Our study in bioengineering
arrived at the main conclusion that a plasma physics based coating technology
used to modify a material surface, can be applied to existing commercially
available cobalt chromium stents, for improved biocompatibility. Research
included experimental work in engineering, plasma physics and biochemistry to
address primary aims, completed at the University of Sydney, Australia. The development
of cardiovascular stents to treat coronary artery disease (CAD), was a significant
early innovation (1). The challenge of addressing
sub-optimal biocompatibility of cardiovascular stents via reproducible,
multidisciplinary research, led to my interest in bioengineering cobalt
chromium stent material alloy L605 (2).
Previous research work in plasma activated coating (PAC) technology were first conducted on a stainless steel biomaterial alloy 316L (3) (4) (5), followed by feasible translation of the optimized PAC recipe to a stainless steel coronary stent platform of the same alloy (6). The PAC technique creates a free radical reservoir on any solid material, to enable favourable biological interactions (5). Primarily cardiovascular stent modification technologies aim to lessen clinical complications of thrombosis, neointimal hyperplasia (inflammatory response resulting in artery wall thickening) and restenosis (re-narrowing of artery wall after stent implantation) (7). The intended outcome of stent modification is biofunctionalization (8), i.e., development of a stent material that displays biological function and elicits a biologically favourable response in vitro and in vivo (9).
The study was a first to deposit and characterise the PAC-L605
modification (10). Briefly, the salient
features of PAC technology observed include; surface hydrophilicity, nanoscale surface
thickness, microscale roughness, difference in surface chemistry compared to
bare metal alloy L605 and non-delamination of PAC under increasing force of nanoindentation (10) (11). The study was also
a first to visualize PAC-L605 interface via focused ion beam milling (FIB) and
high resolution transmission electron microscopy (HR-TEM) to demonstrate the robust, nanoscale
ionic stitching at plasma coating-material interface (Figure 1). Two PAC recipes (PAC1/PAC2) were then developed on cobalt chromium alloy L605 and used simultaneously to study surface biofunctionalization by replicating in vitro assays (4) (12).
Figure 1. The HRTEM image (A) and corresponding inverse fast Fourier transform (IFFT) pseudo colour image (B) show the PAC-L605 interface. Notice nanoscale ionic stitching seen in gray (A) and green (B) in the corresponding images at 1.2 M x magnification and 1.1 Å resolution. Figure reproduced from reference (10).
Confirmatory results proved covalent protein binding capacity on PAC-L605 with extra cellular matrix protein tropoelastin (TE) (9). The recombinant human tropoelastin used in the study was synthesized and provided by the lab of Professor Anthony Weiss, School of Biochemistry and Molecular Biotechnology, University of Sydney (13). The outcome resulted in the use of PAC-L605 with TE (PAC-L605-TE) as another variable of surface modification, for in-lab tests conducted to understand cell-material and blood-material interactions. In total I used five different biomaterial surfaces to first observe cell on material interactions. After 5 days of cell culture, homogeneous anchorage of human coronary artery endothelial cell (hCAEC) cytoskeleton was observed on PAC2 surfaces with or without tropoelastin (Figure 2).
Figure 2. The filamentous actin (F-actin) cytoskeleton of HCAECs on biomaterials identified and visualized with immunofluorescent staining at 40x magnification. F-actin were homogeneously spread on PAC2 surfaces with/without tropoelastin (highlighted in red) and chosen for further experiments in cell-material culture (9). Figure reproduced from reference (9).
Next, to understand blood-material interactions, I first conducted static platelet rich plasma (PRP) assays and observed that protein in PRP prevented platelet/fibrin adherence to hydrophilic PAC and PAC/TE surfaces, while fibrinogen fibrils and platelets adhere to alloy L605 (Figure 3) (9). The PAC surfaces were exposed to air over a period of time, to induce surface oxidation/ageing and reassessed with static PRP, to show that PAC retained hemocompatibility with age (9). Blood-material contact was also conducted under modified flow conditions with 5 different biomaterials, as with cell culture before, to demonstrate lower clot formation specifically on PAC1 recipe surfaces, with or without tropoelastin. Similar to static assays, flow assays too did not show reduced hemocompatibility of PAC with age (or time of exposure to air) (9). After blood flow tests, I conducted immunofluorescent staining of material surfaces to quantify surface fibrin, and results showed significantly lower deposition of fibrin on the hemocompatible PAC (Figure 4).
Figure 3. Surface hemocompatibility tested with static PRP assays: platelet/fibrin adherence was not observed on modified surfaces (A,B) whilst aggregating on alloy L605 cobalt chromium surfaces (C), images at 1.2 K x magnification obtained via scanning electron microscopy (SEM). Figure reproduced from reference (9).
Figure 4. Comparatively low surface fibrin clot deposition (green) with nuclei (blue) observed on hemocompatible PAC surfaces with immunofluorescence (IF), after blood flow assay (9). Figure reproduced from reference (9).
To complete the biofunctionalization study, I carried out
standardized tests laid down in ISO 10993 part 4 for the biological evaluation
of medical device materials in contact with blood (12). Since guidelines were specific for the evaluation of a final, finished medical device (12), tests
conducted were considered a pre-analytical optimization step, to be reassessed
in-lab after translation of surface coating to a cardiovascular stent platform. Briefly, biochemical events occurring
after blood-material contact, activate biomarkers of coagulation and
inflammation quantified in the study (Figure 5). Individual tests were conducted to specifically identify and quantify 5 biomarkers; 1) Thrombin Antithrombin complex (TAT), 2) Beta thromboglobulin
(β-TG), 3) soluble P-selectin (sP-selectin), 4) soluble terminal complex C5b9
(SC5b-9) and polymorphonuclear neutrophil elastase (PMN elastase) (9). Results showed comparatively lower plasma biomarkers of TAT, β-TG,
sP-selectin and SC5b-9 on modified PAC surfaces (9).
Figure 5. A highly simplified summary of the biochemical reactions triggered after blood-biomaterial contact. The highlighted components were quantified in the study. Figure reproduced from reference (9).
To recap, the study demonstrated biofunctionalization of plasma modified cobalt chromium surfaces (PAC-L605) in vitro for the first time, with applications at the artery-stent material interface for vascular compatibility of cardiovascular stents. A notable observation in bioengineering material surfaces for biofunctionalization is that isotropic nanoscale structures developed with micron-/sub-micron scale roughness facilitate cell anchorage, proliferation and differentiation (10)(14). The cell cytoskeleton is theorized to play an integral role in biomechanical sensing for coordinated attachment to a substrate (15) as inferred in the study. The in vitro models of hemocompatibility were an oversimplification of a very complex physiological system, based on previous experimental standardization (16) (17). The hemocompatibility of PAC surfaces (with/without tropoelastin) observed during flow, static and standardized blood assays were related to surface hydrophilicity (10)(18), furthermore, the properties of surface modification retained overtime regardless of surface oxidation/ageing. The multidisciplinary study arrived at the main conclusion that PAC technology used to modify alloy L605, can be applied to existing commercially available cobalt chromium stents for further in lab tests, prior to pre-clinical investigations (9).
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