Study of 3,4-dihydroxyphenylalanine (DOPA) as anchoring unit to functionalize biomaterials

!! ! ! MASTER’S!THESIS!-!EEIGM! Materials)Science)and)Engineering) STUDY)OF)L-3,4-DIHYDROXYPHENYLALANINE)(DOPA))AS) ANCHORING)UNIT)TO)FUNCTIONALIZE)METALLIC,)CERAMIC) AND)POLYMERIC)BIOMATERIALS.) ) ) Report) ) Student:! ! Nathan!Chebre! ! Director:! ! Carles!Mas!Moruno! Department:!! BBT!! Convocation:!February!2018!! ! ! ! 1! ! ! ! ! 2! Summary [EN]!This!project!aims!at!studying!and!comparing!the!efficiency!and!stability!of!the!binding!of!a!model! peptide!to!a!diverse!range!of!biomaterials!using!L-3,4-dihydroxyphenylalanine!(DOPA)!which!contains! catechol!groups,! !as! anchoring! unit.! The! capacity! of! DOPA! to! bind! to! metallic! biomaterials! such! as! titanium!is!well!described;!however,!the!binding!to!other!types!of!materials,!i.e.!ceramics!and!polymers,! needs! to! be! explored.! In! particular,! the! functionalization! of! alpha-tricalcium! phosphate! (α-TCP),! polymethylpentene!or!poly(4-methyl-1-pentene)!(PMP),!poly(lactic!acid)!(PLA),!chitosan!and!alginate!will! be!studied.!! --! [CAS]! El! objetivo! de!este!trabajo! es! comparar! la! eficiencia!y!la! estabilidad! del! enlace! de! un! péptido! modelo!a!un! variedad!de! biomateriales!usando!la! L-3,4-dihidroxifenilalanina!(DOPA)! que! contiene!un! grupo!catecol!como! unidad!de!anclaje.!La! capacidad! de! la! DOPA! de!unirse!a!biomateriales!metálicos! como!el!titanio!está!bien!descrita!en!investigaciones!previas,!pero!su!aplicación!en!materiales!de!tipo! cerámico!o!polimérico!no!ha!sido!estudiada.!En!particular,!la!funcionalización!de!alfa!trifosfato!de!calcio! (α-TCP),!polimetilpenteno!o!poli(4-metil-1-penteno)!(PMP),!ácido!poliláctico!(PLA),!quitosano!y!alginato! serán!estudiadas.!! Glossary α-TCP!:!alpha-tricalcium!phosphate! ATR!:!Attenuated!Total!Reflectance! CA!:!contact!angle! CF!:!5(6)-carboxyfluorescein!molecule! DOPA!:!L-3,4-dihydroxyphenylalanine! ECM!:!extracellular!matrix! FTIR!:!fourier!transformation!infrared!! PBS!:!phosphate!buffered!saline! PLLA!:!poly-L-lactic!acid!! PMP!:!polymethylpentene!or!poly(4-methyl-1-pentene)! PTF!:!platform! RGD!:!Arg-Gly-Asp!tripeptide! SEM!:!scanning!electron!microscopy! Tris!:!2-amino-2-(hydroxymethyl)-1,3-propandiol! XPS!:!X-Ray!photoelectron!spectroscopy! ! ! ! 3! Acknowledgements I!would!like!to!thank!the!people!who!helped!me!working!on!this!project.!! Dr.!Carles!Mas!Moruno,!Ramón!y!Cajal!researcher,!my!academic!tutor!at!the!Biomaterials,!Biomechanics! and! Tissue! Engineering! Group! during! this! project! for! his! help,! availability,! constant! involvement! and! expertise.!! Dr.!Montserrat!Espanol,!Researcher!at!Biomaterials,!Biomechanics!and!Tissue!Engineering!Group,!for!her! kindness,!availability!and!for!helping!me!several!time!with!the!confocal!microscopy!observations!and!her! wise!advice!on!alginate!films!preparation.!! Dr.!Giuseppe!Scionti,!Juan!de!la!Cierva!Postdoctoral!Researcher,!Biomaterials,!Biomechanics!and!Tissue! Engineering!Group,!for!his!help!with!the!3D-Printing!technique!for!Titanium!3D!scaffolds,!alginate!and! hydrogels!materials.!! Dr.! Daniel! Rodriguez,! associate! professor!and! researcher!at! Biomaterials,! Biomechanics! and! Tissue! Engineering!Group!for!his!help!and!advices!about!chitosan-based!polymer!film!fabrication.!! Dr.! Cristina! Canal,! Ramón! y! Cajal! researcher,! for! her! help! with! PLA! commercial! films! materials! and! allowing!me!to!use!her!material.!! Elia!Vidal,!PhD!Student!at!Biomaterials,!Biomechanics!and!Tissue!Engineering!Group,!for!her!help!on!3Dprinted!titanium-based!scaffolds.!! Anna!Diez-Escudero,!PhD!Student!at!Biomaterials,!Biomechanics!and!Tissue!Engineering!Group,!for!her! help!and!expertise!on!α-TCP!materials!fabrication.!! Dr.!Sabrina!Schmeckebier,!researcher!at!Leibniz!Research!Laboratories!for!Biotechnology!and!Artificial! Organs,! for! providing! PMP! commercial! films! (ordered! from! Goodfellow! company)! used! during! this! project.!! Montserrat!Dominguez,!Technical!Support!to!Research!Team!at!the!Multiscale!Center!(CRCEMB),!for!her! formation!and!help!on!the!FTIR-ATR!technique.! Trifon! Todorov,! Technical! Support! to! Research! Team! at! the! Multiscale! Center! (CRCEMB),! for! his! formation!and!help!on!the!SEM!technique.! Angelica!Santos,!PhD!Student!at!Biomechanics!and!Tissue!Engineering!Group!,!for!her!help!on!titanium! polishing!technique.! ! ! ! ! ! 4! Summary)................................................................................................................................................)2! Glossary)..................................................................................................................................................)2! Acknowledgements)................................................................................................................................)3! Introduction)............................................................................................................................................)5! I.!State)of)the)art).................................................................................................................................)6! 1.!Bioactivity,)Osseointegration)and)Implants).................................................................................................)6! i.!Process!of!osseointegration!around!implant!...................................................................................................!6! ii.!Issues!with!foreign!materials!as!implants!.......................................................................................................!8! iii.!Factors!influencing!osseointegration!.............................................................................................................!8! 2.!Biomaterials)for)bone)and)other)tissues)......................................................................................................)9! i.!Metals!................................................................................................................................................................!9! ii.!Glass!and!Ceramics!........................................................................................................................................!10! iii.!Polymers!.......................................................................................................................................................!11! 3.!Strategies)to)increase)bioactivity)and)osseointegration)............................................................................)12! i.!Methods!to!physically!functionalize!biomaterials!.........................................................................................!12! ii.!Methods!to!chemically!functionalize!biomaterials!.......................................................................................!13! 4.!DOPA)Chemistry)........................................................................................................................................)17! i.!Generalities!.....................................................................................................................................................!17! ii.!Binding!of!DOPA!to!biomaterials:!Previous!studies!......................................................................................!19! II.!Objectives).....................................................................................................................................)24! III.!Experimental)Section)..................................................................................................................)26! 1.!Materials)and)methods).............................................................................................................................)26! 2.!Immobilization)of)biomolecules)on)materials)............................................................................................)30! 3.!Characterization)methods).........................................................................................................................)32! IV.!Results)and)Discussion)................................................................................................................)34! 1.!Functionalization)of)Ti)disks).......................................................................................................................)34! i.!Preliminary!tests!on!Ti!disks!...........................................................................................................................!34! ii.!Oxidation!state!influence!of!catechol!...........................................................................................................!34! 2.!Platform-functionalization)of)commercial)PLLA)Films)...............................................................................)39! 3.!Platform-Functionalization)of)PMP)Films)..................................................................................................)42! 4.!Platform-Functionalization)of)α-TCP)samples)...........................................................................................)45! 5.!Alginate)and)Chitosan)functionalization)....................................................................................................)47! Achievements)and)Work)Perspectives)..................................................................................................)50! Conclusion)............................................................................................................................................)52! Economic)Impact)..................................................................................................................................)53! Environmental)Impact)..........................................................................................................................)54! References)............................................................................................................................................)55! ! ! ! 5! Introduction Biomaterials!can!be!defined!as!materials!used!as!devices!that!interact!with!biological!systems!for! application! in! diverse! areas! of! medicine.! It! is! important! to! know! the! biocompatibility! and! integration/reabsorption!capacity!of!the!material!so!as!not!to!imply!unwished!reactions!at!the!tissuematerial!interface.!According!to!the!9th!European!Conference!on!Biomaterials,!biocompatibility!is!“the! ability!of!a!material!to!perform!with!an!appropriate!host!response!in!a!specific!application”.!! !Materials!can!be!classified!in!four!different!families:!metals,!ceramics,!polymers!and!composites.! Each!family!contains!biomaterials!as!well.!This!project!will!mainly!focus!on!biomaterials!used!for!bone! applications.!In!this!regard,!there!are!two!different!kinds!of!biomaterials;!on!the!one!hand!those!used!for! bone!regeneration,!and,!on!the!other!hand,!those!used!for!bone!replacement/substitution.!Nonetheless,! in!this!project!biomaterials!used!for!other!medical!applications!will!be!studied!as!well.! Surface! functionalization! is! the! main! topic! of! this! project.!Functionalization! is! a! technique! of! surface! modification! that! allows! the! introduction! of! chemical! functionalities! on! the! biomaterial!to! improve!its! biocompatibility! and! bioactivity.! ! In! this! project! we! will! explore! the! functionalization! of! diverse!biomaterials!with!a!peptidic!multifunctional!platforms!containing!the!bioactive!peptides!DWIVA! and!RGD.!The!anchoring!unit!of!this!molecule!is!L-3,4-dihydroxyphenylalanine)(DOPA),!an!amino!acid! that!contains!a!catechol!functionality.!Catechols!are!known!to!be!very!efficient!binders!to!a!wide!range! of!surfaces.!! Thus,! this!project! aims! at! comparing! the! efficiency! of! the! RGD/DWIVA/DOPA! platform! covering/immobilization!on!various!biomaterials!such!as!commercially!pure!titanium,!synthetic!polymers! (PMP,!PLA),!natural!polymers!(chitosan!and!alginate),!and!calcium!phosphate!cements!(α!-TCP).!! Methodology:!The!biomaterials!will!be!prepared!according!to! their!different!chemical!nature.!Others! will!be!used!as!provided!by!commercial!suppliers!(Titanium!disks,!PLLA!and!PMP!films).!All!biomaterials! will!be!initially!characterized!by!contact!angle!(CA),!IR!(FTIR-ATR)!and!optical!and/or!electron!microscopy.! The!samples!will!be!functionalized!via!a!simple!dip-coating!(immersion)!process.!The!functionalization!of! the!samples!and!the!stability!of!the!coatings!will!be!evaluated!by!a!combination!of!different!techniques,! including!CA,!FTIR-ATR,!fluorescence!microscopy,!and!XPS.! ! ! ! ! ! 6! I. State of the art 1. Bioactivity, Osseointegration and Implants i. Process of osseointegration around implant It!can!be!important!to!have!in!mind!some!mechanism!that!are!involved!in!a!healing!process!and! foreign!body!integration.!The!concept!of!bioactivity!relies!to!the!capacity!to!form!a!direct!bond!with!the! bone!without!the!interposition!of!a!fibrous!capsule.!It!is!a!four!steps!process,!described!in!fig(1-1).![1]!! !! ! ! ! ! ! ! ! ! Fig!1-1:!Simplified!four!steps!of!bioactivity!on!a!biomaterial!implant.!a)!Inflammation!process!is!the!first! reaction,! it! results! of! the! interaction! between! the! blood! and! the! biomaterial,! a! provisional! matrix! is! formed.! b)! Chemical!interactions!with!the!environment!make!a!surface!modification!and!promote!adsorption!of!proteins!and! cellular!activity.!!c)!Then!mineralization!starts,!creating!a!carbonated!apatite!layer!on!surface.!d)!This!layer!grows! consistently!before!letting!the!formation!of!mineralized!collagen!matrix!appear!on!it. [1]! Hemostasis.!After!a!fracture,!trauma!or!surgery,!a!wide!number!of!reactions!take!place.!First!of! them!is!bleeding!and!the!arrival!of!wound-healing!mediators!to!the!site!of!injury.!This!is!the!beginning!of! hemostasis,!which!may!last!from!minutes!to!hours.!The!first!tissue!in!contact!with!the!implant!is!blood.!A! hemostatic!plug!forms,!consisting!of!platelet!aggregates!and!fibrin.!Platelets!are!the!key!agents!of!the! early!steps!of!healing!but!also!rule!the!long-term!regeneration!process.!Macroscopic!coagulation!occurs! at!the!wound!site.!Immune!response!occurs!after!a!few!hours!of!implantation,!started!by!leukocytes.! The! process! of! creation! of! new! blood! vessels! that! replace! those! affected! by! trauma! is! named! angiogenesis.!(fig!1-2)!This!is!a!very!important!step!in!new!bone!formation!because!osteoblasts,!which! b c d ! ! ! 7! are!the!cells!responsible!of!osteogenesis,!cannot!survive!if!not!surrounded!by!blood!vessels.!During!this! period!some!specific!proteins!can!establish!cell!attachment!sites.![2]! Inflammation.!Inflammation!starts!within!15!minutes!from!trauma!and!lasts!for!some!days.!It! starts! with! platelets! activation! during! hemostasis.! The! first! hours! are! marked! by! the! arrival! of! polymorphonuclear!leukocytes!(PMN),!then!followed!by!monocytes!that!will!turn!into!macrophages.!In! the!late!inflammatory!phase!those!macrophages!secrete!fibrogenic!and!angiogenic!growth!factors.!They! can! promote! the! formation! of! a! new! extracellular! matrix.! A! provisional! matrix! is! thus! formed! and! adheres!around!the!implant,!composed!by!activated!platelets!that!aggregate!within!the!fibrin!matrix.! Differentiated!osteoblasts!spread!on!the!implant!surface.!(fig!1-2)! ! Fig!1-2:!Scheme!representing!the!first!two!steps!of!wound!healing![3]! Proliferation.!Proliferation!starts!after!more!than!two!weeks.!Once!the!osteoblasts!attach!to!the! implant!surface,!the!extracellular!matrix!begins!to!be!secreted.!Collagens!form!a!primary!matrix,!which! has! an! unorganized! bone! structure! called! woven! bone.! Woven! bone! stabilizes! the! fracture! and! mineralization!starts!with!the!former!bone!of!the!patient.!(fig!1-3)! Remodeling.!Remodeling!could!start!after!six!weeks!and!extent!during!several!years,!this!is!the! last! stage! in! the! process! of! osteogenesis! and! it! is! a! continuous! process! in! life.! It! leads! to! a! mature! lamellar!bone,!which!has!an!enhanced!mechanical!resistance!than!before.!Osteoclasts!differentiation!is! mostly!responsible!of!this!process.!(fig!1-3)! ! Fig!1-3:!Scheme!representing!the!last!two!steps!of!wound!healing![3]! The! majority! of! cell! adhesive! processes! that! take! place! during! implant! osseointegration! are! mediated!through!integrin-RGD!binding!mechanisms.![2]! ! ! ! 8! ) ii. Issues with foreign materials as implants Knowing!the!large!number!of!steps!involved!in!biomaterial!biointegration,!it!is!clear!that!various! issues!can!occur!during!the!process.! !An! incomplete! osteointegration! of! the! implant!is! a! major!problem.! This!means! that! the! interface!between!the!metal!and!bone!is!covered!with!a!fibrous!tissue,!which!will!not!provide!sufficient! mechanical!and!biochemical!properties!for!the!prosthesis!to!be!optimally!integrated.!Bacterial!infections! can! also! occur! after! the! operation,! due! to! the!exposure! of! the!surface! of! an!implant! that! is! not! osseointegrated.!! These! problems! might!be! solved! functionalizing! the! surface! of! metallic! implants!with! biomolecules! of! the! extracellular! matrix! of! bone,! like! proteins! and! peptides,! which! may! mimic! the! physiological!process!of!bone!formation.!Such!biomimetic!strategy!pushes!scientists!to!understand!at! how!nature!works.!Implant!functionalization!with!biomolecules!may!provide!better!cell!adhesion,!bone! growth!or!antibacterial!properties!to!the!biomaterial.!! The! first! notable! reaction! after! implantation! is! the! non-specific! protein! adsorption! (except! interaction!with!water!and!ions).!Then!the!implant!is!interrogated!by!neutrophils!and!macrophages.!This! interaction!with!macrophages!and!cytokines,!which!are!released!by!macrophages,!initiates!the!attraction! of! fibroblasts! and! the! encapsulation! process.! The! osseointegration! can! be! improved! by! favoring! the! interaction! of! osteoblasts!with!the! surface.!A!faster! integration!of!the!implant!will!block!the!immune! response!and!the!formation!of!a!fibrous!and!unstable!capsule!around!the!implant.![4]! Mechanical!aspects!should!be!considered!too,!in!terms!of!Young’s!modulus,!tensile!strength,! fracture! or! yield! strength! of! the! implanted! material.! Trying! to! reach! a! comparable! Young’s! modulus! between!bone!and!implant!is!a!major!goal,!knowing!that!compact!bone’s!modulus!ranges!10-30!GPa.!If! the!difference!is!too!high,!stress!shielding!could!occur!and!lead!to!a!mechanical!insulation!of!the!implant! from!the!tissue!and!finally!collapse!the!prosthetic!device.!! ! iii. Factors influencing osseointegration Osseointegration!can!be!characterized!as!a!structural!and!functional!connection!between!newly! formed! bone! and! the! implant! surface.! In! order! to! optimize! osteoblastic! migration,! adhesion,! proliferation!and!differentiation,!a!great!deal!of!research!has!been!performed!on!the!design!of!novel! surface!geometries! and!topographies! for! implants.! The! use! of! surface! modifications! is! explored! with! great!detail!later!in!Section!“Strategies!to!increase!bioactivity!and!osseointegration”.! Microtopography!/!microroughness!involves!machined!surfaces,!which!have!been!turned,!milled! or!polished!during!the!manufacturing!process.!Microrough!surfaces!have!shown!a!higher!level!of!bone- ! ! ! 15! c) Methods of Immobilization !Physical! adsorption! is! one! of! the! various! methods! introduced! to! immobilize! biomolecules! on! biomaterials!surfaces.!This!adsorption!is!induced!via!van!der!Waals!forces!or!electrostatically.!It!can!be! obtained! by! a! simple! dipping! method.! Physical! entrapment! requires! a! barrier! system! or! hydrogels.! Successful!treatment!depends!on!weak!physisorption,!but!also!on!external!parameters!such!as!micromovements!of!the!implants,!pH,!temperature!and!solvent!conditions!in!the!host!tissue.!Some!studies! revealed!that!such!surface!modifications!have!enhanced!bone!formation!around!the!implant.![7]! There!are!several!methods!to!functionalize!inorganic!materials!with!organic!molecules.!Covalent! immobilization!is!based!on!the!use!of!a!reactive!linker,!which!allows!a!covalent!binding!of!biomolecules! to! surfaces.! This! is! in! principal! more! complex! than! physical! adsorption! described! before.! It! aims! at! inducing!specific!tissue!response!by!immobilization!of!selected!biomolecules!on!the!surfaces.!! The!use!of!self-assembled!monolayers!is!possible.!They!carry!molecules!with!polar!head!group! such!as!-SH,!-NH2,!-COOH,!are!likely!to!bind!with!charge!surfaces!or!interact!by!van!der!Waals!forces.! What!interests!researchers!is!a!strong!linkage!to!the!substrate,!a!good!example!is!the!reaction!between! silanes!or!silanols!attached!to!oxide!surfaces!(for!instance!TiO2)!thanks!to!a!condensation!reaction.!This! process!is!named!silanization.!(fig!1-7)! ! Fig!1-7:!Schematic!illustration!of!the!attachment!of!silanes!to!hydroxyde!terminated!surfaces!(example!of!TiO2)! Surface! functionalization! through! silanization! is! normally! a! multi-step!step! procedure.! For! examples!the!use!of!3-aminopropyltriethoxysilane!(APTES)!requires:!1)!Activation!of!the!Ti!surface!with! plasma!or!an!acidic/basic!treatment;!2)!silanization!with!APTES;!3)!cross-linking!with!N-succinimidyl-3maleimidoproionate!(SMP);!and!4)!Addition!of!the!biomolecules!(e.g.!an!RGD!peptide).!! RGD!could!also!be!grafted!onto!Ti!surfaces,!which!were!previously!covered!with!PEG!terminated! by!–COOH!or!–NH2!groups.!The!carboxylic!acid!function!is!used!to!bind!with!peptides!that!contain!–NH2!! groups.! For! example,! bone! healing! test! carried! in! rabbit! tibia! showed! better!mineralization! on! RGD/PEG/Ti!than!on!RGD/Ti!grafted!implants.![12]! ! ! ! ! 16! Multifunctional systems: Peptide-based platforms ! Another!approach!to!improve!cell-material!interactions!and!the!biointegration!of!implants!is!to! use! multifunctional! coatings! that! combine! diverse! biofunctionalitites! at! the! same! time.!This! was! achieved!on!Ti!in!a!recent!work!using!a!peptide-based!divalent!platform.!! The!platform!was!made!of!two!distinct!bioactive!sequences,!spacer!units!to!provide!an!adequate!access! to!interact!with!integrin!receptors,!a!Lys!residue,!and!an!anchoring!group.!(fig!1-8)![15] [14]! ! Fig!1-8:!Schematic!representation!of!the!peptide-based!platform![14]! The!anchoring!group!used!was!the!amino!acid!cysteine!(Cys),!which!has!a!thiol!group!that!can!be! used!for!silanization!treatment.!The!silanization!with!APTES!provides!immobilization!of!the!platform!on! the!Ti!surface!thanks!to!linkages!made!with!TiO2.!This!molecule!contains!two!integrin-binding!peptides! (RGD!and!PHSRN),!which!mimic!the!biological!cell!ahdeisve!function!of!fibronectin.!Functionalization!of! Ti! with! this! molecule! improved! the! adhesion! and! differentation! of! osteoblasts!in! vitro! and! bone! formation!in!vivo!(Ref!11!and!12).! ! ! ! ! 17! 4. DOPA Chemistry i. Generalities The!interest!in!DOPA!and!catechol!coatings!comes!from!the!observation!of!the!ability!of!marine! mussels!to!anchor!to!foreign!surfaces.!(fig!1-9)!This!anchoring!property!is!conferred!by!adhesive!proteins! rich!in!3,4!dihydroxyphenylalanine!(DOPA)!(fig1.10),!which!contain!a!catechol!group!as!side!chain.!They! were!identified!by!Waite!and! Tanzer! in! 1981! as!providers!of!versatile!adhesion!of! marine!mussels!to! both!organic!and!inorganic!surfaces!under!inhospitable!regions!(harsh!and!wet!conditions).![16]! ! Fig!1-9!:!The!byssus!of!marine!mussels!that!allows!them!to!attach!to!inorganic!surfaces!such!as!rocks!or!wood![17]! Such! strong! bond! is!made!possible! through! the! formation! of! reversible! non-covalent! or! irreversible!covalent!interactions.!Nowadays,!the!specific!mechanisms!involved!in!those!small!molecules! adhesion!to!a!large!panel!of!surfaces!is!still!not!entirely!understood,!but!lots!of!researches!are!driven!on! various!materials!so!as!to!learn!more!about!it.!! ! Fig!1-10!:!3,4!Dihydroxyphenylalanine!(DOPA)!molecule! Some! reports! showed! a! very! high! strength,! fully! reversible! and! non-covalent! interaction! between!a!single!DOPA!residue!and!a!metal!oxide!surface.![18]! It!is!interesting!for!the!project!to!have!a!closer!look!on!the!different!types!of!interactions!that! could! be! generated! between! the! catechol! group! and! inorganic! compounds.! Studies! about! DOPA! anchoring!on!biomaterials!have!been!resumed!to!write!this!part.!! ! ! ! 18! First,!the!dihydroxy!functionality!of!catechol!can!form!strong!hydrogen!bonds!(H-bonds)!with!a! substrate.! This! could! promote! its! absorption! to! mucosal! tissues! and! hydroxyapatite! surfaces! for! example.! (fig! 1-11-A)! π-π! interactions! are! provided! by! the! benzene! ring! of! the! catechol! group.! This! allows! the! protein! to! bind! with! compounds! rich! content! in! aromatic! groups! and! gold! substrates.! Positively! charged! ions! can! also! form! cation-π! interactions! with! the! benzene! ring.! This! increases! the! adhesion! of! the! catechol! group! to! surfaces! that!both! contain!aromatic! groups! and! are! positively! charged.!(fig!1-11-B)! Catechols!can!create!strong,!reversible!complexes!with!various!metals!ions!such!as!Ti3+,!Ti4+,!Fe3+.! The!complexation!chemistry!has!been!used!for!self-healing!hydrogels!or!pH-responsive!drug!carrier!for! example.!(fig!1-11-C)! Other! complexes! can! be! formed! by! catechols.! The! most! interesting! one! is! with! metal! oxide! surfaces!such!as!TiO2.!Separatig!a!single!DOPA!molecule!from!a!titanium!surface!requires!a!pull-off!force! around! 800! pN,! which! represents! 40%! the! strength! of! a! covalent! bond! (for! example! 2000! pN! for! a! carbon-silicon! bond).! It! makes! catechol! an! ideal! anchoring! group! for! surface! modification! for! various! metal!substrates!!(Au2O3,!Al2O3,!NiTi,!TiO2…).!(fig!1-11-E)![19]! ! Fig!1-11!:!Various!reversible!chemical!bondings!between!DOPA!and!other!inorganic!materials.!A)!Hydrogen!bonding! with!hydroxy!functions!of!catechol.!B)!π-π!electron!interaction!between!benzene!rings.!C)!Cation-π!interaction!with! positively!charged!ions.!D)!Complex!formed!with!various!catechol!groups!and!metallic!ion.!E)!Co-ordination!bonding! with!metal!oxide!surface![19]! It!appears!that!the!adhesive!capability!of!the!catechol!group!is!highly!dependent!on!its!oxidation! state.! The! reduced! form! of! catechol,! existing! at! acidic! pH,! has! high! adhesive! strength! to! inorganic! surfaces.!But!when!catechol!is!oxidized!to!the!quinone!form!(fig!1-12),!the!adhesion!potential!is!greatly! reduced.! Indeed! the! required! force! to! separate! a! single! molecule! of! DOPA!when! the! side! chain!is! oxidized!is!reduced!to!80%.![19]! ! ! ! 19! ) Fig!1-12:!DOPA!oxidation!to!quinone!form!(pKa!=!9,2)! But!this!behavior!is!associated!to!titanium!and!inorganic!materials,!it!appears!that!oxidation!of! DOPA!under!elevated!pH!promotes!covalent!bonds!with!organic!surfaces.!In!fact,!the!quinone!function! can!react!with!different!nucleophilic!functional!groups!such!as!primary!amines!–NH2!or!thiols!–SH!that! can!be!found!on!biological!substrates.![19]!! AFM!force!linking!measurements!were!carried!at!pH!9.7!between!DOPA!and!Si!modified!surface! with!amine!groups!(fig!1-13).!This!study!showed!a!dramatic!increase!of!pull-of!force!at!the!interface!with! the!organic!surface,!2.2!nN!in!this!case,!in!comparison!to!800!pN!with!Ti-catechol!interfaces!at!neutral! pH.! This! is! consistent! enough! to! confirm! a!covalent! adhesion.! This! information! could! be! relevant! for! chitosan!functionalization,!as!this!polymer!contains!amine!groups. [18]!) ! ! Fig!1-13:!Example!of!oxidized-DOPA!platform!with!quinone!state!reacting!with!amine-functionalized!Si!surface![18]! These!findings! suggest! the! possibility! of! using! DOPA! as! anchor! on! a! wide! range! of! inorganic! materials!but!also!on!its!oxidized!form!of!quinone!to!organic!surfaces.!! ii. Binding of DOPA to biomaterials: Previous studies Regarding!L-DOPA!chemistry!and!its!ability!to!functionalize!a!wide!range!of!materials,!a!series!of! studies!were!made!in!order!to!test!the!viability!of!DOPA-coated!or!catechol-functionalized!biomaterials! ! ! ! 20! to! improve! their! biological! properties.! The! following!part! presents!a! non-exhaustive! summary! of! published!studies!that!could!be!interesting!for!the!project.!! ! a) Metals (i.e. Titanium) A! successful! anchoring! of! catechol! moieties! and! DOPA! derivatives! on! titanium! surfaces! was! demonstrated!in! previous! studies.! For! example,! the! rupture! force! of! DOPA! and! related! amino! acids! residues! to! a! chemically! well-defined! TiO2!surface! was! measured! thanks!to! single-molecule! force! spectroscopy!(AFM).! As! previously! introduce,! it! appears! that! DOPA! might! bind! to! TiO2!by! strong! coordination!bonds.!It!is!known!that!catechol!moieties!can!form!strong!complexes!with!metal!atoms!and! their! oxides.!The! dependence! of! pH! environment,! and! oxidation! state! of! DOPA! appeared! to! be! important,!as!quinone!and!semiquinone!present!in!basic!medium!(pH=9.8)!did!not!adhere!as!strongly!as! DOPA!did.!The!study!also!showed!that!increasing!the!quantity!of!–OH!group!in!the!synthetized!DOPA! molecule,! for! example! with! N-Boc-6-hydroxy-DOPA,! provided!a! stronger! interaction! with! the! metal! surface.!![20]! b) Ceramics According! to! observations! that! revealed! unsuficient! adhesion! forces!for! dental! and! medical! adhesives! in! wet! environments!some! studies! have! been! carried.!Using! catechol! coatings! on! hydroxyapatite!(HA)!has! been! investigated!to! achieve! adhesion! in! an! aqueous! environment.! For! instance,!one!study!compared!the!adsorption!of!catechol!molecules!to!HA!and!other!chemical!groups! such! as! alcohol.! Catechol! group! showed!a! superior! adsorptive! property! related!to! the! other! representative! solutes.! These! observations! were! done! with!contact! angle! measurements. [21]!This! observation!opens!the!door!to!the!potential!use!of!catechol/DOPA!in!adhesive!systems!on!HA.!! Various!mechanisms!were!proposed!by!researchers!on!the!absorption!of!catechol!groups!on!HA! surfaces.! Misra!and! collaborators!propose!that! this!adhesion!is! due! to! hydrogen! bonding,!although! electrostatic!interactions!and!the!effect!of!aromatic!rings!should!be!taken!into!account!too.!![21]! Zirconia!is!an!ideal!material!for!implants!in!dentistry!and!orthopedics!thanks!to!its!very!good! esthetic!properties!and!limited!plaque!adhesion.!But!it!has!a!very!high!physicochemical!stability,!which! can!dramatically!slow!down!its!biointegration.!Some!studies!were!made!about!L-DOPA!coatings!on!ZrO2! so! as! to! improve! its! biocompatibility.! ZrO2!disks! were! immersed! in! a! solution! of! L-DOPA! (2mg/ml)! dissolved!in!10mM!Tris-HCl!(fixed!at!pH!8,5).!A!pH-induced!oxidative!polymerization!occurs!on!surface,! notable! thanks! to! a! change! of! color! in! the! solution,! this! leads! to! the! spontaneous! growth! of! a! selfpolymerization!layer!on!the!surface!of!the!zirconia.!Results!clearly!showed!that!L-DOPA!coated!zirconia! had! a! better! cyto-compatibility! regarding! cell! spreading! and! proliferation! than! uncoated! one.! This! statement!was!made!thanks!to!a!scanning!electron!microscope!(SEM)!with!chemical!analysis,!but!also! regarding!a!change!in!surface!wettability!observed!in!contact!angle!measurements.!XPS!confirmed,!with! ! ! ! 21! the! presence! of! N! 1s! peaks,! that! the! surface! was! successfully! functionalized! with! L-DOPA.! Exact! mechanisms! remain! unclear,! but! some! studies! said! that! the! L-DOPA! coating! involves! simultaneously! adsorption!and!polymerization!on!surface.!It!was!shown!that!the!N/Zr!ratio!increased!with!temperature! (tests! were! carried! at! 22! and! 37°C),!which!suggests! that! adding! energy! to! the! system! improves! polymerization!rate.![22]! ! c) Conjugation to polymers Catechol-based!polymers!were!studied!and!synthesized,!as!described!in!some!publications.)[23]) Three!cases!were!discussed!in!which!catechols!were!present!in!the!polymer!main!chain,!or!as!pendant! groups!or!incorporated!at!the!extremity!of!already!well-defined!polymers.!(fig!1-14)! ! Fig!1-14:!Different!cases!of!catechol-functionalized!polymers![23]! Autopolymerization of polydopamine To!polymerize!dopamine!(catechol-containing!molecules),!catechols!have!to!be!converted!first! into! highly! reactive! quinones.! It! can! be!done! easily! by! a! strong! oxidant! agent,! an! enzyme! or! by! an! aerated!aqueous!solution!at!neutral!to!alkaline!pH.!Autopolymerization!of!catecholamines!can!be!driven! in!aerated!basic!solutions.!This!!was!first!demonstrated!by!Messersmith!et!al.!for!dopamine!(reference).! The! polydopamine!network! is! supposed! to! be! formed! by! Schiff! base! formation! and/or! Michael! type! addition,! due!to! the! reaction! involving! quinone! groups! and! its! primary! amino! groups.!(fig! 1-15)! This! molecule!can!self-polymerize!and!lead!to!a!complex!macromolecular!structure!including!free!catechol! units.!! ! ! ! 22! ! Fig!1-15:!Mechanisms!of!self-polymerization!of!dopamine!to!poly(dopamine) [23]! Those! units! make! possible! the! strong! adhesion! of! the! polymer! network! to! different! kind! of! surfaces!as!described!before.!Some!studies!exploited!the!ability!of!polydopamine!to!adhere!on!organic! and!inorganic!substrates!such!as!glass,!polystyrene!or!poly(dimethylsiloxane)!(PDMS).! Poly(catecholamines)!were!used!as!coatings!which!act!as!anchoring!layers!for!polymer!brushes! and!other!(bio)macromolecules.!PEG!polymers!have!been!functionalized!at!chain-ends!with!catechols! bearing-molecules.!These!conjugates!were!designed!for!the!biomedical!field,!in!order!to!stabilize!metal! nanoparticles!(FePt,!Fe3O4).!Catechols!were!grafted!by!means!of!chemical!ligation.!! Preparation!of!catechol!side!chain!polymers!can!be!achieved!by!radical!polymerization!of!vinyl! monomers!that!incorporate!an!unprotected!catechol!unit.!Those!polymers!can!bear!catechol!groups!on! each!monomer!unit!and!that!can!provide!a!strong!adhesive!property,!which!was!tested!on!wood!with! homopolymerized!dopamine!acrylamide.![23]! Alginate functionalization Alginate!is!a!polysaccharide!made!of!two!homopolymers!blocks!of!(1,4)-linked!β-D-mannuronate! and!α-L-guluronate.!(fig!1-16)!It!is!a!natural!polymer!that!can!be!extracted!from!seaweed!and!is!widely! used!as!biomaterial!for!hydrogels.!Hydrogels!made!with!alginate!and!rich!in!guluronate!HGA!are!rigid,! porous! and! can! take! its! integrity! for! long! periods! of! time.! Its! applications! are!drug! and! cells-delivery! systems,!wound!dressings!and!dental!implants.! ! Fig!1-16:!Structure!of!β-D-mannuronate!and!α-L-guluronate,!two!blocks!that!form!the!alginate!homopolymer [11]! ! ! ! 23! Previous! studies! showed! the! possibility! of! using!DOPA-coatings! on! alginate! materials! or! hydrogels!thanks!to!strong!chemical!bonds.!L-DOPA!can!have!a!redox!activity!allowing!oxidative!crosslinking!to!form!protein!networks.!(fig!1-17)!This!chemical!ability!has!been!used!to!generate!3D!hydrogel! networks.!In!other!studies!catechol!moieties!were!combined!to!polymers!such!as!PEG!and!chitosan.!An! alginate-catechol! hydrogel! was! synthesized! and! showed! good! physical! and! mechanical! properties! assorted!with!low!immunogenicity!and!no!cytotoxicity.!Preparation!of!the!hydrogel!was!made!possible! by!cross-linking!via!oxidative!catechol!polymerization.![24]! ! Fig!1-17:!Preparation!of!catechol!functionalized!alginate!hydrogels![24]! Chitosan Chitosan!is!a!chitin-derived!polysaccharide,!which!is!a!principal!component!of!the!exoskeletal! structure! of! insects! and!crustaceans.! It! has! biocompatible,! biodegradable,! bioadhesive! and! hemocompatible!properties.!It!can!be!used!as!hemostatic!agent!in!order!to!activate!the!formation!of! coagulants,!or!in!scaffolds!for!drug-delivering!systems.![11]! ! Fig!1-18:!Chitosan!chemical!structure![25]! ! Catechol-conjugated! chitosan! was! used! in! thiolated! Pluronic! hydrogels! so! as! to! include! new! desirable!properties!to!it.!The!mixture!is!initially!a!viscous!solution! but! becomes!a!cross-linked! gel!at! body!temperature!and!physiological!pH.!Some!notable!new!properties!were!introduced!thanks!to!the! catechol!groups!such!as!a!strong!adhesiveness!to!organic!matrix!(soft!tissues,!mucous!layers),!and!better! hemostatic! behavior! than! before! (compared! to!non-functionalized! chitosan).! At! theoretical! level,! primary!amine!groups!in!the!chitosan!as!well!as!thiolated!Pluronic!were!supposed!to!be!in!situ!crosslinked!by!catechol!groups.!Experimentally!catechol!moieties!were!conjugated!onto!chitosan!thanks!to! standard!EDC!chemistry.![26] ! ! ! 24! II. Objectives Functionalize!materials!with!peptide-platforms!could!allow!them!to!have!better!cell-adhesion! properties!and!provide!a!better!osseointegration.!As!mentioned!before,!the!main!limitation!of!methods! similar!to!silanization!is!that!it!requires!a!multi-step!preparation,!which!includes!activation!of!the!metal! surface,!silanization,!and!the!addition!of!biomolecules.!In!some!cases!it!could!be!also!necessary!to!add! an!extra!step!with!a!crosslinker.!This!coating!is!water-sensitive!and!the!process!is!highly!dependent!on! the! silane! concentration,! temperature,! time! and! humidity.! Regarding! the! environmental! impact,! silanization!requires!the!use!of!organic!solvents!like!toluene.![12]!! To!overcome!these!limitations,!the!use!of!a!functional!group!that!could!provide!a!single!and!one! step!stable! binding,! with!less!parameters!to!control,!and! in!aqueous! solutions,! would!be!a!preferred! alternative.!This!can!be!achieved!with!the!chemical!group!catechol,!present!in!the!amino!acid!DOPA.!! DOPA!is!an!interesting!molecule!that!could!be!used!as!anchoring!unit!in!this!kind!of!platforms.! This!project!aims!at!adhering!peptide-platform!carrying!a!DOPA!molecule!as!anchoring!unit!(figure!2-1)! to! a! wide! range! of! materials!(metals,! ceramics! and! polymers)!used! in! biomedical! applications! and! specially!for! bone! implants.!The!process!used!is!the!immobilization!of!biomolecule!with!a!single!step! functionalization,! which!implies! lots! of! advantages! of! cost,! time! and! toxicity! comparing! to! other! methods!such!as!silanization.! Regarding! the! past! studies,! it! appears! that! the! adhesion! of! DOPA! and! catechol! moieties! to! titanium!surfaces!is!effective.![20]!Oxidation-state!of!catechol!unit!was!described!as!an!important!factor! that!can!alterate!the!strength!of!the!adhesion!between!the!molecule!and!the!biomaterial!surface.![19]! The!peptide-platform!will!be!placed!in!different!solvent,!with!neutral!and!basic!pH!in!order!to!compare! the!adhesion!of!quinone-state!and!catechol-state!units.!! ! ! ! ! ! ! ! ! Treball Final de Grau - Clàudia Huertas Martínez 11 FN is formed by three types of repeating motifs, which are organized in at least seven domains. RGD, discovered in 1984, is the sequence that binds fibronectin, and many other molecules of the ECM, to cells via a transmembrane receptor protein which belongs to the integrin class of proteins. Integrins (Figure(4) are heterodimers that contain several types of α and β polypeptide chains, and there are different integrin subtypes that recognize and bind to the RGD with varying degree of affinity. Integrin interactions can transfer signals in both directions, outside-in or inside-out the cell and the extracellular surrounding. In this way they coordinate a variety of cellular activities, including cell adhesion, migration, ECM assembly, proliferation, survival, and differentiation [17]. (Figure(4) Specifically, FN receptor interacts indirectly with actin filaments present in the cytosol of the cells. Attachment proteins, and among them vinculin and talin, are involved in this process. Talin links the integrin to actin via vinculin which interacts with the actin Those large intracellular protein complexes form focal adhesions, which apart from anchoring cells in the ECM, send signals from the exterior and this further influence cell behavior [17] Fig! 2-1:!A)! Chemical! structure! of! the! peptide-platform! carrying! DOPA! molecule! as! anchoring! unit.! B)!General!scheme!of!the!platform! structure! ! ! ! 31! ! ! ! ! ! ! ! Fig!3-9:!Example!of!immobilization!experiment!preparation!on!PLLA!films!placed!in!a!multi-well!plate.!a)! Negative!control!with!dH2O!b)!PTF-CF!dH2O!c)!PTF-CF!Tris! ! The!α-TCP!disks!synthetized!in!the!BBT!laboratory!are!porous!materials!that!can!absorb!a!certain! quantity! of! liquid.! This! makes! them! hard! to! functionalize! with! the! same! method! used! for! the! other! materials!(addition!of!a!drop).!Because!of!this,!a!different!protocol!was!used.!α-TCP!samples,!both!fine! and!coarse,!were!functionalized!with!only!50!μL!of!the!peptide-platforms.!In!this!case,!a!drop!of!solution! was!carefully!put!on!each!sample,!making!sure!that!the!solution!was!absorbed!by!the!surface!and!no! liquid!went!out!of!the!disk.!The!coating!was!carried!overnight.!Then!samples!were!cleaned!with!distilled! water!under!orbital!agitation!at!100!rpm,!during!2!minutes!and!repeated!6!times.!The!samples!were! dried!at!ambient!temperature!under!hood!aspiration!for!a!day.! a)) b)) c)) ! ! ! 32! 3. Characterization methods ! i. Contact Angle Measurements !When!a!liquid!spreads!on!a!solid!substrate!the!induced!process!is!called!wettability.!A!surface! can! be! hydrophobic,! which! means! that! the! surface! induces! a! preferable! contact! with! itself! than! an! interface!with!the!liquid!used!for!the!test.!The!other!case!is!a!hydrophilic!material,!which!is!the!inverse! situation.!! Chemical!groups!present!on!the!surface,!which!create!polar!and!electrostatic!interactions,!are! responsible!of! the! solid/liquid! interaction.! Surface! topology,! that! includes! porosity,! roughness! and! crystallography,!is!the!second!parameter!that!changes!the!contact!angle.!The!type!of!liquid!used!for!the! measurements!also!changes!the!obtained!values!due!to!its!functionality!and!structure.!Contact!angle!is! basically!the!angle!between!the!deposited!drop’s!profile!and!the!tangent!to!the!surface,!at!the!meeting! point!of!atmosphere,!liquid!and!solid. [28]!(fig!3-10)!! ! ! ! Fig!3-10:!Contact!angle!scheme [28]!! ! Static! contact! angle! measurements! on! Ti! disks! and! polymer! films! were! performed! using! a! contact! angle! OCA15! plus! with! the! sessile! drop! method.! All! measurements! were! carried! at! room! temperature!using!mQ!water!as!wetting!liquid!(1!µL!drop).!Static!contact!angle!were!calculated!using!an! ellipse-fitting! with! SCA20! software.! Three! measurements! were! made! per! disk! for! three! samples! replicates.!Data!described!were!obtained!doing!a!mean!and!standard!deviation!calculation!of!the!series! of!9!measurements!per!condition.!! ! ii. FTIR – ATR The!attenuated!total!reflection!(ATR)!with!infrared!spectroscopy!(IR)!enables!to!directly!analyze! solid!or!liquid!samples!without!any!preparation.!An!infrared!wave!is!directed!to!an!optically!dense!crystal! (in! this! case! a! germanium! crystal)! with! a! high! refraction! rate! with! a! certain! angle.! This! creates! an! evanescent!wave,!which!spreads!on!the!crystal!surface!and!inside!the!sample!that!is!put!in!contact!with! the! crystal.! Transmittance! spectra! are! obtained! with!this! technique,! which!can! theoretically! allow! ! ! ! 33! identifying!functional! groups! and! certain! bindings! modes! according! to! the! presence! of! characteristic! peak!at!a!defined!wavenumber!(cm-1).!!These!characteristic!peaks!are!well!described!in!the!literature.!! An!FT-IR!spectroscope!Nicolet!6700!from!Thermoscientific!was!used!to!carry!the!experiments.! Acquisition!was!realized!with!the!software!OMNIC.!Ti!disks!were!characterized!using!512!scans!with!a! resolution!of!4.!PLLA!and!PMP!films!were!characterized!using!512!scans!with!a!resolution!of!2.!Before! testing,!the!samples!were!cleaned!with!distilled!water!under!agitation!and!dried!with!N2.!! ! iii. Fluorescence observations Fluorescence!images!were!obtained!with!a!confocal!laser!scanning!microscope!device!(Zeiss!LSM! 800).!10x!objective!was!used.!Fluorescence!visualization!was!made!possible!to!the!carboxyfluorescein! molecule!present!on!the!platform!(PTF-CF).!Acquiring!of!images!was!made!with!Zen!2.3!software!from! Zeiss!company.!At!least!three!images!per!simple!were!taken.!Fluorescence!Intensity!of!images!(mean! pixel!value!and!arbitrary!unit)!was!measured!by!image!treatment!with!the!software!ImageJ.!! iv. X-Ray Photoelectron Spectroscopy (XPS) ! Photoelectron! spectroscopy! consists! in! analyzing! the! energy! of! emitted! electrons! from! a! substance!irradiated!by!electromagnetic!UV!or!X-Ray.!It!provides!information!about!bonding!energy!that! can!be!correlated!to!the!chemical!composition!of!the!samples.!Thanks!to!this!technique!results!such!as! elemental!composition!at!the!parts!per!thousand!range,!empirical!formula,!chemical!state!and!electronic! state!of!the!elements!that!exist!within!a!material!can!be!obtained.!Theoretically,!XPS!scans!of!negative! uncoated! samples! could! be! compare! to! a! PTF-coated! one,! so! as! to! identify! changes! of! element! composition!on!the!surface!that!could!be!attributed!to!the!adhesion!of!peptide!platforms.!This!is!a!nondestructive!analyzing!method!applicable!to!all!type!of!materials.!Only!a!thin!width!of!solid!samples!can! be!scanned!(0,5!to!5!nm)!and!measurements!are!carried!under!vacuum.![29]!Experiments!were!carried! in! duplicate,! putting! two! samples! for! each! type! of! treatment.! Measurements! were! made! at! the! Barcelona! Research! Center! in! Multiscale! Science! and! Engineering,! with! a! XPS! machine! from! Specs! (Germany),! with! a! Magnesium! Kα!XR50! source! at! 200! W! for! each! sample.! The! analysis! device! was! Phoibos!150!MCD!9!(9!multi!channel!electron!detector)!and!the!vacuum!pressure!was!at!2.10-8!mbar.!! ! ! ! 34! IV. Results and Discussion 1. Functionalization of Ti disks i. Preliminary tests on Ti disks !The! first! studies! of! functionalization!with! the! PTF!were! done! on! Ti! using!PBS!as!solvent,! according!to!the!methods!described!in!the!experimental!section.!! ! Fig!4-1!:!Distilled!H2O!contact!angle!measurements!on!functionalized!Ti!disks! ! Thanks!to!contact!angle!characterization,!a!difference!between!negative!control!samples!and! functionalized! samples! was! shown.! A! significant! decrease! between! polished! Ti! and! PTFfunctionalized! Ti! contact! angle! was! observed.! In! fact! CA! value! was! around! 82°! before! any! immobilization!and!was!around!68°!after.!(fig!4-1)!But!the!samples!that!were!only!covered!with!PBS! solution!also!showed!an!important!decrease!till!70°.!The!decision!was!made!to!change!the!solvent!for! further!tests,!to!dissolve!PTF!in!dH2O!and!Tris!buffer!and!avoid!the!important!effect!of!PBS!buffer!on! the!surface!state!of!the!samples.!! ii. Oxidation state influence of catechol As!previously!explained,!the!oxidation!state!of!the!catechol!group!(catechol!or!quinone)!has!an! important!effect!on!its!capacity!to!bind!to!material!surfaces.!Thus,!the!immobilization!of!the!PTF!was! done!in!various!solvents!so!as!to!test!the!influence!of!the!oxidation!state!of!Dopa/catechol!on!surface! adhesion.!To!that!extent,!platforms!were!dissolved!in!either!distilled!water!(pH!7),!which!stabilizes!the! “catechol-form”,!or!Tris-base!buffer!(fixed!at!pH!9,7),!which!stabilizes!the!“quinone-state”.!! Ti!polished!! Ti!ctrl!in!PBS! Ti!PTF!PBS! Ti!CF-PTF!PBS! Séries1! 82,6! 70,2! 68,3! 69,2! 50,0! 55,0! 60,0! 65,0! 70,0! 75,0! 80,0! 85,0! 90,0! Contact)Angle)(°)) dH2O)CA)Measurements)on)Ti)disks) ! ! ! 35! Differences! were! observed! between! samples! thanks! to! contact! angle! measurements.! Ti! ctrol! had!a!mean!of!83,2°,!while!Ti!PTF!H2O!had!the!lowest!value!66,1°!and!Ti!PTF!Tris!77,3°.!(fig!4-2)!A!lower! hydrophobicity!of!the!samples,!visualized!on!fig!4-3,!could!be!associated!to!a!biofunctionalization!of!the! surface!and!an!adhesion!of!platforms.!A!higher!change!in!PTF!H2O!samples!compared!to!PTF!Tris!is!due! to!a!better!adhesion!of!peptide!platforms!on!the!surface!of!Ti!disks.!! ! Fig!4-2!:!distilled!H2O!contact!angle!measurements!on!Ti!disks! ! ! ! Fig!4-3:!Contact!angle!measurements.!a)!Sessile!drop!on!Ti!H2O!PTF!b)!Sessile!drop!on!Ti!Tris!PTF! ! Ti!ctrol!(H2O)! Ti!PTF!H2O! Ti!PTF!Tris! Séries1! 83,2! 66,1! 77,3! 50,0! 55,0! 60,0! 65,0! 70,0! 75,0! 80,0! 85,0! 90,0! 95,0! Contact)Angle)(°)) dH2O)CA)Measurements)on)Ti)c.p.) a b ! ! ! 36! ! Fig!4-4:!Fluorescence!intensity!measurements!on!functionalized!Ti!disks! Regarding!results!in!fluorescence!observations,!platforms!in!Tris!solution!(pH!9,7)!did!not!adhere! on!Ti!surfaces.!In!fact!the!mean!pixel!value!with!PTF-Tris!(7,7u)!is!close!to!ctrol!samples!(7,1u).!PTF-H2O! showed!an!important!change!of!pixel!value!(13,5u).!(fig!4-4)!That!would!confirm!the!hypothesis!about! the!quinone!negligible!adhesion!on!metallic!substrates!compared!to!catechol.!! ! Fig!4-5:!Fluorescence!images!of!functionalized!Ti!samples.!a)!Ti!ctrol.!b)!Ti!dH2O!PTF-CF.!c)!Ti!Tris!PTF-CF.! Ti#ctrol# Ti#PTF#Tris# Ti#PTF#H2O# Séries1# 7,1# 7,7# 13,5# 0,0# 2,0# 4,0# 6,0# 8,0# 10,0# 12,0# 14,0# 16,0# 18,0# 20,0# Fluorenscence*Intensity*(a.u.)* Fluorescence*intensity*Ti*disks* b c a ! ! ! 37! !FTIR-ATR! observations! didn’t! show! significant! results.! In! fact,! control! Ti! c.p.! without! functionalization!had!a!flat!spectra!without!any!characteristic!peak!as!expected.!(fig!4-6)!But!further!tests! with!functionalized!samples!couldn’t! be! analyzed! because! the! obtained! spectras! showed! unexpected! peaks! that! could! be! associated! to! contamination! or! ethanol! that! was! used! to! clean! the! germanium! cristal!between!each!test!sample.!! ! Fig!4-6:!Ti!c.p.!infrared!spectra!control! XPS!measurements!on!Ti!samples!allowed!obtaining!%atomic!concentration.!(fig!4-7)!The!results! showed!a!significant!increase!of!N!atomic!percentage!between!control!and!PTF-functionalized!samples.! In!fact!%N!is!4,8%!of!total!atomic!content!for!PTF!in!dH2O!samples,!and!was!1,1%!for!Ti!control.!!These! changes!were!less!important!with!PTF!Tris!but!N!atomic!%!reached!a!2,8%.!(fig!4-7)! Thanks!to!peak!deconvolution!of!XPS!results,!other!information!has!been!analyzed.!For!instance,! the! quantity! of! aliphatic! carbons! (apolar)! can! be! compared! to! polar! carbons.! Likewise,! the! type! of! oxygen!can!be!studies,!e.g.!oxide!bonds!or!OH/polar!bonds.!After!functionalization!of!Ti!with!PTF,!an! increase! of! polar! C! %! (more! energetic)! was! observed,! together! with! a! decrease! of! aliphatic! %! (less! energetic).!The!platform!adhesion!implies!the!appearance!of!CO!bonds!on!the!surface!that!are!polar!and! more! energetic! than! aliphatic! bonds.! The! same! analysis! can! be! done! with! O%.! Initially,! on! control! samples,!the!oxide!O!is!associated!with!TiO2!elements,!and!OH/polar!bonds!should!be!due!to!the!Ti-OH! groups!at!the!surface.!A!decrease!from!64,4%!to!47,6%!of!O!oxide!content!between!control!and!PTF! dH2O! samples! was! observed.! Simultaneously,! the! polar! percentage! increases.! This! is! also! due! to! CO! bonds!brought!by!the!peptide-platform!adhesion.!(fig!4-8)!! ! ! ! ! ! 38! ! Fig!4-7:!Graphical!draw!of!atomic%!for!functionalized!Ti!c.p.!samples!! ! ! Fig!4-8:!At%!from!XPS!measurements!of!different!types!of!C!and!O!after!peak!deconvolution!for!Ti!samples! Discussion According!to!the!presented!results,!it!is!possible!to!say!that!a!significant!adhesion!of!peptideplatforms!in!dH2O!solvent!was!observed!on!Ti!c.p.!surface.!The!XPS!observations!with!an!increase!of!N! atomic!percentage!confirmed!this!statement.!Concerning!the!oxidized-state!platforms,!in!Tris!solvent,! they!also!showed!different!results!comparing!to!control,!that!can!also!be!associated!to!an!adhesion,!but! with!a!lower!intensity!than!unoxidized!catechol.!! ! ! ! C" N" O" Ti" Ti"ctrol" 52,5" 1,1" 34,5" 11,9" Ti"PTF"dH2O" 63,1" 4,8" 25,0" 7,2" Ti"PTF"Tris" 63,7" 2,8" 25,8" 7,8" 0,0" 10,0" 20,0" 30,0" 40,0" 50,0" 60,0" 70,0" 80,0" %"At"concentra+on" XPS"Measurements"-"Ti"%At"concentra+on" alphatic polar oxide OH///polar Samples C/285 C286 C289 C/total O/530 O/532 O/total Ti 57,1 35,3 7,7 100 64,4 35,6 100 PTF 35,4 51,8 12,8 100 47,6 52,4 100 PTF/+/Tris 50,0 40,0 10,0 100 53,0 47,0 100 ! ! ! 39! 2. Platform-functionalization of commercial PLLA Films ! PLLA!samples!were!functionalized!as!described!in!Methods.!! ! Fig!4-9!:!Distilled!H2O!contact!angle!measurements!on!PLLA!films! Contact! angle! measurements! show! a! clear! difference! between! PTF! H2O! and! PTF! Tris.! Dopabased!platforms!seem!to!adhere!better!in!neutral!than!in!basic!solvent.!In!fact,!a!clear!decrease!of!CA! value!was!observed!between!negative!control!and!PTF!in!dH2O!(from!111,7°!to!98,9°)!while!the!platform! in!Tris!coating!was!similar!to!control!(110,8°).!(fig!4-9)! ! Fig!4-10:!Fluorescence!intensity!measurements!on!functionalized!PLLA!films! Samples!showed!high!variability!at!fluorescence!microscopy,!but!after!repeating!a!second!time,! correct!results!could!have!been!isolated!to!prove!a!good!adhesion!of!platforms!in!dH2O!solvent!on!PLLA! surfaces.!(fig!4-10)!Indeed!fluorescence!intensity!increase!from!0,9!a.u.!to!23,8!a.u.!between!ctrol!and! PLLA$ctrol$ PLLA$PTF$H2O$ PLLA$PTF$Tris$ Séries1$ 111,7$ 98,9$ 110,8$ 90,0$ 95,0$ 100,0$ 105,0$ 110,0$ 115,0$ 120,0$ !Contact!Angle!(°)! dH2O!CA!measurements!on!PLLA! PLLA$ctrol$ PLLA$PTF$dH2O$ PLLA$PTF$Tris$ Séries1$ 0,9$ 23,8$ 19,5$ 0,0$ 5,0$ 10,0$ 15,0$ 20,0$ 25,0$ 30,0$ 35,0$ Fluorescence*Intensity*(a.u.)* Fluorescence*Intensity*PLLA* ! ! ! 40! PTF-dH2O!functionalized!samples,!some!notable!difference!was!also!observed!with!a!value!of!19,5!a.u.! for!PTF-Tris!samples.!! As!shown! in!fig!4-10,!the! standard! deviation! of!the! fluorescence!microscopy!data!was!rather! high.! This! could! be! due! to! various! factors,! including! heterogeneity! of! the!surface! between! different! samples,!or!inefficient!washing!of!the!samples.!However,!a!notable!difference!between!negative!control! and!functionalized!samples!can!be!observed.!(fig!4-11)! ! Fig!4-11:!Fluorescent!images!of!functionalized!and!control!PLLA!samples.!a)!PLLA!negative!control.!b)!PLLA!dH2O! PTF-CF.!c)!PLLA!Tris!PTF-CF! The!FTIR-ATR!technique!appeared!to!be!unsuccessful!to!notice!any!difference!between!samples.! In!fact,!negative!control!and!platform-functionalized!PLLA!did!not!show!any!peak!differences!although! significant! results! and! notifications! of! PTF! presence! were! noticed! with! fluorescence! microscopy! and! contact!angle!measurements.!(fig!4-12)!Moreover,!only!one!double!peak!was!observed!between!1150! cm-1!and!1206!cm-1,!which!could!be!associated!to!C-O!stretching!region.!!A!strong!peak!region!should!be! present!around!1750!cm-1!corresponding!to!the!carbonyl!stretching!region!but!wasn’t!observed!in!any! sample!curve!fitting.!!! ab c ! ! ! 47! Discussion α-TCP! samples! showed! retention!of! the! platforms! that! could! be! observed! by! the! yellow! coloration! of! samples! that! were! functionalized! by! PTF-CF! molecules.! But! this! couldn’t! be! well! characterized!at!fluorescence!microscopy.!In!further!tests,!a!better!protocol!should!be!investigated!to! functionalize!the!cement!and!adapted!characterization!methods!should!be!found.!! ! 5. Alginate and Chitosan functionalization Alginate!and!Chitosan-based!films!were!synthetized!at!the!BBT!laboratory,!as!explained!in!the! Experimental! Section.! These! films!were! meant! to! be! functionalized! with!the!peptide-based! platform! similarly! to! the! other! biomaterials.!However,! both! materials! have! particular!swelling! properties! that! made! impossible! to! carry! the! experiment! with! the! same! parameters! and! steps! used! for! the! other! materials.!! Alginate! films! showed! a! very! important! diminution! of! volume! after! cross-linking! and! casting.! This! retraction!implies! brittle! properties! for! the! film! and! requires! their! re-hydration! to! be! used! for! functionalization!tests.!Two!different!preparations!were!made!to!try!to!reduce!this!effect:!first!1%!wt/v! alginate!solution!with!100mM!of!CaCl2!(fig!4-23!and!4-24),!and!then!increasing!the!quantity!of!alginate!to! 5%!wt/v,!as!this!was!supposed!to!induce!a!more!ductile!film!(fig!4-25).!But!this!method!was!not!effective! and!both!films!showed!the!same!type!of!volume!reduction.!In!particular,!the!5%wt/v!alginate!film!was! even!more!brittle!after!casting.!(fig!4-26)! ! ! ! ! Fig!4-23:!Alginate!film!(1%!wt/v)!preparation!at!the!BBT!laboratory,!right!after!removal!from!CaCl2!crosslinking!solution! ! ! ! ! ! 48! ! ! ! ! ! Fig!4-24:!Alginate!films!(1%wt/v)!preparation!at!the!BBT!laboratory,!after!1!day!and!overnight!casting!at!room! temperature!and!air.! ! Fig!4-25:!Alginate!film!(5%!wt/v)!preparation!at!the!BBT!laboratory,!right!after!removal!from!CaCl2!cross-linking! solution! ! ! ! Fig!4-26:!Alginate!film!(5%wt/v)!preparation!at!the!BBT!laboratory,!after!1!day!and!overnight!casting!at!room! temperature!and!air.! ! ! Further!re-hydration!of!the!synthetized!alginate!films!was!investigated!in!order!to!make!them! suitable! for! functionalization! and! characterization.! Re-hydration! was! carried! on! 1%! wt/v! films,! which! were!submerged!20!seconds!in!distilled!water!in!a!Petri!dish!and!then!removed!from!water.!This!allowed! the!film!to!recover!a!flat!shape!(fig!4-27),!the!properties!obtained!were!suitable!for!preparing!samples!in! order! to! functionalize! them.! This! means! that! the! re-hydrated! films! were! flat,! with! sufficiently! good! mechanical!properties!to!handle!them.!!5%!wt/v!films!had!to!be!submerged!during!2!minutes!in!distilled! water!in!order!to!recover!a!flat!shape!and!be!suitable!for!functionalization.!(fig!4-28)! ! ! ! 49! ! Fig!4-27:!!Alginate!film!(1%!wt/v)!preparation!at!the!BBT!laboratory,!right!after!removal!from!20!seconds!immersion! in!distilled!water!(re-hydration)! ! ! ! ! ! Fig!4-28:!!Alginate!film!(5%!wt/v)!preparation!at!the!BBT!laboratory,!right!after!removal!from!2!minutes!immersion! in!distilled!water!(re-hydration)! ! Re-hydration!was!less!successful!with!chitosan!films,!which,!in!fact,!showed!very!low!mechanical! properties!and!resistance!after!removal!from!distilled!water.!The!films!could!not!be!manipulated!after! re-hydration!to!be!cut!or!moved!without!being!damaged.!! ! Discussion !Alginate! and! chitosan! were! not! functionalized! for! the! moment! because! of! their! lack! of! maniability.!Other!conditions!should!be!tested!in!order!to!find!an!acceptable!protocole!considering!their! swelling!properties!that!are!sufficient!to!prevent!from!a!further!analisis!and!characterization!with!the! same!methods!used!for!the!other!biomaterials!during!the!project.!! ! ! ! ! 50! Achievements and Work Perspectives Immobilization!of!peptide-platform!using!DOPA/catechol!as!anchoring!molecule!was!carried!on! various! biomaterials! surfaces.! Table! 5-1! summarizes! the! results! obtained! according! to! the! characterization!methods!explained!before!in!the!report.!! ! Material) DOPA)with)catechol)state) adhesion)(pH)<)9,7)) DOPA)with)quinone)state) adhesion)(pH)=)9,7)) Ti!c.p.! STRONG! WEAK! PLLA!commercial!films! STRONG! WEAK! PMP!commercial!films! WEAK! WEAK! α-TCP! ?! ?! Alginate!films! ?! ?! Chitosan!films! ?! ?! Table!5-1:!Results!table!summarizing!the!peptide-platform!adhesion!on!various!biomaterials!tested!during!the! project.! As! described! before! in! the! literature,! Ti! c.p.! was! successfully! functionalized! using! DOPA! as! anchoring! unit.! This! was!verified! by! various! techniques.! Every! characterization! method! showed! comparable! results! for! Ti! surfaces.! In! particular,!XPS,! which! is! the! most! precise! one,! confirmed! that! DOPA!is!a!successful!anchoring!unit!for!peptide-platforms!to!Ti.!It!also!confirmed!that!the!oxidation!state! of!the!catechol!unit!is!a!key!factor,!proving!that!fewer!molecules!adhere!in!Tris!solution!(pH!=!9,7)!that! implies!a!quinone!oxidation!state.!! PLLA!also!appeared!to!be!a!successful!substrate!for!catechol-bearing!platforms.!The!fact!that!the! interactions!between!those!chemical!species!were!not!described!in!previous!articles!is!interesting,!and! converts! this! work! in! the! first! report! that! describes! DOPA! as! a! binder! to! PLLA! biomaterials.! The! differences!of!adhesion!between!the!oxidized!and!neutral-form!of!DOPA!observed!on!PLLA!were!similar! to!those!of!Ti!c.p.!samples.!Hydrogen!bonding!could!hypothetically!explain!the!adhesion!between!DOPA! and!the! surfaces!according!to!the! possible!interactions!between!catechol!and! other!chemical!species! described!in!literature.!! The!possible!reaction!on!surface!with!PMP!polymer-type!films!was!not!described!or!studied!in! previous!works.!According!to!the!results!obtained,!the!functionalization!with!DOPA!anchoring!unit!did! ! ! ! 51! not!show!a!significant!adhesion!on!PMP!commercial!films.!This!is!a!consistent!with!the!fact!that!any!of! the!interactions!previously!described!in!B.P.!Lee’s!and!P.!Kord!Forooshani’s!article!(reference)!and!in!fig! 1-11!correspond!to!the!chemistry!of!PMP.!! α-TCP! coated! samples! were! impossible! to! analyze! at! fluorescence! microscopy! because! of! an! important!retention!of!CF!molecule!that!led!to!very!fluorescent!surfaces.!It!could!be!interesting!to!use! this! technique! with! another! perspective! in! order! to! test! the! covalent! binding! of! platforms! and! have! analyzable!results.!! Treated! samples! could! not! be! characterized! with! the! sessile! drop! method! for! contact! angle! measurements! because! of!its! important! absorption! of! water.! Some! articles! describe! well! that! the! captive!bubble!method!is!an!effective!technique!to!measure!the!surface!energy!and!contact!angle!of! cements!and!solids!on!which!liquids!spread!out.!In!this!case,!a!bubble!of!air!is!injected!beneath!a!solid,! on!the!surface,!which!is!submerged!in!a!liquid.!This!is!possible!to!do!this!kind!of!measurements!at!the! BBT!laboratory!and!it!could!be!interesting!to!do!it!in!order!to!have!one!more!characterization!results.!!! FTIR-ATR!technique!was!not!efficient!and!it!could!be!due!to!various!parameters,!some!passed! studies!also!had!this!problem!in!order!to!identify!the!adhesion!of!a!small!quantity!of!peptides!on!surface.! RAMAN!spectroscopy!could!be!tested,!considering!that!it!is!more!sensitive!to!bonds!involving!nitrogen! atoms!for!example.!! ! ! ! ! 52! Conclusion Peptide-based! platforms! carrying!a! DOPA/catechol! as! anchoring! unit! were! adhered! to! biomaterials!thanks!to!a!direct!coating!immobilization.!Ti!c.p.!and!PLLA!showed!consistent!results!that! proved!an!adhesion!of!the!platforms!on!surface!of!the!materials!at!neutral!pH,!but!not!at!basic!pH,!at! which!catechols!are!oxidized!to!quinones.!PMP!commercial!films!could!not!be!functionalized.!! As!a!potential!further!continuation!of!the!project,!PLLA-functionalization!should!be!repeated!to! ensure!a!better!reproducibility!and!a!better!understanding!of!the!interactions!that!are!created!between! the!surface!and!the!DOPA/catechol!anchoring!unit.!The!functionalization!method!could!be!transferred!to! 3D! structures! in! order! to! compare! the!efficiency! of!platform-adhesion! between! 2D/films!and! 3D! scaffolds.!It!is!well!known!that!the!porosity,!morphology!and!geometry!of!biomaterials!play!an!important! role!in!the!attachment!of!biomolecules!and!subsequent!cell!behavior.!It!will!be!thus!interesting!to!test! this!in!further!studies!with!3D!structures.!Although!this!was!originally!planned!at!the!beginning!of!the! project,!it!was!finally!discarded!because!of!the!lack!of!time.!In!the!BBT!laboratory,!established!protocols! already!exist!in!order!to!synthetize!3D!scaffolds!of!titanium,! alginate! and!chitosan!(as!hydrogels! with! Pluronic!or!Gelatin!addition).!! In! future! works,!some! perspectives! may! be! explored.! On! the! basis! of!the! observed!swelling! properties!of!chitosan!films,!it!is!now!unsure!whether!it!will!be!possible!to!functionalize!this!material!and! carry!a!good!characterization.!This!might!be!easier!with!alginate!films,!which!showed!better!handling! and!mechanical!properties.!Thus,!first!efforts!should!be!focused!on!the!functionalization!of!alginate.!! ! ! ! ! ! 53! Economic Impact Different! kind! of! resources! were! used! during! this! project,! their!approximative!costs! are! described!in!the!next!tables.!Main!expenses!come!from!workers!involved!and!the!equipment!used!to! characterize!the!results!obtained!during!the!project.!The!overall!cost!of!this!6-month!master!thesis!was! estimated! at! 9006,76€.! Noteworthy! some! expenses! weren’t! taken! into! account! because! came! from! other!projects,!for!instance!the!platforms!stocks!that!were!used!or!the!α-TCP!powder.!! ! ! Table!6-1:!Costs!of!workers!involved!in!the!project! ! Table!6-2:!Costs!of!equipment!used!during!the!project! ! Table!6-3:!Costs!of!reagents!used!for!cleaning!and!as!solvents!during!the!project! Quantity((h) Price((€/h) Cost((€) Junior(Engineer 720 0 0 PhD(Student 10 30 300 Technician( 5 45 225 Senior(Engineer 80 50 4000 Total(cost 4525 Work(involved Quantity((h(or(u.) Price((€/h) Cost((€) Contact(angle(device 20 40 800 Fluorescence(microscopy 15 60 900 FTIR(-(ATR 12 35 420 SEM 240 80 XPS 8 150 1200 Safety(clothes,(pipettes,(containes,(etc.( - - 200 Total(cost 3600 Equipment(used( Quantity Price,(€/L) Cost,(€) Acetone 1 16,7 16,7 Ethanol 1 25,5 25,5 Cyclohexane 0,2 37,2 7,44 Distilled,Water 5 0,1 0,5 Acetic,Acid,Glacial, 0,0005 37,2 0,02 Total,cost 50,16 Reagents,for,cleaning,and,solvents ! ! ! 54! ! Table!6-4:!Costs!induced!by!sample!preparation!during!the!project! ! Table!6-5:!Costs!of!powder!and!materials!employed!during!the!project! Environmental Impact !Environmental!impact!has!also!to!be!taken!into!account!at!the!end!of!the!project.!Every!reagent,! chemical! specie! used! all! along! the! project! was! treated! with! an! environmental! friendly! approach! and! recycled!or!selective!sorted!in!the!laboratory!in!order!to!fulfill!specific!norms.!Strict!residue!management! is! followed! by! the! laboratory! and! all! the! departments! of! the! Diagonal! Bèsos! Campus! in! Barcelona.! Chemical! components! were! stored! in! specific! labelled! containers! and! then! taken! by! ECOCAT! to! be! treated.!No!chemical!reagent!or!piece!of!equipment!was!used!abusively,!or!without!necessity!so!as!to! prevent!from!energy!waste!and!undesirable!additional!residues.!! ! ! Quantity Price, Cost,(€) Titanium,bar,of,10,mm,width 12cm 368,5,€/m 44,22 Bakelite,embedment,LaboPress 4 10,€/h 40 Automatic,Polishing,Machine,Buehler 15 30,€/h 450 305mm,diameter,P800 4 150,€/100 6 305mm,diameter,P1200 4 100,€/100 4 305mm,diameter,P2500 4 450,€/100 18 305mm,diameter,velvet,grind,paper 2 500,€/25 40 Colloidal,alumina 0,5 60,€/L 30 24-samples,well,plates 10 65€/5 130 Total,cost 762,22 Preparation,of,samples Quantity((g) Price((€/g) Cost((€) Na2HPO41,25 0,706 0,88 α-TCP(coarse 6 ? α-TCP(fine 4,5 ? Tris(base(powder( 0,25 0,148 0,04 Chitosan(medium(molecular(weight(powder 0,5 1,32 0,66 Alginic(Sodium(Salt 3 0,1196 0,36 CaCl21,25 0,299 0,37 PMP(commercial(films( 15cm2107(€/100cm216,05 PLLA(commercial(films 15cm2102/225cm26,80 Titanium(bar(of(10(mm(width 12cm 368,5(€/m 44,22 Total(cost 69,38 Powder(and(materials ! ! ! 55! References [1]! V.!Amigo-Borras,!Asignatura!Biomateriales!estructurales.!Valencia,!Spain:!Universitat!Politecnica!de!Valencia,! 2015-2016.! [2]! R.! 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