Fucoidan and Hyaluronic Acid Modified ZE21B Magnesium Alloy for Better Hemocompatibility and Vascular Cell Response

Abstract

Magnesium alloy stents exhibit significant potential in the treatment of cardiovascular and cerebrovascular diseases due to their remarkable mechanical support and biodegradability. However, bare magnesium alloy stents often degrade too quickly and exhibit inadequate biocompatibility, which severely restricts their clinical applicability. Herein, a composite coating consisting of an MgF₂ conversion layer, a polydopamine (PDA) layer, fucoidan, and hyaluronic acid was prepared to enhance the corrosion resistance and biocompatibility of ZE21B alloy for a vascular stent application. The modified ZE21B alloy exhibited relatively high surface roughness, moderate wettability, and better corrosion resistance. Moreover, the modified ZE21B alloy with a low hemolysis rate and fibrinogen adsorption level confirmed improved hemocompatibility for medical requirements. Furthermore, the ZE21B alloy modified with fucoidan and hyaluronic acid enhanced the adhesion, proliferation, and NO release of endothelial cells (ECs). Simultaneously, it inhibits the adhesion and proliferation of smooth muscle cells (SMCs), promoting a competitive advantage for ECs over SMCs due to the synergistic effects of fucoidan and hyaluronic acid. The incorporation of fucoidan and hyaluronic acid markedly improved the corrosion resistance and biocompatibility of the ZE21B magnesium alloy. This development presents a straightforward and effective strategy for the advancement of biodegradable vascular stents.

1. Introduction

The global prevalence of cardiovascular disease is alarming, as it continues to be one of the leading causes of mortality worldwide. Interventional therapies, such as percutaneous coronary interventions, can rapidly alleviate blockages in blood vessels by restoring the blood flow through balloon dilation and stent placement [1]. Traditional non-degradable vascular stents, including 316L stainless steel, nickel–titanium alloy, and cobalt-based alloy, can elicit local inflammatory responses when retained in the body for extended periods [2]. These responses may promote the proliferation of vascular smooth muscle cells (SMCs), contribute to intimal hyperplasia, and ultimately lead to in-stent restenosis. Magnesium alloy vascular stents have excellent biodegradability and mechanical support properties, which can overcome the many shortcomings of traditional non-degradable stents and have great potential in the field of cardiovascular disease intervention [3,4]. However, magnesium alloys degrade at an excessive rate and do not possess the necessary biological functions to fulfill the clinical requirements for vascular stents. Consequently, employing various surface modification techniques to enhance the corrosion resistance and biological functionality of magnesium alloys represents an effective strategy for addressing the current challenges associated with magnesium alloy stents [5,6,7].

Currently, a variety of methods are available to enhance the corrosion resistance of magnesium alloys. One approach involves modifying the alloy composition by adding elements such as aluminum and rare earths to refine the grain structure and reduce impurities, thereby promoting element homogenization and improving corrosion resistance [8]. Another strategy is the application of surface protection treatments [9], including chemical conversion processes to create protective passive films [10], and micro-arc oxidation to produce a controllable thickness of a ceramic layer that offers the resistance to both corrosion and wear [11]. The magnesium fluoride (MgF₂) layer serves a crucial function as a chemical conversion coating, significantly enhancing the corrosion resistance of magnesium alloys [12,13]. The chemical reaction between magnesium alloy and hydrofluoric acid solution results in the formation of a MgF₂ layer. This MgF₂ layer is insoluble in water, allowing it to adhere easily to the surface of the magnesium alloy and create a smooth, dense film. The MgF₂ layer effectively prevents corrosive media from contacting the magnesium substrate, thereby reducing the corrosion rate. In corrosive environments, such as bodily fluids, the MgF₂ layer significantly enhances the corrosion resistance of magnesium alloy.

Magnesium alloy stents should have excellent biocompatibility in addition to appropriate corrosion resistance. The MgF₂ layer can significantly enhance the corrosion resistance of magnesium alloys; however, it lacks the biological functionality necessary for vascular stents. Polydopamine (PDA) coatings exhibit excellent biocompatibility and low cytotoxicity, characterized by their strong adhesion capabilities, allowing them to adhere firmly to a variety of substrates [14,15,16]. Furthermore, PDA coatings are rich in functional groups and facilitate convenient secondary functional modifications. The application of a PDA coating to the surface of magnesium alloy stents not only protects the magnesium alloy from rapid degradation but also acts as an intermediate layer, offering reactive sites for subsequent modifications of the magnesium alloy. As a naturally occurring marine polysaccharide, fucoidan exhibits a range of biological functions, including anticoagulant, antithrombotic, and anti-inflammatory properties [17]. Additionally, it modulates vascular cell behavior, enhances endothelial cell (EC) adhesion and proliferation on vascular scaffolds, and decreases the risk of restenosis [18,19]. Hyaluronic acid serves as a crucial component of the vascular extracellular matrix and significantly influences vascular cell behavior, including promoting cell adhesion, proliferation, migration, and differentiation [20,21]. Additionally, hyaluronic acid enhances angiogenesis, emulates natural mechanisms, creates a supportive microenvironment for EC growth, diminishes platelet activation and adsorption capacity, and facilitates vascular endothelialization [22,23,24]. The combination of fucoidan and hyaluronic acid can inhibit thrombosis, promote cell adhesion, and significantly enhance the biocompatibility and pro-endothelialization ability of magnesium alloys. Moreover, the negative electrical properties of fucoidan and the chain structure of hyaluronic acid can jointly inhibit the release of corrosion products and delay the degradation rate of magnesium alloys. The PDA layer can provide binding sites for the immobilization of fucoidan and hyaluronic acid on magnesium alloy. Therefore, it was expected that a composite coating consisting of an MgF₂ layer, a polydopamine (PDA) layer, fucoidan, and hyaluronic acid could significantly enhance the corrosion resistance and biocompatibility of Mg-Zn-Y-Nd (ZE21B) magnesium alloy for a vascular stent application.

This study presents the development of a composite coating composed of an MgF₂ layer, a PDA layer, fucoidan, and hyaluronic acid, applied to ZE21B magnesium alloy to enhance its corrosion resistance and biocompatibility for vascular stent applications. Initially, the ZE21B magnesium alloy was passivated using hydrofluoric acid to create a dense and uniform MgF₂ layer. Subsequently, the alloy was immersed in a dopamine hydrochloride solution to form a PDA coating, followed by the immobilization of natural fucoidan and hyaluronic acid. The surface micromorphology, chemical composition, and structure, surface wettability, and corrosion resistance of the modified ZE21B alloy were characterized. Hemolysis and fibrinogen adsorption assays were performed to evaluate the hemocompatibility of the coated alloy. Furthermore, the adhesion, proliferation, and NO release of ECs, along with the adhesion and proliferation of SMCs, were systematically investigated. Competitive growth behavior between ECs and SMCs in co-culture experiments was also assessed to determine the cytocompatibility of the modified ZE21B alloy.

2. Materials and Methods

2.1. Materials

Mg-Zn-Y-Nd alloy (ZE21B, 2.00 wt% Zn, 0.46 wt% Y, 0.50 wt% Nd) was self-developed by Henan Key Laboratory of Advanced Light Alloy (Zhengzhou, China). Hydrofluoric acid (HF) was bought from the Haohua Chemical Reagent Co., Ltd. (Luoyang, China). Dopamine (DA) hydrochloride was purchased from the Beijing HWRK Chemical Technology Co. (Beijing, China). Fucoidan and hyaluronic acid were purchased from the Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Human umbilical artery smooth muscle cells (SMCs) and venous endothelial cells (ECs) were provided by the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). A nitric oxide (NO) assay kit and cell counting kit-8 (CCK-8) were obtained from the Shanghai Biyuntian Biotechnology Co. (Shanghai, China). FITC-phalloidin and DAPI and TMB were purchased from the Solarbio Biotechnology Co., Ltd. (Beijing, China). An anti-human fibrinogen-HRP conjugated antibody produced in goat was purchased from the Beijing Solepol Co. (Beijing, China). Anti-human fibrinogen γ-chain antibody was purchased from the Shanghai Huzheng Biotechnology Co. (Shanghai, China).

2.2. Preparation of Fucoidan and Hyaluronic Acid Modified ZE21B Alloy

Magnesium alloy (ZE21B) samples (10 mm in diameter, 3 mm in thickness) were polished with progressively finer sandpapers (200#, 400#, 600#, 800#, and 1000#) to obtain the optimal surface quality. After drying, these as-prepared samples were labeled as ZE21B. Subsequently, the polished ZE21B samples were immersed in a 40% (v/v) HF solution for 48 h at an ambient temperature to form a uniform MgF₂ protective coating, with the resulting samples labeled as ZF. These fluoridated samples were then rinsed copiously with deionized water and dried for further use. To dissolve DA, a Tris-HCl buffer solution was initially prepared by mixing tris (hydroxymethyl) aminomethane (Tris) and hydrochloric acid (HCl). The pH value of the buffer solution was adjusted to 8.5 by adding the appropriate amount of HCl solution. The ZF samples were deposited in DA solution (2 mg/mL, pH = 8.5) at room temperature for 24 h to produce the polydopamine (PDA) coating for further immobilization of fucoidan and hyaluronic acid. The PDA deposited samples were then incubated in the mixed solution at 30 °C for 4 h, with a fixed fucoidan concentration (0.5 mg/mL) and varying hyaluronic acid concentration (1.0 mg/mL, 3.0 mg/mL, and 5.0 mg/mL), and the corresponding samples were denoted as ZFFHI, ZFFHII, and ZFFHIII, respectively.

2.3. Characterization of Fucoidan and Hyaluronic Acid Modified ZE21B Alloy

The surface micromorphology of unmodified and modified ZE21B alloy samples was examined using a scanning electron microscope (SEM, ZEISS Sigma 360, Oberkochen, Germany) at an accelerating voltage of 5 kV. The surface chemical composition was analyzed with an X-ray photoelectron spectrometer (XPS, Thermo ESCALAB 250XI, Cheshire East, UK) operating at a pressure of 5 × 10⁻¹⁰ Torr, utilizing Al Kα radiation as the source. Additionally, the surface chemical structure of the samples was characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet iS 10, Madison, WI, USA) over a wavenumber range of 4000 to 600 cm⁻¹, with data collected from 32 scans. The wettability of the samples was assessed using an easy-drop goniometer (Powereach JC2000C, Shanghai, China), with a droplet volume of 3 μL; the average water contact angle was determined from five repeated measurements. The corrosion resistance of the samples in a Hanks’ solution was evaluated through a three-electrode electrochemical system (Risetest RST5200F, Shanghai, China), employing a platinum auxiliary electrode, a saturated calomel electrode (SCE) as the reference, and the sample as the working electrode. Kinetic potential polarization curves were recorded at a scan rate of 0.001 V/s within the range of −2.0 V to −1.0 V (vs. SCE). The corrosion potential and corrosion current density were derived from the polarization curves obtained from the three electrochemical systems.

2.4. Hemolysis Rate of Fucoidan and Hyaluronic Acid Modified ZE21B Alloy

A hemolysis assay was conducted to assess the hemocompatibility of the ZE21B alloy modified with fucoidan and hyaluronic acid. Physiological saline served as the negative control, while ultrapure water was utilized as the positive control. Fresh whole blood was collected from healthy volunteers and diluted with physiological saline in a 4:5 ratio. Initially, ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples were placed into separate 15 mL centrifuge tubes, to which 10 mL of physiological saline was added. For the control groups, 10 mL of physiological saline and ultrapure water were added, respectively. All tubes were subsequently incubated in a water bath at 37 °C for 30 min. Following this, 200 μL of the diluted blood was introduced into each tube, which was then incubated in the 37 °C water bath for 1 h. After the incubation period, 6 mL from each tube was transferred to a new centrifuge tube, and the samples were centrifuged at 2500 rpm for 15 min. Finally, 100 μL of the supernatant from each sample was transferred to a 96-well plate, and absorbance at 540 nm was measured using a microplate reader. The hemolysis rate was calculated using the following formula:

$$\mathrm{Hemolysis}\mathrm{rate} (\%)={\displaystyle \frac{{\mathit{OD}}_t-{\mathit{OD}}_n}{{\mathit{OD}}_p-{\mathit{OD}}_n}}\times 100\%$$

(1)

OD_t, OD_n, and OD_p are the absorbance values of the experimental group, negative control group, and positive control group, respectively.

2.5. Fibrinogen Adsorption of Fucoidan and Hyaluronic Acid Modified ZE21B Alloy

Non-specific protein adsorption induces thrombosis and reduces the hemocompatibility of vascular stents. Fibrinogen, an important coagulation protein in plasma, is often chosen as a model protein to study the hemocompatibility of vascular stents. Platelet-poor plasma (PPP) was obtained by centrifuging fresh blood from healthy volunteers at 3000 rpm for 15 min. Subsequently, 80 µL of PPP was applied to the surfaces of the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples, which were then incubated at 37 °C for one hour. After washing the samples with physiological saline, 50 µL of a 5% bovine serum albumin (BSA) blocking solution was added to the surfaces and incubated at 37 °C for 30 min. The samples were washed again with physiological saline. Following this, 20 µL of an HRP-conjugated goat anti-human fibrinogen adsorption antibody solution (diluted 1:500 in saline) was added to the sample surfaces and incubated at 37 °C for 1 h. After additional washing to remove any residual liquid, 100 µL of tetramethylbenzidine (TMB) solution was added to the sample surfaces and left in the dark for 10 min. Finally, 50 µL of H₂SO₄ solution was added to halt the chromogenic reaction, and the absorbance of the final solution was measured at 450 nm using a microplate reader.

2.6. Growth Behavior of ECs and SMCs on ZE21B Alloy Modified with Fucoidan and Hyaluronic Acid

ECs were cultured on ZE21B, ZF, ZFPCI, ZFPCII, and ZFPCIII substrates at a density of 1.0 × 10⁴ cells/mL to assess their growth behavior. At predetermined time points (1 day and 3 days), samples were transferred to a new 24-well plate, washed with phosphate-buffered saline (PBS), and fixed in a 4% paraformaldehyde solution for 30 min. Subsequently, samples were treated with a 0.5% Triton X-100 solution for 5 min and then sealed with a 5% bovine serum albumin (BSA) solution for 30 min. The ECs on the samples were stained with 5 µL/mL of fluorescein isothiocyanate (FITC)-labeled phalloidin for 50 min and 5 µL/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 4 min in the dark. Finally, fluorescence images of the ECs on each sample were captured using a multifunctional confocal laser scanning microscope (CLSM, Nikon C2 Plus, Tokyo, Japan).

To assess the proliferation of ECs on the samples, a CCK-8 assay was conducted at specified time points of 1 day and 3 days. The cell culture procedures were consistent with those described previously. On days 1 and 3, CCK-8 solution was added to the 24-well culture plates and incubated for an additional 3 h. Subsequently, 100 µL of the solution was transferred to a 96-well plate, and the absorbance of each well was measured at 450 nm using a microplate reader.

The release of NO from ECs cultured on the samples was measured using a nitric oxide detection kit. ECs were seeded onto the samples at a density of 1.0 × 10⁴ cells/mL. After 1 and 3 days, 50 µL of supernatant from each well was transferred into a 96-well plate. Subsequently, 50 µL of Griess I solution and 50 µL of Griess II solution were added to each well. Finally, the absorbance of the resultant mixture was quantified at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

The growth behavior of SMCs on the samples was investigated using cell fluorescence staining assays and the CCK-8 assay. The seeding density of SMCs was set at 8.0 × 10³ cells/mL. The culture and experimental procedures for SMCs were comparable to those employed for ECs.

2.7. Coculture of ECs and SMCs on ZE21B Alloy Modified with Fucoidan and Hyaluronic Acid

The competitive growth of SMCs and ECs on the samples was assessed through a co-culture experiment. ECs and SMCs were stained with the fluorescent dyes CMFDA and CFDA, respectively, resulting in green and red fluorescence. An equal number of ECs and SMCs, specifically 5 × 10³ cells per well, were seeded onto the sample surfaces. Following a 24-h incubation period, the samples were fixed in a 4% paraformaldehyde solution for 30 min and subsequently rinsed three times with phosphate-buffered saline (PBS). Fluorescence images of the ECs and SMCs on the samples were captured using a confocal laser scanning microscope (CLSM). The cell densities of ECs and SMCs, along with the ratios of ECs to SMCs, were calculated from the acquired fluorescence images.

2.8. Statistical Analysis

The data utilized for all statistical analyses were derived from a minimum of three independent experiments and are presented as the mean ± standard deviation. Statistical significance was assessed using one-way analysis of variance (ANOVA), with values of * p < 0.05 indicating statistically significant differences between the datasets.

3. Results and Discussion

3.1. Characterization of Fucoidan and Hyaluronic Acid Modified ZE21B Alloy

A multilayer coating consisting of an MgF₂ layer, a PDA layer, fucoidan and hyaluronic acid was prepared on the ZE21B alloy for better corrosion resistance, hemocompatibility, and vascular cell response. The combination of the PDA layer, fucoidan, and hyaluronic acid formed an outer organic layer that enhanced the hemocompatibility of the ZE21B alloy and promoted the regulation of vascular cell growth. The MgF₂ layer, serving as the inner inorganic layer, primarily protected the ZE21B alloy from corrosion caused by aggressive solutions while enhancing the alloy’s overall corrosion resistance. The SEM images of ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples at different magnifications (3000× and 10,000×) were shown in Figure 1a. The ZE21B sample exhibited a flat and smooth surface at both low and high magnification, except for the presence of some scratches due to the polishing processes. After fluoridation, the surface of the ZF sample was smooth and flat without obvious defects at low magnification, while a large number of fine particulate matter and a small number of holes appeared on the surface of the ZF sample at high magnification. The fine particulate matter may have been formed by the MgF₂, while the holes may have resulted from the leaching of second-phase precipitates from the ZE21B alloy during the fluoridation reaction. After the deposition of PDA and the immobilization of fucoidan and hyaluronic acid, many micro-/nanoparticles appeared on the surfaces of ZFFHI, ZFFHII, and ZFFHIII samples under the observation at low magnification, and the roughness of these sample surfaces became larger compared with that of the ZF sample. Under high magnification, numerous granular aggregates were clearly observed on the surfaces of the ZFFHI, ZFFHII, and ZFFHIII samples. Additionally, a uniformly dense layer of PDA film was distinctly visible adhering to the surfaces of these samples. It is important to emphasize that there were no significant differences in the surface micromorphology of the ZFFHI, ZFFHII, and ZFFHIII samples. This lack of variation could primarily be attributed to the different grafting amounts of hyaluronic acid applied to these samples, which did not result in substantial changes in micromorphology.

Figure 1b presents the XPS survey spectra for the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples, with the corresponding surface chemical compositions summarized in Table S1. The analysis reveals that the primary elements present on the sample surfaces were Mg, C, O, F, S, and N. For the ZE21B sample, the three primary elements detected on the surface were Mg, C, and O. The presence of oxygen was likely attributed to metal oxides of magnesium, while the carbon may result from external contamination. Following fluoridation, a distinct F 1s photoelectronic peak at 685.4 eV was observed in the spectrum of the ZF sample. This peak resulted from the formation of an MgF2 layer on the ZE21B alloy. After the immobilization of fucoidan and hyaluronic acid, the new S 2p and N 1s photoelectronic peaks at 175.0 eV and 400.0 eV appeared in the spectra of ZFFHI, ZFFHII, and ZFFHIII samples because of the sulfur element in the fucoidan and nitrogen element in the hyaluronic acid. As listed in Table S1, the relative atomic contents of the sulfur element on the ZFFHI, ZFFHII, and ZFFHIII samples were 0.53%, 0.40%, and 0.33%, respectively, which exhibited a gradual decrease with the increasing content of hyaluronic acid on the sample surfaces. Moreover, the C/N molar ratios of ZFFHI, ZFFHII, and ZFFHIII samples also presented an increasing tendency along with the increase in hyaluronic acid on the sample surfaces. The XPS results prove the successful immobilization of fucoidan and hyaluronic acid onto the ZE21B magnesium alloy.

The ATR-FTIR spectra of ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples are shown in Figure 1c. The spectra of the ZE21B and ZF samples did not exhibit any visible infrared characteristic absorption peaks, which can be attributed to the absence of organic compounds on their surfaces. For fucoidan and hyaluronic acid modified samples, some obvious characteristic adsorption peaks could be observed due to the introduction of organic natural polymer compounds. The prominent and broad adsorption peak observed at 3343 cm⁻¹ could be attributed to the stretching vibrations of O-H and N-H bonds present in fucoidan and hyaluronic acid. Additionally, the sharp and distinctive peak at 1673 cm⁻¹ corresponds to the stretching vibrations of C=O bonds in the amide groups of hyaluronic acid, while the adsorption peak associated with the stretching vibrations of C-N bonds was observed at 1483 cm⁻¹. Furthermore, the strong characteristic adsorption peak at 1132 cm⁻¹ was primarily due to the stretching vibrations of S=O and C-O bonds found in the sulfonic acid groups, carboxylic acid groups, and amide groups of both fucoidan and hyaluronic acid. The alteration in chemical functional groups on the surface of the samples effectively demonstrates the successful modification of fucoidan and hyaluronic acid onto the ZE21B magnesium alloy.

The wettability of material surfaces significantly affects cell adhesion and growth by modulating cell–interface interactions. Moderately hydrophilic surfaces can facilitate initial cell adhesion and spreading [25,26]. The water contact angles of ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples are shown in Figure 1d. The ZF sample showed the best surface wettability, with the lowest water contact angle of 27.0 ± 1.4°. The water contact angles of ZFFHI, ZFFHII, and ZFFHIII samples were 35.6 ± 1.9°, 36.9 ± 4.5°, and 31.0 ± 0.7°, respectively, which were slightly higher than that of the ZF sample and displayed moderate surface wettability. This was mainly because both fucoidan and hyaluronic acid were hydrophilic polymers. Notably, there were no statistically significant differences in the surface wettability between the ZFFHI, ZFFHII, and ZFFHIII samples. This suggests that varying concentrations of hyaluronic acid did not produce distinct changes in surface hydrophilicity.

Figure 1e presents the polarization curves for the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples. The corresponding corrosion potentials and corrosion current densities are summarized in Table S2. The corrosion resistance of magnesium alloys is crucial to their use as vascular scaffolds. Poor corrosion resistance in magnesium alloy stents can lead to rapid degradation in physiological environments, resulting in premature loss of mechanical support and compromised vascular patency. Conversely, stents that exhibit good corrosion resistance will degrade more slowly and be gradually absorbed by the body after fulfilling their role in vascular remodeling. In general, the lower the corrosion current is, the better the corrosion resistance is. The ZF sample exhibited the highest corrosion resistance, characterized by a corrosion current density of 1.8 × 10⁻⁸ A/cm², which was significantly lower than that of the ZE21B sample, which displayed a corrosion current density of 1.1 × 10⁻⁶ A/cm². The corrosion current densities for the ZFFHI, ZFFHII, and ZFFHIII samples were 7.8 × 10⁻⁷ A/cm², 9.6 × 10⁻⁷ A/cm², and 7.5 × 10⁻⁷ A/cm², respectively, indicating better corrosion resistance than that of the ZE21B alloy. This enhanced performance could be attributed primarily to the protective roles of the MgF₂ layer, PDA layer, fucoidan, and hyaluronic acid, which collectively act as a physical barrier to mitigate rapid corrosion of the ZE21B alloy in the corrosive medium.

3.2. In Vitro Hemocompatibility of Fucoidan and Hyaluronic Acid Modified ZE21B Alloy

Hemocompatibility is a critical indicator of the safety and efficacy of blood–contact medical devices, significantly influencing both the clinical risks associated with the device and patient prognosis [27,28]. Direct contact between medical devices and blood can precipitate serious adverse reactions if the surface properties or chemical composition of the materials are not compatible with blood components. Specifically, vascular stents with inadequate blood compatibility are at a heightened risk of thrombus formation on their surfaces, which can result in vessel occlusion or heart failure. Hemolysis and fibrinogen adsorption assays were performed to assess the hemocompatibility of the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples. The results of these assays are illustrated in Figure 2. The blank ZE21B sample exhibited the highest hemolysis rate at 4.6 ± 0.4%, caused by the release of substantial quantities of metal ions and alterations in the local microenvironmental pH value. The formation of an MgF₂ layer led to a reduction in the hemolysis rate of the ZE21B alloy to 3.8 ± 0.2%. This decrease could be attributed to the protective effect of the MgF₂ layer, which minimized the leaching of metal ions. The hemolysis rates of the ZFFHI, ZFFHII, and ZFFHIII samples were 2.9 ± 0.3%, 2.3 ± 0.5%, and 1.6 ± 0.4%, respectively, much lower than those of the ZE21B and ZF samples. This could be primarily attributed to the excellent compatibility of natural fucoidan and hyaluronic acid with red blood cells, which prevents their rupture. Notably, the hemolysis rates of the ZFFHI, ZFFHII, and ZFFHIII samples exhibited a decreasing trend as the concentration of hyaluronic acid on the modified ZE21B alloy increased. This trend suggests a reduction in the risk of hemolysis for the ZE21B magnesium alloy when utilized in vascular stent applications. The hemolysis rates of the ZFFHI, ZFFHII, and ZFFHIII samples conformed to the safety threshold (below 5%) established by ISO 10993-4 for blood–contact medical devices [29].

Non-specific protein adsorption significantly affects the performances of blood–contact medical devices. Proteins that adsorb onto device surfaces can alter the surface properties of the materials, enhance platelet adhesion and activation, and contribute to thrombus formation, ultimately resulting in vascular occlusion or device malfunction [30,31]. Fibrinogen was selected as a model protein to assess non-specific protein adsorption in samples ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII. As illustrated in Figure 2b, the levels of fibrinogen adsorption in the ZE21B and ZF samples were significantly higher than those in the ZFFHI, ZFFHII, and ZFFHIII samples. The absence of specific binding sites on the ZE21B and ZF samples hindered the effective repulsion of non-target proteins, resulting in significant adsorption of non-specific proteins. Following modification with natural fucoidan and hyaluronic acid, these two agents demonstrated a synergistic effect in inhibiting non-specific protein adsorption on the ZE21B alloy. In physiological environments, proteins typically exhibit a negative charge; therefore, the negatively charged fucoidan and hyaluronic acid can reduce non-specific protein adsorption by means of electrostatic repulsion. Taking the fibrinogen adsorption level of the ZE21B sample as a reference, the fibrinogen adsorption levels of the ZFFHI, ZFFHII, and ZFFHIII samples were 0.26, 0.25, and 0.23 times higher than that of the ZE21B sample, respectively. The relatively low hemolysis rates (less than 5%) and the minimal fibrinogen adsorption levels observed in the ZFFHI, ZFFHII, and ZFFHIII samples demonstrate the exceptional hemocompatibility of the fucoidan and hyaluronic acid modified ZE21B alloy, making it a suitable candidate for vascular stent applications.

3.3. Adhesion, Proliferation, and NO Release of ECs on Modified ZE21B Alloy

The growth behavior of ECs, including cell adhesion, proliferation, and NO release, was investigated on the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples through fluorescence staining assays, CCK-8 assays, and NO detection assays. The fluorescence images, proliferation levels, and NO release from ECs cultured on these samples for 1 and 3 days are presented in Figure 3. As illustrated in Figure 3a, the substantial accumulation of metal ions resulting from the degradation of the ZE21B alloy led to apoptosis in the surrounding cells. Consequently, after 1 day and 3 days of cell culture, there were nearly no ECs in a healthy condition on the ZE21B alloy surface. In the case of the ZF, ZFFHI, ZFFHII, and ZFFHIII samples, the ECs typically exhibited growth, spreading, and proliferation on day 1 and day 3. On day 1, the ECs on the samples displayed either rounded or irregular morphologies, accompanied by a limited coverage area. This observation suggests that the cells were in a growth status that had not yet reached full spreading. The number of ECs in the ZFFHI and ZFFHIII groups was significantly higher than that in the other groups. On day 3, the ECs on the ZF, ZFFHI, ZFFHII, and ZFFHIII samples exhibited a flattened polygonal or cobblestone-like morphology. The cells displayed clearly defined margins and abundant pseudopods, indicating effective spreading across the sample surfaces. In addition to a substantial expansion in the coverage area of ECs, there was a notable increase in the quantity of ECs present in the samples, particularly within the ZFFHI, ZFFHII, and ZFFHIII groups.

The results of CCK-8 assays and cell fluorescence staining assays are basically consistent. As shown in Figure 3b, after 1 day of cell culture, the ECs on each sample were at a low proliferation level and the ZFFHI group showed the highest level of cell proliferation. After 3 days of cell culture, the proliferation levels of ECs were significantly elevated in all experimental groups. Specifically, the proliferation levels of ECs in the ZFFHI, ZFFHII, and ZFFHIII groups were markedly higher than those in the ZE21B and ZF groups. When taking the cell proliferation level of the ZF group as a reference, the proliferation levels of the ZFFHI, ZFFHII, and ZFFHIII groups were found to be 2.46, 2.67, and 2.73 times greater, respectively. Moreover, no statistically significant difference in the proliferation levels of ECs was observed between the ZFFHII and ZFFHIII groups. The growth rate of ECs in the ZFFHI, ZFFHII, and ZFFHIII groups were distinctly higher than that in ZF group, suggesting the natural fucoidan and hyaluronic acid could promote the rapid proliferation of ECs on the modified ZE21B alloy.

No gas signaling molecule significantly affects ECs and SMCs. It can maintain the normal physiological functions of ECs, promote the release of vasodilatory factors, and preserve endothelial integrity. Additionally, it inhibits the excessive proliferation of SMCs and prevents vascular stenosis [32,33]. Figure 3c illustrates the NO release levels of ECs cultured on the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples after 1 and 3 days. After 1 day of culture, the NO release levels of ECs in the ZFFHI, ZFFHII, and ZFFHIII groups were significantly higher than those in the ZE21B and ZF groups, although all groups exhibited lower overall levels. After 3 days of culture, NO release levels were markedly elevated in all experimental groups except for the ZE21B group, suggesting that ECs were in a favorable growth microenvironment. Specifically, the NO release levels in the ZFFHI, ZFFHII, and ZFFHIII groups were 2.69, 3.10, and 2.37 times greater than that of the ZF group, respectively. Among the ZFFHI, ZFFHII, and ZFFHIII groups, the ZFFHII groups presented the highest NO release level, suggesting that the optimal composition of fucoidan and hyaluronic acid on the ZE21B alloy could be more favorable for providing a good microenvironment for ECs growth, activating nitric oxide synthase in the ECs, and enhancing cell growth activity.

3.4. Adhesion and Proliferation of SMCs on Modified ZE21B Alloy

Vascular SMCs significantly influence the performance of vascular stents. Following stent implantation, SMCs migrate from the medial layer to the intima and undergo extensive proliferation, with their phenotypic transformation being associated with in-stent restenosis. Excessive proliferation of SMCs can result in neointimal thickening, which narrows the vessel lumen and compromises stent patency [34,35]. The adhesion and proliferation of SMCs on ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples were investigated using fluorescence staining assays and CCK-8 assays after 1 and 3 days of culture, with the results presented in Figure 4. Fluorescence images of SMCs on the samples at the specified time points are shown in Figure 4a. Increased concentrations of metal ions released from the degradation of the ZE21B alloy resulted in apoptosis in SMCs, leading to a significant reduction in viable SMCs on the ZE21B surface after both 1 and 3 days of culture. On day 1, SMCs demonstrated normal adhesion and growth on the ZF, ZFFHI, ZFFHII, and ZFFHIII samples; however, both the coverage area and the number of SMCs on these samples remained relatively low. Notably, the number of SMCs in the ZFFHI, ZFFHII, and ZFFHIII groups was significantly higher than that in the ZF group. After 3 days of culture, there was a marked enhancement in both the spreading area and the number of SMCs on the ZF, ZFFHI, ZFFHII, and ZFFHIII samples. These SMCs exhibited an elongated, shuttle-like shape on the surfaces, displaying distinct cell outlines and maintaining stable morphology.

The proliferation levels of SMCs determined by CCK-8 assays on the samples after 1 day and 3 days of cell culture are shown in Figure 4b. On day 1, the proliferation levels of SMCs in the ZFFHI, ZFFHII, and ZFFHIII groups were much higher than those in the ZE21B and ZF groups. When the cell culture time was extended to 3 days, the number of SMCs in the ZF, ZFFHI, ZFFHII, and ZFFHIII groups increased, especially for the ZF group. The ZFFHIII groups demonstrated the highest proliferation levels of SMCs. However, there was no statistically significant difference in the number of SMCs between the ZF, ZFFHI, and ZFFHII groups. In contrast to the ZF group, the proliferation of SMCs in the ZFFHI, ZFFHII, and ZFFHIII groups was notably inhibited, despite an overall increase in the number of SMCs. This phenomenon could be primarily attributed to the inhibitory effects of fucoidan and hyaluronic acid on SMC proliferation. It should be noted that due to the short cell culture time, it was difficult to determine what roles and effects fucoidan and hyaluronic acid had on ECs and SMCs, respectively.

3.5. Competitive Growth of ECs and SMCs on Modified ZE21B Alloy

The competitive growth of SMCs and ECs significantly influences the performance of vascular stents [36,37]. Excessive proliferation of SMCs can lead to their migration to the intima, where they secrete an extracellular matrix, resulting in neointimal thickening. This process may trigger in-stent restenosis and compromise vessel patency. Conversely, well-developed ECs can effectively prevent thrombosis by properly covering the stent’s surface. When these two cell types compete for growth, a scenario in which ECs are inhibited and SMCs dominate can diminish the stent’s anti-thrombotic and anti-restenosis properties. In contrast, when ECs predominate, normal vascular function is maintained, thereby ensuring the long-term effectiveness of the stent. The competitive growth behavior of ECs and SMCs on the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII samples was assessed after 1 day of coculture using cell fluorescence staining assays within the same culture system. The fluorescence images of ECs stained with green and SMCs stained with red on the samples are shown in Figure 5a, and the corresponding cell densities and ratios of ECs/SMCs are presented in Figure 5b. The ECs and SMCs in the ZE21B, ZF, ZFFHI, ZFFHII, and ZFFHIII groups could normally grow and proliferate after 1 day of culture. The total number of ECs and SMCs in the ZFFHI, ZFFHII, and ZFFHIII groups was significantly greater than that in the ZE21B and ZF groups. For example, the cell densities of ECs and SMCs in the ZF group were measured at 75.0 ± 2.4 cells/mm² and 47.5 ± 2.3 cells/mm², respectively. In contrast, the ZFFHII group demonstrated significantly elevated cell densities, with 212.5 ± 3.1 cells/mm² for ECs and 115.0 ± 2.4 cells/mm² for SMCs. The ECs/SMCs ratios for the ZFFHI, ZFFHII, and ZFFHIII groups were 1.75, 1.85, and 1.56, respectively. The ZFFHII group possessed the highest ratio of ECs/SMCs, which reveals that an appropriate composition of fucoidan and hyaluronic acid on the ZE21B alloy could significantly enhance the competitive growth advantage of ECs over SMCs. The enhanced growth of ECs compared to SMCs on the fucoidan and hyaluronic acid-modified ZE21B alloy demonstrates potential for promoting rapid endothelialization while inhibiting intimal hyperplasia.

4. Conclusions

In summary, we developed a composite coating composed of an MgF₂ layer, a PDA layer, fucoidan, and hyaluronic acid to enhance the corrosion resistance and biocompatibility of the ZE21B alloy for vascular stent applications. The combined MgF₂ and PDA layers effectively protected the ZE21B alloy from excessive degradation. Furthermore, the composite coating results in a modified ZE21B alloy with a lower corrosion current density, demonstrating superior corrosion resistance compared to the uncoated ZE21B alloy. The inclusion of natural fucoidan and hyaluronic acid in the coating significantly enhanced the hemocompatibility of the ZE21B alloy and influenced the cellular behavior of vascular cells on the alloy surface. The modified ZE21B alloy promoted the adhesion, proliferation, and NO release of ECs, thereby enhancing the competitive growth advantage of ECs over SMCs. This research presents a straightforward and effective strategy for the development of biomacromolecule-based functional coatings for biodegradable vascular stents.