1 IN VITRO BIOCOMPATIBILITY EVALUATION OF SURFACE-MODIFIED TITANIUM ALLOY Alyson Schlieper, Venkat Shankarraman, Sang-Ho Ye, and William R. Wagner McGowan Institute for Regenerative Medicine, Department of Bioengineering INTRODUCTION Ventricular Assist Devices (VADs) have become a means to both preserve and prolong the lives of patients with heart failure. By providing circulatory support, VADs lessen the load of the heart. Compared to traditional medical management of heart failure, VADs have a higher patient survival rate [1]. VADs are used as both a bridge to transport as well as form of destination therapy for patients requiring long-term or permanent circulatory support. While VADs do increase survival rate for heart failure patients, survival percentage of patients using this type of mechanical circulatory support for permanent or long-term support decreases significantly the longer the patient is reliant upon the device. In a study by Lietz K. et al., it was seen that the percent of patients still alive one-month post VAD implantation was 86.1%, but only 30.9% of patients with VADs were still alive twenty four months after implantation [2]. VADs do pose risks to patients since this implanted medical device can be thrombogenic. Blood cells in contact with the VAD can become activated, forming cell aggregates, which can lead to thrombosis, as well as other complications. To improve patient survival rates, many studies focus on improving the biocompatibility of the VAD material in contact with the blood. Platelet activation, since it is the initial step to thrombus formation is often used as a way to quantify material biocompatibility. A good indicator as to whether or not a material will cause the blood to clot, low platelet activation in blood samples corresponds to a biocompatible material. Material coatings or surface modifications have shown an increase in the biocompatibility of materials, as the coated materials display lower levels of platelet activation. A previous study was done by Ye et al. to assess the biocompatibility of a siloxane functionalized phosphorylcholine polymer coating. The study revealed the incorporation of the coating resulted in decreased platelet deposition on the coated titanium alloy surface as well as lower platelet activation levels than the unmodified titanium alloy. The results of this study suggests that an incorporation of a siloxane functionalized phosphorylcholine polymer coating onto a titanium alloy can improve the material’s biocompatibility [3]. OBJECTIVE The goal of this study is to evaluate whether the incorporation of a siloxane functionalized phosphorylcholine polymer (MPCMPSi) coating onto a titanium alloy (Ti 6 Al 4 V) will improve the biocompatibility of the titanium alloy. Using flow cytometry, the study will compare platelet activation levels of blood samples exposed to a coated titanium alloy, as well as a non-coated titanium to test and see if the titanium surface modification will result in lower levels of platelet activation. SUCCESS CRITERIA The MPCMPSi coating can be considered successful in increasing the biocompatibility of the titanium alloy if the coated titanium shows platelet activation levels in the 8 – 10% range. METHOD The study evaluated three different materials: titanium alloy (Ti 6 Al 4 V), titanium alloy coated with a siloxane functionalized phosphorylcholine polymer (MPCMPSi), and alumina (Al 2 O 3 ). Alumina was chosen as a positive control. Samples were polished and cut to 1 x 2.5cm dimensions. Titanium samples were coated with MPCMPSi using a simple silanization technique after the surfaces were passivated with a 35% nitric acid for 1 hr [3]. All materials were sterilized by alternating washing between unadulterated acetone and ethanol. Following cleansing, samples were stored in 70% ethanol. Whole ovine blood was collected by jugular venipuncture using an 18 gauge 1 ½” needle directly into a syringe after discarding the first 3 ml. The blood was withdrawn into a 60 ml syringe which contained 6 ml of 0.1 M sodium citrate. Three sterilized samples (uncoated titanium, coated titanium, and alumina) were fixed in three separate tubes, and each tube was subsequently filled with 5.2 ml of citrated whole ovine blood. A tube with no material was also included to provide a baseline of how activated the blood sample was. After submersion, the samples were incubated at 37° C and rocked for 45 min. Once incubation time was complete, blood samples were prepared for flow cytometry using a platelet activation assay. Blood (5 μl) was transferred from the incubation tubes into 5ml round bottom polystyrene tubes with 35 μl of Tyrode’s buffer with BSA and citrate, 5 μl CAPP2A (Serotec, USA) at a concentration of 7.5 μg/ml, 5 μl of GαM IgG-PE (Serotec, USA) at a concentration of 60 μg/ml. The samples were incubated for 20 minutes, and then washed with 1 ml of Tyrode’s buffer with BSA and citrate. After the first wash, 5 μl of MCA 2418 (Serotec, USA) at a concentration of 25 μg/ml was added to each sample. The samples were incubated for a second time, again, for 20 minutes and washed for a final time with 1 ml of Tyrode’s buffer with BSA and citrate. Samples were then fixed with 500 ml of 1% Paraformaldehyde. After fixation, samples were run through a flow cytometer to count the number of fluorescently labeled platelets in each sample. RESULTS After three trials, blood exposed to no material averaged a 17.34% activation. Uncoated titanium displayed a 14.48% activation level, while the coated titanium displayed an average platelet activation level of 16.88% activation. Alumina showed a 15.91% activation level.