Biofunktionalisierung oxidischer Titanoberflächen über eine spezifische Anbindung von biotinyliertem Fibronektin für die medizinische Anwendung

In der hier vorliegenden Dissertation wird die Entwicklung und Charakterisierung einer biomimetischen Beschichtung für Titanimplantatoberflächen, insbesondere Dentalimplantate, beschrieben. Ziel war es, die Adhäsion und Aktivität von Osteoblasten auf Titanoberflächen zu steigern und so eine Beschleunigung der Implantatintegration in das Knochengewebe zu erreichen. Hierfür wurde eine spezielle Art der biomimetischen Beschichtung entwickelt, bei der biotinyliertes Fibronektin (bFn) über Streptavidin auf eine biotinylierte TiOX-Modelloberfläche immobilisiert wurde. Die Biotinmodifizierung der TiOX-Oberfläche erfolgte hierbei über einen „Self-Assembly-Prozess“ durch sequenzielle Chemiesorption von N-(6-aminohexyl)aminopropyltrimethoxysilan sowie verschiedenen Sulfo-NHS-Biotin-Derivaten, welche den Aufbau einer Streptavidin-Monolage ermöglichten. Als ein wichtiges Resultat zeigte sich, dass die Streptavidin-Monolage effektiv die unspezifische Adsorption von Proteinen an die TiOX-Oberfläche unterbindet und hierdurch die Adhäsion von Osteoblasten auf dieser unterdrückt. Dies hat den Vorteil, dass auf eine antiadhäsive Basisbeschichtung, welche für eine spezifische Zellreaktion wichtig ist, verzichtet werden kann. Dieses osteoblastere Adhäsionsverhalten änderte sich signifikant nach Anbindung von bFn an die Streptavidin-Monolage, mit dem Ergebnis, einer drastischen Steigerung der Osteoblastenadhäsion. Weiterhin besaßen Osteoblasten auf diesen Oberflächen ein Proteinexpressionsmuster, das auf eine erhöhte Osteoinduktion schließen lässt. Es zeigte sich darüber hinaus eine verstärkte Zelladhäsion sowie eine Steigerung des osteoinduktiven Effekts auf Substraten, bei denen bFn über eine Streptavidin-Monolage immobilisiert wurde, gegenüber mit nativem Fibronektin (Fn) modifizierten TiOX-Oberflächen. Ein wesentlicher Schwerpunkt bestand daher in der Analyse der Zusammensetzung und Struktur der biomimetischen Beschichtung über „Surface Plasmon Spectroscopy“ und „Atomic Force Microscopy“. Diese ergab, dass bFn und natives Fn auf den jeweiligen Oberflächen eine unterschiedliche Konformation einnimmt. Im Gegensatz zu nativem Fn, das bei der Adsorption unter physiologischen Bedingungen auf TiOX-Oberflächen eine kompakte Konformation besitzt, nimmt bFn auf einer Streptavidin-Monolage eine entfaltete Konformation ein. Bei letzterer handelt es sich um dieselbe, welche Fn in vivo innerhalb der extrazellulären Matrix besitzt. Sie unterscheidet sich von der kompakten Fn-Konformation dahingehend, dass entlang der Fn-Achse weitere Proteinbindestellen zugänglich werden und hierdurch die Zellaffinität von Fn gesteigert wird. Die nachgewiesene Konformationsänderung kann somit als Grund für die gesteigerte Osteoblasten-Adhäsion und Aktivität auf Oberflächen mit bFn angenommen werden. Diese Kenntnisse konnten weiterhin für die Optimierung des biomimetischen Schichtsystems genutzt werden. So war es möglich, durch alternierendes Inkubieren der Biotin-aktivierten Oberfläche mit Streptavidin und bFn, ein Multilayersystem gezielt aufzubauen. Der Vorteil dieses Multilayersystems gegenüber einer einfachen Monolage aus bFn besteht in einer erhöhten Stabilität der biomimetischen Beschichtung, wodurch eine Anwendung in der Praxis erleichtert würde.

Studio della superficie degli impianti dentali in titanio: La nanotecnologia nella valutazione delle nuove superfici implantari in rapporto all'osteointegrazione dei mascellari

Il presente lavoro parte dalla descrizione dei processi di rimodellamento osseo mascellare a seguito della perdita di elementi dentari e la successiva riabilitazione mediante impianto dentale osteointegrato. Approfondiremo proprio i complessi aspetti dell’osteointegrazione su superfici implantari in titanio sia a livello micro che macroscopico. Nel campo dell’implantologia, infatti, il titanio risulta essere il materiale maggiormente impiegato in virtù della sua eccellente biocompatibilità e resistenza. Successivamente prenderemo in analisi i trattamenti di superficie implantare ad oggi più diffusi, lavorati prevalentemente a livello microscopico e infine confronteremo una sistematica trattata tradizionalmente con una innovativa trattata superficialmente a livello nanometrico. Il confronto avverrà in vivo, paragonando i risultati ottenuti clinicamente e radiograficamente tra le 2 sistematiche implantari, utilizzate per ripristinare la funzione masticatoria nei pazienti arruolati.

Bioengineered periodontal tissue formed on titanium dental implants

The ability to use autologous dental progenitor cells (DPCs) to form organized periodontal tissues on titanium implants would be a significant improvement over current implant therapies. Based on prior experimental results, we hypothesized that rat periodontal ligament (PDL)-derived DPCs can be used to bioengineer PDL tissues on titanium implants in a novel, in vivo rat maxillary molar implant model. Analyses of recovered implants revealed organized PDL tissues surrounding titanium implant surfaces in PDL-cell-seeded, and not in unseeded control, implants. Rat PDL DPCs also exhibited differentiative potential characteristic of stem cells. These proof-of-principle findings suggest that PDL DPCs can organize periodontal tissues in the jaw, at the site of previously lost teeth, indicating that this method holds potential as an alternative approach to osseointegrated dental implants. Further refinement of this approach will facilitate the development of clinically relevant methods for autologous PDL regeneration on titanium implants in humans.

Influence of various surface treatments on reosseointegration around contaminated implants

The similarity of periodontitis and peri-implantitis demands for the utilization of similar principles for the treatment. Different decontamination methods were available cleaning of implant surfaces contaminated with bacteria. The aim of the present study was to evaluate the effects of various decontamination methods on reosseointegration on contaminated implants. Six mongrel dogs were used. The mandibular 1st molars and all premolars were removed bilaterally. Three months later, experi- mental implants with different surface characters were installed in each sides of the mandible. The implant consisted of two parts; the implant body and an exchangeable intraosseous implant cylinder. After osseointegration, experimental peri-implantitis was induced by cotton ligatures until the bone loss reached the junction of the two segments of the implant. After debridement of the bone defects, three treatment models were performed; (i) contaminated cylinders were removed, pristine cylinders were placed; (ii) contaminated cylinders were cleaned in situ with saline and (iii) contaminated cylinders was removed, cleaned with saline, sterilized by autoclaving. All implants were covered with membranes. After 3 months, histological evaluations were accomplished. The results indicated that in situ saline therapy demonstrated a significant difference at SLA surfaces in bone-implant-contact. Treatment of contaminated implants in situ with saline resulted in resolution of peri-implantitis and bone fill in defects.

Enhancement of titanium implants via bioengineered periodontal tissues

Osseointegration of titanium dental implants into the jaw bone, which is required for maintenance of the implant in the jaw, results in ankylosis. Dental implants are therefore very unlike natural teeth, which exhibit significant movement in response to mechanical forces. The ability to generate periodontal ligament (PDL) tissues onto dental implants would better mimic the functional characteristics of natural teeth, and would likely improve implant duration and function. OBJECTIVES: The objective of this study was to investigate the feasibility of bioengineering PDL tissues onto titanium implant surfaces. METHODS: Bilateral maxillary first and second molars of 8-week old rats were extracted and used to generate single cell suspensions of PDL tissues, which were expanded in culture. Immunohistochemistry and RT-PCR were used to identify putative PDL progenitor/stem cell populations and characterize stem cell properties, including self-renewal, multipotency and stem cell maker expression. Cultured rPDL cells were harvested at third passage, seeded onto Matrigel-coated titanium implants (1.75 mm x 1 mm), and placed into healed M1/M2 extraction sites. Non-cell seeded Matrigel-coated titanium implants served as negative controls. Implants were harvested after 8, 12, or 18 weeks. RESULTS: Cultured rPDL cells expressed the mesenchymal stem-cell marker STRO-1. Under defined culture conditions, PDL cells differentiated into adipogenic, neurogenic and osteogenic lineages. While control implants were largely surrounded by alveolar bone, experimental samples exhibited fibrous PDL-like tissues, and perhaps cementum, on the surface of experimental implants. CONCLUSIONS: PDL contains stem cells that can generate cementum/PDL-like tissue in vivo. Transplantation of these cells might hold promise as a therapeutic approach for the bioengineering of PDL tissues onto titanium implant. Further refinement of this method will likely result in improved dental implant strategies for use of autologous PDL tissue regeneration in humans. This research was supported by CIMIT, and NIH/NIDCR grant DE016132 (PCY), and TEACRS (YL).

Laser wavelengths and oral implantology

In modern implant dentistry there are several clinical indications for laser surgery. Different laser systems have a considerable spectrum of application in soft and hard peri-implant tissues. The literature was searched for clinical application of different laser wavelengths in peri-implant tissues: second-stage surgery of submerged implants, treatment of infrabony defects, removal of peri-implant hyperplastic overgrowths, and, possibly, the preparation of bone cavities for implant placement. This report describes the state-of-the-art application of different laser systems in modern implant dentistry for the treatment of peri-implant lesions and decontamination of implant surfaces. Our study evaluated in vitro examinations, clinical experience and long-term clinical studies. The exact selection of the appropriate laser system and wavelength was dependent on the scientific evaluation of recent literature and the level of changes in implant and tissue temperatures during laser application. The significant reduction in bacteria on the implant surface and the peri-implant tissues during irradiation and the cutting effects associated with the coagulation properties of the lasers are the main reasons for laser application in the treatment of peri-implant lesions and the successful long-term prognosis of failing oral implants. The various applications of lasers in implant dentistry are dependent on the wavelength and laser-tissue interactions.

Wear particles and surface topographies are modulators of osteoclastogenesis in vitro

Prosthetic and osteosynthetic implants from metal alloys will be indispensable in orthopedic surgery, as long as tissue engineering and biodegradable bone substitutes do not lead to products that will be applied in clinical routine for the repair of bone, cartilage, and joint defects. Therefore, the elucidation of the interactions between the periprosthetic tissues and the implant remains of clinical relevance and several factors are known to affect the longevity of implants. Within this study, the effects of metal particles and surface topography on the recruitment of osteoclasts was investigated in vitro in a coculture of osteoblasts and bone marrow cells. The cells were grown in the presence of particles of different sizes and chemical composition or on metal discs with polished or sandblasted surfaces, respectively. At the end of the culture, newly formed osteoclasts were counted. Osteoclastogenesis was reduced when particles were added directly to the coculture. The effect depended on the size of the particles, small particles exerting stronger effects than larger ones. The chemical composition of the particles, however, did not affect the development of osteoclasts. In cocultures grown on sandblasted surfaces, osteoclasts developed at higher rates than they did in cultures on polished surfaces. The data demonstrate that wear particles and implant surfaces affect osteoclastogenesis and thus may be involved in the induction of local bone resorption and the formation of osteolytic lesions, leading eventually to the loosening of orthopedic implants.

In vitro adhesion of fibroblastic cells to titanium alloy discs treated with sodium hydroxide.

OBJECTIVE Adhesion of osteogenic cells on titanium surfaces is a prerequisite for osseointegration. Alkali treatment can increase the hydrophilicity of titanium implant surfaces, thereby supporting the adhesion of blood components. However, it is unclear if alkali treatment also supports the adhesion of cells with a fibroblastic morphology to titanium. MATERIALS AND METHODS Here, we have used a titanium alloy (Ti-6AL-4V) processed by alkali treatment to demonstrate the impact of hydrophilicity on the adhesion of primary human gingival fibroblast and bone cells. Also included were the osteosarcoma and fibroblastoma cell lines, MG63 and L929, respectively. Cell adhesion was determined by scanning electron microscopy. We also measured viability, proliferation, and protein synthesis of the adherent cells. RESULTS Alkali treatment increased the adhesion of gingival fibroblasts, bone cells, and the two cell lines when seeded onto the titanium alloy surface for 1 h. At 3 h, no significant changes in cell adhesion were observed. Cells grown for 1 day on the titanium alloy surfaces processed by alkali treatment behave similarly to untreated controls with regard to viability, proliferation, and protein synthesis. CONCLUSION Based on these preliminary In vitro findings, we conclude that alkali treatment can support the early adhesion of cells with fibroblastic characteristics to a titanium alloy surface.

Antimicrobial effect of polymeric biomaterials for bone infection treatment

Dissertação de mestrado em Bioquímica, apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2016.

Controlled surface nanotopography and oxygen plasma treatment of PEEK to improve cellular response

Poly(aryl-ether-ether-ketone) (PEEK) is a semi crystalline polymer which exhibits properties that make it an attractive choice for use as an implant material. It displays natural radiolucency, and MRI compatibility, as well as good chemical and sterilization resistance, both of which make it of particular interest in orthopaedic implants. However, PEEK has demonstrated poor cellular adhesion both in vitro and in vivo. This is problematic as implant surfaces that do not develop a layer of adhesive cells are at risk of undergoing fibrous encapsulation, which in turn leads to lack of a strong interface between the implant device and the patient tissue, which can in turn lead to failure of the implant and revision surgery . As incorporating nanotopography into a polymer surface has been demonstrated to be able to direct the differentiation behaviour of stem cells, a possible solution to PEEKs underlying issues with poor cellular response would be to incorporate specific nanoscale topography into the material surface through injection moulding, and then analysing if this is a viable method for addressing PEEKs issues with cellular response. In addition to nanoscale topography, the experimental PEEK surfaces were treated with oxygen plasma to address the underlying cytophobicity of the material. As this type of treatment has been documented to be capable of etching the PEEK surface, experiments were carried out to quantify the effect of this treatment, both on the ability of cells to adhere to the PEEK surface, as well as the effect it has upon the nanotopography present at the PEEK surface. The results demonstrated that there were a range of plasma treatments which would significantly improve the ability of cells to adhere to the PEEK surface without causing unacceptable damage to the nanotopography. Three different types of cells with osteogenic capacity were tested with the PEEK surfaces to gauge the ability of the topography to alter their behaviour: SAOS-2, osteoprogenitors and 271+ MSCs. Due to PEEKs material properties (it is non transparent, exhibits birefringence and is strongly autofluorescent) a number of histological techniques were used to investigate a number of different stages that take place in osteogenesis. The different cell types did display slightly different responses to the topographies. The SAOS-2 cells cultured on surfaces that had been plasma treated for 2 minutes at 200W had statistically significantly higher levels of von Kossa staining on the NSQ surface compared to the planar surface, and the same experiment employing alizarin red staining, showed a statistically significantly lower level of staining on the SQ surface compared to the planar surface. Using primary osteoprogenitor cells designed to look into if whether or not the presence of nanotopography effected the osteogenic response of these cells, we saw a lack of statistically significant difference produced by the surfaces investigated. By utilising HRP based immunostaining, we were able to investigate, in a quantitative fashion, the production of the two osteogenic markers osteopontin and osteocalcin by cells. When stained for osteocalcin, the SQ nanotopography had total percentage of the surface with stained material, average area and average perimeter all statistically significantly lower than the planar surface. For the cells that were stained for osteopontin, the SQ nanotopgraphy had a total percentage of the surface with stained material, average area and average perimeter all highly statistically significantly lower than those of the planar surface. Additionally, for this marker the NSQ nanotopography had average areas and average perimeters that were highly significantly higher than those of the planar surface. There were no significant differences for any of the values investigated for the 271+ MSC’s When plasma treatment was varied, the SAOS-2 cells demonstrated an overall trend i.e. increasing the energy of plasma treatment in turn leads to an increase in the overall percentage of staining. A similar experiment employing stem cells isolated from human bone marrow instead of SAOS-2 cells showed that for polycarbonate surfaces , used as a control, mineralization is statistically significantly higher on the NSQ nanopattern compared to the planar surface, whereas on the PEEK surfaces we observe the opposite trend i.e. the NSQ nanotopography having a statistically significantly lower amount of mineralization compared to the planar surface at the 200W 2min and 30W 1min plasma treatments. The standout trend from the PEEK results in this experiment was that the statistically significant differences on the PEEK substrates were clustered around the lower energy plasma treatments, which could suggest that the plasma treatment disrupted a function of the nanotopograhy which is why, as the energy increases, there are less statistically significant differences between the NSQ nanotopography and the Planar surface This thesis documents the response of a number of different types of cells to specific nanoscale topographies incorporated into the PEEK surface which had been treated with oxygen plasma. It outlines the development of a number of histological methods which measure different aspects of osteogenesis, and were selected to both work with PEEK, and produce quantitative results through the use of Cell Profiler. The methods that have been employed in this body of work would be of interest to other researchers working with this material, as well as those working with similarly autofluorescent materials.

Effects of implant design on marginal bone changes around early loaded, chemically modified, sandblasted Acid-etched-surfaced implants: a histologic analysis in dogs

A minimal marginal bone loss around implants during early healing has been considered acceptable. However, the preservation of the marginal bone is related to soft tissue stability and esthetics. Implant designs and surfaces were evaluated to determine their impact on the behavior of the crestal bone. The purpose of this study is to evaluate histologic marginal bone level changes around early loaded, chemically modified, sandblasted acid-etched-surfaced implants with a machined collar (MC) or no MC (NMC).

Clinical field trial examining an implant with a sand-blasted, acid-etched surface

BACKGROUND: Conventionally, endosseous dental implants have required 3 to 6 months of uninterrupted healing based on observations for dental implants that were characterized by a relatively smooth machined surface. Many studies have since demonstrated that implants with a roughened surface resulted in greater bone apposition, earlier bone contact, and a stronger bond between the implant and the bone, suggesting that implants with roughened surfaces could be loaded earlier than 3 to 6 months. Formal clinical studies confirmed that implants with rough surfaces can have abutments placed and be loaded occlusally as early as 6 weeks postplacement. The purpose of this prospective, human clinical investigation was to evaluate a large number of implants with a specific rough surface (sand-blasted acid-etched [SLA]) placed in everyday practice under routine private-practice conditions. METHODS: A prospective, multicenter, human clinical observational study was initiated with the goal of recruiting a minimum of 500 patients and 800 implants. The implants were to be placed and restored in predominantly private-practice settings around the world. Ninety-two practitioners in 16 countries agreed to participate, and 86 followed the study design. Patients had to be in good health, have sufficient bone to encase the implant, and agree to return for recall appointments. Exclusion criteria included heavy smoking (>10 cigarettes a day) and bone augmentation procedures at the implant site. All implants were two-piece (an abutment was to be placed after 6 weeks of healing) and were characterized by the presence of a transmucosal polished collar. Each implant had an SLA surface. All implants were positioned using a non-submerged (single-stage) surgical technique. Survival and success rates were calculated by life-table analyses. RESULTS: A total of 706 patients were enrolled and 1,406 implants were placed. In the final analyses, 590 patients with 990 implants (70.4% of those enrolled) met all inclusion criteria, including placement of an abutment and provisional restoration within 63 days of surgical placement. The majority of implants were 10 and 12 mm long (78.7%) and were placed in type II and III bone (87%). Seventy-three percent of the implants were placed in the mandible, and 27% were placed in the maxilla. The cumulative survival rate was 99.56% at 3 years and 99.26% at 5 years. The overall success rate was 99.12% at 3 years and 97.38% after 5 years. CONCLUSIONS: Under private-practice conditions, implants with an SLA surface could be placed and restored predictably within 6 to 8 weeks. Data from this prospective, multicenter, human observational study reinforced the results of more formal clinical studies and demonstrated that implants with the SLA surface can be restored in patients in approximately half of the time of conventional healing periods.

Fatigue and Fluoride Corrosion on Streptococcus mutans Adherence to Titanium-Based Implant/Component Surfaces

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)