Hydrothermally induced nanostructured TiO2 coatings - with special reference to biologic events of peri-implant tissue integration

Soft tissue integration between the oral implant and the surrounding tissue is considered crucial for implant success. Various surface modifications have been used to obtain bioactive TiO2 coatings on the implant surface to improve osseointegration, bioactivity, and antibacterial properties. Among these methods, the hydrothermal (HT) coating technique has recently gained attention to produce anatase crystalline TiO2 coating for improved bioactivity and enhanced osteoconductivity. However, little is known about HT induced TiO2 coatings effect on the peri-implant soft tissue attachment. The objectives of this series of experimental studies were to develop new HT treatment based TiO2 coatings for titanium implants, which promotes wound healing and enhances soft tissue attachment. Another aim was to investigate the effect of UV light treatment on the bioactivity and antibacterial properties of HT induced TiO2 coatings. Hydrothermal induced TiO2 coatings were prepared by mixing titanium dioxide, purified water and tetramethylammonium hydroxide at 150 ± 10 ºC for 48 hours. The HT coatings were characterized using X-ray photoelectron spectroscopy and scanning electron microscope. The surface wettability was determined using contact angle measurements. Blood clotting ability, plasma protein adsorption, platelet adhesion, and activation, were evaluated. Human gingival fibroblast adhesion and proliferation were studied in a cell culture environment. The effect of UV light on the surface wettability, blood coagulation, and cellular response was investigated on coated and non-coated substrates. A novel tissue culture model using pig mandibular block, including alveolar bone and gingival tissues, was used to evaluate the tissue attachment on coated and non-coated titanium implants. Early biofilm formation on the coated and non¬coated titanium substrates was examined in vivo. The effect of UV light treatment on the biofilm formation was also studied. The HT treated titanium surfaces were entirely covered with coating crystals consisting of nearly spherical TiO2 nanoparticles. Higher carbon contents were observed on non-UV treated surfaces compared to UV treated surfaces, and carbon content was noticed to reduce with increasing UV exposure time. TiO2 coated substrates accelerated blood clotting and improved platelet responses compared with non-coated substrates. Coated substrates showed higher surface free energy and better wettability than non-coated ones. UV treatment enhanced the wettability and improved blood clotting of all examined surfaces. Although no differences in protein adsorption was observed. Fibroblast cell adhesion strength was significantly higher on coated substrates. Histological analysis of pig tissue explants showed epithelial, connective, and bone tissue attachment to both coated and non-coated implant surfaces. The peri-implant epithelium appeared to be in close contact with the coated surfaces. Immunohistochemical staining showed CK14 positivity in the basal cell layer of stratified gingival epithelium. TiO2 coating does not enhance salivary microbial adhesion and initial biofilm formation in vivo. The UV treatment provided titanium surfaces with antibacterial properties and showed a trend towards less biofilm formation than non-UV treated surfaces. It can be concluded that HT derived TiO2 coatings enhance biological events related to wound healing and soft tissue integration on the titanium alloy surface. The UV light treatment improved wettability, thrombogenicity and provided the titanium surfaces with antibacterial properties.Hydrotermaalisesti indusoidut nanorakenteiset TiO2 pinnoitteet–tutkimuksia peri-implanttikudosten kiinnittymiseen liittyvistä biologisista tapahtumista. Pehmytkudosten kiinnittyminen hammasimplantin pinnalle on tärkeää implanttihoidon onnistumiselle. Erilaisten pintakäsittelyiden avulla implantteihin on pyritty tuottamaan bioaktiivisia TiO2 pinnoitteita parantamaan osseointegraatiota, bioaktiivisuutta ja antimikrobisia ominaisuuksia. Näistä erityisesti hydrotermaalinen (HT) pinnoitusmenetelmä on herättänyt mielenkiintoa kiteisen anataasimuotoisen TiO2 pinnoitteen teossa paremman bioaktiivisuuden ja osteokonduktiivisuuden saavuttamiseksi. Menetelmän vaikutuksia implanttia ympäröivien pehmytkudosten kiinnittymiseen ei tunneta. Tämän väitöskirjan tutkimusten tavoitteina oli kehittää titaani implantteihin uusi HT menetelmällä valmistettu TiO2 pinnoite, joka edesauttaa haavan paranemista ja parantaa ienkudosten kiinnittymistä. Toisena tavoitteena oli tutkia UV-valo käsittelyn vaikutusta HT menetelmällä tuotettujen TiO2 pinnoitteiden bioaktiivisuuteen ja antimikrobisiin ominaisuuksiin. Hydrotermaaliset TiO2 pinnoitteet valmistettiin sekoittamalla titaanidioksidia, puhdistettua vettä ja tetrametyyliammonium hydroksidia 150 ± 10 ºC 48 tunnin ajan. HT pinnoitteet karakterisoitiin RTG-fotoelektronispektroskopiaa ja pyyhkäisyelektronimikroskopiaa käyttämällä. Pintojen kostumisominai¬suuksia tutkittiin kontaktikulmamittausta käyttäen. Veren reaktioita selvitettiin mittaamalla hyytymistä ja plasmaproteiinien kiinnittymistä sekä tutkimalla verihiutaleiden tarttumista ja morfologiaa. Ihmisen ikenen fibroblastisolujen vaste – kiinnittyminen ja jakautuminen -tutkittiin soluviljelyolosuhteissa. UV-valon vaikutus pinojen kostumiseen, veren hyytymiseen ja solujen käyttäytymiseen tutkittiin pinnoitetuilla ja pinnoittamattomilla näytteillä. Uutta kudosviljelymallia käytettiin tutkittaessa sian alaleuan blokkeihin asetettujen pinnoitettujen ja pinnoittamattomien titaani-implanttien kiinnittymistä luuhun ja implanttia ympäröivään ienkudokseen. Varhaisen biofilmin muodostumista pinnoitettuihin ja pinnoittamattomiin titaaninäytteisiin tutkittiin in vivo olosuhteissa. Lisäksi selvitettiin UV-valo käsittelyn vaikutusta biofilmin muodostumiseen. HT menetelmällä käsitellyt pinnat olivat kokonaan kiteisten lähes pyöreiden TiO2 nanopartikkeleiden peittämät. UV-valolla käsittelemättömillä pinnoilla todettiin suurempi hiilikontaminaatio UV-käsiteltyihin pintoihin verrattuna ja hiilipitoisuuden havaittiin pienenevän UV-käsittelyajan pidentyessä TiO2 pinnoite nopeutti veren hyytymistä ja paransi verihiutaleiden reaktioita pinnoittamattomiin näytteisiin verrattuna. Pinnoitettujen titaaninäytteiden vapaa pintaenergia sekä pintojen kostuminen olivat selvästi pinnoittamattomia paremmat. UV käsittely paransi pintojen kostumista ja nopeutti veren hyytymistä kaikilla pinnoilla. Proteiinin adsorboitumisessa ei kuitenkaan havaittu eroja. Fibroblastit kiinnittyivät selvästi voimakkaammin pinnoitetuille näytteille. Sian kudosnäytteillä tehty tutkimus osoitti epiteeli-, side¬ja luukudoksen kiinnittyvän hyvin sekä pinnoitettuihin että pinnoittamattomiin implantteihin. Peri¬implantti epiteeli oli läheisessä kontaktissa pinnoitettuihin pintoihin. Immunohistokemiallinen värjäys osoitti CK14 positiivisuutta stratifioidun ienepiteelin basaalisolukerroksessa. TiO2 pinnoite ei lisää syljen mikrobien kiinnittymistä tai varhaisen biofilmin muodostumista in vivo. UV-valo käsittely muutti pintoja antimikrobisiksi ja vähemmän biofilmin muodostusta suosivaksi käsittelemättömiin pintoihin verrattuna. Tulokset osoittivat, että HT menetelmällä valmistetut TiO2 pinnoitteet tehostavat haavan paranemisen biologisia tapahtumia ja pehmytkudosten kiinnittymistä titaanin pinnalle. UV-valo käsittely lisää pintojen kostumista, trombogeenisyyttä ja muuttaa pintojen ominaisuuksia antimikrobisiksi

Organotypic in vitro block culture model to investigate tissue-implant interface. An experimental study on pig mandible

In vitro studies of implant-tissue attachment are primarily based on two-dimensional cell culture models, which fail to replicate the three-dimensional native human oral mucosal tissue completely. Thus, the present study aimed to describe a novel tissue culture model using pig mandibular block including alveolar bone and gingival soft tissues to evaluate the tissue attachment to titanium implant provided with hydrothermally induced TiO2 coating. Tissue attachment on TiO2 coated and non-coated implants were compared. Ti-6Al-4V alloy posts were used to function as implants that were inserted in five pig mandibles. Implants were delivered with two different surface treatments, non-coated (NC) titanium and hydrothermal induced TiO2 coated surfaces (HT). The tissue-implant specimens were cultured at an air/liquid interface for 7 and 14 days. The tissue-implant interface was analyzed by histological and immunohistochemical stainings. The microscopic evaluation suggests that pig tissue explants established soft and hard tissue attachment to both implant surfaces. The epithelial cells appeared to attach to the coated implant. The epithelium adjacent to the implant abutment starts to change its phenotype during the early days of the healing process. New bone formation was seen within small pieces of bone in close contact with the coated implant. In conclusion, this in vitro model maintains the viability of pig tissue and allows histologically and immunohistochemically evaluate the tissue-implant interface. HT-induced TiO2 coating seems to have a favorable tissue response. Moreover, this organotypic tissue culture model is applicable for further studies with quantitative parameters to evaluate adhesion molecules present at the implant-tissue interface.[GRAPHICS]

Early Biofilm Formation on UV Light Activated Nanoporous TiO2 Surfaces In Vivo

Purpose. To explore early S. mutans biofilm formation on hydrothermally induced nanoporous TiO2 surfaces in vivo and to examine the effect of UV light activation on the biofilm development. Materials and Methods. Ti-6Al-4V titanium alloy discs (n = 40) were divided into four groups with different surface treatments: noncoated titanium alloy (NC); UV treated noncoated titanium alloy (UVNC); hydrothermally induced TiO2 coating (HT); and UV treated titanium alloy with hydrothermally induced TiO2 coating (UVHT). In vivo plaque formation was studied in 10 healthy, nonsmoking adult volunteers. Titanium discs were randomly distributed among the maxillary first and second molars. UV treatment was administered for 60 min immediately before attaching the discs in subjects' molars. Plaque samples were collected 24h after the attachment of the specimens. Mutans streptococci (MS), non-mutans streptococci, and total facultative bacteria were cultured, and colonies were counted. Results. The plaque samples of NC (NC + UVNC) surfaces showed over 2 times more often S. mutans when compared to TiO2 surfaces (HT + UVHT), with the number of colonized surfaces equal to 7 and 3, respectively. Conclusion. This in vivo study suggested that HT TiO2 surfaces, which we earlier showed to improve blood coagulation and encourage human gingival fibroblast attachment in vitro, do not enhance salivary microbial (mostly mutans streptococci) adhesion and initial biofilm formation when compared with noncoated titanium alloy. UV light treatment provided Ti-6Al-4V surfaces with antibacterial properties and showed a trend towards less biofilm formation when compared with non-UV treated titanium surfaces

Early Biofilm Formation on UV Light Activated Nanoporous TiO2 Surfaces In Vivo

Purpose. To explore early S. mutans biofilm formation on hydrothermally induced nanoporous TiO2 surfaces in vivo and to examine the effect of UV light activation on the biofilm development. Materials and Methods. Ti-6Al-4V titanium alloy discs (n = 40) were divided into four groups with different surface treatments: noncoated titanium alloy (NC); UV treated noncoated titanium alloy (UVNC); hydrothermally induced TiO2 coating (HT); and UV treated titanium alloy with hydrothermally induced TiO2 coating (UVHT). In vivo plaque formation was studied in 10 healthy, nonsmoking adult volunteers. Titanium discs were randomly distributed among the maxillary first and second molars. UV treatment was administered for 60 min immediately before attaching the discs in subjects’ molars. Plaque samples were collected 24h after the attachment of the specimens. Mutans streptococci (MS), non-mutans streptococci, and total facultative bacteria were cultured, and colonies were counted. Results. The plaque samples of NC (NC + UVNC) surfaces showed over 2 times more often S. mutans when compared to TiO2 surfaces (HT + UVHT), with the number of colonized surfaces equal to 7 and 3, respectively. Conclusion. This in vivo study suggested that HT TiO2 surfaces, which we earlier showed to improve blood coagulation and encourage human gingival fibroblast attachment in vitro, do not enhance salivary microbial (mostly mutans streptococci) adhesion and initial biofilm formation when compared with noncoated titanium alloy. UV light treatment provided Ti-6Al-4V surfaces with antibacterial properties and showed a trend towards less biofilm formation when compared with non-UV treated titanium surfaces

Effect of Atomic-Layer-Deposited Hydroxyapatite Coating on Surface Thrombogenicity of Titanium

This study aimed to evaluate the surface characteristics of a nanocrystalline hydroxyapatite coating made through atomic layer deposition (ALD-HA) on titanium surfaces and to investigate its effect on blood coagulation and platelet adhesion. Grade 2 square titanium discs (0.7 cm, 1 mm thick) were used (n = 108). Half of the substrates (n = 54) were coated with ALD-HA, and the other half were used as the non-coated control. Surface free energy (SFE), contact angle (CA), surface roughness (Ra), and chemical composition were evaluated. Blood thrombogenic properties were assessed on ALD-HA and non-coated surfaces using the kinetic clotting time method. The platelets’ adhesion and morphology were also evaluated. The ALD-HA-coated surfaces demonstrated significantly higher polar SFE (p < 0.001) and lower CA (p < 0.001) values compared to the non-coated surfaces. In addition, the surface roughness was significantly lower for the ALD-HA (p < 0.001) than for the non-coated surfaces. Platelets adhered to both surfaces; however, there was variability in platelet morphologies in different areas with higher platelet density on the ALD-HA surfaces. There was no significant difference in the overall absorbance values of the hemolyzed hemoglobin for both substrates, and the total clotting time was achieved at 60 min. It can be concluded that the ALD-HA coating of titanium can enhance surface wettability, increase surface free energy, and support blood coagulation and platelet adhesion