**3.3 Discussion**

Thus, the IR spectra of the samples immersed in SBF for 1 month showed different behaviour *in vitro* as a function of composition and crystallization state.

The inertness of BG1 structures might be determined by their chemical composition. High contents of SiO2 have influences both in bioactivity and the thermal expansion coefficient of the glass. In general, SiO2-rich glasses with silica contents higher than 60 wt.% have a better mechanical stability and adhesion to the metallic substrate, but are not soluble in body fluids (Lopez-Esteban et al., 2003). Decreasing the silica content is therefore mandatory for improving the bioactivity and partial dissolution of the glass surface. For the BG1 structures one can notice an important silicon (~50 at.%) and phosphorous content (~10 at.%). In silicabased glasses phosphorous acts as a network former producing structural rearrangement where the silicate network is more interconnected by creating P-O-Si cross-links. At higher content of phosphorus, the glass is increasing in connectivity. The glass compositions with higher content of network formers, do not bond either to bone or to soft tissues and elicit

Fig. 9. SEM micrographs of BG3 films after immersion in SBF for 30 days. Left side, SEM-top

The SEM microstructures of BG3 films immersed for 30 days clearly asserted the growth of thick and rough coatings with randomly distributed irregular spherulitic shaped microsized agglomerates on top of all three types of samples, evidencing the good biomineralization capability of all coatings (*Fig. 9-top row*). The thicknesses of the chemical grown HA layers were determined by tilt-SEM (*Fig. 9-bottom row*). One can observe that the BG3-3 and BG3-4 coatings led to the thickest chemical growths: total film thickness ~1 μm, and ~1.3 μm, respectively, compared to the as-deposited film thickness of ~ 0.4 μm. The HA chemically grown layers had in case of BG3-1 and BG3-2 structures a lower average thickness of ~0.7 μm. Thus, the best biomineralization, correlated with the thickest HA

Thus, the IR spectra of the samples immersed in SBF for 1 month showed different

The inertness of BG1 structures might be determined by their chemical composition. High contents of SiO2 have influences both in bioactivity and the thermal expansion coefficient of the glass. In general, SiO2-rich glasses with silica contents higher than 60 wt.% have a better mechanical stability and adhesion to the metallic substrate, but are not soluble in body fluids (Lopez-Esteban et al., 2003). Decreasing the silica content is therefore mandatory for improving the bioactivity and partial dissolution of the glass surface. For the BG1 structures one can notice an important silicon (~50 at.%) and phosphorous content (~10 at.%). In silicabased glasses phosphorous acts as a network former producing structural rearrangement where the silicate network is more interconnected by creating P-O-Si cross-links. At higher content of phosphorus, the glass is increasing in connectivity. The glass compositions with higher content of network formers, do not bond either to bone or to soft tissues and elicit

layer growth, was obtained for the BG3-4 coating (G.E. Stan et al., 2011).

behaviour *in vitro* as a function of composition and crystallization state.

view; Right side, ESEM-cross view

**3.3 Discussion** 

formation of a non-adherent fibrous interfacial capsule. No surface changes during SBF tests were evidenced for the annealed BG1 structure. Unlike in case of BG2, the crystallization had no effect on BG1's surface reactivity. The nucleated Na2Mg(PO3)4 phase proved to be inert in SBF. It can be concluded that the chemical composition, bonding configuration and crystallinity could play important roles in BG films' reactivity in SBF.

In case of as-deposited BG2 samples a continuous dissolution process occurs, after 30 days the entire film is dissolved into the surrounding fluid. One can notice the high content of Ca (~20 at.%) and Na atoms (~43 at.%) in the BG2 film (*Table 3*). It can be speculated that when very high numbers of alkali and alkali-earth ions are released in SBF, the pH will rapidly and drastically change at the film-fluid interface, producing a chemical unbalance which will suppress the polymerization of silanols and the further formation of the SiO2-rich surface layer. The bioactive process is thus disrupted, leading to continuous dissolution of the BG amorphous film. On the other hand the annealing of BG1 films followed by slow cooling promotes formation of combeite, wollastonite and Na3PO4, which have smaller solubility, and consequently a decreased leaching rate of Na+ and Ca2+ into the SBF solution. This will determine the conditions at the surface of the film to be more stable, therefore allowing polymerisation of soluble Si(OH)4 in a SiO2-rich layer which will act as a nucleation site for apatite (L.L. Hench & J. Wilson, 2003). During 30 days of SBF soaking, dissolution-reprecipitation processes took place, the annealed BG2 layer is partially dissolved and finally we obtain a multilayer structure containing a bottom BG layer coated by an amorphous SiO2-rich thin film and at the top a Ca-P type layer. Thus in case of BG2 annealed samples our FTIR results are consistent with Hench's theory. Previous studies have also reported that both the combeite (Chen et al., 2006) and wollastonite (Xue et al., 2005) can generate on their surface Ca/P rich layers when in contact with SBF. The growth of the bioapatite layer is essential for bone generation and bonding ability. However, the combeite was proved to rapidly transform into amorphous calcium phosphate phase in contact with SBF, but it delays the process of crystallization into a hydroxyapatite phase (L.L. Hench & J. Wilson, 2003). Besides biocompatibility, these structures could actively improve proteins and osteocytes adhesion, significantly shortening the osteointegration time.

Regarding the higher biomineralization rates of BG3-3 and BG3-4 films one can hypothesize that the chemical processes involved in the bioactivity mechanism are accelerated, because of the increased sodium content of these films and a propitious bridging oxygen/nonbridging oxygen ratio (*Table 4*), speeding up the ionic exchange and the chemical growth of HA. Hench's theory states that the first stage of SBF immersion of a bioglass involves the rapid exchange of Na+ ions from the glass for H+ and H3O+ ions from the solution, which shall initialize the formation of silica-rich layer known to favour the nucleation of Ca-P type layers. Therefore, we can suppose that a higher number of sodium ions released into SBF solution will change the chemical equilibrium of the precipitation reaction, thereby catalyzing the CHA chemical growth mechanism.

Therefore, for achieving bioactive properties, an optimal ratio of network formers/network modifiers is required, but not sufficient. The disruption of the Si-O-Si bonds and creation of Si-O-NBO bonds, determined by increased network modifiers content, is mandatory in the first steps of Ca-P chemical growth mechanism. The disruption of the Si-O-Si bonds evidenced by the increasing content of Si-O-NBO groups plays an important role in the

Magnetron Sputtered BG Thin Films: An Alternative Biofunctionalization

coatings in simulated body fluid (SBF) was studied.

**4. Conclusions** 

implantology.

to obtain the best biological outcome.

new perspectives in regenerative medicine.

Pasuk for the professional assistance with GIXRD measurements.

**5. Acknowledgments** 

**6. References** 

Approach – Peculiarities of Bioglass Sputtering and Bioactivity Behaviour 93

The effects of sputtering pressure, composition of working atmosphere and deposition parameters on the compositional, morpho-structural and mechanical properties of the coated implants were emphasized, and their influence on the *in vitro* behaviour of the

The adhesion strength at the coating – substrate interface is a critical factor in successful implantation and long-term stability of such implant-type structures. The chapter presented few technological RF-MS derived approaches to surpass the low adherence drawback when dealing with BG having high CTE: by introducing intermediate buffer layers [BG1-xTix (x=0-1)] with compositional gradient, or by strong inter-diffusion phenomena induced via heat-treatments, with the aim in the nucleation of BG-Ti mix phases at the substrate-coating interface. The importance of a proper *in situ* substrate cleaning, performed by argon ion sputtering processes which will enhance the ad-atoms diffusion, should be also emphasized. Also when using BG cathode target with CTE closely matching the Ti substrates, the RF-MS deposition conditions exert great influence on the adherence of films as well as on their composition. The application of the above mentioned technological algorithms conducted to pull-out adhesion values higher than the usual ones reported in literature, opening new perspectives in oral and orthopaedic

Peculiarities of the *in vitro* behaviour of the BG thin films were discussed in correlation with the widely accepted Hench bioactivity mechanism. Based on our *in vitro* studies it was concluded that the chemical composition, bonding configuration and crystallinity could play important roles in BG films' reactivity in SBF. For achieving bioactive properties an optimal ratio of network formers/network modifiers is a necessary, but not sufficient, condition. BG compositions with very high network modifier content have a poor native stability, and their *in vitro* behaviour can be tailored by thermal treatments of crystallization

By varying the sputtering pressure and working atmosphere we attempted to tailor the films' composition and structure, hinting optimal mechanical performance and bioactive behaviour. Understanding these correlations is important for biomaterials science, implantology, as well as from an applied physics and technological point of view, opening

The financial support of Centre for Research in Ceramics and Composites (CICECO) – University of Aveiro and of the Romanian Ministry of Education, Research, Youth and Sport (Core Program – Contract PN09-45) is gratefully acknowledged. Authors thank Dr. Iuliana

Agathopoulos, S.; Tulyaganov, D.U.; Ventura, J.M.G.; Kannan, S.; Karakassides, M.A. &

Ferreira J.M.F. (2006). Formation of hydroxyapatite onto glasses of the CaO–MgO–

dissolution rate of silica in the first steps of Hench's chemical growth mechanism, favouring the bioactivity processes of the films by a higher rate of biomineralization (Serra et al., 2002; Liste et al., 2004). This hypothesis is in agreement with our results, the stronger biomineralization, expressed by the thickest CHA film grown, was obtained for the BG3 sample. Although BG compositions with very high network modifier content have a native poor stability, we can tailor their in-vitro behaviour by thermal treatments of crystallization to obtain the best bioactive outcome (G.E. Stan et al., 2010b).

The mechanism of calcium phosphates formation onto the surface of bioactive glasses is widely accepted, and involves the dissolution of cations from the surface of bioactive glasses and the consequent increase of the supersaturation degree in the surrounding fluid, with respect to HA components. Hench's theory states that the first stage of a bioglass mineralization upon immersion in SBF involves the rapid exchange of Na+ ions from the glass for H+ and H3O+ ions from the solution, which shall initialize the breaking of the Si-O-Si bonds of the glass structure and the subsequent formation of silanol groups. In a next step the silanol groups polycondensate forming a silica-rich layer at the bioglass surface, which favours the growth of Ca-P type layers. Homogenous nucleation (precipitation) of biomimetic apatite occurs spontaneously in a solution supersaturated with respect to hydroxyapatite. Heterogeneous precipitation, on the other hand, takes place on the sample's surface. Both, homogenous and heterogeneous nucleation, could be considered in competition during the SBF immersion.

Due to their designed low thickness (lower than 700 nm), even in case of total dissolution, the amount of BG film that could be released in the SBF solution is around few micrograms. Thus, intuitively the bioactivity processes will not be governed in case of thin BG films preponderantly by the supersaturation of SBF solution in Na, Ca and P and further precipitation of Ca-P phases, but by the pH evolution at the sample-solution interface, surface energy and electrostatic interactions between electrically negative charged sample surface and the cations and anions from stagnant solution in the proximity of the BG surface.

A reinterpretation of the classical biomineralization process based on the electrical double layer effect will be presented. The electrical double layer refers to two parallel layers differing in charge surrounding an object (BG sample). The increased number of surface hydroxyl groups could act as nucleation sites for HA. Thus the first layer, with negative surface charge, comprises Si–OH- groups primarily formed during chemical interactions with the fluid. Ca2+ ions and with them also anions, such as (HPO4)2-, are present in the electrolyte space charge facing the sample surface enriched in polymerising silanol groups. Thus, the second layer is composed of calcium ions attracted to the surface charge via the Coulomb force, electrically screening the first layer. This second layer could be seen in the first stages as a diffuse layer being loosely connected with the silica-rich one, as it consists of free calcium ions which move in the fluid under the influence of electric attraction and thermal motion. As the (HPO4)2- anions interact and react with this second layer, the heterogeneous nucleation of CaO–P2O5 layer starts, which is anchoring progressively into the sample's surface, resulting in a hydrated precursor cluster consisting of calcium hydrogen phosphate, and in time by consuming and incorporating various ions [(OH)-, F- or (CO3)2-] from the surrounding media crystallize into a carbonated-flour-hydroxyapatite mixed layer. Consequently in case of BG thin films one should consider the heterogeneous nucleation as the dominant mechanism of HA growth.
