Effect of surface characteristics on cellular adherence and activity

• Ersättning av förlorade tänder med tandimplantat är numera en vanlig behandlingsmetod med generellt goda resultat. Antalet implantatsystem på den internationella marknaden har ökat kraftigt under senare år. Stora forskningsinsatser har lagts på att utveckla nya modifierade titanmaterial med ytegenskaper som påskyndar integreringen av implantat i benvävnad. Exempel på sådana modifieringar är hög ytråhet och kemiska ytförändringar som förstärker bioaktiva karaktärsdrag hos titan. Vissa modifieringar har visat sig kunna stimulera benceller att bilda benvävnad och därmed skapa bättre förutsättningar för inläkning av implantat. Klinisk forskning pekar på att även mjukvävnad runt tandimplantat, liksom slemhinnan runt naturliga tänder, har avgörande betydelse för skydd mot mikrobiella angrepp. En funktionell mjukvävnadsbarriär anses vara nödvändig för att tandimplantat skall fungera livet ut. Emellertid är kunskapen enbart sporadisk om hur celltyper såsom mjukvävnads- celler och orala bakterier, reagerar när de kommer i kontakt med modifierade titanytor i munhålan.På tandimplantat i den orala miljön, liksom på naturliga tänder, finns salivproteiner (pellikel) och sådana munhålebakterier som har förmåga att kolonisera fasta ytor som titan. Bakterier som fäster till implantat bildar med tiden komplexa bakteriesamhällen som är inbäddade i en matrix, en så kallad biofilm. Under vissa förhållanden kan dessa mikrobiella biofilmer ge upphov till kroniska infektioner. Flera kliniska uppföljningsstudier pekar på att kroniska infektioner i anslutning till tandimplantat är vanligare än man tidigare trott. Sådana infektioner kan vara svåra att behandla och leda till så omfattande benförlust att tandimplantat förloras. Syftet med denna avhandling var att förbättra förståelsen för hur orala streptokocker, fibroblaster och keratinocyter påverkas då de kommer i kontakt med moderna tandimplantat som har modifierade ytstrukturer.Resultaten i denna avhandling visade att titanytor med skrovliga strukturer på mikrometernivå, framtagna för bättre benläkning, främjade även adhesion av orala streptokocker vilket kan tolkas som att biofilmer lättare ackumuleras på sådana ytor. Vidare framkom det att streptokockers vidhäftningsförmåga och metabola aktivitet ökade avsevärt när en naturlig salivfilm fanns på plats på titanytan vilket ytterligare skulle påskynda bilningen av biofilmer på tandimplantat. Karaktärisering av proteinsammansättningen i salivfilmen på titan visade att sekretoriskt IgA, amylas, cystatin och prolactin-inducible protein var de dominerande proteinerna. S. oralis är en bakterie som ofta förekommer i samband med kroniska infektioner runt tandimplantat. Denna bakterie visade sig ha ett adhesin som interagerade speciellt bra med salivproteiner på titan. Detta var första gången som detta adhesin identifierades.Vad gäller adhesion av mjukvävnadsceller som fibroblaster och keratinocyter visade det sig att dessa celler kunde fästa väl till en titanyta som modifierats med hjälp av anodisk oxidering. Vid en karaktärisering av ytan framkom det att den var rik på anataskristaller och hade också nanoporösa strukturer. Orala streptokocker visade sig binda sämre till den anatasrika ytan än till kommersiellt rent titan. Sammantaget förefaller denna ytmodifikation vara en lämplig kandidat för nya implantat då den minskar inbindningen av streptokocker samtidigt som den tillåter fibroblaster och keratinocyter att växa på ytan.Sammanfattningsvis visar resultaten i denna avhandling att ytmodi- fierat titan påverkar såväl adhesion av mjukvävnadsceller som bakterie- celler och deras aktivitet. Studierna visar också att salivpellikeln på implantat spelar en viktig roll för bakteriernas kolonisation och metabolism. Vi föreslår därför att beroende på tillämpningsområde i praktiken, experimentell utvärdering av nya implantatytor ska i så stor sträckning som möjligt innefatta granskning av effekten av ytornas egenskaper på olika vävnadsceller såväl som bakterier i närvaro av vävnadsvätskor.

• After insertion of the dental implant into the jaw, the neck of the implant protruding through the mucosa (implant abutment) will be exposed to the complex environment of the mouth. This results in the formation of a conditioning protein coat (pellicle) derived from saliva and/or gingival crevicular fluid. Microorganisms in saliva are transported to the surfaces where they initiate biofilm (plaque) formation. Over time, early colonizers promote co-aggregation of later colonizers, leading to development of complex plaque, which can include hundreds of different bacterial species. Continuous undisturbed growth of plaque has been reported to trigger inflammatory responses in the periodontal tissues, which can compromise the integration of the implant abutment with the surrounding oral mucosa and eventually progress to breakdown of supporting bone tissue (peri-implant disease).Key elements in the long-term success of dental implants are the formation of a stable connection between the sub-crestal anchoring part of the implant (fixture) and the host bone tissue (osseointegration) and integration of the abutment with the surrounding soft tissues. Consequently, much research has been focused on development of surfaces that may optimize osseointegration as well as support the formation of a healthy cuff of keratinized mucosa around the implant abutment, providing a barrier that prevents the passage of microorganisms into the underlying connective tissues. Reports from a large number of studies have shed light upon the positive effects of surface modifications on osseointegration. However, the effect of such modifications on development of oral biofilms and soft-tissue cells is not understood.The overall aim of this thesis was to obtain a better understanding of the adaptive processes occurring at the implant-host tissue interface. Thus the effects of surface characteristics on formation of pellicles as well as adherence and activity of early colonizing bacteria were examined. Furthermore, we investigated adherence of epithe- lial cells and fibroblasts to nano-porous titanium surfaces in order to identify surface characteristics that may facilitate improved soft tissue attachment.In paper I, the effects of surface roughness as well as the effect of a saliva- or serum- derived coating on adherence of different strains of Streptococcus oralis (an early colonizer of mouth that is also recovered from implant surfaces in vivo) to titanium was examined. Titanium plates with smooth (average height deviation (Sa) < 0.5 μm) or moderately rough (Sa 1-2 μm) surface topography were used together with a flow-cell model and confocal laser scanning microscopy (CLSM) with Live/Dead BacLight staining kit. Microbial adherence to moderately rough surfaces was greater overall than that to smooth surfaces, suggesting that implants with moderately rough surfaces, developed to improve osseointegration, have a greater propensity for retention of adhered bacteria. Furthermore, a saliva pellicle promoted binding of S. oralis although different strains varied in their binding capacity. Adherence could be attributed to specific binding, involving bacterial adhesins and salivary molecules in the pellicle. The presence of potential adhesins was investigated by comparing cell-wall protein preparations from the different strains using two-dimensional gel electrophoresis (2DE) and mass spectros- copy (MS/MS). This showed that S. oralis strains that bound well to saliva-coated surfaces expressed an adhesin (SOR_0366) that was not found in the non-adherent strain. To our knowledge this is the first time that this putative adhesin in S. oralis has been identified at the protein level.In paper II, the effects of surface contact on bacterial activity were studied by comparing metabolic activity of planktonic and surface-associated bacteria. Biofilms were formed on smooth titanium surfaces, either uncoated or coated with saliva, for 2 hours using the same flow-cell model and one of the S. oralis strains in study I. Metabolic activity was assessed using CLSM with the BacLight CTC vitality kit. The dominant proteins in the salivary pellicles on the titanium surfaces in vitro were identified using 2DE and MS/MS. Metabolic activity in S. oralis cells was shown to be up-regulated upon surface contact and this effect was enhanced in the presence of a salivary pellicle. Pellicle characterisation indicated selective adsorption of salivary proteins to titanium, with the enrichment of prolactin-inducible protein, secretory IgA, alpha-amylase and cystatins on the surfaces.Paper III compared the early stages of biofilm formation on modified titanium surfaces to that on commercially pure titanium (CpTi) control surfaces (C) in the presence of a salivary pellicle. Modified surfaces were prepared by anodic oxidation on CpTi (N1) or titanium alloy (N2). A 2 hour adhesion assay of mono- cultures and mixed-cultures of four early colonizing oral streptococci (Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis and Streptococcus sanguinis) was used. All surfaces showed similar mean surface roughness values (Sa 0.2 μm), while increased anatase content and oxide layer thickness were recorded on the two modified surfaces compared to control. Fluorescence microscopy and Live/Dead BacLight staining were used for visualization of bacteria. Results demonstrated high levels of viability for bacteria on all surfaces, with reduced surface coverage on modified surfaces compared to control. It was concluded that the anatase-rich surfaces could contribute to reduced biofilm formation, possibly through altered conformation of the absorbed salivary pellicle proteins.In paper IV, adherence of soft-tissue cells to the same surfaces as in paper III was investigated. The surfaces were characterised using scanning electron microscopy (SEM) and adherence of oral keratinocytes and gingival fibroblasts was then investigated using a 24 hour adhesion assay and fluorescence microscopy with Live/ Dead BacLight staining. Cell adhesion strength was assessed using a standardized washing technique. Since dental implant abutments are placed in a bacteria-rich environment, the effect of consortium of commensal oral streptococci on keratinocyte function was evaluated. SEM revealed both the N1 and N2 surface to have a nano-porous structure, with pores in the range of 50 nm superimposed on the turned structure. The pores on N2 were more sparsely distributed, with larger pore-free areas, than on the N1 surface. Only minor differences were seen between adhesion levels for keratinocytes and fibroblasts on the nano-porous surfaces compared to the control. While keratinocytes exhibited greater adhesion strength than fibroblasts to all surfaces, no differences in adhesion strength were observed for either cell types between the modified and the control surfaces. The presence of bacteria reduced adherence of keratinocytes to all surfaces as well as causing damage to the cells.In summary, the results presented in this thesis show that surface modification of titanium affects adhesion of soft-tissue cells as well as adherence and activity of oral bacteria. In particular, anatase- rich, nano-porous surfaces appear to have promising properties for use in dental implant abutments since they reduce binding of oral streptococci while at the same time allowing fibroblasts and keratinocytes to attach to the surface. In addition, the studies show that the salivary pellicle formed on implant abutment surfaces plays an important role in bacterial colonization and metabolism. This work thus demonstrates that surfaces designed to improve implant success rates should be tested in models that include host-tissue cells, bacteria and pellicle proteins.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Klinisk medicin -- Odontologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Clinical Medicine -- Dentistry (hsv//eng)

Nyckelord

Bacteria

microbial biofilm

dental implant

streptococci

oral bacteria

salivary pellicle

early colonizers

titanium oxide

microbial activity

oral keratinocytes

gingival fibroblasts

cell attachment

surface modification

anodic oxidation

anatase

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vet (ämneskategori)

dok (ämneskategori)