Matteo Santin

Osteointegration of titanium and its alloys by anodic spark deposition and other electrochemical techniques: A review

Direct osteointegration of titanium and titanium alloy implants is one of the main goals of biomaterial research for den- tal and orthopedic applications. Chemical, mechanical or biological treatments have been investigated to obtain a fast and durable implant to bone bonding. In recent years, to improve osteointegration scientists have focussed their attention on the interface intere- actions between the implant surface and the bone. In particular, many efforts have been concentrated on the modification of the thin oxide layer spontaneously formed on the surface of titanium. This stable oxide layer has been considered responsible for the titanium biocompatibility and for the ability of this material to osteointegrate. The stability of the oxide layer in air is well known. However, when titanium is placed in vivo the titanium oxide layer changes its properties because of the electrolytic nature of the body fluids, whose components are able to interact and bind to the metal surface ox- ide. In particular, titanium dioxide exhibits hydroxyl group formation which, in their deprotonated form, can bind calcium thus promoting osteointegration. Therefore, the rate of osteointegration can be modulated tailoring the oxide layer properties before im- plantation. The introduction of new techniques capable of changing the physicochemical and morphological properties of the oxide film can be a step forward towards developing a new generation of highly compatible surfaces suitable to induce a strong and durable bonding to the bone. Recently, an electrochemical technique, discovered in the early 1930s and widely investigated in the 1960s, has been applied to titanium and its alloys to enhance implant osteointegration. This technique, initially employed to produce a corro- sion-resistant thick oxide film on valve metals, is known as Anodic Spark Deposition or Anodic Spark Discharge (ASD). It consists of a high voltage anodization of titanium in electrolyte solutions, whose ions become embedded in the thickened dioxide layer as a consequence of the melting process at the surface induced by the high plasma temperature. The high temperature is generated by ran- domly produced sparks originated during the process by the dielectric breakdown of the thickened oxide layer. By this technique it is possible to obtain relatively thick oxide layers enriched with the electrolytes originally dissolved in the medium and bringing a micro- porous and oxygen-rich surface. These properties can be finely modulated controlling each parameter of the reaction. Several research groups studied ASD and explored its potential, sometimes by coupling this technique with further treatments such as hydrothermal, thermal and chemical treatments, with the goal of obtaining a new generation of biocompatible and osteoinductive or osteoconduc- tive surfaces. The first studies on ASD were performed by Kurzes research group, who paved the way for the first really effective sur- face treatment developed by Ishizawa et al. Ishizawa patented a new method to coat titanium with thydroxyapatite, through the en- richment of titanium oxide layer with calcium and phosphorus ions via ASD process. In this method, after ASD process titanium oxide layer is treated to a hydrothermal process, which introduces on the surface a thin layer of hydroxyapatite crystals. Kokubo et al considered the ASD technique to improve the titanium dioxide bioactivity, and demonstrated the capability of this modified oxide lay- er to enhance calcium-phosphate nucleation after soaking in SBF. Recently, the same authors of this paper functionalized the Ca and P enriched titanium dioxide by a final alkali treatment, achieving a nano-structured surface that proved to exhibit high mineraliz- ing potential and selective adsorption of protein (fibronectin). ASD can now be considered an effective method for the modification of titanium surfaces. By this method a new generation of implantable surfaces can be therefore designed, which may improve the per- formances of orthopedic and dental implants. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 91-107)

Effect of the urine conditioning film on ureteral stent encrustation and characterization of its protein composition

The goal of this study was to characterize the protein composition of the conditioning film deposited onto the surface of ureteral stents during in vivo implantation and to relate its presence to the precipitation of calcium crystals. The protein pattern of the conditioning film of implanted nonencrusted and encrusted urological stents was assessed by SDS-PAGE and Western blot of the desorbed species. The results obtained highlighted different electrophoresis profiles between nonencrusted and encrusted stents. Western blot showed the ubiquitous presence of albumin, while Tamm-Horsfall Protein and alpha1-microglobulin adsorption was limited to nonencrusted devices. By an in vitro dynamic model in which artificial urine was flowed through the lumen of control and retrieved nonencrusted stents, we demonstrated that the organic layer remarkably enhanced crystal precipitation and aggregation events on the surface.

In vitro host response assessment of biomaterials for cardiovascular stent manufacture

The deployment of a vascular stent during angioplasty has greatly reduced the risks of restenosis. However, the presence of the device still induces a host response as well as a mechanical action on the blood vessel wall and an alteration of the haemodynamics. Platelet and inflammatory cells can adhere on the stent surface and be activated to produce biochemical signals able to stimulate an excessive proliferation of the smooth muscle cells with the consequent obstruction of the vessel lumen. For these reasons, the host response to two of the materials used in stent manufacture, stainless steel and diamond-like carbon, was investigated in vitro. The data showed that stainless steel induced a higher level of host response both in terms of platelet aggregation and macrophage activation. However, the spreading of inflammatory cells was more accentuated on diamond-like carbon. The inflammatory cells produced levels of platelet-derived growth factor, a key signal in smooth muscle cell proliferation, similar to stainless steel thus suggesting that carbon coatings may not be able to prevent restenosis.

Soybean-based biomaterials: preparation, properties and tissue regeneration potential

Future successes in regenerative medicine will depend on the development of new biodegradable biomaterials able to control tissue regeneration in vitro and in vivo. None of the products currently available to surgeons can combine all the essential characteristics for biodegradable biomaterials, which are tunable degradation rate, controlled inflammatory reaction, no toxicity and stimulation of tissue regeneration. These clinical features should be provided, together with ease of handling during surgery and cost-effective production. Here, an overview is presented of a novel class of soybean-based biomaterials, which can be manufactured as different hydrogel formulations, all tailored for specific clinical applications. ln vitro and in vivo studies have ascertained their activity on various biochemical and cellular components of regenerating tissues. Beyond their use, the ascertained bioactivity of some of the soybean components may open new investigations and commercial routes in regenerative medicine.

Substrate-induced phenotypical change of monocytes/macrophages into myofibroblast-like cells: a new insight into the mechanism of in-stent restenosis.

Stented coronary angioplasty is the procedure of choice to re-establish patency in obstructed coronary arteries. However, the stent implantation procedure often leads to in-stent restenosis, a process that is characterized by stent strut colonization by macrophages and smooth muscle cells and by neointima formation. The present in vitro study investigates the effect of stent materials on the phenotypical features of monocyte/macrophages. Human peripheral blood monocytes from healthy donors (n = 7) were cultured up to 7 days on substrates mimicking: (i) the stent surface (i.e., electropolished stainless steel), (ii) the de-endothelialized vessel wall (collagen-based extracellular matrix gel), and (iii) thrombus (i.e., fibrin gel). The cells were analyzed by immunocytochemistry for their ability to express alpha-actin, a typical myofibroblast marker, by ELISA to determine PDGF-BB and TGF-beta1 secretion and by PCR to evaluate hyaluronan synthase 1, 2, and 3 genes expression. Data were statistically analyzed by ANOVA (Dunnetts test) and data considered significantly different at p </= 0.05. The data demonstrated that mononuclear cells adhering to stainless steel acquire a phenotype capable of expressing alpha-actin while secreting significantly higher levels of PDGF-BB and TGF-beta. The expression of the three hyaluronan synthase isoforms was also altered by the metal substrate, where cells expressed genes only for the isoforms synthesizing high molecular weight hyaluronan. This study therefore suggests that mononuclear cells adhering on the stent metal surface undergo phenotypical transformation into myofibroblast-like cells that are able to contribute to neointimal tissue synthesis.

A novel biomimetic treatment for an improved osteointegration of titanium

Direct osteointegration of titanium and titanium alloys implants is one of the main goals of biomaterials research for dental and orthopedic applications. Chemical, mechanical or biological treatments are investigated searching for fast and durable implant to bone bonding. The aim of the present work is to assess the in vitro mineralisation capabilities and to investigate the mechanical and physico-chemical properties of a new biomimetic treatment on titanium. The new surface treatment was obtained using Anodic Spark Deposition technique, and consists of a first ASD treatment performed in solutions containing phosphate ions followed by a second ASD treatment in a solution rich in calcium ions. The resulting surface is finally treated by alkali etching. The physio-chemical and mechanical properties of this material are analyzed and the mineralization potential is considered by surface analysis after soaking it in different solutions of simulated body fluid (SBF). The developed biomimetic treatment was then compared to other treatments from the literature. The proposed treatment was found to possess a very high mineralization capaci-ty, that makes its application very interesting in terms of speed and strength of direct implant osteointegration. (Journal of Ap-plied Biomaterials & Biomechanics 2003; 1: 33-42).

Fibrinogen adsorption and platelet adhesion to metal and carbon coatings

In order to study the haemocompatibility of metal and carbon coatings, fibrinogen adsorption and platelet adhesion to various coatings have been investigated. Two metallic coatings--titanium and zirconium, and two carbon coatings - isotropic diamond-like and isotropic graphite-like coatings, were prepared by plasma vapour deposition onto stainless steel substrate. It has been shown that the adsorption of fibrinogen to metal and carbon coatings and its post-adsorptive transition are dependent on both the material properties and the fibrinogen environment. The adsorption of fibrinogen from human plasma on titanium and zirconium coatings is similar to that on uncoated stainless steel surface. Both carbon coatings adsorb much greater amount of fibrinogen from plasma, and fibrinogen retention by carbon surfaces is also greater than by metal surfaces. Increased numbers of adhered platelets have been found on both carbon coatings in comparison to the metal materials, although this does not correlate with the amount of adsorbed fibrinogen.

Serum protein absorption on silk fibroin fibers and films: surface opsonization and binding strength

Fibroin, the core of the silk filament protein, has been proposed as a biomaterial for different biomedical applications since it can be engineered as a thread, fabric or film. Infrared spectroscopy suggests that the dissolution of the fibroin filaments and subsequent casting of the fibroin solution into films followed by treatment with methanol alters the protein structure leading to an increase in amorphous domains. The adsorption ofserum proteins on fabrics and films showed different hydrophobic binding strengths for the two materials with the protein binding being greatest for the more hydrophobic fibroin fibers. Differences between the materials were also observed in the adsorption of key immunoproteins. Although the C3 fragment of the complement system was adsorbed on both the surfaces, it appeared to be activated preferentially on the fibroin films and not on the fibroin fibers, whereas the Bb and C1q factors were only significantly present on the fibroin fabric. IgG appeared to be adsorbed, although to different extents, on both types of fibroin substrates. These results suggest that the biocompatibility of the silk fibroin may be affected by changes in protein structure induced by processing the material.

Self-hardening calcium deficient hydroxyapatite/gelatine foams for bone regeneration

In this work gelatine was used as multifunctional additive to obtain injectable self-setting hydroxyapatite/gelatine composite foams for bone regeneration. The foaming and colloidal stabilization properties of gelatine are well known in food and pharmaceutical applications. Solid foams were obtained by foaming liquid gelatine solutions at 50°C, followed by mixing them with a cement powder consisting of alpha tricalcium phosphate. Gelatine addition improved the cohesion and injectability of the cement paste. After setting the foamed paste transformed into a calcium deficient hydroxyapatite. The final porosity, pore interconnectivity and pore size were modulated by modifying the gelatine content in the liquid phase.

Nanoparticles of a different source induce different patterns of activation in key biochemical and cellular components of the host response

Nanoparticulate materials are produced by industrial processing or engineered for specific biomedical applications. In both cases, their contact with the human body may lead to adverse reactions. Most of the published papers so far have focused on the cytotoxic effects of nanoparticles (NPs). Instead, the present in vitro study investigates the effect of different types of NP on key components of the host response such as clot formation and the inflammatory cells. The different NPs were pre-conditioned with platelet-rich human plasma for 30 min and then incubated with the blood mononuclear cells for 20 hours. The potential of the different NPs to induce clot formation, platelet activation and monocyte/macrophage differentiation was assessed by morphological analysis, immunocytochemistry and biochemical assays. The data showed that nanoparticulate materials based on antimony, silver and nickel were capable of promoting the polymerization of fibrin and the aggregation and fragmentation of platelets, leading to a moderately activated monocyte phenotype. This process was more pronounced in the case of antimony- and silver-based NPs that share a similar size and round-shaped morphology. Conversely, NPs of cobalt, titanium and iron appeared to stimulate cells to acquire a macrophage phenotype able to secrete higher levels of tumour necrosis factor α, a pro-inflammatory cytokine. Therefore, the present study provides clear indications about the subtle and adverse effects that the invasion of these materials may produce in the cardiovascular system and in vital organs.