldehyde (Sigma, Germany) in PBS was be added and placed for 12h at 4oC. Cells were then dehydrated in a graded ethanol solution (50%, 60%, 70%, 80%, 90% ethanol in water) each for 10min, and finally in pure ethanol twice for 10min each. Finally, the cells were dried and coated with layer of platinum for SEM imaging [109]. 55 Chapter 4 Results & Discussions CHAPTER 4 RESULTS & DISCUSSIONS 4.1 Polymer Characterization After the polymers were synthesized, the products were analyzed and characterized in detail. FTIR was used to confirm the existence of the proper functional groups and bonds in the substituted polyphosphazene polymers by comparing the absorption spectra with reference absorption peaks for the polymers. For the polymerization of polydichlorophosphazene from (NPCl2)3 via ring opening polymerization, the FTIR spectra were obtained and shown in Fig. 4.1, showing that the infrared bands for (NPCl2)n (1300, 1230 (P-N) ) shift to lower wavelength side after polymerization, compared to (NPCl2)3 at 1190, 1220 which have been reported by Allcock [75]. 56 Chapter 4 Results & Discussions 80 (NPCl2)3 (NPCl2)n 70 60 Transmittance 50 40 30 20 10 1748.3 1061.3 0 644.53 -10 872.24 0 300 600 900 2022.3 1200 1500 1800 2100 2400 2700 3000 3300 Wavenumber(cm-1) Figure 4. 1: FTIR spectrum of the monomer (hexachlorocyclotriphosphazene) and poly(dichlorophosphazene) The characteristics IR peaks for PGP are 3200-3400 (m, NH), 2900-3000(w, CH), 1740 (s, C=O ester), 1100-1250 (s, P=N) and 900(P-NH). For PMPP, they are 29603050 (w, aromatic stretch), 1500 (s, aromatic absorb), 1100-1250 (s, P=N), 900 (s, PNH), 590 (m, aromatic stretch). FTIR results show that PPHOS1, PPHOS2 and PPHOS3 have similar characteristic peaks but with different strengths: 3200-3400 (NH), 2900-3000 (CH), 1740(C=O ester), 1500 (Aromatic absorb), 1100-1250 (P=N), 900 (P-NH). The spectra show that the polymer possesses a combination of the two different chemical groups of ethyl glycinate and methyl phenoxy. 57 Chapter 4 Results & Discussions NMR was used to further characterize and identify the polymer by determining the absorptions that arise because of different nuclear spins interacting through the intervening bonding electrons. The different chemical shifts from 1H NMR represent the hydrogen in different chemical functional groups of the polymer as shown in Table 4.1 and Figure 4.2. Table 4.1 shows the results of 1H NMR for all of the substituted polyphosphazenes. PGP possesses chemical shift at 1.1-1.2ppm, 3.3-3.7ppm and 4.0-4.3ppm which are related to the ethyl glycinato group in PGP. Meanwhile PMPP possesses aromatic group with chemical shift at around 6.7-7.1ppm and its methyl protons of the methyl phenoxy group was represented by a chemical shift around 1.9-2.2ppm. PPHOS1 [85], PPHOS2 and PPHOS3 showed both the chemical shifts of PMPP and PGP but with different chemical integrations (area under peaks). This was caused by the different compositions of the side group in the polymer. Table 4.1: 1H NMR of substituted polyphosphazenes Chemical Shift (ppm)1.1-1.2a 1.9-2.2b 3.3-3.7c 4.0-4.3d 6.7-7.1e _____________________________________________________________________ PGP 1.6304 - 1.1879 1.000 - PPHOS1 1.253 0.755 1.355 0.979 1.000 PPHOS2 0.4211 0.9213 0.3332 0.2364 1.1279 PPHOS3 0.2411 0.874 0.185 0.160 1.000 PMPP - 1.000 - - 1.3075 Note at a. Methyl protons of the ethyl group, b. Methyl protons of the methyl phenoxy side unit, c. Methyl protons of the ethyl group & N-H proton, d. Methylene protons next to nitrogen on glycinato groups, e. Protons of the aromatic group from phenoxy. 58 Chapter 4 Results & Discussions Figure 4.2: 1H NMR spectrum of PGP Figure 4.3: 1H NMR spectrum of PMPP 59 Chapter 4 Results & Discussions Figure 4.4: 1H NMR spectrum of PPHOS2 Table 4.2: 31 P NMR of substituted polyphosphazenes _____________________________ 31 Polymer P NMR _____________________________ PGP 0.8 PPHOS1 9.6 17 PPHOS2 18 PPHOS3 17.4 PMPP 22 _____________________________ The value of the phosphorous peak from 31 P NMR results is similar to the literature review besides for sign [85]. This might be due to operation error or the storage of the sample under d-chloroform for several weeks before testing. P-OH moieties and hydrogen bonding between the hydroxyl group might be formed and affect the results [110]. 60 Chapter 4 Results & Discussions The copolymer composition can be determined by calculating the ratios of peak integrals to obtain the ratios of side groups to the number of repeat units. Table 4.3 shows the side group ratio as determined from the 1H NMR integral calculations, CHN compositions, average molecular weight and glass transition temperature of the polymers. Side group ratio of the polymer was calculated based on H NMR integration and also CHN composition by trial and error. From these results, it can be seen that PPHOS1 possesses 25% methyl phenoxy groups and 75% ethyl glycinato group. While PPHOS2 has nearly equal amounts of the two different side groups and PPHOS3 has the highest compositions (78%) of methyl phenoxy and the lowest compositions (22%) of ethyl glycinato. Gel Permeation Chromatographic (GPC) was carried out to determine the molecular weight and also the polydispersity of the polymers. All of the substituted polyphosphazene substitution has molecular weight in the range 1x104 daltons with low polydispersities, of about 1-1.5. The molecular weight and polydispersity obtained were lower than previously reported values [100]. This might be due to the slightly modified synthesis procedures that were employed. 61 Chapter 4 Results & Discussions Table 4.3: Characterization data for substituted polyphosphazenes by using elemental analyzer, GPC and DSC Polymers Ratio Elemental analyzer (wt%) Mw(Da) Tg(oC) ____________________________________________________________ xa ya C N H PGP 1 0 38.24 16.08 6.44(found) 38.53 16.85 6.48(calc.) 5.18e4 -18 PPHOS1 0.75 0.25 44.10 12.56 6.60(found) 45.33 13.91 6.21(calc) 2.14e4 -14.14 PPHOS2 0.40 0.60 54.975 9.175 5.88(found) 51.93 11.01 5.96(calc) 3.74e4 -7 PPHOS3 0.22 0.78 59.81 7.29 58.44 8.18 6.66(found) 5.70 (calc) 2.32e4 -5.55 PMPP 0 1 64.03 5.04 64.82 5.40 5.27(found) 5.45(calc) 3.68e4 -0.3 a The compositions of the side group of substituted polyphosphazenes were determined by 1H NMR and elemental analysis results. The glass transition temperature of the polymers were obtained from differential scanning calorimeter (DSC) and was found to be in the range of -18oC to 0oC. The glass transition temperature increased from PGP to PMPP with the increasing content of methyl phenoxy group. This is due to the presence of methyl phenoxy side group which possesses a stiff benzene ring that reduces chain flexibility and increases the glass transition temperature. All of the polymers were casted as film to microscope slides for SEM investigation and the images are shown in Figure 4.5. The results reveal that PGP film (Fig 4.5(a)) has the smoothest surface among the five polymers, while PPHOS1, PPHOS3 and PMPP films (Figure 4.5 (b), (d) and (e) ) show a regular and orderly pattern. However, the morphology of the PPHOS2 films (Fig. 4.5 (c)) was random. The difference in film 62 Chapter 4 Results & Discussions morphologies of the polymers can be explained by the crystallinity of the polymers, of which the polymer molecular structure is a key controlling factor. PMPP which have smaller side groups compared to PGP might be packed into crystals easily and increase the crystallinity. In addition, the tendency of aromatic rings to stack together in an orderly fashion also increases the alignment of the polymer. The results also revealed that molecular weight and polydispersity are also factors that can affect the morphology as observed by G. Gruenwald [111]. Lower molecular weight might lead to perfect crystallite formation compared to high molecular weight polymer. Therefore, similar polymers of higher molecular weight as synthesized before would not form the same morphology [85]. On the other hand, the substitution pattern of PPHOS2 was presumed to be random and therefore did not form a regular and orderly pattern. Similar morphology have been also observed for NP(OPhCH3)0.40(NHCH2COOCH2CH3)0.60 (image not shown) which have nearly equal compositions of the two different side groups. The polyphosphazene copolymers with equal or nearly equal amounts of both side groups therefore did not facilitate the alignment and formation of crystalline structures that can be clearly observed. 63 Chapter 4 Results & Discussions (a) (b) (c) (d) (e) Figure 4.5: SEM of substituted polyphosphazene films on microscope slide: (a) PGP, (b) PPHOS1, (c) PPHOS2, (d) PPHOS3, (e) PMPP 64 Chapter 4 Results & Discussions To further investigate this hypothesis, the samples were analyzed by XRD to determine their crystallinity. The crystallinity of the polymers was supported by XRD results as shown in Fig. 4.6. X-Ray Diffraction (XRD) produces very distinctive patterns for crystalline and amorphous materials. The relatively sharp peaks are due to the scattering from the crystalline regions and the non-crystalline materials will form broader underlying peaks. The percentage of crystallinity or fraction of crystal is then given by the ratio Xc=Ac/(Aa+Ac) Where Aa is the area under amorphous base and Ac is the area under the crystalline peaks [112]. As observed in Figure 4.6, PGP and PPHOS2 only show an amorphous base without any crystalline peak. Meanwhile, PPHOS1 and PPHOS3 show quite similar crystalline patterns and PMPP possesses the highest crystallinity among the polymers. These XRD results are in agreement with the SEM results and thus, it can be concluded that the different crystallinities of the polymers result in different surface morphologies. 65 Chapter 4 Results & Discussions PGP PPHOS2 PPHOS3 PPHOS1 PMPP Intensity 2000 0 20 2theta 40 Figure 4.6: XRD diagram of the five different polyphosphazenes polymer films From the SEM results, distinct intermolecular phase separation can also be observed. This was strongly supported by Energy Dispersive X-ray Analysis (EDX). The X-rays for EDX are generated in a region about 2 microns in depth, and thus EDX is not a surface analytical technique. By moving the electron beam across the material an image of the elements within the sample, except for proton, can be acquired as shown in Figure 4.7. 66 Chapter 4 Results & Discussions Figure 4.7: EDX spectrum for PPHOS2 (a) (b) (c) Figure 4.8: SEM of (a) PPHOS1, (b) PPHOS2 and (c) PPHOS3 with application of EDX Table 4.8 shows that different areas of the same samples have different chemical composition. For example, the smooth surface in the white box of Fig. 4.8 presented higher O atomic percentage and lower C atomic percentage than the rough surface. A higher O atomic percentage and lower C atomic percentage is indicative of ethyl glycinato-rich domain and lower O atomic group and higher C atomic percentage is indicative of a methyl phenoxy-rich domain. 67 Chapter 4 Results & Discussions Table 4.4: C and O atomic percentage from EDX results for PPHOS for different areas as shown in Fig. 4.8 Polymer Rough Atomic (%) Smooth Atomic (%) PPHOS 1 C 41 C 31 O 33 O 50 ______________________________________________ PPHOS 2 C 49 C 27 O 24 O 58 ______________________________________________ PPHOS 3 C O 50 23 C O 15 68 It seems that the copolymer blocks segregate into ethyl glycinato-rich and methyl phenoxy-rich domains that form periodic arrays termed microphases. Methy phenoxyrich domains is observed to be morphologically rougher than ethyl glycinato-rich domains. This type of the structure is shown schematically in Figure 4.9 where the two different chemical groups are anchored in the different domains. 68 Chapter 4 Results & Discussions Figure 4.9: Schematic representation of the structure of a PPHOS copolymer. The thicker lines represent the methyl phenoxy-rich domain and the thinner lines the ethyl glycinato-rich group domain. Figure 4.10 and Figure 4.11 show the resulting images of PPHOS 1 and PPHOS2 surfaces obtained from AFM using the tapping mode. Both of the images exhibited a small area which is dragged by the tip even in tapping mode. This can be explained by the fact that polyphosphazenes with different chemical functional groups form separate regions; one is softer and the other one is harder. The softer area is composed of amorphous, ethyl glycinato group and the harder area is mainly composed of crystalline methyl phenoxy group. The AFM tip ground through the softer area and formed the unclear image. However, further analysis and study are necessary to confirm this hypothesis. 69 Chapter 4 Results & Discussions Figure 4.10: AFM images of PPHOS1 Figure 4.11: AFM image of PPHOS2 70 Chapter 4 Results & Discussions Thermogravimetric analysis (TGA) scans (Figure 4.12) show distinct thermal history for the substituted polyphosphazenes depending on its structure and composition. Previous works have observed the different thermal processes of the polymer include random chain cleavage of the polyphosphazene at temperatures between 120oC to 250oC and depolymerization to form small molecule cyclic phosphazenes at temperatures around 300oC [113, 114] PPHOS3 PGP PMPP 100 Weight Remaining (%) 80 60 40 20 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 o Temperature ( C) Figure 4.12: The TGA scans of the polymers The polymer with pure ethyl glycinato group (PGP) was more prone to undergo skeletal cleavage of chain polymer as shown by the greater weight loss (36%) at 120o C to 250oC, compared with PPHOS3 (30%) and PMPP (20%). In contrast, the pure 71 Chapter 4 Results & Discussions methyl phenoxy side group PMPP shows a greater tendency to depolymerize to monomer or small cyclic oligomers at around 300oC as shown by a sharp weight loss (60%). It is therefore observed that nonpolar substituent polymer is more likely to undergo random chain cleavage of the backbone than depolymerization. All the polymers ultimately form a nonvolatile residue which were black and stable up to 800oC. It is probable that cross-linking of these polymers occurs by cleavage of the PN backbone. The surface wettability of the polymer films was investigated by using dynamic water contact angle. From Figure 4.13, PGP was seen as the most hydrophilic polymer among the five polymers due to its lowest advancing contact angle, θa (20o) and receding contact angle, θr. Meanwhile PPHOS1, PPHOS2 and PPHOS3 possess advancing water contact angles in the range of 30o to 70o. In contrast, PMPP was the most hydrophobic polymer because of its highest advancing contact angle (82o). Therefore, it can be observed that substitution of PPHOS with higher composition of methyl phenoxy reduced surface wettability due to the fact that the ethyl glycinato group which possesses the oxygen and nitrogen group is more polar and hydrophilic than the methyl phenoxy side group. It was also observed that the higher substitution of methyl phenoxy groups, the greater the hysteresis (θa-θr) which may be due to the surface chemical heterogeneities or the surface roughness. SEM micrographs of PPHOS1, PPHOS2, PPHOS3 and PMPP film samples were uneven, having rough topographical features while PGP was smooth. Basically, thermodynamic and mechanistic are two views points that have been espoused in the literature on the effect of surface roughness effects. However, 72 Chapter 4 Results & Discussions observations are dependent on how the experiments are performed and the quantification of the effects is difficult and somewhat controversial [115-116]. In addition, the increase in contact angle hysteresis may be due to the larger surface inhomogeneities, in agreement with the findings by the other research groups [117]. The hysteresis is also used as a probe for the sign of functional-group segregation at the surface [118]. The large hysteresis of the polymer films might be also attributed to the mobility of the functional group located near the surface of the material to form segregated domains, or chemical inhomogeneities. 90 80 70 60 50 40 30 20 10 0 A n g le Advancing angle Receding angle PGP PPHOS1 PPHOS2 PPHOS3 PMPP Polymers Figure 4.13: Water contact angles for substituted polyphosphazenes. Values are the mean ±standard deviations (n=6) 73 Chapter 4 Results & Discussions 4.2 In Vitro Cytotoxicity Testing All of the synthesized polyphosphazenes were tested for their cytotoxicity effect in an effort to ensure their biocompatibility. The ISO 10993-5 guide lines stipulate 3 approaches for a standard cytotoxicity test as shown in Figure 4.14. Cytotoxicity Testing Direct Contact Indirect Contact Extraction Figure 4.14: Three approaches for cytotoxicity testing For the indirect contact test, an intermediary such as agar or filter is required to isolate the cells from any physical contact with the samples. During incubation, extracts from the samples will diffuse through the agar to the underlying cells. This method is particularly useful in comparing polymers that are surface modified. The main advantage of the indirect and direct contact test is that minimal amounts of the tested materials are required. However, direct contact and indirect contact are subjective evaluation as compared to the extraction method [119, 120]. A preliminary cytotoxicity test was carried out by firstly casting the polymer as a film onto the Petri dish before seeding with L929 cells to study their interaction with polyphosphazenes. Changes in cell morphology and the detachment of cells from the Petri dish were used as indicator of cell survival by microscopic observations before the tests were carried out. From these results, cell proliferation was the highest on PGP, 74 Chapter 4 Results & Discussions followed by PPHOS2 and PMPP thin film as shown in Figure 4.15. The number of cells on PGP increased significantly from day 1 to day 7 but the number of cells on PPHOS2 and PMPP were only maintained at low levels. Thus, the cytotoxicity level can be ranked as follow: PMPP> PPHOS2> PGP. Since there are several factors that might influence the result such as the surface wettability and surface topology, a more detailed test was carried out based on extraction method to avoid a biased evaluation. Cells/cm 2 L929 Cell Proliferation 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 PGP PPHOS2 PMPP 1 2 3 6 9 Days Figure 4.15: L929 cell proliferations at 1,2,3,6 and 9 days after seeding on PGP, PPHOS2 and PMPP thin film. 4.2.1 In vitro cytotoxicity testing based on extraction The in vitro cytotoxicity testing was carried out by including the pH dependent effects as well by excluding the pH dependent effects. For both cases, the cytotoxicity level after 24h and 48h was evaluated using the MTT assay and observed with a phase contrast microscope. By including the pH dependent effect, the different functional group compositions of polyphosphazenes shows a varied level of cytotoxicity as seen in Fig. 4.16. The highest cell viability was maintained with the negative control of 75 Chapter 4 Results & Discussions HDPE, which is represented as a non-response for cytotoxicity. PGP maintained higher cell viability compared to PPHOS2 and PMPP after 48h of exposure to the polymer extract. The lowest cell viability (30%) was observed on the positive control c e ll v ia b ility (% ) of polyurethane films containing 0.1% ZDEC at both of the test times. 150 100 50 0 PGP PPHOS2 PMPP positive negative polymers after 24h Include After 48h Include Figure 4.16: MTT results of pH dependent effect on L929 after 24h and 48h The optical micrographs images (Figure 4.17) were captured to study the morphology of the cells cultured in the polymer extract after 48h. A confluent monolayer of cells on the negative control could still be observed. However, the number of cell decreases from PGP to PMPP with the increasing content of the methyl phenoxy group. Therefore PGP possessed higher cell viability than PPHOS2 and PMPP. The number of cells was also observed to be higher on PPHOS2 than PMPP and the lowest cell viability was seen on the positive control. The cells with the positive control have a grainy morphology and lack cytoplasmic space which might be due to cell lysis (integration). These reduced the cell viability and increase the cytotoxicity level. In 76 Chapter 4 Results & Discussions short, the results from the micrographs are consistent with the finding of the MTT assay. (a) (c) (b) (d) (e) Figure 4.17: Light microscope of cells after exposure to (a) negative control (b) PGP (c) PPHOS2 (d) PMPP (e) positive control 77 Chapter 4 Results & Discussions To exclude the pH effect, the polymer extracts were adjusted to pH 7.4 by adding fresh medium. MTT assay was performed to investigate the cell viability after 24h and 48h of the addition of polymer extract and the results are shown in Figure 4.18. c e ll v ia b ilit y ( % ) 200 150 100 50 0 PGP PPHOS2 PMPP positive negative polymers after 24h exclude After 48h Exclude Figure 4.18: MTT results of exclude pH dependent effect on L929 after 24h and 48h The results showed that the cell viability for PGP, PPHOS2 and PMPP were not significantly different at 24h; however, after 48h, the highest cell number was observed on the negative control, followed by PGP, PPHOS2, PMPP and the positive control. The trend observed was similar to the tests that included the pH effect. In addition, the light micrograph images (Figure 4.19) also showed that the cell viability was reduced via the introduction of methyl phenoxy group. It can be summarized that cytotoxicity level was increased by introducing methyl phenoxy groups for both cases, 78 Chapter 4 Results & Discussions whether pH effect was included or excluded. Furthermore, the result is also consistent with the initial cytotoxicity test. (a) (b) (a) (b) (c) (d) Figure 4.19: Light micrograph of cells after exposure to (a) negative control (b) PGP (c) PPHOS2 (d) PMPP extraction after 48h 79 Chapter 4 Results & Discussions 4.3 Interaction between polyphosphazenes and Hep3B O D 560n m 4.3.1 Cell proliferation 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 PGP PPHOS1 PPHOS2 PPHOS3 PMPP PLL/laminin TCPS * * * * * * 3 * * * 5 7 incubation day Figure 4.20: MTT assays of Hep3B cells measured as optical density (OD) function of culture time on polymers.*Statistically significant differences in data (calculated using one-way ANOVA with p PPHOS> PGP. Additionally, the synthesized polyphosphazenes were also used to study the influence of hydrophilic and hydrophobic surface characteristics on cell adhesion and cell functionality, especially for a liver cancer cell line, Hep3B. Although PGP represents the lowest cytotoxicity level from the cytotoxicity testing, but it did not show the highest liver cell viability with the substituted polyphosphazenes thin film. It is clear that the overall biological processes of cell adhesion and growth are sensitive to the outermost or surface functional groups and also to the wettability of the polymers. Of all samples analyzed, the greatest cell proliferation was observed on PPHOS2, with moderate hydrophilicity. Moderate surface wettability (60-70o) is the major factor in promoting high levels of cell attachment but not cell functionality. The morphology of the cells showed profound influence on cell functionality and proliferation. When Hep3B cells were cultured on PPHOS2, they were spread and flattened. However, cells on PGP were more rounded and spheroidal in morphology with clustering of the cells. In short, our results suggested that the more hydrophilic surfaces did not enhance cell spreading but supported significantly greater cell 96 Chapter 5 Conclusions & Suggestions functionality such as albumin secretion and cytochrome P450 activities. The morphology of the cells was also found to be dependent on surface composition which has a direct influence on cell functionality and proliferation. Mitosis and differentiation are not parallel but subsequent events within a cell [133]. This can explain why the cells on PPHOS2 with high cell proliferation did not show high cell functionality while cells on PGP showed high cell functionality but low proliferation. Our finding also imply that in order to engineer material surfaces with optimal properties for maintaining call differentiation, one must understand the complex interrelationships among material surface properties and cellular responses. In conclusion, the wettability material has a strong influence on the attachment and morphologies of hepatocytes while the effects of surface properties and chemical functional group still remain to be elucidated. Polyphosphazenes would appear to be an interesting candidate as scaffolds for tissue engineering, as it is shown to be easily modified and tailored to the specific requirements for the applications. 5.2 Suggestions 1. Different side groups of new substituted polyphosphazenes should be synthesized. 2. Surface modification on the substituted polyphosphazenes should be carried out to increase the cell proliferation and functionality of the polymers. 3. 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Polymer Edn. 6. 2001. pp. 613-627. 113 [...]... engineering such as the characteristics of the cell, cell activities, cell source, cell interaction and cell morphology Generally the cells sources can be categorized into primary cells, cell lines and stem cells 2.2.3.1 Primary cells Freshly isolated cells that are obtained from animal or human liver are called primary liver cells [20] Cells are cultured under specific environment to promote cell growth [9]... epithelial cells or hepatocytes [24] However, the question of why, how and what makes the stem cell decide to become a particular cell type and differentiate is still under investigation 2.2.4 Cells seeding and cell behaviors The methods of cell seeding and cell behaviors play an important role in tissue engineering Cell behavior includes cell adhesion, cell spreading, cell migration and cell morphology 1 Cell. .. biodegradability and surface properties Firstly, the ideal structure of scaffold is to have an open and ample space for cell growth and proliferation [2], and porous and three dimension form to maximize diffusion parameters [24] Having a porosity of at least 95 % is another important characteristic 21 Chapter 2 Literature Review that allow for vascular and cellular growth [45], exchange of nutrients and waste... Stem cells Stem cells are self-renewing and undifferentiated cell that can be found in an embryo or adult They can undergo unlimited division and can give rise to one or several different cell types [21] The liver epithelial stem cells, also referred to as oval cells, are easily propagated in culture and thus are a potential source of hepatocytes for liver tissue engineering [21-23] The liver stem cells... produce tissue for liver and pancreas 2.Lack of interconnected channel 3 Acidic degradation Cell 1 Angiogenesis 2 Cell source Limitations Scaffold Construct Assembly 1 Non-uniform cell distribution 2 Poor mechanical properties Figure 2.2: Current limitations and issues of tissue engineering 2.2 Liver and cells 2.2.1 The liver The liver (Figure 2.3) [17] is one of the most sophisticated and complicated... properties and stability 1 Chapter 1 Introduction Polyphosphazene, which is a broad class of inorganic polymer with the general formula of [NPR]2, was studied in this thesis as it has different chemical side group which can be easily modified and used to control the chemical and physical properties of the polymer Therefore, this bioerodible polymer has shown promise for use in drug delivery and tissue... multiple cell types and is highly vascularized [13] The liver performs a variety of functions necessary for survival, i.e it is responsible for production of a number of the proteins, 95% of which are found in plasma such as serum albumin It is also well known for detoxification of compounds and is a center of the storage of the vitamins A, B, D and K [16] The liver is so unique in that it has an amazing... viability for xenogeneic transplantation 3 Scaffolds Scaffolds, also known as matrices or templates, play a crucial role for cell attachment A significant feature of the scaffold is the ability to control cell adhesion, growth and functionality [7] The use of scaffolds in tissue engineering sometimes involves cell seeding onto the scaffold prior to the implantation, but in other cases, the scaffolds. .. Review Figure 2.3: Cellular arrangement within the liver lobules 2.2.2 Liver tissue engineering A main reason for the development of liver tissue engineering is due to the problems and inconsistencies in the use of hepatic support systems for liver failure [18] This has led to the identification of the fundamental steps in liver tissue engineering (Figure 2.4), i.e the growth of the cell in polymers... therefore require a substratum to survive and function [32] They are polar cell with membrane domains, and are responsible for the detoxification activity of the liver During liver failure, a large number of hepatocyctes, estimated to be 10-20% of the liver mass, must be delivered and engrafted, for implantation to successfully replace the liver function [33] As hepatocytes must be seeded within a few .. .HYDROPHOBIC AND HYDROPHILIC SUBSTITUTED POLYPHOSPHAZENES AS SCAFFOLDS FOR LIVER CELL GROWTH KO CHOON YING (B Eng (Hons.), UTM) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING... the cell, cell activities, cell source, cell interaction and cell morphology Generally the cells sources can be categorized into primary cells, cell lines and stem cells 2.2.3.1 Primary cells... analysis, such as skin, heart valves and bone since 1989 Also, cell processing has been consolidated whilst stem cell research has expanded In contrast, bioartificial organs and encapsulated cell therapy