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Mass Spectrometry-Based Proteomics Analysis of Bioactive Proteins in EMD That Modulate Adhesion of Gingival Fibroblast to Improve Bio-Integration of Dental Implants

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Mass Spectrometry-Based Proteomics Analysis of Bioactive Proteins in EMD That Modulate Adhesion of Gingival Fibroblast to Improve Bio-Integration of Dental Implants Western University Scholarship@Western Electronic Thesis and Dissertation Repository 3-28-2019 3:00 PM Mass Spectrometry-Based Proteomics Analysis Of Bioactive Proteins In EMD That Modulate Adhesion Of Gingival Fibroblast To Improve Bio-Integration Of Dental Implants David Zuanazzi Machado Jr The University of Western Ontario Supervisor Siqueira, Walter L. The University of Western Ontario Graduate Program in Biochemistry A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © David Zuanazzi Machado Jr 2019 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Biochemistry Commons, Dental Materials Commons, Oral Biology and Oral Pathology Commons, and the Periodontics and Periodontology Commons Recommended Citation Zuanazzi Machado, David Jr, "Mass Spectrometry-Based Proteomics Analysis Of Bioactive Proteins In EMD That Modulate Adhesion Of Gingival Fibroblast To Improve Bio-Integration Of Dental Implants" (2019). Electronic Thesis and Dissertation Repository. 6064. https://ir.lib.uwo.ca/etd/6064 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. i ABSTRACT Titanium (Ti) implants are used in dental practice to replace damaged or lost teeth. For effective treatment, the dental implant needs to integrate with the surrounding hard and soft tissues on the implant site. Despite improvements in bone-implant integration that have been achieved through surface modifications, the integration with the soft tissues is still deficient. The oral mucosa that embraces the transmucosal component of the implant only contacts the surface without making a strong attachment with the connective tissue. The lack of attachment offers no protection against bacterial invasion that can lead to local infection and implant loss. Among different modification applied to Ti surfaces, coating the surface with specific proteins is a new area in biomedical research aiming to improve implant biointegration. To this end, proteins that comprise the enamel matrix derivative (EMD) would be good candidates to be used as surface coatings. EMD is a rich protein mixture used as a biomaterial to promotes tissue regeneration by modulating many cells, including gingival fibroblasts, the most abundant cell type of oral connective tissue. However, many proteins containing in EMD are yet to be identified. Considering that surface features are important to modulate protein adsorption, we worked with the hypothesis that we could use the characteristics of different Ti surfaces in a surface-affinity approach to creating unique coatings with EMD. Therefore, it would allow us to identify bioactive proteins within EMD that could be further used as a coating on the implant to promote adhesion of gingival fibroblast to the surface, improving the implant biointegration. Since it is not well-established whether the surface attracts or binds specific proteins from complex mixtures, saliva was used as a ii model in combination with mass spectrometry to investigate surface specificity for protein binding of three different Ti surfaces (PT, SLA, and SLActive). By applying this approach, we showed that the Ti surfaces had a low specificity for protein binding due to high similarity on the pellicle composition despite differences in characteristics between surfaces. The lack of binding specificity led us to explore the EMD composition utilizing another strategy. Through the MudPIT methodology, we fractionated EMD in 32 fractions to characterize its proteome. We identified 2000 proteins through tandem mass spectrometry (MS/MS) including novel proteins that are associated with EMD biological activity, i.e. biomineralization, wound healing and biological adhesion. The obtained EMD fractions were then applied to human gingival fibroblast (HGF) to evaluate their capability to promote cell adhesion on a coated surface. The adhesion assay indicated that two EMD fraction (F23 and F24), which contained the adhesion proteins fibrillin-1 and tenascin C, showed a significantly higher response than native EMD and other EMD fractions. Overall, the work presented herein indicated the need for more studies on surface-proteins interaction given the low surface specificity presented by each Ti surface. Also, this thesis provided an in-depth insight on the complexity of EMD protein composition, including the identification of novel proteins that are related to EMD biological activity such as the adhesion of gingival fibroblasts. Key Words: Dental implants, titanium, size exclusion chromatography, mass spectrometry, proteomics, peptides, gene ontology, fibroblasts, cell adhesion. iii DEDICATION First and foremost, I would like to dedicate this thesis to God and to my beautiful family that always provided me with endless encouragement and support during my academic endeavors. To my lovely wife Maura whose everyday-presence and unconditional love have inspired me to be a better husband and father. Without you, I would never be the person I am today. To my amazing children, Giovanna and Enzo, who have shaped my heart with their hugs, kisses, and words. Holding you in my arms is always breathtaking. Secondly, I would like to thank my parents who raised me with love, values, and discipline. Words are not enough to demonstrate my gratitude for supporting my decision throughout my life, particularly the hardest ones. To my sister Rebeca, thank you for our never-ending friendship and your presence in my life. Thank you all for your companionship during this chapter of my life. Surely, without you, none of this would be possible. iv ACKNOWLEDGEMENT First, I would like to express my sincere gratitude to my supervisor Dr. Walter Siqueira for his continuous support during my Ph.D. study. Thank you for your guidance during my development as a scientist and for providing inspiration and encouragement to continue working on dental research. Also, I want to say a big thanks to the past and present members of the Siqueira lab for the time spent together whether working in the lab or drinking coffee. Every one of you has contributed in many different ways during these five years we shared. Cindy, thank you for always being available to assist me with the mass spectrometer and for providing useful insights during our conversations. You have been a real helper. Thank you to all member of the Junop lab. I genuinely appreciate the support I have received, especially Dr. Murray Junop for generously opening his lab to process my samples and Chris Brown for his patience while showing me how to operate the FPLC system properly. Likewise, I want to extend my gratitude to Dr. Douglas Hamilton and members of the Hamilton Lab for the warm welcome and incredible support while I was using the cell culture room. Thank you, Georgia, Adam, JT, Sarah, and Karrie, for providing the fibroblasts and space and time to culture the cells. At last but not least, I want to thank Dr. Andrew Leask and members of Leask Lab for useful insights and for make yourselves available when needed and for providing the PrestoBlue reagent that was critical to finish my work. v TABLE OF CONTENTS ABSTRACT ......................................................................................................... i DEDICATION …………………………………………………………………………. iii ACKNOWLEDGEMENT ..…………..................................................................... iv TABLE OF CONTENTS ...................................................................................... v LIST OF TABLES ................................................................................................ ix LIST OF FIGURES ............................................................................................. x LIST OF APPENDICES ..................................................................................... xii LIST OF ABBREVIATIONS …………………………………………..................... xiii Chapter 1 Introduction .................................................................................. 1 1.1 Titanium Dental implant ......................................................................... 1 1.2 Osseointegration ................................................................................... 3 1.3 Titanium Surfaces …….......................................................................... 4 1.4 Biomimetic Technology .…..................................................................... 7 1.5 Enamel Matrix Derivative (EMD) ........................................................... 8 1.5.1 Influence of EMD in hard and soft tissues ………...............………. 9 1.6 Mass spectrometry-based proteomics .…........................................... 12 1.6.1 Top-down approach ………………………….………….…...……... 15 1.6.2 Bottom-up approach …………………………...……….…....……... 15 1.6.2.1 Protein/peptides Separation Methods ……….………..…... 16 1.6.2.2 Ionization and proteins identification …..….…………..…... 17 1.7 Thesis rationale ................................................................................... 18 1.8 Scope of thesis .................................................................................... 19 1.9 References ……................................................................................... 22 vi Chapter 2 Salivary Pellicle Proteome Formed onto Three Different Titanium Surfaces ……………………………………………………….
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