Polydopamine (PDA) coatings with endothelial vascular growth factor (VEGF) immobilization inhibiting neointimal formation post zinc (Zn) wire implantation in rat aortas

doi:10.21203/rs.3.rs-3060311/v1

Download PDF

Research article

Polydopamine (PDA) coatings with endothelial vascular growth factor (VEGF) immobilization inhibiting neointimal formation post zinc (Zn) wire implantation in rat aortas

https://doi.org/10.21203/rs.3.rs-3060311/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 04 Sep, 2023

Read the published version in Biomaterials Research →

You are reading this latest preprint version

Background Bioresorbable stents are designed to provide temporary mechanical support to the coronary arteries and then slowly degrade in vivo to avoid chronic inflammation. Zinc (Zn) is a promising material for bioresorbable stents; However, it can cause inflammation and neointimal formation after being implanted into blood vessels.

Methods To improve biocompatibility of Zn, we first coated it with polydopamine (PDA), followed by immobilization of endothelial vascular growth factor (VEGF) onto the PDA coatings. Adhesion, proliferation, and phenotype maintenance of endothelial cells (ECs) on the coated Zn were evaluated. Then, a wire aortic implantation model in rats mimicking endovascular stent implantation in humans was used to assess vascular responses to the coated Zn wires in vivo. Thrombosis in aortas post Zn wire implantation, degradation of Zn wires in vivo, neointimal formation surrounding Zn wires, and macrophage infiltration and extracellular matrix (ECM) remodeling in the neointimas were examined.

Results In vitro data showed that PDA-coated Zn encouraged EC adhesion, spreading, proliferation, and phenotype maintenance on its surfaces. VEGF functionalization on PDA coatings further enhanced the biocompatibility of Zn to ECs. Implantation of PDA-coated Zn wires into rat aortas didn’t cause thrombosis and showed a faster blood flow than pure Zn or the Zn wires coated with VEGF alone. In addition, the PDA coating didn’t affect the degradation of Zn wires in vivo. Besides, the PDA-coated Zn wires reduced neointimal formation, increased EC coverage, decreased macrophage infiltration, and declined aggrecan accumulation in ECM. VEGF immobilization onto PDA coatings didn’t cause thrombosis and affect Zn degradation in vivo as well, and further increased the endothelization percentage as compared to PDA coating alone, thus resulting in thinner neointimas.

Conclusion These results indicate that PDA coatings with VEGF immobilization would be a promising approach to functionalize Zn surfaces to increase biocompatibility, reduce inflammation, and inhibit neointimal formation after Zn implantation in vivo.

Polydopamine (PDA)

Endothelial vascular growth factor (VEGF)

Neointimal formation

Zinc (Zn)

Degradation

Endovascular stent placement post coronary angioplasty is known to prevent acute vascular cloture and chronic artery narrowing arising from vascular remodeling. Unfortunately, non-degradable stents can remain in blood vessels for lengthy periods and trigger chronic inflammation via immune cells as foreign bodies. The inflammation then stimulates the local vascular smooth muscle cell (SMC) proliferation and eventually leads to in-stent restenosis [1]. Bioresorbable stents are then designed to provide temporary mechanical support to the coronary arteries and degrade slowly in vivo to avoid chronic inflammation caused by non-degradable stents. Zinc (Zn) is a promising bioresorbable stent material as it can degrade slowly to provide sufficient mechanical support to blood vessels before vascular remodeling is finished and then quickly after complete vascular remodeling [2–6]. Furthermore, Zn is essential for numerous enzymatic reactions in vivo and has unique cardioprotective roles against atherosclerosis [7]. Mechanical strength of Zn is also much higher than bioresorbable polymers, such as poly-L-lactic acid [3]. Nonetheless, Zn can induce inflammation and neointimal formation in blood vessels post implantation [8–12]. Meanwhile, the excessive Zn²⁺ released when Zn degrades can hinder cell adhesion and mobility [13]. Therefore, biocompatibility of Zn still needs to be improved to increase the efficacy of the bioresorbable stents and hence the clinical outcome of patients implanted with the stents.

To overcome the challenge of poor cell adhesion and growth on Zn substrates, surface modifications are usually required. Manipulation of surface topography on Zn substrates is one strategy. It has been shown that topography in microscales on Zn plates can reduce macrophage inflammatory polarization and enhance adhesion and differentiation of bone cells as compared to topography in nanoscales [14]. Another study shows that zinc phosphate coatings on Zn surfaces can improve the viability, adhesion and proliferation of the seeded cells [15]. Although the in vitro data show promising results, further studies are needed to validate the efficacy of these coatings in vivo.

Dopamine can spontaneously polymerize under alkaline conditions to deposit a thin and adherent polydopamine (PDA) coating onto various substrates [16]. Catechol and quinone functional groups in PDA coatings make nucleophiles can be covalently coupled to them as ad-layers [17]. In addition, the coating method is simple, which just needs immersing objects in an aqueous dopamine solution for dip-coating. Meanwhile, dopamine coating alone has been shown to increase biocompatibility of implanted materials [18]. PDA can also eliminate reactive oxygen species (ROS) as radical scavengers because of abundant phenol groups [19]. Due to these unique properties, PDA is widely used for surface functionalization of medical implants. For example, hyaluronic acid and PDA coatings are fabricated on 316L stainless steels or cobalt-chromium to improve their hemocompatibility and reduce the activation of macrophages on their surfaces [20–22]. A similar strategy is also used to modify surfaces of poly(dimethylsiloxane) (PDMS) or poly(urethane) (PU) substrates, which shows an enhanced hemocompatibility and anti-inflammation effects as compared to those unmodified PDMS or PU [21, 23]. Vascular endothelial growth factors (VEGFs) immobilized on of PDA coatings increase endothelial cell (EC) attachment, viability, and proliferation onto 316L stainless steels and titanium substrates [24, 25]. PDA coating constructed on titanium substrates can also be used to immobilize heparin/poly L-lysine nanoparticles to enhance hemocompatibility and anti-inflammatory properties of the titanium substrates [26, 27]. However, most of the above results were obtained through in vitro studies, and the effects of the PDA-based coating in vivo are lacking. In addition, whether PDA-based coating can improve biocompatibility of Zn-based stents remains unknown.

In this study, we systematically investigated PDA-based coatings for surface functionalization of Zn, a new material for bioabsorbable stents (Fig. 1). We first coated Zn with PDA and VEGF to increase its biocompatibility toward ECs. The adhesion, proliferation, and phenotype maintenance of ECs on the coated Zn were evaluated. Then, a wire aortic implantation model in rats mimicking endovascular stent implantation in humans was used to assess vascular responses to the coated Zn wires in vivo. Thrombosis in aortas post Zn wire implantation, degradation of Zn wires in vivo, neointimal formation surrounding Zn wires, and macrophage infiltration and extracellular matrix (ECM) remodeling in the neointimas were examined.

2.1 Surface functionalization

Zn wires (0.125 mm in diameter) or sheets (0.05 mm in thickness, Goodfellow, UK) were immersed in dopamine hydrochloride solutions (Sigma, 1 mg/mL in 10 mM Tris buffer, pH 8.5) at room temperature overnight for dopamine polymerization. After that, the Zn wires or sheets were washed with deionized (DI) water to remove unbound dopamine. The polydopamine (PDA)-coated wires or sheets were further immersed in VEGF¹⁶⁵ protein solutions (MCE, China, 0.5 mg/mL in 10 mM Tris buffer, pH 8.5) at 37 ℃ overnight for VEGF¹⁶⁵ deposition. The Zn wires or sheets that were coated with PDA or VEGF165 alone were used as a comparison. The human recombinant VEGF¹⁶⁵ was used for cell culture in vitro, and the rat recombinant VEGF¹⁶⁵ was used for animal experiment in vivo.

2.2 Surface characterization

To visualize proteins immobilized on surfaces of Zn wires or sheets, rhodamine B isothiocyanate (RBITC) modified bovine serum albumin (RBITC-BSA, Zhong Ke Chen Yu Life Science, Beijing, China) was used as a model molecule. The diluted RBITC-BSA solution (0.57 mg/mL in 10 mM Tris buffer, pH 8.5) was added to the PDA-coated Zn wires or sheets and incubated with them overnight at 37 ℃. After washing three times with phosphate buffered saline (PBS), the Zn sheets and wires were observed with fluorescence microscopes (IX73, Olympus, Japan) and confocal fluorescence microscope (FV1200, Olympus, Japan), respectively. BCA Protein Assay Kit (Pierce, Thermo Scientific) was used to quantify the protein adsorbed onto the surfaces of the Zn substrates. To reveal the microstructures of the surfaces of the coated Zn substrates, the coated Zn sheets were washed with PBS, and fixed in 2.5% glutaraldehyde at room temperature for 30 min. After dehydration in gradient ethanol, samples were coated with gold and observed under a field-emitting scanning electron microscope (SEM, COXEM, Korea). To track the release of the adsorbed RBITC-BSA from the surfaces of the Zn materials, the coated Zn sheets were incubated in PBS at 37 ℃ for 7 days. At the designated timepoints, the sheets were read under IVIS® Lumina LT In Vivo Imaging System (IVIS, PerkinElmer) and the fluorescence intensity was quantified.

2.3 Cell culture

Human umbilical vein endothelial cells (HUVECs, Meisen, China) were maintained in endothelial cell growth medium (ECM, ScienCell, USA) at 37 ℃ in a humidified atmosphere containing 5% CO₂ with medium replaced every day. Upon 80% confluence, HUVECs were dissociated from tissue culture flasks with trypsin and seeded onto coated Zn sheets. Cells with a passage number between 2 to 8 were used in this study.

2.4 Cell adhesion tests

HUVECs were labeled with CMFDA (CellTracker™ Green, Invitrogen) as manuals for visualization. After that, the labeled HUVECs were seeded on different Zn substrates. 12 hours later, cells were imaged with a fluorescence microscope.

2.5 Cell proliferation assays

Cell proliferation was evaluated with cell counting kit-8 (CCk-8, Dojindo, Japan) as instructions. Briefly, HUVECs were incubated in ECM containing 10% CCk-8 at 37 ℃ for 2 hours. Then, the OD value of the supernatants was measured with a microplate reader at a wavelength of 450 nm.

2.6 Cell skeleton staining

After 3, 5, and 7 days of culture on different Zn surfaces, HUVECs were washed with PBS, fixed with 4% paraformaldehyde (PFA, HaoKe Biotechnology, Hangzhou, China), and permeabilized with 0.1% Triton X-100 for 10 minutes respectively at room temperature. After that, the cells were stained with Rhodamine phalloidin (Invitrogen) for visualization. Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI, Sigma). The stained cells were imaged with a fluorescence microscope for cellular morphology and quantity evaluation.

2.7 NO probe detection

The NO levels in cells were detected with a fluorescence NO probe, 3-Amino,4-aminomethyl-2',7'-difluorescein diacetate (DAF-FM DA, Beyotime Biotechnology, China) as manuals. Briefly, 5µM DAF-FM DA was incubated with HUVECs after 7 days of culture on different Zn substrates for 20 minutes. After that, cells were gently washed with PBS to remove free probes and imaged with a fluorescence microscope.

2.8 Immunocytochemistry

HUVECs were cultured on different Zn substrates for 7 days. After that, cells were washed with PBS and fixed in 4% PFA solution for 10 minutes at room temperature. After being permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, cells were incubated with the following primary antibodies in 5% bovine serum albumin (BSA) solution for 1 hour at room temperature: endothelial nitric oxide synthase antibody (eNOS, ab5589, Abcam) and von Willebrand Factor antibody (VWF, sc-365712, Santa Cruz Biotechnology). Then, the samples were washed with PBS again and incubated with goat anti-rabbit secondary antibody (A-11037, Invitrogen) for 1 hour at room temperature. Nuclei were counterstained with DAPI. The stained samples were then imaged with a fluorescence microscope.

2.9 Aortic implantation

All the animal experiments were approved by the Chinese Institutional Animal Care and Use Committee at Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University.

Male young SD rats (age: 6–8 weeks, body weight: 300–400 grams, Laboratory Animal Center in Zhejiang Province, Hangzhou, China) were used for aortic implantation. Four different groups of samples were implanted into animals, including the Zn group (Zn wires without any coating), the VEGF group (Zn wires coated with VEGF alone), the PDA group (Zn wires coated with PDA alone), and the PDA + VEGF group (Zn wires coated with PDA and VEGF). 5 animals were used for each group (n = 5). Totally, 20 rats were used in this study. Aortic implantation was performed as described previously [5, 12]. Briefly, a midline incision was made on the abdomens of rats to expose the abdominal aorta. The aorta was separated from the inferior vena cava and the blood flow in the aorta was blocked with a microvascular clamp. Zn wires were then inserted into the aorta followed by the removal of the microvascular clamp to recover the blood flow. The surgical site was closed with sutures lastly. No anticoagulation or antiplatelet treatments were administrated pre- or post-operatively.

2.10 Patency monitoring with ultrasound

VisualSonics high resolution ultrasound imaging system (Vevo 3100, FUJIFILM) was used to monitor blood flow in the abdominal aortas 4 weeks after implantation. Animals were anesthetized by isoflurane inhalation (5% for induction, and then 1.5% for maintenance). The abdomens were opened to expose the aortas and ultrasound gels were applied to the aortas. Cross section images of the aortas in B mode, color mode, and PW mode were acquired. The diameters of the aortas were evaluated with B mode images. The patience of the abdominal aorta was determined by color mode images and parameters regarding the blood flow, such as velocity time integral (VTI), mean velocity and mean gradient, were measured by PW mode images.

2.11 Micro-computed tomography (micro-CT) and SEM scanning

1 month after implantation, the rats were sacrificed, and the aortas implanted with Zn wires were explanted out for high resolution micro-CT (Skyscan 1275, Bruker, Belgium) scanning to evaluate the degradation of the Zn wires. The explanted samples were scanned with micro-CT at a resolution of 75 µm/pixel. The volume and the surface areas of each wire was calculated from the 3-dimensional (3D) reconstruction of the scanned images. To characterize the surface erosion of the implanted Zn wires, the tissues surrounding the wires were carefully peeled off and the wires were dehydrated in gradient ethanol. After being coated with gold, the samples were observed under field-emitting SEM (Nova Nano 450, Czech).

2.12 Chemical staining

The aortas implanted with Zn wires were fixed in 4% PFA at 4°C overnight. The fixed samples were further soaked in 30% sucrose solution (Sigma, China) at 4°C for 24 hours and embedded into the optimal cutting temperature compound (OCT, Sakura), snap-frozen at -80°C, and cryo-sectioned at 10 µm in thickness. Slides were then stained with hematoxylin and eosin (H&E), Masson's trichrome (MTC), Elastic Verhoeff-Van Gieson (EVG) and alizarin red S (ARS). All the stained slides were captured with an inverted microscope (IX73, Olympus, Japan).

2.13 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and reactive oxygen species (ROS) staining

To evaluate cell apoptosis surrounding the Zn wires, the tissue sections were stained with TUNEL using In Situ Cell Death Detection Kit (Roche) as manuals. To assess ROS levels, the samples were stained with an ROS detection kit (BestBio, China) as instructed. Nuclei were counterstained with DAPI. The stained slides were then imaged with a fluorescence microscope (IX73, Olympus, Japan).

2.14 Immunofluorescence staining

Samples were washed with PBS three times and then blocked with 5% BSA solution for 30 minutes, followed by incubation with the primary antibodies diluted in 5% BSA solution overnight at 4°C: eNOS (ab5589, Abcam), alpha smooth muscle actin antibody (αSMA, ab7817, Abcam), CD68 (MCA341GA, BioRad), Cyclin D1 (A19038, ABclonal), Aggrecan (13880-1-AP, Proteintech). After that, samples were washed with PBS three times and incubated with goat anti-rabbit secondary antibody or goat anti-mouse antibody for 1 hour at room temperature. Nuclei were counterstained with DAPI. Tissue sections without primary antibody incubation were used as negative controls. The stained samples were observed with an inverted microscope (IX73, Olympus, Japan).

2.15 Statistics

All data were presented as mean ± standard deviation. Each test had at least three replicates and was repeated 3 times independently. One-way ANOVA followed by Turkey’s post-Hoctest was used to analyze the data. P < 0.05 was considered statistically significant.

3.1 Coating construction on Zn surfaces

Dopamine polymerizes spontaneously in alkaline conditions to form a PDA coating on substrate surfaces, which has the ability to immobilize proteins through reaction between amine groups of the proteins and the PDA-coated substrates [17, 28]. BSA was used as a model protein to study protein deposition on the PDA-coated Zn surfaces. To visualize the BSA, RBITC labeled BSA was used in this study. As shown in Fig. 2a, BSA could not be adsorbed onto the surfaces of Zn. However, when the surfaces of Zn were pretreated with PDA, BSA could well deposit on the surfaces, as evidenced by the strong red fluorescence. The deposited BSA on Zn surfaces were further quantified with the BCA Protein Assay Kit. The BSA on the Zn surfaces pretreated with PDA was significantly higher than that on the pure Zn surfaces (Fig. 2b). Although there was no protein coated on the Zn surfaces in the PDA group, the test also showed a positive result. This is probably due to the amine groups of the PDA which can also reduce Cu²⁺ to Cu¹⁺ to induce a color change of the reaction, similarly to the amine groups of the proteins. Confocal fluorescence microscope observation further validated the bi-layered coatings: one PDA layer, which exhibited green autofluorescence; the other BSA layer, which showed red fluorescence (Fig. 2c). SEM examination demonstrated that the surfaces of pure Zn and PDA-treated Zn were relatively smoother than those of the BSA and PDA + BSA coated Zn (Fig. 2d). The PDA + BSA group had rougher surfaces than the BSA group, suggesting a higher level of BSA deposition. Stability of the coatings were assessed by incubating the coated Zn plates in PBS at 37 ℃ for 7 days, and fluorescence of the residual BSA was measured. After 1 day of PBS incubation, BSA on the pure Zn surfaces was almost invisible, while the Zn plates in the PDA + BSA group still showed strong fluorescence (Fig. 2e). With the time of incubation, there was a gradual reduction of the fluorescence on the surfaces of PDA-coated Zn plates, which, however, was still stronger than that on the surfaces of pure Zn plates from day 1 to day 7 (Fig. 2f).

3.2 Adhesion and proliferation of HUVECs on the PDA and VEGF-coated Zn surfaces

After characterizing PDA-based coatings, we next evaluated behaviors of HUVECs on different Zn surfaces. To facilitate the growth of the HUVECs, VEGF was coated on PDA-treated Zn surfaces. Figure 3a showed that there was no difference in cell adhesion on the VEGF-, PDA-, or PDA + VEGF-coated Zn surfaces 12 hours after cell seeding, while there was less cell adhesion on the pure Zn surfaces. Quantification of adhered cells also confirmed this result (Fig. 3b). After 7 days of culture, the HUVECs on the Zn surfaces coated with PDA and VEGF spread well with flat morphologies. However, the HUVECs on the pure Zn surfaces and the Zn surfaces coated with VEGF alone still showed round morphologies. The PDA coating also favored cell spreading but was less effective than the PDA and VEGF coating (Fig. 3a). The PDA group had higher cell covered areas than the VEGF group did, whereas the VEGF plus PDA coating further increased the cell covered areas (Fig. 3c). The HUVECs on the pure Zn and VEGF-coated Zn surfaces had relatively low proliferation capacities from day 3 to day 5 (Fig. 3d-g), indicating an unfavorable microenvironment for HUVECs. The PDA coating improved the biocompatibility of Zn surfaces, as evidenced by the increased cell proliferation from day 3 to day 5. However, cell proliferation decreased on day 7. In contrast, the PDA coating combined with VEGF maintained the proliferation of HUVECs at a relatively high level (Fig. 3d-g). These results indicate that PDA and VEGF coating greatly enhance the biocompatibility of Zn surfaces.

3.3 Phenotype maintenance of HUVECs on PDA and VEGF-coated Zn surfaces

eNOS is constitutively expressed in endothelial cells (ECs), which is responsible for NO synthesis. To evaluate the phenotypes of the HUVECs on different Zn surfaces, the levels of NO and eNOS expression in cells were detected. As shown in Fig. 4a and c, the HUVECs on both PDA- and PDA + VEGF-coated Zn surfaces had higher levels of NO and eNOS than those on the pure Zn surfaces and VEGF-coated Zn surfaces. Quantification of fluorescence intensity of NO and eNOS confirmed these results (Fig. 4b and d). Meanwhile, there was no difference in expression of NO and eNOS between the PDA group and the PDA + VEGF group. VWF is another biomarker of endothelial cells. The expression of vWF in the HUVECs on the PDA-coated Zn surfaces was comparable to those on the pure Zn surfaces and VEGF-coated Zn surfaces, which, however, was lower than the PDA + VEGF group (Fig. 4e and f). These results imply that PDA coating alone could dramatically improve the phenotype maintenance of HUVECs on Zn surfaces, and VEGF further enhanced cellular functions of HUVECs.

3.4 Patency of aortas post PDA and VEGF-coated Zn wire implantation

After verification of the efficacy of PDA and VEGF coating in promoting cell growth and functions on Zn surfaces in vitro, vascular responses to this coating were further evaluated in vivo using an aortic implantation model (Fig. 5a). The implanted Zn wires could be well identified with ultrasound in B mode (arrow heads, Fig. 5b). Color mode showed a greater blood flow in the PDA and PDA + VEGF groups compared to the pure Zn and VEGF groups, though the aortas implanted with Zn wires in all the groups were patent (dashed circles, Fig. 5b). Accordingly, PW mode showed high and sharp peaks in the PDA and PDA + VEGF groups, whereas low and flat peaks were observed in the pure Zn and VEGF groups (dashed squares, Fig. 5b). Detailed analysis of the peaks in PW mode revealed that compared to the pure Zn and VEGF groups, the PDA and PDA + VEGF groups had longer VTI, faster mean velocity, and higher gradient (Fig. 5c-e). Nonetheless, there was no difference in diameter of the aortas implanted with Zn wires among different groups (Fig. 5f).

3.5 Degradation of PDA and VEGF-coated Zn wires in vivo

Zn as a new material for bioabsorbable stents has desired degradation rate in vivo [2]. To investigate whether PDA and VEGF coatings would affect the degradation of Zn, the Zn wires post implantation were scanned with micro-CT. Macroscopic views of the explanted Zn wires showed that the middle parts of the wires were inside the aortas and both ends were outside (Fig. 6a). Micro-CT scanning showed that there were no obvious degradation signs in any group (Fig. 6b). The analysis of the middle part of the wires (region of interests, ROI) exhibited that there was no significant difference either in volume or surface areas between the different groups (Fig. 6c). SEM examination also confirmed these data (Fig. 6d). The VEGF-, PDA- and PDA + VEGF-coated Zn wires had relatively smooth surfaces, which were comparable to those of the pure Zn wires. These results indicate that the PDA-based coatings didn’t affect the degradation of Zn wires in vivo.

3.6 Intimal hyperplasia surrounding PDA and VEGF-coated Zn wires in vivo

As shown in Fig. 7a, vascular responses to Zn wires in different groups differed dramatically. Both non-coated and VEGF-coated Zn wires had a thick layer of neointimas, while PDA-coated Zn wires had a thinner one. PDA and VEGF coatings further decreased the neointimal formation. Quantification data showed that the neointima in the PDA group was only 50% thick as compared to the pure Zn group and the neointima thickness in the PDA and VEGF group was only 14.57% (Fig. 7b). Immunofluorescence staining revealed that there were quite a lot of Cyclin D1⁺ or Ki67⁺ cells in the neointimas of the non-coated and VEGF-coated Zn wires, whereas such positive cells were rather few in the neointimas of the PDA-coated and PDA and VEGF-coated Zn wires (Fig. 7c). Quantification of the Cyclin D1⁺ or the Ki67⁺ cells demonstrated a similar trend, with more Cyclin D1⁺ or Ki67⁺ cells in the Zn and VEGF groups than those in the PDA and PDA + VEGF groups (Fig. 7d and e).

3.7 EC coverage on PDA and VEGF-coated Zn wires in vivo

Rapid endothelization can efficiently prevent thrombosis after stent implantation in blood vessels. Therefore, EC coverage on Zn wires post implantation was evaluated in this study. Immunofluorescence staining showed that outer layers of neointimas consisted of smooth muscle cells (SMCs, αSMA⁺ cells) and endothelial cells (ECs, eNOS⁺ cells, Fig. 8a). There was no difference in αSMA positive areas among different groups (Fig. 8b). However, PDA coatings and PDA and VEGF coatings significantly increased percentage of the EC coverage from 2.73% in the pure Zn group and 5.29% in the VEGF group to 57.37% and 76.78%, respectively (Fig. 8a and c). These results demonstrate that PDA coatings can improve biocompatibility of Zn to ECs and accelerate the endothelization in vivo. Immobilization of VEGF on PDA coatings can further enhance the biocompatibility of Zn.

3.8 Macrophage infiltration in neointimas of PDA and VEGF-coated Zn wires in vivo

Besides SMCs and ECs, we also evaluated macrophage infiltration in neointimas surrounding Zn wires in different groups. CD68 was used as a marker of macrophages in this study. Both non-coated and VEGF-coated Zn wires had infiltration of numerous CD68 positive cells (Fig. 9a). In contrast, the PDA group had limited infiltration of CD68 positive cells, and the PDA and VEGF group had even fewer CD68 positive cells. Quantification data also confirmed this result, with significantly fewer CD68 positive cells in the PDA and PDA + VEGF groups than those in the Zn and VEGF groups (Fig. 9b). Meanwhile, the Zn and VEGF groups showed significantly higher ROS levels than the PDA and PDA + VEGF groups (Fig. 9c and d). TUNEL staining also showed more cell apoptosis in the Zn and VEGF groups than that in the PDA and PDA + VEGF groups (Fig. 9c and e). The improved biocompatibility of Zn wires after PDA coating induced less macrophage infiltration and thus low levels of ROS and cell apoptosis.

3.9 Extracellular matrix (ECM) remodeling surrounding PDA and VEGF-coated Zn wires in vivo

Finally, we characterize ECM components of neointimas surrounding Zn wires. The MTC staining showed that collagens were the main component of the ECM (Fig. 10a). The Zn and VEGF groups had higher collagen contents than the PDA and PDA + VEGF groups did (Fig. 10b). Elastin was less expressed in the neointimas surrounding Zn wires as compared to the collagens. Besides, the level of elastin in the PDA + VEGF group was lower than any other three groups (Fig. 10c). The ARS staining showed obvious calcification in the Zn group, whereas the other three groups had no obvious calcification (Fig. 10a and d). An increase in aggrecan expression is indicative of osteoblast-like differentiation of SMCs [29, 30]. The neointimas surrounding the pure Zn wires and the Zn wires coated with VEGF were positively stained for aggrecan, whereas the PDA and PDA + VEGF groups had quit few aggrecan-positive areas (Fig. 10a). Compared to the pure Zn wires, the neointimas in the PDA and PDA + VEGF groups had only 7.14% and 5.24% aggrecan-positive areas of the Zn group, respectively (Fig. 10e).

Inspired by compositions of adhesive plaques of mussels, self-polymerization of dopamine, containing both catechol and amine groups, is utilized to form a thin and adherent layer of PDA coatings on diverse substrate surface [16]. Under alkaline conditions, surface-tethered catechol groups shift toward the quinone groups, holding a latent reactivity toward nucleophiles, such as amine groups of proteins, for bioconjugation reactions [17]. Bioactive molecules then can be immobilized onto the PDA coatings through covalent binding to create a bioactive surface. Consequently, compared to the non-PDA-treated Zn surfaces, the PDA-coated Zn surfaces exhibited more BSA immobilization (Fig. 2), although there was a dramatic decline in the BSA fluorescence intensity on both PDA-coated Zn surfaces and the pure Zn surfaces on day 3 (Fig. 2e and f). This observation is probably due to the quenching of the RBITC that was used to label the BSA. On the other hand, since endothelial cells can efficiently inhibit thrombosis and neointimal formation [31, 32], we thus chose VEGF as ad-layers to promote attachment, spreading, proliferation, and phenotype maintenance of ECs on Zn surfaces. Indeed, ECs had optimal behaviors on the PDA and VEGF-coated Zn surfaces (Figs. 3 and 4), which is consistent with previous studies [24, 25]. However, in contrast to cobble-like structures of ECs on substrates of stainless steels and titanium, ECs displayed a round morphology even on the PDA and VEGF coated Zn surfaces, probably due to a high concentration of Zn²⁺ released into cell culture media (over 10 µg/ml/day) during Zn degrades [13, 15, 33]. Generally, Zn degrades 2–3 times faster under in vitro conditions than in vivo ones [5]. Because of a lower concentration of Zn²⁺ released during slower degradation of Zn in vivo, Zn usually exhibits a better biocompatibility in vivo scenario.

In this study, a wire aortic implantation model in rats was used to evaluate vascular responses of surface-modified Zn in vivo, which allows a reliable study of hemocompatibility, biodegradation, and intima hyperplasia of materials for stents under a real vascular environment, including the blood- and vascular wall-material interfaces (Fig. 5a) [2, 5, 9, 12, 34–36]. The diameter of the Zn wires used in this study was around 125 µm, falling within the range of structs of traditional metal stents (60–140 µm, made from stainless steels or cobalt-chromium alloy) [37]. Therefore, the interfaces between the Zn wires and blood can well represent those between stents and blood, although the Zn wires don’t assume mechanical stress that stents are subjected to when deployed in coronary arteries [5]. In addition, due to real blood-material interfaces created in this model, hemocompatibility of materials or coatings for stents can be precisely evaluated via ultrasound imaging (Fig. 5b-f). Similarly, degradation of materials for stents in blood can also be accurately assessed with micro-CT and SEM (Fig. 6). Moreover, the wire aortic implantation model provides us an opportunity to observe intimal hyperplasia surrounding materials for stents in vivo (Fig. 7). Intimal hyperplasia is the main reason for in-stent restenosis and limited intimal hyperplasia post stent implantation is a golden standard to judge whether stents have good biocompatibility [1]. Because of complicated mechanisms for intimal hyperplasia, it will be difficult to recapitulate intimal hyperplasia in vitro and judge biocompatibility of stents only through in vitro experiments. Therefore, the wire aortic implantation model used in this study is a valuable platform to evaluate new materials or coatings for stents in vivo which can greatly reduce cost for new stent development. Of course, after screening out candidate materials or coatings for stents, real stent manufacturing and coronary artery implantation in large animals, such as pigs, are still needed before clinical trials.

In this study, we found that the PDA coatings alone could greatly improve biocompatibility of Zn to HUVECs in vitro as compared to the non-coated or VEGF-coated Zn (Figs. 3 and 4). These results are consistent with previous studies which show that PDA can effectively improve EC attachment, proliferation, and migration because of rich amino groups in PDA [38, 39]. Deposition of VEGF on PDA layers further improved the biocompatibility of Zn, probably because a more favorable microenvironment was constructed on Zn surfaces by VEGF, which play an important role in vascular development [40]. Accordingly, in vivo data supported the improved hemocompatibility, reduced neointimal formation, increased EC coverage, decreased macrophage infiltration, and declined aggrecan accumulation in ECM after PDA coating (Fig. 5, and 7–10). We also observed a decreased ROS level in the neointimas surrounding the Zn wires coated with PDA, probably because abundant phenol groups in PDA coatings can eliminate ROS as a radical scavenger [19]. Our findings, for the first time, highlight that PDA coatings alone increase hemocompatibility of Zn and decrease neointimal formation post Zn wire implantation in vivo. Although PDA and VEGF coatings further inhibited intimal hyperplasia possibly due to an accelerated reendothelialization in vivo (Fig. 7), but the improvement is limited. Considering the high costs of VEGF for commercial use, PDA is probably a better choice due to its lower price and simplicity in coating diverse materials. Of course, optimization of PDA coatings for Zn is still needed to achieve minimal neointimal hyperplasia after post Zn implantation in vivo.

In this study, we coated Zn, a new material for bioabsorbable stents, with PDA and VEGF to increase its biocompatibility. PDA-coated Zn showed an increased EC adhesion, spreading, proliferation, and phenotype maintenance on its surfaces. VEGF immobilization onto PDA coatings further enhanced the biocompatibility of Zn. Implantation of PDA-coated Zn into rat aortas showed an improved hemocompatibility, reduced neointimal formation, increased EC coverage, decreased macrophage infiltration, and declined aggrecan accumulation in ECM. VEGF and PDA coatings further increased the EC coverage as compared to the PDA coatings alone and hence resulted in thinner neointimas. These results indicate that functionalization of Zn surfaces with PDA and VEGF is a promising strategy to inhibit neointimal formation post Zn implantation in vivo.

Smooth muscle cell SMC

Zinc Zn

Polydopamine PDA

Poly(dimethylsiloxane) PDMS

Poly(urethane) PU

Endothelial cell EC

Extracellular matrix ECM

Rhodamine B isothiocyanate RBITC

Scanning electron microscope SEM

In Vivo Imaging System IVIS

cell counting kit-8 CCk-8

Paraformaldehyde PFA

3-Amino,4-aminomethyl-2',7'-difluorescein diacetate DAF-FM DA

Bovine serum albumin BSA

Endothelial nitric oxide synthase antibody eNOS

Von Willebrand Factor antibody VWF

Vascular endothelial growth factor VEGF

Velocity time integral VTI

Micro-computed tomography Micro-CT

3-dimensional 3D

Optimal cutting temperature compound OCT

Hematoxylin and eosin H&E

Masson's trichrome MTC

Elastic Verhoeff-Van Gieson EVG

Alizarin red S ARS

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling TUNEL

reactive oxygen species ROS

Alpha smooth muscle actin antibody αSMA

Human umbilical vein endothelial cell HUVEC

phosphate buffered saline PBS

Ethics approval and consent to participate

All the animal experiments were approved by the Chinese Institutional Animal Care and Use Committee at Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by Zhejiang Provincial Natural Science Foundation of China (LQ22H180001), the Science and Technology of Medicine and Health program of Zhejiang Province (2023RC028), the Natural Key Research and Development Project of Zhejiang Province (2018C03015) and the National Natural Science Foundation of China (81901402).

Authors' contributions

JF: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Funding acquisition. QZ: Validation, Methodology, Software, Formal analysis, Investigation, Writing - Review & Editing. ZC: Investigation. JZ: Writing - Review & Editing. SW: Investigation. MZ: Investigation. SX: Investigation. DL: Supervision, Project administration, Funding acquisition. GF: Supervision, Project administration, Funding acquisition. WZ: Supervision, Project administration, Funding acquisition. All authors have read and approved the manuscript.

Acknowledgements

Not applicable.

Authors' information

Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province, Department of Cardiology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, 310016, China

Diaz-Rodriguez S, Rasser C, Mesnier J, Chevallier P, Gallet R, Choqueux C, et al. Coronary stent CD31-mimetic coating favours endothelialization and reduces local inflammation and neointimal development in vivo. Eur Heart J. 2021;42:1760–9.
Bowen PK, Drelich J, Goldman J. Zinc exhibits ideal physiological corrosion behavior for bioabsorbable stents. Adv Mater. 2013;25:2577–82.
Cockerill I, See CW, Young ML, Wang Y, Zhu D. Designing Better Cardiovascular Stent Materials - A Learning Curve. Adv Funct Mater. 2021;31.
Brar HS, Wong J, Manuel MV. Investigation of the mechanical and degradation properties of Mg-Sr and Mg-Zn-Sr alloys for use as potential biodegradable implant materials. J Mech Behav Biomed Mater. 2012;7:87–95.
Fu J, Su Y, Qin YX, Zheng Y, Wang Y, Zhu D. Evolution of metallic cardiovascular stent materials: A comparative study among stainless steel, magnesium and zinc. Biomaterials. 2020;230:119641.
Hermawan H, Dube D, Mantovani D. Developments in metallic biodegradable stents. Acta Biomater. 2010;6:1693–7.
Haase H, Rink L. Zinc signals and immune function. BioFactors. 2014;40:27–40.
Yang H, Wang C, Liu C, Chen H, Wu Y, Han J, et al. Evolution of the degradation mechanism of pure zinc stent in the one-year study of rabbit abdominal aorta model. Biomaterials. 2017;145:92–105.
Zhao S, Seitz JM, Eifler R, Maier HJ, Guillory RJ 2nd, Earley EJ et al. Zn-Li alloy after extrusion and drawing: Structural, mechanical characterization, and biodegradation in abdominal aorta of rat. Materials science & engineering C, Materials for biological applications. 2017;76:301 – 12.
Jin H, Zhao S, Guillory R, Bowen PK, Yin Z, Griebel A et al. Novel high-strength, low-alloys Zn-Mg (< 0.1wt% Mg) and their arterial biodegradation. Materials science & engineering C, Materials for biological applications. 2018;84:67–79.
Guillory RJ 2nd, Bowen PK, Hopkins SP, Shearier ER, Earley EJ, Gillette AA, et al. Corrosion Characteristics Dictate the Long-Term Inflammatory Profile of Degradable Zinc Arterial Implants. ACS biomaterials science & engineering. 2016;2:2355–64.
Su Y, Fu J, Zhou J, Georgas E, Du S, Qin YX, et al. Blending with transition metals improves bioresorbable zinc as better medical implants. Bioactive Mater. 2023;20:243–58.
Shearier ER, Bowen PK, He W, Drelich A, Drelich J, Goldman J, et al. In Vitro Cytotoxicity, Adhesion, and Proliferation of Human Vascular Cells Exposed to Zinc. ACS biomaterials science & engineering. 2016;2:634–42.
Cockerill I, Su Y, Lee JH, Berman D, Young ML, Zheng Y, et al. Micro-/Nanotopography on Bioresorbable Zinc Dictates Cytocompatibility, Bone Cell Differentiation, and Macrophage Polarization. Nano Lett. 2020;20:4594–602.
Su Y, Wang K, Gao J, Yang Y, Qin YX, Zheng Y, et al. Enhanced cytocompatibility and antibacterial property of zinc phosphate coating on biodegradable zinc materials. Acta Biomater. 2019;98:174–85.
Lee H, Dellatore SM, Miller WM, Messersmith PB. Mussel-inspired surface chemistry for multifunctional coatings. Science. 2007;318:426–30.
Lee H, Rho J, Messersmith PB. Facile Conjugation of Biomolecules onto Surfaces via Mussel Adhesive Protein Inspired Coatings. Adv Mater. 2009;21:431–4.
Wang JL, Li BC, Li ZJ, Ren KF, Jin LJ, Zhang SM, et al. Electropolymerization of dopamine for surface modification of complex-shaped cardiovascular stents. Biomaterials. 2014;35:7679–89.
Ma W, Zhang X, Liu Y, Fan L, Gan J, Liu W, et al. Polydopamine Decorated Microneedles with Fe-MSC-Derived Nanovesicles Encapsulation for Wound Healing. Adv Sci. 2022;9:e2103317.
Wu F, Li J, Zhang K, He Z, Yang P, Zou D, et al. Multifunctional Coating Based on Hyaluronic Acid and Dopamine Conjugate for Potential Application on Surface Modification of Cardiovascular Implanted Devices. ACS Appl Mater Interfaces. 2016;8:109–21.
You I, Kang SM, Byun Y, Lee H. Enhancement of blood compatibility of poly(urethane) substrates by mussel-inspired adhesive heparin coating. Bioconjug Chem. 2011;22:1264–9.
Lih E, Choi SG, Ahn DJ, Joung YK, Han DK. Optimal conjugation of catechol group onto hyaluronic acid in coronary stent substrate coating for the prevention of restenosis. J tissue Eng. 2016;7:2041731416683745.
Xue P, Li Q, Li Y, Sun L, Zhang L, Xu Z, et al. Surface Modification of Poly(dimethylsiloxane) with Polydopamine and Hyaluronic Acid To Enhance Hemocompatibility for Potential Applications in Medical Implants or Devices. ACS Appl Mater Interfaces. 2017;9:33632–44.
Luo R, Tang L, Wang J, Zhao Y, Tu Q, Weng Y, et al. Improved immobilization of biomolecules to quinone-rich polydopamine for efficient surface functionalization. Colloids and surfaces B Biointerfaces. 2013;106:66–73.
Poh CK, Shi Z, Lim TY, Neoh KG, Wang W. The effect of VEGF functionalization of titanium on endothelial cells in vitro. Biomaterials. 2010;31:1578–85.
Liu T, Liu Y, Chen Y, Liu S, Maitz MF, Wang X, et al. Immobilization of heparin/poly-(L)-lysine nanoparticles on dopamine-coated surface to create a heparin density gradient for selective direction of platelet and vascular cells behavior. Acta Biomater. 2014;10:1940–54.
Liu T, Zeng Z, Liu Y, Wang J, Maitz MF, Wang Y, et al. Surface modification with dopamine and heparin/poly-L-lysine nanoparticles provides a favorable release behavior for the healing of vascular stent lesions. ACS Appl Mater Interfaces. 2014;6:8729–43.
Schomig A, Dibra A, Windecker S, Mehilli J, Suarez de Lezo J, Kaiser C, et al. A meta-analysis of 16 randomized trials of sirolimus-eluting stents versus paclitaxel-eluting stents in patients with coronary artery disease. J Am Coll Cardiol. 2007;50:1373–80.
Lei Y, Sinha A, Nosoudi N, Grover A, Vyavahare N. Hydroxyapatite and calcified elastin induce osteoblast-like differentiation in rat aortic smooth muscle cells. Exp Cell Res. 2014;323:198–208.
Leopold JA. Vascular calcification: Mechanisms of vascular smooth muscle cell calcification. Trends Cardiovasc Med. 2015;25:267–74.
Yau JW, Teoh H, Verma S. Endothelial cell control of thrombosis. BMC Cardiovasc Disord. 2015;15:130.
Fernandes A, Mieville A, Grob F, Yamashita T, Mehl J, Hosseini V, et al. Endothelial-Smooth Muscle Cell Interactions in a Shear-Exposed Intimal Hyperplasia on-a-Dish Model to Evaluate Therapeutic Strategies. Adv Sci. 2022;9:e2202317.
Jablonska E, Vojtech D, Fousova M, Kubasek J, Lipov J, Fojt J et al. Influence of surface pre-treatment on the cytocompatibility of a novel biodegradable ZnMg alloy. Materials science & engineering C, Materials for biological applications. 2016;68:198–204.
Bowen PK, Seitz JM, Guillory RJ 2nd, Braykovich JP, Zhao S, Goldman J, et al. Evaluation of wrought Zn-Al alloys (1, 3, and 5 wt % Al) through mechanical and in vivo testing for stent applications. J biomedical Mater Res Part B Appl biomaterials. 2018;106:245–58.
Pierson D, Edick J, Tauscher A, Pokorney E, Bowen P, Gelbaugh J, et al. A simplified in vivo approach for evaluating the bioabsorbable behavior of candidate stent materials. J biomedical Mater Res Part B Appl biomaterials. 2012;100:58–67.
Bowen PK, Guillory RJ 2nd, Shearier ER, Seitz JM, Drelich J, Bocks M et al. Metallic zinc exhibits optimal biocompatibility for bioabsorbable endovascular stents. Materials science & engineering C, Materials for biological applications. 2015;56:467 – 72.
Foin N, Lee RD, Torii R, Guitierrez-Chico JL, Mattesini A, Nijjer S, et al. Impact of stent strut design in metallic stents and biodegradable scaffolds. Int J Cardiol. 2014;177:800–8.
Cheng C, Li S, Nie S, Zhao W, Yang H, Sun S, et al. General and biomimetic approach to biopolymer-functionalized graphene oxide nanosheet through adhesive dopamine. Biomacromolecules. 2012;13:4236–46.
Chen J, Li Q, Xu J, Zhang L, Maitz MF, Li J. Thromboresistant and rapid-endothelialization effects of dopamine and staphylococcal protein A mediated anti-CD34 coating on 316L stainless steel for cardiovascular devices. J Mater Chem B. 2015;3:2615–23.
Eichmann A, Simons M. VEGF signaling inside vascular endothelial cells and beyond. Curr Opin Cell Biol. 2012;24:188–93.

Download PDF

Journal Publication

published 04 Sep, 2023

Read the published version in Biomaterials Research →

Editorial decision: Major revision
19 Jul, 2023
Reviewers agreed at journal
27 Jun, 2023
Reviewers invited by journal
27 Jun, 2023
Submission checks completed at journal
21 Jun, 2023
Editor invited by journal
21 Jun, 2023
Editor assigned by journal
19 Jun, 2023
First submitted to journal
14 Jun, 2023

You are reading this latest preprint version

Polydopamine (PDA) coatings with endothelial vascular growth factor (VEGF) immobilization inhibiting neointimal formation post zinc (Zn) wire implantation in rat aortas

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.1 Surface functionalization

2.2 Surface characterization

2.3 Cell culture

2.4 Cell adhesion tests

2.5 Cell proliferation assays

2.6 Cell skeleton staining

2.7 NO probe detection

2.8 Immunocytochemistry

2.9 Aortic implantation

2.10 Patency monitoring with ultrasound

2.11 Micro-computed tomography (micro-CT) and SEM scanning

2.12 Chemical staining

2.13 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and reactive oxygen species (ROS) staining

2.14 Immunofluorescence staining

2.15 Statistics

3. Results

3.1 Coating construction on Zn surfaces

3.2 Adhesion and proliferation of HUVECs on the PDA and VEGF-coated Zn surfaces

3.3 Phenotype maintenance of HUVECs on PDA and VEGF-coated Zn surfaces

3.4 Patency of aortas post PDA and VEGF-coated Zn wire implantation

3.5 Degradation of PDA and VEGF-coated Zn wires in vivo

3.6 Intimal hyperplasia surrounding PDA and VEGF-coated Zn wires in vivo

3.7 EC coverage on PDA and VEGF-coated Zn wires in vivo

3.8 Macrophage infiltration in neointimas of PDA and VEGF-coated Zn wires in vivo

3.9 Extracellular matrix (ECM) remodeling surrounding PDA and VEGF-coated Zn wires in vivo

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1