Improving bioimplants: Identifying proteins on modified titanium surfaces

Implant imperfections

Biomedical implants constructed from titanium and its alloys are routinely used as dental implants, artificial joints and bone pins, driven by the ability of this metal to bind with bone in a process referred to as osseointegration. The fact that titanium is lightweight and non-magnetic adds to its popularity for medical applications.

As later implants were developed, they were treated with coatings designed to improve the biointegration and prevent reversal of the metal-bone binding. One of the most common coatings comprises molecules associated with the extracellular matrix (ECM) or a peptide (Arg-Gly-Asp) representing the minimum adhesion motif of ECM molecules.

The ECM supports living cells, providing the physical and chemical conditions that enable the development of tissues. It is a complex mixture of proteins and polysaccharides secreted by the cells, so coating foreign bodies like implants with ECM can fool the cells into accepting them. However, binding problems remain in the long term.

As a result, a team of scientists in France has been experimenting with poly(sodium styrenesulphonate) (polyNaSS) as an alternative coating and found that it can interact with a broader range of proteins due to the negative sulphonate sites. They postulated that pre-treatment of a polyNaSS-titanium composite with a mixture of ECM molecules such as cell-matrix proteins, growth factors and cytokines before implantation would improve osseointegration.

In order to be able to test this, the researchers devised a procedure for assessing the extent of protein adsorption and identifying the adsorbed proteins. Didier Lutomski and colleagues from the University of Paris, the Armed Forces Blood Transfusion Centre, Clamart, and the French Blood Establishment, Lille, used platelet-rich plasma as the protein source for adsorption on polyNaSS-titanium.

Proteomics of adsorbed proteins on polymer-titanium surfaces

The team employed oxidised titanium granules coated with polyNaSS rather than a solid surface like that used in real implants to increase the contact area between titanium and the proteins. The granules were packed into a chromatographic column for affinity chromatography of the platelet-rich plasma.

The flow-through fraction was retained and the adsorbed proteins were eluted. Both fractions were subjected to 2D gel electrophoresis and the protein patterns on the gels were compared. The flow-through and eluted fractions contained 283 and 221 proteins spots, respectively, indicating that many proteins are adsorbed on the surfaces.

A comparison with the gel patterns resulting from affinity chromatography of the plasma on non-coated oxidised titanium granules showed that surface modification with polyNaSS modified the adsorption of 19 polypeptides. They were identified by mass spectrometry following isolation and enzymatic digestion, using the conventional proteomics techniques.

Five of the polypeptides were found to be completely adsorbed on the polyNaSS-titanium surface whereas 14 had low affinity. Good adsorption appeared to be independent of the plasma concentration of the individual proteins.

For instance, 10 high-abundance plasma proteins with typical concentrations in the range 15-130 µmol/L, including immunoglobulin G, transthyretin and alpha-1 antitrypsin, were not adsorbed. Conversely, complement factor B which is present in plasma at 2-5 µmol/L was completely adsorbed.

So, surface modification with the polysulphonate changed the selectivity of the titanium surface towards protein adsorption. Just as importantly, the study shows that proteomics methods can be applied to the study of proteins adsorbed on alloys, and possibly on other biomaterials like ceramics.

"This work will provide a basis for further investigations aiming at the development of a polyNaSS/titanium surface pre-treated with an appropriated mixture of proteins to improve the bioreactivity of the implant," said Lutomski.

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