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Gingival!Health!Transcriptome! ! ! ! Gingival!Health!Transcriptome! ! Thesis! ! Presented!in!Partial!Fulfillment!of!the!Requirements!for!the!Degree!Master!of! Science!in!the!Graduate!School!of!The!Ohio!State!University! ! By! Christina!Zachariadou,!DDS! Graduate!Program!in!Dentistry! The!Ohio!State!University! 2018! ! ! Thesis!Committee:! Angelo!J.!Mariotti,!DDS,!PhD,!Advisor! Thomas!C.!Hart,!DDS,!PhD! John!D.!Walters,!DDS,!MMSc! ! ! ! 1! ! ! ! ! ! ! ! ! ! ! Copyright!by! Christina!Zachariadou! 2018! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 2! ! ! ! Abstract! ! Introduction:!In!the!field!of!periodontology,!a!satisfactory!definition!of!periodontal! health!is!lacking.!Instead,!clinicians!use!surrogate!measures,!such!as!color,!texture,! consistency,!probing!depths!and!bleeding!on!probing!to!examine!periodontal!tissues! and! diagnose! disease,! or! the! absence! of! it,! which! they! define! as! “clinical! health”.!! Additionally,!it!has!been!shown!that!age!progression!is!accompanied!by!changes!in! the!periodontium.!As!a!result,!understanding!the!gene!expression!in!healthy!gingiva,! through!the!field!of!transcriptomics,!could!provide!some!insight!on!the!molecules! that!contribute!to!gingival!health.!Also,!comparing!the!transcriptome!of!young!and! older!subjects,!taking!into!consideration!the!effect!of!sex/gender,!can!shed!light!on! differential! gene! expression! with! age! progression! and! on! individual! differences! between! sexes,! and! may! provide! future! therapeutic! endpoints! of! periodontal! treatment.!The!main!focus!of!this!study!was!to!ascertain!collagen!(COL)!and!matrix! metalloproteinase!(MMP)!gene!expression!in!clinically!healthy!gingival!tissues.! ! Material! and! Methods:! ! Gingival! biopsies! were! obtained! from! nineteen! nonZ smoking!individuals!with!clinically!healthy!gingiva.!!The!location!of!biopsy!collection! was! the! interpapillary! attached! gingiva! between! the! second! premolar! and! first! ! ii! molar! of! the! two! maxillary! quadrants.! Immediately! after! harvesting,! the! tissues! were! immersed! and! stored! in! RNAlater! solution.! Two! age! groups! were! included:! “young”! (18Z35! years! old)! and! “older”! individuals! (≥60! years! old).! RNA! was! extracted! and! NextZGeneration! RNA! Sequencing! was! performed,! with! the! use! of! Unique! Molecular! Identifiers! (UMIs).! Collagen! and! MMP! gene! expression! was! described.!Comparisons!were!performed!between!young!and!older!individuals!as!a! total,!as!well!as!between!young!versus!older!females,!and!young!versus!older!males.! Statistical!significance!was!evaluated!using!the!student’s!tZtest!with!a!PZvalue!≤!0.05! set!as!the!statistically!significant!level.! ! Results:! RNAZsequencing! analysis! with! Unique! Molecular! Identifiers! (UMIs)! showed!that!1790!genes!were!differentially!expressed!between!the!young!and!old! groups;! 347! genes! were! upregulated! and! 1443! genes! were! downregulated! with! aging.! Expression! of! fortyZeight! collagen! genes! was! seen! in! the! gingiva! of! participating!individuals.!!The!top!four!expressed!collagen!genes,!with!high!number! of!transcripts,!were!COL1A2,!COL17A1,!COL3A1!and!COL1A1.!Fifteen!collagen!genes! were!downregulated!with!aging,!and!one!(COL4A6)!was!upregulated.!Decreases!in! collagen! gene! expression! seemed! to! be! sexZspecific,! primary! affecting! the! female! aging!group,!while!only!one!collagen!gene!(COL16A1)!was!downregulated!in!aging! males.!!TwentyZfive!MMP!genes!were!expressed!in!our!sample.!The!five!genes!with! the! highest! expression! were! MMP2,! MMP28,! MMP24ZAS1,! MMP16! and! MMP14.! ! iii! MMP2!and!MMP15!expression!was!downregulated!in!aging!females,!while!MMP16! was!downregulated!in!aging!males.!! ! Conclusion:! Transcriptomic! analysis! showed! that! fortyZeight! COL! genes! and! twentyZfive! MMP! genes! are! expressed! in! clinically! healthy! gingiva.! The! results! of! this!study!indicate!that!collagen!gene!expression!decreases!with!aging.!This!effect!is! more! prominent! in! females.! MMP! expression! seems! to! be! stable! throughout! life,! with!only!MMPZ2!and!MMPZ15!decreasing!in!aging!females!and!MMP16!decreasing! in!aging!males.! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! iv! ! ! ! ! ! ! ! ! Dedication! ! Dedicated!to!my!parents,!Nikolaos!and!Vasiliki,!and!my!sister,!Lydia!for!providing! me!with!all!necessary!love,!values!and!perseverance!so!that!one!day!I!have!the! chance!to!become!an!excellent!human!being! ! ! ! ! ! ! ! ! ! ! v! ! ! ! Acknowledgements! ! I!would!like!to!thank!my!advisor,!Dr.!Angelo!Mariotti,!for!giving!me!the!opportunity! to! work! with! him! in! this! project! and! throughout! my! residency.! His! help! and! guidance!were!invaluable.! ! I! thank! Dr.! Thomas! Hart! for! his! pivotal! role! in! the! process! and! analysis! of! my! biopsies,!his!unique!ideas,!and!for!being!a!member!of!my!committee.! ! I! thank! Dr.! John! Walters! for! providing! insightful! suggestions! on! this! project,! as! a! member!of!my!committee,!and!for!his!emotional!support!during!the!stressful!course! of!residency.! ! I!thank!Dr.!Dimitris!Tatakis!for!his!constructive!criticism!and!dedication!to!push!me! to!work!harder!and!expand!my!limits.! ! I!thank!Deborah!Hooper!for!all!her!help!with!data!collection!and!advice!throughout! my!project.! ! ! vi! I!thank!my!fellow!residents!in!the!Department!of!Periodontology!for!their!assistance! in!recruiting!patients!for!my!study.! ! Finally,! I! would! like! to! thank! my! family! for! all! their! unconditional! love,! help! and! emotional!support.! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! vii! ! ! ! Vita! ! 2008Z2013!...................................................!DDS,!Aristotle!University!of!Thessaloniki,!Greece! 2014Z2015……………………………………………………….Private!Practice!/!General!Dentistry! 2015Z2018!................................................................!PostZDoctoral! Training! in! Periodontics,! School!of!Dentistry,!The!Ohio!State!University,!Columbus,!Ohio,!USA! 2016Z2018…………………Teaching!Associate,!Department!of!Periodontology,!School!of! Dentistry,!The!Ohio!State!University,!Columbus,!Ohio,!USA! 2017Z2018………Chief!Resident,!Department!of!Periodontology,!School!of!Dentistry,! The!Ohio!State!University,!Columbus,!Ohio,!USA! ! ! ! Fields!of!Study! ! Major!Field!of!Study:!!Dentistry! ! ! ! ! viii! ! ! ! Table!of!Contents! ! Abstract!!......................................................................................................................................!ii! Dedication!..................................................................................................................................!v! Acknowledgements!................................................................................................................!vi! Vita!.................................................................................................................................................!viii! ! Chapters! ! Chapter!1:!Introduction!........................................................................................................!1! Chapter!2:!Materials!and!Methods!...................................................................................!18! Chapter!3:!Results!...................................................................................................................!23! Chapter!4:!Discussion!............................................................................................................!30! Chapter!5:!Conclusion!...........................................................................................................!44! References!..................................................................................................................................!46! Appendix!A:!Distribution!of!subjects!in!sex!and!age!groups!................................!53! Appendix!B:!General!Patient!Characteristics!!.............................................................!55! Appendix!C:!Antibiotic!Intake!............................................................................................!59! Appendix!D:!Clinical!Parameters!......................................................................................!61! ! ix! Appendix!E:!NextZGeneration!Sequencing!Reads!......................................................!63! Appendix!F:!Collagen!Genes!–!Total!Number!of!Transcripts!...............................!65! Appendix! G:! Number! of! Collagen! Gene! Transcripts! Expressed! in! the! Young! Male! Group!............................................................................................................................................!68! Appendix!H:!Number!of!Collagen!Gene!Transcripts!Expressed!in!the!Old!Male!Group !.........................................................................................................................................................!71! Appendix!I:!Number!of!Collagen!Gene!Transcripts!Expressed!in!the!Young!Female! Group!............................................................................................................................................!74! Appendix! J:! Number! of! Collagen! Gene! Transcripts! Expressed! in! the! Old! Female! Group!............................................................................................................................................!77! Appendix!k:!Differential!Collagen!Gene!Expression!with!Aging!........................!80! Appendix!L:!Differential!Collagen!Gene!Expression!between!Sexes!.................!83! Appendix!M:!MMP!Genes!–!Total!Number!of!Transcripts!.....................................!86!
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