Poster U5 - Enhanced Hemocompatibility of Titania Nanotube Surfaces with Tanfloc/Carboxymethyl-Kappa-Carrageenan Polyelectrolyte Multilayers

Introduction: Cardiovascular diseases are a leading cause of mortality globally and in the United States. Coronary artery disease, primarily due to atherosclerosis, often results in myocardial infarction. Vascular stents are used to treat severe cases, but restenosis, caused by smooth muscle cell proliferation, remains a challenge. Drug-eluting stents help prevent restenosis but can inhibit endothelialization, increasing late stent thrombosis risk. Promoting rapid and complete endothelialization of implant surfaces is vital to improving hemocompatibility and preventing complications like thrombosis and restenosis.
This study addresses the need for sustainable antithrombotic coatings for titanium-based medical devices. Traditional heparin (HEP) coatings, while effective, pose sustainability and adverse effect challenges. We propose using renewable biopolymers, carboxymethyl-kappa-carrageenan (CMKC) and Tanfloc (TAN), for surface modification. CMKC, a modified polysaccharide similar to heparin, offers comparable antithrombogenic properties without the drawbacks of animal-derived heparin. This shift to plant-based sources, such as algae-derived CMKC, represents a significant step towards ethical and sustainable medical material science.
Our study demonstrates enhanced blood compatibility and anticoagulant effects of CMKC and TAN-coated surfaces compared to uncoated controls and similar coatings with heparin. We evaluate these coatings' impact on endothelial cells and smooth muscle cell behavior, focusing on cell adhesion and proliferation. The findings aim to reduce thrombosis risks, promote endothelialization, while supporting environmental sustainability goals.

Materials and

Methods: This study proposes an approach to enhance the hemocompatibility of titania nanotube (TiNT) surfaces using biopolymer multilayers. Specifically, polysaccharide-based CMKC and condensed tannin derivative TAN were used to create PEMs. The TAN-CMKC PEMs were compared with similar PEMs containing kappa carrageenan (TAN-KC), hyaluronan (TAN-HA), and heparin (TAN-HEP) to assess their impact on blood-material interactions.
Titanium sheets were anodized to create TiNT surfaces. Subsequently, PEMs were deposited using a layer-by-layer approach. Hemocompatibility was assessed by determining fibrinogen adsorption, platelet adhesion and activation, and whole blood clotting kinetics. Fibrinogen adsorption was quantified using an ELISA. Platelet adhesion and activation were analyzed through fluorescence microscopy and scanning electron microscopy. Blood clotting kinetics were assessed by measuring the absorbance of free hemoglobin.
Human aortic endothelial cells (HAECs) and human aortic smooth muscle cells (HASMCs) were cultured in respective growth media and seeded on the sterilized TiNT surfaces at a concentration of 2.0 × 104 cells/ml. Adhesion and proliferation were investigated using fluorescence microscopy, and cell morphology was examined using SEM.
Immunofluorescent staining was used to evaluate the expression of specific marker proteins in HAECs and HASMCs. The HAECs were immunostained for VE-cadherin and vWF after 7 and 10 days of culture, and the HASMCs were immunostained for calponin after 4 and 7 days of culture. For EC migration studies, a scratch assay was performed on confluent HAEC monolayers, and the migration was observed using fluorescence microscopy.
These methods collectively aimed to assess the hemocompatibility and cellular behavior on biopolymer-modified TiNT surfaces.

Results, Conclusions, and Discussions:

Results:
TiNT surfaces modified with CMKC and TAN PEMs demonstrated superior hemocompatibility compared to other surfaces studied. This was evidenced by a significant decrease in fibrinogen adsorption compared to control (Figure 1) and a reduction in platelet adhesion and activation (Figure 2). The kinetics of whole blood clotting showed substantial inhibition of clot formation on CMKC and TAN modifications (Figure 3). Comparative analyses revealed that CMKC-containing surfaces had performance comparable to, if not better than, TAN-HEP PEMs in reducing clot formation. These findings underscore CMKC's potential as a sustainable and effective heparin alternative.
SMC behavior on various modified TiNT surfaces over 1, 4, and 7 days is illustrated in Figure 4. On day 1, SMCs showed minimal adhesion and spreading across all surfaces. By day 4, HASMC adhesion and spreading remained limited. By day 7, HASMC proliferation remained low on TAN-CMKC surfaces, indicating that these coatings effectively inhibit HASMC proliferation. Conversely, TAN-KC surfaces promoted SMC proliferation, suggesting that TAN-CMKC PEMs are more effective in preventing HASMC growth than other coatings. The presence of green-stained calponin in HASMCs suggests maintained smooth muscle cell phenotype, further confirming that the coatings limit HASMC growth.
HAEC adhesion and proliferation were enhanced on TAN-CMKC surfaces. SEM and fluorescence microscopy confirmed increased cell spreading and coverage. Immunofluorescence results indicated higher VE-cadherin and vWF expression on TAN-CMKC surfaces, demonstrating maintained HAEC functionality. The HAEC migration study revealed that TAN-CMKC surfaces showed improved migration compared to other surfaces, indicating superior support for endothelialization.
Discussions:
The findings highlight the potential of CMKC as a sustainable and effective antithrombotic coating for blood-contacting medical devices. The enhanced HAEC adhesion, proliferation, and migration, along with reduced SMC growth, indicate that these surfaces could mitigate complications such as thrombosis and restenosis.

Conclusions:
The study demonstrates that CMKC and TAN PEMs enhance the hemocompatibility of TiNT surfaces. The results indicate significant reductions in clot formation and improved HAEC proliferation, functionality, and migration, suggesting that CMKC is a viable heparin alternative. The combination of biopolymer coatings and nanotopography plays a critical role in maintaining blood compatibility and supporting endothelialization while inhibiting HASMC proliferation.

Acknowledgements (Optional): National Institutes of Health (NIH), grant number 1R21EB033511