Poster N10 - Interaction of Erythrocytes with Nanostructured Polymeric Surfaces

Introduction: The medical sector relies extensively on implant devices to address cardiovascular and orthopedic disorders. Metals such as titanium and its alloys are profoundly used as implant materials because of their advantageous mechanical and chemical properties. However, these implants can lead to issues like thrombosis and inflammation due to unfavorable blood-material interactions. Despite advancements, no surface has achieved complete hemocompatibility. Research endeavors focus on modifying surface characteristics like topography, chemistry, and wettability to enhance compatibility with blood and its constituents.

Current studies highlight the importance of altering surface properties to reduce protein absorption and platelet adhesion, thus mitigating blood clot formation. However, the interaction of erythrocytes (Red Blood Cells) with various surfaces remains relatively unexplored, despite their crucial role in thrombosis processes. Understanding these interactions is vital as improper erythrocyte-surface interactions can lead to further complications for patients.

This study aims to evaluate the hemocompatibility of nanostructured surfaces such as Nanofibers and Nanowires, particularly focusing on the polymer, polycaprolactone (PCL). Utilizing cell cytotoxicity assays, fluorescence microscopy, and scanning electron microscopy, this study assesses erythrocyte behavior and potential hemolytic effects on the nanostructured polymeric surfaces.

The findings from this research could significantly contribute to the development of nanostructured surfaces for enhanced blood-contacting medical devices. By addressing critical issues of thrombosis and inflammation, these surfaces hold promise for improving patient outcomes in cardiovascular and orthopedic interventions.

Materials and

Methods: For this study, the control used was 3D printed Polycaprolactone (PCL) disk. Nanofibers (NF) and Nanowires (NF), made out of PCL, were the two types of nanostructured surfaces, which were the first level of surface modification. The Nanofibers were fabricated using a technique called electrospinning. The PCL disk surface was modified to make PCL Nanowires (NW) via a nano-templating process, using nanopore alumina membranes. The surfaces of both the NF and NW were chemically modified by coating them with just Tanfloc (TN) and Tanfloc + Carboxymethyl-kappa-carrageenan (TN+CMKC).

The morphology of these surfaces were characterized using scanning electron microscopy (SEM). The wettability of the surfaces were determined by water contact angle measurements obtained using goniometer. X-ray photoelectron spectroscopy (XPS) was used to find the elemental composition of each group. X-ray diffraction (XRD) was used to analyze physical properties such as phase composition and crystal structure of the material surface. To examine the anti-bacterial properties, Staphylococcus Aureus and Pseudomonas Aeruginosa were seeded on these surfaces. Their adhesion and proliferation were investigated after 6 hours and 24 hours using SEM and fluorescence microscopy.

Isolated Erythrocytes (Red Blood cells) from whole human blood were seeded on the control and modified nanostructured surfaces. Erythrocytes interaction and morphological changes on these surfaces were studied after 1.5 hours and 6 hours of incubation using SEM and fluorescence microscopy. Cytotoxicity of the surfaces was characterized using lactase dehydrogenase (LDH assay). Whole blood clotting assay was conducted to understand the blood clotting behavior on all of these different surfaces.

Results, Conclusions, and Discussions: SEM results show that NW surfaces present uniform and vertically oriented wire like structures while NF surfaces are composed of unaligned, thin thread like structures spread on a planar level. SEM results also confirm that these surfaces coated with TN and TN+CMKC do not change the NF and NW topography. XPS indicates the successful modification of NF and NW surfaces with TN and CMKC by indicating traces of Nitrogen and Sulphur respectively. The contact angle results show that NW surfaces are hydrophilic and NF surfaces are super-hydrophilic.

Adhesion and proliferation studies show that NF and NW surfaces coated with TN+CMKC had significantly lower number of bacteria after 24 hours in comparison with control PCL. In addition, both surfaces coated with TN and TN+CMKC promoted enhanced antibacterial properties when compared to nanostructured surfaces without any chemical modification.

Percentage of Erythrocytes adhesion and morphological retainment was high for chemically modified nanostructured surfaces than the control surfaces. NW surfaces has higher percentage of free hemoglobin (Absorbance) compared to NF surfaces. None of the nanostructured (modified) surfaces were cytotoxic.

This study helps in understanding the interaction of Erythrocytes with nanostructured polymeric surfaces. This information is vital as improper erythrocyte-surface interactions can lead to further complications in patients with biological implants. The results indicate that the modified surfaces were non-toxic to the Erythrocytes and can be favorable for surfaces of medical devices that come in contact with blood.

Acknowledgements (Optional): The presenting author would like to thank Liszt Madruga for his valuable guidance in this research and Abhishek Bhattacharjee, Somayeh Baghersad and Aniruddha Savargaonkar for all their support. Also, the author thanks Dr. Paulo Soares from Pontifical Catholic University of Paraná, for his help with XRD.