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!

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Christina!Zachariadou!

2018!

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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.!

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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!

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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.!

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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!

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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!

Appendix!N:!Number!of!MMP!Gene!Transcripts!Expressed!in!the!Young!Male!Group

!...... !88!

Appendix! O:! Number! of! MMP! Gene! Transcripts! Expressed! in! the! Old! Male! Group

!...... !90!

Appendix! P:! Number! of! MMP! Gene! Transcripts! Expressed! in! the! Young! Female!

Group!...... !92!

Appendix!Q:!Number!of!MMP!Gene!Transcripts!Expressed!in!the!Old!Female!Group

!...... !94!

Appendix!R:!Differential!MMP!Gene!Expression!with!Aging!...... !96!

! x! Appendix!S:!Differential!MMP!Gene!Expression!between!Sexes!...... !98!

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List!of!Tables!

Table!1:!Collagen!gene!expression!...... !28!

Table!2:!MMP!gene!expression!!...... !29!

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List!of!Figures!

Figure!1:!Clinical!photos!of!biopsy!areas!...... !21

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Chapter!1!

Introduction!

Anatomy!of!the!Periodontium!

According!to!the!Glossary!of!Periodontal!Terms,!periodontium!is!defined!as! the! group! of! tissues! that! invest! and! support! the! teeth.! These! tissues! include! the! gingiva,!periodontal!ligament,!root!cementum!and!alveolar!bone!proper1.!!

As! the! present! work! will! focus! on! gingiva,! we! consider! it! necessary! to! provide!more!detailed!information.!Gingiva!is!the!tissue!that!is!visible!clinically;!it! covers!the!alveolar!process!and!cervical!portion!of!the!teeth1.!It!is!unique,!in!a!sense! that!intact!mucosa!(gingiva)!is!penetrated!by!calcified!tissues!(teeth),!at!the!same! time! maintaining! its! ability! to! protect! the! host! from! potential! microbial! attacks2.!

Gingiva! is! the! first! line! of! defense! and! confers! protection! to! the! underlying! periodontal! tissues! with! its! function! as! a! barrier! and! provision! of! rapid! epithelial! cell!turnover,!as!well!as!its!ability!to!respond!to!microbial!challenge!with!production! of! antimicrobial! peptides! and! inauguration! of! the! innate! and! acquired! immune! responses3.!!

! 1! During!clinical!examination,!three!parts!of!the!gingiva!can!be!identified:!free,! attached!and!interdental.!Attached!gingiva!extends!from!the!mucogingival!junction! to!the!base!of!the!gingival!sulcus!and!is!bound!to!the!underlying!periosteum4.!!Free! gingiva! is! located! coronally,! is! not! attached! to! each! tooth! and! extends! from! the! gingival! margin! to! the! bottom! of! the! sulcus1,4.! Interdental! gingiva! presents! as! the! papilla!between!two!adjacent!teeth!and!forms!a!col!with!buccal!and!lingual!peaks!at! the!interproximal!area5.!!

Microscopically,! gingiva! consists! of! an! epithelial! layer! and! underlying! connective! tissue! or! lamina! propria.! More! specifically,! the! external! surface! of! the! gingiva! (free! and! attached)! is! covered! by! keratinized! stratified! squamous! epithelium,! termed! oral! epithelium.! This! consists! of! four! epithelial! strata:! basale,! spinosum,! granulosum! and! corneum! and! can! be! either! orthokeratinized! or! parakeratinized4.! Free! gingiva! is! characterized! by! the! presence! of! two! additional,! unique! types! of! epithelium:! sulcular! epithelium! and! junctional! epithelium.! ! The! sulcular! epithelium! covers! the! internal! surface! of! the! gingival! sulcus! and! extends! from!the!free!gingival!margin!to!the!junctional!epithelium.!It!exhibits!the!typical!four! epithelial! layers! and! is! impermeable,! but! it! is! also! nonZkeratinized.! Finally,! the! junctional!epithelium!is!located!apically!to!the!sulcular!epithelium!and!provides!the! specialized! attachment! of! epithelium! to! the! tooth! through! the! internal! basement! membrane.!It!is!comprised!of!basal!and!suprabasal!cell!layers,!and!is!thicker!at!the! coronal!portion!(20Z30!cell!layers)!and!thinner!at!the!apical!portion!(3Z4!cell!layers).!

It! is! nonZkeratinized! and! permeable1,! 4.! The! main! cell! noted! at! the! gingival!

! 2! epithelium! is! the! keratinocyte! (90%! of! the! total! cell! population).! Additional! cell! populations! include! melanocytes! and! Langerhans! cells! –otherwise! referred! to! as!

“clear!cells”,!as!well!as!sensory!and!inflammatory!cells,!such!as!polymorphonuclear! leucocytes!(PMNs).!!1,!4!

The! connective! tissue! of! the! gingiva! or! lamina! propria! comprises! cells,! ground! substance,! vessels! and! nerves.! The! major! cell! population! is! fibroblasts,! which! are! responsible! for! the! generation! and! degradation! of! matrix! components.!

Additionally,! endothelial! cells,! mast! cells! and! inflammatory! cells,! such! as! PMNs,! lymphocytes,! macrophages! and! plasma! cells! are! seen1.! Ground! substance! or! extracellular! matrix! does! not! only! passively! support! and! fill! the! spaces! between! cells,!but!it!exhibits!the!fundamental!role!of!aiding!in!cellZcell!and!cellZextracellular! matrix!interactions,!thus!maintaining!the!normal!function!of!the!connective!tissue2.!

The!most!prevalent!protein!in!the!gingiva!is!collagen,!accounting!for!approximately!

60%! of! the! total! protein! content.! Collagen! Type! I! is! abundantly! seen! and! forms! collagen!fibers!providing!tensile!strength!to!the!gingiva.!The!fibrillar!collagens!Type!

III!and!V!are!also!present,!although!in!smaller!amounts.!Finally,!collagen!Type!IV!and!

VI!are!identified,!located!primarily!in!relation!to!the!basement!membrane2.!Except! for! collagen,! lamina! propria! includes! a! variety! of! other! molecules,! such! as! nonZ collagenous! proteins,! glycoproteins! and! proteoglycans.! More! specifically,! elastic,! oxytalan!and!reticulin!fibers!are!mainly!seen!in!association!with!blood!vessels!or!the! basement!membrane.!Fibronectin!is!an!abundant!glycoprotein!extensively!present! in!the!connective!tissue!and!laminin!is!associated!with!the!basement!membrane.!An!

! 3! important!component!of!the!lamina!propria!is!proteoglycans,!such!as!decorin!and! versican.! Those! molecules! consist! of! a! proteinZcore! and! a! covalentlyZbound! negativelyZcharged! glycosaminoglycan! chain.! Proteoglycans! are! necessary! for! the! regulation! of! the! fluidity! and! consistency! of! the! connective! tissue.! The! most! prevalent! glycosaminoglycans! in! the! gingival! tissues! are! dermatan! sulfate! and! chlondroitinZ4Zsulfate,!but!heparan!sulfate!and!hyaluronic!acid!are!also!present.!1,!2!

The!interface!between!epithelium!and!connective!tissue!is!characterized!by! the!presence!of!the!basement!membrane.!This!basal!lamina!consists!of!two!distinct! layers,! the! lamina! lucida! and! lamina! densa.! Lamina! lucida! connects! with! the! epithelial!cells!via!hemidesmosomes,!while!lamina!densa!attaches!to!the!underlying! connective!tissue!through!anchoring!fibrils.!4,!6!

Collagen!and!Matrix!Metalloproteinases!(MMPs)!

! Collagen!proteins!are!structural!components!of!the!connective!tissues.!Each! molecule!consists!of!three!polypeptide!chains!(αZchains)!that!form!a!triple!helix!in!at! least! one! region.! The! aminoZacid! sequence! of! the! triple! helical! domain! plays! a! pivotal!role!in!collagen’s!formation!and!structure.!More!specifically,!repetition!of!the! aminoZacid!triplet!Glycine!–!X!–Y!is!seen!in!every!collagen!molecule,!where!X!and!Y! represent! proline! and! hydroxyproline! 30%! of! the! time2,! 7.! According! to! the! literature,!28!different!collagen!types!have!been!identified,!and!assigned!to!groups7.!!

! 4! Fibrillar! collagens! represent! the! first! category.! They! are! characterized! by! their!quality!of!forming!perfect!glycine!–!XZ!Y!triplet!structures,!and!are!organized!in! a!linear!fashion.!Collagens!type!I,!II,!III,!XI,!XXIV,!XXVII!belong!in!this!group.!Fibrillar! collagens! are! usually! accompanied! by! the! next! category,! namely! collagens! associated! with! collagen! fibrils.! Members! of! this! group! are! the! “fibrilZassociated! collagens!with!interrupted!triple!helices!(FACITs)”,!including!collagens!type!IX,!XII,!

XIV,! XX.! These! collagens! do! not! form! fibrils,! but! instead! they! connect! fibrillar! collagens! to! matrix! through! their! nonhelical! globular! region.! Additionally,! FACITZ like!collagens,!types!XVI,!XIX,!XXI,!XXII,!are!mainly!localized!at!tissue!interfaces!and! basement! membranes2,! 7.! As! a! result,! type! VII! collagen,! which! is! the! major! constituent!of!the!anchoring!fibrils!at!the!epitheliumZconnective!tissue!interface!of! the!gingiva6,!is!assigned,!by!some!authors,!to!this!category7.!

! NetworkZforming!collagens!include!types!IV,!VI,!VIII,!and!X.!Collagen!type!IV! is! a! major! component! of! the! basement! membrane! and! forms! a! branching! sheet! structure2,!while!types!VIII!and!X!form!hexagonal!lattices7.!Collagen!type!VI!has!been! described!as!a!collagen!forming!beaded!filaments;!the!molecule!forms!dimers!that! aggregate! in! tetramers,! finally! forming! end! to! end! associations! giving! the! appearance!of!a!beaded!string2,!7.!!

! Transmembranous!collagens!have!also!been!described!and!include!types!XIII,!

XVII,! XXIII! and! XXV.! It! is! imperative! to! note! that! collagen! type! XVII! is! a! major! component!of!the!hemidesmosomes,!extending!from!the!cell!membrane!to!the!basal! lamina7.!!

! 5! Collagens!types!XV!and!XVIII!constitute!the!endostatin!precursor!collagens.!

These! proteoglycan! core! proteins! can! be! cleaved! to! form! antiangiogenic! peptides.!

Finally,!collagens!XXVI!and!XXVIII!have!been!localized!in!testis/ovaries!and!dorsal! root! ganglia/! peripheral! nerves,! respectively,! and! have! not! been! assigned! to! a! specific!group7.!!!

Collagen! degradation! is! important! for! tissue! homeostasis! and! can! be! performed!through!two!pathways,!one!extracellular!and!one!intracellular.!Through! the! extracellular! pathway,! enzymes! called! matrix! metalloproteinases! (MMPs)! are! released!from!cells,!such!as!fibroblasts,!neutrophils,!and!macrophages,!and!degrade! collagens.! Intracellular! degradation! of! collagen! involves! its! phagocytosis! by! cells!

(mainly!fibroblasts)!and!subsequent!lysosomal!degradation!of!fragments!of!collagen! fibrils8,!9.!

Matrix!metalloproteinases!are!enzymes!whose!activity!depends!on!zinc!in!the! active!site.!They!are!released!in!latent!forms!as!zymogens.!Their!activation!requires! disruption! of! the! bond! between! the! active! site! zinc! and! a! cysteine! residue,! by! introduction!of!a!molecule!of!water!through!a!mechanism!called!“cysteine!switch”10.!

Their!major!classes!include!collagenases,!gelatinases,!stromelysins,!matrilysins,!and! membraneZtype! metalloproteinases10,! 11.! Tissue! inhibitors! of! metalloproteinases!

(TIMPs)!regulate!their!function.!

! 6! Defining!Health!and!Disease!

According!to!the!World!Health!Organization!(WHO),!health!is!defined!as!“a! state!of!complete!physical,!mental!and!social!wellZbeing!and!not!merely!the!absence! of! disease! or! infirmity”.! Unfortunately,! the! above! definition! –simultaneous! acquisition!of!“physical,!mental!and!social!wellZbeing”Z!could!establish!health!as!the! privilege!of!few!individuals,!even!then,!lasting!for!only!a!limited!amount!of!time.!In! the! field! of! periodontology,! complete! and! accurate! understanding! of! health! is! lacking,! and! health! is! defined! as! “the! condition! of! a! patient! when! there! is! normal! function! without! evidence! of! disease! or! abnormality”! (Glossary3 of3 Periodontal3

Terms).! As! a! result,! one! could! reason! that! defining! disease! before! health! is! contradictory;!instead,!we!should!understand!what!health!constitutes!before!trying! to!diagnose!any!deviation!of!that,!or,!in!other!words,!trying!to!diagnose!disease.!!

As!this!shortcoming!has!been!increasingly!acknowledged!in!the!literature,!a! new! approach! has! been! recently! introduced.! A! model! suggested! by! Mariotti! and!

Hefti12! takes! into! consideration! both! patientZcentered! and! objective! factors! and! defines! periodontal! health! through! the! satisfaction! of! four! characteristics:! 1)! functional!dentition,!2)!painless!function!of!the!dentition!3)!periodontal!attachment! apparatus! in! a! state! consistent! with! longZterm! stability! and! 4)! psychological! and! social!wellZbeing!of!the!individual.!Although!the!majority!of!these!characteristics!is! mainly! influenced! by! the! patient,! it! is! the! clinician’s! responsibility! to! diagnose! stability! or! deterioration! of! the! periodontium.! Under! this! model,! accurate!

! 7! differentiation! of! sites! with! active! or! inactive! disease! and! identification! of! active! sites!at!risk!for!future!disease!progression!would!be!important.!!

In!current!clinical!practice,!a!variety!of!surrogate!measurements!are!used!to! diagnose! and! classify! periodontal! disease! or! the! absence! of! it.! The! most! common! surrogate!values!include!changes!in!color,!texture!and!shape,!bleeding!on!probing,! probing!depths!(PD),!clinical!attachment!loss!(CAL),!furcation!involvement,!mobility! and!findings!of!the!radiographic!examination,!while!gingival!crevicular!fluid!(GCF)! and! temperature! measurements! have! also! been! used1.! Each! one! of! these! measurements! comes! with! advantages! and! shortcomings,! which! will! briefly! be! discussed!here.!!Evaluation!of!erythema,!edema!and!changes!in!texture!with!loss!of! stippling!has!been!consistently!used.!These!changes!are!considered!to!be!the!result! of! increase! in! number! and! permeability! of! blood! vessels,! epithelial! atrophy! and! greater! percentage! of! cell! rich/collagen! poor! connective! tissue13,! thus! reflecting! presence! of! ongoing! inflammation.! However,! clinical! presentations! usually! ensue! disease!initiation,!and!early!stages!of!disease!frequently!lack!profound!alterations!in! texture! and! shape14.! In! addition,! these! evaluations! are! highly! subjective! and! susceptible!to!examiner!variability15.!Bleeding!on!probing!has!been!considered!an! early! sign! of! inflammation! preceding! visual! signs16.! Although! it! is! true! that! continuous! absence! of! bleeding! is! a! reliable! predictor! for! maintenance! of! periodontal!health!with!a!positive!predictive!value!of!98%,!studies!have!indicated! the! inability! of! this! measurement! to! predict! future! disease! progression,! with! sensitivity! of! frequent! bleeding! of! 29%17.! In! addition,! bleeding! on! probing!

! 8! measurements! are! highly! susceptible! to! falseZpositive! readings! due! to! trauma,! if! forces!above!0.25N!are!applied18.!

As! far! as! measurements! of! probing! depth,! attachment! loss,! furcation! involvement,! mobility,! as! well! as! radiographic! examination! are! considered,! examining!those!values!on!one!single!examination!(a!correctional!exam)!offers!only! a!snapshot!in!time,!and!cannot!differentiate!between!activity!or!history!of!disease.!

Diagnosis! of! ongoing! disease! with! progressive! attachment! loss! would! require! longitudinal!assessment;!in!other!words,!measurements!at!two!separate!time!points! with! apparent! changes! of! these! values! between! the! two! appointments.!

Unfortunately,! this! approach! means! that! destruction! of! periodontal! tissues! has! already!taken!place,!thus!raising!concerns!about!whether!the!disease!is!still!in! an! active!state.!The!situation!becomes!more!perplexed!if!one!considers!potential!errors! inherent! in! these! measurements.! Probing! force18,! angulation19,! probe! characteristics20,!21,! tooth! and! site! characteristics22! and! inflammatory! stage! of! the! tissues23!have!also!been!shown!to!affect!the!accuracy!of!probing!and,!subsequently,! attachment!level!measurements.!!

Gingival! crevicular! fluid! (GCF)! flow! and! identification! of! gingival! fluid! components! have! also! been! used! in! an! effort! to! distinguish! between! health! and! disease! and/or! identify! sites! at! risk! of! disease! progression.! Indeed,! it! has! been! shown! that! sampling! of! measurable! amounts! of! GCF! increased! with! increasing! severity! of! inflammation24.! Additionally,! it! has! been! suggested! that! biological! mediators!present!in!the!GCF,!such!as!alkaline!phosphatase,!elastase,!collagenaseZ2!

! 9! etc.,! could! be! beneficial! in! diagnosing! disease.25! However,! according! to! the! same! authors,!currently!there!is!no!single!or!combination!of!gingival!crevice!fluid!markers! to! determine! whether! periodontal! treatment! is! sufficient! and/or! necessary! to! prevent! further! periodontal! breakdown.! Also,! problems! inherent! in! GCF! sampling! and! processing,! such! as! contamination,! sampling! duration,! patient! and! site! variability!limit!the!utilization!of!this!tool!in!routine!clinical!practice26.!!

Another! important! aspect! to! consider! is! the! disparity! between! clinical! and! histological! health.! In! conditions! of! strict! histological! health,! any! type! of! inflammatory! reaction! should! be! absent! from! the! periodontium.! However,! this! is! rarely!the!case.!Periodontal!tissues!continuously!respond!to!the!bacterial!challenge! in!the!oral!cavity,!presenting!with!subclinical!inflammation!even!in!cases!of!clinically! diagnosed! health.! More! specifically,! in! the! classic! paper! by! Page! and! Schroeder27,! four! stages! of! periodontal! disease! were! established:! initial,! early,! established! and! advanced!lesion.!The!initial!lesion,!although!not!detectable!clinically,!presented!with! increased!permeability,!vascular!density!and!GCF!volume;!infiltration!of!PMNs!and! monocytes! in! the! junctional! epithelium! and! lymphocytes! in! the! connective! tissue! was!also!seen.!Similar!results!were!identified!by!Brecx!et!al.28!In!this!study,!clinically! health!gingiva!(GI=0)!was!populated!by!numerous!PMNs!and!lymphocytes.!!Thus,!it! seems! that! even! if! we! could! collect! a! biopsy! from! each! and! every! patient! and! examine!this!tissue!under!the!microscope,!histology!would!be!insufficient!to!provide! definite!answers!on!what!constitutes!periodontal!health.!

! 10! Finally,! in! our! quest! for! periodontal! health,! we! should! consider! the! wellZ known!variability!of!individual!response!to!inflammation.!Studies!have!shown!that! certain!people!are!recalcitrant!to!disease!(“low!responders”),!while!others!exhibit!an! intense! inflammatory! reaction! to! the! accumulation! of! similar! amounts! of! plaque!

(“high!responders”)29.!The!etiology!of!this!variability!has!been!attributed!to!local!or! systemic!factors,!including!metabolic,!genetic!and!environmental!components15.!For! example,!the!effect!of!smoking30,!Down!syndrome31!and!diabetes32!on!periodontal! tissues! has! extensively! been! studied! and! shown! to! affect! the! periodontium.!

Furthermore,! it! seems! that! our! ability! to! maintain! health! is! influenced! by! the! interaction! of! two! systems:! our! genome! and! how! environmental! factors! affect! genetic!responses,!or,!in!other!words,!our!epigenetic!code.!

Our! genetic! code,! encrypted! in! the! specific! nucleotide! sequence! that! characterize!each!one!of!us,!does!not!usually!change!with!time!and!is!identical!for!all! our!cells.!This!primary!sequence!of!DNA!contains!much!of!the!information!needed! for! the! functions! of! the! human! body.33! However,! in! addition! to! this! primary! nucleotide! code,! our! genetic! information! is! also! regulated! by! our! epigenetic! code,! which! is! defined! as! “a! group! of! acquired! or! inherited! and! potentially! transgenerational! dynamic! molecular! mechanisms! that! are! affected! by! the! environment!and!act!directly!upon!the!genome!and!genetic!machinery!throughout! life!to!regulate!gene!expression”33.!The!epigenetic!code!provides!the!mechanism!for! cells!to!receive!and!retain!environmentally!induced!signals,!thus!affecting!our!ability! to!adapt!to!environmental!changes!and!achieve!wellZorchestrated!body!functions,!or!

! 11! respond! with! disease! initiation! and! progression.! This! occurs! at! several! different! levels,!through!the!processes!of!DNA!methylation,!histone!modification!or!regulation! through! nonZcoding! RNA33,!34.! Regarding! the! periodontium! specifically,! a! range! of! external!factors,!such!as!smoking35,!36,!diet35,!37,!stress38,!or!bacteria39,!40,!can!cause! epigenetic! changes! in! genes! expressing! for! proZinflammatory! mediators.! For! example,! challenge! with! T.3 denticola! resulted! in! upregulation! of! MMPZ2Zrelated! genes! and! chronic! activation! of! MMPZ2! in! PDL! cells.! This! change! was! probably! mediated!through!epigenetic!mechanisms40.!!

From! the! preceding! introduction,! it! is! clear! that! periodontal! health! can! be! characterized! in! different! ways,! in! terms! of! macroscopic! changes! of! the! gingiva,! histologic!examination!of!periodontal!tissues!or!general!analysis!of!the!genetic!code.!

Each!approach!has!strengths!and!limitations.!To!help!clinicians!develop!an!objective,! quantifiable! approach! that! can! be! implemented! to! determine! health,! some! have! proposed! evaluating! expression! of! genes! and! proteins! through! the! fields! of! transcriptomics!and!proteomics.!Working!towards!this!direction,!a!variety!of!animal! and!human!studies!have!looked!at!the!transcriptome!of!healthy!and!periodontally! compromised!gingiva.!!

Studies! from! the! Papapanou! group! have! examined! differential! gene! expression!between!health!and!periodontitis.!In!a!study!by!Demmer!et!al.41,!gingival! samples! from! diseased! and! healthy! papillae! were! collected! from! ninety! periodontitis! patients.! Microarray! analysis! reported! that! periodontitis! specimens! were! characterized! by! an! upregulation! of! genes! belonging! to! inflammatory!

! 12! pathways,!such!as!apoptosis,!humoral!response,!antigen!presentation,!angiogenesis! and! immunoglobulinZrelated! genes.! For! example,! matrix! metalloproteinases! and! their! inhibitors! were! significantly! upregulated! in! disease.! Similar! results! were! obtained! by! Davanian! et! al42.! In! this! study,! diseased! and! healthy! tissues! from! periodontitis! patients! were! analyzed! through! RNAZsequencing.! Health! was! characterized!by!a!nonZinflammatory!profile,!while!diseased!samples!expressed!high! amounts!of!inflammatory!mediators!(ILZ1β,!ILZ6,!ILZ8,!TNFZα,!RANTES!and!MCPZ1).!!

Other! studies! have! used! transcriptomics! to! evaluate! differentially! gene! expression!in!healthy!gingiva!with!aging.!Most!of!these!studies!have!been!conducted! in! nonZhuman! primates,! and! only! a! small! number! have! examined! humans.! In! a! series! of! studies! on! monkeys! by! Gonzalez! et! al,! buccal! gingival! samples! from! the! premolar/molar!maxillary!region!were!collected!from!animals!of!different!ages.!The! results! reported! that! young! animals! present! with! higher! antiZinflammatory! and! decreased! proZinflammatory! cytokine/! chemokine! profile! compared! to! older! animals43.! Additionally,! expressions! of! genes! of! the! Major! Histocompatibility!

Complex!Class!II!(MHCZII)!pathway!were!up!regulated,!whereas!the!MHCZI!pathway! was!down!regulated.!The!affected!genes!mainly!related!to!Natural!Killer!(NK)!cell! interactions! with! Antigen! Presenting! Cells! (APCs)! leading! to! functional! downZ regulation,! with! minimal! to! no! effects! for! CD8+! and! CD4+! cells! respectively44.!

Additionally,!as!shown!in!a!study!by!Ebersole!et!al45,!gingival!aging!in!monkeys!is! associated!with!gene!expression!leading!to!antigenZdependent!activation!and!B!cell! differentiation/maturation!processes.!!

! 13! In!a!human!study!conducted!by!Kim!et!al46,!gingival!biopsies!were!collected! from! adults! without! periodontal! disease.! The! sample! included! three! young! individuals! aged! 17Z20! years! old! and! three! old! individuals! ≥60! years! old.! Their! results! mainly! focused! on! MMPs! and! reported! that! MMP3,! MMP9,! MMP12,! and!

MMP13!were!significantly!upregulated!in!older!adults.!Concomitant!upregulation!of!

ILZ1β!indicated!a!potential!regulatory!role!of!this!cytokine!in!MMP!expression.!

The! results! of! these! studies! are! interpreted! to! indicate! an! immune! dysregulation!in!gingival!tissues!with!progressing!age,!which!is!consistent!with!the! theories!of!“inflammaging’’!and!“immunosenescence’’.!According!to!these!theories,! aging!tissues!are!characterized!by!persistently!elevated!levels!of!cytokines!and!proZ inflammatory!markers!leading!to!a!chronic!state!of!lowZgrade!inflammation!in!aged! individuals47.!The!impact!of!a!hyperZinflammatory!phenotype!is!further!aggravated! by! the! concomitant! presence! of! compromised! innate! and! acquired! immune! responses,! a! process! termed! “immunosenescence”48.! If! these! observations! are! accurate,! aging! can! predispose! healthy! gingival! tissues! to! inflammation! and! breakdown,! thus! having! a! contributory! role! to! the! increased! prevalence! of! periodontal!diseases!in!older!individuals49.!

In! our! opinion,! a! major! shortcoming! of! the! methodology! of! most! of! these! studies!is!that!they!have!used!ontology!approaches!to!try!to!fit!gene!expression!in! predetermined!pathways.!It!is!true!that!this!type!of!analysis!gives!a!simplified!and! easyZtoZunderstand! perspective! of! differences! in! gene! expression! between! study! groups.!However,!we!feel!that!a!lot!of!bias!can!be!inherent!in!these!approaches.!!One!

! 14! reason!is!that!the!underlying!pathways!involve!many!genes,!and!specific!genes!are! involved!in!many!pathways.!As!a!result,!we!cannot!determine!with!certainty!which! of! these! pathways! are! activated! and! how! the! rest! of! the! unaffected! genes! are! regulating!the!response.!Furthermore,!variances!in!gene!expression!are!not!usually! mentioned.!Knowing!the!exact!number!of!transcripts!of!each!gene!in!each!individual! could!give!us!a!better!understanding!of!the!genes!with!high!or!low!expression!in!the! tissues,! and! how! individual! responses! can! affect! the! statistical! significant! differences.! Consequently,! although! gene! ontology! approaches! give! the! illusion! of! strong!support!of!the!data,!they!can!often!identify!a!specific!pathway!based!on!very! weak!evidence!of!gene!expression.!Additionally,!given!the!very!limited!input!data,! these! approaches! cannot! necessarily! identify! the! correct! pathway! and! merely! determine!which!of!the!pathways!evaluated!are!most!likely.!Therefore,!results!are! commonly! over! interpreted! and! presented! as! definitive! evidence! of! increased! or! decreased!activation!of!a!biologic!pathway.!!!

Taking!all!of!the!above!into!consideration,!the!present!study!aims!to!evaluate! the! transcriptome! of! gingival! tissues! collected! from! a! sample! of! young! and! older! individuals! with! clinically! healthy! gingiva! and! no! history! of! periodontitis.! More! specifically,! we! have! decided! to! focus! our! examination! on! gene! expression! of! collagens! and! their! degradation! enzymes,! MMPs.! In! this! study,! periodontal! health! was! defined! as! visually! nonZinflamed! gingiva! (coral! pink! color,! no! change! in! texture),!tissue!tightly!conformed!to!the!tooth!(no!changes!in!consistency,!absence! of!edema),!absence!of!bleeding!on!probing!and!probing!depths!≤3mm.!As!part!of!our!

! 15! analysis,! the! exact! number! of! transcripts! for! the! collagen! and! MMP! genes! will! be! reported.!Additionally,!comparisons!between!the!young!and!older!age!group,!taking,! also,!under!consideration!the!effect!of!sex/gender,!will!be!performed.!

! 16! Aims:!

1. Describe!the!quantitative!expression!of!collagen!genes!in!clinically!healthy!

gingiva!

2. Describe!the!quantitative!expression!of!matrix!metalloproteinases!(MMPs)!

genes!in!clinically!healthy!gingiva!

3. Describe! the! differential! collagen! gene! expression! with! aging! in! clinically!

healthy!gingiva,!taking!into!consideration!the!effect!of!sex/gender!!

! 17! !

Chapter!2!

Materials!and!Methods!

Subjects!

Nineteen!subjects!with!clinically!healthy!gingiva!(10!males!and!9!females)!were! recruited! from! the! Ohio! State! University! College! of! Dentistry! between! December!

2016!and!February!2018.!!

! Patients!were!included!if!they!were!≥!18!years!of!age,!medically!healthy!or! presenting! with! medically! controlled! systemic! disease,! periodontally! healthy!!

(defined! as:! PD! ≤! 3mm,! visually! nonZinflamed! papilla! tissue,! tissue! tightly! conformed! to! tooth! with! no! bleeding! on! probing),! nonZcurrent! users! of! tobacco! products,! and! had! both! the! second! premolar! and! first! molar! of! the! right! and! left! maxillary!quadrants.!

The!exclusion!criteria!included!women!who!were!pregnant!at!the!time!of!biopsy! collection,! patients! who! needed! antibiotic! prophylaxis! before! dental! appointment,! patients! who! had! antibiotics! within! the! past! 6! months,! patients! with! history! of! periodontal! disease! or! systematic! periodontal! therapy! other! than! occasional!

! 18! prophylaxis,!and!patients!with!diabetes!or!any!systemic!condition!that!is!entailed!as! a! diagnosis! of! ! “Gingival! Diseases! modified! by! medications”! or! “Periodontitis! as! a! manifestation!of!systemic!diseases”.!

In! addition,! patients! had! to! belong! to! one! of! the! following! age! groups:! 18Z35! years!old!(young!age!group)!or!≥60!years!old!(older!age!group).!

Signed!informed!consent!was!obtained!prior!to!enrollment.!The!study!protocol! and!informed!consent!was!approved!by!the!Institutional!Review!Board!of!the!Ohio!

State!University!(IRB#!2015H0261)!

Clinical!Examination!

Patients!that!met!the!study!inclusion!criteria!were!evaluated!in!the!Graduate!

Periodontics!Clinic!at!the!Ohio!State!University.!FullZmouth!examination!confirmed! the!clinical!absence!of!periodontal!inflammation!and!PD>3mm!on!all!teeth!present!

(6!sites/tooth).!Emphasis!was!given!on!the!first!molar!and!second!premolar!of!each! maxillary! quadrant,! as! these! were! the! sites! of! biopsy! collection.! The! following! variables! were! recorded! on! these! sites:! Probing! Pocket! Depth! (PPD),! Bleeding! on!

Probing!(BOP),!Redness!and!Swelling.!

Additionally,!information!on!age,!sex,!systemic!diseases!and!medications!of! each!subject!was!recorded.!

! 19! Biopsy!Harvest!

Two!biopsies!were!collected!from!each!patient.!The!biopsies!were!harvested! from!the!buccal!papillary!area!between!the!first!molar!and!second!premolar!of!each! maxillary! quadrant.! Anesthesia! was! secured! by! infiltration! of! the! buccal! vestibule! with! the! minimum! amount! of! anesthetic! needed! to! provide! painless! procedure! to! the! patient! (Lidocaine! 2%,! epi:! 1:10000).! Biopsy! punches! were! used! to! ensure! surgical! removal! of! standardized! fullZthickness! specimens! of! 3mm! in! diameter.!

Attention! was! given! to! harvest! the! biopsies! from! attached! gingiva,! avoiding! the! sulcular! epithelium! or! oral! mucosa.! After! excision! of! the! biopsies,! pressure! was! applied! on! the! sites! until! hemostasis! was! achieved.! Clinical! photos! of! the! biopsy! areas!are!shown!in!Figure!1.!

The! tissue! specimens! were! immediately! irrigated! with! saline,! sectioned! in! two! halves! and! submerged! in! RNAlater! solution! (sectioning! was! performed! to! ensure!maximal!diffusion!of!RNAlater!solution!through!the!biopsied!tissue).!Biopsies! were!incubated!overnight!in!the!reagent!at!2Z8°C.!Then,!tissues!were!transferred!in! the!reagent!to!Z20°C!for!storage.!

Patients!were!called!back!a!week!later!to!ensure!proper!healing!of!the!biopsy! sites.!

! 20! Figure!1.!Clinical!photos!of!biopsy!areas!

! !

RNA!Isolation,!cDNA!and!Library!Construction!

The!method!was!performed!through!Qiagen's!service!using!the!specific!kit.!!

RNA! isolation,! cDNA! production! and! library! construction! was! conducted! using!the!QIAseq!UPX!3’!Transcription!kit!(Qiagen)!according!to!the!manufacturer’s! instructions.!!

The! RNA! integrity! level! was! measured! for! each! sample! using! the! Agilent!

TapeStation!System!in!order!to!obtain!an!RNA!Integrity!Number!(RINe)!values.!RINe! values! higher! than! 7! indicate! RNA! of! suitable! quality! for! NextZGeneration!

Sequencing! (NGS)! analysis.! Contamination! with! salts! or! organic! compounds! were! evaluated! by! spectrophotometric! measurements! (Abs! 260/230! and! Abs! 260/280! ratios).!!

! 21! Each! RNA! molecule! was! tagged! with! a! Unique! Molecular! Index! (UMI)! incorporated!during!the!reverse!transcription!step.!

NextPGeneration!RNA!sequencing!

NextZgeneration!sequencing!was!conducted!on!an!Illumina!MiSeq.!Primary! analysis!of!the!data!generated!UMIZbased!gene!expression!estimates!from!the!reads.!

Statistical!Analysis!

Comparisons! between! young! (males! –! females! combined)! versus! older!

(males! –! females! combined)! groups! were! performed.! In! an! effort! to! better! understand! the! sex! effect! on! differential! gene! expression! with! aging,! focused! comparisons!between!young!males!versus!old!males!and!young!females!versus!old! females!were!performed.!The!young!and!the!male!group!served!as!controls!in!our! analyses.! Statistical! significance! was! evaluated! using! the! student’s! tZtest! to! determine! if! two! sets! of! data! differ! significantly.! PZvalue! ≤! 0.05! was! set! as! the! statistically!significant!level.!

! 22! !

Chapter!3!

Results!

Clinical!Data!

Nineteen! patients! participated! in! this! study.! Ten! patients! (5! males! and! 5! females)!belonged!to!the!young!age!group!with!ages!ranging!between!24Z35!years! old.! Nine! patients! (5! males! and! 4! females)! belonged! to! the! older! age! group,! with! ages!ranging!between!62Z81!years!old!(Appendix!A).!!

Appendix!B!shows!the!general!patient!characteristics.!Subjects!belonging!to! the!young!age!group!were!in!the!majority!systemically!healthy.!Asthma!and!thyroid! nodule! were! noted! in! two! individuals,! treated! with! albuterol! inhaler! and! T4! respectively.! A! third! individual! presented! with! hypothyroidism! and! anxietyZ depression.! The! same! individual! was! taking! contraceptives! at! the! time! of! biopsy! collection,! a! fact! that! she! disclosed! at! the! postZoperative! appointment.! Controlled! systemic!diseases!were!noted!in!most!of!the!older!individuals.!The!most!common! illnesses!were!hypertension!and!hypercholesterolemia.!The!most!commonly!taken! medications!were!antihypertensive!drugs,!statins!and!aspirin!81mg.!One!patient!had!

! 23! received! antibiotics! (amoxicillin! 500mg)! four! months! before! biopsy! collection! (as! shown!in!Appendix!C).!

Intraoral! clinical! data! were! collected! at! the! day! of! biopsy! harvest.! Probing! depth! and! bleeding! on! probing! measurements! were! performed! at! 6! sites/tooth!

(mesiobuccal,! midbuccal,! distobuccal,! mesiopalatal,! midpalatal,! distopalatal).!

Probing! depths! around! the! first! molar! and! second! premolar! of! the! maxillary! quadrants! (teeth! #3Z4! and! #13Z14)! were! ≤3mm.! Tissues! around! the! same! teeth! were! coral! pink! in! color! for! all! individuals.! Edema! and! BOP! were! absent.! This! information!is!shown!in!Appendix!D.!

RNA!sequencing!Data!

ThirtyZeight! biopsies! (two! biopsies! from! each! individual)! were! analyzed! in! this! study.! Quality! control! was! performed! using! the! Agilent! 2200! TapeStation! system,! which! assigns! an! RNA! Integrity! Number! (RIN)! to! each! sample.! Within! a! scale!of!1Z10,!10!represents!high!quality!RNA,!while!1!represents!low!quality.!All,! except! one! of! the! submitted! samples,! were! assigned! RIN! numbers! higher! than! 7,! indicating!high!quality!RNA.!Although!an!RIN!value!of!5.7!was!assigned!to!one!of!the! samples,!it!was!still!deemed!adequate!for!sequencing.!

NextZGeneration! Sequencing! (NGS)! was! performed! and! yielded! a! total! of!

113,925,455! reads.! After! excluding! part! of! the! reads! due! to! reasons! shown! in!

Appendix!E,!57,344,811!reads!were!used!(aligned!to!genome,!unique!loci).!A!total!of!

! 24! 37,085!genes!were!identified!and!demonstrated!to!be!expressed!when!grouping!all! the!samples.!!

Samples! #8L,! 14L,! 15L,! 15R,! 17R! and! 18L! were! dropped! from! further! analysis,!as!they!generated!a!low!Z!very!low!number!of!reads.!Subject!#3!(samples!

#3L!and!#3R)!was,!also,!excluded,!as!she!was!taking!contraceptives!during!biopsy! collection.! Comparisons! between! young! and! older! groups! were! performed,! taking! into!consideration!the!effect!of!sex/gender.!!

1790!genes!were!differentially!expressed!between!the!young!and!old!groups;!

347!genes!were!upregulated!and!1443!genes!were!downregulated!with!aging.!When! looking! specifically! at! the! male! group,! expression! of! 662! genes! were! significantly! altered!with!aging,!with!428!genes!being!upregulated!and!234!being!downregulated.!

For!the!female!group,!312!genes!were!upregulated!and!1538!were!downregulated,! giving!a!total!of!1850!genes!differentially!expressed!with!aging.!

Collagens!

We!decided!to!focus!on!collagen!gene!expression!due!to!the!importance!of! this! protein! for! the! extracellular! matrix! of! the! gingiva.! Appendix! F! depicts! the! number!of!transcripts!for!each!collagen!gene!as!a!total,!while!Appendices!GZJ!show! the!number!of!transcripts!for!each!subject!group!and!per!individual.!Appendices!K! and!L!depict!differential!gene!expression!with!aging!and!between!sexes!respectively.!

! 25! ! FortyZeight! distinct! collagen! genes! were! identified! as! expressed! in! healthy! gingiva! from! young! and! older! individuals.! The! top! four! expressed! collagen! genes,! with! high! number! of! transcripts! among! individuals,! were! COL1A2,! COL17A1,!

COL3A1!and!COL1A1.!The!collagen!genes!with!their!total!number!of!transcripts!are! shown!in!Table!1.!

Statistically!significant!differences!in!gene!expression!with!aging!were!seen! for! 16! different! collagen! transcripts.! Except! for! one! (COL4A6),! all! of! the! collagen! genes!were!downregulated.!Looking!at!the!general!comparison!between!young!and! old!individuals,!seven!collagen!genes!(COL1A1,!COL1A2,!COL3A1,!COL5A2,!COL9A1,!

COL14A1!and!COL16A1)!were!downregulated.!!However,!a!number!of!collagen!gene! changes!appear!to!be!sexZspecific,!as!the!altered!gene!expression!was!mainly!seen!in! females.! Except! for! the! genes! mentioned! above,! females! presented! with! downregulation! of! eight! additional! collagen! genes! with! aging! (COL6A1,! COL6A2,!

COL6A3,!COL12A1,!COL18A1,!COL19A1,!COL23A1!and!COL24A1).!COL16A1!was!the! only!collagen!gene!significantly!downregulated!in!males.!

Matrix!Metalloproteinases!(MMPs)!

For! our! analysis,! we! evaluated! expression! of! MMP! genes,! as! the! protein! products!of!these!genes!play!an!important!role!in!the!extracellular!degradation!of! the! collagen! matrix.! Appendix! M! depicts! the! number! of! transcripts! for! each! MMP! gene! as! a! total,! while! Appendices! NZQ! show! the! number! of! transcripts! for! each!

! 26! subject! group! and! per! individual.! Appendices! R! and! S! depict! differential! gene! expression!with!aging!and!between!sexes!respectively.!

! TwentyZfive!MMP!genes!were!identified!in!our!study!samples.!The!five!genes! with!the!highest!expression!were!MMP2,!MMP28,!MMP24ZAS1,!MMP16!and!MMP14.!

The!MMP!genes!with!their!total!number!of!transcripts!are!shown!in!Table!2.!

Looking!at!differential!gene!expression,!MMPs!do!not!seem!to!be!significantly! altered! with! aging.! Among! all,! MMP2,! MMP15! and! MMP16! were! the! only! MMPs! significantly!changing!with!aging.!MMP2!and!MMP15!was!downregulated!in!females,! while!MMP16!was!downregulated!in!males.!

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! 27! Table!1.!Collagen!gene!expression.!The!total!number!of!transcripts!for!each!collagen! gene!is!shown!in!parentheses.!Number!of!collagen!gene!transcripts!per!individual!is! shown!in!detail!in!appendices!FZJ.! !

COL1A2!(8995)! COL8A1!(417)! COL24A1!(151)! COL6A6!(28)!

COL17A1!(6583)! COL6A2!(338)! COL11A1!(149)! COL9A1!(23)!

COL3A1!(5347)! COL7A1!(327)! COL23A1!(141)! COL9A3!(22)!

COL1A1!(5078)! COL5A1!(297)! COL25A1!(89)! COL13A1!(17)!

COL4A3BP!(927)! COL16A1!(255)! COL4A3!(73)! COL22A1!(10)!

COL14A1!(780)! COL15A1!(246)! COL28A1!(64)! COL6A4P2!(9)!

COL6A3!(731)! COL4A6!(237)! COL10A1!(57)! COL11A2!(7)!

COL6A1!(688)! COL27A1!(215)! COL4A4!(52)! COL9A2!(6)!

COL5A2!(680)! COL4A2!(213)! COL8A2!(43)! COL4A2PAS2!(2)!

COL4A5!(667)! COL21A1!(207)! COL19A1!(42)! COL2A1!(1)!

COL18A1!(613)! COL4A1!(173)! COL6A5!(31)! COL5A1PAS1!(1)!

COL12A1!(506)! COL5A3!(168)! COL26A1!(28)! COL6A4P1!(1)!

! 28! ! !

! ! ! ! ! !

! MMP7!(2) MMP21!(1) MMP10!(3) MMP13!(2) MMP23A!(1) ! ! ! ! ! ! ! ! ! ! ! ! MMP1!(3) MMP3!(11) MMP20!(7) MMP11!(10) MMP25!(10) ! ! ! ! ! ! ! ! ! ! AS1!(12) P MMP15!(30) MMP19!(26) MMP17!(19) MMP12!(13) MMP25 ! ! ! ! !

! ! ! ! ! ! MMP9!(49) MMP24!(46) MMP27!(41) MMP26!(36) MMP23B!(66) ! ! ! ! ! ! Number!of!MMP!gene!transcripts!per!individual!is!shown!in!detail!in! Q. Z (303) ! ! ! ! AS1!(285) P MMP14!(98) MMP2!(453) MMP28! MMP16!(108) MMP24 Table!2.!MMP!gene!expression.!The!total!number!of!transcripts!for!each!MMP!gene!is!shown! in!parentheses.! appendices!M

! 29! !

Chapter!4!

Discussion!

! Transcriptomics!is!the!study!of!an!organism’s!transcriptome,!the!sum!of!all!

RNA! transcripts.! Earlier! transcriptomic! studies! have! used! microarrays! for! their! analysis.! These! approaches! use! oligonucleotide! probes,! complementary! to! the! sequence!of!the!genes!under!evaluation.!The!probes!are!arrayed!on!a!solid!substrate! and! fluorescentZlabeled! transcripts! are! expected! to! hybridize! to! their! complementary!sequences50.!The!basic!shortcoming!with!this!approach!is!that!the! genes! under! evaluation! need! to! be! predetermined,! so! that! relative! probes! can! be! selected!and!incorporated!onto!the!gene!array.!In!other!words,!microarrays!do!not! offer! the! possibility! of! discovering! new! genes! in! a! specific! tissue,! detect! SNPs,! or! splice! variants50,! 51.! ! RNAZsequencing! is! a! newer! approach! that! overcomes! the! problem! above.! Generally,! RNA! is! fragmented,! converted! to! cDNA,! amplified! and,! finally,! aligned! to! the! genome50.! Thus,! the! need! to! know! transcript! products! beforehand!is!eliminated51.!Potential!errors!lie!in!the!amplification!process,!as!many! times! it! is! impossible! to! know! if! the! variance! or! expression! levels! seen! are! true! biologic!phenomena!or!amplification!errors52.!

! 30! The!basic!strength!of!the!present!study!is!the!use!of!targeted!DNA!sequencing! with! enrichment! panels! to! accurately! detect! lowZfrequency! transcripts! that! have! been! amplified.! According! to! this! approach,! random! oligonucleotide! barcodes,! named! Unique! Molecular! Indices! (UMIs),! are! used! to! tag! each! fragmented! RNA! molecule! before! amplification;! ultimately,! unique! DNA! sequences! can! be! distinguished!from!PCR!amplification!errors.!As!a!result,!we!are!confident!that!the! number!of!transcripts!noted!in!this!study!is!a!highly!accurate!determination!of!the! transcripts!present!in!the!gingiva!at!the!specific!time!point!of!biopsy!collections.!!

Nineteen!patients!providing!thirtyZeight!biopsies!were!included!in!this!study.!

Biopsies!#8L,!14L,!15L,!15R,!17R!and!18L!yielded!a!very!low!number!of!reads,!and! were! excluded! from! further! statistical! analysis.! Although! a! low! number! of! transcripts! can! and! do! provide! information! for! what! genes! are! expressed! in! the! tissues,!as!well!as!their!sequence,!looking!at!relative!differences!between!transcripts! expressed! in! low! numbers! could! compromise! the! results.! We! believe! that! RNA! degradation!had!already!occurred!in!these!tissues.!Unfortunately,!we!cannot!know! with!certainty!the!reason!for!the!low!quality!of!the!specific!biopsies,!as!the!clinical! procedure!and!biopsy!storage!was!the!same!for!all!subjects.!Fibrotic!tissues!that!did! not! allow! proper! RNAlater3 diffusion! may! explain! the! low! reads! for! both! left! and! right! biopsies! of! subject! #15,! but! this! explanation! does! not! shed! light! on! the! unilateral! lowZquality! biopsies! for! the! rest! of! the! subjects.! Although! transfer! of! samples!from!the!storage!area!to!the!location!for!laboratory!analysis!was!performed! with! caution,! following! the! manufacturer’s! instructions,! we! cannot! exclude! that!

! 31! some! of! the! vials! potentially! shifted! position! leading! to! tissues! staying! out! of!

RNAsolution! for! some! time.! Regarding! the! rest! of! the! samples,! subject! #3! was! a! young! female,! and! she! was! taking! contraceptives! at! the! time! of! biopsy! collection.!

Although!we!are!aware!of!studies!reporting!that!new!generation!contraceptives!do! not! have! an! effect! on! the! periodontium53,! contradictory! reports! do! exist! in! the! literature54.!As!a!result,!we!decided!to!avoid!any!potential!confounders!and!exclude! this!subject!from!further!statistical!analysis.!

This!study!provides!a!comprehensive!report!of!the!collagen!genes!expressed! in!the!interpapillary!attached!gingiva!of!nineteen!individuals!with!clinically!healthy! periodontium.!These!collagen!genes!can!be!reviewed!in!Table!1!and!Appendices!FZJ.!

It!is!interesting!to!note!the!variety!of!collagen!genes!that!gingival!cells!can!produce.!

Defining!the!circumstances!or!the!genetic!and!environmental!factors!that!can!trigger! their! expression! would! be! extremely! useful.! Some! of! these! genes,! such! as!

COL6A4P1,! COL5A1ZAS1,! COL2A1,! COL4A2ZAS2,! COL9A2,! COL11A2,! COL6A4P2,!

COL22A1!provided!only!a!limited!number!of!reads!in!a!limited!subset!of!individuals,! and!the!importance!of!their!function!in!the!gingiva!should!be!evaluated!with!caution.!

Collagens!type!I,!III,!IV,!V,!VI!and!VII!(anchoring!fibrils)!have!been!previously! reported!in!gingival!tissues2,!6.!As!a!result,!expression!of!their!respective!genes!does! not! come! as! a! surprise.! Collagens! types! I! and! III! have! been! reported! as! the! most! abundant2!in!the!gingiva.!Having!said!that!and!keeping!in!mind!that!gene!expression! does!not!always!correlate!with!protein!abundance,!it!is!interesting!to!note!that!the! expression! of! COL17A1! is! higher! than! COL3A1.! Collagen! Type! XVII! is! a!

! 32! transmembrane! protein! and! a! structural! component! of! hemidesmosomes7,! 55.!

Although! studies! have! failed! to! localize! it! at! the! dentoZepithelial! junction56,! it! is! present!at!the!epithelialZconnective!tissue!junction.!Evidence!of!this!is!provided!by! the! fact! that! this! collagen! –reported! as! BP180Z! is! one! of! the! major! pemphigoid! antigens! and! seems! to! be! targeted! by! autoantibodies! in! patients! with! mucous! membrane!pemphigoid!affecting!the!oral!cavity57.!

COL4A3BP! was! highly! expressed! in! gingival! tissues.! This! gene! encodes! a! kinase!that!phosphorylates!the!NZterminal!region!of!the!nonZcollagenous!domain!of! the! alpha! 3! chain! of! type! IV! collagen.! One! isoform! of! this! protein! plays! a! role! in! ceramide! intracellular! trafficking.! Ceramide! is! one! of! the! intermediates! in! the! process! of! glycosphingolipid! biosynthesis,! a! pivotal! pathway! for! membrane! production!in!mammalian!cells.!Its!transfer!from!the!endoplasmic!reticulum!to!the!

Golgi!apparatus!seems!to!be!highly!important,!and!could!explain!the!high!expression! of!COL43BP!in!the!gingival!tissues58.!

Expression!of!COL8A1,!encoding!one!of!the!two!alpha!chains!of!collagen!type!

VIII! was! seen.! This! collagen! forms! hexagonal! lattices! and! is! highly! expressed! in! endothelial! cells7.! Synthesis! of! type! VIII! collagen! has! also! been! reported! in! the! gingiva59.!Junctional!epithelial!cells!produce!this!collagen,!which!seems!to!play!a!role! in!the!attachment!of!epithelial!cells!to!the!root!surface.!Although,!in!our!study,!we! tried!not!to!include!any!part!of!the!sulcus!or!junctional!epithelium!in!our!biopsies,!it! is! possible! that! we! inadvertently! did! so.! An! alternative! explanation! is! that! endothelial!cells!present!in!the!connective!tissue!expressed!this!collagen.!

! 33! Collagen! types! XII! and! XIV! belong! to! the! FibrilZAssociated! Collagens! with! interrupted! Triple! Helices! (FACITs).! Collagen! XII! has! previously! been! reported! in! the! periodontal! ligament2,! 60.! The! presence! of! fibrillar! types! of! collagen! is! a! preZ requisite!for!the!expression!of!those!collagens!in!tissues61,!and!the!high!expression! of!collagen!I!in!periodontal!tissues!probably!explains!their!concomitant!expression.!!

Another! collagen! belonging! to! the! FACIT! group! is! type! IX.! Although! not! highly!expressed,!collagen!genes!COL9A1,!COL9A2!and!COL9A3!were!found!in!the! gingival!tissues!at!the!present!study.!This!collagen!has!been!mainly!associated!with! cartilaginous! tissues,! where! it! crossZlinks! with! collagen! type! II7.! However,! it! is! interesting!to!note!that!the!same!collagen!is!deemed!necessary!for!restoration!and! remodeling! of! intramembranous! trabecular! bone.! More! specifically,! Col9a1Znull! mice!presented!with!abnormal!and!disorganized!healing!of!the!trabecular!bone!after! alveolar!bone!wounding62.!

Collagen!genes!COL27A1,!COL24A1,!COL11A1,!COL11A2,!and!COL2A1!encode! for! fibrillar! collagens.! It! is! noteworthy! that! these! collagens! are! associated! with! osseous!or!cartilaginous!functions,!such!as!calcification!of!cartilage!(type!XXVII)63,! osteoblast!differentiation!and!bone!matrix!deposition(type!XXIV)64,!and!nucleation! of!type!II!collagen!(type!XI)7.!Additionally,!COL2A1,!encoding!for!collagen!type!II!(a! cartilage! component)2,! was! found! expressed! with! only! one! transcript! in! one! individual;!thus,!the!importance!of!this!gene!in!the!gingiva!is!under!question.!It!is! possible! that! expression! of! these! collagen! genes! was! performed! by! cells! at! the! deeper!portion!of!the!biopsies,!closer!to!the!periosteum!and!alveolar!bone.!Although!

! 34! our! biopsies! may! have! included! part! of! the! periosteum,! we! tried! to! limit! this! phenomenon!by!using!a!scalpel!blade!to!dissect!the!tissue!from!the!underlying!bone.!

We!should,!also,!note!that!collagen!type!XXVII!is!expressed!by!epithelial!cell!layers!in! developing!tissues,!such!as!stomach,!lung,!skin,!tooth,!gonad!and!cochlear65.!

To! our! knowledge,! expression! of! collagen! genes! COL16A1,! COL19A1,!

COL21A1!and!COL22A1!has!not!been!previously!described!in!gingiva.!These!genes! encode! for! FACIT! type! collagens.! As! their! function! is! associated! with! basement! membranes,! adhesion! of! epithelial! cells! and! fibroblasts,! and! integrity! of! the! extracellular!matrix66Z69,!their!presence!in!gingiva!is!not!surprising.!

Expression! of! COL18A1! was! also! seen! in! high! amounts.! Collagen! XVIII! has! been! described! both! as! an! endostatin! precursor! collagen! and! heparin! sulfate! proteoglycan7! and! seems! to! play! an! important! role! in! skin! elasticity.! In! skin,! it! is! produced!by!keratinocytes,!endothelial!cells,!epithelial!cells!of!the!sweat!glands!and! adipocytes! and! its! function! is! related! to! the! basement! membrane70.! ! Collagen! XV! belongs! to! the! same! category! of! endostatin! precursor! collagens! and! has! a! chondroitin!sulfate!side!chain7.!This!collagen!is!localized!at!the!basement!membrane! of! most! epithelial! and! all! nerve,! muscle,! fat! and! endothelial! tissues,! connecting! it! with!the!adjacent!fibrillar!collagens71.!

The! genes! for! transmembrane! collagens! XIII,! XXIII! and! XXV! were,! also,! expressed!in!gingival!tissues!in!the!current!study.!The!function!of!collagen!type!XIII! is! unknown,! but! has! been! associated! with! basement! membrane! interactions,! osteoblast! differentiation,! vascular! integrity! and! immunity72.! Collagen! XXIII! has!

! 35! been!found!on!the!surface!of!basal!keratinocytes!in!epithelial!tissues73,!and!has!been! shown!to!play!a!role!in!tumor!metastasis74,!while!collagen!XXV!is!a!component!of! amyloid!plaques,!potentially!playing!a!role!to!Alzeheimer’s!disease7,!75.!

Other! collagens! expressed! in! gingiva! include! type! X,! a! network! forming! collagen!associated!with!hypertrophic!chondrocytes7,!type!XXVI,!which!is!expressed! in! testis! and! ovaries76,! and! type! XXVIII,! whose! function! is! associated! with! the! nervous!system7.!Finally,!COL6A4P1!and!COL6A4P2!are!pseudogenes!occurring!due! to!a!chromosome!break!at!the!location!of!the!gene!encoding!for!COL6A477.!

Matrix! Metalloproteinases! (MMPs)! gene! expression! was! also! avaluated! in! this!study.!The!identified!MMPs!and!their!levels!of!expression!are!noted!in!Table!2! and!Appendices!MZQ!.!TwentyZfive!distinct!MMP!genes!were!identified.!MMP2!and!

MMP28!were!the!ones!with!the!highest!expression.!!

MMP2!is!one!of!the!gelatinases.!Its!substrates!include!type!IV,!V,!VII,!X,!and!XI! collagen,! denaturated! collagens! and! elastin11.! MMP28! or! epilysin! seems! to! be! expressed!in!normal!tissues,!suggesting!a!role!in!homeostasis.!Enhanced!expression! of!this!molecule!in!basal!keratinocytes!has!been!reported!during!wound!healing78.!!

Describing! collagen! and! MMP! gene! expression! in! gingiva! is! important.!

However,! we! need! to! remember! that! this! is! a! transcriptomics! study,! and! mRNA! levels!do!not!always!correlate!with!protein!abundance!in!tissues.!Factors,!such!as!

RNA! secondary! structures,! regulatory! proteins! and! sRNAs! acting! as! translational! modulators,!codon!bias,!ribosomal!density!and!ribosomal!occupancy,!protein!halfZ lives!and!degradation!times,!as!well!as!shortcomings!of!the!laboratory!methods!used!

! 36! for!the!analyses!are!factors!that!can!complicate!the!association!between!mRNA!and! protein!levels51,!79.!In!a!study!by!Schwanhäusser!et!al!on!mouse!fibroblasts,!mRNA! and! protein! levels! correlated! with! a! coefficiency! (R2)! of! 0.4180.! It! seems! that! consistency!of!gene!expression!during!the!cell!cycle!and!functional!characteristics!of! mRNA!and!proteins!could!play!a!role!in!this!association.!According!to!Greenbaum!et! al,! fluctuating! RNA! levels! correlate! well! with! protein! amounts,! while! steady! gene! expression!has!a!weaker!correlation79.!Additionally,!in!their!study,!Schwanhäusser! et! al! report! that! regulatory! genes,! such! as! transcription! factors! or! chromatin! modifying!enzymes!have!unstable!mRNA!and!protein!levels.!Genes!related!to!RNA! functions,! such! as! processing! of! mRNAs,! tRNAs! and! nonZcoding! RNAs! have! stable! proteins! and! unstable! mRNAs,! while! genes! associated! with! extracellular! proteins! have!stable!mRNAs!and!unstable!proteins80.!!

Another! aspect! to! consider! is! that! the! RNA! levels! studied! in! this! report! represent!the!total!RNA!of!all!cells!present!in!the!gingival!tissues.!Unfortunately,!this! means!that!we!cannot!infer!how!gene!expression!varies!between!different!tissues,! and! cells! within! these! tissues,! such! as! epithelium,! connective! tissue! and! endothelium.!Additionally,!we!cannot!address!if!expression!of!specific!genes!in!this! study! is! the! result! of! a! transient! cell! in! the! gingiva! at! that! specific! time! point! of! biopsy!collection,!such!as!neutrophils,!macrophages,!etc.!!

Taking! the! above! limitations! under! consideration,! we! further! aimed! to! identify!differential!gene!expression!with!aging.!Aging!or!the!process!of!becoming! older! is! “a! ubiquitous! complex! phenomenon! that! results! from! environmental,!

! 37! stochastic,!genetic,!and!epigenetic!events!in!different!cell!types!and!tissues!and!their! interactions! throughout! life”47.! Degenerative! changes! in! multiple! organs! of! the! human! body! accompany! age! progression.! Periodontal! tissues! are! no! exception.!

Looking! specifically! at! the! gingiva,! it! has! been! reported! that! the! epithelium! undergoes!atrophy81!and!diminished!keratinization,!ultimately!becoming!thinner82.!

Additionally,!the!lamina!propria!of!older!individuals!presents!as!a!denser!and!more! coarsely!textured!tissue.82!These!changes!are!the!result!of!an!increase!in!the!number! and! thickness83! of! fiber! bundles! and! decrease! in! cellularity81.! After! all,! as! age! progresses,!fibroblasts!gradually!lose!their!ability!to!migrate!and!remodel!collagen! gels84.!At!the!same!time,!the!rate!of!collagen!synthesis!decreases82!and!the!rate!of! conversion!of!salt!soluble!to!saltZinsoluble!collagen!increases82.!

The! results! of! this! study! demonstrate! decreased! collagen! gene! expression! with!aging.!These!changes!are!seen!for!COL1A1,!COL1A2,!COL3A1,!COL5A2,!COL9A1,!

COL14A1! and! COL16A1,! and! are! consistent! with! decreases! in! collagen! gene! expression! (COL1A1)! noted! with! aging! in! skin85.! On! the! contrary,! MMP! gene! expression! levels! seem! to! be! unaffected,! with! only! MMP2,! MMP15! and! MMP16! decreasing!with!age.!MMP15!and!MMP16!have!been!implicated!in!the!activation!of! proMMP286;! thus! the! simultaneous! decrease! of! their! respective! genes! could! potentially! be! associated.! Whether! these! decreases! in! gene! expression! are! accompanied!by!commensurate!changes!in!protein!levels!cannot!be!determined!at! this! point.! However,! looking! at! the! literature,! it! seems! that! this! would! not! be! the! case.! More! specifically,! in! a! study! by! Gagliano! et! al87,! gingival! biopsies! were!

! 38! harvested!from!5!young!and!7!aged!subjects.!Although!mRNA!levels!of!collagen!type!

I!decreased!in!elderly!individuals,!collagen!I!and!III!protein!levels!in!the!tissues!were! similar!between!the!two!groups.!Additionally,!MMP1!mRNA!levels!were!unaffected! with!aging,!as!were!the!protein!levels!of!MMP1!and!MMP2.!Decreased!expression!of!

COL1A1!has!been!shown!by!other!authors!too!and!has!been!attributed!to!epigenetic! effects!leading!to!hypermethylation!changes!in!the!promoter!region!of!this!gene88.!

This!discrepancy!between!mRNA!and!protein!levels!could!perhaps!be!attributed!to! the! fact! that! insoluble! collagen! increases! with! age! in! gingival! tissues82.! Although! nothing! more! than! a! speculation! at! this! point,! one! could! assume! that! increased! amounts! of! collagen! that! cannot! be! easily! degraded! in! gingival! tissues! work! as! a! negative!feedback,!signaling!cells!to!produce!less!collagen.!

Regarding!MMP!gene!expression,!our!results!do!not!corroborate!the!findings! by! Kim! et! al46.! In! their! study,! healthy! gingival! tissues! were! obtained! from! three! young!and!three!old!individuals.!RNAZsequencing!was!used!for!analysis!and!pointed! towards! an! upregulation! in! the! gene! expression! of! MMP3,! MMP9,! MMP12! and!

MMP13!and!a!downregulation!of!MMP27.!The!discrepancies!between!their!and!our! results! could! potentially! stem! from! the! smaller! sample! size! in! their! study.!

Additionally,! evaluation! of! clinically! healthy! gingiva! is! a! highly! subjective! measurement;!thus,!the!gingival!condition!of!their!patients!cannot!be!corroborated.!

Location! of! biopsy! collection! would! also! be! expected! to! play! a! role,! as! during! harvesting,! we! strived! to! exclude! sulcular! and! junctional! epithelium! from! our! biopsies.! These! are! the! areas! where! initial! inflammatory! changes! are! primarily!

! 39! seen27,!as!a!response!to!microbial!attacks.!!!In!any!case,!the!lack!of!increased!MMP! gene!expression!noted!in!our!study!is!not!consistent!with!the!hyper!inflammatory! profile!and!dysregulated!immune!response!in!aging!gingiva!noted!in!the!literature.!

This!is!further!corroborated!by!the!absence!in!our!biopsies!of!other!markers!known! to! increase! with! inflammation! in! periodontal! tissues,! such! as! TNFZα,! ILZ8,! PGE2,!

MMP8,!ILZ2!and!ILZ1289.!

Looking!at!the!effect!of!aging!on!gingival!gene!expression!without!examining! these! responses! separately! in! males! and! females! would! be! nothing! more! that! an! oversimplification.! After! all,! the! literature! reports! that! anatomy! and! disease! of! tissues!present!with!different!characteristics!between!males!and!females90.!As!a!part! of!this!effort!the!terms!“gender”!and!“sex”!need!to!be!understood!and!differentiated.!

More! specifically,! “sex”! refers! to! biological! and! physiological! characteristics! that! define! males! and! females,! as! this! is! seen! in! the! different! chromosomes,! organs,! organ! systems! and! anatomy.! Females! present! with! two! X! chromosomes,! one! of! which! forms! a! heterochromatic! structure! and! is! inactive91,! in! order! to! achieve! commensurate! expression! of! XZlinked! genes! between! males! and! females! (“dosage! compensation”)92.! An! RNAZcoding! gene,! the! Xist! gene,! is! responsible! for! the! initiation!of!X!inactivation!and!leads!to!epigenetic!changes,!such!as!DNA!methylation! and!histone!modification,!that!stabilize!the!inactive!X!chromosome!(Xi)93.!“Gender”! refers! to! the! roles,! behaviors,! activities! and! attributes! that! individual! societies! consider!appropriate!for!men!and!women94.!Gender!influences!in!the!periodontium! are!mainly!related!to!risk!perception!and!cultural!norms,!and!are!expressed!in!the!

! 40! lower! percentage! of! men! utilizing! preventive! care! services95! and! adequately! following!oral!hygiene!practices96.!On!the!other!hand,!sex!and!genetic!influences!can! manifest! through! the! effects! of! sex! chromosomes! on! immunity90,! as! well! as! the! presence!of!steroid!receptors!in!the!periodontium.!More!specifically,!studies!of!the! gingiva! reveal! that! estrogen! and! androgen! receptors! are! seen! in! epithelium,! connective!tissue!fibroblasts!and!endothelial!cells97,!98!while!progesterone!receptors! have!been!characterized!in!human!gingival!fibroblasts,!although!in!small!amounts99.!

As! a! result,! sex! hormones! have! been! shown! to! affect! the! epithelium,! gingival! fibroblasts! and! vasculature! of! the! periodontium53! whereas! potential! effects! on! periodontal!pathogens!have!not!been!substantiated100.!!

Trying! to! integrate! the! above! in! our! study,! we! evaluated! the! changes! in! collagen!and!MMP!gene!expression!focusing!separately!on!males!and!females.!The! collagenZrelated!results!were!very!interesting,!as!fourteen!collagen!genes!decreased! with!aging!in!the!female!group,!but!only!one!collagen!gene!was!decreased!in!males.!

Thus,!it!seems!that!altered!collagen!gene!expression!with!aging!is!an!effect!primarily! stemming!from!the!female!group.!Similar!results!were!not!obtained!for!MMPs.!After! all,!changes!in!this!group!with!aging!were!limited!and!showed!only!a!small!sex!bias;!

MMP2!and!MMP15!expression!was!significantly!reduced!in!the!female!group,!while!

MMP16!expression!was!reduced!in!the!aging!male!group.!

In!an!effort!to!explain!these!differential!responses!between!sex!groups,!we! looked! at! the! literature! for! effects! of! sex! hormones! on! collagen! production.!!

According!to!Mariotti53!et!al,!estradiol!decreases!collagen!production!from!gingival!

! 41! fibroblasts.! Same! results! have! been! shown! in! other! tissues.! More! specifically,! incubation!of!human!aortic!smooth!muscle!cells!with!physiologic!concentrations!of!

17βZestradiol,!progesterone!or!testosterone!showed!that!production!of!collagen!I,!II! and!III!were!reduced!by!all!sex!steroids,!but!the!effect!of!estradiol!and!progesterone! was! more! intense.! MMPZ2! expression! remained! unaffected101.! Although! these! studies! provide! mechanisms! to! explain! variations! in! collagen! production! between! sexes,!their!findings!probably!point!to!the!opposite!direction.!After!all,!estrogen!and! progesterone! concentrations! sharply! decrease! after! menopause;! if! the! mechanism! for! the! altered! gene! expression! could! simply! be! explained! by! hormones,! the! decreased!presence!of!estrogens!and!progesterone!in!our!menopausal!older!female! group,! would! lead! to! an! upregulation! of! collagen! gene! expression.! On! the! other! hand,! studies! in! skin! are! more! consistent! with! our! findings,! as! estrogens! seem! to! enhance!collagen!synthesis102.!!

The! basic! strength! of! this! study! is! the! analytical! approach! used.! RNAZ sequencing!with!UMIs!provided!us!with!the!comprehensive!trasncriptome!of!each! participant,!and!with!precise!knowledge!of!the!number!of!individual!transcripts!for! each!gene,!minimizing!amplification!errors.!Investigation!of!the!transcriptome!will! continue,!in!an!effort!to!identify!differential!expression!of!additional!genes!between! age!and!sex!groups.!Additionally,!the!investigation!will!extend!to!analyze!RNAs!with! regulatory!roles!on!the!genome,!such!as!miRNAs.!Finally,!succeeding!in!identifying! the! proteome! (entire! complement! of! proteins)! of! these! individuals! can! clarify! the! relationship!between!gene!expression!and!protein!levels.!

! 42! On! the! other! hand,! one! of! the! limitations! of! this! study! is! potentially! the! sample! size.! However,! although! nineteen! subjects! is! not! a! high! number,! it! is! definitely!more!than!what!other!transcriptomic!studies!conducted!on!humans!have! used46,103.!After!all,!we!have!to!keep!in!mind!that!harvesting!gingival!biopsies!from! people! with! healthy! gingiva! does! not! offer! a! direct! benefit! to! them,! thus! raising! ethical! concerns! for! studies! with! a! high! number! of! participants.! Additionally,! identifying!people!≥!60!years!old!with!healthy!periodontium!is!no!mean!feat,!due!to! the!high!prevalence!of!periodontal!diseases!and!edentulism!with!increasing!age.!!In! this!study,!we!tried!to!recruit!systemically!healthy!patients.!However,!this!was!not! achievable!in!the!older!age!group,!so!we!expanded!our!selection!to!people!with!wellZ controlled! systemic! diseases.! Although! the! systemic! illnesses! that! we! accepted! in! this! study! are! not! part! of! the! reported! diseases! affecting! the! periodontium,! we! cannot! exclude! that! these! diseases! or! the! medications! used! to! treat! them! do! not! have! effects! on! the! epigenetic! profile,! and! hence! gene! expression! of! the! periodontium.!Finally,!as!this!is!a!transcriptomics!study,!any!correlations!between! gene!expression!and!protein!levels!must!be!interpreted!with!caution.!

! !!

! 43! !

Chapter!5!

Conclusions!

Gingival!biopsies!were!harvested!from!the!right!and!left!interproximal!area! between!the!maxillary!second!premolar!and!first!molar!from!nineteen!individuals! with!clinically!healthy!gingiva.!Four!groups!of!patients!were!analyzed,!namely!young! males,! young! females,! old! males! and! old! females.! RNA! sequencing! with! UMI! identifiers! was! used! for! the! analysis.! This! study! focuses! on! collagen! and! matrix! metalloproteinase!gene!expression.!

! Expression!of!fortyZeight!distinct!collagen!genes!was!identified!in!the!gingiva! of!the!participating!individuals.!!The!top!four!expressed!collagen!genes,!with!high! number!of!transcripts,!were!COL1A2,!COL17A1,!COL3A1!and!COL1A1.!Regarding!the! effect! of! age,! fifteen! collagen! genes! were! determined! to! be! significantly! downregulated!with!aging,!and!one!(COL4A6)!was!upregulated.!These!changes!were! not!seen!in!the!male!aging!group,!but!were!mostly!expressed!by!females.!!

! TwentyZfive! distinct! MMP! genes! were! identified! in! our! sample.! The! five! genes!with!the!highest!expression!were!MMP2,!MMP28,!MMP24ZAS1,!MMP16!and!

MMP14.! The! effect! of! age! on! MMP! gene! expression! is! not! as! intense! as! the! one!

! 44! observed! in! collagen! genes.! More! specifically,! MMP2! and! MMP15! expression! was! downregulated!in!aging!females!and!MMP16!was!downregulated!in!aging!males.!!

! !

! 45! !

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! 51! 97.! Vittek! J,! Hernandez! MR,! Wenk! EJ,! Rappaport! SC,! Southren! AL.! Specific! estrogen! receptors! in! human! gingiva.! J3Clin3Endocrinol3Metab! 1982;54:608Z 612.! 98.! OjanotkoZHarri! A,! Forssell! H,! Laine! M,! Hurttia! H,! Blauer! M,! Tuohimaa! P.! Immunohistochemical! detection! of! androgen! receptors! in! human! oral! mucosa.!Arch3Oral3Biol!1992;37:511Z514.! 99.! Kawahara! K,! Shimazu! A.! Expression! and! intracellular! localization! of! progesterone!receptors!in!cultured!human!gingival!fibroblasts.!J3Periodontal3 Res!2003;38:242Z246.! 100.! Kumar!PS.!Sex!and!the!subgingival!microbiome:!do!female!sex!steroids!affect! periodontal!bacteria?!Periodontol32000!2013;61:103Z124.! 101.! Natoli! AK,! Medley! TL,! Ahimastos! AA,! et! al.! Sex! steroids! modulate! human! aortic! smooth! muscle! cell! matrix! protein! deposition! and! matrix! metalloproteinase!expression.!Hypertension!2005;46:1129Z1134.! 102.! Kanda!N,!Watanabe!S.!Regulatory!roles!of!sex!hormones!in!cutaneous!biology! and!immunology.!J3Dermatol3Sci!2005;38:1Z7.! 103.! Wang!Y,!Tatakis!DN.!Human!gingiva!transcriptome!during!wound!healing.!J3 Clin3Periodontol!2017;44:394Z402.! ! !

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! 52! ! ! ! !

Appendix!A:!Distribution!of!Subjects!in!Sex!and!Age!Groups!

! 53! ! Appendix!A:!Distribution!of!subjects!in!sex!and!age!groups! ! !

! ! ! 18Z35!years!old! ≥60!years!old! ! ! Subject!#4! Subject!#2! ! Subject!#7! Subject!#15! Male! Subject!#8! Subject!#17! Subject!#11! Subject!#18! Subject!#13! Subject!#19! ! Subject!#1! Subject!#9! ! Subject!#3! Subject!#10! Female! Subject!#5! Subject!#12! Subject!#6! Subject!#16! Subject!#14! Z! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! 54! ! ! !

Appendix!B:!General!Patient!Characteristics!

! 55! Appendix!B.!General!Patient!Characteristics!

! ! ! ! ! Subject! Age1! Sex! Systemic!Illnesses! Medications! ! ! ! ! ! ! #1! 25! Female! Z! Z! ! ! ! ! ! ! ! #2! 66! Male! Hypertension! ACE!Inhibitor! Coronary!Artery!Stent! Atorvastatin! Hypercholesterolemia! ! ! ! ! ! ! #3! 35! Female! Hypothyroidism! Levothyroxine! Anxiety!/!Depression! Liothyronine! ! Bupropion! Fluticasone! Contraceptives! ! ! ! ! ! ! #4! 25! Male! Asthma! Albuterol!Inhaler! ! ! ! ! ! ! #5! 27! Female! Thyroid!Nodule! T4! ! ! ! ! ! ! #6! 29! Female! Z! Z! ! ! ! ! ! ! #7! 24! Male! Z! Z! ! ! ! ! ! ! #8! 28! Male! Z! Z! ! Continued! !

! 56! Appendix!B!continued!

! ! ! ! ! Subject! Age1! Sex! Systemic!Illnesses! Medications! ! ! ! ! ! ! #9! 65! Female! Hypertension! Angiotensin!Receptor! ! Osteoarthritis! Antagonist! Ibuprofen! ! ! ! ! ! #10! 62! Female! Hypertension! ΒZblocker/Diuretic! Hypercholesterolemia! Atorvastatin! GERD! Omeprazole! Vitamin!D! Calcium! Probiotic! ! ! ! ! ! #11! 26! Male! Z! Z! ! ! ! ! ! ! #12! 78! Female! Hypertension! ACE!Inhibitor! COPD!(prevention)! Leukotriene!Receptor! C2!Fusion! Antagonist! ! Ambien! ! ! ! ! ! #13! 26! Male! Z! Z! ! ! ! ! ! ! #14! 26! Female! Z! Z! ! ! ! ! ! ! #15! 71! Male! Hypertension! ACE!Inhibitor! Hypercholesterolemia! Aspirin!81mg! Arthritis! Rosumvastatin! Sleep!Apnea! Multivitamin! Hep!A!(in!childhood)! FishOil! CPAP! ! Continued!

! 57! !

Appendix!B!continued!

! ! ! ! ! Subject! Age1! Sex! Systemic!Illnesses! Medications! ! ! ! ! ! ! #16! 81! Female! Hypothyroidism! Levothyroxine! Mild!Heart!Attack! Angiotensin!Receptor! Metal!Stent! Antagonist! ! ΒZblocker! ! ! ! ! ! #17! 70! Male! Asthma! Topical!Corticosteroid! Albuterol! Omeprazole! ACE!Inhibitor! Simvastatin! ! ! ! ! ! #18! 64! Male! Hypertension! Angiotensin!Receptor! Antagonist! Multivitamin! Calcium! Coenzyme!Q10! ! ! ! ! ! #19! 62! Male! Hypertension! ACE!Inhibitor! Hypercholesterolemia! Aspirin!81mg! Pravastatin! ! ! 1!Age!in!years!

! ! !

! 58! !

Appendix!C:!Antibiotic!Intake!

! 59! !!!!!!Appendix!C.!Antibiotic!Intake! !

! ! ! Subject! Antibiotic!Intake! ! ! #1! No!Antibiotic!Intake!for!≥6!months! #2! No!Antibiotic!Intake!for!≥6!months! ! #3! No!Antibiotic!Intake!for!≥6!months! #4! No!Antibiotic!Intake!for!≥6!months! ! #5! No!Antibiotic!Intake!for!≥6!months! ! #6! No!Antibiotic!Intake!for!≥6!months! #7! No!Antibiotic!Intake!for!≥6!months! ! #8! No!Antibiotic!Intake!for!≥6!months! #9! No!Antibiotic!Intake!for!≥6!months! ! #10! No!Antibiotic!Intake!for!≥6!months! #11! No!Antibiotic!Intake!for!≥6!months! ! #12! Antibiotic!Intake!4!months!before!Biopsy!Harvesting! #13! No!Antibiotic!Intake!for!≥6!months! ! #14! No!Antibiotic!Intake!for!≥6!months! #15! No!Antibiotic!Intake!for!≥6!months! ! #16! No!Antibiotic!Intake!for!≥6!months! #17! No!Antibiotic!Intake!for!≥6!months! ! #18! No!Antibiotic!Intake!for!≥6!months! #19! No!Antibiotic!Intake!for!≥6!months! !

! 60! !

Appendix!D:!Clinical!Parameters!

! 61! Appendix!D.!Clinical!Parameters!

! ! ! ! ! Subject! Probing!Depth!(PD)2! Redness! Edema! BOP2! ! #1! ≤3mm! Negative! Negative! Negative! #2! ≤3mm! Negative! Negative! Negative! #3! ≤3mm! Negative! Negative! Negative! #4! ≤3mm! Negative! Negative! Negative! #5! ≤3mm! Negative! Negative! Negative! #6! ≤3mm! Negative! Negative! Negative! #7! ≤3mm! Negative! Negative! Negative! #8! ≤3mm! Negative! Negative! Negative! #9! ≤3mm! Negative! Negative! Negative! #10! ≤3mm! Negative! Negative! Negative! #11! ≤3mm! Negative! Negative! Negative! #12! ≤3mm! Negative! Negative! Negative! #13! ≤3mm! Negative! Negative! Negative! #14! ≤3mm! Negative! Negative! Negative! #15! ≤3mm! Negative! Negative! Negative! #16! ≤3mm! Negative! Negative! Negative! #17! ≤3mm! Negative! Negative! Negative! #18! ≤3mm! Negative! Negative! Negative! #19! ≤3mm! Negative! Negative! Negative! ! 2!Measurements!for!PD!and!BOP!were!performed!at!6!sites/tooth!(mesiobuccal,! midbuccal,!distobuccal,!mesiopalatal,!midpalatal,!distopalatal)! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! 62! !

Appendix!E:!NextZGeneration!Sequencing!Reads!

! 63! Appendix!E.!NextZGeneration!Sequencing!Reads

!Cell!ID! Not! Matching! Not! All! Cell!ID! a!Used! Mapped! Total!Reads! NNNNNN! Not! Less!Than! Oligo! 25!bp! to! 113,925,455! Sequence! Extracted! Within! 4,683,587! Genome! 0! 71,062! Edit! Distance! 4,371,229! 1!bp! 7,094,573!

Cell!Has! Reads! !Aligned! No!Genes! Used,! to! !Aligned! With! to!ERCC,! Aligned!to! !Not! Genome,! Annotated! More! Multiple! Genome,! 37,085! Multiple! genes! 37,549,875! than!5! Loci! Unique! Loci! UMIs! Loci! 0! 2,806,789! 3,529! 57,344,811!

! 64! !

Appendix!F:!Collagen!Genes!–!Total!Number!of!Transcripts!

! 65! Appendix!F.!Collagen!genes!expressed!in!clinically!healthy!gingival!tissues.!The!last! column!shows!the!total!number!of!expressed!transcripts!when!combining!the! biopsies!of!all!subjects.! !

Stran Chro Total$ Gene$ID$ Gene$ LOC$5'$ LOC$3'$ d$ m$ Count$ ENSG00000164692.17- COL1A2- 1- chr7- 94394561- 94431232- 8995- ENSG00000065618.16- COL17A1- 81- chr10- 104086002- 104031286- 6583- ENSG00000168542.12- COL3A1- 1- chr2- 188974320- 189012746- 5347- ENSG00000108821.13- COL1A1- 81- chr17- 50201632- 50183289- 5078- ENSG00000113163.15- COL4A3BP- 81- chr5- 75512138- 75368486- 927- ENSG00000187955.11- COL14A1- 1- chr8- 120059780- 120372036- 780- ENSG00000163359.15- COL6A3- 81- chr2- 237414375- 237324003- 731- ENSG00000142156.14- COL6A1- 1- chr21- 45981737- 46005050- 688- ENSG00000204262.11- COL5A2- 81- chr2- 189179879- 189031896- 680- ENSG00000188153.12- COL4A5- 1- chrX- 108439844- 108697545- 667- ENSG00000182871.14- COL18A1- 1- chr21- 45405137- 45513720- 613- ENSG00000111799.20- COL12A1- 81- chr6- 75206051- 75084326- 506- ENSG00000144810.15- COL8A1- 1- chr3- 99638475- 99799226- 417- ENSG00000142173.14- COL6A2- 1- chr21- 46098097- 46132849- 338- ENSG00000114270.15- COL7A1- 81- chr3- 48595267- 48564073- 327- ENSG00000130635.15- COL5A1- 1- chr9- 134641774- 134844843- 297- ENSG00000084636.17- COL16A1- 81- chr1- 31704319- 31652247- 255- ENSG00000204291.10- COL15A1- 1- chr9- 98943179- 99070792- 246- ENSG00000197565.15- COL4A6- 81- chrX- 108439497- 108155607- 237- ENSG00000196739.14- COL27A1- 1- chr9- 114155560- 114312511- 215- ENSG00000134871.17- COL4A2- 1- chr13- 110305812- 110513027- 213- ENSG00000124749.16- COL21A1- 81- chr6- 56394094- 56056590- 207- ENSG00000187498.14- COL4A1- 81- chr13- 110307149- 110148963- 173- ENSG00000080573.6- COL5A3- 81- chr19- 10010471- 9959561- 168- ENSG00000171502.14- COL24A1- 81- chr1- 86156943- 85729233- 151- ENSG00000060718.18- COL11A1- 81- chr1- 103108496- 102876467- 149- ENSG00000050767.15- COL23A1- 81- chr5- 178590555- 178237618- 141- ENSG00000188517.14- COL25A1- 81- chr4- 109302657- 108810721- 89- ENSG00000169031.18- COL4A3- 1- chr2- 227164565- 227314792- 73- ENSG00000215018.9- COL28A1- 81- chr7- 7535853- 7356203- 64- ENSG00000123500.9- COL10A1- 81- chr6- 116158747- 116118923- 57- ENSG00000081052.11- COL4A4- 81- chr2- 227164113- 227002711- 52------Continued!

! 66! Appendix!F!continued! ------ENSG00000171812.10- COL8A2- 81- chr1- 36125220- 36095236- 43- ENSG00000082293.12- COL19A1- 1- chr6- 69866571- 70209976- 42- ENSG00000172752.14- COL6A5- 1- chr3- 130345516- 130484844- 31- ENSG00000160963.13- COL26A1- 1- chr7- 101362820- 101559024- 28- ENSG00000206384.10- COL6A6- 1- chr3- 130560334- 130678155- 28- ENSG00000112280.15- COL9A1- 81- chr6- 70303083- 70215061- 23- ENSG00000092758.15- COL9A3- 1- chr20- 62816244- 62841159- 22- ENSG00000197467.13- COL13A1- 1- chr10- 69801931- 69964275- 17- ENSG00000169436.16- COL22A1- 81- chr8- 138914006- 138588235- 10- ENSG00000228252.8- COL6A4P2- 1- chr3- 130212823- 130273806- 9- ENSG00000204248.10- COL11A2- 81- chr6- 33192499- 33162681- 7- ENSG00000049089.13- COL9A2- 81- chr1- 40317816- 40300487- 6- COL4A28 ENSG00000224821.5- AS2- 81- chr13- 110463287- 110456396- 2- ENSG00000139219.17- COL2A1- 81- chr12- 48004486- 47972965- 1- COL5A18 ENSG00000204011.4- AS1- 81- chr9- 134652843- 134649385- 1- ENSG00000230524.8- COL6A4P1- 81- chr3- 15205959- 15151833- 1- !

! 67! !

Appendix!G:!Number!of!Collagen!Gene!Transcripts!Expressed!in!the!Young!Male!

Group!

! 68! !

Appendix! G.! Number! of! collagen! gene! transcripts! expressed! in! the! young! male! group.!The!first!column!shows!the!gene!name,!while!the!remaining!columns!show! the!number!of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers! #4,!7,!8,!11!and!13!correspond!to!subjects,!while!the!letters!“L”!and!“R”!symbolize! the!biopsies!harvested!from!the!left!and!right!side!of!the!maxilla!respectively.! !

Gene$ 4L- 4R- 7L- 7R- 8R- 11L- 11R- 13L- 13R- Total$ COL1A2- 179- 389- 342- 308- 72- 152- 103- 65- 31- 1641- COL17A1- 184- 366- 223- 126- 33- 23- 63- 46- 28- 1092- COL3A1- 84- 176- 208- 189- 31- 61- 72- 19- 13- 853- COL1A1- 79- 156- 190- 222- 27- 75- 68- 22- 9- 848- COL4A3BP- 20- 38- 42- 13- 3- 3- 17- 2- 17- 155- COL14A1- 8- 34- 27- 16- 6- 4- 6- 1- 4- 106- COL6A3- 9- 23- 16- 18- 4- 4- 9- 2- 1- 86- COL6A1- 13- 41- 21- 15- 4- 3- 4- 6- 3- 110- COL5A2- 15- 23- 26- 15- 6- 1- 14- 2- 10- 112- COL4A5- 14- 22- 16- 4- 9- 3- 7- 3- 1- 79- COL18A1- 7- 32- 12- 8- 2- 5- 4- 10- 1- 81- COL12A1- 5- 24- 25- 21- 1- 5- 3- 3- 0- 87- COL8A1- 6- 27- 16- 2- 2- 2- 4- 0- 1- 60- COL6A2- 5- 15- 12- 11- 1- 5- 7- 5- 0- 61- COL7A1- 9- 15- 9- 11- 1- 2- 2- 4- 0- 53- COL5A1- 6- 18- 10- 4- 1- 6- 4- 2- 1- 52- COL16A1- 8- 11- 5- 9- 1- 5- 2- 4- 6- 51- COL15A1- 2- 10- 8- 4- 1- 2- 7- 4- 1- 39- COL4A6- 4- 13- 4- 5- 2- 0- 6- 1- 3- 38- COL27A1- 2- 6- 2- 1- 0- 5- 8- 1- 1- 26- COL4A2- 4- 12- 9- 5- 0- 0- 1- 1- 0- 32- COL21A1- 5- 10- 10- 0- 1- 0- 4- 1- 0- 31- COL4A1- 1- 6- 2- 3- 0- 0- 4- 0- 4- 20- COL5A3- 4- 7- 9- 3- 0- 1- 4- 1- 1- 30- COL24A1- 7- 7- 5- 1- 1- 0- 0- 0- 1- 22- COL11A1- 3- 3- 10- 6- 0- 2- 2- 0- 0- 26- COL23A1- 1- 10- 9- 1- 0- 0- 0- 1- 0- 22- COL25A1- 2- 3- 3- 0- 0- 0- 0- 1- 2- 11------Continued-

! 69! Appendix-H-continued------COL4A3- 0- 2- 6- 0- 1- 0- 1- 0- 0- 10- COL28A1- 0- 4- 2- 1- 0- 0- 1- 1- 1- 10- COL10A1- 1- 1- 0- 0- 1- 1- 1- 0- 0- 5- COL4A4- 2- 0- 1- 0- 0- 0- 0- 0- 1- 4- COL8A2- 0- 9- 3- 1- 0- 0- 0- 2- 0- 15- COL19A1- 0- 2- 2- 0- 0- 0- 0- 0- 1- 5- COL6A5- 2- 7- 0- 1- 0- 0- 0- 0- 0- 10- COL26A1- 0- 1- 0- 0- 0- 1- 0- 0- 1- 3- COL6A6- 0- 1- 1- 0- 0- 0- 0- 0- 0- 2- COL9A1- 0- 0- 2- 0- 0- 0- 0- 0- 0- 2- COL9A3- 0- 3- 2- 1- 0- 0- 1- 0- 0- 7- COL13A1- 0- 0- 1- 0- 0- 0- 0- 0- 0- 1- COL22A1- 0- 0- 1- 0- 0- 0- 0- 0- 0- 1- COL6A4P2- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL11A2- 0- 0- 1- 0- 0- 0- 0- 0- 0- 1- COL9A2- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL4A28AS2- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL2A1- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL5A18AS1- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL6A4P1- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- !

! 70! !

Appendix!H:!Number!of!Collagen!Gene!Transcripts!Expressed!in!the!Old!Male!Group!

! 71! Appendix!H.!Number!of!collagen!gene!transcripts!expressed!in!the!old!male!group.! The! first! column! shows! the! gene! name,! while! the! remaining! columns! show! the! number!of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers!#2,! 17,! 18! and! 19! correspond! to! subjects,! while! the! letters! “L”! and! “R”! symbolize! the! biopsies!harvested!from!the!left!and!right!side!of!the!maxilla!respectively.! !

Gene$ 2L$ 2R$ 17L$ 18R$ 19L$ 19R$ Total$ COL1A2- 272- 319- 690- 37- 22- 212- 1552- COL17A1- 543- 517- 172- 48- 26- 103- 1409- COL3A1- 222- 196- 430- 23- 20- 110- 1001- COL1A1- 126- 125- 480- 27- 37- 94- 889- COL4A3BP- 64- 56- 13- 16- 10- 24- 183- COL14A1- 27- 36- 30- 1- 2- 18- 114- COL6A3- 49- 41- 41- 3- 3- 16- 153- COL6A1- 47- 26- 54- 9- 4- 11- 151- COL5A2- 30- 29- 18- 8- 1- 19- 105- COL4A5- 90- 81- 12- 3- 8- 8- 202- COL18A1- 58- 57- 19- 5- 3- 8- 150- COL12A1- 34- 20- 30- 3- 2- 16- 105- COL8A1- 25- 15- 3- 2- 6- 10- 61- COL6A2- 18- 16- 30- 5- 2- 4- 75- COL7A1- 39- 30- 7- 1- 3- 4- 84- COL5A1- 18- 20- 14- 1- 3- 5- 61- COL16A1- 17- 21- 9- 1- 1- 1- 50- COL15A1- 25- 16- 5- 2- 4- 6- 58- COL4A6- 26- 36- 6- 5- 7- 6- 86- COL27A1- 23- 19- 12- 1- 2- 7- 64- COL4A2- 13- 19- 11- 2- 0- 11- 56- COL21A1- 6- 8- 8- 6- 5- 3- 36- COL4A1- 5- 13- 10- 1- 1- 12- 42- COL5A3- 9- 17- 8- 0- 0- 2- 36- COL24A1- 7- 8- 2- 1- 2- 4- 24- COL11A1- 8- 5- 3- 2- 0- 1- 19- COL23A1- 13- 11- 1- 2- 1- 3- 31- COL25A1- 7- 9- 0- 1- 1- 1- 19- COL4A3- 4- 9- 0- 0- 0- 5- 18- COL28A1- 5- 2- 3- 0- 0- 1- 11- COL10A1- 2- 5- 0- 0- 1- 2- 10------Continued-

! 72! Appendix-H-continued------COL4A4- 3- 2- 2- 1- 0- 0- 8- COL8A2- 3- 1- 0- 0- 0- 1- 5- COL19A1- 2- 1- 1- 0- 1- 2- 7- COL6A5- 0- 1- 0- 1- 0- 1- 3- COL26A1- 1- 1- 0- 1- 0- 0- 3- COL6A6- 1- 0- 0- 0- 1- 0- 2- COL9A1- 0- 0- 1- 0- 0- 0- 1- COL9A3- 3- 0- 1- 0- 0- 0- 4- COL13A1- 4- 1- 1- 0- 0- 0- 6- COL22A1- 0- 2- 0- 0- 0- 0- 2- COL6A4P2- 0- 0- 0- 0- 0- 0- 0- COL11A2- 2- 1- 1- 0- 0- 0- 4- COL9A2- 0- 0- 0- 0- 0- 0- 0- COL4A28AS2- 0- 0- 0- 0- 0- 0- 0- COL2A1- 0- 0- 0- 0- 0- 0- 0- COL5A18AS1- 0- 0- 0- 0- 0- 0- 0- COL6A4P1- 0- 0- 0- 0- 1- 0- 1- !

! 73! !

Appendix!I:!Number!of!Collagen!Gene!Transcripts!Expressed!in!the!Young!Female!

Group!

! 74! Appendix! I.! Number! of! collagen! gene! transcripts! expressed! in! the! young! female! group.!The!first!column!shows!the!gene!name,!while!the!remaining!columns!show! the!number!of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers! #1,!5,!6!and!14!correspond!to!subjects,!while!the!letters!“L”!and!“R”!symbolize!the! biopsies!harvested!from!the!left!and!right!side!of!the!maxilla!respectively.! !

Gene$ 1L$ 1R$ 5L$ 5R$ 6L$ 6R$ 14R$ Total$ COL1A2- 943- 844- 301- 388- 804- 1424- 136- 4840- COL17A1- 795- 903- 124- 424- 380- 365- 28- 3019- COL3A1- 583- 555- 169- 220- 447- 814- 65- 2853- COL1A1- 559- 509- 125- 158- 501- 988- 79- 2919- COL4A3BP- 67- 81- 24- 105- 66- 52- 4- 399- COL14A1- 62- 69- 38- 70- 85- 111- 7- 442- COL6A3- 103- 77- 27- 37- 78- 88- 2- 412- COL6A1- 86- 53- 19- 39- 53- 77- 3- 330- COL5A2- 65- 69- 21- 26- 67- 128- 4- 380- COL4A5- 37- 44- 17- 40- 76- 59- 2- 275- COL18A1- 63- 73- 7- 34- 45- 55- 6- 283- COL12A1- 42- 44- 14- 31- 46- 63- 4- 244- COL8A1- 65- 73- 11- 25- 20- 46- 0- 240- COL6A2- 23- 37- 5- 20- 30- 42- 1- 158- COL7A1- 47- 49- 1- 22- 13- 9- 3- 144- COL5A1- 19- 37- 9- 15- 30- 36- 1- 147- COL16A1- 23- 29- 7- 13- 16- 25- 0- 113- COL15A1- 21- 19- 7- 18- 21- 19- 0- 105- COL4A6- 9- 16- 1- 9- 21- 19- 0- 75- COL27A1- 14- 10- 4- 17- 15- 29- 0- 89- COL4A2- 10- 11- 4- 16- 15- 33- 1- 90- COL21A1- 18- 18- 3- 19- 25- 27- 0- 110- COL4A1- 16- 14- 7- 16- 15- 20- 0- 88- COL5A3- 9- 20- 7- 15- 13- 12- 0- 76- COL24A1- 8- 9- 2- 10- 20- 31- 4- 84- COL11A1- 14- 17- 1- 4- 12- 28- 4- 80- COL23A1- 10- 10- 3- 14- 19- 14- 0- 70- COL25A1- 2- 6- 0- 9- 15- 17- 0- 49- COL4A3- 2- 7- 1- 6- 9- 8- 2- 35- COL28A1- 3- 7- 2- 1- 14- 3- 0- 30- COL10A1- 4- 3- 4- 8- 9- 3- 0- 31------Continued-

! 75! Appendix-I-continued------COL4A4- 3- 3- 3- 5- 4- 10- 0- 28- COL8A2- 4- 0- 0- 3- 2- 5- 0- 14- COL19A1- 2- 5- 0- 3- 1- 16- 0- 27- COL6A5- 3- 1- 1- 1- 2- 1- 0- 9- COL26A1- 0- 1- 0- 4- 6- 4- 1- 16- COL6A6- 1- 2- 0- 4- 7- 2- 0- 16- COL9A1- 3- 2- 0- 0- 5- 9- 0- 19- COL9A3- 3- 1- 0- 2- 1- 1- 0- 8- COL13A1- 0- 0- 0- 3- 1- 2- 0- 6- COL22A1- 0- 0- 0- 1- 3- 2- 0- 6- COL6A4P2- 5- 1- 0- 0- 0- 2- 0- 8- COL11A2- 0- 0- 0- 0- 0- 0- 0- 0- COL9A2- 0- 0- 0- 1- 2- 2- 0- 5- COL4A28AS2- 0- 0- 0- 0- 0- 1- 0- 1- COL2A1- 1- 0- 0- 0- 0- 0- 0- 1- COL5A18AS1- 0- 1- 0- 0- 0- 0- 0- 1- COL6A4P1- 0- 0- 0- 0- 0- 0- 0- 0- !

! 76! !

Appendix!J:!Number!of!Collagen!Gene!Transcripts!Expressed!in!the!Old!Female!

Group!

! 77! Appendix!J.!Number!of!collagen!gene!transcripts!expressed!in!the!old!female!group.! The! first! column! shows! the! gene! name,! while! the! remaining! columns! show! the! number!of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers!#9,! 10,! 12! and! 16! correspond! to! subjects,! while! the! letters! “L”! and! “R”! symbolize! the! biopsies!harvested!from!the!left!and!right!side!of!the!maxilla!respectively.! !

Gene$ 9L$ 9R$ 10L$ 10R$ 12L$ 12R$ 16L$ 16R$ Total$ COL1A2- 20- 154- 166- 75- 129- 79- 13- 13- 649- COL17A1- 26- 126- 124- 38- 149- 126- 11- 20- 620- COL3A1- 20- 74- 101- 59- 69- 63- 11- 15- 412- COL1A1- 14- 63- 80- 43- 42- 34- 2- 8- 286- COL4A3BP- 4- 22- 19- 7- 17- 35- 3- 4- 111- COL14A1- 2- 28- 29- 3- 7- 14- 1- 1- 85- COL6A3- 1- 16- 18- 0- 9- 8- 1- 2- 55- COL6A1- 0- 10- 6- 3- 12- 14- 1- 3- 49- COL5A2- 2- 9- 11- 6- 8- 8- 1- 4- 49- COL4A5- 2- 23- 19- 6- 11- 10- 3- 6- 80- COL18A1- 0- 10- 11- 4- 8- 14- 1- 0- 48- COL12A1- 1- 12- 13- 5- 6- 5- 0- 1- 43- COL8A1- 0- 7- 9- 3- 13- 5- 2- 0- 39- COL6A2- 0- 10- 6- 0- 8- 5- 0- 0- 29- COL7A1- 0- 7- 6- 0- 6- 4- 1- 2- 26- COL5A1- 0- 5- 10- 3- 5- 3- 0- 1- 27- COL16A1- 0- 1- 9- 3- 7- 7- 1- 0- 28- COL15A1- 0- 8- 6- 2- 5- 5- 4- 0- 30- COL4A6- 2- 3- 2- 5- 0- 6- 3- 7- 28- COL27A1- 4- 2- 6- 0- 2- 10- 0- 0- 24- COL4A2- 0- 4- 10- 4- 4- 4- 0- 0- 26- COL21A1- 0- 3- 6- 3- 5- 0- 7- 0- 24- COL4A1- 0- 5- 6- 0- 5- 3- 0- 0- 19- COL5A3- 0- 8- 4- 2- 5- 1- 0- 0- 20- COL24A1- 0- 3- 1- 1- 1- 1- 0- 3- 10- COL11A1- 0- 6- 1- 5- 2- 3- 1- 0- 18- COL23A1- 0- 0- 3- 0- 4- 2- 0- 0- 9- COL25A1- 0- 4- 0- 0- 1- 0- 2- 0- 7- COL4A3- 0- 1- 2- 0- 3- 0- 0- 0- 6- COL28A1- 0- 2- 1- 0- 5- 1- 0- 1- 10- COL10A1- 1- 3- 0- 1- 0- 2- 2- 1- 10------Continued-

! 78! Appendix-J-continued------COL4A4- 0- 0- 4- 0- 0- 4- 0- 0- 8- COL8A2- 0- 1- 1- 1- 2- 2- 0- 0- 7- COL19A1- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL6A5- 0- 0- 0- 0- 1- 6- 0- 0- 7- COL26A1- 0- 0- 0- 0- 1- 1- 0- 0- 2- COL6A6- 0- 0- 0- 0- 1- 4- 0- 1- 6- COL9A1- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL9A3- 0- 0- 0- 0- 0- 2- 0- 0- 2- COL13A1- 0- 1- 0- 0- 2- 1- 0- 0- 4- COL22A1- 0- 0- 0- 0- 0- 1- 0- 0- 1- COL6A4P2- 0- 0- 0- 0- 1- 0- 0- 0- 1- COL11A2- 0- 0- 0- 0- 0- 2- 0- 0- 2- COL9A2- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL4A28 AS2- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL2A1- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL5A18 AS1- 0- 0- 0- 0- 0- 0- 0- 0- 0- COL6A4P1- 0- 0- 0- 0- 0- 0- 0- 0- 0- !

! 79! !

Appendix!K:!Differential!Collagen!Gene!Expression!with!Aging!

! 80! Appendix!K.!Differential!collagen!gene!expression!with!aging.!Fold!regulations!and! pZvalues! are! shown! for! each! comparison! (Young! Z! Old,! Male! Young! Z! Male! Old,! Female!Young!Z!Female!Old).!Statistical!significant!changes!are!shown!in!bold!letters.! The! young! and! the! male! group! served! as! controls! in! our! analyses.! As! a! result,! positive! changes! correspond! to! increases,! while! negative! changes! correspond! to! decreases!in!older!individuals!and!female!subjects.! !

Male-Young-8-Male- Female-Young-8- Young-8-Old-- Old- Female-Old- Collagens-- Fold- Fold- Fold-Regul- p8value- p8value- p8value- Regul- Regul- COL1A1- A1.88$ 0.001$ 81.18- 0.466- A3.37$ 0.00003$ COL1A2- A1.94$ 0.0003$ 81.48- 0.092- A2.69$ 0.0003$ COL2A1- 81.38- 1- 1- 1- 81.69- 1- COL3A1- A1.55$ 0.016$ 81.15- 0.49- A2.15$ 0.0057$ COL4A1- 81- 0.787- 1.65- 0.478- 81.52- 0.174- COL4A2- 1.15- 0.75- 1.49- 0.328- 81.1- 0.578- COL4A28AS2- 81.38- 1- 1- 1- 81.697- 1- COL4A3- 81.17- 0.415- 1.36- 0.867- 81.85- 0.0897- COL4A3BP- 81.12- 0.446- 81.12- 0.4795- 81.12- 0.673- COL4A4- 81.14- 0.688- 1.37- 1- 81.16- 0.509- COL4A5- 1.17- 0.5- 1.45- 0.359- 81.02- 1- COL4A6- 1.64$ 0.036$ 1.57- 0.3997- 1.63- 0.056- COL5A1- 81.32- 0.164- 81.06- 0.9195- 81.68- 0.063- COL5A18AS1- 81.38- 1- 1- 1- 81.698- 1- COL5A2- A1.79$ 0.001$ 81.43- 0.086- A2.24$ 0.007$ COL5A3- 81.16- 0.573- 81.09- 0.958- 81.19- 0.492- COL6A1- 81.24- 0.227- 1.24- 0.5299- A2.11$ 0.0066$ COL6A2- 81.27- 0.157- 1- 0.977- A1.75$ 0.025$ COL6A3- 81.37- 0.107- 1.33- 0.332- A2.41$ 0.001$ COL6A4P1- 1.62- 1- 1.74- 0.948- 1- 1- COL6A4P2- 82.49- 0.626- 1- 1- 82.02- 0.528- COL6A5- 81.07- 1- 82.82- 0.294- 2.16- 0.506- COL6A6- 81.13- 0.785- 81.14- 1- 1.21- 0.775- COL7A1- 81.21- 0.291- 1.05- 1- 81.65- 0.092- COL8A1- 81.44- 0.159- 81.05- 0.989- 81.79- 0.0582- COL8A2- 81.42- 0.394- 83.11- 0.068- 1.56- 0.7796------Continued-

! 81! Appendix-K-continued- $ $ - - $ $ COL9A1- A5.04$ 0.046$ 81.64- 1- A14.09$ 0.0297$ COL9A2- 82.89- 0.5- 1- 1- 84.49- 0.436- COL9A3- 81.42- 0.509- 81.97- 0.706- 81.19- 0.875- COL10A1- 1.02- 0.754- 1.37- 1- 1.11- 0.554- COL11A1- 81.58- 0.113- 81.75- 0.1799- 81.361- 0.371- COL11A2- 3.43- 0.25- 2.01- 0.947- 5.53- 0.569- COL12A1- 81.33- 0.0884- 1.007- 0.834- A1.82$ 0.0335$ COL13A1- 1.97- 0.453- 2.76- 0.449- 1.94- 0.816- COL14A1- A1.52$ 0.0193$ 81.144- 0.463- A1.87$ 0.013$ COL15A1- 81.01- 0.826- 1.026- 0.739- 81.03- 1- COL16A1- A1.47$ 0.0325$ A1.74$ 0.0177$ 81.23- 0.572- COL17A1- 81.29- 0.128- 81.15- 0.494- 81.48- 0.127- COL18A1- 81.25- 0.13- 1.19- 0.828- A1.92$ 0.009$ COL19A1- 82.2- 0.107- 1.18- 1- A19.13$ 0.007$ COL21A1- 1.01- 0.7135- 1.21- 0.69- 81.11- 0.971- COL22A1- 81.28- 1- 1.26- 0.947- 81.59- 0.9001- COL23A1- 81.38- 0.273- 1.14- 0.832- A2.33$ 0.036$ COL24A1- 81.73- 0.057- 81.099- 1- A2.53$ 0.0263$ COL25A1- 81.34- 0.488- 1.17- 1- 81.898- 0.374- COL26A1- 81.98- 0.0994- 81.23- 0.709- 82.22- 0.164- COL27A1- 1.191- 0.665- 1.54- 0.525- 81.11- 0.909- COL28A1- 81.1- 0.736- 81.19- 0.636- 1.08- 1- !

! 82! !

Appendix!L:!Differential!Collagen!Gene!expression!Between!Sexes!

! 83! Appendix! L! Differential! collagen! gene! expression! between! sexes.! Fold! regulations! and!pZvalues!are!shown!for!each!comparison!(Male!Young!–!Female!Young,!Male!Old! –! Female! Old).! Statistical! significant! changes! are! shown! in! bold! letters.! The! male! group!served!as!control!in!our!analyses.!As!a!result,!positive!changes!correspond!to! increases,!while!negative!changes!correspond!to!decreases!in!female!subjects.! !

Male-Young-8- Male-Old-8-Female- Female-Young- Old- Collagens-- Fold- Fold- p8value- p8value- Regul- Regul- COL1A1- 1.64- 0.0509- 81.74- 0.075- COL1A2- 1.42- 0.161- 81.28- 0.441- COL2A1- 1.67- 1- 1- 1- COL3A1- 1.62$ 0.044$ 81.15- 0.77- COL4A1- 1.75- 0.136- 81.435- 0.395- COL4A2- 1.38- 0.132- 81.194- 0.909- COL4A28AS2- 1.67- 1- 1- 1- COL4A3- 1.63- 0.144- 81.53- 0.653- COL4A3BP- 1.09- 0.586- 1.09- 0.49- COL4A4- 2.73- 0.115- 1.64- 0.384- COL4A5- 1.46- 0.124- 81.02- 0.675- COL4A6- 81.26- 0.524- 81.21- 0.661- COL5A1- 1.21- 0.507- 81.31- 0.497- COL5A18AS1- 1.67- 1- 1- 1- COL5A2- 1.41- 0.1298- 81.11- 0.7698- COL5A3- 1.12- 0.753- 1.03- 1- COL6A1- 1.39- 0.1399- A1.88$ 0.045$ COL6A2- 1.04- 0.851- 81.68- 0.103- COL6A3- 2.02$ 0.0037$ 81.58- 0.162- COL6A4P1- 1- 1- 81.73- 1- COL6A4P2- 6.39- 0.1396- 2.34- 0.941- COL6A5- 82.11- 0.3393- 2.86- 0.326- COL6A6- 2.87- 0.129- 3.58- 0.317- COL7A1- 1.15- 0.4198- 81.49- 0.4399- COL8A1- 1.84$ 0.021$ 1.08- 0.822- COL8A2- 82.15- 0.333- 2.28- 0.321- - - - - Continued------

! 84! Appendix-L-continued- - - - - COL9A1- 3.37- 0.0691- 81.75- 1- COL9A2- 4.37- 0.339- 1- 1- COL9A3- 81.86- 0.583- 81.06- 1- COL10A1- 2.42- 0.183- 1.88- 0.077- COL11A1- 1.44- 0.14- 1.85- 0.107- COL11A2- 82.56- 1- 81.06- 1- COL12A1- 1.36- 0.1496- 81.35- 0.645- COL13A1- 1.97- 0.738- 1.18- 0.912- COL14A1- 2.02$ 0.0029$ 1.23- 0.3399- COL15A1- 1.097- 0.861- 1.03- 0.566- COL16A1- 81.23- 0.417- 1.15- 0.465- COL17A1- 1.33- 0.192- 1.03- 0.736- COL18A1- 1.52- 0.075- 81.51- 0.293- COL19A1- 2.198- 0.127- 86.52- 0.312- COL21A1- 1.58- 0.121- 1.17- 0.359- COL22A1- 1.98- 0.738- 81.05- 1- COL23A1- 1.53- 0.119- 81.72- 0.464- COL24A1- 1.85$ 0.022$ 81.23- 1- COL25A1- 1.78- 0.2096- 81.24- 1- COL26A1- 2.06- 0.373- 1.15- 1- COL27A1- 1.23- 0.747- 81.39- 0.589- COL28A1- 1.28- 0.727- 1.62- 0.286- !

! 85! !

Appendix!M:!MMP!Genes!–!Total!Number!of!Transcripts!

! 86! Appendix!M.!MMP!genes!expressed!in!clinically!healthy!gingival!tissues.!The!last! column!shows!the!total!number!of!expressed!transcripts!when!combining!the! biopsies!of!all!subjects.! ! Total- Gene-ID- Gene- Strand- Chrom- LOC-5'- LOC-3'- Count- ENSG00000087245.12- MMP2- 1- chr16- 55389700- 55506691- 453- ENSG00000271447.5- MMP28- 81- chr17- 35795707- 35756249- 303- MMP248 ENSG00000126005.15- AS1- 81- chr20- 35278131- 35216462- 285- ENSG00000156103.15- MMP16- 81- chr8- 88328025- 88032009- 108- ENSG00000157227.12- MMP14- 1- chr14- 22836557- 22849027- 98- ENSG00000189409.12- MMP23B- 1- chr1- 1632095- 1701810- 66- ENSG00000100985.7- MMP9- 1- chr20- 46008908- 46016561- 49- ENSG00000125966.9- MMP24- 1- chr20- 35226654- 35277000- 46- ENSG00000137675.4- MMP27- 81- chr11- 102705806- 102691487- 41- ENSG00000167346.7- MMP26- 1- chr11- 4704927- 4992429- 36- ENSG00000102996.4- MMP15- 1- chr16- 58025566- 58046901- 30- ENSG00000123342.15- MMP19- 81- chr12- 55842966- 55835433- 26- ENSG00000198598.6- MMP17- 1- chr12- 131828393- 131851783- 19- ENSG00000262406.2- MMP12- 81- chr11- 102875034- 102862736- 13- MMP258 ENSG00000261971.6- AS1- 81- chr16- 3059370- 3051096- 12- ENSG00000149968.11- MMP3- 81- chr11- 102843803- 102835801- 11- ENSG00000099953.9- MMP11- 1- chr22- 23768226- 23784316- 10- ENSG00000008516.16- MMP25- 1- chr16- 3046681- 3060726- 10- ENSG00000137674.3- MMP20- 81- chr11- 102625332- 102576835- 7- ENSG00000196611.4- MMP1- 81- chr11- 102798160- 102789920- 3- ENSG00000166670.9- MMP10- 81- chr11- 102780628- 102770503- 3- ENSG00000137745.11- MMP13- 81- chr11- 102955734- 102942995- 2- ENSG00000137673.8- MMP7- 81- chr11- 102530753- 102520508- 2- ENSG00000154485.4- MMP21- 81- chr10- 125775821- 125766453- 1- ENSG00000215914.4- MMP23A- 1- chr1- 1699942- 1701782- 1- ! !

! 87! !

Appendix!N:!Number!of!MMP!Gene!Transcripts!Expressed!in!the!Young!Male!Group!

! ! !

! 88! Appendix!N.!Number!of!MMP!gene!transcripts!expressed!in!the!young!male!group.! The! first! column! shows! the! gene! name,! while! the! remaining! columns! show! the! number!of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers!#4,! 7,!8,!11!and!13!correspond!to!subjects,!while!the!letters!“L”!and!“R”!symbolize!the! biopsies!harvested!from!the!left!and!right!side!of!the!maxilla!respectively.! !

Gene- 4L- 4R- 7L- 7R- 8R- 11L- 11R- 13L- 13R- Total- MMP2- 10- 20- 16- 20- 2- 8- 5- 7- 1- 89- MMP28- 4- 8- 11- 3- 1- 1- 2- 8- 2- 40- MMP248AS1- 3- 17- 6- 12- 1- 0- 3- 3- 1- 46- MMP16- 2- 13- 5- 0- 2- 0- 1- 0- 2- 25- MMP14- 3- 1- 8- 2- 3- 1- 0- 0- 0- 18- MMP23B- 3- 5- 3- 0- 0- 1- 0- 0- 0- 12- MMP9- 0- 2- 0- 1- 0- 0- 0- 0- 0- 3- MMP24- 2- 0- 0- 0- 0- 0- 0- 0- 0- 2- MMP27- 0- 2- 1- 0- 0- 0- 0- 0- 0- 3- MMP26- 0- 2- 1- 0- 0- 0- 0- 1- 1- 5- MMP15- 0- 1- 1- 0- 0- 0- 0- 1- 0- 3- MMP19- 0- 0- 2- 0- 2- 0- 0- 0- 1- 5- MMP17- 1- 3- 1- 1- 0- 0- 0- 1- 0- 7- MMP12- 0- 0- 0- 1- 0- 0- 0- 0- 0- 1- MMP258AS1- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP3- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP11- 0- 1- 0- 0- 0- 0- 0- 0- 0- 1- MMP25- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP20- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP1- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP10- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP13- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP7- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP21- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP23A- 0- 0- 0- 0- 0- 0- 0- 0- 0- 0- !

! 89! !

Appendix!O:!Number!of!MMP!Gene!Transcripts!Expressed!in!the!Old!Male!Group!

! 90! Appendix!O.!Number!of!MMP!gene!transcripts!expressed!in!the!old!male!group.!The! first!column!shows!the!gene!name,!while!the!remaining!columns!show!the!number! of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers!#2,!17,!18! and!19!correspond!to!subjects,!while!the!letters!“L”!and!“R”!symbolize!the!biopsies! harvested!from!the!left!and!right!side!of!the!maxilla!respectively.! !

Gene- 2L- 2R- 17L- 18R- 19L- 19R- Total- MMP2- 16- 26- 34- 3- 0- 9- 88- MMP28- 50- 55- 8- 8- 5- 0- 126- MMP248AS1- 21- 19- 17- 1- 3- 1- 62- MMP16- 8- 2- 2- 1- 0- 0- 13- MMP14- 6- 7- 4- 0- 0- 3- 20- MMP23B- 3- 3- 2- 1- 2- 0- 11- MMP9- 6- 6- 1- 0- 0- 0- 13- MMP24- 3- 4- 0- 3- 0- 1- 11- MMP27- 4- 3- 1- 0- 0- 2- 10- MMP26- 0- 1- 2- 0- 0- 0- 3- MMP15- 6- 1- 5- 1- 0- 0- 13- MMP19- 4- 3- 1- 0- 0- 0- 8- MMP17- 2- 0- 0- 0- 0- 2- 4- MMP12- 3- 5- 1- 0- 0- 0- 9- MMP258AS1- 1- 2- 0- 0- 0- 1- 4- MMP3- 0- 2- 1- 0- 0- 0- 3- MMP11- 0- 0- 1- 0- 0- 0- 1- MMP25- 0- 0- 0- 0- 0- 0- 0- MMP20- 1- 0- 0- 0- 0- 0- 1- MMP1- 0- 0- 0- 0- 0- 0- 0- MMP10- 0- 0- 0- 0- 0- 0- 0- MMP13- 0- 0- 0- 0- 0- 0- 0- MMP7- 0- 0- 0- 0- 0- 0- 0- MMP21- 0- 0- 0- 1- 0- 0- 1- MMP23A- 1- 0- 0- 0- 0- 0- 1- !

! 91! !

Appendix!P:!Number!of!MMP!Gene!Transcripts!Expressed!in!the!Young!Female!

Group!

! 92! Appendix!P.!Number!of!MMP!gene!transcripts!expressed!in!the!young!female!group.! The! first! column! shows! the! gene! name,! while! the! remaining! columns! show! the! number!of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers!#1,! 5,! 6! and! 14! correspond! to! subjects,! while! the! letters! “L”! and! “R”! symbolize! the! biopsies!harvested!from!the!left!and!right!side!of!the!maxilla!respectively.! !

Gene- 1L- 1R- 5L- 5R- 6L- 6R- 14R- Total- MMP2- 54- 35- 14- 26- 33- 49- 9- 220- MMP28- 21- 12- 2- 13- 20- 15- 3- 86- MMP248AS1- 20- 23- 8- 27- 25- 26- 1- 130- MMP16- 8- 6- 4- 5- 16- 16- 0- 55- MMP14- 8- 7- 4- 12- 8- 7- 1- 47- MMP23B- 3- 2- 2- 10- 14- 4- 0- 35- MMP9- 4- 3- 1- 1- 4- 7- 0- 20- MMP24- 2- 2- 1- 5- 5- 11- 0- 26- MMP27- 2- 6- 1- 0- 5- 6- 0- 20- MMP26- 2- 3- 0- 1- 5- 11- 0- 22- MMP15- 2- 5- 0- 1- 1- 4- 0- 13- MMP19- 2- 3- 2- 1- 2- 1- 0- 11- MMP17- 0- 1- 2- 2- 1- 0- 0- 6- MMP12- 0- 0- 1- 0- 1- 0- 0- 2- MMP258AS1- 1- 1- 0- 1- 3- 1- 0- 7- MMP3- 1- 0- 1- 0- 0- 3- 0- 5- MMP11- 1- 2- 2- 1- 0- 0- 1- 7- MMP25- 0- 3- 2- 0- 1- 2- 0- 8- MMP20- 0- 0- 0- 0- 1- 5- 0- 6- MMP1- 1- 0- 0- 1- 0- 0- 0- 2- MMP10- 0- 2- 0- 0- 0- 0- 0- 2- MMP13- 0- 0- 0- 0- 0- 1- 0- 1- MMP7- 0- 0- 0- 1- 1- 0- 0- 2- MMP21- 0- 0- 0- 0- 0- 0- 0- 0- MMP23A- 0- 0- 0- 0- 0- 0- 0- 0- !

! 93! !

Appendix!Q:!Number!of!MMP!Gene!Transcripts!Expressed!in!the!Old!Female!Group!

! 94! Appendix! Q.! Number! of! MMP! gene! transcripts! expressed! in! the! old! female! group.! The! first! column! shows! the! gene! name,! while! the! remaining! columns! show! the! number!of!transcripts!in!each!biopsy!and!the!total!for!the!whole!group.!Numbers!#9,! 10,! 12! and! 16! correspond! to! subjects,! while! the! letters! “L”! and! “R”! symbolize! the! biopsies!harvested!from!the!left!and!right!side!of!the!maxilla!respectively! ! Gene- 9L- 9R- 10L- 10R- 12L- 12R- 16L- 16R- Total- MMP2- 0- 8- 17- 3- 3- 7- 0- 1- 39- MMP28- 2- 9- 7- 4- 6- 10- 0- 2- 40- MMP248AS1- 2- 5- 5- 2- 7- 12- 0- 0- 33- MMP16- 0- 5- 2- 1- 2- 1- 0- 1- 12- MMP14- 0- 4- 1- 1- 1- 2- 0- 0- 9- MMP23B- 0- 1- 2- 0- 2- 2- 0- 1- 8- MMP9- 0- 2- 0- 0- 0- 0- 1- 0- 3- MMP24- 0- 2- 2- 0- 0- 2- 0- 0- 6- MMP27- 0- 2- 1- 0- 1- 0- 0- 0- 4- MMP26- 0- 1- 1- 0- 2- 0- 0- 1- 5- MMP15- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP19- 0- 0- 0- 0- 2- 0- 0- 0- 2- MMP17- 0- 1- 0- 0- 0- 0- 0- 0- 1- MMP12- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP258AS1- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP3- 0- 0- 1- 0- 0- 0- 0- 0- 1- MMP11- 0- 0- 0- 0- 1- 0- 0- 0- 1- MMP25- 0- 0- 0- 0- 0- 1- 0- 0- 1- MMP20- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP1- 0- 0- 0- 0- 1- 0- 0- 0- 1- MMP10- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP13- 0- 0- 0- 1- 0- 0- 0- 0- 1- MMP7- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP21- 0- 0- 0- 0- 0- 0- 0- 0- 0- MMP23A- 0- 0- 0- 0- 0- 0- 0- 0- 0- ! ! ! ! ! ! ! !

! 95! !

Appendix!R:!Differential!MMP!Gene!Expression!with!Aging!

! 96! Appendix!R.!Differential!MMP!gene!expression!with!aging.!Fold!regulations!and!pZ values!are!shown!for!each!comparison!(Young!Z!Old,!Male!Young!Z!Male!Old,!Female! Young!Z!Female!Old).!Statistical!significant!changes!are!shown!in!bold!letters.!The! young!and!the!male!group!served!as!controls!in!our!analyses.!As!a!result,!positive! changes!correspond!to!increases,!while!negative!changes!correspond!to!decreases!in! older!individuals!and!female!subjects.! ! Female-Young-8- Young-8-Old-- Male-Young-8-Male-Old- Female-Old- MMPs-- Fold-Regul- p8value- Fold-Regul- p8value- Fold-Regul- p8value-

MMP1- 81.08- 1- 1- 1- 1.36- 1- MMP2- A1.67$ 0.0024$ 81.37- 0.117- A2.09$ 0.005$ MMP3- 1.2- 1- 3.23- 0.947- 81.375- 0.9- MMP7- 81.76- 1- 1- 1- 82.395- 0.933- MMP9- 1.07- 1- 2.5- 0.323- 81.886- 0.455- MMP10- 81.76- 1- 1- 1- 82.395- 0.933- MMP11- 81.79- 0.375- 81.13- 1- 81.8- 0.162- MMP12- 3.08- 0.375- 3.89- 0.213- 82.395- 0.933- MMP13- 1.175- 1- 1- 1- 1.92- 0.5689- MMP14- 81.41- 0.137- 81.21- 0.491- 81.65- 0.127- MMP15- 1.25- 0.784- 2.73- 0.183- A9.87$ 0.049$ MMP16- A1.85$ 0.0473$ A2.35$ 0.037$ 81.41- 0.395- MMP17- 81.45- 0.34500- 81.97- 0.444- 81.59- 0.528- MMP19- 81.06- 0.401- 1.08- 0.794- 81.59- 0.578- MMP20- 82.02- 0.252- 1.74- 1- 85.19- 0.203- MMP21- 1.62- 1- 1.74- 0.947- 1- 1- MMP23A- 1.62- 1- 1.74- 1- 1- 1- MMP23B- 81.35- 0.514- 81.25- 0.893- 81.33- 0.521- MMP24- 1.08- 1- 3.14- 0.189- 81.35- 0.499- MMP248AS1- 81.13- 0.451- 1- 0.936- 81.25- 0.438- MMP25- 82.49- 0.626- 1- 1- 82.02- 0.297- MMP258AS1- 81.05- 1- 3.99- 0.947- 85.88- 0.436- MMP26- 81.68- 0.364- 81.77- 0.714- 81.29- 0.811- MMP27- 1.01- 1- 2.16- 0.553- 81.49- 0.446- MMP28- 1.67- 0.0896- 1.81- 0.19- 1.47- 0.394- ! !

! 97! !

Appendix!S:!Differential!MMP!Gene!Expression!Between!Sexes!

! 98! Appendix!S.!Differential!MMP!gene!expression!between!sexes.!Fold!regulations!and! pZvalues!are!shown!for!each!comparison!(Male!Young!–!Female!Young,!Male!Old!–! Female! Old).! Statistical! significant! changes! are! shown! in! bold! letters.! The! male! group!served!as!control!in!our!analyses.!As!a!result,!positive!changes!correspond!to! increases,!while!negative!changes!correspond!to!decreases!in!female!subjects.! ! Male-Young-8-Female- Male-Old-8-Female-Old- Young- MMPs-- Fold-Regul- p8value- Fold-Regul- p8value-

MMP1- 2.348- 0.824- 2.34- 0.941- MMP2- 1.15- 0.414- 81.31- 0.629- MMP3- 4.37- 0.339- 81.36- 1- MMP7- 2.35- 0.824- 1- 1- MMP9- 2.59- 0.146- 81.86- 1- MMP10- 2.35- 0.824- 1- 1- MMP11- 2.23- 0.193- 1.35- 0.941- MMP12- 81.09- 1- 87.54- 0.297- MMP13- 1.67- 1- 2.34- 0.941- MMP14- 1.15- 0.715- 81.18- 1- MMP15- 1.698- 0.635- 810.41- 0.0898- MMP16- 1.04- 0.947- 1.74- 0.1895- MMP17- 82.36- 0.583- 81.67- 1- MMP19- 81.07- 1- 81.76- 1- MMP20- 5.04- 0.1396- 81.73- 1- MMP21- 1- 1- 81.73- 1- MMP23A- 1- 1- 81.73- 1- MMP23B- 1.31- 0.446- 1.23- 1- MMP24- 4.4$ 0.0321$ 81.06- 1- MMP248AS1- 1.27- 0.304- 1.01- 0.755- MMP25- 6.39- 0.058- 2.34- 0.941- MMP258AS1- 5.72- 0.339- 83.91- 1- MMP26- 1.76- 0.386- 2.36- 0.322- MMP27- 2.59- 0.067- 81.29- 1- MMP28- 81.09- 1- 81.36- 0.766- ! !

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