**11. Seeking novel targets in the pursuit of rational stent design.**

Given the fact that the urinary conditioning film has been directly implicated in causing both bacterial adhesion and associated/non-associated encrustation, it becomes important to switch our focus from designing a biomaterial that inhibits direct bacterial and ion/mineral deposition to one that inhibits conditioning film components. It is important to focus on understanding this biological target further and the first step is to identify components of the conditioning film. Santin et al have previously identified human serum albumin as well as Tamm-Horsfall Protein (THP) as major conditioning film components found on four stents removed from patients (Santin, Motta et al. 1999). More recently, Canales et al have studied the conditioning films of stents removed from 27 patients, identifying hemoglobin alpha and beta chain, albumin, calgranulin B, fibrinogen beta chain, vitronectin, annexin A1, calgranulin A, fibrinogen gamma chain, and THP as the ten most common adherent components (Canales, Higgins et al. 2009). In addition, this group also hypothesized that the presence of histones likely contribute to stent encrustation given their unique net positive charge. Despite the fact that these papers have contributed to a large extent to the identification of conditioning film components, it still needs to be determined whether urinary conditioning films differ between stent types or patients, as the molecules targeted in stent design should be "universal" and need to be common between patients and stent types.

Our group has recently compared the composition of conditioning films found on certain stents from Boston Scientific (Polaris) to those on Bard stents (Inlay) after they have been removed from patients (Lange et al, unpublished data). Both of these stents differ in their biomaterials, as the Polaris stent is made of an olefinic copolymer, while the InLay stent is made of polyurethane. To date, there does not appear to be a significant difference in the conditioning film composition from patients with the same stent type or between the two different stent types, indicating that conditioning film deposition is not affected by different stent biomaterials or patients. Similar results have also been obtained by Tieszer et al, who showed via X-ray photoelectron spectroscopy that the elemental composition of conditioning film components was unaffected by stent biomaterial or patient characteristics (Tieszer, Reid et al. 1998). Our study found that the fifteen most common proteins include cytokeratins, serum albumin, hemoglobin subunits alpha and beta, THP, fibrinogen gamma chain, protein S100A9, vitronectin and apolipoprotein. Interestingly, the majority of the fifteen most commonly found proteins are binding sites for bacteria and thus facilitate bacterial adhesion and biofilm formation. In addition to this, the presence of calcium binding proteins such as the S100 proteins or THP may act as a nidus for encrustation. We found that significantly less Polaris stents contained THP and fibrinogen gamma chain compared to the InLay stent, eliminating these two proteins as potential targets. Overall these results validate specific conditioning film components as targets for future stent biomaterial design as they appear to play a role in stent associated infection and encrustation. Further analysis will have to be performed to determine whether commonalities exist between the physical characteristics of these components and whether they can be targeted to inhibit their deposition.

Our current experiments are aimed at studying the temporal deposition of urinary components onto the surface of stent pieces, as some proteins such as serum albumin are known to bind to other proteins rather than the surfaces themselves. In the context of urinary component deposition, it is possible that certain proteins with a higher affinity to the stent surface form a base layer to which other proteins such as serum albumin attach. Such a mechanism of deposition would be favorable for the purpose of rational stent design, as the proteins forming the base layer would make excellent targets for adhesion prevention. If temporal aspects can be determined in addition to the various layers of proteins, potential targets could be identified to prevent the initiating events of encrustation and infection.
