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Control of Marine Biofouling and Medical Biofilm Formation with Engineered Topography

Permanent Link: http://ufdc.ufl.edu/UFE0021311/00001

Material Information

Title: Control of Marine Biofouling and Medical Biofilm Formation with Engineered Topography
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Schumacher, James F
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: algae, antifouling, bacteria, barnacle, biofilm, biofouling, biomaterials, microtopography, silicone, topography
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Biofouling is the unwanted accumulation and growth of cells and organisms on clean surfaces. This process occurs readily on unprotected surfaces in both the marine and physiological environments. Surface protection in both systems has typically relied upon toxic materials and biocides. Metallic paints, based on tin and copper, have been extremely successful as antifouling coatings for the hulls of ships by killing the majority of fouling species. Similarly, antibacterial medical coatings incorporate metal-containing compounds such as silver or antibiotics that kill the bacteria. The environmental concerns over the use of toxic paints and biocides in the ocean, the developed antibiotic resistance of bacterial biofilms, and the toxicity concerns with silver suggest the need for non-toxic and non-kill solutions for these systems. The manipulation of surface topography on non-toxic materials at the size scale of the fouling species or bacteria is one approach for the development of alternative coatings. These surfaces would function simply as a physical deterrent of settlement of fouling organisms or a physical obstacle for the adequate formation of a bacterial biofilm without the need to kill the targeted microorganisms. Species-specific topographical designs called engineered topographies have been designed, fabricated and evaluated for potential applications as antifouling marine coatings and material surfaces capable of reducing biofilm formation. Engineered topographies fabricated on the surface of a non-toxic, polydimethylsiloxane elastomer, or silicone, were shown to significantly reduce the attachment of zoospores of a common ship fouling green algae (Ulva) in standard bioassays versus a smooth substrate. Other engineered topographies were effective at significantly deterring the settlement of the cyprids of barnacles (Balanus amphitrite). These results indicate the potential use of engineered topography applied to non-toxic materials as an environmentally friendly coating for antifouling applications in the ocean. In addition, a biomaterial-grade silicone modified with a tailored engineered topography significantly inhibited the bacterial biofilm growth from Staphylococcus aureus for up to 14 days exposure without the use of bactericidal agents. Mature biofilms were present on equivalently exposed smooth silicone surfaces. Engineered surface topographies present a promising means of blocking biofilm development on medical surfaces and reducing the rate of related infections.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by James F Schumacher.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Brennan, Anthony B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021311:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021311/00001

Material Information

Title: Control of Marine Biofouling and Medical Biofilm Formation with Engineered Topography
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Schumacher, James F
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: algae, antifouling, bacteria, barnacle, biofilm, biofouling, biomaterials, microtopography, silicone, topography
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Biofouling is the unwanted accumulation and growth of cells and organisms on clean surfaces. This process occurs readily on unprotected surfaces in both the marine and physiological environments. Surface protection in both systems has typically relied upon toxic materials and biocides. Metallic paints, based on tin and copper, have been extremely successful as antifouling coatings for the hulls of ships by killing the majority of fouling species. Similarly, antibacterial medical coatings incorporate metal-containing compounds such as silver or antibiotics that kill the bacteria. The environmental concerns over the use of toxic paints and biocides in the ocean, the developed antibiotic resistance of bacterial biofilms, and the toxicity concerns with silver suggest the need for non-toxic and non-kill solutions for these systems. The manipulation of surface topography on non-toxic materials at the size scale of the fouling species or bacteria is one approach for the development of alternative coatings. These surfaces would function simply as a physical deterrent of settlement of fouling organisms or a physical obstacle for the adequate formation of a bacterial biofilm without the need to kill the targeted microorganisms. Species-specific topographical designs called engineered topographies have been designed, fabricated and evaluated for potential applications as antifouling marine coatings and material surfaces capable of reducing biofilm formation. Engineered topographies fabricated on the surface of a non-toxic, polydimethylsiloxane elastomer, or silicone, were shown to significantly reduce the attachment of zoospores of a common ship fouling green algae (Ulva) in standard bioassays versus a smooth substrate. Other engineered topographies were effective at significantly deterring the settlement of the cyprids of barnacles (Balanus amphitrite). These results indicate the potential use of engineered topography applied to non-toxic materials as an environmentally friendly coating for antifouling applications in the ocean. In addition, a biomaterial-grade silicone modified with a tailored engineered topography significantly inhibited the bacterial biofilm growth from Staphylococcus aureus for up to 14 days exposure without the use of bactericidal agents. Mature biofilms were present on equivalently exposed smooth silicone surfaces. Engineered surface topographies present a promising means of blocking biofilm development on medical surfaces and reducing the rate of related infections.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by James F Schumacher.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Brennan, Anthony B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021311:00001


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1 CONTROL OF MARINE BIOFOULING AND MEDICAL BIOFILM FORMATION WITH ENGINEERED TOPOGRAPHY By JAMES FREDERICK SCHUMACHER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 James Frederick Schumacher

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3 To my wife Iris

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4 ACKNOWLEDGMENTS I must express my sincere gratitude to my advisor, colleague, and friend, Dr. Anthony Brennan. I appreciate the advice and guidance provided by my doctoral committee consisting of Dr. Scott Berceli, Dr. William Ditto, Dr. Nam Ho Kim, and D r. Roger Tran Son Tay. I must acknowledge and thank my research collaborators Dr. Maureen Callow, Dr. James Callow, Dr. Nick Aldred, Dr. Anthony Clare, Dr. Patrick Antonelli, and Edith Sampson. I extend a special thanks to Al Ogden for providing access t o the UF Nanofabrication Center facilities, and for his training and assistance on their equipment. I must also thank Dr. Ronald Baney for his support and interesting discussions on science and life. I also acknowledge Jennifer Wrighton and April Derfiny ak for their friendship and administrative assistance throughout my graduate studies. Graduate students, past and present, have been vital for my progression and success throughout this entire process. First, I must thank my mentors, Dr. Adam Feinberg and Dr. Thomas Estes, for getting me started with my research and teaching me the culture of graduate school. I must also acknowledge the senior members of the ONR team, Dr. Leslie Wilson and Dr. Michelle Carman, who provided their guidance and support when I first joined the ONR project. The ONR team has changed quite a bit since I first started graduate school and I have now played a role as a senior member for the past 2 years. During this time, I have had the pleasure of working with two great colleague s, Kenneth Chung and Christopher Long, who have both been vital in providing experimental assistance and feedback during the completion of my dissertation research. I also sincerely thank the many undergraduate research assistants and technicians that hav e aided in the preparation of samples for assay analysis, including Paul Robinson, Carlos Inguanzo, Joanna Sanford, Cristina Fernandez, Sean Royston, Scott Long, and Greg Gamewell.

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5 I extend a special acknowledgement to the many friends that I have made whi le pursuing my doctoral degree including Dr. Brian Hatcher, Robin Hatcher, Dr. Clay Bohn, Ayelet Feinberg, Dr. Brett Almond, Erika Kennedy, Dr. Cliff Wilson, Nancy Estes, Victoria Salazar, Jim Seliga, Shema Freeman, Dr. Amin Elachchabi, Dave Jackson, and J ulian Sheats. I could have not completed my research and dissertation without the support, encouragement, and unconditional love of my family including my wife, Iris, my parents Fred and Sue, my sister Jennele, and last, but certainly not least, my wonderf ul grandparents Carole and Bob Bevilacqua and Vera and Frederick Schumacher. I extend a very special thanks to my in laws, Pedro, Iris, and Laura Susana Enriquez who have provided me with a tremendous amount of love and support. In addition, I thank the rest of my family whose love and encouragement were always close by.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ........... 4 LIST OF TABLES ................................ ................................ ................................ .................... 10 LIST OF FIGURES ................................ ................................ ................................ .................. 11 ABSTRACT ................................ ................................ ................................ ............................. 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ............. 17 Surfaces in Biological Systems ................................ ................................ ........................... 17 Scope of Research ................................ ................................ ................................ .............. 18 Research Goal and Specific Aims ................................ ................................ ....................... 19 Specific Aim 1 High Fidelity Fabrication of Engineered Topography ....................... 20 Specific Aim 2 Reduce Algal Spore Settlement with Engineered Topography .......... 20 Specific Aim 3 Reduce Barnacle Cyprid Settlement with Engineered Topography ... 21 Specific Aim 4 Inhibit Bacterial Biofilm Formation with Engineered Topography ... 21 Definition of Engineered Topography ................................ ................................ ................ 22 2 BACKGROUND ................................ ................................ ................................ ............... 24 Introduction ................................ ................................ ................................ ........................ 24 Marine Biofouling ................................ ................................ ................................ .............. 26 Process of Fouling, Biofouling, and Marine Biofouling ................................ ............... 26 Sh ip Hull Fouling ................................ ................................ ................................ ........ 27 Non Toxic Coatings and Topography ................................ ................................ .......... 28 Medical Bacterial Biofilms ................................ ................................ ................................ 30 Prevention and Consequences ................................ ................................ ..................... 31 Non Toxic Strategies and Topography ................................ ................................ ........ 31 3 MATERIALS AND METHODS ................................ ................................ ........................ 33 Overview ................................ ................................ ................................ ........................... 33 Fabrication of Engineered Topography ................................ ................................ ............... 33 Pattern Design and Ph otomask Generation ................................ ................................ .. 33 Pattern design limitations ................................ ................................ ..................... 33 Photomask specifications ................................ ................................ ..................... 34 Pattern design digitization with AutoCAD ................................ ............................ 34 Photomask fabrication ................................ ................................ .......................... 35 Process of Photolithography ................................ ................................ ........................ 35 Pre treatment of silicon wafers ................................ ................................ ............. 36 Coating photoresist on silicon wafers ................................ ................................ ... 36

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7 Pre exposure bake ................................ ................................ ................................ 36 Photomask alignment and photoresist exposure ................................ .................... 36 Development of transferred pattern ................................ ................................ ....... 37 Hard bake ................................ ................................ ................................ ............. 37 Etching of Patterned Silicon Wafer ................................ ................................ .............. 37 Replication of Topographically Mod ified Silicon Wafers ................................ ............ 38 Pattern Fidelity Evaluation of Engineered Topography ................................ ....................... 38 4 BIORESPONSE OF ALGAL SPORES TO ENGINEERED TOPO GRAPHY AND DEVELOPMENT OF ENGINEERED ROUGHNESS INDEX ................................ .......... 48 Introduction ................................ ................................ ................................ ........................ 48 Experimental Design ................................ ................................ ................................ .......... 49 Engineered Roughness Index ................................ ................................ ............................. 50 Materials and Methods ................................ ................................ ................................ ....... 51 Materials ................................ ................................ ................................ ..................... 51 Pattern Designs ................................ ................................ ................................ ........... 52 Pattern Fabrication ................................ ................................ ................................ ...... 53 Topographical Replication ................................ ................................ ........................... 53 Sample Preparation for Ulva Settlement Assay ................................ ............................ 53 Ulva Zoospore Settlement A ssay ................................ ................................ ................. 53 Stat istical M ethods ................................ ................................ ................................ ...... 54 Correlating Spore Settlement to Engineered Roughness Index ................................ ..... 55 Results ................................ ................................ ................................ ............................... 55 Discussion ................................ ................................ ................................ .......................... 56 5 BIORESPONSE OF BARNACLE CYPRIDS TO ENGINEERED TOPOGRAPHY AND CORRELATION TO ALGAL SPORES ................................ ................................ ... 65 Introduction ................................ ................................ ................................ ........................ 65 Experimental Design ................................ ................................ ................................ .......... 67 Effect of Topographic Feature Height on Settlement of Ulva Zoospores ...................... 67 Design of Barnacle Specific Engineered Topographies ................................ ............... 68 Design and Testing of First Generation Hierarchical Structures ................................ ... 68 Materials and Methods ................................ ................................ ................................ ....... 69 Materials ................................ ................................ ................................ ..................... 69 Pattern Fabrication Methods ................................ ................................ ........................ 69 Topographical Replication ................................ ................................ ........................... 70 Sample Preparation for Ulva Settlement Assay ................................ ............................ 70 Ulva Zoospore Settlement Assay ................................ ................................ ................. 70 Statistical Methods for Settlement Assay with Ulva Zoospores ................................ .... 71 Sample Preparation for Settl ement Assay with Balanus amphitrite Cyprids ................. 71 Settlement Assay with Balanus amphitrite ................................ ................................ .. 72 Statistical Methods for Settlement As say with Balanus amphitrite .............................. 72 Correlating Settlement of Spores of Ulva and Cyprids of Balanus amphitrite to Topographical Aspect Ratio ................................ ................................ ..................... 73 Results ................................ ................................ ................................ ............................... 73

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8 Topographical Aspect Ratio and Ulva Settlement ................................ ........................ 73 Topographical Aspect Ratio and Balanus amphitrite Settle ment ................................ .. 74 Correlations between the Settlements of Ulva and Balanus amphitrite with Topographical Aspect Ratio ................................ ................................ ..................... 75 Hierarchical Stru ctures and Ulva Settlement ................................ ................................ 76 Discussion ................................ ................................ ................................ .......................... 76 6 INHIBITION OF MEDICAL BIOFILM FORMATION WITH ENGINEERED TOPOGRAPHY ................................ ................................ ................................ ................. 88 Introduction ................................ ................................ ................................ ........................ 88 Materials and Methods ................................ ................................ ................................ ....... 89 Materials ................................ ................................ ................................ ..................... 89 Sharklet AF Design and Fabrication of Topographical Molds ................................ ...... 89 Sample Preparation ................................ ................................ ................................ ..... 90 Bac terial Biofilm Growth Assay ................................ ................................ .................. 90 Characterization of Bacterial Coverage ................................ ................................ ........ 91 Statistical Analysis ................................ ................................ ................................ ...... 92 Results ................................ ................................ ................................ ............................... 92 Discussion ................................ ................................ ................................ .......................... 93 7 ENGINEERED NANO FORCE GRADIENTS FOR THE INHIBITION OF SETTLEMENT OF SWIMMING ALGAL SPORES ................................ ....................... 101 Introduction ................................ ................................ ................................ ...................... 101 Theory ................................ ................................ ................................ ............................. 103 Experiment al Design ................................ ................................ ................................ ........ 105 Design of Ulva S pecific Nano Force Gradients ................................ ......................... 10 5 Materials ................................ ................................ ................................ ................... 106 Engineered Topography Mold Fabrication ................................ ................................ 106 Engineered Topography Replication ................................ ................................ .......... 107 Contact Angle Characterization ................................ ................................ ................. 107 Sample Preparation for Ulva Settlement Assay ................................ .......................... 107 Statistical Methods ................................ ................................ ................................ .... 108 Results ................................ ................................ ................................ ............................. 108 Discussion ................................ ................................ ................................ ........................ 110 8 CONCLUSIONS AND FUTURE WORK ................................ ................................ ........ 118 Marine Biofouling ................................ ................................ ................................ ............ 118 Medical Biofilm Formation ................................ ................................ .............................. 119 Engineered Nano Force Gradients ................................ ................................ .................... 120 Protruding and Recessed Features ................................ ................................ .................... 120 APPENDIX A SINUSOIDAL DESCRIPTION OF DESIGNED PATTERNS USED AS TEMPLATES OF ENGINEERED TOPOGRAPHY ................................ ................................ ............... 123

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9 LIST OF REFERENCES ................................ ................................ ................................ ........ 133 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ... 144

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10 LIST OF TABLES Table page 3 1 Process conditions used to etch patterned silicon wafers. ................................ ............... 43 3 2 Process conditions used for oxygen plasma etch. ................................ ........................... 43 4 1 ................................ ................................ ................................ ............ 62 4 2 Calculated engineered roughness index (ERI) values for the studied topographical surfaces fabricated in PDMSe. ................................ ................................ ....................... 64 5 1 ANOVA for Ulva riable ................................ ................................ ................................ ............ 83 5 2 Kruskal Wallis analysis for percent settlement of barnacle cyprids ( B. amphitrite ) on smooth and topographically modified PDMSe surfaces. ................................ ................ 84 5 3 ANOVA for Ulva ................................ ................................ ................................ ....... 87 7 1 Values used to calculate end defl ection forces using Equation 7 1 for topographical features pictured in Figure 7 2. ................................ ................................ .................... 112 7 2 Sessile drop water contact angle for PDMSe surfaces. ................................ ................. 115

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11 LIST OF FIGURES Figure page 1 1 Surface model considering the surface chemistry, surface topography, mechanical properties, and surface energy of a material and their influence on th e settlement, adhesion, and growth of living organisms. ................................ ................................ ..... 23 2 1 Microorganisms of bacteria and algae settling and colonizing on a silicone elastomer surface. ................................ ................................ ................................ ......................... 32 3 1 Process of e beam lithography used to print a photomask from a CAD drawing of a designed pattern. ................................ ................................ ................................ ............ 40 3 2 Photolithography process used to transfer the patt ern on a photomask to a photoresist coated silicon wafer. ................................ ................................ .................... 40 3 3 Etch processing of patterned silicon wafers using deep reactive ion etching. .................. 41 3 4 Designed patterns drawn in AutoCAD. ................................ ................................ .......... 41 3 5 Karl Suss mask aligners used to transfer designed patterns from a photomask to a photoresist coated wafer. ................................ ................................ ............................... 42 3 6 A 4 in. by 4 in. photomask created in AutoCAD. ................................ ........................... 42 3 7 Patterned silicon wafer being loaded into a Surface Technology Systems (STS) Multiplex r eactive ion etcher. ................................ ................................ ........................ 43 3 8 SEM images of a silicon wafer etched for a total time of 55 seconds using optimized process parameters. ................................ ................................ ................................ ........ 44 3 9 SEM images of the surface of PDMSe replicated from the etched silicon wafer pictured in Figure 3 8. ................................ ................................ ................................ ... 44 3 10 SEM image of an engineered topography on the surface of PDMSe containing f lopped features. ................................ ................................ ................................ ............ 45 3 11 SEM images of a set of engineered topographies on the surface of PDMSe produced from the same pattern showing the effect of overexposed features. ................................ 45 3 12 SEM images of engineered topographies on the surface of PDMSe with missing features. ................................ ................................ ................................ ......................... 46 3 13 SEM images of engineered topographies on the sur face of PDMSe with no defects across the viewable area. ................................ ................................ ............................... 46

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12 3 14 Light micrograph image of a rejected sample of an engineered topography on the surface of PDMSe taken during the final pattern f idelity assessment before biological testing. ................................ ................................ ................................ ......................... 47 3 15 Light micrograph image of a high fidelity sample (> 99%) of an engineered topography on the surface of PDMSe taken during the final patte rn fidelity assessment before biological testing. ................................ ................................ .............. 47 4 1 SEM images of engineered topographies on a PDMSe surface. ................................ ...... 60 4 2 Photog raph and light micrograph of sample configuration used for Ulva settlement assays. ................................ ................................ ................................ ......................... 61 4 3 Ulva spore settlement data on PDMSe surfaces represented as mean spore density (spores/mm 2 ) standar d error (n = 3). ................................ ................................ ........... 62 4 4 Representative light micrographs, obtained by a mixture of epifluorescence and transmitted light, of spores settled on PDMSe surfaces. ................................ ................. 63 4 5 Correlation between Ulva spore settlement and engineered roughness index (ERI) at a fixed feature spacing of 2 m. ................................ ................................ ..................... 64 5 1 SEM images of the Ulva specific Sharklet AF topography produced on the surface of PDMSe fabricated at three different feature heights. ................................ ...................... 80 5 2 SEM images of the barnacle specific engineered topographies p roduced on the surfac e of PDMSe ................................ ................................ ................................ ......... 81 5 3 Light micrograph and SEM images of hierarchical PDMSe topographies. ..................... 82 5 4 Ulva spore settlement data on PDMS e surfaces represented as mean spore density (spores/mm 2 ) standard error (n=3). ................................ ................................ ............. 83 5 5 Mean barnacle cyprid settlement (% standard error) after 48 hours on PDMSe surfaces ................................ ................................ ................................ ........................ 84 5 6 Graphical representation of the linear correlation for the percent reduction in settlement of Ulva and B. amphitrite with topographical aspect ratio relative to a smooth PDMSe surface.. ................................ ................................ ............................... 85 5 7 Light micrographs showing Ulva spores settled against the vertical walls of the channels with and without the presence of the Ulva specific Sharklet AF topography. ... 86 5 8 Ulva spore settlement data on PDMSe surfaces represented as mean spore density (spores/mm 2 ) standard error (n = 3). ................................ ................................ ........... 87 6 1 Sharklet AF topography on polydi methylsiloxane elastomer (PDMSe) with a 2 ................................ ........................ 96

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13 6 2 Configuration and dimensions of the circular PDMSe samples used for bacterial biofilm gro wth experiments. ................................ ................................ .......................... 96 6 3 Position of randomized PDMSe samples placed within a 3 inch Petri dish for bacterial biofilm growth testing. ................................ ................................ .................... 97 6 4 Locations of SEM analysis on samples exposed to Staphylococcus aureus ................... 97 6 5 Image processing of SEM images to quantify bacterial coverage on PDMSe surfaces exposed to Staphylococ cus aureus ................................ ................................ ................ 98 6 6 SEM images of bacterial coverage on smooth and Sharklet AF PDMSe surfaces. .......... 99 6 7 Mean value of percent cover age of S. aureus on Smooth and Sharklet AF PDMSe surfaces at various time points. ................................ ................................ ................... 100 7 1 Hemisphere representing a settling cell/microorganism contacting two dissimilar topographical feature s. ................................ ................................ ................................ 111 7 2 Estimated lateral forces required to cause a 10% end deflection of micrometer sized topographical features in PDMSe modeled as cantil ever beams ................................ .. 112 7 3 Pattern designs of 2 element engineered topographies representing a range of modeled nano force gradients ................................ ................................ ..................... 113 7 4 SEM images of force gradient engineered t opographies fabricated in PDMSe by replication of silicon wafer molds. ................................ ................................ ............... 114 7 5 Mean spore ( Ulva ) density ( standard error, n = 3, counts = 30 per n) measured and calculated for each PDMSe sur face studied (3 m feature height for all engineered topographies). ................................ ................................ ................................ .............. 115 7 6 Representative light micrographs, obtained by a mixture of epifluorescence and transmitted light, of spores ( Ulva ) s ettled on PDMSe surfaces. ................................ .... 116 7 6 Magnified light micrographs showing the preferred settlement location of spores relative to the topographical features on GR0 and GR5. ................................ ............... 117 7 7 Magnified light micrographs showing the preferred settlement location of spores relative to the topographical features on GR1 GR4 and SK. ................................ ........ 117 8 1 Microscope images of algal zoospores of Ulva and bacterial cells of Staphylococcus aureus preferentially attached to the recessed area between protruding features. .......... 122 8 2 SEM images of the Sharklet AF pattern fabricated as both protruding features and recessed features in PDMSe. ................................ ................................ ........................ 122 A 1 Designed Sharklet AF pattern used to create the Sharklet AF engineered topography. 126

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14 A 2 Sine wave (blue line) applied to a periodic row of the Sharklet AF pattern. .................. 127 A 3 Cosine wave (red line) applied to the periodi c row above the sine wave (blue line) on the Sharklet AF pattern. ................................ ................................ ............................... 127 A 4 Sharklet AF pattern described by three sets (N = 0, 1, and 2) of sine and cosine waves (Eqs. 4 1 and 4 2) for A = 8, B = 20, c = 24, and w = ( / 12). .......................... 128 A 5 Two element designed pattern represented by two sinusoidal waves. ........................... 128 A 6 Five element designed pattern represented by two sinusoidal waves. ........................... 129 A 7 Two arbitrary elements selected for the creation of a designed pattern based on the derived sinusoidal waves. ................................ ................................ ............................ 129 A 8 Definition and location of sinusoidal wave variables including S D X D L D centroid ( ) of smaller feature, and topmost point ( ) of larger feature. ................................ ...... 130 A 9 Definition and location of sinusoidal wave variable P S ................................ ................ 130 A 10 Arbitrary shapes used to fill the space between the periodic elements. ......................... 131 A 11 Designed pattern from Figure A 10 arrayed as defined by the derived sinusoids (Eqs. A 8 and A 9) for N = 0 and x = 0 to ................................ ................................ 131 A 12 Designed pattern from Figur e A 10 arrayed as defined by the derived sinusoids (Eqs. A 8 and A 9) for N = 0, 1, and 2 and x = 0 to 3 ................................ ................ 132

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CONTROL OF MARINE BIOFOULING AND MEDICAL BIOFILM FORMATION WITH ENGINEE RED TOPOGRAPHY By James Frederick Schumacher August 2007 Chair: Anthony B. Brennan Major: Biomedical Engineering Biofouling is the unwanted accumulation and growth of cells and organisms on clean surfaces. This process occurs readily on unprotected surf aces in both the marine and physiological environments. Surface protection in both systems has typically relied upon toxic materials and biocides. Metallic paints, based on tin and copper, have been extremely successful as antifouling coatings for the hu lls of ships by killing the majority of fouling species. Similarly, antibacterial medical coatings incorporate metal containing compounds such as silver or antibiotics that kill the bacteria. The environmental concerns over the use of toxic paints and bi ocides in the ocean, the developed antibiotic resistance of bacterial biofilms, and the toxicity concerns with silver suggest the need for non toxic and non kill solutions for these systems. The manipulation of surface topography on non toxic materials at the size scale of the fouling species or bacteria is one approach for the development of alternative coatings. These surfaces would function simply as a physical deterrent of settlement of fouling organisms or a physical obstacle for the adequate formatio n of a bacterial biofilm without the need to kill the targeted microorganisms. Species specific topographical designs called engineered topographies have been designed, fabricated and evaluated for potential applications as antifouling marine coatings and material surfaces capable of reducing biofilm formation.

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16 Engineered topographies fabricated on the surface of a non toxic, polydimethylsiloxane elastomer, or silicone, were shown to significantly reduce the attachment of zoospores of a common ship fouling green algae ( Ulva ) in standard bioassays versus a smooth substrate. Other engineered topographies were effective at significantly deterring the settlement of the cyprids of barnacles ( Balanus amphitrite ). These results indicate the potential use of engi neered topography applied to non toxic materials as an environmentally friendly coating for antifouling applications in the ocean. In addition, a biomaterial grade silicone modified with a tailored engineered topography significantly inhibited the bacteria l biofilm growth from Staphylococcus aureus for up to 14 days exposure without the use of bactericidal agents. Mature biofilms were present on equivalently exposed smooth silicone surfaces. Engineered surface topographies present a promising means of blo cking biofilm development on medical surfaces and reducing the rate of related infections.

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17 CHAPTER 1 INTRODUCTION Surfaces in Biological Systems The initial biological response to a material placed within a living environment is controlled by the surface characteristics of the material. This biological surface interaction influences the efficac y of biomaterials used in medicine [1,2] and the degree of biofouling on marine surfaces [3] Surface properties can be both structural and chemical. Surface structure can be random or highly ordered and range in length from the atomic to the macroscopic level. The lower end of this range includes atomic defects that may interact with water, ions, and other biomolecules [4,5] as well as nanometer scale surface roughness that influence the adhesion and structure of proteins, both free [6] and membrane bou nd [7] The upper end includes micrometer scale surface irregularities and intentionally produced microtopographies which can directly modulate cell and microorganism behavior such as cellular alignment [8,9] and microorganism attachment [10,11] Surface chemistry generally describes the outermost atomic layer of the material and can be uniform or heterogeneous. Surface chemistry is associated with the chemical properties of the material and very short range interactions (a few ngstrm) between the surfa ce and the constituents of the biological environment [12] However, these short range interactions influence multiple dimensional scales spanning from the molecular level when considering intermolecular forces in biology (e.g., ligand receptor binding) [ 13] to the macroscopic influence on surface energy and its relationship to the adhesion of microorganisms [14,15] Material surfaces are thus composed of any number of unique chemistries in combination with physical structures across many length scales, al l of which contribute to the biological response of the living environment to the material.

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18 Scope of Research The complexity of a material surface requires the consideration of numerous design parameters when engineering a surface for a desired biological response. A surface model has been developed by the Brennan Research Group at the University of Florida which considers the topography, chemistry, mechanics, and surface energy and their influence on the settlement, adhesion, and growth of living organism s (Figure 1 1). The versatility of this model is that both main effects and interactions of the surface parameters can be studied using design of experiments. The biological systems that are currently being studied using this model include the marine env ironment with the goal of reducing the fouling on submerged surfaces and various infectious microbial systems to create antibacterial surfaces that reduce bacterial adherence or deter biofilm formation. My research addresses the design and fabrication of d iscrete, periodic surface structures on polydimethylsiloxane elastomer (PDMSe) that are tailored to elicit a specific response of a targeted organism (e.g., algal spore) or influence a specific biological event (e.g., bacterial biofilm formation). This ty pe of ordered and biologically tailored surface topography has been associated with the geometric dimensions and spatial arrangement of the individual features of the surface. Engineered topographies were designed, fabricated on the surface of PDMSe, and evaluated for applications as antifouling marine surfaces and as medical surfaces that reduce biofilm formation. Current solutions for the marine environment rely on m etallic based paints and biocide releasing coatings that function by killing the local fouling species [16,17] Similarly, antibacterial medical coatings incorporate metal containing compounds such as silver and antibiotics that kill the bacteria [18,19] The environmental concerns over the use of toxic paints

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19 and biocides in the ocean [16,17] the antibiotic resistance of bacteria [20] and toxicity concerns with silver [18] suggest the need for non toxic and non kill solutions for these systems. The us e of engineered topography represents an antifouling and antibacterial strategy that is both non toxic and non killing. Engineered topography can be fabricated onto the surface of most non toxic polymeric materials without any change to the surface chemis try or addition of any chemicals. The non toxic, topographically modified surface would function simply as a physical deterrent of settlement of fouling organisms and a physical obstacle for the adequate formation of a bacterial biofilm without the need t o kill the targeted microorganisms. The potential for marine applications was evaluated with biological assays in collaboration with Dr. Maureen Callow and Professor James Callow at the University of Birmingham, UK, who specialize in ship fouling algae and Dr. Nick Aldred and Professor Anthony Clare of the University of Newcastle, UK, experts in the field of barnacle settlement and adhesion. A bioassay for biofilm formation was developed in collaboration with Ms. Edith Sampson and Dr. Patrick Antonelli of the Department of Otolaryngology at the University of Florida. Research Goal and Specific Aims The overall goal of my research was to identify feature dimensions of engineered topography to reduce the settlement (i.e., attachment) of zoospores of the alga Ulva and cyprids of the barnacle Balanus amphitrite and inhibit the formation of bacterial biofilms of Staphylococcus aureus Completion of this goal required the ability to fabricate high fidelity (> 95% feature replication), micrometer resolution surfac e topographies with precise control ( 0.5 m) of feature dimensions including feature width and length, spacing between features, and feature height. Precise control of these dimensional parameters allowed for the development of an empirical model based off experimental settlement data, known as the engineered roughness index (ERI), to relate the design parameters of engineered topography to an inhibitory

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20 settlement response. A theoretical model based on the differential bending moments among geometrical ly dissimilar surface features, known as engineered forces gradients, was developed to design novel antifouling surface topographies and predict settlement behavior. Novel engineered topographies were designed based on this model and the efficacy of this model was tested with settlement assays on the fabricated surfaces. Specific aims for my research were developed at the start of the project with special emphasis placed on my focus on the use of engineered topography for biofouling applications. Specific Aim 1 High Fidelity Fabrication of Engineered Topography The objective of this aim was to fabricate high fidelity (> 95% feature replication), micrometer scale structures on the surface of PDMSe. It is necessary to have precise control ( 0.5 m) of fe ature dimensions such as feature width, feature length, and spacing between features. Pattern fidelity was evaluated using light microscopy and scanning electron microscopy (SEM) of the finished, topographically modified PDMSe surface. Completion of this aim was essential to evaluate the antifouling efficacy of fabricated engineered topographies as the presence of a high ratio of pattern defects could influence bioassay results. Specific Aim 2 Reduce Algal Spore Settlement with Engineered Topography An appropriately designed engineered topography fabricated on the surface of PDMSe will significantly reduce algal spore settlement by at least 50% compared to a smooth PDMSe surface as evaluated by an independent settlement assay using the spores of the gree n alga Ulva Investigated engineered topographies were designed and fabricated by methods outlined in my research pursuant with specific aim 1. The Ulva assay protocol and collection of the raw data of spore settlement were performed by Dr. Maureen Callow and Dr. John Finlay at the University of Birmingham, UK. Effective engineered topographies were identified and an

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21 empirical model relating the significant intrinsic properties of engineered topography and Ulva settlement was developed based on regression analysis of experimental data. Specific Aim 3 Reduce Barnacle Cyprid Settlement with Engineered Topography An engineered topography designed specifically for a barnacle cyprid and fabricated on the surface of PDMSe will reduce cyprid settlement by at le ast 50% compared to a smooth PDMSe surface as evaluated by an independent settlement assay using cyprids of B. amphitrite Engineered topographical designs were fabricated using methods described in my research. The collection of the raw data of cyprid se ttlement was performed by Dr. Nick Aldred and Professor Anthony Clare at the University of Newcastle, UK. Barnacle specific engineered topographies significantly reduced cyprid settlement and a correlation between topographical aspect ratio and the settle ment behavior of both barnacle cyprids and the spores of Ulva was identified. Specific Aim 4 Inhibit Bacterial Biofilm Formation with Engineered Topography A biomaterial grade PDMSe material, surface modified with a tailored engineered topography will re duce the formation and growth of bacterial biofilm by at least 50% compared to a smooth PDMSe surface after 14 days of continuous exposure to Staphylococcus aureus An analysis technique was developed to quantify and statistically compare the amount bacter ial coverage on tested samples. The biofilms were cultured by Ms. Edith Sampson in Florida. An engineered topography originally designed for algal spores ( Ulva ) wa s shown to have a significant inhibitory effect on the formation and growth of biofilm from Staphylococcus aureus up to 21 days.

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22 Definition of Engineered Topography The terms used to describe a topographically modified surface are numerous. These terms in clude: roughness, topography, pattern, architecture, structure, and texture. They are often used e topography, nanoroughness, micro architecture, and micro scale surface structure. Although the prefixes define and narrow the size scale of the surface, it does not provide a distinction between the root words. A new term, developed by myself and collea gues in the Brennan Research Group, is distinguishable in both definition and design from the topographies, patterns, and structures presented in previous research and paten t art relating to the effect of surface characteristics on biological organisms and events. defined topography. The desired shape, arrangement, and dimensions of the feature s that comprise the topographical surface are defined before a fabrication technique is even considered. Therefore, an engineered topography is not the consequence of a surface modification or fabrication technique. Surface features are selected based on the consideration of a specific biological response such as the inhibition of microorganism settlement or the increase in attachment of proteins. When a particular organism is selected, an engineered topography becomes a species specific topography of wh ich the geometry, size, and arrangement of the features are tailored to elicit a specific response from the targeted organism. Engineered topographies contain features that are 3 dimensional structures that are based on a designed 2 dimensional pattern of geometries. The topographical features are restricted to

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23 the shape and arrangement of the 2 dimensional patterned geometries, but are not limited in the third dimension (e.g., height or depth). When certain dimensional restrictions are placed on engineer ed topography, some of these designed patterns and associated engineered topographies are patentable [21] A general numerical description of restricted, designed patterns is derived using sinusoidal waves to further extend the coverage of patentable art associated with these surface designs (Appendix A). designed size, shape, an d periodicity [22] mechanical features that exhibits randomness and polydispersity in terms of size, shape, and per iodicity [22] mechanical topography tailored for a specific biological response. Figure 1 1. Surface model considering the surface chemistry, surface topography, mechanical properties, and surface energy of a material and their influence on the settlement, adhesion, and growth of living organisms. Additional parameters associated with the surface topography, including feature dimensions and tortuosity, are included in the mo del as it i s the focus of my research

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24 CHAPTER 2 BACKGROUND Introduction The field of biomaterials is typically associated with materia ls used for medical devices, biomedical implants and recently, tissue engineering For most synthetic biomaterials, the surface is designed to minimize the bi ological surface interaction (e.g., minimize growth of bacteria/biofilm on catheters, reduce platelet adhesion and thrombosis on vascular grafts, and maintain clean pacemaker lead surfaces) and remain inert or integrate favorably with the surrounding tissu e. Other biomaterials used for tissue engineering and other specialized applications are designed to facilitate the adhesion of preferred cells that promote the growth of replacement tissue and function. T he study of marine biofouling seemingly unrelated to biomaterials, focuses on the biological response to materials placed within the marine environment Ideal marine s urfac es are antifouling and foul releasing An antifouling surface is defined as one that inhibits biological settlement and adhesion. A foul releasing surface refers to one with adhered organisms which release under operation hydrodynamic forces. The common thread that ties the field of biomaterials and the study of marine biofouling is the interaction between biological systems and mat erial surfaces. Solutions for the marine environment have typically relied on the use of toxic paints; however, with the recent environmental restrictions placed on toxic marine materials [16] non toxic biomaterials and concepts may be useful to explore f or marine applications. Equally, novel non toxic antifouling marine materials may have applications as non fouling biomedical surfaces (e.g., antithrombogenic, antibacterial). Thus, similarities should exist among the desired material surface properties and fundamental scientific principles used to develop antifouling/non fouling

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25 surfaces in both the physiological and marine environments. Both scientific disciplines are traditionally kept separate in books and journals, and, in most cases, are treated as completely different areas of science. Consequently, these similarities have yet to be reconsolidated and compared. The background information regarding marine biofouling and biofilm formation on biomaterials is written from the perspective to highlight these similarities. Additionally, specific biological responses to a common material in the marine and physiological environments may be similar. For example, zoospores of the most common marine fouling alga Ulva and bacteria of an infectious strain of S taphylococcus aure us both show gregarious settlement (i.e., settling in colonies) on the same elastomeric substrate material (Figure 2 1). Both organisms gain structural support by settling in groups rather than in isolation. Spores secrete an adhesive t o provide permanent attachment and stability to the colony [23] Similarly, a colony of bacteria will secrete an adhesive polysaccharide to provide connectivity and serve as the matrix for the formation of a bacterial biofilm [24] These biological respo nses can occur in hours, as in the case of the spores of algae, or days as in the case of bacteria. The manipulation of surface topography on non toxic surfaces has been investigated for both biomaterials and marine biofouling to control and/or direct the settlement, adhesion, and growth of cells and microorganisms. Common non toxic materials used in both applications are silicone elastomers based on polydimethylsiloxane (PDMS). A preferred and widely used type is a Silastic brand polydimethylsiloxane ela stomer (PDMSe) manufactured by the Dow Corning Corporation. The focus of my research is the design and fabrication of engineered topography on the surface of a Silastic T 2 PDMSe to decrease fouling by marine organisms and reduce bacterial biofilm formati on on biomaterials.

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26 Marine Biofouling Process of Fouling, Biofouling, and Marine Biofouling Fouling is the accumulation of matter on a surface. This is simplest and most general definition by which all other associated terms are derived. The word fouling carries a negative connotation which implies that this accumulation is undesirable. Fouling is generally divided into three categories which include inorganic fouling, organic fouling, and biofouling. Inorganic fouling, sometimes called mineral fouling o r scaling, involves the precipitation of inorganic salts such as CaCO 3 Ca(OH) 2 NaSO 4 MgSiO 3 and Li 2 SO 4 [25] This type of fouling occurs frequently on superheated surfaces like heat transfer equipment [26] but can also occur in hard water or aqueous solutions under moderate heating [25] The deposition of organic material on a surface is known as organic fouling. This may include proteins, polysaccharides, oils, and other organic molecules. Organic fouling occurs readily both in the physiological [1 ,2] and marine [3,17] environments and typically begins on the material surface within minutes of biological exposure. This short term accumulation of organic fouling components leads to the formation of a conditioning layer or film resulting in a conditi oned surface [27,28] This conditioning layer alters the surface properties of the material such as surface chemistry and wettability [29,30] Thus, this is an important process to consider when correlating the surface properties of a clean material to t he attachment of biological organisms. Biofouling, or biological fouling, is the undesirable settlement, attachment, or growth of living organisms and cells to surfaces. This phenomenon can have an adverse impact on a wide range of applications such as me dical devices and implants [31] dentistry and oral hygiene [32] water treatment facilities [33] heat transfer equipment [25] and naval military operations (e.g., ship hull biofouling) [34] Thus, specialized disciplines of research and science have de veloped

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27 around these areas to address specific applications, all sharing the common theme of controlling biofouling. The study of marine biofouling and its prevention is one these specialized disciplines. Marine b iofouling is the unwanted accumulation of living organisms on a n ocean submerged surface including bacteria, fungi, protozoa, algae and invertebrates The process of fouling on an unprotected surface in seawater happens in a series of distinct deposition, accumulation, and biological events. Org anic fouling, consisting mainly of proteins, proteoglycans, and polysaccharides, occurs within minutes and its continued accumulation leads to the formation of an organic conditioning film [35,36] Soon after, typically 1 to 2 hours, the process of marine biofouling begins with the attachment and colonization of marine bacteria [37] formation of a bacterial biofilm [38] The growing bacterial biofilm composed of dead and living cells and secreted slime, together with the conditioning film, define the primary film [37] The establishment of this primary film provides a suitable substrate for additional biofouling by diatoms, spores of macroalgae, protozoa, and the larvae of barnacles [36] Ship Hull Fouling Marine biofouling incurs large functional and monetary costs to both military and commercial marine vessels by increasing fuel costs due to drag, the cost of dry docking for cleaning, and los s of hull strength f rom biocorrosion [17,34] Tin and copper based paints applied to the ship hull are ef fective at reducing biofouling [39] ; however, an alternative is needed to address the environment restrictions on tin and to replace the current copper / co biocide formu lations [16,40,41] The ideal coating is non biocidal, but with both anti fouling (anti settlement) and foul releasing (low adhesion) properties [42,43]

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28 Specific to military operations, m arine biofouling adversely impacts the efficiency of naval forces. The rapid deployment of naval vessels requires that all surfaces be free from biological films, organisms or structures that can impede peak operational conditions. The United States Navy and the Of fice of Naval Research (ONR) ha ve been purs uing the development of non biocidal, antifouling and foul release marine coatings for over ten years. There has yet to be an effective solution that complies with the current and anticipated federal and international regulations regarding marine coatings [17] Combating the marine biofouling process is complicated by the diversity of marine organisms and environmental conditions. The diversity o f settlement behavio rs and range of bioadhesives employed by such a consortium of organ isms [44] c omplicates th e ability to control marine biofouling by non biocidal strategies and necessitates an experimental examination of each fouling organism for species specific fouli ng control [23,45] The succes sful design of antifouling and foul releasing coatings is conti ngent on developing a more complete understanding of the interaction between marine orga nisms and manmade surfaces. Non Toxic Coatings and Topography Polydim ethylsiloxane elastomers, or silicones, are one of the current standard materials in non toxic mari ne coatings. While these coatings are known to be foul releasing due to their combination of low surface energy and low modulus [46 49] they are not inherently antifouling and bioaccumulation will occur under static and low f low conditions [16] One app r oach to reduce organism settlement on PDMSe utilizes the application of topography to the surface. The approach is based on the hypothesis that a defined and periodic surface structure will exert a differential tension across the body (or sensing foot) o f the settling organism resulting in a therm odynamically unstable surface.

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29 Hills and Th omason have reported that the settlement of the barnacle larvae of Semibalanus balanoides correlates with changes in the Euclidean geometry of surface texture as opposed to a Fractal dimension [54] Berntsson et al. has shown that topography with profile heights of 30 45 m reduces settlement of the barnacle Balanus improvisus [55] In addition to barnacles, t he settlement and adhesion behavior of zoospores of the major ship fouling alga Ulva (syn. Enteromorpha ) has been studied in laboratory bioassays on topographically modified PDMSe. Re sults from Callow et al. indicate d that surface topography containing 5 m wide ridges (separated by 5, 10, or 20 m channels) or 5 m diameter pillars (5, 10, or 20 m spacing) significantly increased spore settlement for all channel separations and pillar spacing (5 m feature height) [10] For the topography containing 5 m wide ridges, spores primarily settled in the channels betwe en ridges with the majority of these spores against the ridge wall [10] Likewise, for the topography containing 5 m wide pillars, spores attached preferentially to the walls of the pillars, typically found in groups surrounding the feature [10] It was therefore hypothesized that narrower channels and pillar spacing (2 to 3 m) may be effective at reducing the settlement of the ~ 5 m in diameter spore [10] Nature can provide inspiration for the design of antifouling surface topographies. R esearchers have investi gated the an tifouling properties of the surface of foul free organisms that live in the marine environment [58] Sc ardino et al. have studied the antifouling qualities of the mussel Mytilus galloprovincialis and showed that it is due in part t o its surface microstructure [59,60] Baum et al. examined the nanoroughness of skin from the pilot whale Globicephala melas [61] It was hypothesized that t he nanotopography in conjunction with a cleaning prope rties [61]

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30 S urface topography is clearly an import ant modulator of surface coloniz ation by sessile marine organisms, including fouling species [50 54] More recently, antifouling strategies that exploit surface topography have typically been based on a c onsideration of the length scale of the settling body of each targeted fouling organism [10,55 57] This length scale can range over several orders of magnitude from bacteria (0.25 1 m) and green algal spores (5 7 m for Ulva ) up to tubeworm larvae (~ 20 0 m for Hydroides elegans ) and barnacle cypris larvae (cyprids) (~ 500 m for Balanus amphitrite ). Surface modification techniques have greatly improved in recent years with the ability to produce discrete, highly ordered su rface features on the nano/mic ro scale to more accurately define this biological surface interface. This allows for the isolation of specific dimensions of feature geometry that may be dominant factors for inhi biting settlement of organisms. The focus of my research is the identifica tion of these dimensions for selected fouling organisms with engineered topographies as evaluated by standard bioassays. Medical Bacterial Biofilms Bacterial biofilms are a major concern in the development of biomaterials, ultrafiltration systems, and unde rwater vessels. In the biomedical arena, bacterial colonization of surfaces compromises the effectiveness of implanted materials and can result in persistent infections [62,63] B iomaterial surfaces for tissue constructs and implants are subject to a com petition between bacterial adhesion and tissue integration, where an ideal surface would prevent the former while promoting the l atter [64] However, the chemical mechanical and physica l properties of tissue construct mate rials are inherently meant to e nhance all forms of biological attachment, making it equally likely that such a surface would submit to bacterial colonization Microorganisms colonize biomedical implants by developing biofilms, structured communities of microbial cells embedded in an ext racellular polymeric matrix that are adherent to the implant and/or the host tissues [63,65] Biofilms contribute to microorganism resistance to

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31 host defenses and antibiotic therapy [66,67] The current clinical means of managing biofilms has been preven tion. Prevention and Consequences Preventing biofilm associated infections has traditionally been through the use of antibacterial agents such as antibiotics and silver that can be delivered systemically or released directly from the biomaterial [31] Pha rm acokinetics and toxicity of some of these agents incorporated within coatings on biomaterials have limited the effectiveness of such therapies [31,68 70] Antibiotics, antibodies, and phagocytes can clear planktonic cells released by the biofilm, but th e sessile communities themselves are resistant to such agents [71,72] Antibiotic therapy resulting in incomplete eradication of biofilm has been linked with the emergence of antibiotic resistant bacteria, which may compromise the effectiveness of these a gents for even non biofilm mediated infections [73 76] Non Toxic Strategies and Topography Another strategy for preventing the development of biofilms has been to alter the biomaterial surface properties. Surface modification techniques to tailor the sur face energy via surface chemistry and surface topography have been developed to study the effects on biofilm formation [77,78] Bacterial adhesion has been investigated on surface topographies that range from random structures to ordered arrays. There ap pears to be a trend toward increased bacteria l coverage as the R a roughness values increased on electropolished stainless steel [79] Pseudomonas aeruginosa was less likely to foul hydrophilic, electrically neutral, smooth polymeric surfaces [80] Bacter ial adhesion was reduced on stainless steel surface microtopographies that were generated by a one directional polishing relative to smooth surfaces [81] The effects of a non random topography consisting of etched grooves of varying widths in silicon cou pons with P. aeruginosa and P. fluorescens showed that rates of attachment were

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32 independent of groove size and greatest on the downstream edges of grooves [82] More recently, a defined microtopography in the form of etched pits was shown to have the high est microbial retention on pit sizes similar to the size of the bacteria [83] Silastic brand and equivalent PDMSe materials have been popular for use in medical coatings and tubing including catheters (e.g., Foley and central venous), drains (e.g., kidney dialysis tubing), and shunts (e.g., brain fluid shunt) [84] PDMSe is the material of choice for mammary prostheses, testicular implants, and orthopedic finger joints [84] Device related infection risk due to the attachment and colonization of bacteria remains a significant problem for bladder catheters (10 30%), central venous catheters (3 8%), and penile implants (2 10%) [85] The application of engineered topography, as opposed to roughness, to the surface of existing PDMSe surfaces on these devices provides an interesting non toxic strategy to control bacterial colonization and biofilm formation that reduces infection and prolongs the device life. Figure 2 1. Microorganisms of bacteria and algae settling and colonizing on a silicone elastomer su rface. A) SEM image of the bacteria of Staphylococcus aureu s (~ 1 m in diameter) settling in groups on the surface of a PDMSe substrate. B) Fluorescent light micrograph of zoospores of the green alga Ulva settling in groups on the surface of PDMSe subst rate (Image courtesy of Dr. Maureen Callow). Spores in these images appear as red diameter due to the autofluorescence of chlorophyll

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33 CHAPTER 3 MATERIALS AND METHOD S Overview Engineered topographies are created on the surface of a polydimethylsiloxane elastomer (PDMSe) by replication of microfabricated silicon molds. Silicon molds are created using lithographic and dry etching technique s. First, a two dimensional pattern is designed, digitized and printed to a photomask using electron beam (e beam) lithography (Figure 3 1). Photolithographic techniques are then used to transfer the pattern present on the photomask to a photoresist coat ed silicon wafer (Figure 3 2). Next, the patterned silicon wafer is etched to a desired depth using deep reactive ion etching (Figure 3 3). As a final step, the remaining photoresist is removed from the wafer by an oxygen plasma etch. The processed sili con wafer contains three dimensional features etched within the wafer surface in the designed pattern. This wafer serves as a mold for topographical replication. The replication of topographical features to PDMSe is accomplished via an iterative casting method. The specific materials, equipment, and methods to complete this fabrication process are described. This fabrication process was used to produce engineered topographies specific for the control of marine biofouling and the reduction of medical bio film formation. Fabrication of Engineered Topography Pattern Design and Photomask Generation Pattern design limitations A pattern consists of two dimensional geometric shapes arrayed over a specified area. Patterns are not to contain any dimensions smalle r than 1 m. This dimensional limitation was defined based on the known resolution limit of ultra violet (UV) photolithography (~ 0.5 m under ideal conditions) [86] The arrayed pattern is not to exceed the boundaries of a 3 in. by

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34 3 in. square. This a rea restriction was based on the equipment capabilities at the time my research was being conducted. Silicon wafers larger than 4 in. in diameter could not be processed. The pattern must be able to be drawn and stored in a computer aided design (CAD) pro gram. This is necessary so that the pattern can be uploaded to computer controlled lithography equipment and printed on a photomask. Also, patterns must not be too complex or robust such that digitized computer files exceed 1 gigabyte as storage and uplo ading becomes a problem. Most geometric patterns that follow these guidelines can be successfully fabricated into three dimensional topographical features. Some examples of designed patterns are included in Figure 3 4. Photomask specifications The mask a ligners available at the time of my research included two Karl Suss brand mask aligners, models MJB3 (Figure 3 5A) and MA4 (Figure 3 5B). The specifications of a photomask are defined by the available mask holders on each piece of equipment. The MJB3 mod el mask aligner supported 3 in. by 3 in. to 4 in. by 4 in. square photomasks. The MA4 model mask aligner supported 4 in. by 4 in. to 5 in. by 5 in. square photomasks. Photomasks used in my research were designed as 4 in. by 4 in. squares so that they cou ld be used on either mask aligner. This specification must be considered when defining the layout of digitized pattern designs. Multiple pattern designs can be printed to a single photomask using strategic layout configurations (Figure 3 6). Pattern desi gn digitization with AutoCAD A student version of AutoCAD 2004 software package was used to generate and digitize designed patterns. AutoCAD was also used to define the layout of multiple patterns for photomask generation. Once complete, the AutoCAD draw ing was stored in the proper file

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35 format to be transferred to a computer controlled e beam lithography system to be directly printed to a photomask. To begin, the default drawing units in AutoCAD are set to micrometers. Drawings are produced following the photomask specifications and pattern limitations. The layout of the photomask, including the shape of the photomask and areas where patterns will be arrayed, was drawn directly in the CAD design (Figure 3 6). This drawn layout was generated in a separat e drawing layer than the designed pattern. Also, if multiple patterns were placed on the same photomask, each pattern was contained in a separate drawing layer. Once complete, the CAD drawing of the photomask containing arrayed patterns in separate layer s was saved as a .dwg file type. Photomask fabrication The CAD drawing (.dwg file format) of a completed photomask with arrayed patterns was electronically transferred to Advance Reproductions Corporation in North Andover, MA, to be printed to a photomask using their in house e beam lithography equipment. The fabricated photomask was made of quartz (4 in. by 4 in. and 0.09 in. thick) with a thin layer of chrome (< 100 nm) on one side of the quartz surface. The designed pattern is etched within this chrome layer such that the features of the pattern are clear (no chrome) surrounded by a dark background (chrome). Process of Photolithography The process of photolithography involves the transfer of the pattern present on the fabricated photomask to the surface of a silicon wafer. Prime silicon wafers, 4 in. diameter, were purchased from University Water in South Boston, MA. The entire photolithography process was performed using equipment and materials available at the Nano Fabrication Facility

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36 (previously th e Micro electronics Laboratory) located in Benton Hall at the University of Florida in a 1000 class clean room. Pre treatment of silicon wafers Prime silicon wafers were placed in an oven at 150 C for at least 15 minutes to completely dry the wafers. Onc e cooled, hexamethyldisilazane (HMDS) was vapor deposited on each wafer for 5 minutes. HMDS promotes the adhesion of photoresist to the silicon surface. Coating photoresist on silicon wafers Photoresist (Microposit S1813) was coated on silicon wafers by a spin coating process. A Headway spinner model PWM32, operating at 4000 RPM, was used to complete this process. A silicon wafer was placed on the vacuum chuck and a small amount of photoresist (~ 10 ml), enough to cover at least half the wafer, was depos ited in the center of wafer. The spin coater was then started and the wafer was spun at 4000 RPM for 40 seconds. Pre exposure bake Photoresist coated silicon wafers are then placed in an oven at 90 C for 30 minutes to drive out all the solvent in the pho toresist. This produces a completely dry photoresist layer in preparation for ultraviolet exposure. This pre exposure bake is sometimes referred to as a pre bake or softbake. The time and temperature of the pre exposure bake are specified by the photore sist manufacturer. Photomask alignment and photoresist exposure Patterns contained on the photomask are transferred to the photoresist coated silicon wafer using a mask aligner. Karl Suss mask aligners, models MJB3 and MA4, were used for pattern transfer via photoresist exposure. The same procedure was used whether using the MJB3 or MA4. The photomask was attached to the mask holder, placed above the loading stage, and aligned over the center of the wafer chuck. The silicon wafer was placed on the wafer chuck and

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37 loaded into the mask aligner beneath the mask holder containing the photomask. The wafer chuck was raised so that the silicon wafer makes direct contact with the photomask. The photoresist was then exposed through the photomask using ultraviol et light (405 nm wavelength) for 6 to 7 seconds (Figure 3 2). The photoresist was exposed only in the areas of the pattern present on the photomask. Development of transferred pattern The exposed silicon wafer was developed by immersion in developer solut ion to rinse away exposed areas of the photoresist. AZ MIF312 developer solution manufactured by Clariant was used for silicon wafers coated with Microposit S1813 photoresist. Exposed wafers were completely immersed and agitated in developer solution for 40 seconds, removed, and immediately immersed and agitated in de ionized water for 60 seconds. The wafer was then dried with compressed nitrogen. Hard bake The developed and dried silicon wafer was then placed in an oven at 120 for 30 minutes. This pro cess is typically referred to as a hard bake. The hard bake enhances the adhesion and increases the etch resistance of the remaining photoresist on the silicon wafer. Etching of Patterned Silicon Wafer Patterned silicon wafers are etched to a desired feat ure depth using deep reactive ion etching. Deep reactive ion etching was accomplished using a Multiplex reactive ion etcher model SP001 manufactured by Surface Technologies System (STS, Figure 3 7). This system ws for the creation of high aspect ratio features. The process conditions for an isotropic etch are listed in Table 3 1. The etch depth was controlled by the total time of the process. Once etched, photoresist was cleaned from the wafer with an oxygen p lasma etch on the STS system using process conditions outlined in Table 3 2. The

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38 cleaned silicon wafer contained features etched within the surface and served as a negative mold for topographical replication in PDMSe (Figure 3 8). Replication of Topograph ically Modified Silicon Wafers Hexamethyldisilazane was vapor deposited on the processed silicon in order to prevent adhesion to PDMSe. A platinum catalyzed PDMSe, Silastic T 2 (Dow Corning Corporation), was used as the base material for topographical mod ification. The elastomer was prepared, as specified by the manufacturer, by mixing ten parts of the resin and one part of the curing agent by weight for 5 minutes. The mixture was degassed under vacuum (95 102 kPa) for 30 minutes, removed, and poured ove r the processed silicon wafer. After curing for 24 hours, the PDMSe film was carefully removed from the silicon wafer. The resultant topography on the PDMSe surface contained features projecting from the surface at heights respective of the etch depth (F igure 3 9). Pattern Fidelity Evaluation of Engineered Topography The evaluation of pattern fidelity was assessed on the PDMSe engineered topographical surface replicated from the silicon master. Scanning electron microscopy (SEM) was used to evaluate shor t range pattern fidelity (~ 40 m by 40 m field of view) and to inspect individual features to define approximate dimensions such as feature width, length, and spacing. Feature height was visualized with SEM images of cross sectional samples. SEM was co nducted using a JOEL JSM 6400 scanning electron microscope, manufactured by JOEL USA, located in the Major Analytical Instrumentation Center (MAIC). PDMSe samples to be imaged were mounted on 10 mm diameter aluminum stubs (5 mm thickness) using double sid ed tape. Mounted PDMSe samples were then sputter coated with Au/Pd for 1 min by a MAIC technician before SEM imaging. Long range pattern fidelity (~ 350 m by 350 m field of view) was assessed using light microscopy. A Zeiss Axioplan 2 light microscope was used for imaging.

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39 SEM was useful when new patterns were first being fabricated or process modifications were investigated. Gross defects such as flopped features (Figure 3 10), overexposed features (Figure 3 11), and missing features (Figure 3 12) co uld be identified quickly and modifications to the process were made. When a random sampling of SEM images from a particular engineered topography on the surface of PDMSe using specified process parameters showed no evidence of gross defects (Figure 3 13) iterative casting was used to create an inventory of topographically modified films. Pattern fidelity (%) was defined as (1 defective features / total features) 100 for a given field of view. Light microscopy was used as a final pattern fidelity ass essment on engineered topography samples prepared for biological assays. Five random areas on each sample were imaged at 400X magnification. Samples were rejected and not tested if any of the five random areas had a pattern fidelity value lower than 97% (Figure 3 14). Most of the samples that passed the final pattern fidelity inspection had pattern fidelity values of > 99% (Figure 3 15).

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40 Figure 3 1. Process of e beam lithography used to print a photomask from a CAD drawing of a designed pattern. A) Electron beam source. B) Electron beam used to print a pattern to a photomask. C) Photomask with printed features. Figure 3 2. Photolithography process used to transfer the pattern on a photomask to a photoresist coated silicon wafer. A) Ultraviolet light source. B) Photomask containing a pattern of clear features on a black background. C) Photoresist coated silicon wafer.

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41 Figure 3 3. Etch processing of patterned s ilicon wafers using deep reactive ion etching. A) Cross section of a patterned silicon wafer with bare silicon features surrounded by photoresist. Silicon covered in photoresist is not etched during the process. B) Scanning electron micrograph of the c ross section of an etched silicon wafer showing features within the silicon surface. Figure 3 4. Designed patterns drawn in AutoCAD. A) 10 m equilateral triangles surrounded by 2 m (diameter) circles spaced by 2 m. B) 20 m (base to top) hexagons spaced by 2 m. C) Sharklet AF design composed of 2 m wide ribs of varying lengths including 4, 8, 12, and 16 m. Ribs are spaced by 2 m in all directions.

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42 Figure 3 5. Karl Suss mask aligners used to transfer designed patterns from a photomask to a photoresist coated wafer. A) Model MJB3. B) Model MA4. Figure 3 6. A 4 in. by 4 in. photomask created in AutoCAD. The six smaller squares represent the area of coverage of six individual designed patterns. With this layout, six different pattern s can be transferred directly to a single silicon wafer.

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43 Figure 3 7. Patterned silicon wafer being loaded into a Surface Technology Systems (STS) Multiplex reactive ion etcher. The machine is located in the Micro electronics Laboratory in Benton Hall There are plans to move this piece of equipment to the University of Florida Nano F abrication Facility which was being constructe d at the time my research was conducted Table 3 1. Process conditions used to etch patterned silicon wafers. Process para meters Passivation Etching C 4 F 8 85 sccm 0 sccm SF 6 0 sccm 130 sccm RF power at stage 0 W 12 W RF power from coil 600 W 600 W Cycle time 5.0 s 7.0 s Delay time 0.5 s 0.5 s sccm: standard cubic centimeter per minute. Table 3 2. Process conditions used for oxygen plasma etch. Process parameters Plasma etch O 2 45 sccm RF power at stage 12 W RF power from coil 800 W Total time 5 min

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44 Figure 3 8. SEM image s of a silicon wafer etched for a total time of 55 seconds using optimized process parameters A) Tilted SEM image at 2000X magnification. B) Tilted SEM image at 4000X magnification. Figure 3 9. SEM image s of the surface of PDMSe replicated from the etched sili con wafer pictured in Figure 3 8 A) Tilted SEM images at 2000X magnification. B) Tilted SEM image at 4000X magnification.

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45 Figure 3 10. SEM image of an engineered topography on the surface of PDMSe containing flopped features. Flopped features are a result of too high of an aspect rati o (feature height / feature width). Figure 3 11. SEM images of a set of engineered topographies on the surface of PDMSe produced from the same pattern showing the effec t of overexposed features. A) 6 m diameter pillars with a 2 m diameter hole in th e center B) Same pattern in subpart A that was overexposed during photolithography. The overexposure results in an increase in the exposed feature diameter in all directions causing wider features and a washing out of the inner diameter hole

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46 Figure 3 12. SEM images of engineered topographies on the surface of PDMSe with missing features. A) Arrow indicates a single missing feature on the surface. The surface was tilted at 35 when imaged. B) Multiple missing features on the surface indicated by t he dark, irregular spots across the viewable area. This is an example of pattern fidelity of < 70%. Figure 3 13. SEM images of engineered topographies on the surface of PDMSe with no defects across the viewable area. These are representative images o f engineered topographies with pattern fidelity of > 99% for the relative field of view

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47 Figure 3 14. Light micrograph image of a rejected sample of an engineered topography on the surface of PDMSe taken during the final pattern fidelity assessment bef ore biological testing. The defects appear as dark spots in the image indicating that those features had flopped over during fabrication and sample preparation. This is an example of an engineered topography of pattern fidelity of < 97%. Figure 3 15. Light micrograph image of a high fidelity sample (> 99%) of an engineered topography on the surface of PDMSe taken during the final pattern fidelity assessment before biological testing. Unlike Figure 3 14, no defects are visible within the field of view

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48 CHAPTER 4 1 BIORESPONSE OF ALGAL SPORES TO ENGINEERED TOPOGRAPHY AND DEVELOPMENT OF ENGIN EERED ROUGHNESS INDE X Introduction Biofouling is the accumulation of living organisms on a surface including bacteria, fungi, protozoa, algae and invertebrates. The di versity o f settlement behavio rs and range of bioadhesives employed by such a consortium of organ isms [44] complicates the ability to combat marine biofouling by non biocidal strategies and necessitates an experimental examination of each fouling organism f or species specific fouli ng control [23,45] Marine biofouling incurs large functional and monetary costs to both military and commercial vessels by increasing fuel costs due to drag, the cost of dry docking for cleaning, and loss of hull strength conseque nt on biocorrosion. Tin and copper based paints are ef fective at reducing biofouling [39] ; however, an alternative is needed to address the environment restrictions on tin and replace the current copper / co biocide formulations [16,40,41] The ideal coa ting is non biocidal, but with both anti fouling (anti settlement) and foul releasing (low adhesion) properties [42,43] Polydimethylsiloxane elastomer (PDMSe), or silicone, comprise s the major type of material currently marketed as non toxic marine coating s. While these coatings ar e known to release hard fouling [46 49] and soft fouling [88] under suitable hydrodynamic conditions, they are not inherently antifouling Bioaccumulation will occur under static and low f low conditions [16] My approach to redu ce organism settlement on PDMSe utilizes engineered surface topographies. The settlement and adhesion behavior of zoospores of the major ship fouling alga 1 Portions of this chapter were previously published by the author in the journal Biofouling [87] and are reprinted here with full permission f rom Taylor & Francis.

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49 Ulva (syn. Enteromorpha ) has been studied in laboratory bioassays on topographically modified PDMSe. Topographic surface f eatures that enhance [10] and reduce [89] settlement of Ulva spores relative to a smooth PDMSe surface have been identified Re sults from Callow et al. indicate d that surface topography containing 5 m wide ridges (separated by 5, 10 or 20 m channels) or 5 m diameter pillars (5, 10, or 20 m spacing) significantly increased spore settlement for all channel separations and pillar spacing (5 m feature height) [10] For the topography containing 5 m wide ridges, spores primarily se ttled in the channels between ridges with the majority of these spores against the ridge wall [10] Likewise, for the topography containing 5 m wide pillars, spores attached preferentially to the walls of the pillars, typically found in groups surroundin g the feature [10] It was therefore hypothesized that narrower channels and pillar spacing (2 to 3 m) may be effective at reducing the settlement of the approximately 5 m in diameter spore [10] Myself and colleagues presented a biomimetically inspired surface topography (Sharklet AF) containing 2 m wide rectangular like (ribs) periodic features (4, 8, 12, and 16 m in length) spaced at 2 m that reduced Ulva settlement by 86% [89] This was the first example of a topographic inhibition of settlement of marine alga It was unclear at this point which feature aspects (i.e., size, spacing, and geometry) of the Sharklet AF topography, all of which influence the calculated roughness factor, were important for the dramatic inhibitory effects. However, we suggested that feature size and spacing at or above 5 m should be avoided to ach ieve this inhibitory response [89] For my research, I examined the effect of topographic feature size, geometry, and roughness on settlement of Ulva zoospores using engineer ed topographies. Experimental Design Surface topographies containing 2 m diameter circular pillars (2 m spacing) and 2 m wide ridges separated by 2 m wide channels were tested for Ulva settlement against the

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50 Sharklet AF to evaluate the inhibitory effec ts of other periodic topographies with a 2 m feature resolution. I expected that if feature width and spacing were the dominant factors for reducing Ulva settlement, each of these surfaces would exhibit an equivalent inhibitory effect. Differences in se ttlement would then be attributed to the feature geometry or roughness properties of these topographies. I also included a surface topography that contained both 2 m diameter circular pillars and 10 m equilateral triangles. This surface represented a mu lti feature topography containing differing geometries and sizes. Features were arrayed so that spacing between all features was approximately 2 m. This spacing was designed to inhibit spores from settling in the flat areas between features. It may be expected that the top surface of the 10 m equilateral triangle would provide a large enough surface area for the spore body to settle. Based on this assumption, this surface topography would be predicted to be less effective than a complete array of 2 m pillars. Engineered Roughness Index The roughness of engineered surface topography was characterized using a newly described dimensionless ratio called the engineered roughness index (ERI, Eq. 4 1). ( 4 1) The ERI encompasses three variables asso ciated with the size, geometry, and spatial arrangement [90] depressed surface fraction (f D ), and degree of freedom for movement (df). ctual surface area to the projected planar surface area [90] The actual surface area includes areas associated with feature tops, feature walls, and depressed areas between features [90] The projected planar surface area includes just the feature tops and depressions [90]

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51 The depressed surface fraction (f D ) is the ratio of the recessed surface area between protruded features and the projected planar surface area. This depressed surface fraction term is S 1 where S is the surface solid fraction of a rough surface [91,92] 1 is the solid liquid interface term of the Cassie Baxter relationship for wetting on a rough surface [93] The degree of freedom for movement (df) relates to the tortuosity of the surface and refe rs to the ability of an Ulva spore to follow recesses (i.e., grooves) between features within the topographical surface. It has been previously observed that Ulva spores preferentially settle along the recessed areas within the surface topography. If the recesses form a continuous and intersecting grid, movement in both the x and y coordinates is permitted and the degree of freedom is 2. Alternatively, if the grooves are individually isolated (e.g., as in channel topographies) then movement is only allow ed in one coordinate direction and the degree of freedom is 1. The ERI was developed to provide a more comprehensive quantitative description of alone does not a dequately capture the tortuosity of the engineered topographies studied (see r values in Table 4 2). The values included in the ERI were based on the preferential settle tendencies of Ulva spores and the hypothesis that increasing the tortuosity of surfac e topography will make the surface less favorable for settlement. As such, larger ERI values should be indicative of reduced settlement. Materials and Methods Materials Silastic T 2 (Dow Corning Corporation), a platinum catalyzed PDMSe, was used as the ba se material for topographical modification. The elastomer was prepared by hand mixing

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52 (5 minutes) ten parts of the resin and one part of the curing agent by weight. The mixture was degassed under vacuum (95 102 kPa) for 30 minutes and allowed to cure for 24 hours at ~ 22C. Pattern Designs Pattern designs included geometric features of 2 m wide ribs of various lengths (4, 8, 12, and 16 m), 2 m diameter circular pillars, 2 m wide continuous ridges, and 10 m equilateral triangles. The 2 m ribs of vari ous lengths were combined centered and in parallel at a feature spacing of 2 m. The features were aligned in the following order as indicated by feature length (m): 4, 8, 12, 16, 12, 8, and 4. This combination of features formed a diamond and was the repeat unit for the arrayed pattern. The spacing between each diamond unit was 2 m. Similar to that of the skin of a shark in terms of feature arrangement [94] this pattern was designed such that no single feature is neighbored by a feature similar to itself (Figure 4 1A). This unique pattern has been named Sharklet AF [89] Patterns of 2 m pillars and 2 m ridges were designed at an analogous feature spacing of 2 m. The pillars were hexagonally packed so that the distance between any two adjacent p illars was 2 m (Figure 4 1C). Ridges were continuous in length and spaced by 2 m channels (Figure 4 1D). A multi feature pattern was designed by combining 10 m triangles and 2 m pillars (Figure 4 1B). Pillars were arranged in the same hexagonal packi ng order as in the uniform structure. At periodic intervals, a 10 m equilateral triangle replaced a set of six 2 m pillars forming the outline of a 10 m triangle. Thus, this design maintained a 2 m feature spacing between each edge of the triangle an d pillars.

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53 Pattern Fabrication The pattern designs were transferred to photoresist coated silicon wafers. Patterned silicon wafers were reactive ion etched, utilizing the Bosch process, to a depth of approximately 3 m creating a topographical negative. Wafers were then stripped of photoresist and cleaned with an oxygen plasma etch. Hexamethyldisilazane was vapor deposited on the processed silicon wafers to methylate the surfaces in order to prevent adhesion to PDMSe. Topographical Replication Topographi cal surfaces were transferred to PDMSe from replication of the patterned silicon wafers. The resultant topographies contain features projecting from the surface at a height of approximately 3 m. Pattern fidelity was evaluated with light (Figure 4 2B) an d scanning electron (Figure 4 1) microscopy. Sample P reparation for Ulva Settlement A ssay Ulva spore settlement assays were conducted with 76 mm by 25 mm glass microscope slides coated with smooth and topographically modified PDMSe surfaces. Glass slides coated with PDMSe topographies were fabricated using a two step curing process as prev iously described [89] The resultant slide (~ 1 mm thickness ) contained an adhered PDMSe film with a 25 mm by 25 mm area containing topography bordered on both sides by 25 mm by 25 mm smooth (n o topography) areas (Figure 4 2A). Ulva Zoospore Settlement Assay Three replicates of each topographically modified PDMSe sample, permanently adhered to glass microscope slides, were shipped to the University of Birmingham, United K ingdom, to be evaluated for settlement of Ulva spores The Ulva assay and collection of the raw data of spore settlement were performed by Dr. Maureen Callow and Dr. John Finlay at the University of Birmingham. Topographies included the Sharklet AF, 2 m diameter circular pillars, 2 m wide

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54 ridges, and a multi feature topography containing 10 m equilateral triangles and 2 m diameter circular pillars. A u niformly smooth PDMSe sample was included in the assay and served as a control for direct comparison Fertile plants of Ulva linza were collected from Wembury beach, UK ( Ulva z oospores were released and prepared for attachment experiments as documented p reviously [23] Topographical samples were pre soaked in n ano pure water for severa l days prior to the a ssay in order for the surfaces to fully wet. Sample s were transferred to artificial seawat er (Tropic Marin ) for 1 hour prior to exp erimentation without exposure to air. Samples were then rapidly transferred to assay dishes to minimiz e any dewetting of the t opographical areas. Ten m l of spore suspension (adjusted to 2 x 10 6 ml 1 ) w ere added to each dish and placed in darkness for 60 minutes. The slides were then rinsed and fixed with 2% glutaraldehyde in artificial seawater as desc ri bed in Callow et al. [23] Spore counts were quantified using a Zeiss epifluorescence microscope attached to a Zeiss Kontron 3000 image analysis system [10] Thirty images and counts we re obtained from each of three replicates at 1 mm intervals along both the vertical (15) and hori zontal (15) axes of the slide. Statistical Methods Spore density was reported as the mean number of settled spores per mm 2 from 30 counts on each of three replicate slides standard error (n = 3). Statistical differences betwee n surfaces were evaluated using a nested analysis of variance (ANOVA) followed by the Student Newman Keuls (SNK) test for multiple comparisons [95] Replicate slides (3) of each surface (5) were treated as a nested variable within each surface.

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55 Correlatin g Spore Settlement to Engineered Roughness Index The mean spore density measured for each of the studied PDMSe surfaces was plotted against the calculated engineered roughness index to determine if any correlations existed. It must be noted that these ERI values are for a fixed feature spacing of 2 m and depth of 3 m. Results Spores were calculated to settle at a mean density of 671 66 spores/mm 2 on the smooth PDMSe surface. All topographies showed a statistically significant reduction in spore densit y relative to this smooth surface as evaluated by ANOVA analysis followed by the SNK multiple comparison test (Table 4 1, Figure 4 3). A lower mean spore density was measured on the triangles/pillars, 279 66 spores/mm 2 compared to both the pillars, 430 81 spores/mm 2 and ridges, 460 54 spores/mm 2 The Sharklet AF topography had the lowest spore density, 152 32 spores/mm 2 compared to all other surfaces. An examination of light micrographs of settled spores (Figure 4 4) indicated some general trends regarding settlement preference on topographic features. For 2 m wide ridges, the majority of the settled spores were bridged between the top edges of neighboring ridges. A few smaller spores were found squeezed within the 2 m wide channels between ri dges. Spores remained atop the hexagonal packed 2 m diameter pillars. No settled spores were observed on flat areas between pillars. For the multi feature topography containing both 10 m triangles and 2 m pillars, spores completely avoided settling o n the flat top surface of the triangle. Most spores appeared to have settled on top of a pillar while leaning against the edge of the triangle feature. As previously observed with the Sharklet AF [89] the majority of settled spores were located around t he edges of the diamond repeat unit. A lower number of spores were observed to be bridged between the features within the repeat unit.

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56 The mean spore density measured on each tested PDMSe surface was plotted against the calculated engineered roughness ind ex (Table 4 2, Figure 4 5). A correlation was observed and a linear regression model was fit to the data. A fairly strong (R 2 = 0.71, p < 0.001) inverse linear relationship existed between mean spore density and ERI (Eq. 4 2). (4 2) The Sharklet AF had the highest ERI (8.6) and lowest mean spore density. Following the trend, the triangles/pillars topography had the second highest ERI (7.0) and the second lowest mean spore density. Both the uniform ridges and pillars topographies had lower ERI valu es (5.0 and 6.1 respectively) and higher mean spore densities than both the Sharklet AF and triangles/pillars. There were no statistical differences in the mean spore densities of uniform ridges and pillars topographies. Since all topographies were desig ned at a feature spacing of 2 m and a depth of 3 m, it must be noted that this relationship applied only for these fixed dimensions. Discussion In common with the dispersal stages of many other sessile fouling organ isms, motile spores (zoospores) of Ulv a (syn. Enteromorpha ) need to quickly locate and attach to a surface in order to complete their life history. Ulva s pores are not however, inert particles that settle on any surface irrespective of its properties. These s a behavior in relation to surface pro perties such as wettability [96] chemistry [14,97] presence of other organisms [98,99] and topography [89] Once the Ulva spore has detected a suitable asting only a few minutes, involving the loss of the four flagella, the secretion of an adhesive which anchors the settled spore to the substratum and the initiation of production of a new cell wall [23,100]

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57 The effect of non network forming PDMS oils inc orporated within the particular PDMSe material used for my study on Ulva spore settlement has been examined [101] The addition of PDMS oils to smooth PDMSe substrates had no effect on the settlement behavior of Ulva spores. In relation to surface topogra phy, myself and colleagues have previously reported that spore settlement density was a function of the channel spacing (5, 10, and 20 m) between 5 m wide and tall ridges on PDMSe surfaces [10,101] For all topographies studied, the number of spores tha t settled was increased relative to a smooth PDMSe surface. For all ridge separations, settlement occurred almost entirely in channels between ridges, preferentially against the ridge walls, as opposed to the tops of the ridges. This previous observation coincides with results equilateral triangles. Thus, it is suggested that spores respond differently to a protruded feature area versus a recessed area betw een protruded features. Although it has been previously suggested that feature sizes at or above 5 m should be avoided to deter settlement, this needs to now be clarified to distinguish between features that either: a) create a protruded area (e.g., rid ges) or b) create a recessed area between protruded features (e.g., channels). The analyses of results presented here and previously, confirm that the recessed area between protruded features must remain below 5 m; however, features that create a protrud ed area greater than 5 m (e.g., 10 m equilateral triangles) were shown to be antifouling features. A surface energy model, correlating wettability to bioadhesion, was developed to characterize previously studied settlement enhancing topographies and to d esign new and potentially successful antifouli ng coatings [89] The application of this model in conjunction with the examination of natural topographic surfaces [94,102] subsequently led to the design concepts of the Sharklet AF and the multi feature tri angles/pillars topography. The model

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58 predicted these surfaces to be antifouling versus a smooth substrate and the results presented here have confirmed such a trend. The engineered roughness index was developed as an extension of this model to further ch aracterize and distinguish unique engineered topographies. It must be noted that the correlations identified only applied when the spacing between topographical features was designed at 2 m. Surfaces of an equivalent roughness factors to the Sharklet AF are being designed, fabricated, and tested for spore settlement to further evaluate the effectiveness of this model. Periodic topographies of hexagonally packed pillars and continuous ridges were designed at the same 2 m lateral resolution (i.e., feature width and spacing) as the Sharklet AF. The Sharklet AF had statistically significant lower spore settlement (approximately 65% less) than both the pillars and ridges. A correlation was detected between the mean spore density and the engineered roughness indices associated with these surfaces. Since feature width and spacing were the same for all these topographies, differences in ERI values were associated only with differences in feature geometry and tortuosity. This indicated that the geometr ic shape and arrangement of the individual features of Sharklet AF was critical because it enhanced anti settlement effectiveness over topogr aphies of equivalent dimensions. Not all topographically modified or roug hened surfaces have anti settlement properties for Ulva spores. Spore settlement results on topographies presented here and previously have indicated that a critical interacti on must be achieved between individual topographical features and the spore for the entire surface to be an effective inhibitory s urface. Although trends with ERI values and spore settlement have been ascertained, it was only after topographic surfaces were designed at a feature spacing of 2 m. This indicated that an interaction exists between

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59 roughness measures and feature spacin g that must be considered when designing topographic surfaces. My proposed criteria to identify topographical feature limits of a surface are as follows: 1) the Ulva spore must be forced to remain on top of the protruded topographical features and not be able to settle between features (feature spacing), 2 ) the Ulva spore must not be able to stabilize its entire mass on one single feature (feature size), and 3) if the Ulva spore is bridged between two topographical features, the spore must not be able to c ontact the floor between features i.e., the number of attachment points must be minimized [57] It was only after surface topographies were designed around these feature limits that the trend between spore settlement and ERI values was identified.

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60 Fi gure 4 1. SEM images of engineered topographies on a PDMSe surface. A) 2 m ribs of lengths 4, 8, 12, and 16 m com bined to create the Sharklet AF design. B) 10 m equilateral triangles combined with 2 m diameter circular pillars. C) Hexagonally packed 2 m diameter circular pillars. D) 2 m wide ridges separated by 2 m wide channels.

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61 Figure 4 2. Photograph and light micrograph of sample configuration used for Ulva settlement assays. A) P DM Se film containing an engineered topography ad hered to th e center of a microscope slide. B) Light micrograph image showing the surface of the topogr aphically modified area (Sharklet AF) of the slide. The long axes of the ribs of this particular engineered topography are aligned with the long axis of the micros cope slide.

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62 Figure 4 3. Ulva spore settlement data on PDMSe surfaces represented as mean spore density (spores/mm 2 ) standard error (n = 3). All surfaces were tested with a single batch of spores allowing direct comparisons between samples. PDMSe su rfaces included: uniformly smooth surface (Smooth), 2 m wide ridges spaced by 2 m wide channels (Ridges), hexagonally packed 2 m diameter circular pillars (Pillars), 10 m equilateral triangles combined with 2 m diameter circular pillars (Triangles/Pi llars), and Sharklet AF. Solid horizontal bars represent statistically different groups (ANOVA p = 0.001, SNK test p < 0.05). Table 4 1. df MS F p Surface 4 34 60891 12.93 0.001 Slide(Surface) 10 267678 48.04 <0.001 Error 435 5572 (Figure 4 3). Three replicates slides are nested within each surface. Thirty cou nts are obtained for each slide.

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63 Figure 4 4. Representative light micrographs, obtained by a mixture of epifluorescence and transmitted light, of spores settled on PDMSe surfaces. SM) Smooth. R) 2 m wide ridges separated by 2 m wide channels. P) H exagonally packed 2 m diameter circular pillars. T) 10 m equilateral triangles combined with 2 m diameter cir cular pillars. SK) Sharklet AF Spores in these images appear as red spots ~ 5 m diameter due to the autofluorescence of chlorophyll.

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64 Table 4 2. Calculated engineered roughness index (ERI) values for the studied topographical surfaces fabricated in PDMSe. Feature geometry Engineered roughness index Depth Spacing Width r df f D ERI (r df)/f D Ridges 3 2 2 2.5 1 0.50 5 Pillars 3 2 2 2.3 6 2 0.77 6.1 Triangles/Pillars 3 2 2 or 10 2.23 2 0.63 7 Sharklet AF 3 2 2 2.5 2 0.58 8.6 Smooth n/a n/a n/a 1 2 1 2 Figure 4 5. Correlation between Ulva spore settlement and engineered roughness index (ERI) at a fixed feature spacing of 2 m. Plotted is the calculated ERI for the tested PDMSe surfaces against the experimental mean spore density standard error (n = 3). The tested PDMSe surfaces included: Smooth ( ), Ridges ( ), Pillars ( ), Triangles/Pillars ( ), and Sharklet AF ( ). The eq uation of the linear regression (R 2 = 0.71) is as follows: Spore density (spores/mm 2 ) = 847 78.1 (ERI).

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65 CHAPTER 5 2 BIORESPONSE OF BARNA CLE CYPRIDS TO ENGIN EERED TOPOGRAPHY AND CORRELATION TO ALGAL SPORES Introduction Surface topography is considered an import ant modulator of surface coloniz ation by sessile marine organisms, including fouling species [51 54] More recently, antifouling strategies that exploit surface topography have typically been based on a consideration of the length scale of the settling body of each targeted fouling organism [10,55 57] This length scale can range over several orders of magnitude from bacteria (0.25 1 m) and green algal spores (5 7 m for Ulva ) up to tubeworm larvae (~ 200 m for Hydroides elegans ) and barnacle cypris larvae (cyprids) (~ 500 m for Balanus amphitrite ). Surface modification techniques have greatly improv ed in recent years with the ability to produce discrete, highly ordered surface features on the micro/nano scale to more accurately define this biological surface interface. This allows for the isolation of specific dimensions of feature geometry that may be dominant factors for inhibiting settlement of organisms. However, dimensional differences between target species makes combating marine fouling a challenging task, and necessitates individual examination of each fouling organism for species specific f ouling control. Besides the overall dimensions of target organisms, the scale and function of their settlement sensory organs, which in most cases are used to probe, navigate, and sense a surface, must also be taken into consideration ( e.g., barnacle cypr id antennules) I investigated the effect the topographical aspect ratio (feature height / feature width) of engineered topographies on the settlement of cyprids of Balanus amphitrite and spores of Ulva linza Topographical surfaces of fixed feature width and spacing were produced at various 2 Portions of this chapter were previously published by the author in the journal Biofouling [103] and a re reprinted here with full permission from Taylor & Francis.

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66 feature heights to study antifouling effectiveness with regard to aspect ratio. Trends, identified in the analysis, were compared across species ( B. amphitrite and Ulva ) to test for the existence of associated correla tions. Cyprids are the final larval stage of barnacles and are highly specialized for their role of locating and attaching to suitable surfaces for adult growth; an activity that, in many species, must be completed within a month of metamorphosis from the first nauplius stage [104] Cyprids are capable of temporary attachment and exploration on immersed surfaces whilst in search of a suitable settlement site. The rapidly reversible adhesion mechanism of cyprids is poorly understood at present [105] but i nvolves secretion of a viscous [106,107] tool and have been studied in great detail [108 110] Each antennule is divided into four segments, the third and fourth of which are of p rimary importance to my study. The fourth segment has a sensory function and is almost entirely filled with neuron dendrites [108,111] The third segment b e ars the antennular attachment disc of which the surface is covered by micro scale cuticular villi, surrounded by an encircling velum. The oval attachment disc of B. amphitrite measures approximately 25 30 m in the long axis by approximately 15 m in width and the entire antennule has a total length of around 200 m [110,112] Dispersal and colonisation by the green fouling alga Ulva is achieved through the production of vast numbers of zoospores that swim in the water column until a suitable surface for colonisation is located. In common with the cypris larvae o f barnacles, zoospores are able to [97] Surface attributes that moderate settlement include wettability [113,114] chemistry [14] biofilms [98,100] and topography [10,101] Location of a

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67 suitable settlement site is critical for both Ulva spores and barnacle cyprids, since once committed to settlement by secretion of adhesive(s), motility is lost and the spore or larva is permanently attached to the substratum [44] The sensory structures of the zoospore are not u [23] but a sensory role for the four flagella is also possible. At the molecular level, thigmotropic mechanosensors in the spo re plasma membrane are presumably involved, but direct evidence for their presence is currently lacking. The critical topographical length scale (feature width and spacing) for effective antifouling correlates with approximately one half the size of the Ul va spore body [87] and approximates that of the antennular disc of B. amphitrite [115] This emphasizes the point by Genzer and Efimenko that surface topography containing only one length scale may not function effectively as a universal antifouling marin e coating [42] Therefore, novel hierarchical designs that super impose the smaller anti alga l topography onto the larger anti barnacle topography are fabricated The hierarchical structure was intended to provide insight into the effect of multi length scale topography on multiple settling organisms. The settlement response of the spores of Ulva on first generation hierarchical surfaces is included in my study. Experimental D esign Effect of Topographic Feature Height on S ettlement of Ulva Z oospores Ulva specific antifouling surface topographies have been correlated with a previously defined engineered roughness index or ERI [87] ERI is a dimensionless ratio based on depressed surface fraction and the degree of freedom of spo re movement. This model was correlated to topographies of a fixed feature height of 3 m. However, the roughness limit and associated trend (i.e., lower feature heights) of this model have not been evaluated and defined. Therefore, the most effective an tifouling Ulva specific

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68 engineered topography, Sharklet AF [87,89] was modified to evaluate and define this trend with decreasing feature heights. Engineered surface topographies of the Sharklet AF geometry at a 2 m feature resolution (width and spacing ) were tested for spore settlement at feature heights of 1 m (+1SK2x2), 2 m (+2SK2x2), and 3 m (+3SK2x2) (Figure 5 1). All topographical surfaces were fabricated in PDMSe and compared against a smooth PDMSe surface. The smooth PDMSe surface has been c haracterized elsewhere [116] It would be expected that there is a limiting feature height at which the antifouling properties of an engineered topography diminish and begin to approach settlement values of a smooth surface. Design of Barnacle Specific En gineered T opographies Surface topographies, engineered for inhibition of settlement of barnacle cyprids ( B. amphitrite ), were designed at 20 m feature width and spacing by re developing the most successful antifouling surface geometry for Ulva spores, Sh arklet AF [87,89] to a 20 m feature resolution (i.e., 20 m feature width and spacing) (Figure 5 2A). Channels of equivalent feature resolution (i.e., spaced by 20 m wide ridges) were also created (Figure 5 2D). These barnacle specific topographies we re fabricated at feature heights of 20 m (+20SK20x20, Figure 5 2B; +20CH20x20, Figure 5 2E) and 40 m (+40SK20x20, Figure 5 2C; +40CH20x20, Figure 5 2F) to evaluate the effect of feature height on antifouling efficacy. All topographies were created in PD MSe and settlement results were compared against a smooth PDMSe surface. Design and T esting of First Generation Hierarchical S tructures It has been shown that settlement of Ulva spores is enhanced on feature dimensions that are effective at decreasing sett lement of barnacle cyprids [10,101] Spores preferentially settle against the vertical walls of ridges separating channels as opposed to a smooth surface. As such, any effective antifouling topography to target multiple species would be required to conta in discrete features of multiple length scales.

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69 Engineered topographies containing multiple length scales have been fabricated to test the efficacy of an Ulva specific anti settlement topography when combined in a hierarchical structure with a barnacle spe cific anti settlement topography. The Ulva specific Sharklet AF surface (2 m feature width and spacing; 3 m feature height) was superimposed on 20 m wide channels separated by 20 m wide ridges (+3SK2x2 / +5CH20x20; Figures 5 3A, 5 3B) and 200 m wide channels separated by 200 m wide ridges (+3SK2x2 / +5CH200x200; Figures 5 3C, 5 3D). I hypothesized that if the anti settlement properties of the Sharklet AF surface were dominant factors over the attractiveness of the vertical ridges walls, spore settle ment of Ulva would be reduced relative to a smooth surface. If the latter was the case, settlement of spores would be increased relative to a smooth surface with an increased settlement on 20 m channels compared to 200 m channels due to the higher densi ty of vertical ridge walls available to spores. Materials and Methods Materials A platinum catalyzed PDMSe, Silastic T 2 (Dow Corning Corporation), was used as the base material for topographical modification. The elastomer was prepared by mixing ten part s of the resin and one part of the curing agent by weight for 5 minutes. The mixture was degassed under vacuum (95 102 kPa) for 30 minutes, removed, and allowed to cure for 24 hours at ~ 22C. Pattern Fabrication Methods The pattern designs were created o n photoresist coated silicon wafers using my techniques. Patterned silicon wafers were reactive ion etched to depths between 1 and 3 m for Ulva specific topographies and to 20 and 40 m for barnacle specific topographies. Wafers were then stripped of ph otoresist and cleaned with an oxygen plasma etch. Hexamethyldisilazane was vapor

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70 deposited on the processed silicon wafers to methylate the surfaces in order to prevent adhesion. These processed wafers served as negative molds for topographical replicati on. For hierarchical structures, processed silicon wafers ( i.e., cleaned and containing topography) were re coated with photoresist and the seconda ry topography using my techniques Topograp hical R eplication Topographical surfaces were transferred to PDMS e from replication of the patterned silicon wafers. The resultant topographies contain features projecting from the surface at heights respective of the etch depth. Pattern fidelity was evaluated using light and scanning electron microscopy. Sample P repa ration for Ulva Settlement A ssay Ulva spore settlement assays were conducted with 76 mm by 25 mm glass microscope slides coated with smooth and topographically modified PDMSe surfaces. Glass slides coated with PDMSe topographies were fabricated using a tw o step curing process as prev iously described [89] The resultant slide (~ 1 mm thickness ) contained an adhered PDMSe film with a 25 mm by 25 mm area containing topography bordered on both sides by 25 mm by 25 mm smooth (n o topography) areas. Ulva Zoospor e Settlement Assay Three replicates of each topographically modified PDMSe sample, permanently adhered to glass microscope slides, were shipped to the University of Birmingham, United Kingdom, to be evaluated for settlement of Ulva spores The Ulva assay and collection of the raw data of spore settlement were performed by Dr. Maureen Callow and Dr. John Finlay at the University of Birmingham Topographies included the Ulva specific Sharklet AF topography at feature heights of 1 m (+1SK2x2), 2 m (+2SK2x2 ), and 3 m (+3SK2x2). A u niformly smooth PDMSe sample was included in the assay and served as a control for direct comparison.

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71 Fertile plants of Ulva linza were collected from Wembury beach, UK ( Z oospores were released and prepared for attachment experiments as documented p reviously [23] Topographical samples were pre soaked in n ano pure water for several days prior to the a ssay in order for the surfaces to fully wet. Sample s were transferred to artificial seawat er (ASW; Tropic Marin ) for 1 hour prior to exp erimentation without exposure to air. Samples were then rapidly transferred to assay dishes to minimize any dewetting of the t opographical areas. Ten m l of spore suspension (adjusted to 2 x 10 6 ml 1 ) w ere added to each dish and pla ced in darkness for 60 minutes. The slides were then rinsed and fixed with 2% glutaraldehyde in artificial seawater as d escribed in Callow et al. [23] Spore counts were quantified using a Zeiss epifluorescence microscope attached to a Zeiss Kontron 3000 image analysis system [10] Thirty images and counts we re obtained from each of three replicates at 1 mm intervals along both the vertical (15) and hori zontal (15) axes of the slide. Statistical Methods for Settlement A ssay with Ulva Zoospores Spore densi ty was calculated as the mean number of settled spores per mm 2 from 30 counts on each of three replicate slides standard error (n = 3). Statistical differences between surfaces were evaluated using a nested analysis of variance (ANOVA) followed by the S tudent Newman Keuls (SNK) test for multiple comparisons [95] Replicate slides (3) of each surface were treated as a nested variable within each surface. Statistical computations were completed using Minitab 14 statistical software package. Sample Prepar ation for Settlement Assay with Balanus a mphitrite C yprids C yprid settlement assays were conducted on circular disks of topographically modified PDMSe films measuring 3 cm in diameter and approximately 1 mm thick Surfaces included

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72 20 m wide channels spa ced by 20 m wide ridges at feature heights of 20 m (+20CH20x20) and 40 m (+40CH20x20) and the barnacle specific Sharklet AF surface (20 m feature width and spacing) at feature heights of 20 m (+20SK20x20) and 40 m (+40SK20x20). The samples were ship ped to the University of Newcastle upon Tyne, United Kingdom, to be evaluated for cyprid settlement. Settlement Assay with Balanus a mphitrite The B. amphitrite assay and collection of the raw data of cyprid settlement were performed by Dr. Nick Aldred at the University of Newcastle upon Tyne. The c yprids of B. amphitrite were cultured as described in Hellio et al. [117] All surfaces were stored in ASW (Tropic Marin) for six hours prior to exp erimentation, followed by ultra sonication in a MSE Soniprep 150 ultrasonicator operating at 3 m amplitude for thirty seconds to ensure total wetting and the expulsion of air from the surface features. Settlement assays took the form of drop tests on the PDMSe substrata. One drop (volume = 1.0 ml) of ASW was depos ited onto each test surface (n = 6 replicates per surface) followed by the addition of 20 three day old cyprids in 500 l of ASW Each PDMSe disk with associated 1.5 ml droplet was placed with in a 3 cm non vented polystyrene Petri dish. All six dishes p er surface type were then sealed in larger Petri dishes containing water soaked tissue to raise the humidity and prevent evaporation of the droplets during incubation. The dishes were incubated at 28 o C in the dark and settlement counts were taken at 48 hr s Cyprids were considered to be settled when they had expelled their perm anent cement [118] Statistical Methods for Settlement Assay with Balanus a mphitrite The number of cyprids settled was determined for each replicate of each surface type at 48 hours Mean ( standard error, n = 6) and median settlement values, expressed as a

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73 percentage of cyprids settled, were determined for each surface type. Since n was low and the variability heterogeneous between surface types, median settlement values were com pared using Kruskal Wallis multiple comparison analysis [119] Statistical computations were completed using Minitab 14 statistical software package. Correlating Settlement of Spores of Ulva and Cyprids of B alanus a mphitrite to Topographical Aspect Ratio The mean spore density and mean number of settled cyprids measured on each topographically modified surface was expressed as a percent reduction in settlement relative to the smooth PDMSe surface. This allowed for the direct comparison of the effect of to pographical aspect ratio (feature height / feature width) on the settlement behavior of the spores of Ulva and cyprids of B. amphitrit e The 2 m wide Ulva specific Sharklet AF topography had topographical aspect ratios of 0.5 for +1SK2x2, 1.0 for +2SK2x2 and 1.5 for +3SK2x2. A previously reported reduction of 85% relative to the smooth PDMSe surface on a 2.0 aspect ratio topography (+4SK2x2) was included in the analysis [89] The 20 m wide barnacle specific topographies had topographical aspect ratios of 1.0 for +20SK20x20 and +20CH20x20 and 2.0 for +40SK20x20 and +40CH20x20. The smooth PDMSe surface was characterized as having an aspect ratio of 0. Linear regression analyses were completed on each species specific settlement with respect to topograp hical aspect ratio. Regression analysis incorporated all raw data points including the variance associated with settlement on a smooth surface. Results Topographical Aspect Ratio and Ulva Settlement The anti settlement effectiveness of the Ulva specific S harklet AF (2 m feature width and spacing) topography in PDMSe was strongly dependent on feature height (Table 5 1, Figure 5

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74 4). The mean spore density on the smooth PDMSe surface was 191 27 spores/mm 2 The Sharklet AF topography, fabricated at a 3 m feature height (+3SK2x2), significantly reduced the mean spore density by 63% compared to the smooth surface (SNK test, p < 0.05). As the feature height on the Sharklet AF topography was decreased to 2 m (+2SK2x2), the mean spore density was also signif icantly reduced but by a lesser amount, 44%, compared to the smooth surface (SNK test, p < 0.05). At the lowest feature height of 1 m (+1SK2x2), the mean spore density was significantly reduced by the least amount, 25%, compared to the smooth surface (SN K test, p < 0.05). Although all feature heights significantly reduced settlement compared to the smooth PDMSe surface, the degree of reduction decreased with a decreasing feature height. Additionally, the +3SK2x2 had a significantly lower mean spore dens ity than both the +2SK2x2 and +1SK2x2 (SNK test, p < 0.05). There was no significant difference in mean spore densities between the +2SK2x2 and +1SK2x2. Topographical Aspect Ratio and Balanus a mphitrite Settlement The engineered topographies designed for barnacles (20 m feature width and spacing) reduced cyprid settlement compared to a smooth PDMSe surface (Table 5 2, Figure 5 5). The mean percent cyprid settlement on the smooth PDMSe surface after 48 hours was 33.5 4.2%. Topographies fabricated at a 20 m feature height reduced the mean percent cyprid settlement by 47% for +20CH20x20 (Mean = 17.8 8.3%) and 63% for the +20SK20x20 (Mean = 12.4 5.0%) compared to the smooth surface. Topographies fabricated at a 40 m feature height reduced the mean p ercent cyprid settlement by 84% for +40CH20x20 (Mean = 5.5 3.8%) and 97% for +40SK20x20 (Mean = 1.1 1.1%) compared to the smooth surface. At both feature heights, the barnacle specific Sharklet AF reduced settlement of cyprids by a larger degree than the channels. Median values were used for statistical comparisons to the control smooth surface. Topographies of +20CH20x20 (median = 14.3%), +20SK20x20

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75 (median = 11.3%), +40CH20x20 (median = 3.0%) and +40SK20x20 (median = 0.0%) were significantly differ ent than the smooth surface (median = 30.5%) at confidence levels of 91%, 97%, > 99%, and > 99% respectively (Kruskal Wallis multiple comparisons). For comparisons among topographies, the +40SK20x20 was significantly different compared to +20CH20x20 and + 20SK20x20 at confidence levels of 96% and 92% respectively. Correlations between the Settlements of Ulva and Balanus a mphitrite with Topographical Aspect Ratio The percent reduction in spore density ( Ulva ) and cyprids settled ( B. amphitrite ) relative to a smooth PDMSe was plotted against topographical aspect ratio (feature height / feature width) for the engineered topographies studied. Ulva specific topographies included aspect ratios of 0.5, 1.0, 1.5, and 2.0 (+1SK2x2, +2SK2x2, +3SK2x2, and +4SK2x2 respe ctively). Barnacle specific topographies included aspect ratios of 1.0 (+20CH20x20), 1.0 (+20SK20x20), 2.0 (+40CH20x20), and 2.0 (+40SK20x20). Trends were observed in both cases and a linear regression model was fitted to each set of raw data (Figure 5 6 ). A direct linear relationship existed between percent reduction in the density of settled spores and aspect ratio (Eq. 5 1, R 2 = 0.66, p < 0.001 for slope) as well as percent reduction in cyprids settled and aspect ratio (Eq. 5 2, R 2 = 0.52, p < 0.001 f or slope). (5 1) (5 2) The y intercepts in each equation were not significantly different to 0 (p = 0.96 for spores, p = 0.66 for cyprids) and therefore not included in the regression equations. For Ulva spore density was reduced by 42% with each unit increase in aspect ratio. Similarly for B. amphitrite the number of cyprids settled was reduced by 45% with each unit increase in aspect ratio.

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76 Hierarchical Structures and Ulva Settlement Hierarchical structures combining an Ulva specific Shar klet AF surface with 20 m wide channels separated by 20 m wide ridges (+3SK2x2 / +5CH20x20) or 200 m wide channels separated by 200 m wide ridges (+3SK2x2 / +5CH200x200) were evaluated for Ulva spore settlement compared to a smooth PDMSe surface. The preference of Ulva spores to settle against the vertical wall of a ridge was a dominant factor over the anti settlement properties of the Ulva specific Sharklet AF surface (Figure 5 7). For this assay, the mean spore density on a smooth PDMSe surface was 241 39 spores/mm 2 The mean spore density was increased to 291 34 spores/mm 2 on the +3SK2x2 / +5CH200x200 surface and was further increased to 354 43 spores/mm 2 on the +3SK2x2 / +5CH20x20 (Figure 5 8). The mean spore density on the +3SK2x2 / +5CH20 x20 surface was significantly higher than both the smooth and +3SK2x2 / +5CH200x200 PDMSe surfaces (Table 5 3). The presence of the anti settlement Ulva specific Sharklet AF surface did not deter Ulva spores from settling against the vertical walls of the ridges. Subsequently, as expected, the higher density of ridge walls on the +3SK2x2 / +5CH20x20 compared to +3SK2x2 / +5CH200x200 resulted in a higher mean spore density. Discussion Successful nontoxic, anti fouling surface topographies fabricated in poly dimethylsiloxane elastomer (PDMSe) have be en developed for Ulva spores [87,89] and new surfaces presented in my study also demonstrate anti settlement properties for barnacle ( B. amphitrite ) cyprids. These engineered surface topographies have defined and ordered structural features that are tailored to the identified critical dimension s (i.e., feature width and spacing) of the fouling plant or an imal of interest; 2 m for algal ( Ulva ) spores [87] and 20 m for barnacle ( B. amphitrite ) cyprids [115] In ad dition to feature width and spacing, topographical feature heights in the range of 30

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77 45 m in PMMA [55] and 46 69 m in PDMSe [56] have been shown to be critical antifouling dimensions for the barnacle Balanus improvisus The experiments in my study exami ned the isolated effect of feature height, or topographical aspect ratio, on antifouling effectiveness. For zoospores of Ulva the anti settlement properties of the Ulva specific Sharklet AF surface were significantly diminished when the topographical fea ture height fell below 3 m. Additionally, a 1 m feature height approached the spore density value of a smooth surface. These results indicated that in order to obtain substantial reductions in spore settlement with an engineered topography in PDMSe, su rfaces containing a feature height below 3 m should be avoided. Furthermore, the trend with feature height suggested that feature heights above 4 m may provide an enhanced reduction in spore settlement. This may be true, but cannot be experimentally ve rified with the current low modulus PDMSe system due to pattern fidelity issues with aspect ratios above 2 [89] Other non toxic, higher modulus material systems are being investigated to confirm this predicted trend. A new barnacle specific Sharklet AF s urface was designed based on the original Ulva specific geometry. This surface differed from its Ulva counterpart in that the feature dimensions were increased by one order of magnitude to a 20 m feature width and spacing. In a study similar to that car ried out with Ulva the effect of feature height on the settlement of barnacle cyprids ( B. amphitrite ) was examined. Engineered topographies of feature heights of 40 m showed enhanced anti settlement properties against cyprids compared to feature heights of 20 m. The best performing surface, the barnacle specific Sharklet AF fabricated at a 40 m feature height, reduced cyprid settlement by 97% compared to the smooth PDMSe surface.

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78 Similar linear correlations with topographical aspect ratio and organism settlement were identified for Ulva and B. amphitrite It must be noted that these trends only existed when surface feature dimensions of width and spacing were fabricated at species specific critical dimensions. The dimensionless value of aspect ratio was used to compare the isolated effect of topographical feature height across species. When normalized to organism settlement on a smooth PDMSe, the percent reduction in settlement associated with aspect ratio for Ulva (42 aspect ratio) and B. amphitri te (45 aspect ratio) was analogous. When considering the complexity and differences in the sensing and adhesion modes between these two organisms, this comparable trend suggests there may be a unique, unidentified anti settlement property of engineered topography that is organism independent. Some current theories exist such as attachment point theory [57] underwater superhydrophobicity [43,120] and engineered roughness index [87] ; any of which may influence settlement behavior. Engineered topography in conjunction with appropriately designed experiments and measurement techniques provides the opportunity to further examine and validate the aforementioned theories. For example, these identified trends and associated correlations can be used to estima te the minimal level of roughness required to reduce settlement of fouling organisms [42] Similarly, it is also important to recognize that settlement differences related to variations with topographical aspect ratio may also be related to the predicted critical limits of roughness ratio required for underwater superhydrophobicity [120] Achieving underwater superhydrophobicity with a surface of a sufficient roughness ratio is theorized to be the mechanism by which antifouling/anti settlement behaviour i s observed on topographically modified surfaces [43] The settlement behavior of Ulva spores on first generation hierarchical structures provided valuable information regarding the design of multi length scale engineered structures that are

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79 effective for a range of organisms. The preference of spores to settle in the angle between the channel floor and ridge wall was a dominant factor over the presence of the antifouling engineered topography for this organism. This was emphasized by the fact that higher settlement was measured on 20 m wide channels spaced by 20 m wide ridges (higher density of ridge walls available) than on the 200 m wide channels spaced by 200 m wide ridges both of which contained the Ulva specific Sharklet AF surface. Vertical ridg e walls with an adjacent space (i.e., channel) of 5 m or greater should therefore be avoided when targeting multiple biofouling organisms including Ulva spores with hierarchical engineered topographies. For example, the single length scale sawtooth like [56] for the barnacle Balanus improvisus combined with the Ulva specific Sharklet AF would be a more appropriate design based on these findings. Conceptually, there would be no ridge walls in this hiera rchical structure against which spores could settle. New micro fabrication techniques are being developed to produce these types of ridge less hierarchical structures that maintain antifouling properties against the targeted biofouling organisms.

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80 Figu re 5 1. SEM images of the Ulva specific Sharklet AF topography produced on the surface of PDMSe fabricated at three different feature heights. A) Top down image of the surface. B) 1 m feature height (+1SK2x2). C) 2 m feature height (+2SK2x2). D) 3 m feature height (+3SK2x2).

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81 Figure 5 2. SEM images of the barnacle specific engineered topographies produced on the surface of PDMSe. A) Top down image of the b arnacle specific Sha rklet AF topography. B) Cross sectional image of the 20 m feature heig ht Sharklet AF (+20SK20x20). C) Cross sectional image of the 40 m feature height Sharklet AF (+40SK20x20). D) Top down image of 20 m c hannels spaced by 20 m ridges. E) Channels at a 20 m feature height (+20CH20x20). F) Channels at a 40 m feature h eight (+40CH20x20).

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82 Figure 5 3. Light micrograph and SEM images of hiera rchical PDMSe topographies. A ) Light micrograph of Ulva specific Sharklet AF topography on 20 m wide channels spaced by 20 m wide ridges (+3SK2x2 / +5CH20x20). B) SEM image of +3SK2x2 / +5CH20x20. C ) Light micrograph of the Ulva specific Sharklet AF topography on 200 m wide channels spaced by 200 m wide ridges (+3SK2x2 / +5CH200x200). D) SEM image of +3SK2x2 / +5CH200x200.

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83 Figure 5 4. Ulva spore settlement data on PDMSe surfaces represented as mean spore density (spores/mm 2 ) standard error (n=3). All surfaces were tested with a single batch of spores allowing direct comparisons between samples. PDMSe surfaces included a uniformly smooth surface (Smooth) and the Ulva s pecific Sharklet AF topography at three different feature heights: 1 m (+1SK2x2), 2 m (+2SK2x2), and 3 m (+3SK2x2). Asterisks indicate statistically different groups (ANOVA p < 0.001, SNK test p < 0.05). Table 5 1. ANOVA for Ulva spore density for th with the nested variable df MS F p Surface 3 230276 24.53 <0.001 Slide(Surface) 8 9386 5.08 <0.001 Error 348 1847 different feature heights (+1SK2x2, +2SK2x2, and +3SK2x2). Three replicate slides were nested within each surface. Thirty spore counts were obtained for each slide.

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84 Figure 5 5. Mean barnacle cyprid settlement (% standard error) after 48 hours on PDMSe surfa ces. Surfaces included smooth (Smooth), 20 m wide channels spaced by 20 m wide ridges at feature heights of 20 m (+20CH20x20) and 40 m (+40CH20x20) and the barnacle specific Sharklet AF surface (20 m feature width and spacing) at feature heights of 2 0 m (+20SK20x20) and 40 m (+40SK20x20). Asterisks indicate statistically different groups (Kruskal Wallis multiple comparison test, > 90% confidence levels). Table 5 2. Kruskal Wallis analysis for percent settlement of barnacle cyprids ( B. amphitrite ) on smooth and topographically modified PDMSe surfaces. Surface Sample size Median Average Rank z Smooth 6 30.5 22.2 3.16 +20CH20x20 5 14.3 14.5 0.33 +20SK20x20 6 11.3 12.8 0.27 +40CH20x20 4 3.0 9.5 1.14 +40SK20x20 5 0.0 6.2 2.37 H = 13.5 df = 4 p = 0.01 H = 14.1 df = 4 p = 0.01 (adjusted for ties)

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85 Figure 5 6. Graphical representation of the linear correlation for the percent reduction in settlement of Ulva and B. amphitrite with topographical aspect ratio relative to a smooth PDMSe surface. Linear regressions were calculated using raw data represented as the mean percent reduction in spore density for Ulva ( dark red regression line) and the percent reduction in cyprids settled for B. amphitrite black regression line).

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86 Figure 5 7. Light micrographs showing Ulva spores settled against the vertical walls of the channels with and without the presence of the Ulva specific Sharklet AF topography. A) Spores settled against the walls of a 20 m wide channel with the prese nce of the Sharklet AF topography (+3SK2x2 / +5CH20x20). B) Spores settled against the ridge wall of a 200 m wide channels without the presence of the Sharklet AF surface (+5CH200x200). C) Spores settled against the ridge wall of a 200 m wide channel w ith the presence of the Sharklet AF topography (+3SK2x2 / +5CH200x200).

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87 Figure 5 8. Ulva spore settlement data on PDMSe surfaces represented as mean spore density (spores/mm 2 ) standard error (n = 3). All surfaces were tested with a single batch of spores allowing direct comparisons between samples. PDMSe surfaces included a uniformly smooth surface (Smooth) and the Ulva specific Sharklet AF topography combined into a hierarchical structure with 200 m wide channels spaced by 200 m ridges (+3SK2x2 / +5CH200x200) and 20 m wide channels spaced by 20 m wide ridges (+3SK2x2 / +5CH20x20). Asterisks indicate statistically different groups (ANOVA p<0.001, SNK test p<0.05). Table 5 3. ANOVA for Ulva ted Df MS F p H. Surface 2 287452 9.69 0.013 Slide(H. Surface) 6 29662 7.57 <0.001 Error 261 3918 The two hierarchical struct ures included the Ulva specific Sharklet AF surface (2 m feature width and spacing; 3 m feature height) combined with 20 m wide channels spaced by 20 m wide ridges (+3SK2x2 / +5CH20x20) or 200 m wide channels spaced by 200 m wide ridges (+3SK2x2 / +5 CH200x200). Three replicate slides were nested within each surface. Thirty spore counts were obtained for each slide.

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88 CHAPTER 6 3 INHIBITION OF MEDICA L BIOFILM FORMATION WITH ENGINEERED TOPO GRAPHY Introduction A surface of uniform chemistry with an engineered microtopography was investigated for the inhibition of bacterial biofilm formation. The most successful design tha t has been investigated to date for similarly sized microorganisms (zoospores of algae) is the Sharklet AF design This particular microtopography is unique in that it has non random, clearly defined surface features that are tailored to the critical dime nsions of the fouling organism. Recent width and spacing have shown a st rong correlation between the engineered roughness index (ERI) and the inhibition of settle ship fouling alga, Ulva [87] In addition, the Sharklet AF feature width and spacing has been demonstrated to be a strong inhibitor of the settlement of barna cle cyprids of B. amphitrite [103] 25 [112] T he Ulva specific Sharklet AF surface was selected for my study for its potential to inhibit biofilm formation of Staphylococcus aureus based on the approximate match between critical the bacteria (Figure 6 1). I hypothesized that the dimensions of the topography would be slightly too large to effectively reduce the attachment of the bacteria in the size range of ~ 1 2 b ut that it would be effective at physically disrupting the colonization of additional bacteria and subsequent formation of biofilm. S. aureus was selected as the bacterial pathogen due to both its 3 Portions of this chapter were previously published by the author in the journal Biointer phases [121] and are reprinted here with full permission from the American Vacuum Society.

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89 size and its association with nosocomial infections in implanted devices, such as cochlear implants, sutures, and heart valves [70,122] Materials and Methods Materials Dow Corning Silastic T 2, a platinum catalyzed polydimethylsiloxane elastomer (PDMSe), was used for its low modulus, low surface energy, and propensity for minimal bioadhesion [101] Silastic brand silicone elastomers are biomaterials used in numerous medical devices including tubing, catheters, and pacemaker leads [84] The elastomer was prepared by mixing one part by weight of curing agent with ten parts by weight of resin, then degassing under vacuum (28 30 in Hg) for 30 minutes. The mixture was cured at ~22C for 24 hours. Sharklet AF Design and F abrication of Topographical M olds The Sharklet AF design [87,89] tested consists of 2 m wide rectangular ribs of varying lengths ranging from 4 m to 16 m. The ribs of varying lengths are combined into a periodic, diamond like array at a fixed spacing of 2 m between neighboring features (F ig ure 6 1 B ). The final, resultant Sharklet AF topography in PDMSe was created by replication of silicon wafer molds. Silicon wafer molds were fabricated by first transferring the Sharklet AF design to photoresist coated silicon wafers using photoli thogra phic techniques Next, the patterned silicon photoresist with an O 2 plasma etch. The etched silicon wafer surfaces were then methylated (via vapor deposition) with hexamethyldisilazan e to prevent adhesion. These wafers served as negative molds for topographical replication of the Sharklet AF topography at a feature height of 3 m onto a PDMSe surface.

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90 Sample Preparation Silicon molds were replicated into PDMSe to produce ~ 0.4 mm thic k films containing protruding topographical features. Briefly, the PDMSe material components were prepared and mixed as described above poured over the silicon wafer molds, and the entire system was pressed between two larger glass plates with the approp riate spacers to produce a 0.4 mm thick film. After curing for 24 hours, the PDMSe film was removed from the silicon wafer yielding protruding topographical features forming the Sharklet AF topography on the PDMSe surface (Figure 6 1 C ). PDMSe samples of smooth (replicated from an unmodified silicon wafer ) and Sharklet AF were punched out with a circular die 8 mm in diameter (Figure 6 2) An edge notch (~ 1 2 mm radius) was punched using a 3 mm in diameter die to serve as a consistent handling location (F igure 6 2). Five replicates each of smooth PDMSe and Sharklet AF PDMSe disks were statistically randomized among ten defined locations of 3 inch diameter Petri dishes (Figure 6 3). One dish was prepared and labeled for each day to be examined (0, 2, 7, 1 4, and 21) during the bacterial growth assay. An additional dish was prepared that would be incubated with growth medium containing no bacteria for 21 days. Bacterial Biofilm Growth Assay The samples prepared for the bacterial biofilm assay were transferr ed to the research group of Dr. Patrick Antonelli, Professor & Chair of the Department of Otolaryngology, at the University of Florida. The bacterial culturing and biofilm growth assay were conducted by Edith Sampson, Scientific Research Manager, Departme nt of Otolaryngology. Staphylococcus aureus (ATCC 29213) was subcultured in tryptic soy broth (TSB) growth medium and incubated at 37C (5% CO 2 ) overnight in static conditions. Optical absorbance was measured, serial dilutions were performed, and a linear growth curve was regressed by plotting the linear optical density versus colony forming units (CFU). Bacterial concentration was

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91 determined via spectrophotometry by interpolating CFU per ml from the linear optical density CFU regression. A bacterial sus pension solution ( S. aureus + TSB) of approximately 10 7 CFU per ml was prepared. Approximately 20 ml of this bacterial solution was added to each 3 inch Petri dish containing five randomized replicates of smooth and Sharklet AF PDMSe disks (Figure 6 3). Dishes labeled days 2, 7, 14, and 21 were placed in a 37C (5% CO 2 ) incubator. The dish labeled day 0 was removed at this time. Another dish exposed only to sterile medium was incubated with the samples for 21 days and served as a negative control. Ever y 24 hours, dishes were put on an orbital rocker for 1 minute at 30 RPM and the medium was replaced to allow for continued bacterial growth. Each respective dish was removed on days 0, 2, 7, 14, and 21. Areas surrounding the samples on the placement grid were rinsed with deionized water using a Pipet Aid and gently aspirated to eliminate non adherent cells for each dish removed at the specified time point. This rinsing procedure was repeated for a total of 3 times, and the dish was then put on an orbital rocker for 1 minute at 30 RPM. Each sample in the dish was then treated with 20 ml of 10mM cetyl pyridinium chloride fixative and allowed to air dry overnight. Characterization of Bacterial Coverage Samples were dehydrated in a graded ethanol series of 2 5, 50, 75, 95, and 100% at 10 minute intervals. Samples were washed twice with hexamethyldisilazane with a 5 minute interval between washings, followed by drying using a vacuum desiccator. Each sample was attached to a 25 mm in diameter aluminum disc (~ 7 mm thickness) an d sputter coated with Au/Pd. Surfaces were imaged with a JEOL 6400 scanning electron microscope (SEM). SEM images at 2000X from areas A, B, and C for each replicate were used to quantify bacteria l growth on each surface (Figure 6 4 ). Bi ofilms were identified by the presence of a colony of microorganisms and /or exopolymeric matrix. Biofilm growth was estimated and quantified by measuring the percent

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92 area of coverage of bacteria. This value was obtained by processing the obtained SEM ima ges (Figures 6 5A B) by first using Macromedia Fireworks software to outline and blacken the area containin g bacteria and/or biofilm (Figure s 6 5 C D ). Processed SEM i mages were analyzed for percent coverage using ImageJ software [123] Areas of coverage were numbered, outlined, and the total summation of area c overed was measured (Figure 6 5E F). T he results for areas A, B, and C are combined to reflect the percentage coverage of bacter ia for that specific replicate. Statistical Analysis The mean value ( standard error replicates = 5 ) of percent are a coverage of bacteria on both s mooth and Sharklet AF PDMSe surfaces at days 0, 2, 7, 14, and 21 was calculated. Statistical differences were evaluated by a two way analysis of variance for the factors of multiple comparisons. Statistical differences were considered at the 95% confidence level. Statistical calculations were completed using Minitab 14 statistical so ftware package. Results Individual cells were randomly distributed on the surface of smooth PDMSe (Figure 6 6A) for the day 0 time point, while individual cells on the Sharklet AF PDMSe surface were isolated to the recesses between the pro truding features (Figure 6 6 B). M icro colonies of bacteria began to f orm on the smooth surface (Figure 6 6C ) on day 2 and the Sharklet AF surface continued to have isolated cells ac crete between features (Figure 6 6D) Growth of the micro colonies increased on day 7 for the smooth surface into early stage biofilm (Figure 6 6 E). The Sharklet AF surface (day 7) continued to exhibit small sized clusters of bact eria, with no evidence of early stage biofilm development; the clusters were positioned similar to day 2 in the re cesses between protrud ing topographical features (Figure 6 6 F). The smooth surface had the first evid ence of mature biofilm (Figure 6 6G ) on day 14 while the Sharklet AF surface had an

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93 increase in the number of small clusters of bacterial cells compared t o day 7, but still no evidence of early biofilm development or f ormation (Figure 6 6H). A significant portion of the smooth PDMSe sur faces were colonized by biofilm at day 21 (Figure 6 6 I), and biofilms first appeared in isolated areas on the Sharklet AF PDMSe surfaces (Figure6 6 J). Areas surrounding the large bacterial colonies of biofilm on the topographical surfaces for day 21 were virtu ally devoid of additional biofilms SEM images of the negative control samples exposed to only TSB media showed no c ells. Image and statistical analysis incorporating all time points indicat ed that the smooth PDMSe surface had an overall significant increase in bacter ial coverage versus the Sharklet AF PDMSe surface < 0.05), with the first eviden ce of b iofilm on day 7 samples. Further statistical analysis indicated that the Sharklet AF surface had significantly lower values < 0.05), with biofilm colonies not appearing until day 21. B iofil m colonies covered only isolated areas on Sharklet AF samples, with little evidence of biofil ms in other areas. The mean value ( standard error) of percent are a coverage of bacteria on both s mooth and Sharklet AF PDMSe surfaces at days 0, 2, 7 14, and 2 1 was calculated and graphically displayed (Figure 6 7 ). Discussion Most in vitro studies involving S. aureus have examined the adhesion behavior over the course of a few hours [11,81,124] However, for most transcutaneous devices such as catheters, the t ime frame for use and biofilm formation is typically 14 days [31] Thus, the focus of my study was to test the effects of an engineered microtopography on bacterial colonization and biofilm formation for a period of time that extended beyond 14 days. The growth assay parameters included optimized conditions for S. aureus colonization and spanned 21 days. My

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94 in vitro study, involving surface topography and S. aureus was designed for a time period approximating that of short term indwelling devices. Mater ial selection for my study was predicated upon the popularity of silicone as the choice material for molded implants, such as cochlear implants and long term catheters, despite being shown to have nearly a 10 fold greater risk of infection than other polym er materials [125] The static culture provided for biofilm growth was chosen to represent the most challenging environment for the material surface in the presence of bacteria, as opposed to the low shear dynamics of indwelling catheters. The results of this experiment strongly suggest that the surface modification of existing silicone devices with the Sharklet AF surface may prolong the service life and improve the efficacy of these devices. It is also encouraging to note that the topographical modific atio n of a surface used in my study involves no chemical changes of the biomaterial surface and does not rely on the release of any antibacterial agents. Both qualitative (i.e., extensive colonization of bacteria ) and quantitative (i.e., percent area cover age of bacteria) measures of biofilm formation revealed inhibition of biofilm development on PDMSe with the Sharklet AF microtopography. Observations of SEM images confirm the hypothesis that bacterial cells can fit in the recessed regions between the pro truded topographical features, but evidence of further bacterial colonization and biofilm formation did not occur on the Sharklet AF features until day 21. It is suggest ed that the protruded features of the topographical surface provided a physical obstac le to deter the expansion of small clusters of bacteria present in the recesses into micro colonies. It was at day 21 when bacteria were observed to form small, multi layered colonies within the recesses in order to extend over the protruding features and connect to other isolated colonies. This phenomenon may be the

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95 exp lanation for the delay of early stage biofilm development to day 21 that was evident on the smooth surface at day 7. engineered surface approach suggests the use of a hierarchy of surface topographies [103] to control bioadhesion. Previous results detailing an engineered pattern capable of promoting of cell growth [126] can be integrated into the Sharklet AF design used i n my study to produce a hierarchical topography for desirable competitive adhesion at the biomaterial surface. One can envision a surface that repels and delays biofilm formation to the extent that host cells, vital to the integration of the biomaterial with the physiological environment, can be established and proliferate on the designed surface. My study was performed using a highly simplified in vitro model. A great deal of work is needed to determine if observations from this in vitro model are cons istent with in vivo performance Current research is evaluating the adhesion and biofilm formation tendencies of other biofilm forming bacteria (e.g., Pseudomonas aeruginosa ) on the Sharklet AF surface Also, the application of the engineered roughness i ndex is being used to predict other engineered topographies that may be effective at inhibiting biofilm formation. Considerations for designing the optimal microtopography for an implantable device will include the interactions with host molecules and the impact on fibrous capsule formation

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96 Figure 6 1. Sharklet AF topography on polydimethylsiloxane elastomer (PDMSe) with a 2 A) Light micrograph with top down view. B) SEM image with top down view C) SEM image taken at 35 degree ti lt to show protruding features. Figure 6 2. Configuration and dimensions of the circular PDMSe samples used for bacterial biofilm growth experiments. The semi circular notch (~ 1 2 mm) was created as a consistent gr asping location (e.g., with forceps) when handling samples.

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97 Figure 6 3. Position of randomized PDMSe sam ples placed within a 3 inch Petri dish for bacterial biofilm growth testing. Samples included five replicates each of smooth and Sharklet AF surfaces. Figure 6 4. Locations of SEM analysis on samples exposed to Staphylococcus aureus Regions of imagi ng are indicated by A, B, and C in the drawing.

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98 Figure 6 5. Image processing of SEM images to quantify bacterial coverage on PDMSe surfaces exposed to Staphylococcus aureus A) Raw image of bacterial coverage on a smooth PDMSe surface obtained by SEM. B) Raw image of bacterial coverage on a Sharklet AF PDMSe surface obtained by SEM. C) SEM image in subpart A processed with Macromedia Fireworks to shade the bacteria covered surface area black. D) SEM image in subpart B processed with Macromedia Firew orks to shade the bacteria covered surface area black. E) Image in subpart C processed with ImageJ software to group and number bacterial colonies on the smooth PDMSe surface. F) Image in subpart D processed with ImageJ software to group and number bacte rial colonies on the Sharklet AF PDMSe surface.

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99 Figure 6 6. SEM images of bacterial coverage on smooth and Sharklet AF PDMSe surfaces. Bacterial coverage is false colored purple to enhance image contrast. A) Smooth day 0. B) Sharklet AF day 0. C) S mooth day 2. D) Sharklet AF day 2. E) Smooth day 7. F) Sharklet AF day 7. G) Smooth day 14. H) Sharklet AF day 14. I) Smooth day 21. J) Sharklet AF day 21.

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100 Figure 6 7. Mean value of percent coverage of S. aureus on Smooth and Sharklet AF PDMSe su rfaces at various time points. Bars represent standard error (n = 5).

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101 CHAPTER 7 ENGINEERED NANO FORCE GRADIENTS FOR THE INHIBITION OF SE TTLEMENT OF SWIMMING ALGAL SPORE S Introduction The biological response to a material placed within a natural aquatic or physiological environment is controlled by the surface ch aracteristics of the material. This biological surface interaction influences the biocompatibility of mater ials used in medicine [1,2] and the degree of biofouling on surfaces in the marine environment [3,17] The field of biomaterials is typically associated with th ose materials used for medical de vices and biomedical implants. For most synthetic biomaterials, the surface is designed to minimize the b iological surface interaction. Other biomaterials, like those used for tissue engineering and other specialized appl ications, are designed to facilitate the adhesion of preferred cells that promote the growth of replacement tissue and function. The study of marine biofouling focuses on the response of the settling or attaching stages of organisms to man made materials placed in the ocean. Ideal surfaces or coatings are antifouling ( i.e., no settlement or attachment of the colonizing larvae, spores or cells ) and /or fouling release ( i.e., organisms release under h ydrodynamic forces because they are weakly adhered). N on b iocidal coatings, biomaterials, and non toxic surface modification techniques are being explored for marine applications with the recent environmental restrictions place on antifouling paints [17] Subsequently, new and promising antifouling materials and designs that are developed may have applications as non fouli ng surfaces in the biomedical field. Similarities exist between the early fouling events on the surface of a synthetic material in both the physiological and marine environments including the f ormation of an organic conditioning film (e.g., proteins), bacterial adhesion and biofilm formation [1 3,17] The micro/macro fouling events in vivo are dominated by mononuclear cells (e.g., macrophages and fibroblasts) [1,2] ;

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102 however, in the more aggress ive and varied marine environment, the microfouling community is more diverse and complex. It includes many types of algal cells whilst a climax marine community comprises a variety of macroalgae and invertebrates such as barnacles, tubeworm and sponges [ 3,17] Since the observations in the early twentieth century (1910s) o n the effect of substrate structure on cellular behavior [127,128] [129] scientis ts and engineers have categorized thre e main factors effective in controlli ng cellular response and function. These surface parameters include chemistry, topography, and mechanics, and have been deem ed as physicochemical cues [9] A direct effect of surface topography and substrate mechanics are the mechanical forces exerted on and sensed by a settling and/or attaching cell. This phenomenon is known as mechanotransduction and its characterization and manipulation have been studied in great detail for applications in cell and tissue engineeri ng [130 132] In the arena of marine biofouling, surface properties have been studied for applications as antifouling and fouling release coatings. Variations in surface chemistry, in relation to surface energy [133 135] and surface topography to create superhydrophobic surfaces [42,43] have been used to control the settlement and adhesion of marine fouling organisms. However, most of these antifouling strategies do not consider the role of mechanotransduction in the design and modeling of non fouling s urfaces. I present a design methodology for the creation of non toxic, antifouling surfaces for use in the marine environment based on mechanotransduction using nano force gradients. The design and fabrication of these gradients incorporate discrete, micr ometer sized features that are associated with the species specific surface design technique of engineered topography [87]

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103 The effectiveness of nano force gradients for antifouling applications was tested by evaluating the settlement behavior of zoospore s of the alga Ulva linza Ulva (syn. Enteromorpha ) is a green macroalga that is common on seashores throughout the world and as a fouling organism on man made structures including ships. Dispersal and colonization of substrata is mainly through the produc tion of vast numbers of motile spores (zoospores). Zoospores are quadriflagellated, naked (i.e., lacking a cell wall), pyriform cells, and the width of spore body being typically 5 7 m in diameter. Settlement involves the swimming spore locating a suita ble surface on which to settle followed by permanent adhesion through the rapid secretion a glycoprotein adhesive that anchors the spore to the substratum [100] Prior to permanent adhesion, the swimming spore undergoes characteristic pre settlement behav ior that involves a 'searching' pattern of exploration close to the substratum [23,136] A number of surface cues have been shown to moderate the way in which a spore interacts with the substratum including wettability [14,113,114] topography [10,87,89,1 01] friction [137] and microbial biofilms [138] A key role for calcium during spore settlement has recently been shown [139] Theory I hypothesize that nano force gradients caused by variations in topographical feature geometry will induce stress gradi ents within the lateral plane of the membrane or body of a settling cell or microorganism during initial contact. This apparent stress gradient and non equilibrium state will function to destabilize and disrupt normal cell/microorganism (C/M) function, sp ecifically settlement (or attachment). In order to settle and gain stability on the surface, the C/M will need to provide energy to adjust its contact area on each topographical feature such that the stresses are equal. However, the energy necessary for the C/M to achieve

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104 this equilibrium state may be thermodynamically unfavorable and it will leave and probe another area to settle. When designed at the appropriate dimensions, the stress on a membrane/body is controlled by the bending moment or stiffness o f the topographical feature of which it is in contact. The geometric dimensions including width, length, and height of the topographical feature as well as the modulus of the base material define its stiffness. By introducing geometric variations in feat ures contained in the engineered topography, an effective force gradient between neighboring topographical features will be developed. If a C/M simultaneously contacts two topographical features of varying geometries, also of inherently different stiffnes s values, the stress exerted on the membrane/body from one feature will differ from the stress exerted on the membrane from the other, geometrically dissimilar feature (Figure 7 1). This difference in forces will cause a stress gradient within the lateral plane of the membrane/body and a non equilibrium state that will cause a C/M to make a choice between providing the energy to create an equilibrium state (i.e., stresses equal) or move to a different area. I postulate that a critical interaction must be a chieved between the topographical surface and the settling C/M for the designed nano force gradients to be sensed by a C/M. The following list highlights the hypothesized key elements of this interaction. 1. The C/M must remain on the topographical featur es and not be able to settle between features. The latter will occur if the protruding topographical features are spaced apart at a greater distance than the width of the C/M. This will allow the C/M to fit in between protruding features and settle (or a ttach) to an apparent flat surface. 2. The C/M must not be able to contact and settle its entire mass on one single feature. If the width of the top of a protruding topographical feature greatly exceeds (i.e., greater than 4 times) the width of the C/M, se ttlement (or attachment) can occur entirely on one individual topographical feature. The force gradient design requires simultaneous contact between two features.

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105 3. If a C/M is bridged between two topographical features, the C/M must not be able to conta ct the floor between features. This will occur if the topographical aspect ratio (feature height / feature width) becomes small (i.e., less than one) so that it no longer has a physical influence on the C/M. When the aspect ratio is small enough such tha t the C/M can contact the recessed floor between protruding features, the maximum amount of surface area contact, even higher than a flat surface, will occur. A set of feature dimensions (feature width = 2 m, spacing = 2 m, and height = 3 m) that have s hown antifouling efficacy against spores of Ulva has been determined [87] With these critical feature dimensions fixed, the length of each engineered topographical feature was varied from 4 m up to 16 m (Figure 7 2). Micrometer sized posts of a polydi methylsiloxane elastomer (PDMSe) have previously been modeled as linearly elastic cantilever beams under pure bending [140] Applying this model, the amount of lateral force required to cause an end deflection of 10% (or 0.3 m) for each topographical fea ture was estimated (Figure 2) using equation 7 1 where F is the applied force, E is the modulus of elasticity, I is the rectangular moment of area, L is the height of the feature, and y is the end deflection distance [141] (7 1) The E for the p articular PDMSe system used in my study is approximately 1.4 MPa [116] Deflection forces ranged from 124 nN for the 4 m length element up to 498 nN for the 16 m length element (Table 7 1, Figure 7 2). Force gradients were created by combining the vari ous modeled elements into a 2 element engineered topography at a fixed feature spacing of 2 m (Figure 7 3). Experimental Design Design of Ulva S pecific Nano Force Gradients Modeled topographical features (Figure 7 2) were combined into 2 element engineere d topographies at the critical feature spacing of 2 m for the spores of Ulva (Figure 7 3). Force gradients were characterized as the difference between the modeled deflection forces for each

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106 topographical feature. Gradient surface 1 (GR1) was designed w ith the 4 m length feature neighbored by the 8 m length feature and an estimated nano force gradient of 125 nN. Gradient surface 2 (GR2) contained the 4 m and 12 m length features (gradient = 249 nN), gradient surface 3 (GR3) included the 4 m and 16 m length features (gradient = 374 nN), and gradient surface 4 (GR4) was designed with the 8 m and 12 m features (gradient = 125 nN). Gradient surface 0 (GR0), 4 m length feature, and gradient surface 5 (GR5), 12 m length feature, were designed to con tain no force gradient as neighboring features were identical. For these engineered topographies fabricated in PDMSe, it was hypothesized that spore settlement would decrease with an increase in the force gradient. A smooth PDMSe surface (SM) was include d in experiment as a positive standard and the most effective engineered topography to date for reducing spore settlement [87] Sharklet AF (SK) was included as an experimental negative standard. Materials A pl atinum catalyzed PDMSe, Silastic T 2 (Dow Cor nin g Corporation ), was used as the base material for engineered topographical modification. The elastomer was prep ared by mixing ten parts resin and one part curing agent by weight for 5 minutes. The mixture was d egassed under vacuum (28 30 in. Hg ) for 3 0 minutes, removed, and allowed to cure for 24 hours at ~ 22C against negative topographical molds. Engineered Topography Mold Fabrication Negative molds of the engineered topographies based on the force gradient pattern designs (Figure 7 3) were fabricat ed in silicon wafers. The gradient pattern designs were transferred to photoresist coat ed silicon wafers and were deep reactive ion etched to a depth of 3 m using my techniques. Wafers were then cleaned (i.e., stripped of photoresist) with an oxygen pla sma etch.

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107 Hexamethyldisilazane was vapor deposited on the processed silicon wafers to methylate the surfaces in order to prevent adhesion to PDMSe during the replication process. Engineered Topography Replication Engineered topographies were transferred t o PDMSe from replication of the patterned and etched silicon wafers. The resultant engineered topographies contained features projecting from the surface at heights respective of the etch depth. Pattern fidelity was evaluated using light and scanning ele ctron microscopy (Figure 7 4). Contact Angle Characterization The three phase water contact angle for each PDMSe surface was measured using a Ram Hart contact angle goniometer with an automated drop dispenser and video capt ure system. T he reported contac t angle ( standard deviation) represents the average contact angle for seven to nine 5 pure water with a cm. Sample Preparation for Ulva Settlement A ssay Three replicates of each PDMSe surface, permanently a dhered to glass microscope slides, were shipped to the University of Birmingham, UK, to be evaluated for settlement of spores of Ulva Engineered topographies included gradient surfaces, GR0 GR5 (Figure 7 4), at a feature height of 3 m. A uniformly smoo th PDMSe sample (SM, Figure 7 4) was included as a positive standard. The Sharklet AF (SK, Figure 7 4) surface at a feature height of 3 m was included as a negative standard. The spore settlement assay and collection of raw data were conducted by Dr. Ma ureen Callow and Dr. John Finlay of the University of Birmingham, UK. Fertile plants of Ulva linza and the zoospores were released and prepared for settlement experiments as previously described [10] Surfaces were pre soaked in nano pure water for four days prior to the assay in order for the surfaces to fully wet. Samples were transferred to artificial seawater (ASW), Tropic Marin,

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108 for 1 hour prior to experimentation without exposure to air. Sampl es were then rapidly transferred to assay dishes. Ten ml of spore suspension (adjusted to 2 x 10 6 ml 1 ) was added to each dish and placed in darkness for 60 minutes. The slides were rinsed to wash away unsettled (or swimming) zoospores and fixed with 2% glutaraldehyde in ASW as previously described [10] Settled spores were quantified using a Zeiss epifluorescence microscope connected to a Zeiss Kontron 3000 image analysis system. Thirty counts were obtained from each of three replicates at 1 mm interval s along both the vertical (15) and horizontal (15) axes of the slide. Statistical Methods Spore density was reported as the mean number of settled spores per mm 2 from 30 counts on each of three replicate slides per surface type standard error (n = 3). S tatistical differences between surfaces were evaluated using a nested analysis of variance (ANOVA) followed by the Student Newman Keuls (SNK) test for multiple comparisons [95] Replicate slides (3) were treated as nested variables within each surface typ e. Each replicate was associated with 30 random spore density counts. Statistical computations were completed using Minitab 14 statistical software package. Results Static surface energy measurements were obtained on each PDMSe surface prior to exposure to the zoospores of Ulva The sessile drop water contact angle measured for each engineered topography, including gradient surfaces 1 5 (GR0 GR5) and Sharklet AF (SK), all fell within the range of 134 4 to 138 3 degrees (Table 7 2). The smooth PDMSe surface had a contact angle of 112 5 degrees. The mean spore ( Ulva ) density ( standard error, n = 3, counts = 30 per n) was determined for each PDMSe surface tested (Figure 7 5). Significantly different groups are represented by

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109 horizontal connecting b ars (Figure 7 5, SNK test p < 0.05). The highest spore density (484 78 spores/mm 2 ) was measured on the smooth (SM) positive standard. The negative standard, Sharklet AF, had the lowest measured spore density (160 33 spores/mm 2 ). Surfaces designed to contain no force gradient (0 nN, GR5 & GR0) had the highest (442 91 spores/mm 2 and 358 63 spores/mm 2 respectively) spore density values among the gradient designs. The lowest spore density among gradient surfaces (229 36 spores/mm 2 53% reduction relative to smooth) was measured on the surface with the highest modeled force gradient (GR3, 374 nN). The remaining gradient surfaces (GR4, 358 58 spores/mm 2 ; GR2, 297 46 spores/mm 2 ; and GR1, 258 46 spores/mm 2 ) with designed force gradients ranging from 125 nN to 249 nN all significantly reduced spore density (26%, 39%, and 47% respectively) relative to the smooth PDMSe surface. The density and distribution of settled spores relative to topographical features can be seen by inspection of light micro graphs obtained by a mixture of epifluorescence and transmitted light (see representative images in Figure 7 6). Spores appear as red spots, approximately 5 m in diameter, due to the autofluorescence of chlorophyll. For gradient surfaces containing no f orce gradient (GR0 & GR5), spores appeared to preferentially settle at the end of the long axis of each feature (4 m for GR0; 12 m for GR5) adhering between the short axes (2 m in width) of neighboring features (Figure 7 7). Furthermore, it was observe d that spores generally made four points of contact in this area as opposed to two points of contact if settled between the long axes of neighboring features. The preferential settlement location of spores to areas of four points of contact on gradient su rface 4 (GR4, Figure 7 8) was similar to gradient surface 5 (GR5, Figure 7 7). Due to the design of gradients surfaces 1 3 (GR1 GR3) and Sharklet AF (SK), spores are only able to make a maximum of three points of contact among the available settlement

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110 loc ations. As expected, spores were observed to be settled in the areas where three points of contact are possible (GR1 GR3 and SK, Figure 7 8). Discussion The gradient surfaces designed with nano force gradients between 125 nN to 374 nN all significantly re duced the settlement of the zoospores of Ulva relative to the smooth PDMSe surface. The highest reduction among gradient surfaces, 53%, was for the surface with the highest modeled force gradient (GR3, 374 nN). The gradient surfaces containing no force g radients had the lowest (GR5, 0 nN) and tied for the 2 nd lowest (GR0, 0 nN) reduction relative to smooth. The other gradient surfaces with modeled force gradients between 125 nN and 249 nN all fell within these two limits in terms of reduction relative to the smooth PDMSe surface. These results indicated that a force gradient approach, considering the mechanotransduction response of a settling C/M, may be a conceptual methodology for the design of non fouling engineered topographies. By designing surface topography that considers the local micrometer scale effects on the C/M rather than the overall surface property of the material (e.g., superhydrophobicity and surface energy [43,42] ), more effective non toxic antifouling surfaces and models can be develo ped. For example, the engineered topographies studied, including gradient surfaces and the Sharklet AF, all had static water contact angles within the range of 134 to 138 degrees, yet yielded significant differences in spore settlement behavior. The Shark let AF surface was not designed based on force gradients [87,89] The design was inspired by nature and modeled after the skin of a shark. Coincidently, the Sharklet AF does contain a variety of discrete nano force gradients across the surface. By apply ing the same model to estimate a force gradient between features as with the gradient designs, a nano force gradient of 125 nN was estimated in three different areas of the topography. These areas

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111 occurred between the 4 and 8 m length features, the 8 and 12 m length features, and the 12 and 16 m length features. Although this modeled force gradient was not the highest of the gradient designs, the Sharklet AF surface had the lowest measured spore density than any of the gradient surfaces. This, however could be due to the fact that the Sharklet AF was a more complex, 4 element engineered topography, compared to the 2 element gradient surfaces. The 4 element, Sharklet AF may have presented a more tortuous surface for the zoospore as they explored the s urface. In addition, other engineered topographies, not designed based on force effectiveness against the spores of Ulva [87] There still is much to be stud ied and understood in terms of the unique properties of the Sharklet AF surface which make it such an effective antifouling surface. Figure 7 1. Hemisphere representing a settling cell/microorganism contacting two dissimilar topogr aphical features. Du ring initial contact and deformation, this dissimilarity theoretically creates a stress gradient within the lateral plane of the membrane/body due to the difference in bending moments between the two geometrically dissimilar features and the strain on the membrane induced by the separation distance between the features.

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112 Table 7 1. Values used to calculate end deflection forces using Equation 7 1 for topographical features pictured in Figure 7 2. End deflection (10%) Feature (length width) E (MPa) I (m 4 ) L (m) Calculated force (nN) 0.3 m 4 m 2 m 1.4 2.7 10 24 3 10 6 124 0.3 m 8 m 2 m 1.4 5.3 10 24 3 10 6 249 0.3 m 12 m 2 m 1.4 8.0 10 24 3 10 6 373 0.3 m 16 m 2 m 1.4 1.1 10 23 3 10 6 498 Figure 7 2. Estimated lateral forces required to cause a 10% end deflection of micrometer sized topographical features in PDMSe modeled as cantilever beams (3 m feature height).

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113 Figure 7 3. Pattern designs of 2 element engineered topographies representing a range of model ed nano force gradients. Gradient surface 1 (GR1), 4 m and 8 m length features, and gradient surface 4 (GR4), 8 m and 12 m length features, contain an estimated force gradient of 125 nN. Gradient surface 2 (GR2), 4 m and 12 m length features, inclu de a force gradient of 249 nN. Gradient surface 3 (GR3), 4 m and 16 m length features, was designed at a force gradient of 374 nN. Gradient surface 0 (GR0), 4 m length feature, and gradient surface 5 (GR5), 12 m length feature, contain no force gradi ent as neighboring features are the same.

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114 Figure 7 4. SEM images of force gradient engineered topographies fabricated in PDMSe by replication of silicon wafer molds. GR0 and GR5 contain no force gradients. GR1 and GR4 contain estimated force gradient s of 125 nN. GR2 contains a force gradient of 249 nN. GR3 contains a force gradient of 374 nN. The Sharklet AF (SK) engineered topography was included as a negative standard. A smooth PDMSe surface (SM) was included as a positive standard.

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115 Table 7 2. Sessile drop water contact a ngle for PDMSe s urfaces Surface type Mean contact angle (degrees SD ) GR0 138 3 GR1 134 3 GR2 136 1 GR3 134 4 GR4 135 2 GR5 137 4 SK 135 2 SM 112 5 GR0 GR5 = gradients surfaces 0 5; SK = Sharklet AF ; SM = Smooth PDMSe surface SD = standard deviation Figure 7 5. Mean spore ( Ulva ) density ( standard error, n = 3, counts = 30 per n) measured and calculated for each PDMSe surface studied (3 m feature height for all engineered topographies). The experimental design included a positive (smooth PDMSe surface, SM) and negative (Sharklet AF PDMSe surface, SK) standard. Gradient surfaces included forces of 0 nN (GR5 and GR0), 125 nN (GR1 and GR4), 249 nN (GR2), and 374 nN (GR3). Horizontal bars indi cate significantly different groups (SNK test, p<0.05).

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116 Figure 7 6. Representative light micrographs, obtained by a mixture of epifluorescence and transmitted light, of spores ( Ulva ) settled on PDMSe surfaces Surfaces included gradient surfaces 0 5 ( GR0 GR5), Sharklet AF (SK), and uniformly smooth (SM). Spores in these images appear as red spots approximately 5 m in diameter due to the autofluorescence of chlorophyll.

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117 Figure 7 6. Magnified light micrographs showing the preferred settlement locat ion of spores relative to the topographical features on GR0 and GR5. Figure 7 7. Magnified light micrographs showing the preferred settlement location of spores relative to the topographical features on GR1 GR4 and SK.

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118 CHAPTER 8 CONCLUSIONS AND FUTU RE WORK Marine Biofouling Novel, non toxic antifouling technologies are focussing on the manipulation of surface topography to deter settlement of the dispersal stages of fouling organisms. Species specific topographical desi gns called engineered topographies have been designed, fabricated and evaluated for potential applications as antifouling marine coatings and biomaterial surfaces capable of reducing biofilm formation. Design of experiments was used to define specific fea ture characteristics of engineered topography that are most important for effecting the settlement and growth of fouling organisms. The effect of feature size, geometry, and roughness on the settlement of zoospores of the ship fouling alga Ulva was evaluat ed using engineered microtopographies in polydimethylsiloxane elastomer (PDMSe). The engineered topographies studied were designed at a feature spacing of 2 m and all significantly reduced spore settlement compared to a smooth surface. An indirect corre lation between spore settlement and a newly described engineered roughness index (ERI) was identified. fraction, and the degree of freedom of spore movement. Uniform surfac es of either 2 m diameter circular pillars (ERI = 5.0) or 2 m wide ridges (ERI = 6.1) reduced settlement by 36% and 31% respectively. A novel multi feature topography consisting of 2 m diameter circular pillars and 10 m equilateral triangles (ERI = 7. 0) reduced spore settlement by 58%. The largest reduction in spore settlement, 77%, was obtained with the Sharklet AF topography (ERI = 8.6). The effect of the aspect ratio (feature height / feature width) of topographical features engineered in PDMSe, on the settlement of cyprids of Balanus amphitrite and zoospores of Ulva

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119 linza was investigated The correlation of relative aspect ratios to antifouling efficacy was proven to be significant. An increase in aspect ratio resulted in an increase of fouling deterrence for both zoospores and cyprids. The spore density of Ulva was reduced 42% with each unit increase in aspect ratio of the Ulva specific Sharklet AF topography. Similarly, the number of settled cyprids was reduced 45% with each unit increase in aspect ratio. The newly described barnacle specific Sharklet AF topography (40 m feature height, aspect ratio of 2) reduced cyprid settled by 97%. T echniques have been developed to superimpose the smaller Ulva specific topographies onto the barnacle spec ific surfaces into a hierarchical structure to repel both organisms simultaneously. The results for spore settlement on first generation hierarchical surfaces provide insight for the efficacious design of such structures when targeting multiple settling s pecies. Medical Biofilm Formation The surface of an indwelling medical device can be colonized by human pathogens that can form biofilms and cause infections. In most cases, these biofilms are resistant to antimicrobial therapy and eventually necessitate removal or replacement of the device. The engineered surface microtopography effective for the spores of green algae ( Ulva ), Sharklet AF, was investigated for its ability to disrupt the formation of bacterial biofilms without the use of bactericidal agent s. The Sharklet AF was fabricated PDMSe and tested against smooth PDMSe for biofilm formation of Staphylococcus aureus over the course of 21 days. The smooth surface exhibited early stage biofilm colonies at 7 days and mature biofilms at 14 days, while th e topographical surface did not show evidence of early biofilm colonization until day 21. At 14 days, the mean value of percent area coverage of S. aureus on the smooth surface was 54% compared to 7% for the Sharklet AF surface (p < 0.01). These results suggest that surface modification of indwelling

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120 medical devices and exposed sterile surfaces with the Sharklet AF engineered topography may be an effective solution in disrupting biofilm formation of S. aureus Engineered Nano Force Gradients The identific ation of effective antifouling topographies typically occurs through trial and error rather than predictive models. A model and design methodology for the identification of non toxic, antifouling surface topographies for use in the marine environment by t he creation of engineered nano force gradients was developed. The design and fabrication of these gradients incorporate discrete micrometer sized features that are associated with the species specific surface design technique of engineered topography and the concepts of mechanotransduction. The effectiveness of designed nano force gradients for antifouling applications was tested by evaluating the settlement behavior of zoospores of the alga Ulva linza The surfaces with nano force gradients ranging from 125 to 374 nN all significantly reduced spore settlement relative to a smooth substrate with the highest reduction, 53%, measured on the 374 nN gradient surface. These results confirm that designed nano force gradients may be an effective tool and predict ive model for the design of unique non toxic, non fouling surfaces for marine applications as well as biomedical surfaces in the physiological environment. Protruding and Recessed Features It is consistently observed that algal zoospores of Ulva and bacter ial cells of Staphylococcus aureus are settled and attached in the recessed area between protruded features rather than on the top of an equivalent area of a protruded feature (Figure 8 1). Potential antifouling engineered topographies are all fabricated such that features are protruding from the surface rather than recessed within. It would be expected that zoospores and bacterial cells will respond differently to proven antifouling engineered topographies with the pattern recessed within surface rather than protruding, but this hypothesis has yet to be tested experimentally.

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121 This was due to the fact that a fabrication technique to produce the same pattern into two separate engineered topographies; one containing protruding features and another containin g recessed features had not been identified. Recently, however, a method has been developed to produce the same pattern with features in both the protruded and recessed form. An intermediate, elastomeric (Kraton G1657) replication of a microfabricated sil icon wafer was produced via solvent casting that resulted in an engineered topography with protruding features on the surface (Figure 8 2). A solution of 10% Kraton G1657 and 90% toluene (weight per weight) was used for solvent casting. Curing polydimeth ylsiloxane elastomer (PDMSe), Silastic T 2, was then poured directly on the replicated Kraton surface and allowed to cure for 24 hours. The cured PDMSe was easily removed from the Kraton and no evidence of physical adhesion occurred during this replicatio n step. The resultant engineered topography on the PDMSe contained recessed features within the surface (Figure 8 2). Using this technique, bioassays can be conducted that compare the influence of protruding features vs. recessed features of the same pat tern on biological settlement and biofilm growth.

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122 Figure 8 1. Microscope images of algal zoospores of Ulva and bacterial cells of Staphylococcus aureus preferentially attached to the recessed area between protruding features. A) Light microscope image of zoospores of Ulva (~ 5 m diameter) on an engineered topography (20 m wide features spaced by 20 m) in PDMSe. B) SEM image of cells of Staphylococcus aureus (~ 1 m diameter) on an engineered topography (2 m wide features spaced by 2 m) in PDMSe. Figure 8 2. SEM images of the Sharklet AF pattern fabricated as both protruding features and recessed features in PDMSe. A) Top down SEM image of protruding features. B) Tilted (35) SEM image of protruding features. C) Top down SEM image of recesse d features. D) Tilted (35) SEM image of recessed features.

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123 APPENDIX A SINUSOIDAL DESCRIPTI ON OF DESIGNED PATTE RNS USED AS TEMPLATE S OF ENGINEERED TOPOGRAPH Y There exists no numerical description of the designed patterns that are the basis and templates for the creation of engineered topography. Presented here is a numerical description of periodic, designed patterns using two sinusoidal waves. A general equation is developed for the pattern in which the only restrictions are that two features in each periodic pattern have at least one dimension of discrepancy in their geometry, and their spatial arrangement to one another is periodic throughout the pattern. All the elements and features in between and/or around the two periodic features becomes generally selectable in dimension. This numerical description is an attempt to establish a patentable claim to cover a broad enough range of possible pattern designs to protect the intellectual property associated with surfaces such as the Sharklet AF design [21]. existing designed pattern and describing the arrangement of features using a combination of sinusoidal waves. The Sharklet AF design (Figure A 1) will be used for this example. Dimensions are not necessary for this exercise. Dimensions wi ll be applied when a numerical example is worked using the developed generalized equations. By inspection of the periodicity of the features in Figure A 1, a sine wave of the form y(x) = A sin(wx), where A is the amplitude and w is the frequency, can be us ed to describe one row of this periodicity (Figure A 2). Another periodic set of features is present above and below this initial wave and can be described by an additional sine wave that is out of phase from the which defines a cosine wave. This additional row of periodicity can be represented using a cosine wave in the form y(x) = B + A cosine (wx) (Figure A 3).

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124 The entire surface area of the topography can therefore be numerically represented by a combination o f both sinusoidal waves (Equations A 1 and A 2). y(x) N = cN + A sin(wx) (A 1) y(x) N = cN + B + A cos(wx) (A 2) The total area of coverage of the pattern is described by the limits of N and x where N = 0, 1, 2, pattern in Figure A 4 can be numerically represented using N = 0, 1, and 2, A = 8, B = 20, c = 24, and w = ( / 12). Additionally, the parameters can be modified such that patterns containing a different number of total elements such as a 2 element (Figu re A 5) and 5 element design (Figure A 6) can be described. Two arbitrary elements are shown in Figure A 7 differing in both length and width. First, the centroid (center of mass) of the smaller feature (Figure A 8) and the topmost point of the larger fea ture is defined (Figure A 8). If multiple topmost points exist, the point closest to the centroid of the larger feature is selected. The following equations define the values for A, B, and c in equations A 1 and A 2. A = (1/2) (L D ) (A 3) L D : y dimension of the larger of the two elements (Figure A 8) B = (1/2) (S D ) + (P S ) + (1/2) (L D ) (A 4) S D : y dimension of smaller of two elements (Figure A 8) P S : defined y spacing between the two elements after aligning (Figure A 9) c = L D + 2 (P S ) + S D (A 5) (A 6) w: angular frequency (rad) f: frequency (hertz) T: wave period T = 2 X D (A 7) X D : defined x dimension measured from the centroid of smaller feature to the topmost point on the larger feature (Figure A 8)

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125 With the parameters defined, the space between the two features is filled with elements of any geometry and size and can be random or periodic. For this example, the filled elements are periodic throughout the structure (F igure A 10). The start of the first sine wave (Eq. A 1, N = 0) is positioned at the origin of a standard Cartesian coordinate system represented by x and y axes. For all sine waves (Eq. A 1, N = 0, 1, es is positioned at all positive values of x and y such that y N (x) = cN. This relationship occurs within wave periods when sin(wx) = 0. The topmost point of the larger feature is positioned at all positive values of x and y such that y N (x) = cN + A. Thi s relationship occurs within wave periods when sin(wx) = 1. For all cosine waves (Eq. A all positive values of x and y such that y N (x) = cN + B. This relationship occu rs within wave periods when cos(wx) = 0. The topmost point of the larger feature is positioned at all positive values of x such that y N (x) = cN + B + A. This relationship occurs within wave periods when cos(wx) = 1. The variables A, B, c, and w are calcu lated for this example using equations A 3 through A 7. A = (1/2) (18) = 9 B = (1/2) (6) + (3) + (1/2) (18) = 15 c = 18 + (2) (3) + 9 = 33 The calculated variables and combined with equations A 1 and A 2. y N (A 8) y N (A 9)

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126 The example pattern is arrayed according to the Equations A 8 and A 9 for N = 0 and x = 0 to (Figure A 11) and extended over a larger array for N = 0, 1, and 2 and x = 0 to 3 (Figure A 12). This can be further extended to any positive value of N and x to cover a desired surface area. Figure A 1. Designed Sharklet AF pattern used to create the Sharklet AF engineered topography.

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127 Figure A 2. Sine wave (blue line) applied to a periodic row of the Sharklet AF pattern. Figure A 3. Cosine wave (red line) applied to the periodic row above the sine wave (b lue line) on the Sharklet AF pattern.

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128 Figure A 4. Sharklet AF pattern described by three sets (N = 0, 1, and 2) of sine and cosine waves (Eqs. 4 1 and 4 2) for A = 8, B = 20, c = 24, and w = ( / 12). Figure A 5. Two element designed pattern repres ented by two sinusoidal waves.

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129 Figure A 6. Five element designed pattern represented by two sinusoidal waves. Figure A 7. Two arbitrary elements selected for the creation of a designed pattern based on the derived sinusoidal waves.

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130 Figure A 8. Definition and location of sinusoidal wave variables including S D X D L D centroid ( ) of smaller feature, and topmost point ( ) of larger feature. Figure A 9. Definition and location of sinusoidal wave variable P S

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131 Figure A 10. Arbitra ry shapes used to fill the space between the periodic elements. Figure A 11. Designed pattern from Figure A 10 arrayed as defined by the derived sinusoids (Eqs. A 8 and A 9) for N = 0 and x = 0 to

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132 Figure A 12. Designed pattern from Figure A 10 arrayed as defined by the derived sinusoids (Eqs. A 8 and A 9) for N = 0, 1, and 2 and x = 0 to 3

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144 BIOGRAPHICAL SKETCH James Frederick Schumacher, son of Frederick John and Susan Marie Schumacher, was born in Milwaukee, Wisconsin. James lived in the suburbs of Milwaukee for the first 22 years of his life with his parents and sister, Jennele, where he a ttended Elm Dale Elementary School, Greenfield Middle School, and Charles B. Whitnall High School. James maintained a straight A average each semester for most of his early academic career while working part time during the week and on weekends since the age of 14. He worked as a dishwasher, babysitter, busboy, cook, waiter, caterer, and farmhand. In his spare time, James enjoyed golf, bowling, and going After graduating high school in J Distinguished Scholar Award to attend Marquette University to study biomedical engineering. While attending Marquette, James interned with the Kimberly Clark Corporation for four alternating semesters between a cademic studies. He graduated magna cum laude with a BS in Biomedical Engineering in May of 2002. James left Wisconsin at the age of 22 to attend graduate school at the University of Florida. He received the Alumni Fellowship Award and joined the J. Cray ton Pruitt Family Department of Biomedical Engineering to study biomaterials under the guidance of Professor Anthony Brennan. James completed his dissertation and will receive his PhD in Biomedical Engineering in August of 2007. He and his wife, Iris, wi ll be moving to Atlanta to work on medical materials and devices for the Kimberly Clark Corporation.