Porous polyurethane coatings bonded onto surface-modified titanium substrates for the growth of an endothelial cell layer

Cardiovascular diseases refer to the class of diseases that involve the heart or blood vessels (arteries and veins). Examples of medical devices for treating the cardiovascular diseases include ventricular assist devices (VADs), artificial heart valves and stents. Metallic biomaterials such as titanium and its alloy are commonly used for ventricular assist devices. However, titanium and its alloy show unacceptable thrombosis, which represents a major obstacle to be overcome. Polyurethane (PU) polymer has better blood compatibility and has been used widely in cardiovascular devices. Thus one aim of the project was to coat a PU polymer onto a titanium substrate by increasing the surface roughness, and surface functionality. Since the endothelium of a blood vessel has the most ideal non-thrombogenic properties, it was the target of this research project to grow an endothelial cell layer as a biological coating based on the tissue engineering strategy. However, seeding endothelial cells on the smooth PU coating surfaces is problematic due to the quick loss of seeded cells which do not adhere to the PU surface. Thus it was another aim of the project to create a porous PU top layer on the dense PU pre-layer-coated titanium substrate. The method of preparing the porous PU layer was based on the solvent casting/particulate leaching (SCPL) modified with centrifugation. Without the step of centrifugation, the distribution of the salt particles was not uniform within the polymer solution, and the degree of interconnection between the salt particles was not well controlled. Using the centrifugal treatment, the pore distribution became uniform and the pore interconnectivity was improved even at a high polymer solution concentration (20%) as the maximal salt weight was added in the polymer solution. The titanium surfaces were modified by alkli and heat treatment, followed by functionlisation using hydrogen peroxide. A silane coupling agent was coated before the application of the dense PU pre-layer and the porous PU top layer. The ability of the porous top layer to grow and retain the endothelial cells was also assessed through cell culture techniques. The bonding strengths of the PU coatings to the modified titanium substrates were measured and related to the surface morphologies. The outcome of the project is that it has laid a foundation to achieve the strategy of endothelialisation for the blood compatibility of medical devices. This thesis is divided into seven chapters. Chapter 2 describes the current state of the art in the field of surface modification in cardiovascular devices such as ventricular assist devices (VADs). It also analyses the pros and cons of the existing coatings, particularly in the context of this research. The surface coatings for VADs have evolved from early organic/ inorganic (passive) coatings, to bioactive coatings (e.g. biomolecules), and to cell-based coatings. Based on the commercial applications and the potential of the coatings, the relevant review is focused on the following six types of coatings: (1) titanium nitride (TiN) coatings, (2) diamond-like carbon (DLC) coatings, (3) 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer coatings, (4) heparin coatings, (5) textured surfaces, and (6) endothelial cell lining. Chapter 3 reviews the polymer scaffolds and one relevant fabrication method. In tissue engineering, the function of a polymeric material is to provide a 3-dimensional architecture (scaffold) which is typically used to accommodate transplanted cells and to guide their growth and the regeneration of tissue. The success of these systems is dependent on the design of the tissue engineering scaffolds. Chapter 4 describes chemical surface treatments for titanium and titanium alloys to increase the bond strength to polymer by altering the substrate surface, for example, by increasing surface roughness or changing surface chemistry. The nature of the surface treatment prior to bonding is found to be a major factor controlling the bonding strength. By increasing surface roughness, an increase in surface area occurs, which allows the adhesive to flow in and around the irregularities on the surface to form a mechanical bond. Changing surface chemistry also results in the formation of a chemical bond. Chapter 5 shows that bond strengths between titanium and polyurethane could be significantly improved by surface treating the titanium prior to bonding. Alkaline heat treatment and H2O2 treatment were applied to change the surface roughness and the surface chemistry of titanium. Surface treatment increases the bond strength by altering the substrate surface in a number of ways, including increasing the surface roughness and changing the surface chemistry. Chapter 6 deals with the characterization of the polyurethane scaffolds, which were fabricated using an enhanced solvent casting/particulate (salt) leaching (SCPL) method developed for preparing three-dimensional porous scaffolds for cardiac tissue engineering. The enhanced method involves the combination of a conventional SCPL method and a step of centrifugation, with the centrifugation being employed to improve the pore uniformity and interconnectivity of the scaffolds. It is shown that the enhanced SCPL method and a collagen coating resulted in a spatially uniform distribution of cells throughout the collagen-coated PU scaffolds.In Chapter 7, the enhanced SCPL method is used to form porous features on the polyurethane-coated titanium substrate. The cavities anchored the endothelial cells to remain on the blood contacting surfaces. It is shown that the surface porosities created by the enhanced SCPL may be useful in forming a stable endothelial layer upon the blood contacting surface. Chapter 8 finally summarises the entire work performed on the fabrication and analysis of the polymer-Ti bonding, the enhanced SCPL method and the PU microporous surface on the metallic substrate. It then outlines the possibilities for future work and research in this area.

The influence of the gas environment on morphology and chemical composition of surfaces micro-machined with a femtosecond laser

We investigated the influence of different gas environments on the fabrication of surfaces, homogeneously covered with equally sized and spaced micro-structures. Two types of structures have been successfully micro-machined with a femtosecond laser on titanium surfaces in various atmospheres. The surface chemistry of samples machined in oxygen and helium shows TiO2, while machining in nitrogen leads to an additional share of TiN. The actual surface structure was found to vary significantly as a function of the gas environment. We found that the ablated particles and their surface triggered two consecutive events: The optical properties of the gas environment became non-isotropic which then led to the pulse intensity being redistributed throughout the cross section of the laser beam. Additionally, the effective intensity was further reduced for TiN surfaces due to TiN's high reflectivity. Thus, the settings for the applied raster-scanning machining method had to be adjusted for each gas environment to produce comparable structures. In contrast to previous studies, where only noble gases were found suitable to produce homogeneous patches, we obtained them in an oxygen environment.

The significance of brittle reaction layers in fusing of dental ceramics to titanium

This thesis comprises four intercomplementary parts that introduce new approaches to brittle reaction layers and mechanical compatibility of metalloceramic joints created when fusing dental ceramics to titanium. Several different methods including atomic layer deposition (ALD), sessile drop contact angle measurements, scanning acoustic microscopy (SAM), three-point bending (TPB, DIN 13 927 / ISO 9693), cross-section microscopy, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS) were employed. The first part investigates the effects of TiO2 layer structure and thickness on the joint strength of the titanium-metalloceramic system. Samples with all tested TiO2 thicknesses displayed good ceramics adhesion to Ti, and uniform TPB results. The fracture mode was independent of oxide layer thickness and structure. Cracking occurred deeper inside titanium, in the oxygen-rich Ti[O]x solid solution surface layer. During dental ceramics firing TiO2 layers dissociate and joints become brittle with increased dissolution of oxygen into metallic Ti and consequent reduction in the metal plasticity. To accomplish an ideal metalloceramic joint this needs to be resolved. The second part introduces photoinduced superhydrophilicity of TiO2. Test samples with ALD deposited anatase TiO2 films were produced. Samples were irradiated with UV light to induce superhydrophilicity of the surfaces through a cascade leading to increased amount of surface hydroxyl groups. Superhydrophilicity (contact angle ~0˚) was achieved within 2 minutes of UV radiation. Partial recovery of the contact angle was observed during the first 10 minutes after UV exposure. Total recovery was not observed within 24h storage. Photoinduced ultrahydrophilicity can be used to enhance wettability of titanium surfaces, an important factor in dental ceramics veneering processes. The third part addresses interlayers designed to restrain oxygen dissolution into Ti during dental ceramics fusing. The main requirements for an ideal interlayer material are proposed. Based on these criteria and systematic exclusion of possible interlayer materials silver (Ag) interlayers were chosen. TPB results were significantly better in when 5 μm Ag interlayers were used compared to only Al2O3-blasted samples. In samples with these Ag interlayers multiple cracks occurred inside dental ceramics, none inside Ti structure. Ag interlayers of 5 μm on Al2O3-blasted samples can be efficiently used to retard formation of the brittle oxygen-rich Ti[O]x layer, thus enhancing metalloceramic joint integrity. The most brittle component in metalloceramic joints with 5 μm Ag interlayers was bulk dental ceramics instead of Ti[O]x. The fourth part investigates the importance of mechanical interlocking. According to the results, the significance of mechanical interlocking achieved by conventional surface treatments can be questioned as long as the formation of the brittle layers (mainly oxygen-rich Ti[O]x) cannot be sufficiently controlled. In summary in contrast to former impressions of thick titanium oxide layers this thesis clearly demonstrates diffusion of oxygen from sintering atmosphere and SiO2 to Ti structures during dental ceramics firing and the following formation of brittle Ti[O]x solid solution as the most important factors predisposing joints between Ti and SiO2-based dental ceramics to low strength. This among other predisposing factors such as residual stresses created by the coefficient of thermal expansion mismatch between dental ceramics and Ti frameworks can be avoided with Ag interlayers.

Study of superficial properties of titanium treated by PIIID

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

Bioactive coating on titanium implants modified by Nd:YVO4 laser

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Streptococcus mutans attachment on a cast titanium surface

This study examined by means of scanning electron microscopy (SEM), the attachment of Streptococcus mutans and the corrosion of cast commercially pure titanium, used in dental dentures. The sample discs were cast in commercially pure titanium using the vacuum-pressure machine (Rematitan System). The surfaces of each metal were ground and polished with sandpaper (#300-4000) and alumina paste (0.3 μm). The roughness of the surface (Ra) was measured using the Surfcorder rugosimeter SE 1700. Four coupons were inserted separately into Falcon tubes contained Mueller Hinton broth inoculated with S. mutans ATCC 25175 (109 cuf) and incubated at 37 °C. The culture medium was changed every three days during a 365-day period, after which the falcons were prepared for observations by SEM. The mean Ra value of CP Ti was 0.1527 μm. After S. mutans biofilm removal, pits of corrosion were observed. Despite the low roughness, S. mutans attachment and biofilm formation was observed, which induced a surface corrosion of the cast pure titanium.

Nanometer Scale Titanium Surface Texturing Are Detected by Signaling Pathways Involving Transient FAK and Src Activations

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

How do titanium and Ti6A14V corrode in fluoridated medium as found in the oral cavity? an in vitro study

The purpose of this work was to evaluate the corrosion of commercially pure (CP) titanium and Ti6Al4V in vitro at different F- concentrations regularly found in the oral cavity by using different electrochemical tests and surface analysis techniques. electrochemical impedance spectroscopy (EIS), open circuit potential (OCP) and potentio-dynamic polarization tests were associated to advanced characterization techniques such as SEM, EDS, AFM, ICP-MS and XPS. OCP tests revealed a higher reactivity of both CP titanium and Ti6Al4V at 12,300 ppm F- concentration than that recorded at 227 ppm F-. Also, a significant decrease of the corrosion resistance of both materials was noticed by EIS in fluoride solutions. Material loss caused by corrosion was noticed on titanium surfaces by SEM and AFM in the presence of high F- concentration. CP titanium degraded by pitting corrosion while Ti6Al4V suffered from general corrosion showing micro-cracks on surface. Furthermore, a high release of metallic ions from the test samples after immersion at high F concentrations was detected by ICP-MS, that can be potentially toxic to oral tissues. (C) 2014 Elsevier B.V. All rights reserved.

Influence of corrosion on lipopolysaccharide affinity for two different titanium materials

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Osteoblast differentiation is enhanced by a nano-to-micro hybrid titanium surface created by Yb:YAG laser irradiation

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

Wear and Corrosion Interactions on Titanium in Oral Environment: Literature Review

The oral cavity is a complex environment where corrosive substances from dietary, human saliva, and oral biofilms may accumulate in retentive areas of dental implant systems and prostheses promoting corrosion at their surfaces. Additionally, during mastication, micromovements may occur between prosthetic joints causing a relative motion between contacting surfaces, leading to wear. Both processes (wear and corrosion) result in a biotribocorrosion system once that occurs in contact with biological tissues and fluids. This review paper is focused on the aspects related to the corrosion and wear behavior of titanium-based structures in the oral environment. Furthermore, the clinical relevance of the oral environment is focused on the harmful effect that acidic substances and biofilms, formed in human saliva, may have on titanium surfaces. In fact, a progressive degradation of titanium by wear and corrosion (tribocorrosion) mechanisms can take place affecting the performance of titanium-based implant and prostheses. Also, the formation of wear debris and metallic ions due to the tribocorrosion phenomena can become toxic for human tissues. This review gathers knowledge from areas like materials sciences, microbiology, and dentistry contributing to a better understanding of bio-tribocorrosion processes in the oral environment.

Potential of chemically modified hydrophilic surface characteristics to support tissue integration of titanium dental implants

In the past, several modifications of specific surface properties such as topography, structure, chemistry, surface charge, and wettability have been investigated to predictably improve the osseointegration of titanium implants. The aim of the present review was to evaluate, based on the currently available evidence, the impact of hydrophilic surface modifications of titanium for dental implants. A surface treatment was performed to produce hydroxylated/hydrated titanium surfaces with identical microstructure to either acid-etched, or sand-blasted, large grit and acid-etched substrates, but with hydrophilic character. Preliminary in vitro studies have indicated that the specific properties noted for hydrophilic titanium surfaces have a significant influence on cell differentiation and growth factor production. Animal experiments have pointed out that hydrophilic surfaces improve early stages of soft tissue and hard tissue integration of either nonsubmerged or submerged titanium implants. This data was also corroborated by the results from preliminary clinical studies. In conclusion, the present review has pointed to a potential of hydrophilic surface modifications to support tissue integration of titanium dental 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.

Pro-osteogenic topographical cues promote early activation of osteoprogenitor differentiation via enhanced TGFβ, Wnt, and Notch signaling

Objectives Titanium implant surfaces with modified topographies have improved osteogenic properties in vivo. However, the molecular mechanisms remain obscure. This study explored the signaling pathways responsible for the pro-osteogenic properties of micro-roughened (SLA) and chemically/nanostructurally (modSLA) modified titanium surfaces on human alveolar bone-derived osteoprogenitor cells (BCs) in vitro. Materials and methods The activation of stem cell signaling pathways (TGFβ/BMP, Wnt, FGF, Hedgehog, Notch) was investigated following early exposure (24 and 72 h) of BCs to SLA and modSLA surfaces in the absence of osteogenic cell culture supplements. Results Key regulatory genes from the TGFβ/BMP (TGFBR2, BMPR2, BMPR1B, ACVR1B, SMAD1, SMAD5), Wnt (Wnt/β-catenin and Wnt/Ca2+) (FZD1, FZD3, FZD5, LRP5, NFATC1, NFATC2, NFATC4, PYGO2, LEF1) and Notch (NOTCH1, NOTCH2, NOTCH4, PSEN1, PSEN2, PSENEN) pathways were upregulated on the modified surfaces. These findings correlated with a higher expression of osteogenic markers bone sialoprotein (IBSP) and osteocalcin (BGLAP), and bone differentiation factors BMP2, BMP6, and GDF15, as observed on the modified surfaces. Conclusions These findings demonstrate that the activation of the pro-osteogenic cell signaling pathways by modSLA and SLA surfaces leads to enhanced osteogenic differentiation as evidenced after 7 and 14 days culture in osteogenic media and provides a mechanistic insight into the superior osseointegration on the modified surfaces observed in vivo.

Oral Actinomyces Species in Health and Disease: Identification, Occurence and Importance of Early Colonization

The genus Actinomyces consists of a heterogeneous group of gram-positive, mainly facultatively anaerobic or microaerobic rods showing various degrees of branching. In the oral cavity, streptococci and Actinomyces form a fundamental component of the indigenous microbiota, being among initial colonizers in polymicrobial biofilms. The significance of the genus Actinomyces is based on the capability of species to adhere to surfaces such as on teeth and to co-aggregate with other bacteria. Identification of Actinomyces species has mainly been based on only a few biochemical characteristics, such as pigmentation and catalase production, or on the use of a single commercial kit. The limited identification of oral Actinomyces isolates to species level has hampered knowledge of their role both in health and disease. In recent years, Actinomyces and related organisms have attracted the attention of clinical microbiologists because of a growing awareness of their presence in clinical specimens and their association with disease. This series of studies aimed to amplify the identification methods for Actinomyces species. With the newly developed identification scheme, the age-related occurrence of Actinomyces in healthy mouths of infants and their distribution in failed dental implants was investigated. Adhesion of Actinomyces species to titanium surfaces processed in various ways was studied in vitro. The results of phenotypic identification methods indicated a relatively low applicability of commercially available test kits for reliable identification within the genus Actinomyces. However, in the study of conventional phenotypic methods, it was possible to develop an identification scheme that resulted in accurate differentiation of Actinomyces and closely related species, using various different test methods. Genotypic methods based on 16S rRNA sequence analysis of Actinomyces proved to be a useful method for genus level identification and further clarified the species level identification with phenotypic methods. The results of the study of infants showed that the isolation frequency of salivary Actinomyces species increased according to age: thirty-one percent of the infants at 2 months but 97% at 2 years of age were positive for Actinomyces. A. odontolyticus was the most prominent Actinomyces colonizer during the study period followed in frequency by A. naeslundii and A. viscosus. In the study of explanted dental implants, Actinomyces was the most prevalent bacterial genus, colonizing 94% of the fixtures. Also in the implants A. odontolyticus was revealed as the most common Actinomyces species. It was present in 84% of Actinomyces -positive fixtures followed in frequency by A. naeslundii, A. viscosus and A. israelii. In an in vitro study of titanium surfaces, different Actinomyces species showed variation regarding their adhesion to titanium. Surface roughness as well as albumin coating of titanium had significant effects on adhesion. The use of improved phenotypic and molecular diagnostic methods increased the accuracy of the identification of the Actinomyces to species level. This facilitated an investigation of their occurrence and distribution in oral specimens in both health and disease.