27 research outputs found
Click Reactions: An Efficient Tool Towards Biofunctional Materials
427 p.In 1987, the European Society for Biomaterials coined the term “biomaterial”, defining it as a non-biological material used in medical devices with the specific purpose of interacting with biological systems. Over time, this definition of biomaterial has evolved, adapting to various contexts. Currently, biomaterials are described as materials that actively interact with biological system to assess, treat, promote healing or even replace any tissue or body function. The main characteristic of a biomaterial is its biocompatibility, which refers to the ability of the material to elicit an appropriate response from the host in a specific situation . Again, the interpretation of biocompatibility varies based on the required performance or function of the material. Chen et al. defined biocompatibility as a factor that can be assessed through parameters such as cell viability, tissue response, tumor formation, genetic integrity, immune reaction, and blood clotting potential . Acknowledging this wide spectrum of considerations, the Food and Drug Administration (FDA) agency has stipulated that to consider a material biocompatible [8], it must not cause harm to the patient
Effects of Dual Coating Proteins in Intraosseous Transcutaneous Amputation Prosthesis (ITAP)
Intraosseous transcutaneous amputation prostheses (ITAP) provide an alternative method of attaching artificial limbs for amputees. Conventional stump-socket devices are associated with soft tissue complications including pressure sores, neuroma formation and tissue necrosis. ITAP overcomes these problems by attaching the articial limb transcutaneously to the skeleton. In order for ITAP to be successful, it requires an infection-resistant transcutaneous barrier at the skin-implant interface. Fibronectin (Fn) and Laminin 332 (Ln), are glycoproteins found abundantly in the extracellular matrix. Dual coating proteins 125 I-Fn + Ln and 125 I-Ln +Fn were covalently bonded to Ti6Al4V through silanization. The hypothesis tested was: silanized dual coating protein coatings with fibronectin and laminin, enhances both keratinocyte and fibroblast spreading and increases vinculin focal adhesion plaques on Ti6Al4V in vitro. Both remained stable when immersed in foetal calf serum compared with adsorbed dual coating proteins at all time points up to 72 hours (p<0.05). There was non-competitive binding of laminin on Ti6Al4V in the presence of fibronectin. Keratinocytes and fibroblasts were grown on Ti6Al4V surfaces with single coating Fn, Ln, and dual coating FnLn on adsorbed, silanized with passivation and silanized without passivation discs. Vinculin focal adhesion markers and cell size were quantified. Silanized dual coating proteins without passivation (SiFnLn-) produced the largest number of vinculin markers and biggest cell size at all time points upt to 24 hours (p<0.05). Hydroxyapatite (HA) is a naturally occurring osteoinductive mineral in the body. 125 I-Fn coated on HA discs was assessed for optimal time for loading, concentration and durability. Fibroblasts were grown on polished, HA and Fn coated HA discs. Vinculin markers and cell size were quantified. Fn coated HA discs increased fibroblast attachment compared to uncoated controls of Ti6Al4V discs and HA discs (p<0.05). My thesis demonstrated silanized without passivation dual coating proteins FnLn produced more viculin markers per cell unit and per cell area when compared to uncoated controls and single coating proteins on adsorbed and silanized, passivated discs. Further research is required to establish whether dual coating proteins will produce the same effect in vivo. This can be achieved by silanizing ITAP with dual coating FnLn and implanting them in animals. Histopathological analysis at the skin-implant interface would provide valuable information whether this biochemical and physical modification improve soft tissue integration to percutaneous implants
Bone–Biomaterial Interface : The effects of surface modified NiTi shape memory alloy on bone cells and tissue
AbstractWhenever a foreign material is implanted into a human body an implant–tissue interface area forms between them. In this microenvironment, interactions take place between the implant and the surrounding tissue. The implantation of a biomaterial into tissue results in injury and initiation of the inflammatory response. This host response to biomaterials is an unavoidable series of events that occur when tissue homeostasis is disturbed by the implantation process. In bone tissue, biocompatible implants must initially be capable of strong bone implant contact and subsequently, allow the normal bone remodeling cycle around the implant.NiTi is a metal alloy composed of approximately a 50:50 ratio of nickel and titanium. It possesses shape memory and superelasticity properties, which make it an interesting biomaterial. NiTi has two phases: austenite and martensite. A decrease in temperature or applied stress induce the austenite-to-martensite transformation. Heating or removing the stress restores the parent austenite phase. The alloy in its martensite structure can be reshaped and strained several times more than a conventional metal alloy without irreversible deformation of the material. The alloy returns to its original shape as it changes from martensite-to-austenite. This transformation is seen as the macroscopic shape memory effect.This study further investigated the biocompatibility of NiTi, especially the bone cell response to both austenite and martensite. Different surface treatments were investigated in order to improve and possibly even control NiTi’s bioactivity as a bone implant material.Osteoclasts grew and attached well on the austenite NiTi phase, but the results indicated that the biocompatibility of martensite NiTi was compromised. Oxidation of the NiTi surface improved osteoblast attachment and viability. This was due to the formation of a TiO2 surface layer of moderate thickness. Coating the NiTi surface with the extracellular matrix protein fibronectin was shown to enhance osteoblast proliferation and increase the number of cells in the G1 cell cycle stage. Austenite was more prone to show these effects than martensite. A sol-gel derived titania-silica surface treatment was observed to increase the bone implant contact of functional NiTi intramedullary nails. The surface treatment was most effective with the constant bending load provided by the NiTi nail.Academic dissertation to be presented, with the assent of the Faculty of Medicine of the University of Oulu, for public defence in Auditorium A101 of the Department of Anatomy and Cell Biology, on June 27th, 2008, at 12 noonAbstract
Whenever a foreign material is implanted into a human body an implant–tissue interface area forms between them. In this microenvironment, interactions take place between the implant and the surrounding tissue. The implantation of a biomaterial into tissue results in injury and initiation of the inflammatory response. This host response to biomaterials is an unavoidable series of events that occur when tissue homeostasis is disturbed by the implantation process. In bone tissue, biocompatible implants must initially be capable of strong bone implant contact and subsequently, allow the normal bone remodeling cycle around the implant.
NiTi is a metal alloy composed of approximately a 50:50 ratio of nickel and titanium. It possesses shape memory and superelasticity properties, which make it an interesting biomaterial. NiTi has two phases: austenite and martensite. A decrease in temperature or applied stress induce the austenite-to-martensite transformation. Heating or removing the stress restores the parent austenite phase. The alloy in its martensite structure can be reshaped and strained several times more than a conventional metal alloy without irreversible deformation of the material. The alloy returns to its original shape as it changes from martensite-to-austenite. This transformation is seen as the macroscopic shape memory effect.
This study further investigated the biocompatibility of NiTi, especially the bone cell response to both austenite and martensite. Different surface treatments were investigated in order to improve and possibly even control NiTi’s bioactivity as a bone implant material.
Osteoclasts grew and attached well on the austenite NiTi phase, but the results indicated that the biocompatibility of martensite NiTi was compromised. Oxidation of the NiTi surface improved osteoblast attachment and viability. This was due to the formation of a TiO2 surface layer of moderate thickness. Coating the NiTi surface with the extracellular matrix protein fibronectin was shown to enhance osteoblast proliferation and increase the number of cells in the G1 cell cycle stage. Austenite was more prone to show these effects than martensite. A sol-gel derived titania-silica surface treatment was observed to increase the bone implant contact of functional NiTi intramedullary nails. The surface treatment was most effective with the constant bending load provided by the NiTi nail
Elektrochemisch gestützte Immobilisierung bioaktiver Moleküle an Titanoberflächen
Ein Schlüsselfeld der gegenwärtigen Biomaterialforschung ist die Modifizierung von Oberflächen mit Bestandteilen der extrazellulären Matrix (EZM) oder Molekülen, die bestimmte Funktionen nachahmen.
Trotz einer Reihe positiver Ergebnisse in vitro und in vivo ist es mit den gegenwärtig zur Verfügung stehenden Immobilisierungsmethoden nicht möglich, unterschiedliche Komponenten in einem Prozessschritt zu immobilisieren, definierte Freisetzungscharakteristika für gleiche und/oder unterschiedliche Moleküle zu realisieren und die Beschichtung der Oberflächen nach Sterilisation der Implantate vorzunehmen, um empfindliche bioaktive Substanzen, wie Proteine, vor Schädigung zu bewahren.
An diesem Punkt setzt die vorliegende Arbeit mit dem Ziel an, ein nukleinsäurebasiertes Immobilisierungssystem für Titanwerkstoffe zu entwickeln. Es wird zunächst am Beispiel eines Peptids mit der Aminosäuresequenz Arginin–Glyzin–Asparaginsäure (RGD) nachgewiesen, dass an der Grenzfläche Passivschicht/Elektrolyt von Titanwerkstoffen vorliegende Moleküle in durch anodische Polarisation verdickte Oxidschichten partiell eingebaut werden können und dabei ihre Funktionalität erhalten bleibt.
Diese Immobilisierungsmethode wird zum Immobilisierungssystem erweitert, indem Nukleinsäureeinzelstränge mit der beschriebenen Methode als Ankerstränge (AS) in anodisch formierte Oxidschichten fixiert und in einem zweiten Prozessschritt mit komplementären Gegensträngen (GS) hybridisiert werden.
In der Arbeit wird gezeigt, dass das Peptid in einem weiten Parameterbereich der elektrochemischen Bedingungen immobilisiert werden kann. Demgegenüber führen im Falle des nukleinsäurebasierten Immobilisierungssystems die Bildung reaktiver Sauerstoffspezies, die Photoaktivität der Oxidschicht sowie mehrfache Trocknungen und Wiederbenetzungen zu einer Schädigung gebundener AS bis hin zu einem vollständigen Verlust der Hybridisierbarkeit. Durch Zugabe von Ethanol in hoher Konzentration während des Immobilisierungsschritts, Arbeit unter Lichtausschluss sowie Vermeidung mehrerer Trocknungen und Wiederbenetzungen können die Nebenwirkungen soweit eingeschränkt werden, dass alle immobilisierten AS hybridisierbar sind.
Nach dessen Etablierung im Rahmen dieser Arbeit ist es in nachfolgenden Projekten möglich, das nukleinsäurebasierte Immboilisierungssystem zu einem modularen, nukleinsäurebasierten Immobilisierungssystem zu erweitern, um die eingangs beschriebenen Grenzen etablierter Methoden zu umgehen. Dazu müssen im zweiten Prozessschritt Konjugate aus GS und bioaktiven Molekülen, wie z. B. Peptide oder Wachstumsfaktoren, eingesetzt werden. Weiterhin können durch die Nutzung verschiedener Längen und Basensequenzen die Hybridstabilität und damit die Freisetzungskinetik beeinflusst werden.:1 Einleitung 1
2 Grundlagen 7
2.1 Titanbasislegierungen als Implantatwerkstoff 7
2.2 Biochemische Modifizierung von Titanoberflächen 22
2.3 Modulares Immobilisierungssystem 38
3 Materialien und Methoden 45
3.1 Werkstoffe 45
3.2 Biologisch aktive Moleküle 46
3.3 Elektrochemische Versuchsanordnung 51
3.4 Untersuchungsmethoden 52
4 Experimentelle Ergebnisse 69
4.1 Immobilisierung des RGD-Peptids 69
4.2 Nukleinsäurebasiertes Immobilisierungssystem 118
5 Diskussion der Ergebnisse 143
5.1 Wechselwirkung zwischen Molekülen und Oberfläche 143
5.2 Fixierung adsorbierter Moleküle 155
6 Zusammenfassung und Ausblick 169
Literaturverzeichnis 175
Anhänge
A Kodierung der Aminosäuren 209
B XPS-elementspektren ausgewählter Zustände 211
c Allgemeine Arbeitsvorschriften 215
D Geräte 221Surface functionalization with bioactive molecules is a main field in current biomaterial research. However, in vitro and in vivo results are heterogeneous. This may be at least partially attributed to the limits of the applied immobilization methods. With established immobilization methods possibilities are limited to immobilize different molecules in one step, to implement defined release kinetics for similar and/or different substances, or to carry out the immobilization after sterilization of the implant to save sensitive molecules from damage.
Therefore in this thesis a nucleic acid based immobilization system for bioactive molecules is developed for titanium based materials. Using a peptide with the amino acid sequence arginine–glycine–aspartic acid (RGD) it is demonstrated at first, that small molecules, being present at the interface electrolyte/passive layer, can be immobilized by their partial incorporation in anodically formed oxide layers. The immobilization can be carried out in a wide range of electrochemical parameters and the peptide preserves its biological function under all conditions.
This immobilization method is enhanced by utilizing single-stranded nucleic acids as anchor strands (AS), which can be hybridized by complementary strands (CS) in a second step. Contrary to the peptide, bound AS are damaged by the formation of reactive oxygen species during anodic polarization of the substrate, the photoyctivity of the titanium oxide layer and multiple drying and wetting cycles. These side effects must be constrained by adding ethanol in a high concentration to the electrolyte during the immobilization procedure, excluding light during preparation and avoiding multiple drying and wetting cycles. Applying these conutermeasures, a 100% hybridization of immobilized AS can be achieved.
After establishing the nucleic acid based immobilization system it can be developed further to a modular, nucleic acid based immobilization system to overcome limitations of established immobilization methods. At first, conjugates of CS and bioactive molecules, such as peptides or growth factors, should be used in the hybridization step for a true functionalization of the surface. Furthermore, hybrid stability and thus release kinetics can be adjusted by using CS of different length and base sequences.:1 Einleitung 1
2 Grundlagen 7
2.1 Titanbasislegierungen als Implantatwerkstoff 7
2.2 Biochemische Modifizierung von Titanoberflächen 22
2.3 Modulares Immobilisierungssystem 38
3 Materialien und Methoden 45
3.1 Werkstoffe 45
3.2 Biologisch aktive Moleküle 46
3.3 Elektrochemische Versuchsanordnung 51
3.4 Untersuchungsmethoden 52
4 Experimentelle Ergebnisse 69
4.1 Immobilisierung des RGD-Peptids 69
4.2 Nukleinsäurebasiertes Immobilisierungssystem 118
5 Diskussion der Ergebnisse 143
5.1 Wechselwirkung zwischen Molekülen und Oberfläche 143
5.2 Fixierung adsorbierter Moleküle 155
6 Zusammenfassung und Ausblick 169
Literaturverzeichnis 175
Anhänge
A Kodierung der Aminosäuren 209
B XPS-elementspektren ausgewählter Zustände 211
c Allgemeine Arbeitsvorschriften 215
D Geräte 22
Hydroxyapatite Coating of Magnesium Alloys for the Tailored Degradation of Resorbable Bone Fixation Products
Atmosferik soğuk plazma uygulaması ve RGD peptid konjügasyonu ile yüzey modifikasyonu sağlanan dental implantların osseointegrasyon sürelerinin değerlendirilmesi
xiv, 128 sayfa: resim, şekil29 cm. 1 CDABSTRACTIn this study, modification of the surface energy and physicochemical structure of same SLA surfaced dental implants by ACP, RGD peptid conjugation and the combination of these two technique, histological and biomechanical evaluation of osseointegration of these implants at 2, 4 and 8 weeks were aimed. Today, dental implants are used routinly in treatment of edentuolisim. It is needed a period of 3-6 months for the osseointegration of the inserted dental implants into jaw bone. To reduce this long time of osseointegrationdifferent techniques, implant designs and surface modifications are applied and implants can be provided to jointhe prosthetic functions in an earlier time. In this study, we aim to shorten the 3-6 months osseointegration period. In literature, this study is the first which apply together ACP and RGD peptide to the same implant. At the end of this study, after obtaining favourable results it is aimed to produce new dental implants which are osseointegrated in an earlier time, can solve the patients problems related to edentuolisim in a shorter time and are more economic and prosthetically more convenient for the patientsÖZETBu çalışmada aynı yüzey yapısına sahip SLA yüzeyli dental implantlar kullanarak implantların yüzey enerjileri ve fizikokimyasal yapsını ASP, RGD peptid konjugasyonu ve bu iki tekniğin birlikte uygulamasıyla modifiye edilerek implantların osseointegrasyonunu 2, 4 ve 8 haftalık zaman periyotlarında biyomekaniksel ve histolojik olarak değerlendirmek amaçlanmıştır. Dental implantlar günümüzde eksik dişlerin tedavisinde rutin olarak kullanılmaktadır. Çene kemiğine yerleştirilen implantların osseointegrasyonu için 3-6 ay süreyle beklemek gerekmektedir. Bu süreyi kısaltmak adına birçok teknik, farklı implant dizaynları ve farklı yüzey modifikasyonları uygulanarak implantların protetik fonksiyonlara daha erken katılması sağlanmaktadır. Bu çalışmada 3-6 aylık osseointegrasyon süresini kısaltabilmek hedeflenmiştir. Literatürde aynı implant yüzeyine ASP ve RGD peptidi birlikte uygulayan bir çalışma olmaması bakımından bu çalışma bir ilktir. Çalışmanın sonunda elde edilen olumlu veriler ışığında klinik olarak daha kısa sürede osseointegre olan, hastaların dişsizliğe bağlı sorunlarını dahaerken sürede çözebilen ve protetik olarak daha uygun, daha ekonomik dental implantların üretilebileceği bilimsel bir kaynak ortaya çıkmıştır
Development of osteoinductive Si-based coatings to improve dental implants' performance
290 p.Since Professor Brånemark introduced the new concept of osseointegration and the proposal of titanium as the best choice for implants production, many efforts have been done to modify their surface in order to improve the direct bone-implant contact and in this way accelerate osseointegration process.The aim of this thesis work is on the one hand to accelerate the osseointegration process to avoid the appearance of possible problems during the first stages after implantation, and on the other hand, to promote the osteoinductive ability of implants making them widespread and accessible for every type of patients, moreover under unfavourable conditions. With this purpose, bioactive and osteoinductive coatings based on silicon precursors and obtained through the sol-gel process have been developed
Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture
The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels