Examination of nickel ion release and changes in surface structure of nickel-titanium orthodontic archwires into the CaCO2 cell culture under mechanical loading

Nikl-titanijumski lukovi su postali nezamenljiva komponenta ortodontske terapije fiksnim aparatima, prvenstveno zbog svojih jedinstvenih mehaničkih karakteristika. Iako se NiTi ortodontski lukovi smatraju biokompatibilnim, oni poseduju i jednu negativnu osobinu, a to je otpuštanje nikla, koji je u odgovarajućim koncentracijama dokazani alergen, kancerogen i mutagen. Većina dosadašnjih studija proučavala je otpuštanje nikla u medijum bez simulacije opterećenja koje se javlja u usnoj duplji. Osnovni cilj ove doktorske disertacije je simulacija opterećenja koje se javlja u usnoj duplji, sledstveno identifikovanje otpuštenih jona umedijum, kao i promene koje se dešavaju na površini ovih lukova i promene u njihovojmikrostrukturi. NiTi lukovi su bili postavljeni u komoru koja je sadržala kulturu ćelija CACO-2 i podvrgnuti opterećenju, sličnom onom i u usnoj duplji, pomoću aparata SMAS (simulatora multi-aksijalnog stresa) tokom 24 časa. Nakon eksperimenta NiTi lukovi su analizirani: skening elektronskom mikroskopijom (SEM), Auger elektronskom spektroskopijom (AES) i spektroskopijom fotoelektronima x-zraka (XPS), hemijskom semi-kvantitativnom analizom koja je obuhvatala: TEM (transmisionu elektronsku mikroskopiju), EDX (energetsko-disperzijsku rentgentsku spektroskopiju) i XRD (rendgensku difrakcionu analizu strukture materijala) i dodatno je rađeno merenje električne otpornosti. Dobijeni rezultati i prikazi su poređeni sa kontrolnim lukovima od istog proizvođača, istih osobina i mikrostrukture. Kultura ćelija CACO-2 bila je podvrgnuta optičkoj emisionoj spektrometriji indukcijom spojenom plazmom (ICP-OES) da bi se utvrdila količina oslobođenih jona Ni i Ti sa površine lukova u medijumu. Dobijeni rezultati uspeli su da prikazu značajne mikrostrukturne i promene sastava legure u površinskim nanoslojevima i dublje u matriksu ortodontskih lukova. Ove promene dobijaju na još većem značaju kada se tome dodaju rezultati ICP-OES analize koja je dokazala statistički značajnu koncentraciju otpuštenih jona nikla u iznosu od 1310 ng/L u medijum. Dokazane promene u površinskoj mikrostrukturi ortodontskih lukova, kao i dokazana statistički značajna količina otpustenih jona nikla tokom 24 časa, samo usled mehaničke stimulacije lukova, a u odnosu na lukove kontrolne grupe (koji su analizirani as received, bez opterećenja) predstavljaju veoma bitnu informaciju za ortodonte, upućuju na opreznost i na neophodnost daljih istraživanja. Ključne reči: nikl-titanijum, CACO-2 kultura ćelija, ortodontski lukovi, površinska struktura, simulacija mehaničkog opterećenja, karakterizacija, mikrostruktura, skening elektronska mikroskopija (SEM), Auger elektronska spektroskopija (AES), spektroskopija fotoelektronima x-zraka (XPS), transmisiona elektronska mikroskopija (TEM), energetsko-disperzijskarentgentska spektroskopija (EDX), rendgenska difrakciona analiza strukture materijala (XRD),optička emisiona spektrometrija indukcijom spojenom plazmom (ICP-OES), električna otpornost.Unique mechanical properties made nickel-titanium archwires an irreplaceble part of orthodontic treatment with fixed appliances. Although they are cosidered as biocompatibile, they have one very important negative feature, release of nickel ions, which is proved to be in some concentrations allergenic, cancerogenic and mutagenic. Various studies were examining release of nickel ions into the medium, without loading the wires mechanically, simillar to the loading in the mouth of the patient. The main objective of this PhD is to examine the release of nickel ions into the medium during mechanical loading simillar to the loading in the patient´s mouth and also to identify any changes in the surface microstructure of these archwires. During 24 hours NiTi archwires were placed inside the chamber and immersed in CACO-2 cell culture, and then loaded immediately using the Simulator of Multi-Axial Stress (SMAS). After that the archwires were analysed using: Scanning Electron Microscopy (SEM), Auger Electron Spectroscopy (AES) plus X-ray Photoelectron Spectroscopy (XPS), semi-quantitative analysis that included: Transmission Electron Microscopy (TEM), Energy Dispersive X-ray spectroscopy (EDX) and X-ray Diffraction and Elemental analysis (XRD) and, in the end, measuring electrical resistivity. All results were compared with the control archwires from the same manufacturer, with the same composition and microstructure. CACO-2 cell cultures were examined using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) to determine the amounts of Ni and Ti released from the surface of the archwires. The obtained results showed significant microstructural and compositional changes within the first few nanoleyers of the archwire surface and in the archwire matrix. These results are even more pertinent in combination with the results obtained from ICP-OES, that proved a statistically significant amount of nickel ions released into the medium. Proven surface microstructural changes and the statistically significant amount 1310 ng/L of nickel ions released during 24 hours and mechanical stimulation of the archwires, compared to control group, in as received that were not subjected to mechanical stimulation, are important information for clinical orthodontists, evoke caution and the need for further research

Characterisation of NiTi Orthodontic Archwires Surface after the Simulation of Mechanical Loading in CACO2-2 Cell Culture

Nickel-titanium (NiTi) orthodontic archwires are crucial in the initial stages of orthodontic therapy when the movement of teeth and deflection of the archwire are the largest. Their great mechanical properties come with their main disadvantage-the leakage of nickel. Various in vitro studies measured nickel leakage from archwires that were only immersed in the medium with little or minimal simulation of all stress and deflection forces that affect them. This study aims to overcome that by simulating deflection forces that those archwires are exposed to inside the mouth of a patient. NiTi orthodontic archwires were immersed in CACO2-2 cell culture medium and then immediately loaded while using a simulator of multiaxial stress for 24 h. After the experiment, the surface of the NiTi orthodontic archwires were analysed while using scanning electron microscopy (SEM) and auger electron spectroscopy (AES). The observations showed significant microstructural and compositional changes within the first 51 nm thickness of the archwire surface. Furthermore, the released nickel and titanium concentrations in the CACO2-2 cell culture medium were measured while using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). It was found out that the level of released nickel ions was 1.310 mu g/L, which can be assigned as statistically significant results. These data represent the first mention of the already detectable release of Ni ions after 24 h during the simulation of mechanical loading in the CACO2-2 cell culture medium, which is important for clinical orthodontic praxis

Examination of nickel ion release and changes in surface structure of nickel-titanium orthodontic archwires into the CaCO2 cell culture under mechanical loading

Nikl-titanijumski lukovi su postali nezamenljiva komponenta ortodontske terapije fiksnim aparatima, prvenstveno zbog svojih jedinstvenih mehaničkih karakteristika. Iako se NiTi ortodontski lukovi smatraju biokompatibilnim, oni poseduju i jednu negativnu osobinu, a to je otpuštanje nikla, koji je u odgovarajućim koncentracijama dokazani alergen, kancerogen i mutagen. Većina dosadašnjih studija proučavala je otpuštanje nikla u medijum bez simulacije opterećenja koje se javlja u usnoj duplji. Osnovni cilj ove doktorske disertacije je simulacija opterećenja koje se javlja u usnoj duplji, sledstveno identifikovanje otpuštenih jona umedijum, kao i promene koje se dešavaju na površini ovih lukova i promene u njihovojmikrostrukturi. NiTi lukovi su bili postavljeni u komoru koja je sadržala kulturu ćelija CACO-2 i podvrgnuti opterećenju, sličnom onom i u usnoj duplji, pomoću aparata SMAS (simulatora multi-aksijalnog stresa) tokom 24 časa. Nakon eksperimenta NiTi lukovi su analizirani: skening elektronskom mikroskopijom (SEM), Auger elektronskom spektroskopijom (AES) i spektroskopijom fotoelektronima x-zraka (XPS), hemijskom semi-kvantitativnom analizom koja je obuhvatala: TEM (transmisionu elektronsku mikroskopiju), EDX (energetsko-disperzijsku rentgentsku spektroskopiju) i XRD (rendgensku difrakcionu analizu strukture materijala) i dodatno je rađeno merenje električne otpornosti. Dobijeni rezultati i prikazi su poređeni sa kontrolnim lukovima od istog proizvođača, istih osobina i mikrostrukture. Kultura ćelija CACO-2 bila je podvrgnuta optičkoj emisionoj spektrometriji indukcijom spojenom plazmom (ICP-OES) da bi se utvrdila količina oslobođenih jona Ni i Ti sa površine lukova u medijumu. Dobijeni rezultati uspeli su da prikazu značajne mikrostrukturne i promene sastava legure u površinskim nanoslojevima i dublje u matriksu ortodontskih lukova. Ove promene dobijaju na još većem značaju kada se tome dodaju rezultati ICP-OES analize koja je dokazala statistički značajnu koncentraciju otpuštenih jona nikla u iznosu od 1310 ng/L u medijum. Dokazane promene u površinskoj mikrostrukturi ortodontskih lukova, kao i dokazana statistički značajna količina otpustenih jona nikla tokom 24 časa, samo usled mehaničke stimulacije lukova, a u odnosu na lukove kontrolne grupe (koji su analizirani as received, bez opterećenja) predstavljaju veoma bitnu informaciju za ortodonte, upućuju na opreznost i na neophodnost daljih istraživanja. Ključne reči: nikl-titanijum, CACO-2 kultura ćelija, ortodontski lukovi, površinska struktura, simulacija mehaničkog opterećenja, karakterizacija, mikrostruktura, skening elektronska mikroskopija (SEM), Auger elektronska spektroskopija (AES), spektroskopija fotoelektronima x-zraka (XPS), transmisiona elektronska mikroskopija (TEM), energetsko-disperzijskarentgentska spektroskopija (EDX), rendgenska difrakciona analiza strukture materijala (XRD),optička emisiona spektrometrija indukcijom spojenom plazmom (ICP-OES), električna otpornost.Unique mechanical properties made nickel-titanium archwires an irreplaceble part of orthodontic treatment with fixed appliances. Although they are cosidered as biocompatibile, they have one very important negative feature, release of nickel ions, which is proved to be in some concentrations allergenic, cancerogenic and mutagenic. Various studies were examining release of nickel ions into the medium, without loading the wires mechanically, simillar to the loading in the mouth of the patient. The main objective of this PhD is to examine the release of nickel ions into the medium during mechanical loading simillar to the loading in the patient´s mouth and also to identify any changes in the surface microstructure of these archwires. During 24 hours NiTi archwires were placed inside the chamber and immersed in CACO-2 cell culture, and then loaded immediately using the Simulator of Multi-Axial Stress (SMAS). After that the archwires were analysed using: Scanning Electron Microscopy (SEM), Auger Electron Spectroscopy (AES) plus X-ray Photoelectron Spectroscopy (XPS), semi-quantitative analysis that included: Transmission Electron Microscopy (TEM), Energy Dispersive X-ray spectroscopy (EDX) and X-ray Diffraction and Elemental analysis (XRD) and, in the end, measuring electrical resistivity. All results were compared with the control archwires from the same manufacturer, with the same composition and microstructure. CACO-2 cell cultures were examined using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) to determine the amounts of Ni and Ti released from the surface of the archwires. The obtained results showed significant microstructural and compositional changes within the first few nanoleyers of the archwire surface and in the archwire matrix. These results are even more pertinent in combination with the results obtained from ICP-OES, that proved a statistically significant amount of nickel ions released into the medium. Proven surface microstructural changes and the statistically significant amount 1310 ng/L of nickel ions released during 24 hours and mechanical stimulation of the archwires, compared to control group, in as received that were not subjected to mechanical stimulation, are important information for clinical orthodontists, evoke caution and the need for further research