JASON PAUL MAZZOCCOLI. Submitted in partial fulfillment of the requirements for the degree of Master of Science. Department of Chemical Engineering

Transcription

1 PROPERTIES OF POLY(ETHYLENE GLYCOL) DIACRYLATE BLENDS AND ACOUSTICALLY FOCUSED MULTILAYERED BIOCOMPOSITES DEVELOPED FOR TISSUE ENGINEERING APPLICATIONS by JASON PAUL MAZZOCCOLI Submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemical Engineering CASE WESTERN RESERVE UNIVERSITY August, 2008

2 2

3 This thesis is dedicated to my parents, Ron and Elaine, for their unending support throughout the years and to Sue, for her encouragement. 3

4 Table of Contents List of Tables... 5 List of Figures... 6 Preface... 8 Acknowledgements... 9 Properties of Poly(ethylene glycol) Diacrylate Blends and Acoustically Focused Multilayered Biocomposites Developed for Tissue Engineering Applications Section I: Properties of Crosslinked Low and High Molecular Weight Poly(ethylene glycol) Diacrylate Blends for Tissue Engineering Applications Abstract Introduction Background and Theory Materials and Methods Results and Discussion Conclusions Future Work Section II: Encapsulation and Arrangement of Cells in Poly (ethylene glycol) Diacrylate Hydrogels Using an Acoustic Focusing Process Abstract Introduction Background and Theory Materials and Methods Results and Discussion Conclusions Future Work Overall Summary References

5 List of Tables Table 1 Data set for compressive modulus testing Table 2 Tukey test analyses for comparing average compressive moduli Table 3 Two sample t test results for comparison of moduli of 40/60blend at 20 wt % vs. 40 wt % population and 80/20 blend at 40 wt % vs. 20 wt. % population...33 Table 4 Data set for cell viability measurements Table 5 Tukey test analyses for comparing average cell viability 40 Table 6 Two sample t test results for comparison of average cell viability of 60/40 blend at 20 wt % vs. 40 wt % population and 100/0 blend at 40 wt % vs. 20 wt % population Table 7 Data set for compressive modulus testing and cell viability measurements of 20:80 PEGDA3400/400 blend

6 List of Figures Figure 1 Left, chemical structure of poly(ethylene glycol) diacrylate. Right, chemical structure of Irgacure 2959 photoinitiator Figure 2 The steps of free radical polymerization of PEGDA chains; initiation, propagation, and termination Figure 3 Cross linked polymer system where the wavy lines represent polymer chain and the black circles are crosslinks binding the chains together Figure 4 Mechanical strength vs. molecular weight in polymer systems Figure 5 Photopolymerized PEGDA discs at a total polymer concentration of 20 wt % containing blends of PEGDA3400/400 (wt % ratio). (a) 0/100, (b) 20/80, (c) 40/60, (d) 60/40, (e) 80/20, (f) 100/0. As the wt % of PEGDA 3400 in the blend increases, the discs turn from an opaque white to transparent. Scale bar is equal to 5 mm Figure 6 Photopolymerized PEGDA discs at a total polymer concentration of 40 wt % containing blends of PEGDA3400/400, in wt% ratio. (a) 0/100, (b) 20/80, (c) 40/60, (d) 60/40, (e) 80/20, (f) 100/0. As the wt % of PEGDA 3400 in the blend increases, the discs turn from an opaque white to transparent. Scale bar is equal to 5 mm Figure 7 Typical stress strain curves for discs composed of pure PEGDA3400 (100/0 blend) at 20 and 40 wt % total polymer concentration, with the slope of each line given as the compressive modulus, E. The 20 wt % data was taken from test 4 of 4 on that disc. The 40 wt % data is taken from test 1 of 4 on that disc Figure 8 Compressive modulus of PEGDA3400/400 (wt % ratio of initial polymer solution) blends at 20 and 40 wt % total polymer concentration, with standard error. For both data sets, the compressive modulus reaches a maximum, when the amount of PEGDA3400 in the blend is ~40 wt percent. Sample size is n Figure 9 Cell viability as measured using fluorescent LIVE/DEAD assay of photopolymerized PEGDA3400/400 (wt % ratio) blends after 10 min of UV exposure at 15 W/cm 2 at total polymer concentrations of 20 and 40 wt %, with standard error. The average initial cell viability for all blends at 20 wt % is 78.9% (±7.9%) and is 35.8% (±7.0%) at 40 wt %. Sample size is n 5. Additionally the bioassay was applied to cell suspended in cell media (MEDIA) and cells suspended in media with 10 minutes UV exposure (MEDIA UV) Figure 10 Fluorescent images taken of a photopolymerized PEGDA3400/400 disc, 20/80 blend (wt % ratio) using LIVE/DEAD assay at a total polymer concentration of 20 wt %. The left image is of living cells, and right image is of dead cells. The cell viability is 82%. Scale bar is 100 µm Figure 11 Fluorescent images taken of a photopolymerized PEGDA3400/400 disc, 20/80 blend (wt % ratio) using LIVE/DEAD assay at a total polymer concentration of 40 wt %. The left image is of living cells, and right image is of dead cells. The cell viability is 12%. Scale bar is 100 µm Figure 12 Cell viability and compressive moduli of PEGDA3400/400 (wt % ratio), 20/80 blends, as a function of total polymer concentration, with standard error. Sample size is n 5 for all samples Figure 13 Overall acoustic focusing process. Homogenously dispersed particles are arranged in layers using acoustic fields and the structure is preserved via polymerization Figure 14 Crystal Structure of Lead Zirconate Titanate (PZT) and its reaction to an electrical field

7 Figure 15 Creation of a planar standing wave in an acoustic chamber. The final position of the particle (either at a node or antinode) is determined by its acoustic contrast factor Figure nm silicon dioxide particles layered perpendicular to the surface of the polymer (a,b images are top views); a. SiO 2 particles homogenously dispersed in butyl methacrylate at 0.75 vol %, b. Same system in Fig. 4 a, banded at a 725 khz, unpolymerized; c. Side view of polymerized butyl methacrylate system, at a particle loading of 2 vol %; the thickness of the sample is 3mm. The sound field is applied right to left in these pictures, with the transducer on the right and the glass reflector on the left Figure 17 Side views of 75 µm PVDB beads arranged parallel to the surface, in water at 1 MHz; a. Beads homogenously dispersed in fluid, b. Initial application of sound field, c. Formation of bands, d. Initial response after sound field turned off, e. Agglomerated beads sinking in fluid. The sound field is propagated from top to bottom with transducer located on the top of the image and a glass reflector located on the bottom of the fluid Figure 18 a. 100 nm SiO 2 particle arranged in 2 hydroxyethyl methacrylate at loading of 2 wt % and frequency of 980 khz, perpendicular to polymer surface, b µm carbon particles arranged in two dimensions at a frequency of 540 khz. All images are top views. In Fig 18 a, the sound field is applied right to left. In Fig 18 b, the sound emanates from the top and right hand side of the image Figure 19 Arrangements of cells at radial node positions emanating from a cylindrical sound source Figure 20 Relative pressure of a planar and a cylindrical sound wave traveling in water at a frequency of 2.32 MHz. In the cylindrical case, x=0 represents the center position (axis) of the annular region of the cylindrical transducer, which is the site where the reflection of the sound field takes place and the pressure is maximized. In the planar case, x=0 represents the source of propagation. In the graph, the planar wave is progressive and is not impeded in any manner Figure 21 Schematic of the acoustic chamber comprised of a PZT cylindrical transducer fixed to a glass plate Figure 22 Schematic of sound equipment and acoustic chamber Figure 23 Time progression of acoustic focusing of 3 µm latex beads in water (10 x 10 6 per ml of fluid) at a frequency of 2.32 MHz: a. time = 0, b. time = 3 min., c. time = 10 min., d. time = 15 min., e. time = 40 min, top view images. The distance between bands is about 300 µm Figure 24 3 µm latex beads focused at 7.3 MHz in water at a concentration of 10 million beads per ml of fluid, top view image. The distance between the bands is about 130 µm Figure 25 Focusing of MDA MB 231 cells in 30 wt % PEGDA400 solution at a concentration of 1 million cells per ml, top view images; a. 2 min total, b. 5 min total, c. 10 min total, d. 20 min total, e. 30 min total, f. 60 min total sound field application Figure 26 Microscope image of histology slice of inner rings composed of cancer cells arranged in 30 wt % PEGDA disc at a concentration of 1 x 10 6 cells per ml of fluid. Scale bar is equal to 300 µm Figure 27 Microscope images of fluorescently stained cancer cells encapsulated in a matrix composed of 30 wt % PEGDA400 at a concentration of 5 x 10 6 cells per ml; a b, inner ring shots of live (green) and dead (red) cells respectively. c d, outer ring shots of live and dead cells. Scale bar is equal to 300 µm Figure 28 Fluorescent images of live (Fig. a.) and dead cells (Fig. b) to estimate average ring thickness and average distance between rings. The frequency of the sound field is 2.32 MHz and cell concentration is 5 x 10 6 cells/ml. Assay applied 40 hours after encapsulation and incubation. Scale bar is equal to 500 µm in each picture Figure 29 Live and dead fluorescent images (a.,b.) of cells exposed to no sound field. Live and dead images (c.,d.) of cells exposed to 20 minutes of sound field at 2.32 MHz and 4V RMS. Cell concentration is equal to 5 x 10 6 per ml of fluid

8 Preface The work of this thesis has been organized into two separate sections with the common thread being the utilization of poly(ethylene glycol) diacrylate (PEGDA) based hydrogels for tissue engineering applications. Section I details the development of low and high molecular weight PEGDA blends to create polymeric cellular matrices that have tunable mechanical properties based on the ratio of the high to low molecular weight component in the blend. Properties of the blends were studied at different total polymer concentrations and relationships between cell viability and mechanical strength were explored. Section II describes the technique of acoustic focusing, which used resonant ultrasonic fields to organize cells into multi layered bands within a PEGDA matrix. The combined work of both sections expands the use of PEGDA in unique ways that advances the field of tissue engineering. 8

9 Acknowledgements I would like to thank the Chemical Engineering Department of Case Western Reserve University, and in particular, Dr. Peter Pintauro and Dr. Donald Feke for their advice, assessment, and funding of this work. I would also like to thank Dr. Harihara Baskaran for his guidance regarding many of the biological aspects associated with this work, and for permitting the use of the fluorescence microscope and his laboratory. Furthermore, I would like to thank Dr. Gary Wnek of the Macromolecular Science and Engineering Department for allowing the use of his tissue culture laboratory and Dr. Joseph Mansour of the Mechanical Engineering and Aerospace Department for use of the mechanical testing equipment. Special thanks are extended to Michael Swickrath and Saheli Sarkar for their guidance on tissue culture and microscopy techniques. 9

10 Properties of Poly(ethylene glycol) Diacrylate Blends and Acoustically Focused Multilayered Biocomposites Developed for Tissue Engineering Applications Abstract by JASON PAUL MAZZOCCOLI This research highlights the use poly(ethylene glycol) diacrylate (PEGDA) hydrogels for tissue engineering applications. PEGDA blends with molecular weights of 400 and 3400 were UV crosslinked to photoencapsulate mammalian cells at total polymer concentrations that were varied systematically from 20 to 40 wt %. Initial cell viability was determined via a fluorescent LIVE/DEAD assay and was reduced from 80 to 35% when increasing total polymer concentrations from 20 to 40 wt %. Hydrogels were produced with compressive moduli exceeding 1 MPa; however cell viability decreases as the modulus increases. These preliminary results can be exploited for cartilage tissue development to produce a mechanically strong scaffold. Additional experiments used acoustic fields to create multi layered biocomposites from similar materials. Acoustic focusing should prove useful as a tool to enhance tissue development by optimizing cellcell contact within the polymer matrix, at reduced initial cell loadings, by concentrating cells into bands. 10

11 Section I: Properties of Crosslinked Low and High Molecular Weight Poly(ethylene glycol) Diacrylate Blends for Tissue Engineering Applications 11

12 Abstract Mixtures of commercially available poly(ethylene glycol) diacrylate (PEGDA) with molecular weights of 400 and 3400 were UV crosslinked at total polymer concentrations that varied systematically from 20 to 40 wt %. The compressive moduli of the blends, is maximized when the wt % ratio high and low molecular weight PEGDA is ~ 40:60. The non linear mechanical behavior of the PEGDA blends can be attributed to the crosslinking ability and other characteristics of the polymer network. Cell viability results with a LIVE/DEAD fluorescence assay show an average viability of ~ 80% at a total PEGDA concentration of 20 wt %, for all blends. At a PEGDA3400/400 composition of 20:80 wt %, both the mechanical strength and cell viability were the highest. The compressive modulus for this composition fell from 0.4 to 1.6 MPa. The cell viability varied linearly from 20 to 80 %, when the total polymer concentration was varied from 20 to 40 wt %. Increasing the total polymer concentration increased the compressive modulus of the disc, but adversely affected cell viability for all the PEGDA blend compositions. These early results can be exploited for cartilage tissue engineering applications, where a mechanically strong scaffold is advantageous. 12

13 1. Introduction Poly(ethylene glycol) diacrylate (PEGDA) has been widely used in many biomedical applications due to its cytocompatiblity, non toxicity, and ease of use (1), (2) (3). Some of these applications include hydrogel development for drug delivery, tissue engineering for wound healing, and electrospinning processes for bioapplications (4), (5), (6), (7). Hydrogels are constructed from water soluble polymers or monomers (such as PEGDA or hydroxyethyl methacryate), that are crosslinked into insoluble threedimensional polymer networks that are comprised mostly of water and resemble human tissue (8). Water and nutrients can penetrate into the hydrogel, providing the necessary means to support cellular growth, and ultimately tissue development. It is the particular use of low and high molecular weight crosslinked PEGDA blends as a biocompatible cellular matrix that is of interest in this paper (9), (10), (11). Beyond biocompatibility, the mechanical attributes of a polymeric matrix are equally important and should target the mechanical properties of the tissue that is being replicated. The compressive modulus and other mechanical attributes of crosslinked hydrogel materials can vary greatly (12), (13). In choosing materials that have desirable, intrinsic mechanical properties, altering the processing conditions during polymerization, or combining various polymers, one can achieve a final biomaterial with desired mechanical characteristics (14), (15), (16). For this project, advantage is taken of a simple method of controlling mechanical properties while maintaining cell viability. This was achieved by 13

14 photocrosslinking blends of commercially available low and high molecular weight PEGDA polymers. In combining the different molecular weight species, one takes advantage of the higher concentration of crosslinkable end groups associated with the lower molecular weight polymer, and increased strength of a higher molecular weight polymer. By changing the ratio of the low and high molecular weight species in a polymer blend, one can control the mechanical properties of the polymer matrix. However, the ratio of the different polymers in the blend may have an adverse affect on cell viability, with negative effects attributed specifically to the lower molecular weight component. To assess the relevant trends, human breast cancer cells (MD MBA 231) were encapsulated as analog cells in these PEGDA blends and their viability was determined at fixed total polymer concentrations. It is hoped that these initial experiments will provide the basis for future work involving the use of human mesenchymal stem cells for the development of cartilage tissue, and highlight the use of low and high molecular poly(ethylene glycol) diacrylate blends for tissue engineering applications. 14

15 2. Background and Theory 2.1 The Polymerization Process The diacrylate polymer used in these experiments is a derivative of poly(ethylene glycol) which is available in a wide array of molecular weights. Its structure is shown in Figure 1 (17). PEGDA is a biocompatible, water soluble polymer capable of ultraviolet light induced free radical chain polymerization in the presence of an initiator such as Irgacure 2959 (1 [4 (2 Hydroxyethoxy) phenyl] 2 hydroxy 2 methyl 1 propane 1 one). The chemical structure of the photoinitiator is also given in Figure 1 (18). The active hydroxyl group on the end of the initiator molecule is reactive with suitable functionalized unsaturated organic compounds. The molecular weights of 400 and 3400 of the PEGDA polymers refer to the molecular weight of the ethylene oxide chain, excluding the diacrylate end groups. Figure 1 Left, chemical structure of poly(ethylene glycol) diacrylate. Right, chemical structure of Irgacure 2959 photoinitiator. Free radical polymerization consists of three basic steps, which are initiation, propagation, and termination. In the initiation step, the initiator molecule is cleaved (in this case by UV light or by heat) to produce free radical molecules. These reactive species then attack a monomer molecule by opening its double bond, producing a new radical center on the monomer molecule. The process is repeated many times over as more monomer molecules are added to the reactive center of the original monomer 15

16 molecule (19). The polymerization reaction is terminated by methods such as coupling or disproportination. This general process for the PEGDA reaction is shown schematically in Figure 2. In the initiation reaction, the initiator molecule, I, is cleaved via UV light and produces a radical species, R.. The radical species then attacks a PEGDA chain, producing a new radical center on the vinyl carbon of the PEGDA chain. This radical center is then free to attack another PEGDA chain, creating a crosslink between the chains. Consequently a new, radical center is created on the second PEGDA chain. At any time, two radical centers may combine with one another, to terminate the reaction. 16

17 Figure 2 The steps of free radical polymerization of PEGDA chains; initiation, propagation, and termination. 2.2 Polymer Characteristics that Contribute to Mechanical Strength The creation of the three dimensional PEGDA hydrogel network occurs through a free radical process; however since the base components are polymers themselves, additional structure and strength is added to the PEGDA matrix through the advent of crosslinking between the polymer chains. Crosslinking occurs when individual polymer chains chemically bond with one another to produce, in principle one giant, covalently 17

18 boded molecule, referred to as the polymer network. An idealized representation of this is shown in Figure 3, where the polymer chains are bonded together at specific sites represented by the black dots, forming a continuous molecule. The addition of the crosslinks increases the mechanical strength of the polymer by fusing the individual chains together. Rubber products, such as tires, are crosslinked (vulcanized) to produce strong materials or in other cases, crosslinking is used to impart resistance to melting to materials when exposed to elevated temperature. In the PEGDA matrix, crosslinks increase the compressive modulus of the material. The degree of crosslinking is proportional to the modulus of the polymer sample (20) Figure 3 Cross linked polymer system where the wavy lines represent polymer chain and the black circles are crosslinks binding the chains together. Another way to enhance the strength of a polymer system is to increase its molecular weight. Many important mechanical properties, such as strength, depend on molecular weight as indicated in Figure 4. There is a minimum molecular weight (usually one thousand) required to produce any strength at all, highlighted as point A. Above point A, the mechanical strength increases rapidly until some critical point is 18

19 reached, depicted as point B. Beyond that, the strength reaches some limiting value at point C (21, 22). By crosslinking the chains together, the molecular weight of the overall polymer network increases as well. Figure 4 Mechanical strength vs. molecular weight in polymer systems 2.3 Hydrogel Materials for Tissue Engineering Applications The PEGDA matrices created in these experiments belong to a class of materials called hydrogels. The three dimensional, aqueous nature of the hydrogel permits the targeting of the mechanical and transport properties of human tissue. Crosslinks contained in the structure of the hydrogel permit it to swell in manner that allows the diffusion of nutrients to cells contained within the matrix, but maintains a robust polymer network that holds up to the rigors that human tissues are exposed to. Swelling is a thermodynamic characteristic in which the polymer matrix will absorb a solvent, like water. 19

20 The swelling behavior of a polymer hydrogel can be described by examining the free energy change of mixing, which is a consequence of the polymer chains mixing with the solvent. This thermodynamic process can be described using Equation 1 where, where mixing, Δ Gmixing is the total free energy change of mixing, ΔH mixing ΔSmixing is the entropy change of mixing, and T is the temperature(23). is the change of enthalpy of Δ Gmixing =ΔHmixing TΔ Smixing (1) Enthalpy changes occur because of interactions between the polymer and solvent molecules. For mixing to occur, and the gel to swell, regards to the enthalpy, swelling is favorable when the Δ H mixing ΔGmixing must be negative. In is negative. This will occur if specific interactions (almost bond like interactions) form between the polymer and solvent molecules, to produce a lower energy, mixed state. However, if it is more preferable (a lower energy state) for the polymer and solvent molecules to interact only with themselves, then the change in enthalpy of mixing will be positive and mixing of the two phases will not be favorable. In addition to enthalpy changes associated with mixing, there will also be changes in entropy as well. There is an entropic advantage to mixing since the homogenation of the solvent and polymer molecules creates a more randomized system. However as the polymer and solvent mix, the orientation of the polymer chains changes from a random to a more orderly, elongated state, which incurs an entropy penalty. For mixing to occur, the increase in entropy of the mixing of should be greater than entropy losses from elongation of the polymer chains. In simple terms, the total 20

21 free energy of the entire system is a competing combination mixing and elongation. This relationship is shown in Equation 2, and for mixing (and swelling) to occur, ΔG total, should be negative, so that the process will be spontaneous (24). Δ Gtotal =Δ Gmixing +Δ Gelongation (2) The swelling of the polymer matrix inundates the structure with water, permitting the transport of nutrients to the cells. The diffusion coefficients of nutrients such as sucrose and glucose in water at a temperature of 37 C, is equal to 90 x 10 7 and 71 x 10 7 cm 2 /s (25). Ultimately, the hydrogel should swell to a degree that permits the transport of nutrients at rates that are comparable to those found in the human body. It is difficult to predict how cells will respond to a given polymer, but their overall viability of depends on the total polymer concentration, polymerization process, and cell density, and diffusion of nutrients, amongst other factors (26). In these experiments, the compressive modulus of the various polymer blends is used as an indicator of the overall compressive strength that might be expected from a issue derived in that matrix. However mechanical strength is not the only requirement of a good polymeric matrix. A target compressive modulus was chosen at about 1 MPa which approaches that of human articular cartilage (27). From internal discussions at Case, a cell viability of 80 % would be desirable; however cell viability is often sacrificed for mechanical strength. 21

22 3. Materials and Methods 3.1 Compressive Testing The purpose of these experiments was to assess cell viability in, and the mechanical properties of, crosslinked discs of low and high molecular weight PEGDA. Low molecular weight PEGDA was purchased from Polysciences Inc. (Warrington, PA), with a molecular weight of 400 and a higher molecular weight PEGDA was purchased from Glycosan Biosystems (Salt Lake City, Utah) with a molecular weight of These two components will be referred to as PEGDA400 and PEGDA3400, to describe the low and high molecular weight species, respectively. The PEGDA400 is a viscous liquid and the PEGDA3400 is a white powder and were used as received. Polymer discs were prepared by photocrosslinking PEGDA400, PEGDA3400, or blends of the two. The PEGDA polymer(s) were dissolved in a 1X PBS solution (GIBCO Dulbecco s Phosphate Buffered Saline, Sigma Aldrich, St. Louis, MO), and then agitated in an ultrasonic bath (Fisher Scientific, Model B2200R 1, Pittsburgh, PA) for 15 min. Once the PEGDA polymer was completely dissolved, a photoinitiator solution composed of Irgacure 2959 (CIBA, Basel, Switzerland) dissolved in 1X PBS at a concentration of 0.6 wt %, was added to the PEGDA/PBS solution. For every precursor solution, the final concentration of the photoinitiator was 0.05 wt %, to minimize any adverse affects on cell viability (28). The solution was then mixed again using the ultrasonic bath, and the ph was adjusted to 7.4 using 1M solutions of sodium hydroxide or hydrochloric acid. 22

23 The polymer discs used in the mechanical testing were crosslinked in a 35 mm diameter petrie dish, on a level surface, using a 365 nm Blak Ray long wave ultraviolet lamp (Cole Parmer, Vernon Hills, IL) for min. The intensity of the UV light was approximately 15 W/cm 2 as measured by a Cole Parmer Series 9811 Radiometer (Vernon Hills, IL). The cylindrical shaped samples used in compression tests were obtained by using a 10 mm Acu Punch (Ft. Lauderdale, FL) biopsy punch, to cut out the discs from the petri dish. The discs were then placed in a 1X PBS solution overnight to fully hydrate and then the thickness of each disc was measured using a Chicago digital micrometer (Fremont, CA). Once the thickness of the discs was measured, the compressive modulus of each disc was determined by placing it in a Rheometrics Solids Analyzer II compression device (Piscataway, NJ). A disc was compressed for 3 s at a strain rate of 0.5 s 1. These test conditions were suggested by Dr. Joseph Mansour of the Mechanical and Aerospace Engineering department. A stress strain curve was then recorded by a computer data acquisition system, and the modulus of the sample was calculated by fitting a linear line in Excel. Compression tests were repeated four times per disc, so that an average compressive modulus could be ascertained. For a given PEGDA composition, the number of individual discs used in the determination of the overall modulus for that composition was five at a minimum. Once the compression tests were completed, similar discs were created for determining initial cell viability of photoencapsulated cells. 23

24 3.2 Cell Maintenance and Viability Assessment In these experiments, MDA MB231 human breast cancer cells were used as analog cells for future experiments involving human mesenchymal stems cells. The cancer cells were cultured in a 10 vol % fetal bovine serum comprised of, HyQ DMEM/High Glucose powder (Logan, Utah), penicillin/streptomycin, and sodium bicarbonate media in BD Biosciences T 75 (Sparks, MD) flasks at 37 C in a 5% CO 2 environment. The cells were passaged upon reaching 80% confluence using HyQ Trypsin (Logan, Utah) at a concentration 0.25% (1X). The number of viable cells was then counted by staining a cell solution aliquot with Trypan blue (15 µl stain per 15 µl of cell media) and counting the number of unstained cells using a hemacytomter under 10x magnification. The cells were then separated by centrifugation and added at a concentration of 2 x 10 6 cells per ml of polymer solution to a sterilized filtered (0.45 µm filter) PEGDA solution at a ph of 7.4. The cells were homogenized in the PEGDA solution by pipetting the cell/pegda solution several times. 130 µl of the cell/pegda solution was then added to sterilized molds created from the detached caps of 1.5 ml polypropylene centrifuge tubes (Fisher Scientific, Pittsburgh, PA) and subjected to 10 min of long wave UV light (365 nm) at 15 W/cm 2. The constructs were created three at a time, and had a final thickness of approximately 2 mm and a diameter of 9 mm. After crosslinking, the newly formed constructs were removed from the mold and added to a 48 multi well plate (BD Biosciences, Sparks, MD) and 250 µl of cell media was added to the wells, in preparation for determining the cell viability of the encapsulated cells. 24

25 Cell viability was determined by using a LIVE/DEAD (Molecular Probes, Carlsbad CA) fluorescence bioassay consisting of calcein AM to track living cells, and ethidium homodimer 1 to track dead cells. The time between encapsulation and addition of the dye was about 30 min. 0.5 µl of each dye (concentration of 1 mg/ml) was added to a well containing the photoencapsulated cells. The multiwell plate containing the constructs was placed in a cell incubator for 30 min at 37 C at 5% CO 2. After incubation, half of the cell media was removed, and the discs were examined using an Olympus Model IX71S1F fluorescence microscope (Center Valley, PA) at a magnification of 10x. Green and red fluorescent images were captured using ImagePro software (Media Cybernetics, Inc., Bethesda, MD) and then Adobe Photoshop CS3 (San Jose, CA) was used to count the number of green and red illuminated cells to determine the average cell viability percentage for a given polymer composition. At least five samples were analyzed for each PEGDA blend composition. 25

26 4. Results and Discussion 4.1 Compressive Modulus Testing and Analysis Six different PEGDA compositions at total polymer concentrations of 20 wt % and 40 wt % were used to create the crosslinked constructs used in the compression testing. The specifics of each composition are given in Table 1. Table 1 Data set for compressive modulus testing Blend ratio (wt % ratio of PEGDA3400/400), total polymer concentration (wt %), number of samples, average thickness (mm) (standard deviation) Blend Ratio Total Polymer Concentration Number of Samples Average Disc Thickness 0/ (0.441) 20/ (0.174) 40/ (0.029) 60/ (0.040) 80/ (0.103) 100/ (0.200) 0/ (0.249) 20/ (0.178) 40/ (0.133) 60/ (0.698) 80/ (0.051) 100/ (0.304) 26

27 Photographs of the discs at a total polymer concentration of 20 and 40 wt % are shown in Figures 5 and 6, respectively. Figure 5 Photopolymerized PEGDA discs at a total polymer concentration of 20 wt % containing blends of PEGDA3400/400 (wt % ratio). (a) 0/100, (b) 20/80, (c) 40/60, (d) 60/40, (e) 80/20, (f) 100/0. As the wt % of PEGDA 3400 in the blend increases, the discs turn from an opaque white to transparent. Scale bar is equal to 5 mm. Figure 6 Photopolymerized PEGDA discs at a total polymer concentration of 40 wt % containing blends of PEGDA3400/400, in wt% ratio. (a) 0/100, (b) 20/80, (c) 40/60, (d) 60/40, (e) 80/20, (f) 100/0. As the wt % of PEGDA 3400 in the blend increases, the discs turn from an opaque white to transparent. Scale bar is equal to 5 mm. The discs created from pure PEGDA400 (Fig 5 a and 6 a) are opaque white. As the amount of PEGDA3400 is increased in the blend, the discs become more transparent. In the 20 wt % samples, the discs are transparent at a 40/60 blend (Fig. 5 c.), whereas in the 40 wt % blends the discs are not transparent until the amount of PEGDA3400 reaches 80 wt % (80/20 blend, Fig 6 e). At 40 wt % total PEGDA concentration, a higher ratio of the high molecular weight PEGDA3400 is needed in the blend to create a transparent construct, due to increased overall density of the crosslinked polymer network (relative to the 20 wt % blends). 27

28 Four compression tests were performed on each disc, and a stress strain curve was obtained for each. The stress strain data was recorded via a computer data acquisition system and transferred to Excel for linear extrapolation. Equation 3 describes the relationship between stress (σ) and strain (ε), where the slope of the line is the compressive modulus (E) of the sample, up to a proportional limit, at which the stress and strain are no longer linearly related (29). σ = Eε (3) The strain rate used (0.5 s 1 ) insured that the discs were not permanently deformed from one test to the next. Typical strains were on the order of 5 10% for the samples. The tests were performed in rapid succession to minimze any water loss during the comrpession analysis. The compressive modulus was determined by averaging the slopes of linear fits for each of the samples of a given blend. Figure 7 illustrates two characteristic stressstrain curves for blends comprised of PEGDA3400, at a total polymer concentration of 20 wt % and 40 wt %. For a given polymer blend, increasing the total polymer concentration from 20 to 40 wt % showed marked increases in the compressive modulus, as highlighted in Figure 7, in which the compressive modulus increases from 94 to 711 kpa. 28

29 Figure 7 Typical stress strain curves for discs composed of pure PEGDA3400 (100/0 blend) at 20 and 40 wt % total polymer concentration, with the slope of each line given as the compressive modulus, E. The 20 wt % data was taken from test 4 of 4 on that disc. The 40 wt % data is taken from test 1 of 4 on that disc. When the overall polymer concentration is held constant, it is also possible to increase the compressive modulus of a crosslinked disc by changing the ratio of the low and high molecular weight PEGDA in the uncrosslinked polymer precursor solution. Figure 8 shows the compressive modulus of crosslinked PEGDA blends with total polymer concentrations of 20 and 40 wt %. As one increases the wt percentage of PEGDA 3400 in the blend, a maximum average compressive modulus is achieved when the blend composition is ~ 40/60 wt %, PEGDA3400/400. The moduli at this composition were 0.42 MPa at 20 wt % and 1.70 MPa at 40 wt %. 29

30 Figure 8 Compressive modulus of PEGDA3400/400 (wt % ratio of initial polymer solution) blends at 20 and 40 wt % total polymer concentration, with standard error. For both data sets, the compressive modulus reaches a maximum, when the amount of PEGDA3400 in the blend is ~40 wt percent. Sample size is n 5. ANOVA analyses were performed on the data sets presented in Figure 8 and it was found that the average compressive moduli constituting each population were significantly different based on an overall significance level of The reported F value of the 20 wt % data was 34.8, which was greater than the F 0.05, 5, 27 distribution value of Similarly, the reported F value of the 40 wt % data was 14.2 which was greater than the distribution F 0.05, 5, 26 value of Tukey tests were applied to the compare means of pairs of blends comprised either of the 20 or 40 wt % population to determine significant differences at an overall 30

31 significance level of 0.05 (30). The results of the Tukey analyses for both the 20 and 40 wt % data is given in Table 2. Table 2 Tukey test analyses for comparing average compressive moduli Blend combination for comparison, 20 wt % Tukey results, 40 wt % Tukey results, 0 signifies that the differences between the means of the two blends is not significant, 1 signifies that the difference between the means of the two blends is significant. Blend Combination 20 wt % 40 wt % 20/80, 0/ /60, 0/ /60, 20/ /40, 0/ /40, 20/ /40, 40/ /20, 0/ /20, 20/ /20, 40/ /20, 60/ /0, 0/ /0, 20/ /0, 40/ /0, 60/ /0, 80/ At 20 wt % total PEGDA concentration, the pure PEGDA3400 blend (100/0) was significantly different than all other blends, having the lowest compressive modulus of 0.13 MPa. The 80/20, PEGDA3400/400 blend was statistically different than all the blends, except for the pure PEGDA400, 100/0 blend, which is evident in the arching 31

32 nature of the 20 wt % curve in Figure 8. The blends with the three highest modulus values were the 20/80, 40/60, and 60/40, which had average compressive moduli of 0.42, 0.42, and 0.38 MPa, respectively. These three blends are statistically equivalent according the Tukey analysis at 20 wt %. The pure PEGDA400, 0/100 blend had a compressive modulus of 0.26 MPa and was significantly different than all blends except for the 80/20 blend. The results of the Tukey analysis for the 40 wt % data indicate that the PEGDA400 (0/100) and PEGDA3400 (100/0) blends were statistically equivalent and had compressive moduli of 1.02 and 0.52 MPa. The PEGDA3400 (100/0) had the lowest compressive moduli of the population. The pure PEGDA400 blend was statistically equivalent to all blends except for the 20/80 and 40/60 blends, which had the highest compressive moduli of the group at 1.60 and 1.71 MPa, respectively. These two blends were considered statistically equivalent. This trend is similar to what was observed in the 20 wt % population. A series of two sample t tests were performed to compare the 80/20 blend of the 40 wt % population (lowest modulus at 0.51 MPa) with the entire 20 wt % population and to compare the 40/60 blend of the 20 wt % group (highest modulus at 0.42 MPa) with the entire 40 wt % population. This analysis would determine if there was any statistical overlap amongst the 20 and 40 wt % populations. There results of the two sample t tests are given in Table 3. 32

33 Table 3 Two sample t test results for comparison of moduli of 40/60 Blend at 20 wt % vs. 40 wt % population and 80/20 blend at 40 wt % vs. 20 wt % population. Primary blend comparison against 20 of 40 wt % population, 20 wt % Tukey results, 40 wt % Tukey results, 0 signifies that the differences between the means of the two blends is not significant, 1 signifies that the difference between the means of the two blends is significant. Blend Population at 20 or 40 wt. % Primary Blend 0/100 20/80 40/60 60/40 80/20 100/0 40/60 at 20 wt % vs. 40 wt % population /20 at 40 wt % vs. 20 wt % population When the 80/20 blend at 40 wt % total PEGDA concentration was compared against the 20 wt % data, it was found that the blend as statistically equivalent (denoted by the 0 s in Table 3) to the 20/80, 40/60, and 60/40 blends at the 20 wt % level. Therefore, the increases in compressive strength attained by adding up to 60 wt % PEGDA3400 in the matrix at a total polymer concentration of 20 wt %, is enough to match the strengths of blends that are comprised of 40 wt % PEGDA, twice its own total concentration. The results of the t tests comparing the 40/60 blend at 20 wt % to all the samples at the 40 wt % level show that it is equivalent only to the 80/20 and the 100/0, the weakest blends of that total PEGDA concentration. This shows that discs comprised solely of the longer chained PEGDA3400 material are weaker relative to blends containing some PEGDA400. These 40 wt % blends (80/20, 100/0) approach strengths of constructs that are comprised of only 20 wt % total PEGDA concentration, half their own total concentration. A more detailed explanation of some the trends highlighted in the previous paragraphs are discussed below. 33

34 Increasing the total polymer concentration of the samples increases the number of crosslinks in the polymer matrix due to a larger number of reactive diacrylate groups which ultimately increases the elastic modulus as shown in Figure 7 and 8. The thermodynamic relationship between crosslinking and the elastic modulus is defined in Equation 4, where E is the elastic modulus, n is the crosslink density defined as cross links per unit volume, _ 2 i _ 2 0 r / r represents the ratio of the end to end distance of a polymer chain in a crosslinked and uncrosslinked state, R is the universal gas constant, and T is the temperature. Often times the ratio of the cross linked and uncrosslinked end to end distance is assumed to be one (31). _ 2 r 3 i _ 2 r0 E = n RT (4) The average compressive moduli values of the pure PEGDA400 (0/100, as shown at far left in Fig. 8) are 0.26 and 1.02 MPa at 20 and 40 wt %, respectively. There is also an increase in compressive modulus in the pure PEGDA3400 (100/0 blends, as shown at far right in Fig. 8) from 0.13 and 0.52 MPa at 20 and 40 wt %, respectively. This would be expected according to Equation 4 due to the direct correlation between crosslink density and the elastic modulus. Explaining the non linear mechanical behavior of the blends at a constant total PEGDA concentration in Figure 8 is challenging and may not be only a function of crosslinking. As quoted from Flory, the presence of long chains, although a necessary 34

35 condition for rubberlike (elastic) behavior, is by no means a significant one. These PEGDA blends contain long chains since they are fundamentally composed of polymer chains. Additionally according to Flory, the polymer network requires internal polymer chain mobility, which allows for chain configuration during deformation and the recovery process. The addition of the PEGDA3400 in these blends should help impart this mobility due to the longer (and more flexible) chain length. However, internal motion of the chain molecules is not enough to create a mechanically robust construct as some permanence of structure is also required (32). The addition of the crosslinking permits this permanence of structure. The shorter chain length associated with PEGDA400 means that for a given volume, there should be a high degree of crosslinking since there is an increased concentration of reactive diacrylate groups, relative to systems composed of lower amounts of, or no PEGDA400 at all. The effect of the lower molecular weight species can be seen when the average compressive moduli of the pure PEGDA400 and PEGDA3400 samples are compared at the same total polymer concentration of either 20 or 40 wt %. The modulus increases by a factor of 2 when considering values of 264 vs. 131 kpa (PEGDA400 vs. PEGDA3400 at 20 wt %) or 1022 vs. 516 kpa (PEGDA400 vs. PEGDA3400 at 40 wt %). This data suggests that the pure PEGDA400 increases the amount of crosslinking relative to the pure PEGDA3400 samples. Therefore, a useful property of the low molecular weight species is its ability to increase the number of crosslinks in the blends, which in turn imparts stiffness in the material (33). 35

36 However if the elastic behavior was based solely on the crosslinking, then one might expect a decrease in compressive modulus as the weight fraction of the PEGDA3400 in the blend was increased. This is not the case, since the modulus peaks for both sets of data at ~ 40 wt % PEGDA As PEGDA3400 is added to the blend, some flexible characteristic should be added to the constructs, improving the elastic behavior of the constructs. In addition to the increased flexibility of these chains, chain entanglement plays a role in the elastic behavior of the network which occurs when the polymer chains interweave with one another. This also helps to stiffen the structure. The addition of the longer PEGDA3400 segments would likely increase the amount of entanglement as well. (34). The exact crosslinking behavior of the constructs is difficult to predict. However based on the data presented in Figure 8, it is likely that many factors are contributing to the overall mechanical strength of the discs. These blending of low and high molecular weight PEGDA molecules utilizes crosslinking, chain flexibility, chain entanglement, and possibly other factors, to enhance the compressive modulus of the relative to constructs comprised solely of either pure PEGDA400 or PEGDA3400 constructs. 36

37 4.2 Cell Viability Assessment Once the mechanical testing of the PEGDA blends had been completed, cell viability experiments were executed for the identical polymer compositions, as shown in Table 4. The discs were analyzed for cell viability using a LIVE/DEAD fluorescence bioassay. Table 4 Data set for cell viability measurements Blend ratio (wt % ratio PEGDA3400/400), total polymer concentration (wt %), number of samples for cell viability experiments involving photoencapsulated MDA MB231 cells exposed to fluorescent LIVE/DEAD assay after UV exposure time of 10 min at 15 W/cm 2. Blend Ratio Total Polymer Concentration Number of Samples 0/ / / / / / / / / / / / These results of these experiments are presented in Figure 9. The average initial viability (measured roughly min after addition of the bioassay) for the cells for all blends encapsulated at 20 wt % was 78.9% (±7.9%) and was 35.8% (±7.0%) for the 40 wt % 37

38 series. Additionally two control samples were completed in which the bioassay was applied to cells suspended in media, and then after t cells had been exposed to 10 min of UV light in media to determine any ill effects associated with the UV treatment. An average viability of about 80% would be considered a good target for cell viability as discussed amongst the advisory panel overseeing this project and thesis, however achieving such cell viability at increased polymer concentrations will be challenging as highlighted in the next few paragraphs. Figure 9 Cell viability as measured using fluorescent LIVE/DEAD assay of photopolymerized PEGDA3400/400 (wt % ratio) blends after 10 min of UV exposure at 15 W/cm 2 at total polymer concentrations of 20 and 40 wt %, with standard error. The average initial cell viability for all blends at 20 wt % is 78.9% (±7.9%) and is 35.8% (±7.0%) at 40 wt %. Sample size is n 5. Additionally the bioassay was applied to cell suspended in cell media (MEDIA) and cells suspended in media with 10 minutes UV exposure (MEDIA UV). 38

39 ANOVA analyses were conducted for the 20 and 40 wt % data sets presented in Figure 9. In each instance, the means of the blends were considered significantly different from one another, based on an overall significance level of The calculated F values of 3.68 and 11.1, at 20 and 40 wt %, were higher than F distribution values of (F 0.05, 5, 31 ) and 2.53 (F 0.05, 5, 25 ) and Note the ANOVA analyses did not contain the control experiments for which the cells were suspended in media or the media and UV exposure. Tukey tests were applied to compare the means within the 20 and 40 wt % populations and with the Media and Media UV controls samples. The results of those tests are given in Table 5. 39

40 Table 5 Tukey test analyses for comparing average cell viability Blend combination for comparison, 20 wt % Tukey results, 40 wt % Tukey results, 0 signifies that the differences between the means of the two blends is not significant, 1 signifies that the difference between the means of the two blends is significant. Blend Combination 20 wt % 40 wt % Media UV, Media 0 0 0/100, Media 0 1 0/100, Media UV /80, Media /80, Media UV /80, 0/ /60, Media /60, Media UV /60, 0/ /60, 20/ /40, Media /40, Media UV /40, 0/ /40, 20/ /40, 40/ /20, Media /20, Media UV /20, 0/ /20, 20/ /20, 40/ /20, 60/ /0, Media /0, Media UV /0, 0/ /0, 20/ /0, 40/ /0, 60/ /0, 80/

41 The pure PEGDA3400 (100/0) at the 20 wt % level had an average viability of 84% and was statistically equivalent to all of the other samples. The control samples Media and Media UV were also statistically equivalent with the same viability of 97% indicating that UV light has little effect on the cell viability. The pure PEGDA400 (0/100) discs at 20 wt %, with an average viability of 89%, had the highest viability of all the polymer blends and was equivalent to all samples except for the 60/40 and 80/20 blends. The 60/40 and 80/20 blends were statistically different than the control samples, and had the lowest viability at 70 and 72 %, respectively. However, considering that the pure PEGDA3400 sample (100/0) was statistically equivalent to all of the polymer blends and the controls, and that the PEGDA400 (0/100) sample was equivalent to all blends except the 60/40 and the 80/20 blend, as a population the 20 wt % group, exhibited a good degree of biocompatibility. Tukey tests were also applied to the 40 wt % data. In this case, none of the blends were equivalent to the media controls. This indicates that increasing the total polymer concentration from 20 to 40 wt % as a large, adverse impact on the overall cell viability. The pure PEGDA3400 (100/0) blend had the highest viability at 60% and was statistically different than all of the other polymer blends. The pure PEGDA400 (0/100) had an average viability of 34% and was statistically equivalent to all blends except the 20/80 which had a viability of 20 %, and the 100/0 blend. The 20/80, 40/60, and 60/40 blends were statistically identical and had have the lowest average viability amongst the 40 wt % data at 20%, 27% and 31%. Taking this into account and the fact that the 20/80 and 80/20 blends were statistically different; it would appear that that the addition 41

42 PEGDA3400 to the PEGDA400 up to 60 wt % of the matrix composition was detrimental to the cell viability. However, the viability appears to improve as the amount of PEGDA3400 blend increases to 80%, until finally the entire matrix is composed of only PEGDA3400 as seen in Figure 9. A series of two sample t tests were completed to compare the pure PEGDA3400 blend (100/0) which had the highest viability (60%) of the 40 wt % population with all of the 20 wt % blends and the 60/40 blend of the 40 wt % population (which had the lowest viability of the group at 70 %) with all of the blends of the 20 wt % level to determine if there was any statistical overlap. The results of these t tests are given in Table 6. Table 6 Two sample t test results for comparison of average cell viability of 60/40 blend at 20 wt % vs. 40 wt % population and 100/0 blend at 40 wt % vs. 20 wt % population. Primary blend comparison against 20 of 40 wt % population, Tukey Results; 0 signifies that the differences between the means of the two blends is not significant, 1 signifies that the difference between the means of the two blends is significant. Blend Population at 20 or 40 wt. % Primary Blend 0/100 20/80 40/60 60/40 80/20 100/0 60/40 at 20 wt % vs. 40 wt % population /0 at 40 wt % vs. 20 wt % population The 60/40 blend at 20 wt % was compared to all of the blends at 40 wt %. This blend was not statistically equivalent to any of the blends except for the pure PEGDA /0. As evident in Figure 9, there is a decrease in viability amongst the 20 wt % population as the PEGDA3400 concentration of the blend approaches 60 wt %. 42

43 This reduction is not significant enough to approach the dramatically reduced viability of the majority of the blends at a total 40 wt % level. The results of the t tests regarding the pure PEGDA3400, 100/0, sample at 40 wt % shows that it is statistically equivalent to the 40/60, 60/40, and 80/20 and PEGDA3400/400 blends at the 20 wt % level. This data suggest that the pure PEGDA3400 at 40 wt % exhibits a good degree of biocompatibility, significantly equivalent to blends with half its total polymer content. There appears to be a threshold regarding cell viability and the total polymer concentration. At 20 wt % total PEGDA concentration, there is not much difference amongst the blends with regard to cell viability as denoted by the ANVOA analysis. While larger, the test statistic of 3.68 is fairly close the F distribution value of 2.53 indicating that the differences amongst the means of the 20 wt % population is fairly small. Whereas in the 40 wt % population, the test statistic of 11.1 is much larger the distribution value of Therefore, at 20 wt % the total polymer concentration is low enough, that the ratio of the low and high molecular weight PEGDA in the blend is not that critical to the cell viability (it does make a difference regarding the mechanical strength). However at 40 wt, there appears to be a significant trend, in which increasing the ratio of PEGDA3400 in the blend enhances the cell viability. As mentioned before, part of the reduction in cell viability when comparing samples containing more PEGDA400 than PEGDA3400 (as in the case of the PEGDA400 (0/100 and PEGDA /0 at 40 wt % total PEGDA concentration), can be attributed to an increase free radical concentration that results from the shorter chained 43

44 PEGDA400. Free radicals will adversely affect the cells, as reported by Bryant and Burdick (28, 38). The structure of the polymer network may also be affecting the cell viability as well since the decrease in viability at PEGDA3400 concentrations that reach up to ~60 wt %, coincides with the increase in mechanical strength of the construct. As reported in the literature, there is an optimal polymer network structure that minimizes the infusion of deleterious substances to the cells that may be in the local environment, but does not starve the cells of oxygen and or limit the diffusion of required nutrients (35). The polymer structure of the blends with the highest compressive modulus may diminish cell viability because of restrictions in the diffusion of nutrients due to a tighter, more diffusion restrictive network. Additionally, the composition of the cell/polymer suspension prior to polymerization also plays an important role in overall cell viability. A higher molecular weight species, like the PEGDA3400, will have lower diffusion and permeability rates than the lower molecular weight molecules like the PEGDA400 (36). For example, it was found the that the permeability of polyethylene glycol (PEG) with a MW of 4000 across cell monolayers was 1.44 x 10 6 cm/s, whereas the permeability of a water soluble drug called Propranolol with a MW of ~ 300 had a permeability rate of 30.2 x 10 6 cm/s, a 20 fold increase (37). Therefore it is likely that the PEGDA400 molecules may be permeating into the cells at higher rates, relative to the PEGDA3400, and damaging them. 44

45 Typical images taken from the fluorescence microscope which are used in the analyses of cell viability are given in Figure 10 and 11. These figures highlight the loss of cell viability when the total polymer concentration is increased, as the relative number of illuminated living cells is greatly reduced from Figure 10 to 11 (left hand side of the figures). In this instance, the cell viability drops from 82% to 12%, when the total polymer concentration of a blend containing 20/80 wt % mixture of PEGDA3400/400 was increased from 20 to 40 wt %. Figure 10 Fluorescent images taken of a photopolymerized PEGDA3400/400 disc, 20/80 blend (wt % ratio) using LIVE/DEAD assay at a total polymer concentration of 20 wt %. The left image is of living cells, and right image is of dead cells. The cell viability is 82%. Scale bar is 100 µm. Figure 11 Fluorescent images taken of a photopolymerized PEGDA3400/400 disc, 20/80 blend (wt % ratio) using LIVE/DEAD assay at a total polymer concentration of 40 wt %. The left image is of living cells, and right image is of dead cells. The cell viability is 12%. Scale bar is 100 µm. 45

46 The negative effects of increased polymer concentration is emphasized here in the ~55 % drop in average cell viability across all blends when considering the 20 wt % data set versus the 40 wt% data set. As reported elsewhere in the literature, increases in polymer concentration adversely affect cell viability presumably due to an increase in radical concentration during the polymerization process (38). 4.3 Mechanical Testing and Cell Viability of 20:80 PEGDA3400/400 Blend After comparing Figures 8 and 9, the 20:80 wt %, PEGDA3400/400 blend exhibited strong mechanical properties relative to other blends and good cell viability when the total polymer concentration was 20 wt %. Consequently, additional PEGDA constructs of that composition were created at total polymer concentrations of 25, 30, and 35 wt %. Mechanical and cell viability tests were completed for these constructs as indicated in Table 7. Table 7 Data set for compressive modulus testing and cell viability measurements of 20:80 PEGDA3400/400 blend Blend ratio (wt % ratio of PEGDA3400/400), total polymer concentration (wt %), number of samples (N 1 = number of samples for compression test and N 2 = number of samples for cell viability assessment), average thickness of discs for compression testing (mm) w/standard deviation, R 2 values for linear fits of stress strain curves w/ standard deviation for discs undergoing compression testing. Blend Ratio Total Polymer Concentration N 1, N 2 Average Disc Thickness 20/ , (0.174) 20/ , (0.101) 20/ , (0.105) 20/ , (0.282) 20/ , (0.178) 46

47 ANOVA analyses were performed on the compressive modulus and cell viability data collected for this particular blend. In each instance, the results of the ANOVA indicated that the means of each population are significantly different from one another at an overall significance level of The mechanical strength and cell viability are shown in Figure 12 as a function of the total PEGDA concentration. Figure 12 Cell viability and compressive moduli of PEGDA3400/400 (wt % ratio), 20/80 blends, as a function of total polymer concentration, with standard error. Sample size is n 5 for all samples. The results of Figure 12 show the strong dependence of the cell viability and the compressive modulus on the total polymer content of the PEGDA matrix. The compressive modulus increases from 0.4 to 1.6 MPa, when the total polymer concentration of the precursor solution is increased from 20 to 40 wt. The cell viability 47

48 decreases from ~80% down to 20% when the total polymer concentration increases from 20 to 40 wt %. Consequently, cell viability is sacrificed for compressive strength. The compressive modulus of native articular cartilage is about 1 MPa, which is on par with what can be expected from this PEGDA blend and the others. To reach 1 MPa, the total polymer concentration of the 20/80 blend should be about 25 30%, which would give cell viabilities of ~55 75%. To optimize cell viability, it would make sense to use a lower total polymer concentration when encapsulating the cells. The cells would be more likely to develop into a functional tissue in this matrix of lower polymer content, and deposit extracellular matrix of their own. The extracellular matrix would contribute to the over strength of the polymer, and create a mechanically robust tissue that was initially developed from a more biocompatible polymer with a lower initial loading. Utilizing the natural ability of the cells to increase the strength of the polymer matrix is likely a superior approach to increasing the mechanical strength simply by raising the total PEGDA concentration. 48

49 5. Conclusions Commercially available mixtures of low and high molecular weight poly(ethylene glycol) with molecular weights of 400 and 3400 were photocrosslinked to encapsulate MDA MB231 human breast cancer cells. These cells were used as analogs for future work involving human mesenchymal stem cells. It was the goal of this project to show that low and high molecular weight PEGDA blends could produce a hydrogel system with a compressive modulus on par with of human tissues like articular cartilage, and maintain acceptable levels of initial cell viability. The data suggests that a PEGDA3400 composition of about wt % maximizes the compressive modulus. PEGDA hydrogels with a compressive modulus of 1 MPa, were created at a total polymer concentration of wt % and a cell viability of %, with a PEGDA blend composed of 20 wt % PEGDA3400 and 80 wt % PEGDA400. The cell viability and mechanical strength were strongly dependent on the total polymer concentration. Hydrogels with initial cell viabilities of >80% were obtained as well, at a total polymer concentration of 20 wt %, however their maximum compressive modulus was only about 0.4 MPa. In the future, it should be possible to raise cell viability by reducing the overall polymer concentration, and then allow cells to deposit extracellular matrix, to ultimately increase the compressive modulus of the system. This work provides a good basis for the future development of PEGDA hydrogel molecular weight blends for tissue engineering applications. 49

50 6. Future Work This work confirms that low and high molecular weight PEGDA blends provide a good material for the simple creation of synthetic cellular matrices, capable of reaching mechanical strengths that are useful for tissue engineering purposes. While the experiments performed in this paper studied the initial cell viability and mechanical strength of these matrices, future work must involve the study of cell viability and mechanical strength over long periods of time. This will isolate how cellular propagation and ultimately, tissue development, contributes to the overall characteristics of the PEGDA matrix. If 1 MPa is the target modulus required for cartilage tissue, it may be achieved over time by the subsequent deposition of extracellular matrix in an inherently weaker PEGDA matrix of lower total polymer concentration, but one that is more biocompatible. Additionally, cell viability must be assessed using more functional cells like mesenchymal stem cells or endothelial cells rather than cancer cells serving as analogs. The viability should be assed at regular intervals such as 0, 1, 3, 7 and 21 days to track cellular growth. The effect UV light exposure on these cells lines during the polymerization process should also be reported and the initial cell seeding density should also be optimized to maximize the mechanical properties and function of the tissue. While PEGDA with molecular weights of 400 and 3400 were used in these studies, there may be an advantage in expanding the range of molecular weights of the 50

51 individual PEGDA components. More emphasis should be placed on quantifying the specific contribution of the low and high molecular weight species on mechanical strength and cell viability. While PEGDA appears to be a good candidate for tissue development, molecular weight blends of other polymers should also be explored as well. Through continued research, the use of low and high molecular weight blends may allow researchers to further exploit common polymeric materials currently used in tissue engineering applications. 51

52 Section II: Encapsulation and Arrangement of Cells in Poly (ethylene glycol) Diacrylate Hydrogels Using an Acoustic Focusing Process 52

53 Abstract The goal of this research was to investigate the use of ultrasonic fields to create multi layered composites composed of cells and biocompatible polymers. Cells (or particles) were organized from a dispersed phase using the ultrasonic field, and then the resulting structure was preserved through ultraviolet polymerization of the polymer/cell solution. The geometry of the microstructure was controlled by orientation of the sound field, producing either concentric or planar layers. The bulk of this work involved the layering of MD MBA 231 human breast cancer cells in a concentric ring pattern within crosslinked poly(ethylene glycol) diacrylate (PEGDA) discs, composed of 30 wt % PEGDA with a molecular weight of 400. The operating frequency and voltage of the sound field was 2.32 MHz and 4 V RMS which produced 11 cell layers within the discs, with an average spacing of ~300 µm and thickness of 80 µm. The optimal time of sound field application was determined to be 20 min. The continuity within the cylindrical bands was adequate, but suffered from intermittent breakages due to non uniformities in the sound field. Cell viability studies indicated that the formation process does affects cell viability, mostly through exposure to the polymer solution during application of the sound field. Acoustic focusing may enhance cellular growth by raising cell to cell contact within the polymer matrix, by improving signaling and reception amongst the cells. Additionally the use of ultrasonic fields will concentrate cells in the polymer matrix, which could reduce the initial loading of cells required to form a functional tissue. 53

54 1. Introduction The goal of these experiments was to demonstrate and assess the use ultrasonic fields to arrange cells in layered configurations (or bands) within biocompatible polymers. The application of ultrasonic fields to manipulate particles and biological entities is well documented. Sound fields can be used to fractionate particles of different sizes or densities for separation purposes (39), concentrate particles at the surface of a biosensor for analysis (40), or align particles within polymers to create layered composites (41). While often employed to manipulate solid species, ultrasonic fields are also useful in manipulating droplets within emulsions (42) or to potentially remove air bubbles from liquid streams in low gravity environments (43). For this project, ultrasonic fields are used to arrange cancer cells (serving as analogs to mesenchymal stem cells), dispersed in an aqueous liquid polymer precursor solution composed of 30 wt % poly(ethylene glycol) diacrylate with a molecular weight of MW 400, into banded configurations. After the cells have reached a layered (or banded) state from an initially dispersed phase, the system is crosslinked via ultraviolet light. The crosslinking process preserves the structure of the banded state, whose geometry is dependent on the orientation of the sound field. The distance between the banded regions is controlled by the sound frequency and the banding time is controlled by both the frequency and intensity of the sound field. By organizing the cells and concentrating them into bands, it is theorized that tissue development can be enhanced by increasing cell to cell contact (44), (45). It 54

55 should also be possible to create tissues from lower overall cell loadings, since the final banded structure will compact the cells together, increasing the local concentration. The overall objective of these experiments was to demonstrate that acoustic fields could be used to arrange mammalian cells in a multi layered geometry in a hydrogel matrix and to preserve the structure via free radical polymerization, while maintaining the viability of the cells. 55

56 2. Background and Theory 2.1 Basic Concept This section describes the basic method of creating layered polymer composites using ultrasonic fields. While the focus of the thesis is to study cell arrangement in biocompatible polymers, many non biological particle or polymer systems can be utilized. Creating layered polymer composites using ultrasonic fields is a relatively straight forward process involving little in the way of equipment and only a few processing steps. To create the composite, the first item required is a liquid precursor which can be comprised of a water soluble material like poly(ethylene glycol) diacrylate or 2 hydroxyethyl methacrylate. Other, non biocompatible monomers, such as methyl or butyl methacrylate can be used for non biological applications as well. The second component of the precursor solution is a compound that will initiate a polymerization reaction. This compound could be a water soluble, UV sensitive photoinitiator like Irgacure 2959 or a thermal initiator such as AIBN (azobisisobutyronitrile). Finally, the third component of the precursor solution is particles, which could be a reinforcing agent such as carbon black, a drug for delivery from a degradable polymer matrix, or cells for tissue engineering applications. Regardless, the particles must be dispersed homogenously in the precursor solution, which helps make the each layer of the total multi layered system more uniform since each node will have access to the same number of particles. 56

57 Once the particle/monomer precursor is created, it is processed within an acoustic chamber by subjecting it to standing sound waves created by an ultrasonic transducer and reflector. After a certain period of a time, the particles will travel to fixed positions in the fluid, (nodes or antinodes) as determined by the characteristics of the sound field, creating particle layers or bands at these points. Once the bands have formed in the fluid layer, the entire system is polymerized to freeze the particle bands in place. This process is shown schematically in Figure 13. In this figure, T and R, represent the transducer which produces the sound field, and the reflector which permits the creation of a standing wave by reflecting the incident sound wave. Specifics and images of the acoustic process are discussed in subsequent sections. Figure 13 Overall acoustic focusing process. Homogenously dispersed particles are arranged in layers using acoustic fields and the structure is preserved via polymerization. 2.2 Piezoelectric Materials and the Production of Sound Energy The sound fields produced in these experiments are created using piezoelectric materials. The transducers that create the sound field are constructed from a common ceramic known as lead zirconate titanate (PZT). To provide sound field energy to a medium, the PZT responds to an electrical signal by producing a mechanical strain. Piezoelectric transducers are used to provide sound energy in applications such as non 57

58 contact ultrasonic testing to measure the thickness of materials (46), the monitoring of internal tissue damage in humans (47), and for use as ultrasonic mixers in microfluidic devices (48). Alternatively, a piezoelectric material that is subjected to a mechanical strain, responds by producing an electrical charge. Piezoelectric elements act as receivers in this capacity. Elements operating in this mode are useful in devices such as pressure sensors for online monitoring of chemical processes (49), guitar pickups for acoustic instruments (50), or for as a microphone in microelectronic systems (51). It is the crystal structure such a piezoelectric material that permits the electrical mechanical response (or vice versa). Figure 14 depicts the crystal structure of PZT (52). Figure 14 Crystal Structure of Lead Zirconate Titanate (PZT) and its reaction to an electrical field. A piezoelectric material produces a mechanical response from an electrical signal due to the electrical polarization produced by the applied electrical charge. This results in the back and forth motion of the Zr/Ti atom in Figure 14. As the frequency of the applied electrical charge approaches the natural frequency of the piezoelectric element, the amplitude of vibration will become large. When a PZT element is operated in 58

59 thickness mode, in which the mechanical vibrations are parallel to the applied electrical field, it is possible to reach frequencies of 10 MHz or even as high as 500 MHz, if one uses harmonic modes of vibration (53). The behavior of a piezoelectric material in response to an external stimulus can be described mathematically by constitutive equations, in terms of the mechanical or electrical response (54). Equation 5 defines strain, S, as the compliance (or stiffness) of the material, s, multiplied by a stress T. S = s T (5) Equation 6 describes how the charge density of the PZT element, D, is a function of the permittivity, ε permittivity, multiplied by an electrical field, E. Permittivity is a physical quantity that describes how an electric field effects and is affected by a dielectric medium, and is determined by the ability of a material to polarize in response to an electric field. D = ε E permittivity (6) Equations 5 and 6 can be combined into one equation to describe either the mechanical response or electrical response of the piezoelectric material, as shown in Equations 7 and 8. S = se T + d E (7) D = d T + ε E T (8) 59

60 In Equations 7 and 8, d, is a piezoelectric coupling term, which relates strain and voltage based on the direction of the applied electrical field or mechanical stress. The subscript E on the compliance term means it was measured at a constant or preferably zero, electric field. Similarly, the subscript T on the permittivity term indicates it was measured under zero stress conditions. Using equations 7 and 8 as a fundamental starting point, it is possible to predict the behavior of piezoelectric elements in response to electrical or mechanical stimuli propagated along different axes of the piezoelectric element. In the experiments performed for this study, a PZT plate or cylindrical transducer is used to propagate a sound field into an adjacent fluid layer. The transducer responds to an applied voltage by vibrating, which also vibrates material layers adjacent to the transducer. The layers can be an air, water, or for this application, the liquid polymer precursor solution. The fluid layer vibrates at the same frequency as the transducer and with an intensity that is controlled by the amount of voltage applied to the transducer. 2.3 Planar and Cylindrical Sound Fields When sound is transmitted from a piezoelectric transducer, its energy is emitted as a traveling pressure wave. The relationships used to derive the wave equation for one dimensional sound propagation are given in Equations 9 and 10, which are the linearized Euler s equation and the linearized continuity equation. Equation 9 describes the conservation of momentum and Equation 10 describes the conservation of mass during the propagation of the sound field. Here p, is the pressure, u x particle (air 60

61 molecule for example) velocity in the x direction, ρ 0 is the static density of the medium, and c is the speed of sound through the medium (55). u t x 1 p = ρ x 0 (9) u = 1 p x 2 x ρ0c t (10) By differentiating Equation 9 with respect to x and Equation 10 with respect to t, and subtracting the two resulting equations, the one dimensional wave equation is obtained, as given in Equation p 1 p = x c t (11) Assuming simple harmonic motion, the general solution to Equation 11 can be described as the sum of two waves traveling in opposite directions, which is given in Equation 12. iωt ikx ikx (, ) ( ) p xt = e Ae + Be (12) In equation 12, k is the wave number, and A and B describe the magnitude of the incident and reflected wave. As mentioned in the previous section, the sound field is propagated by a vibrating piezoelectric element. However, this sound field (or wave) is not sufficient in and of itself to drive particles from a homogenously dispersed state into layers. A 61

62 standing wave, which amplifies the intensity of the field, must be created to push the particles into their final banded state. In the acoustic focusing process, a standing wave is produced by reflecting the incident sound wave back towards the transducer. In an idealized system, the amplitude of the of the incident and reflected wave will be identical, meaning that the amplitude of the wave will be equal to 2A, as described in Equation 12. In Figure 15, the acoustic field is propagated by a piezoelectric plate on the left hand side of the acoustic chamber. The incident wave strikes the reflector, which can be made out of glass or stainless steel, and is reflected back towards the transducer. The continuous creation and reflection of the sound field, creates a standing wave, which is characterized by the appearance of fixed node and antinode positions. Node positions occur where the forward and reflected waves destructively interfere and thus the local pressure variation is equal to zero, while and antinode positions describe where the pressure swing reaches a maximum. These positions can be determined using Equation 12. The distance between any two nodes (or antinodes) is equal to the one half the wavelength of the sound field, which ultimately determines the distances between particle bands in the layered polymer composite. The wavelength (λ) is equal to the speed of sound in the material divided by the frequency of the sound field. The distance between an antinode and node is equal to one fourth the wavelength of the sound field. 62

63 Figure 15 Creation of a planar standing wave in an acoustic chamber. The final position of the particle (either at a node or antinode) is determined by its acoustic contrast factor. The primary acoustic radiation force on a solid particle (or liquid droplet or gas bubble) of radius R, suspended in a fluid, under the condition that the particle dimension is much less than the acoustic wavelength in a one dimensional resonant field is given in Equation 13, where F ac is the primary acoustic force, R is the radius of the particle, k is wave number of the sound field, E ac is the energy density of the sound field, F is the acoustic contrast factor and x is the distance away from an antinode (56). ac 3 4π ac sin 2 ( ) F = R ke F kx (13) The acoustic contrast factor is of particular importance, since its sign determines whether a particle will travel to the node or antinode position. The acoustic contrast factor, F, is defined in Equation 14, where Λ is the density ratio of the fluid to the particle and σ is the ratio of the compressibility of the fluid to the particle. If F has a value greater than zero the particle will travel to node, otherwise the particle will rest at 63

64 the antinode position (57). If F, equals zero, the particle will not respond to the acoustic field, since the acoustic force will equal zero as well. ( ) Λ+ 2 Λ 1 /3 1 F = 1 2 3σ 2 + Λ Λ (14) For a given particle, the time it takes to create the layered structure is dependent on the intensity of the acoustic field, the frequency of operation, the viscosity of the fluid the particles are suspended in, and the size of the particle. As the particle travels through the fluid it experiences a drag force that can be defined using Stokes Law which is defined in Equation 15. Here, F D is the drag force, R is the radius of the particle, µ is the viscosity of the fluid, and v is the velocity of the particle as it traverses through the fluid (58). The value of the drag force is negative to indicate that it opposes the acoustic force. FD = 6π Rμv (15) The drag force is proportional to the viscosity of the solution, so doubling the viscosity of the solution will increase the time it takes to band the particle by a factor of two. Increasing the particle size also increases the drag force; however this force is only proportional to the radius, whereas the acoustic force is proportional to the radius cubed. Therefore the increase of the frictional drag force is small when compared to increases of the acoustic force when the particle size is increased. Consequently larger particles band faster than particles of smaller size, when suspended in the same fluid. 64

65 One can predict the time it takes to band a particle by performing a force balance on the particle as it traverses through the fluid. For a particle traveling in the x direction, in which the sound field is propagated in a direction perpendicular to gravity (as case for experiments performed here), the banding time can be estimated by setting the drag force (Stokes Law) and the primary acoustic force equal to one another and then solving for time, t. The result of this force balance is given in Equation 16 in which t is the time it takes it a particle to move certain distance in the x direction, κ is the acoustic wave number, x 0 is the initial position of the particle, and x f is the final position of the particle, usually denoted as the node. The quantity, Γ, describes characteristics of the particle, fluid, and sound field and is given in Equation 17, where μ is the fluid viscosity. 1 tanκ x f ln Γ2κ tanκx0 t = (16) 2 2 Γ= R κ EacF 3μ (17) Using these equations one can predict how long it might take particle so different sizes or densities to travel in various fluid or under several different sound field conditions. Additionally, if the banding time is known, other unknown variables like the fluid viscosity can be determined. Other factors such as gravity also play an important role in the banding process. If the sound field is propagated in a horizontal direction then the particle will travel 65

66 perpendicular to gravity, and sink as it travels through the fluid and rests at the node. If the sound field is propagated vertically, the particle will travel in a direction that is parallel to gravity (vertically to a node or antinode) and then the acoustic force must overcome the net effect force of gravity, which is given in Equation 18, to reach the node. This assumes that the force of gravity acts downward in the negative direction and the acoustic force acts upward in a positive direction. Here, R is the radius of the particle, ρ is the density of the fluid or particle, and g is the gravitational acceleration constant. This force can be positive or negative, depending on the buoyancy of the particle. 4 3 Fg, net = πr ( ρfluid ρparticle ) g (18) 3 Regardless of whether or not the particle is traveling perpendicular or parallel to the sound field, if the net force of gravity is larger than the acoustic force, the particle will not stay fixed at the node. If settling is a problem, one approach to address the issue would be to adjust the density of the fluid so it matches the density of the particle, making it neutrally buoyant. Figure 16 highlights the use of acoustic fields to layer particles within polymers using the setup described in Figure

67 a. b.. c. Figure nm silicon dioxide particles layered perpendicular to the surface of the polymer (a,b images are top views); a. SiO 2 particles homogenously dispersed in butyl methacrylate at 0.75 vol %, b. Same system in Fig. 4 a, banded at a 725 khz, unpolymerized; c. Side view of polymerized butyl methacrylate system, at a particle loading of 2 vol %; the thickness of the sample is 3mm. The sound field is applied right to left in these pictures, with the transducer on the right and the glass reflector on the left. Figure 16 shows the banding of 500 nm amorphous silicon dioxide particles purchased from Alfa Aesar (Cat. #L16985) in butyl methacrylate. Figure 16 a, shows the particles initially dispersed in the butyl methacrylate. The acoustic contrast in the case is about Figure 16 b shows the formation of particle bands (whitish areas) that develop after about 20 minutes, at a frequency of 725 khz at an applied peak to peak voltage of 4V. The particle loading is 0.75% by volume in this instance. The center to center distance between the bands is ~ 1 mm. In the top views in Fig 16 a and b, the sound field is propagated right to left, with the transducer on the right hand side and a glass reflector on the left hand side of the images. The depth of the fluid in each figure is about 3 mm. Figure 16 c, shows a side view of a similar system after polymerization, with a total thickness of 3 mm and a volume loading of 2 %. The position of the transducer and reflector are identical in this picture. The photoinitiator used for the experiments carried out in Figure 16 was 2,2 dimethoxy 2 phenylacetophenone at a concentration of 0.2 wt % purchased from Arcos Organics. 67

68 Changing the orientation of the transducer and reflector such that the sound field will propagate from top to bottom, parallel to the surface of the fluid layer, changes the orientation of the bands. In Figure 17, 75 µm polystyrene divinylbenzene beads are quickly arranged (in less than a second) in water at a frequency of ~ 1MHz. The bands run perpendicular to those presented in Figure 16, since the sound field is propagating from top to bottom rather than right to left. The fast banding time is due to the large particle size. The acoustic contrast factor in this case is about The white strip at the top of the images is the transducer and the bottom white area is a glass reflector, such that the sound field propagates from top to bottom. In Figure 17 a, the beads are dispersed in the water, with no sound field application. After the sound field is activated in Figure 17 b, continuous bands form quickly as shown in Figure 17 c. Figure 17 d shows the initial settling of the bands when the sound field is first turned off nothing is opposing the effect of gravity. Figure 17 e shows the particles after they start to settle more completely. The distance between the bands is about 750 µm as taken from a side view. 68

69 Figure 17 Side views of 75 µm PVDB beads arranged parallel to the surface, in water at 1 MHz; a. Beads homogenously dispersed in fluid, b. Initial application of sound field, c. Formation of bands, d. Initial response after sound field turned off, e. Agglomerated beads sinking in fluid. The sound field is propagated from top to bottom with transducer located on the top of the image and a glass reflector located on the bottom of the fluid. Equation 13 shows that the strength of the acoustic force driving the particles is proportional to the cube of the particle radius. This means that an order of magnitude reduction in particle size reduces the acoustic force by a factor of a thousand. However as indicated in Equation 15, the drag force is proportional to the radius, so that an order of magnitude decrease in radius, means the drag force is also reduced by that amount. Subsequently when particle size is reduced by an order of magnitude, the time it takes to band is increased by two orders of magnitude which is the result of the reduction of the acoustic force, but also a reduction in the frictional drag on the particle as it travels through the fluid on the way to the node. In previous experiments, silicon dioxide particles with a diameter as small as 100 nm (spherical particles purchased from Alfa Aesar, Cat. # 44884) have been aligned in hydrogel composed of 2 hydroxyethyl methacrylate as shown in Figure 18 a. The 69

70 frequency of the sound field was ~ 1 MHz in this case and total particle loading was 2 wt %. It took about 4 hours to band the particle in this case at an applied voltage of 4 V peakto peak. The sound field is propagated right to left in an identical manner described earlier. In addition to arrangement in one direction, particles can also be arranged using two sets of transducers and reflectors to produce bands that travel in twodimensions, or to produce ring like interference patterns as shown in Figure 18 b. Here the particles are spherical carbon particles with diameters ranging from µm focused in butyl methacrylate at a frequency of 540 khz. The collection time was on the order of a few minutes. Figure 18 a. 100 nm SiO 2 particle arranged in 2 hydroxyethyl methacrylate at loading of 2 wt % and frequency of 980 khz, perpendicular to polymer surface, b µm carbon particles arranged in two dimensions at a frequency of 540 khz. All images are top views. In Fig 18 a, the sound field is applied right to left. In Fig 18 b, the sound emanates from the top and right hand side of the image. It is also possible to produce layers in other arrangements by using transducers of different geometries. If the sound field is applied from a cylindrical transducer in the 70

71 radial direction, particles will collect at nodes in a similar fashion as the planar examples cited above. Figure 19 depicts how particles will travel to nodes along the radius of fluid layer that resides in the annular region of a cylindrical or tube style transducer. The band structure for a sound field of this geometry is a series of concentric cylinders that increase in diameter as one travels along the radius of the fluid layer, from the center out towards the inner wall of the transducer. Cells Collect at Node Positions Transducer Inner Wall Top-View of Nodal Cylinders 3-D View of Nodal Cylinders Figure 19 Arrangements of cells at radial node positions emanating from a cylindrical sound source. In this configuration a reflector is not necessary because the sound field constructively interferes with itself. When the electric field is first applied to the cylindrical transducer, a sound wave emanates from the inner wall of the transducer towards (in the radial direction) the center of the fluid region, enclosed by the transducer. Noting the symmetry of this system, at the same time another sound wave is traveling towards the center of the fluid region, from a point directly opposite the first sound wave under consideration. The result of these two converging sound waves meeting at the center of the fluid region is the reflection of the two waves back towards 71

72 the inner wall of the transducer. This continuous propagation and reflection of sound waves in the radial direction results in the production of a standing wave. This highlights the advantages of using cylindrical acoustic chambers which create standing waves without the use of reflectors. The relative pressure profile of the sound field (P rel ) in the cylindrical case can be described using a zero order Bessel Function as defined in Equation 19, where k is the wave number which is equal to 2πf/c where f is the frequency of the sound field and c is the speed of sound in the fluid. In Equation 19, r is the radial position away from the center of the fluid layer (59). P ( ) rel = J0 kr (19) Because the Bessel function in Equation 19 is real, it represents a standing wave. It describes the superposition of two progressive waves, one diverging from the axis of the cylinder, and the other converging on to it (as described in the previous paragraph). The particles collect at node positions where the pressure defined in Equation 19 is equal to zero. Figure 20 shows the difference between planar and cylindrical pressure waves. The amplitude of successive maximums of the cylindrical wave decreases with distance, because the sound energy is spreading energy over cylindrical regions of increasing size. Unlike the planar case, the distance between nodes is not exactly equal to one half the wavelength of the sound field, but approaches that value asymptotically when kr >> π/2 (60). 72

73 The relative pressure of the cylindrical wave is maximized (P rel = 1) at x=0, which is at the center of the cylindrical transducer, due to the convergence (reflection) the two sound wave symmetric sound waves emanating from the inner wall of the transducer as described in the preceding paragraphs. The design of the cylinder itself must be chosen so that it provides a frequency that will provide the desired number or distance between bands. Using Equation 19 it is possible to estimate the number of nodes a given frequency will create in a given fluid. This frequency can then be given to a manufacturer who can then custom make the necessary transducer. x, position Figure 20 Relative pressure of a planar and a cylindrical sound wave traveling in water at a frequency of 2.32 MHz. In the cylindrical case, x=0 represents the center position (axis) of the annular region of the cylindrical transducer, which is the site where the reflection of the sound field takes place and the pressure is maximized. In the planar case, x=0 represents the source of propagation. In the graph, the planar wave is progressive and is not impeded in any manner. 73

74 3. Materials and Methods The human breast cancer (MD MBA231) cells used in these experiments and were cultured in a manner identical to that described in Section I. The cells were suspended in a 30 wt % solution poly(ethylene glycol) with a molecular weight of 400 at a cell concentration of 5 x 10 6 per ml of fluid. The PEGDA solution was filtered, and contained Irgacure 2959 photoinitiator at a concentration 0.1 wt %. In addition to the experiments involving cells, additional experiments were carried out using latex beads with a diameter of 3 µm that were purchased from Polysciences, Inc. These particles are good analogs for cells, and permit testing of the acoustic setup without the complications or safety concerns associated with biological agents. The acoustic chamber used to organize the cells was created from a cylindrical piezoelectric cylinder with a diameter of 8.5 mm and a length of 13 mm, made from a lead zirconate titanate ceramic (EC 64) purchased from EDO Ceramic in Salt Lake City Utah. The wall thickness of the transducer is 1 mm. To complete the acoustic chamber, the transducer is fixed to a standard microscope slide or glass plate using Scotch 3M Superadhesive, and which is allowed to dry for 8 hr. Any excess glue or dirt is removed using acetone. With the transducer firmly fixed to the glass slide, a cavity is created to which the cell (or particle) suspension is added. Once the transducer is fixed to the microscope slide, electrical leads are attached to the inner and outer wall of the transducer, which is connected to the electronic signal source as shown in Figure

75 Leads to Electrical Signal Generator Cell Suspension Cavity Transducer Glass Plate Figure 21 Schematic of the acoustic chamber comprised of a PZT cylindrical transducer fixed to a glass plate. The intensity and frequency of the sound field is controlled by a Fluke 6011A synthesized signal generator as shown in Figure 22. An interface panel lies between the signal generator and acoustic chamber. The typical operating voltage for these experiments was 4 V RMS. This value was chosen to minimize fluid disturbances and is discussed further in the next section of the thesis. Oftentimes a power amplifier is placed between the sound generator and the acoustic chamber to amplify the signal. In this case, a power amplifier was unnecessary due to the larger size of the cells (~ 10 µm), and the relatively high frequency of operation. Additionally, operating at too high of a voltage causes large disturbances in the fluid layer which distorts any layered structures formed during application of the sound field. Heating can also be problematic if the voltage is too high. 75

76 Signal Generator 2.32 MHz, 4V Interface UV Light Acoustic Chamber Figure 22 Schematic of sound equipment and acoustic chamber. Once the cell suspension is added to the acoustic chamber, the sound field is turned on and the cells (or particles) will travel to node positions within the chamber. The amount of time it takes to create a layered structure depends on the characteristics of the sound field and material properties of the particle and fluid, such as size, density, and viscosity. The banding time can be estimated from a force balance on the particle as described in Section 2.3. For the experimental conditions used here, 2.32 MHz and 4 V RMS, the sound field was applied for 20 min, prior to initiating the polymerization process. These conditions were chosen to maximize the effectiveness of the banding process and are discussed in more detail in the next sections. To start polymerization, UV light was applied from a Blak Ray Long Wave UV Lamp (365 nm) for 3 min. The sound field remains on during this process. After 3 min, the UV light is turned off and the polymerized disc is removed from the transducer for further analysis. Images of the 76

77 focusing process for all experiments were captured using an Olympus camera controlled by PIXCI imaging software. 77

78 4. Results and Discussion 4.1 Characterization of the Sound Field and Acoustic Focusing Process Using Latex Beads The fundamental operating frequency of the transducer of 2 MHz was first estimated from material parameters of the EC 64 PZT (54) transducer material, and then was fine tuned by close visual observation of the fluid response to frequency changes using the PIXCI capturing software. A frequency of 2.32 MHz was chosen as the fundamental frequency of operation. At frequencies other than the fundamental frequency which corresponds to a half wave length thickness (61), (or an odd multiple of this frequency), little fluid disturbance is seen even at high voltages. When operated at the fundamental frequency, larger fluid disturbances are observed since the transducer is operating efficiently. An applied voltage of 4 V RMS was chosen to minimize disruptive fluid disturbances and heating. Even so, a clockwise motion of the fluid can be observed during the banding process (by following the particles), which is caused by acoustic streaming due to non uniformities in the sound field. This motion can affect the quality of the banding. For many of the initial experiments, red latex beads with a diameter of 3 µm were used to test the ability of the cylindrical acoustic chamber to band particles. These particles were considered good analogs to cells (which have an average diameter of ~10 20 µm), based on their inherent size and density. The first sets of experiments were designed to determine the length of time needed to create a noticeable band structure from the initial dispersed phase. To this end, the beads were added to water at a 78

79 concentration of 10 x 10 6 per ml of fluid. 0.5 ml of the mixture was added to the acoustic chamber, and the chamber was operated at 2.32 MHz for 40 minutes. The operating voltage was 4 V RMS. The focusing process was captured using a Kodak Easyshare digital camera at various times throughout the focusing process. These images are shown in Figure 23. Figure 23 Time progression of acoustic focusing of 3 µm latex beads in water (10 x 10 6 per ml of fluid) at a frequency of 2.32 MHz: a. time = 0, b. time = 3 min., c. time = 10 min., d. time = 15 min., e. time = 40 min, top view images. The distance between bands is about 300 µm. At time = 0 (Fig. 23 a), the red color of the shows the beads evenly distributed throughout the fluid. At 3 min (Fig. 23 b), the beads have formed concentric rings in the fluid. This structure would be considered a quality macroscopic structure, due to the continuity of all of the bands. At 10 min (Fig. 23 c), increased aggregation of the beads at the center is noticeable by the formation of large red spots. At 15 and eventually at 40 min (Fig. 23 d, e), this phenomenon becomes more pronounced as the size of the red spots increases, and the overall ring structure starts to diminish. The increased collection of the particles at the center is due to the non uniform pressure profile of the cylindrical sound wave, which is strongest at the center due to reflection. Other imperfections in the structure can be caused by scratches on the surface of the transducer and the fact that one face of the transducer is fixed to a surface. 79

80 In the previous paragraphs it was mentioned that the transducer can be operated at an odd multiple of the fundamental frequency. By increasing the frequency of the sound field to 7.3 MHZ, (which if divided by 2.32 MHz is equal to 3.1), the distance between the bands can be reduced by roughly 1/3, since the distance between bands is inversely proportional to the increase in the frequency. Figure 24 show the latex beads focused at a frequency of 7.3 MHz at 4 V RMS. There are about 35 rings present in Figure 24 and about 12 rings present in Figure 23, which reflects the increase in frequency. Consequently, the distance between the rings falls from about 390 µm to 130 µm. Figure 24 3 µm latex beads focused at 7.3 MHz in water at a concentration of 10 million beads per ml of fluid, top view image. The distance between the bands is about 130 µm. 80

81 4.2 Acoustic Focusing Process Using Cells After initial studies using latex beads and water, similar experiments were completed by tracking the focusing process of human breast cancer cells at a concentration of 1 x 10 6 cells per ml of a polymer solution. The polymer solution consisted of a buffered 30 wt % PEGDA400 solution, at physiological ph. Seven separate experiments were completed in which 125 µl of a homogenized cell/polymer solution were added to the transducer. The sound field application varied between total times of 2 and 60 min and the focusing process was recorded for each experiment. Figure 25 shows the last frame of each recording, capturing the final structure for each application time. Again, the voltage of the sound field was 4 V RMS. After examination of the images in Figure 25, 20 min of sound field treatment (Fig. 25 d) produced a good macroscopic structure, while potentially minimizing the exposure of the cells to the unpolymerized PEGDA solution. 81

82 Figure 25 Focusing of MDA MB 231 cells in 30 wt % PEGDA400 solution at a concentration of 1 million cells per ml, top view images; a. 2 min total, b. 5 min total, c. 10 min total, d. 20 min total, e. 30 min total, f. 60 min total sound field application. After the initial experiments, the next step in the experimental process was to look at the banded cell structures on a microscopic level. To this end, cells were photoencapsulated in the PEGDA400 and several constructs were created. These polymerized discs were sent to the histology lab at the Case Western Reserve University Pathology Center for methacrylate embedding and staining using hematoxylin and eosin (H & E). The discs were cut using a microtome to produce 5 µm thick slices, every µm cut perpendicular to the vertical axis of the polymer disc. A few useful images were obtained via this method. One such image is presented in Figure 26 in which the 82

83 innermost ring at the center of the disc can be seen as well as a few of the succeeding rings. Figure 26 Microscope image of histology slice of inner rings composed of cancer cells arranged in 30 wt % PEGDA disc at a concentration of 1 x 10 6 cells per ml of fluid. Scale bar is equal to 300 µm. Unfortunately, the embedding of the PEGDA discs and the microtome process tended to tear the PEGDA, which distorted the ring structure. Another general problem of the acoustic focusing process that was discovered from the histological analysis was cell sedimentation during the application of the sound field. Consequently many of the polymer slices did not contain any cells. This problem will have to be addressed for future tissue engineering applications. The next series of experiments were designed to track the cell viability during the focusing and polymerization processes. There are many commercially available methods to track cell viability. One common assay that was used assay was the MTT 83

84 colorimetric (3 (4, 5 Dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide) which was purchased from Promega. This assay produces purple colored formazan which results from living cells processing the applied tetrazole compound. The purple product is subsequently quantified using a spectrophotometer. While some color change (purple color) was noticeable for the aqueous based MTT assay (an organic assay was also attempted with no success), the purple color did not diffuse from the PEGDA disc, making analysis via spectrophotometry impossible. Therefore, a fluorescent LIVE/DEAD assay was purchased from Molecular Probes to stain both living and dead cells. The advantage of this assay is that the stained (fluorescent) cells can be viewed directly in the disc using a fluorescent microscope, and don t require any spectrometric techniques. With the LIVE/DEAD assay in hand, an effort to create more uniform banding in the discs was attempted by increasing the initial cell loading from 1 x 10 6 to 5 x 10 6 cells per ml of precursor solution. The precursor solution was made from 30 wt % PEGDA400, with the balance being a 1x PBS buffer. Figure 27 show pictures of cells encapsulated in at the increased loadings; with the green indicating living cells and the red showing dead cells immediately after the addition of the LIVE/DEAD assay. The sound field was applied for 20 min at a voltage of 4 RMS. The images were taken at 10x magnification and the measurement bars in each picture are 300 µm long. While there are a significant number of living cells, there are also a large amount of dead cells. From the picture it is estimated that the cell viability is 50% at best. This viability is somewhat low for actual tissue engineering applications, where viabilities of 80% would be more acceptable, 84

85 however, these images indicate that a significant portion of the cells are surviving the acoustic focusing process. From these images, the average distance between rings was 276 µm with a standard deviation of 32 µm. This number is based on 50 measurements amongst the various rings. The average ring thickness was 70 µm with a standard deviation of 20 µm. Figure 27 Microscope images of fluorescently stained cancer cells encapsulated in a matrix composed of 30 wt % PEGDA400 at a concentration of 5 x 106 cells per ml; a b, inner ring shots of live (green) and dead (red) cells respectively. c d, outer ring shots of live and dead cells. Scale bar is equal to 300 µm. Ring distance and thickness measurements were also performed on a different sample operated under the same conditions as the sample in Figure 27. In this instance however, the disc was incubated for 40 hr prior before the LIVE/DEAD assay was performed. The images of this sample are show in Figure

86 Figure 28 Fluorescent images of live (Fig. a.) and dead cells (Fig. b) to estimate average ring thickness and average distance between rings. The frequency of the sound field is 2.32 MHz and cell concentration is 5 x 10 6 cells/ml. Assay applied 40 hours after encapsulation and incubation. Scale bar is equal to 500 µm in each picture. In this experiment, 11 rings were created and the average ring distance and thickness were determined from the green and red fluorescent images. The average thickness of all the bands was 82 µm with a standard deviation of 21 µm. The average distance between the rings was 302 µm with a standard deviation of 33 µm. While the exact speed of sound in the PEGDA solution is unknown, if one assumes that the speed of sound is about that of water, which is 1482 m/s, the distance between nodes should be about 319 µm if approximated using the linear case of one half the wavelength the sound field. The measurement of 302 µm would give a speed of sound of 1400 m/s for 30 wt % PEGDA400 solution. While the images taken of the embedded cells show organized rings that have a significant number of living cells, there were also a large portion of dead cells. However it should be noted that there are a significant number of living cells present in the disc after 40 hr of incubation as indicated by Figure 28 a. It should be noted that this assay is 86

87 not dynamic and cannot predict that the cells will live for longer periods of time, as it only provides a single snapshot of the current cell viability. One notable problem with the fluorescence assay is the existence of bleeding of the fluorescent green color into the disc which reduces the contrast between the living cells and the bulk matrix as shown in Figure 28 a. Additionally, concentrating the cells into the bands makes counting individual cells problematic because the amassed cells blend together. The fluorescent red dead, cells are more distinct however, as in Figure 28 b, due to the nature of the calcien and ethidium homodimer stains. In this assay, the calcien (fluorescent green stain) enters the cellular membrane of all cells and is processed only by living cells to produce a fluorescent product which stains the entire body of the cell. The ethidium (red fluorescent stain) only enters cells with compromised cellular membranes and stains only the nucleus of the cell. When these cells are excited by the light, only the nucleus fluoresces, providing a more pinpointed, precise image. The LIVE/DEAD assay was used in a final series of experiments to try and isolate aspects of the focusing process that may adversely affect cell viability. As indicated in the first section of this thesis, UV exposure seemed to have little effect on cell viability. Those experiments showed that 10 min of UV exposure while the cells were suspended in cell media caused essentially no change in cell viability, when compared to cells that had no UV treatment. The cell viability in each case was over 90%. To study the effects of the sound field on viability, the cells were exposed to the sound field in cell media for 20 min (again at 2.32 MHz and 4V RMS ). Figure 29 a and 29 b 87

88 show the cells that were not subjected any sound fields as a control. There is virtually no cell death. The cells in images in Figure 29 c and 29 d show the cells that were exposed to the sound field. The result of this experiment shows very little cell death from exposure to the sound field. There is noticeable cell aggregation since the cells were banded in the transducer and then removed (cells and media) so the bioassay could be applied. Figure 29 Live and dead fluorescent images (a.,b.) of cells exposed to no sound field. Live and dead images (c.,d.) of cells exposed to 20 minutes of sound field at 2.32 MHz and 4V RMS. Cell concentration is equal to 5 x 10 6 per ml of fluid. The most likely cause of cell death is the detrimental effects of PEGDA exposure as highlighted in Section I of this document. In that section, both PEGDA species (either molecular weight of 400 or 3400) negatively affected the cells. The average cell viability was reduced to 80% from about 95% in pure media, when the total PEGDA concentration was 20 wt %. Cell viability dropped even more dramatically to an average 88