Emiliano Salvagni

Collagen-functionalised titanium surfaces for biological sealing of dental implants: Effect of immobilisation process on fibroblasts response

The clinical success of a dental implant requires not only an optimum osseointegration, but also the development of a biological sealing; i.e., a soft tissue seal around the transmucosal part of the implant. A promising approach to improve the biological seal of dental implants is the biomimetic modification of titanium surfaces with proteins or peptides that have specific cell-binding moieties. In this work we investigated the process of immobilising collagen on smooth and rough titanium surfaces and its effect on human dermal fibroblast (HDF) cell response. Titanium samples were activated by either oxygen plasma or acid etching to generate a smooth or nanorough surface, respectively. Subsequently, collagen grafting was achieved by either physisorption or covalent bonding through organosilane chemistry. The biofunctionalised titanium samples were then tested for stability and characterised by fluorescent labelling, wettability, OWLS and XPS studies. Biological characterisation was also performed through HDF adhesion, proliferation and gene expression. Covalent-bonded collagen showed higher stability than physisorbed collagen. A significant overexpression of the genes involved in fibroblast activation and extracellular matrix remodelling was observed in the collagen-coated surfaces. This effect was more pronounced on smooth than on rough surfaces. Immobilised collagen on the smooth plasma-treated surfaces favoured both fibroblast adhesion and activation. This study provides essential information for the design of implants with optimal biological sealing, a key aspect to avoid peri-implantitis and ensure long-lasting implant fixation.

A bioactive elastin-like recombinamer reduces unspecific protein adsorption and enhances cell response on titanium surfaces.

We present the immobilization on synthetic substrates of elastin-like recombinamers (ELR) that combine a bioactive motif for cell adhesion with protein antifouling properties. Physical adsorption of the recombinamers and covalent-grafting through organosilane chemistry were investigated. The biochemically-modified surfaces were thoroughly characterized and tested for protein absorption in serum by fluorescence-labelling, XPS, Ellipsometry, and OWLS. The ELR were successfully grafted and stable, even upon mechanical stresses; being the covalent bonding favourable over physical adsorption. The coated metal surfaces exhibited excellent reduction of serum protein adsorption (9 ng/cm(2)) compared to the bare metal surface (310 ng/cm(2)). Non-specific protein adsorption may mask the introduced bioactive motifs; therefore, the bioactivated surfaces should display serum-protein antifouling properties. Finally, improved hMSCs response was assessed on the bioactivated substrates. In summary, the coatings simultaneously displayed anti-fouling and bioactive properties. These studies investigated key factors to enhance tissue material interactions fundamental for the design of bioactive devices and future biomedical applications.

A low elastic modulus Ti-Nb-Hf alloy bioactivated with an elastin-like protein-based polymer enhances osteoblast cell adhesion and spreading.

β-type titanium alloys with low Youngs modulus are desirable to reduce stress shielding effect and enhance bone remodeling for implants used to substitute failed hard tissue. For biomaterials application, the surface bioactivity is necessary to achieve optimal osseointegration. In the previous work, the low elastic modulus (43 GPa) Ti-25Nb-16Hf (wt %) alloy was mechanically and microstructurally characterized. In the present work, the biological behavior of Ti-25Nb-16Hf was studied. The biological response was improved by surface modification. The metal surface was modified by oxygen plasma and subsequently silanized with 3-chloropropyl(triethoxy)silane for covalent immobilization of the elastin-like polymer. The elastin-like polymer employed exhibits RGD bioactive motives inspired to the extracellular matrix in order to improve cell adhesion and spreading. Upon modification, the achieved surface presented different physical and chemical properties, such as surface energy and chemical composition. Subsequently, osteoblast adhesion, cell numbers, and differentiation studies were performed to correlate surface properties and cell response. The general tendency was that the higher surface energy the higher cell adhesion. Furthermore, cell culture and immunofluorescence microscopy images demonstrated that RGD-modified surfaces improved adhesion and spreading of the osteoblast cell type.

Study on the use of 3-aminopropyltriethoxysilane and 3-chloropropyltriethoxysilane to surface biochemical modification of a novel low elastic modulus Ti-Nb-Hf alloy.

A biocompatible new titanium alloy Ti-16Hf-25Nb with low elastic modulus (45 GPa) and the use of short bioadhesive peptides derived from the extracellular matrix have been studied. In terms of cell adhesion, a comparative study with mixtures of short peptides as RGD (Arg-Gly-Asp)/PHSRN (Pro-His-Ser-Arg-Asn) and RGD (Arg-Gly-Asp)/FHRRIKA (Phe-His-Arg-Arg-Ile-Lys-Ala) have been carried out with rat mesenchymal cells. The effect of these mixtures of short peptides have already been studied but there are no comparative studies between them. Despite the wide variety of silane precursors available for surface modification in pure titanium, the majority of studies have used aminosilanes, in particular 3-minopropyltriethoxysilane (APTES). Nevertheless, the 3-chloropropyltriethoxysilane (CPTES) is, recently, proposed by other authors. Unlike APTES, CPTES does not require an activation step and offers the potential to directly bind the nucleophilic groups present on the biomolecule (e.g., amines or thiols). Since the chemical surface composition of this new alloy could be different to that pure titanium, both organosilanes have been compared and characterized by means of a complete surface characterization using contact angle goniometry and X-ray photoelectron spectroscopy.

Assessment and comparison of surface chemical composition and oxide layer modification upon two different activation methods on a cocrmo alloy

This study investigated the effect of two different activation methods on the surface chemical composition of a CoCrMo-alloy. The activation was performed with oxygen plasma (OP) or nitric acid (NA). The surface physical–chemical properties were thoroughly characterized by means of several analytical techniques: X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), zinc-complex substitution technique, contact angle, and interferometry. The surface modification was evaluated by assessing contamination removal, the “active” hydroxyl groups (OH-act) present at the surface, the metal oxide ratio (CoyOx−/CryOx−) and changes in the chemical composition and topography of the oxide layer. XPS experimental data showed for both methods (OP and NA) a significant decrease of the carbon contents (C 1s) associated with contaminants and at the same time changes in the atomic composition of the oxide layer (O 1s). In addition, the O 1s XPS spectra showed differences between the percentage of OH− before and after OP or NA treatment, leading to the conclusion that both methods are effective for surface “cleaning” and activation. These results were further investigated and corroborated by ToF-SIMS analysis and zinc complex substitution technique. The general conclusion was that NA is more efficient in terms of contaminants removal and generation of accessible OH-act present at the surface and without altering the native metal oxide ratio (CoyOx−/CryOx−) considered to be essential for biocompatibility.

Biofunctionalization of titanium surfaces for osseintegration process improvement

This study aims to improve the osseointegration of titanium implants through surface immobilization of peptides that induce a beneficial biological response. This was carried out biofunctionalizating titanium surfaces by silanization and subsequent covalent binding of a peptide with a sequence that promotes cell adhesion. Objective: The development of a new technique of immobilization of oligopeptides on the surface of titanium by using 3-chloropropyltrietoxisilane (CPTES) as bonding agent between the surface of titanium and the peptide. Materials and methods: A physicochemical characterization of the surfaces through the techniques of XPS, ToF-SIMS and contact angle was performed. Also cell adhesion studies have been conducted to evaluate in vitro biological response. Results: Through the process of silanization the titanium surface is completely covered with CPTES, which allows the subsequent accession of oligopeptides. The cell adhesion results show a higher cell adhesion and cell extension on biofunctionalized samples. Conclusions: We developed a system of covalent binding of oligopeptides on titanium surfaces that can modify the biological response of the attached cells.

Different Organization of Type I Collagen Immobilized on Silanized and Nonsilanized Titanium Surfaces Affects Fibroblast Adhesion and Fibronectin Secretion

Silanization has emerged in recent years as a way to obtain a stronger and more stable attachment of biomolecules to metallic substrates. However, its impact on protein conformation, a key aspect that influences cell response, has hardly been studied. In this work, we analyzed by atomic force microscopy (AFM) the distribution and conformation of type I collagen on plasma-treated surfaces before and after silanization. Subsequently, we investigated the effect of the different collagen conformations on fibroblasts adhesion and fibronectin secretion by immunofluorescence analyses. Two different organosilanes were used on plasma-treated titanium surfaces, either 3-chloropropyl-triethoxy-silane (CPTES) or 3-glycidyloxypropyl-triethoxy-silane (GPTES). The properties and amount of the adsorbed collagen were assessed by contact angle, X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and AFM. AFM studies revealed different conformations of type I collagen depending on the silane employed. Collagen was organized in fibrillar networks over very hydrophilic (plasma treated titanium) or hydrophobic (silanized with CPTES) surfaces, the latter forming little globules with a beads-on-a-string appearance, whereas over surfaces presenting an intermediate hydrophobic character (silanized with GPTES), collagen was organized into clusters with a size increasing at higher protein concentration in solution. Cell response was strongly affected by collagen conformation, especially at low collagen density. The samples exhibiting collagen organized in globular clusters (GPTES-functionalized samples) favored a faster and better fibroblast adhesion as well as better cell spreading, focal adhesions formation, and more pronounced fibronectin fibrillogenesis. In contrast, when a certain protein concentration was reached at the material surface, the effect of collagen conformation was masked, and similar fibroblast response was observed in all samples.

Biomimetic treatments on dental implants for immediate loading applications

Commercially pure titanium (cp Ti) dental implants have been widely and successfully used with high rates of clinical success in normal situations. However, there is still a lack of reliable synthetic materials to be used either a) when immediate loading of the implant is desired or b) when bone presents compromised conditions due to trauma, infection, systemic disease and/or lack of significant bone volume. Our group has aimed the development of biomimetic strategies of surface modification to obtain metallic implants with osteostimulative capabilities. These surface modifications will provide implants with a rapid rate of newly-formed bone growth and with ossecoalescence, i.e., direct chemical contact with the surrounding tissues. Consequently, the biomimetically-modified implants will be reliably used on those more demanding clinical situations, cp Ti surfaces treated to obtain a combination of an optimal random surface topography (in the micro and nanolevels) with a chemical modification of the naturally-formed titania layer have been proved bioactive. These rough and bioactive surfaces nucleate and grow a homogeneous hydroxyapatite layer both in vitro and in vivo. They stimulate the osteoblasts differentiation and trigger a rapid bone formation that mechanically fixes implants under immediate-loading conditions. A simple process using silane chemistry has been proved specific, rapid, and reliable to covalently immobilize biomolecules on cp Ti surfaces. This methodology can be used to develop biofunc- tionalized implant surfaces with different or combined bioactivities. The biofunctional molecules can be biopolymers, proteins, growth factors, and synthetic peptides specifically designed to be attached to the surface. The bioactive properties of the molecules designed and used can be mineral growing and nucleation, osteoblast differentiation (bone regeneration), fibroblasts differentiation (biological sealing), antibiotic,... Specifically, we have obtained mechanically and thermochemically stable coatings made of recombinant elastin-like biopolymers. The biopolymers bear either a) the RODS peptide, which is a highly-specific cell-adhesion motif present in proteins of the extracellular matrix for different tissues including bone, or b) an acidic peptide sequence derived from statherin, a protein present in saliva with high affinity for calcium-phosphates and with a leading role in the remineralization processes of the hard tissues forming our teeth. Two different biomimetic strategies have been successfully developed combining topographical modification, inorganic treatments and/or biofunctionalization for improving bioactive integrative properties of cp Ti implants.