Introduction

Bioactive glasses can bond to both bone and soft tissue. For transcutaneous implants a proper bonding to the surrounding soft tissue could enhance the functionality. Hence a novel technique, Electron Beam Ablation (ELBA), to coat these implants was introduced [1]. This technology is comparable in performance to pulsed laser deposition (PLD) but has the advantage of lower cost. A commercial bioactive glass (S53P4) was deposited on Ti-substrates by ELBA. As for other bioactive glasses, the bioactivity of the ELBA-S53P4 coating was proven by the in vitro formation of an HCA-layer. For approval of new medical devices, a standard in vitro cytotoxicity test has to be performed.

It is known that the amount of Si leached from bioactive glass is critical regarding the biological response. High Si-concentrations may assist in the formation of the HCA-layer, but can also lead to cell death. Above 230 ppm, leached Si is cytotoxic and below 100ppm it is not [2,3]. In between these values no data is available. It is possible to use a preconditioning step to reduce the Si-release by immersion of the glass in either simulated body fluid or culture medium for a pre-determined time period. It was found that the HCA layer formation was not significantly changed and that bioactivity was not adversely influenced by preconditioning [3].

In order to control the cytotoxicity of the ELBA S53P4 coating several in vitro experiments were performed.

Materials and Methods

Both as-deposited ELBA-S53P4 coatings (3-4µm and >10µm thick) on Ti-substrates and coatings preconditioned by immersion for 72h in a-MEM were subjected to extraction cytotoxicity testing according to ISO-10993-5. After 24h soaking, extracts and diluted (60-70-80%) extracts were added to L929 mouse fibroblast cell cultures for 24h and afterwards a cell count was performed. As a comparison also uncoated Ti-substrates, bulk S53P4 and hydroxyapaptite (HA)-ELBA coated Ti were evaluated. For all experiments the surface–to–volume ratio was 6cm²/ml, assuming that the ELBA coating had a smooth surface. In the non-diluted extraction media, the amount of leached Si was analysed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The reacted ELBA S53P4 coatings were evaluated by ESEM.

Results and Discussion

The table below shows the cytotoxicity and ICP results for the various samples. A reduction in cell number of more than 25% is considered cytotoxic. The ELBA-S53P4 coating showed a cytotoxic behaviour. Both bulk S53P4 and the ELBA-HA coating were not cytotoxic.

Sample		% cell reduction	Cytotoxic	[Si] (ppm)
Bulk S53P4		2	No	36-72
ELBA S53P4 coating	3-4µm	>40	Yes	-
	>10µm	>80	Yes	>800
	72h^a	0.1	No	<90
	60%^b	84	Yes	-
	70%^b	45	Yes	-
	80%^b	13	No	-
ELBA HA coating		19	No	n.a.
Bulk Ti		<6	No	n.a.

^a preconditioning time; ^b dilution of 24h extract

Thus the high Si-concentration is believed to be responsible for the cytotoxicity. Extract dilution showed that at 80% dilution the medium was not cytotoxic anymore. This dilution would reduce the [Si] to <200ppm, which is below the cytotoxicity limit [3]. Preconditioning reduces both the amount of leached Si and the cytotoxicity drastically and thus can be applied to control the cytotoxicity. Thinner coatings are not as cytotoxic. These results, together with an ESEM analysis, support the hypothesis that the interacting bioactive glass surface is controlling the Si-release.

Conclusions

The reaction surface of bioactive glasses is highly important in relation to the biological response due to the potential negative influence of leached components from the glass to the biological environment. Thus the selection of a bioactive glass composition does not only depend on the desired bioactive behaviour, but also on the morphology of the glass. The higher the specific surface of the bioactive glass, the less soluble the glass has to be or, prior to implantation, the glass needs to be preconditioned, to reduce the concentration of leaching elements. Also the surface–to–volume ratio of biomaterials should be quantified carefully and taken into account when performing an in vitro cytotoxicity experiment to generate a controlled stimulation of the desired cell population.

Acknowledgement

This research is part of EU FP5 Growth project G5RD-CT-2001-00533 InCoMeD.

References

[1] Schrooten J., Jaecques S.V.N. et al., Key Engineering Materials Vols. 254-256, 2004, 427-430

[2] Keeting P.E. et al., Journal of Bone and Mineral Research, 7, 11, 1992, 1281-1289

[3] Gough J.E. et al., Biomaterials, 25 (11), 2004, 2039-2046

Introduction

Materials and Methods

Results and Discussion

Bulk S53P4

ELBA HA coating

Conclusions

References