One Produces Multi: A Drug-free Cardiovascular Stent Functionalized with Tailored Collagen Supports in-situ Healing of Vascular Tissues

doi:10.21203/rs.3.rs-3007833/v1

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One Produces Multi: A Drug-free Cardiovascular Stent Functionalized with Tailored Collagen Supports in-situ Healing of Vascular Tissues

https://doi.org/10.21203/rs.3.rs-3007833/v1

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published 25 Jan, 2024

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Drug-eluting stent implantation suppresses the proliferation of vascular endothelial cells and smooth muscle cells and thus intimal hyperplasia. However, drug-eluting stent remains limited by delayed reendothelialization, impaired intimal remodeling, and potentially increased late restenosis. Here, we show a drug-free coating formulation functionalized with meticulously tailored recombinant humanized collagen type III (rhCol III) that performs “one produces multi” behavior in response to injured tissue following stent implantation. In vitro and in vivo experiments have demonstrated that the “one produces multi” coating has the properties of resisting coagulation, reducing inflammatory responses by regulating macrophage polarization, and inhibiting the proliferation of smooth muscle cells. Furthermore, the drug-free coating accelerated neointimal healing probably via cell signaling of CCL5, CEACAM6, GATA3, and XBP, which influences the cell adhesion, proliferation, migration, and apoptosis pathways. Compared with drug-eluting stent, the rhCOL III-coated stent reduced in-stent restenosis and improved vascular neointimal healing 3 months after implantation in both rabbit and porcine models. Collectively, the “one produces multi” drug-free strategy has the potential to serve as a new inspiration for next-generation stent development.

Biological sciences/Biotechnology/Biomaterials/Biomedical materials

Health sciences/Diseases/Cardiovascular diseases/Vascular diseases

Biological sciences/Biotechnology/Biomimetics

Biological sciences/Cell biology/Cell signalling

Recently, cardiovascular disease has surpassed cancer as the number one threat to human health worldwide with the aging of the world's population, and by 2030, it is expected that 23.3 million people would die from it globally (1–4). Stent placement, particularly drug-eluting stent (DES) implantation, has gradually replaced traditional surgical thoracotomy and become the most effective method for treating coronary artery disease (CAD) over the past several decades. (5–7). After implantation, the exposure of stent inevitably damages arterial tissue, triggering a series of pathological responses, including thrombus formation and acute inflammatory response, interrelated and responsible for the excessive deposition of extracellular matrix (ECM) and proliferation of smooth muscle cells (SMCs), which ultimately causes intimal hyperplasia (the key reason for in-stent restenosis (ISR)) (8). Drug-eluting stent (DES) could effectively reduce the incidence of ISR due to locally released anti-hyperplasia drug (e.g., rapamycin, sirolimus, tranilast, and everolimus) (9, 10). However, thus far, delayed healing of endothelial monolayers, late stent thrombosis (LST) risk, and ISR remain major challenges limiting the long-term efficacy of DES (11). Accordingly, the academia “sought to develop drug-free stent that could both avoid LST and promote vascular neointimal healing against the anti-hyperplasia drugs” (12, 13). Considering the multicomplex material/blood interactions, it is a promising approach for the development of stent (whether degradable or nondegradable) to construct a multifunctional surface that endows the vascular stent with anticoagulant, anti-inflammatory, and selectively enhancing endothelialization functions, thus regulating vascular neointimal remodeling and promoting regeneration of injured tissues (14, 15).

Recombinant humanized collagen type III (rhCol III), a class of novel biomaterials, was meticulously tailored by advanced structural biology and genetic engineering technologies (7). The rhCol III possessed enhanced cell adhesion activity and anticoagulant properties, which were attributed to the retention of highly adhesive fragments (Gly-Glu-Arg (GER) and Gly-Glu-Lys (GEK)) of humanized collagen type III and bypassed the hydroxyproline (O) sequence that might induce platelet adhesion and activation. In addition, the properties of rhCol III in terms of good water solubility, low inflammatory response, and promotion of vascular remodeling have been studied (7). The multifunctional rhCol III has demonstrated promise in treating cardiovascular diseases such as coronary atherosclerotic heart, myocardial infarction, and valvular heart disease (3, 16–17). Over the past two decades, it has been confirmed that tissue-inducing biomaterials, a class of optimized designed biomaterials, possess the ability to positively mediate tissue repair or regeneration without the stimulation of growth factors and drugs (18, 19). Inspired by tissue-induced biomaterials, we tested the hypothesis that the single component rhCol III coating (drug-free) can induce multiple functional behaviors in response to the complex microenvironment surrounding the cardiovascular stent implant. Synergistically, the drug-free coating on cardiovascular stent can reduce the LST risk, inhibit ISR, and promote vascular neointimal healing.

In this article, we report the development of “one produces multi” drug-free coating formulation functionalized with meticulously tailored recombinant humanized collagen type III (rhCol III) for modulating vascular remodeling, with the prospect of addressing challenges such as ISR and incomplete endothelialization from drug-eluting stent (Fig. 1). The tailored rhCol III was efficiently and continually linked on the amino-rich surface. We investigated the stability of the “one produces multi” coating in the flowing system simulating blood. In vitro and in vivo assays suggested that the single component rhCol III coating can suppress the inflammatory response by mediating the transition of macrophages to the M2 phenotype, accelerate the endothelialization process, and inhibit the transition of SMCs from a synthetic to a secretory phenotype, thereby reducing ISR and facilitating long-term stent patency in both small-animal and large-animal models. Combining all of the above results into a single, the “one produces multi” strategy (drug-free coating) demonstrated aspirational potential in fulfilling desirable vascular neointimal healing, and was expected to be the direction for next-generation stent development.

Construction and characterization of (rhCol III/PDA-PEI)_n coatings

The (rhCol III/PDA-PEI)_n (n = 0.5, 1, 2, and 4) coatings were prepared on PLA sheets and stents by covalent bonding with EDC and NHS and electrostatic interaction. The immobilization of rhCol III on an amino-rich surface (PDA-PEI coating) could be visualized directly via the fluorescence signal of FITC-labeled rhCol III. As shown in Fig. 2A, the fluorescence signals of the (rhCol III/PDA-PEI)_n (n = 0.5, 1, 2, and 4) coatings prepared on PLA sheets were observed, and the fluorescence signal intensity of (rhCol III/PDA-PEI)_n became stronger as the feeding concentration of rhCol III increased. The results indicated that rhCol III was successfully immobilized onto the PDA-PEI coating. Furthermore, the long-term structural stability of (rhCol III/PDA-PEI)_n coating should be of great concern during the initial stages of implantation, which is the prerequisite for samples to maintain anticoagulant, anti-inflammatory, and enhanced endothelialization capabilities. The stability of the (rhCol III/PDA-PEI)₂ coating prepared on PLA stent was investigated by using a flowing system with bovine blood serum, simulating a complex and changeable physiological environment. As shown in Fig. 2B, compared to that of the fresh coating (the fluorescence signal of the fresh (rhCol III/PDA-PEI)₂ coating was set as 100%), the fluorescence signal of the (rhCol III/PDA-PEI)₂ coating decayed to 89.4% after incubation with bovine blood serum for 1 week. After two weeks, the fluorescence signal on the surface of the (rhCol III/PDA-PEI)₂ coating decayed to 64.5% of the initial value. After 1 month of circulation, approximately 57.2% of the initial fluorescence signal intensity remained in the coating (Figs. 2B and 2C), implying that the coating was relatively stable after implantation. The results above confirmed that higher feeding concentrations of rhCol III induced a higher coverage ratio of the (rhCol III/PDA-PEI)_n coatings, endowing the single component rhCol III coating with a reliable and longer anticoagulant ability in the initial stage of implantation.

The surface micromorphology of the (rhCol III/PDA-PEI)_n coatings (n = 0.5, 1, 2, 4) was observed by SEM. As shown in Fig. S1, the PLA surface was relatively smooth. While the PDA-PEI coating presented a rougher surface with many aggregated nanoparticles that were formed by the self-polymerization of DA into PDA and a simultaneous reaction with PEI via Michael-addition and Schiff-base mechanisms (20). After immobilization of rhCol III, the (rhCol III/PDA-PEI)_n coatings showed a much denser and smoother surface morphology. The reason for this phenomenon was that the carboxyl groups on rhCol III were activated and covalently reacted with the amino groups on PEI under the action of EDC and NHS. In addition to this, electrostatic interaction was also involved, thereby resulting in the formation of an interpenetrating network in the (rhCol III/PDA-PEI)_n coating and reducing the surface roughness. The thickness of the (rhCol III/PDA-PEI)_n coatings increased from 25.1 to 29.2 nm with an increasing concentration of rhCol III from 0.5 to 4 mg/mL. The difference between the (rhCol III/PDA-PEI)_n groups was insignificant, indicating that the coatings became denser and more uniform (Fig. 2D). This phenomenon was consistent with the SEM results. After being modified with the PDA-PEI coating, the PLA substrate exhibited significantly improved hydrophilicity due to the introduction of the abundance of amino groups, with the water contact angle (WCA) decreasing from 92.3° to 24.1°. The hydrophilicity of (rhCol III/PDA-PEI)_n gradually decreased (the WCA ranged from 33.3° to 55.9°) with increasing rhCol III feeding concentration (Fig. 2E). The WCA results further supported the above conclusions.

The chemical structure and composition on the surface of PLA substrates modified with PDA-PEI and (rhCol III/PDA-PEI)_n (n = 0.5, 1, 2, and 4) coatings were investigated by XPS. As shown in Fig. 2F, an obvious peak ascribed to the N1s appeared on the PEI-PDA and (rhCol III/PDA-PEI)_n coatings in contrast to the PLA substrate, providing strong evidence that the PDA-PEI coating was grafted. The successful fabrication of the (rhCol III/PDA-PEI)_n coatings was further confirmed by the C1s high-resolution spectra (Figs. 2G-2L). The following bonding states were observed: 288.3 eV corresponding to carboxyl group C = O bonding, 286.5 eV corresponding to C-N, C-OH, and phenol hydroxyl, and 284.5 eV corresponding to C = C and C–C. When rhCol III was modified on the PDA-PEI coating, a new peak appeared at 288.3 eV, which was assigned to carboxyl group C = O bonding (21). The signal of the COOH peak (the fitting peaks at 288.3 eV) was not visualized on the (rhCol III/PDA-PEI)_n (n = 0.5, 1) coatings, possibly due to the lower amount and uneven distribution of rhCol III on the surface of PLA substrates (7). However, the signal for the COOH peak was displayed on the surface of (rhCol III/PDA-PEI)_n (n = 2, 4) coatings with increasing rhCol III loading, indicating that rhCol III was successfully assembled via the formation of an EDC/NHS active layer.

The QCM-D test is a reliable and effective tool to quantify the amount of rhCol III bounded to the PDA-PEI coating. In this study, the QCM-D experiment was applied to monitor the construction process of the (rhCol III/PDA-PEI)_n (n = 0.5, 1, 2, and 4) coatings and investigate the stability of the coatings with PBS flow. As described in previous work, the decrease in frequency implied that the molecules absorbed on the wafer, while the increase in frequency was caused by the desorption of the molecules (22). The F-time curve of the (rhCol III/PDA-PEI)_n coatings was shown in Fig. 2M. The ΔF gradually increased as the feeding concentration of rhCol III increased. Notably, the rhCol III adsorption amount of the (rhCol III/PDA-PEI)_n coatings (n = 2 and 4) increased significantly compared with that of the (rhCol III/PDA-PEI)_n coatings (n = 0.5 and 1) when the mixed solution pH was adjusted from 5.4 to 7.4, confirming that protonation of the amino group was prevented and the reaction of the carboxyl group of rhCol III with the surface amino group was promoted. After 1.5 h, the adsorption of rhCol III reached a steady state. The strength of attraction and stability of the coating in a physiological environment should be carefully evaluated (23). The value of F slightly increased when PBS was added, suggesting that rhCol III was strongly bonded to the wafer surface against PBS rinsing. The surface zeta potential shift of different groups at approximately pH 7.4 was shown in Fig. 2N. The surface potentials of PLA and PDA-PEI were − 45.89 ± 2.50 mV and 26.52 ± 2.63 mV, respectively. After rhCol III was grafted, the surface potentials of (rhCol III/PDA-PEI)_n decreased gradually, from 5.60 ± 1.52 mV (for the (rhCol III/PDA-PEI)_0.5 group) to -0.46 ± 1.64 mV (for the (rhCol III/PDA-PEI)₄ group). The surface potential of (rhCol III/PDA-PEI)₄ was almost identical to that of pure rhCol III (− 1.72 ± 1.15 mV), indicating that rhCol III covered the PLA substrate uniformly and continuously. In summary, the above results confirmed that the (rhCol III/PDA-PEI)_n coatings were successfully constructed.

Hemocompatibility test

As biomedical devices contact blood after implantation, it is vital to evaluate their antithrombotic properties in vitro and in vivo. The aggregation and activation of platelets on the surface of samples is the key step, closely associated with in-stent restenosis and LST (24). In vitro, the anticoagulant ability of uncoated, PDA-PEI-coated, and (rhCol III/PDA-PEI)_n-coated PLA was evaluated by the platelet adhesion test (Fig. 3A). After 2 h of incubation, there were severe platelets adhered to the surface of bare PLA and the PDA-PEI coating with a full spread shape or a typical pseudopodia shape, indicating that the platelets were highly activated. However, platelet adhesion on the surface of the (rhCol III/PDA-PEI)_n coatings reduced significantly and showed a spherical shape with excellent anticoagulant effect; this effect was related to rhCol III escaping the hydroxyproline (O) sequence, which may induce platelet adhesion and activation (Fig. 3B). Quantitative analysis also confirmed that the (rhCol III/PDA-PEI)_n groups (with concentrations of 0.5, 1, 2, and 4) could significantly inhibit the adhesion and activation of platelets (Figs. 3C and 3D). Notably, the area coverage and the adherent number of platelets on the surface of the (rhCol III/PDA-PEI)_n groups decreased sharply with n ≥ 2, indicating the importance of sufficient rhCol III content for platelet inhibition.

The anti-thrombogenic property was investigated by ex vivo arteriovenous shunt assay after positive in vitro results. The PDA-PEI and (rhCol III/PDA-PEI)_n coatings (n = 0.5, 1, 2, and 4) were prepared on the PVC tubes, which were then carefully connected with the right carotid artery and left external jugular vein of the rabbits (Fig. 3E). After 2 h of ex vivo circulation, there was severe thrombus formed on the surface of the bare PLA and PDA-PEI groups with the network of fibers linked with red blood cells and activated platelets, whereas only a few platelets with round shapes adhered to the surface of the (rhCol III/PDA-PEI)_n coatings (n = 1, 2, 4), implying that these platelets exhibited a resting, nonactivated state (Fig. 3F). Furthermore, we quantitatively evaluated the occlusion rates and thrombus weights of different groups in the circuit. The cross-sectional areas of the groups of PLA and PDA-PEI circuits were reduced by 61.43% and 40.43%, respectively. However, the cross-sectional area of the circuit coated with (rhCol III/PDA-PEI)_n (n = 1, 2, 4) coatings was only reduced by 6.49%, 1.5%, and 0.98%, respectively (Figs. 3G and 3H). The thrombus weight adherent on the surface of the (rhCol III/PDA-PEI)_n (n = 1, 2, 4) groups was significantly decreased compared to that of the bare PLA and PDA-PEI groups, which further supported the above observations (Fig. 3I). In conclusion, the amplified functionalization of the rhCol III coating reduced platelet adhesion and aggregation, exhibiting excellent anticoagulant properties and showing the potential to improve the hemocompatibility of blood-contacting biomaterials. The lower rhCol III loading of the (rhCol III/PDA-PEI)_0.5 coating might attenuate the subsequent biological functions. Therefore, (rhCol III/PDA-PEI)_n (n = 1, 2, 4) coatings were utilized to study other subsequent biological characterizations.

Cell proliferation and morphology of vascular cells

The blood vessel wall is inevitably damaged during vascular stent implantation. This behavior would provoke a series of pathological reactions, including thrombogenesis, inflammatory reactions, and excessive smooth muscle cell (SMC) migration and proliferation, which may lead to in-stent restenosis (ISR). The dense and healthy EC layer could effectively contribute to the excellent thromboresistance of the endothelium and its positive functions in vascular wall remodeling (25). In a previous study, it was demonstrated that rhCol III was bound to stents by stepwise physical LBL assembly to guarantee that the materials possessed great anticoagulant activity, inhibited the adhesion and proliferation of SMCs, and selectively promoted the adhesion and proliferation of HUVECs (7). In this work, we developed an optimized protocol in which an amplified functionalization coating of a single component rhCol III was prepared by covalent bonding, as outlined above in Fig. 1. Based on the above considerations, we first evaluated the growth of vascular cells on different surfaces. The attachment and proliferation of ECs were investigated by fluorescence images. Compared with the bare PLA substrate, the (rhCol III/PDA-PEI)_n (n = 1, 2, 4) coatings loaded with different amounts of rhCol III all showed better coverage of ECs according to the results of rhodamine fluorescence staining and FDA staining after 1 day and 3 days of culture, indicating good EC compatibility (Figs. 4A and S2). The above phenomenon occurred because rhCol III retained the highly adhesive fragments GER and GEK of humanized collagen type III. Moreover, the number of attached ECs was counted. As shown in Fig. 4B, the number of ECs on the surface of (rhCol III/PDA-PEI)_n coatings was higher than that of the control group after 1 day and 3 days of culture. The CCK-8 assay was used to detect cell viability, and the results were shown in Fig. 4C. Importantly, compared with the rest of the samples, the (rhCol III/PDA-PEI)₂ sample showed the best cell activity; however, the cell viability of the (rhCol III/PDA-PEI)₄ group did not increase consistently with the increase in rhCol III concentration from 2 mg/mL to 4 mg/mL. This might be because the surface potential of the (rhCol III/PDA-PEI)₄ coating was approximated and tended to approach 0 mV with the increase of rhCol III loading, which was similar to the zwitterion surface behavior, thus discouraging cell adhesion.

Based on the above results, the (rhCol III/PDA-PEI)₂ coating was selected to further clarify the changes in gene expression after coculture with ECs by global gene expression profiling. Compared with the control group, 62 upregulated genes and 147 downregulated genes were detected in the (rhCol III/PDA-PEI)₂ group, as shown in the volcano plot (Fig. 4D). Multiple databases, including the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes database, were used for gene set enrichment analysis to identify the pathways involved. Compared with the control group, cell adhesion, proliferation, migration, and apoptotic pathways were obviously upregulated in the (rhCol III/PDA-PEI)₂ group (Fig. 4E). Unexpectedly, CCL5 (chemokine (C-C motif) ligand 5), CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6), GATA3 (GATA binding protein 3), and XBP1 (X-box binding protein 1) were involved in all the above-mentioned signaling pathways, suggesting that they might regulate the fate of EC (Figs. 4F-4H). These results suggested that the (rhCol III/PDA-PEI)₂ coating might activate the related signaling of ECs to induce intima formation.

Inhibiting excessive proliferation of SMC early in implantation could effectively prevent subsequent late thrombosis, late restenosis, and material failure. The attachment and proliferation of SMCs were investigated by fluorescence images. Compared with the PLA group, the (rhCol III/PDA-PEI)_n (n = 1, 2, and 4) coatings all suppressed the adhesion and proliferation of SMCs to varying degrees according to the morphology results of rhodamine fluorescence staining after 1 day and 3 days of culture (Fig. 5A), and the number of SMCs adhered to the surface of the (rhCol III/PDA-PEI)_n coatings decreased significantly. The FDA results were consistent with the results of cell fluorescence staining described above (Fig S2). In addition, the results of the cell count and the CCK-8 assay also confirmed that the cell adhesion and proliferation of the PLA group were significantly enhanced compared with those of the (rhCol III/PDA-PEI)_n groups (Figs. 5B and 5C). In particular, the extent of inhibition of SMC proliferation remained almost unchanged between the (rhCol III/PDA-PEI)_n (n = 1, 2, and 4) coatings. This phenomenon was attributed to the surface potentials of the coatings gradually approaching 0 mV with increasing rhCol III loading, as investigated in ECs. The diverse growth trend of (rhCol III/PDA-PEI)_n (n = 1, 2, and 4) coatings on ECs versus SMCs might be due to the different sensitivity and responsiveness that interact between the material surface/interface and cells. The mechanism of rhCol III interaction with SMCs was remained under further investigation.

In summary, the (rhCol III/PDA-PEI)₂ coating not only suppressed the excessive proliferation of SMCs but also supported the growth of ECs, which was beneficial for maintaining the blood vessel patency rate and provided evidence for the blood-contacting devices to achieve neointimal healing.

In vitro and in vivo inflammatory response

After stent implantation, the tissue is damaged, macrophages accumulate and adhere to the stent surface, and distinctively respond to slight alterations in the microenvironment by mediating transformations in the M1- and M2-polarized phenotypes (26, 27). Classical M1 polarization, associated with the proinflammatory response, could be characterized by the expression of CD86. Conversely, M2 polarization could be defined by the expression of CD206, which is associated with anti-inflammatory properties. CD68, as a specific marker, was utilized to define all macrophages (28). Furthermore, activated macrophages secrete a high level of inflammatory cytokines (such as IL-10 and IL-23) to enhance the inflammatory response and cell damage, which could induce subsequent impaired endothelialization, excessive proliferation of SMCs, and intimal hyperplasia, leading to implantation failure (29–31). Therefore, it is essential to reduce the inflammatory response for implant/interventional devices.

To investigate the anti-inflammatory function of tailored rhCol III, the fluorescein diacetate (FDA) was first performed. As shown in Fig. S3A, there were a large number of macrophages with extended pseudopods adhered to the PLA substrate. However, only a few macrophages on the surface of (rhCol III/PDA-PEI)_n (n = 1, 2, and 4) coatings in the inactivated states were observed owing to the anti-inflammatory effect of rhCol III, as investigated in previous work (7). The results of the CCK-8 assay and the cell increase rate also confirmed that the adhesion and proliferation of RAW 264.7 cells on the surface of the (rhCol III/PDA-PEI)_n group were inhibited (Figs. S3B and S3C). In particular, with increasing rhCol III loading, the (rhCol III/PDA-PEI)₂ and (rhCol III/PDA-PEI)₄ coatings showed no significant difference in terms of anti-inflammatory properties. The above phenomenon might be attributed to the fact that as the rhCol III loading increased, the surface of the PLA substrate was completely covered by rhCol III, and accompanied by the surface potential of (rhCol III/PDA-PEI)_n (n = 2 and 4) approximated to 0 mV, thus displaying constant and comparable anti-inflammatory properties.

Immunofluorescence staining of RAW 264.7 cells was conducted to further investigate the mechanisms of inflammatory regulation of the (rhCol III/PDA-PEI)_n coating (Fig. 6A). In the (rhCol III/PDA-PEI)_n (n = 1, 2, and 4) groups, the immunofluorescence signals of CD86 were significantly reduced, while the immunofluorescence signals of CD206 were markedly enhanced compared to that of the control group (Fig. 6B). Correspondingly, quantitative statistical results also revealed that the proportion of M1 polarization and the number of macrophages decreased and the proportion of M2 polarization increased in the (rhCol III/PDA-PEI)_n groups compared to the control group (Figs. 6C and 6D). Furthermore, the anti-inflammatory cytokine IL-10 and the proinflammatory cytokine IL-23 were employed to evaluate the anti-inflammatory behavior of the (rhCol III/PDA-PEI)_n coatings. As expected, compared with the PLA group, the macrophages attached to the surface of (rhCol III/PDA-PEI)_n coatings released more 1L-10 and less IL-23, implying a better anti-inflammatory effect than that of the bare PLA (Figs. 6E and 6F) (32). Combining the above immunofluorescence staining results, it could be concluded that both the (rhCol III/PDA-PEI)₂ and (rhCol III/PDA-PEI)₄ groups exhibited great anti-inflammatory activity with no significant difference, which was ascribed to the loading of sufficient amounts of customized rhCol III, as investigated above. Based on the results of all anti-inflammatory assays in vitro described above, (rhCol III/PDA-PEI)_n (n = 2 and 4) coatings were demonstrated to remarkably suppress the inflammatory response by suppressing macrophage adhesion and proliferation, promoting the polarization of macrophages toward the M2 phenotype, and modulating the expression of inflammation-related proteins.

The in vivo anti-inflammatory ability of the (rhCol III/PDA-PEI)_n (n = 1, 2, and 4) coatings was further performed with subcutaneous implantation in rats. The tissue around the surface of the samples was harvested after 15 and 30 days of implantation. The inflammatory response of the samples was reflected by the thickness of the fibrous encapsulation and the number of infiltrating inflammatory cells. Generally, inflammatory cell infiltration increase, granulation tissue development increase, and a thicker fibrous encapsulation indicate a more severe tissue response (33). After 15 days of implantation, the PLA group exhibited the most severe inflammatory cell infiltration and the thickest fibrous capsule (56.6 ± 5.6 µm) compared with the (rhCol III/PDA-PEI)_n (n = 1, 2, 4) groups (Figs. 7A and 7D). In addition, the relative intensity of CD68 fluorescence on the fibrous capsule was 72.0 ± 4.2 a.u. for control, 37.2 ± 2.9 a.u. for (rhCol III/PDA-PEI)₁, 33.3 ± 2.9 a.u. for (rhCol III /PDA-PEI)₂ and 22.0 ± 1.3 a.u. for (rhCol III/PDA-PEI)₄, respectively (Figs. 7B and 7E). After 30 days of implantation, the inflammatory response of unmodified PLA was further strengthened. While the infiltration of inflammatory cells around the (rhCol III/PDA-PEI)_n samples was still poor, and thin fibrous capsules were visible with thicknesses of 28.3 ± 1.7 µm, 23.6 ± 1.2 µm, and 11.3 ± 0.6 µm. In total, the in vivo anti-inflammatory results, which were consistent with those in vitro, further substantiated that the (rhCol III/PDA-PEI)_n (n = 2 and 4) coatings could effectively improve the in vivo inflammatory response of PLA substrates, displaying good tissue compatibility.

The healing of the injured endothelial layer involves complex interactions between inflammatory cells (including neutrophils, monocytes, and lymphocytes), ECs, and SMCs, and dysregulation of these responses could result in adverse vascular remodeling, neointimal proliferation, and restenosis (34). Combined with the results of in vitro anticoagulation and vascular cell growth (ECs and SMCs) assays, it can be concluded that the (rhCol III/PDA-PEI)₂ coating, in addition to exhibiting superior anticoagulant and anti-inflammatory potential, is also efficient both for rapid endothelialization and suppressing the excessive proliferation of SMCs. The combination of these functions is of great significance to positively induce normal and healthy repair of the damaged endothelium. Therefore, the (rhCol III/PDA-PEI)₂ coating was utilized to further evaluate endothelialization in vivo.

In vivo endothelialization and neointima formation

To further evaluate whether our developed (rhCol III/PDA-PEI)_n coatings could effectively promote in situ endothelialization while inhibiting intimal hyperplasia, long-term stenting tests in rabbit and porcine models were conducted (Fig. 8A). After 3 months of implantation in the abdominal aorta of rabbits, the stents made of bare PLA, modified with rapamycin-eluting (RAPA) and (rhCol III/PDA-PEI)₂ PLA surrounded by the neointima were harvested. As shown in the SEM images (Fig. 8B), the PLA stents were completely covered by the neointima, and the cell morphology on the inner surface of the neointima was randomly distributed, which meant that the cells covered on the bare PLA stents were not oriented. Both the RAPA- and (rhCol III/PDA-PEI)₂-modified PLA stents were completely covered by a layer of regularly arranged cells, with more cells on the surface of the (rhCol III/PDA-PEI)₂-coated PLA stents compared to RAPA group. The cells presented an irregular state on the surface of the PLA, suggesting that the surface of the neointima was composed of different cells and secreted extracellular matrix, while the cells on the surface of RAPA- and (rhCol III/PDA-PEI)₂-coated PLA stents were cobblestone-like, typical of endothelial cells (35).

HE staining and immunohistochemical staining of CD68 and α-SMA were conducted to verify the luminal stenosis rate, the intensity of the inflammatory response, and the SMC phenotype of the neointima (Figs. 8C-8E). The neointima formed around the bare PLA stents was thicker than that around the RAPA and (rhCol III/PDA-PEI)₂ stents, suggesting that both RAPA and (rhCol III/PDA-PEI)₂ coatings could effectively inhibit intimal hyperplasia (Fig. 8C). In addition, quantitative statistics of the lumen stenosis rate and the area of the neointima showed that compared with bare PLA stents, the restenosis rate of RAPA- and (rhCol III/PDA-PEI)₂-coated stents decreased sharply to 30.9 ± 0.25% and 20.4 ± 0.39%, respectively. The area of the neointima of bare PLA stents was 1.7 ± 0.02 mm², higher than that of the RAPA (1.5 ± 0.03 mm²) and (rhCol III/PDA-PEI)₂-coated stents (1.1 ± 0.01 mm²) (Figs. 8F and 8G).

The inflammatory response after stent implantation further exacerbates endothelialization disorders, excessive proliferation of SMCs, and restenosis. As shown in Fig. 8D, the inflammatory response of RAPA-modified stents sharply decreased compared with that of the PLA stents, which was attributed to the anti-inflammatory properties of RAPA. In addition, lower inflammation scores were visible on (rhCol III/PDA-PEI)₂-coated stents owing to the excellent anti-inflammatory activity of rhCol III (Fig. 8H), indicating that its histocompatibility and host integration are optimal.

Some literature has reported that SMCs exhibit a contractile phenotype in healthy mature organisms, which mainly maintains the elasticity and contraction of blood vessels. However, SMCs with a contractile phenotype shift to a synthetic phenotype in some pathological states (4). The in vitro vascular cell growth (including ECs and SMCs) of the single rhCol III component at different concentrations (10, 50, 100, 500 µg/mL) was first investigated. After 1 day of culture, no significant changes in EC and SMC viability were observed in any group. The groups with different concentrations of rhCol III exhibited a significant promotion of vascular cell proliferation compared to that of the PLA group after 3 days, consistent with the enhanced cell adhesion activity of rhCol III (Figs. S4A and S4B). Unexpectedly, the results of in vivo stent implantation assays demonstrated that the expression level of α-SMA-positive around the RAPA-(50.5 ± 1.5 au) and (rhCol III/PDA-PEI)₂-coated PLA stents (66.0 ± 2.7 au) was higher compared with the bare PLA group (32.6 ± 1.5 au), revealing that both RAPA and (rhCol III/PDA-PEI)₂ could promote the contraction SMC phenotype and thus suppress intimal hyperplasia (Figs. 8E and 8I). The above different results of SMC growth in vitro and in vivo might have occurred because, in contrast to the mild and simple cell culture conditions in vitro, the proliferation and the regulation of the phenotype of SMCs in vivo resulted from a series of complex biochemical reactions involving coagulation, inflammation, and delayed healing, especially in the abnormal physiological environment triggered by stent implantation. The above-mentioned results confirmed that the (rhCol III/PDA-PEI)₂ coating possessed improved anticoagulation, reduced anti-inflammation, and accelerated rapid endothelialization, which was responsible for the subsequent suppression of the excessive proliferation of SMCs and intimal hyperplasia in vivo.

To further investigate the cell types growing on the neointima, immunofluorescence staining of CD31, eNOS, and DAPI was conducted (Fig. 8J). The CD31 expression of the (rhCol III/PDA-PEI)₂-coated PLA stents was much higher than that of the PLA and RAPA groups (Fig. 8K). The reason for the above phenomenon was that the RAPA-eluted stents inhibited the growth of SMCs and inhibited the adhesion and proliferation of ECs, resulting in intimal immature healing. Quantitative statistics of the expression of eNOS, which is the key function of endothelial cells, could reflect the degree of total endothelialization (36). As shown in Fig. 8L, the eNOS signal was much lower on PLA (41.8%) and RAPA (78.6%) stents than on (rhCol III/PDA-PEI)₂ stents (88.5%). According to DAPI staining, the cell density on the surface of the PLA (1632 cells/mm²) and RAPA (2917 cells/mm²) stents was lower than that of the (rhCol III/PDA-PEI)₂ group (3114 cells/mm²) (Fig. S5). These results suggested that the (rhCol III/PDA-PEI)_n coatings could achieve in situ endothelialization rapidly and showed potential to realize ideal remodeling of blood vessels.

Our (rhCol III/PDA-PEI)₂ stent illustrated advantages over RAPA-eluted stents in rabbit abdominal aorta stenting experiments. Therefore, we hypothesized that the use of (rhCol III/PDA-PEI)₂-coated stents could be extended to large animal studies or even clinical trials. In response, the (rhCol III/PDA-PEI)₂-coated CoCr stents were implanted in the coronaries of miniature pigs for 3 months to explore the safety and effectiveness of the (rhCol III/PDA-PEI)₂ coating in vivo. As shown in Fig. 8M, the neointimal area of the implanted (rhCol III/PDA-PEI)₂- and RAPA-coated stents was markedly reduced compared with that of bare stents, consistent with the optical coherence tomography (OCT) results (Fig. 8N). As shown in Fig. 8O, HE staining results revealed that the (rhCol III/PDA-PEI)₂-coated stent (1.26 ± 0.02 mm²) markedly reduced the neointimal area compared with the bare stent (4.87 ± 0.2 mm²), while there was no significant change compared with the RAPA-coated stents (1.28 ± 0.1 mm²), which was consistent with the lumen stenosis rate result (Fig. 8P). The results indicated the clinical feasibility of the (rhCol III/PDA-PEI)₂ coating of therapeutics for the healing of the neointima.

In conclusion, in the rabbit model, the (rhCol III/PDA-PEI)₂ coating reduced the inflammatory response, accelerated the endothelialization process, and inhibited the transition of SMCs from a synthetic to a secretory phenotype, thereby reducing restenosis and facilitating long-term stent patency. Hence, the (rhCol III/PDA-PEI)₂ coating exhibited a potential surface modification strategy to realize ideal vascular neointimal healing. Surprisingly, in the porcine animal model, the neointima formed on the surface of drug-free ((rhCol III/PDA-PEI)₂) stents was slightly thinner than commercially available RAPA stents, which further increased the clinical interest in developing next generation drug-free stents.

In conclusion, we proposed and confirmed the following “one produces multi” strategy: a drug-free cardiovascular stent functionalized with tailored recombinant humanized collagen type III (rhCol III) was developed to enhance vascular neointimal healing. Importantly, compared to animal-sourced collagen, customized rhCol III exhibited great anticoagulant properties, anti-inflammatory activity, and cell adhesion activity. Therefore, our single rhCol III component coating was able to endow cardiovascular materials with versatility and meet the expected performance in the stent application field. In our in vitro and in vivo experiments, we demonstrated that the (rhCol III/PDA-PEI)_n coatings improved structural stability, reduced platelet adhesion/activation and aggregation, promoted EC adhesion and proliferation through activation of EC-related signaling and inhibited the growth of SMCs, suppressed the inflammatory response by mediating the transition of macrophages to the M2 phenotype, accelerated endothelialization and inhibited intimal hyperplasia. Of note, in the porcine animal model, the neointima formed on the surface of drug-free ((rhCol III/PDA-PEI)₂) stents was slightly thinner than commercially available RAPA stents. All these results demonstrated the clinical feasibility of the (rhCol III/PDA-PEI)₂ coating of therapeutics for healing the neointima and emphasized the significance and potential applications of tailored multifunctional rhCol III in the surface modification of cardiovascular stents.

Materials

Polylactic acid (PLA) sheets (1 × 1 cm) and stents (Φ2.75 mm × 13 mm) were prepared by Sichuan Xingtai Pule Medical Technology Co., Ltd. for our study. Polyvinyl chloride (PVC) tubes used for the in vitro antithrombotic test were provided by Tuyang Health Products Store. Dopamine hydrochloride (Dopa), polyethyleneimine (PEI, Mw = 25000), 2-(N-morpholino) 2-N-morpholino ethane sulfonic acid (MES), N-ethyl-N’-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma‒Aldrich (Gillingham, UK). Recombinant Humanized Collagen Type III (named rhCol III in this work, average Mw ~ 45 KD) was meticulously tailored based on our requirements using advanced structural biology and genetic engineering technologies. Specifically, rhCol III was synthesized by 16 tandem repeats of the T16WTp (sequence: Ac-GERGAPGFRGPAGPNGIPGEKGPAGERGAP-NH₂). The biological reagents for evaluating blood compatibility and cell compatibility were purchased from professional manufacturers and were mentioned in the experimental section.

Fabrication of the (rhCol III/PDA-PEI)n coatings

Before fabricating the (rhCol III/PDA-PEI)_n coatings, PLA sheets and PLA stents were ultrasonically cleaned with acetone, alcohol, and ultrapure (UP) water in sequence three times (10 min ×3 times) and then dried with N₂ for 2 h before use. The PLA substrates were immersed in Dopa solution (2 mg/mL in a Tris buffer solution of pH 8.5) for 2 h, followed by cleaning with UP water three times (3 min × 3 times). Then, they were immersed in PEI solution (20 mg/mL in tris buffer of pH 8.5) for 2 h, followed by cleaning with UP water three times (3 min × 5 min). The as-prepared coating was rich in amino groups for subsequent rhCol III grafting and was named PDA-PEI. The (rhCol III/PDA-PEI)_n coatings were grafted onto the PDA-PEI-modified substrates via carbodiimide chemistry and electrostatic interaction. Briefly, the rhCol III (0.5, 1, 2, 4 mg/mL, respectively) was immersed in a solution of EDC (0.05 M) and NHS (0.05 M) in MES buffer (0.05 M, pH 5.40), and then the PDA-PEI-treated samples were immersed in the above solution for 1 h. After that, the pH of the above-mentioned solution was adjusted from 5.4 to 7.4, and the reaction was continued for 8 h. The reason for adjusting the pH to 7.4 was to prevent the amino group from protonating and promote the reaction between the carboxyl groups of rhCol III and the amino groups on the surface. When the reaction was completed, the samples were washed with phosphate-buffered saline (PBS) three times (3 min × 3 times) and were denoted as (rhCol III/PDA-PEI)_n, where n referred to the concentration of rhCol III.

Characterization of the (rhCol III/PDA-PEI)_n coatings

The rhCol III loading amount and the stability of (rhCol III/PDA-PEI)_n coatings prepared with FITC-labeled rhCol III were observed by a confocal laser scanning microscope (Leica SP5, Germany). The microstructure of the coating was investigated by SEM (FEI Nova NanoSEM 450). A spectroscopic ellipsometer (M-2000 V, J.A. Woollam, USA) measurement was performed to measure the thickness of the coatings. The wettability of the coating was analyzed by measuring the water contact angle using Attention Theta (Biolin Scientific, Sweden). The surface elemental components of the (rhCol III/PDA-PEI)_n coatings were analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Scientific ESCALAB 250Xi, USA). A quartz crystal microbalance equipped with dissipation (QCM-D, Q-Sense analyzer, Biolin) was applied to efficiently monitor the real-time changes in the grafted molecules' mass. The zeta potentials of the rhCol III and the (rhCol III/PDA-PEI)_n coatings (n = 0.5, 1, 2, and 4) were measured with a Malvern Zetasizer (Nano ZS90, UK) and a SurPASS 3 (Anton Paar, Austria) instrument, respectively.

Real-time monitoring of the construction process using quartz crystal microbalance-dissipation (QCM-D)

The quartz crystal microbalance equipped with dissipation is a powerful tool to efficiently monitor the real-time change in the grafted molecules' mass of the (rhCol III/PDA-PEI)_n coating at room temperature. Briefly, the PDA-PEI film was prepared on gold-coated quartz crystals (AT-cut/5 MHz) as mentioned earlier before monitoring the construction process of the (rhCol III/PDA-PEI)_n coating. Then, the reaction solution was passed into the chamber at a flow rate of 50 µl/min, and the concentration of the above solution was consistent with the experimental concentrations (22, 37). Detailed information on the QCM-D test was shown in Method S1. The data analysis was conducted with Dfind Smartfit modeling in QSense Dfind software.

Stability tests

The stability of the (rhCol III/PDA-PEI)₂ coating on the PLA stents was investigated in a flowing system with bovine blood serum using a peristaltic pump to simulate blood flow velocity. Detailed information on the stability test was shown in Method S2. After one week, two weeks, and one month, the sample was removed. Then, the retention of the FITC-labeled rhCol III signal after immersion in blood serum was obtained by CLSM and quantitatively calculated by Imagepro-plus software. The fluorescence intensity of the fresh (rhCol III/PDA-PEI)₂ coating was set as 100%.

In vitro assessment of the blood compatibility test

Platelet adhesion test

Blood was drawn from a healthy New Zealand White Rabbit and anticoagulated with sodium citrate. The in vitro blood compatibility of the (rhCol III/PDA-PEI)_n coatings was evaluated by morphological observation of the adhered platelets. The (rhCol III/PDA-PEI)_n-coated (n = 0.5, 1, 2, and 4) PLA sheets (1 cm × 1 cm) were placed in 24-well cell culture plates and incubated with 500 µL PRP for 2 h at 37 ℃. Subsequently, the samples were carefully washed with PBS to remove nonadherent platelets. After that, the samples were fixed with 2.5% glutaraldehyde overnight. Then, the morphology of the platelets on the surfaces of the (rhCol III/PDA-PEI)_n coatings was observed by SEM.

Anti-thrombogenicity evaluation by ex vivo arteriovenous shunt assay

All procedures were compliant with the Laboratory Animal Administration Rules of China and Animal Use Regulations of Sichuan University. Three adults New Zealand white rabbits (2.5–3 kg) were used in this experiment (38). The PDA-PEI and (rhCol III/PDA-PEI)_n coatings (n = 0.5, 1, 2, and 4) were prepared on PVC tubes and carefully connected to the right carotid artery and left external jugular vein of the rabbits. After 2 h of blood circulation, the samples were quickly removed and washed carefully with PBS. The thrombus bound to the sample surfaces was collected, weighed, and photographed. The cross-section of the harvested tubes was photographed to analyze the occlusion rates of the samples. Detailed information on the ex vivo study was shown in Method S3.

Cell proliferation and morphology assay

EC proliferation

Human umbilical vein endothelial cells (HUVECs) and human umbilical artery smooth muscle cells (HUVSMCs) were purchased from Service-bio Technology Co. Wuhan, China. Before the in vitro vascular cell compatibility experiment, HUVECs were passaged to a state with better proliferation activity and cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) media supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin at 37℃ under a concentration of 5% carbon dioxide. The samples were placed in a 24-well cell culture plate and sterilized with ethylene oxide at 37°C for 12 h. Then, 1 ml of the EC suspension with a density of 2×10⁴ cells/mL was seeded onto PLA, PDA-PEI, and (rhCol III/PDA-PEI)_n samples (n = 0.5, 1, 2, and 4) for 1 day and 3 days. The samples were washed, fixed, and then stained with fluorescein diacetate (FDA) and rhodamine/DAPI staining. Meanwhile, the cytocompatibility of the single rhCol III component with different concentrations (10, 50, 100, 500 µg/mL) was investigated, and the operational procedures were consistent with those described above. The cell morphology was inspected and visualized by a confocal laser scanning microscope (Leica SP5, Germany). In addition, the cell viability was evaluated by the metabolic CCK-8 assay (a fresh culture medium containing RPMI 1640 and CCK-8 (9:1, v/v)), and the absorbance value at 450 nm was read (39).

RNA extraction and high-throughput RNA sequencing of ECs

When the fusion rate of ECs seeded on the surface of PLA, PDA-PEI, and (rhCol III/PDA-PEI)_n coatings reached approximately 85%, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. RNA integrity was monitored on agarose gels, followed by concentration and purity measurements using a Nanodrop 1000 (Thermo Fisher Scientific, USA) (40, 41). The quality of each library was assessed on an Agilent Bioanalyzer 2100 by performing quantitative PCR. The whole process of RNA sequencing was described in S4. Hierarchical bidirectional clustering analysis was performed to compare the expression patterns of different genes and different samples by using the heatmap R package, and the analysis methods were provided in S5.

SMC proliferation

Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin was used to culture HUASMCs. The other experimental processes and methods were similar to the protocols mentioned for the HUAEC proliferation test.

In vitro anti-inflammatory evaluation

RAW 264.7 mouse macrophages were cultured with RPMI 1640 media supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO₂ atmosphere. The samples were placed in 24-well cell culture plates and sterilized with ethylene oxide at 37°C for 12 h. Then, 1 ml of the RAW 264.7 macrophage suspension with a density of 10 × 10⁴ cells/mL was seeded onto PLA, PDA-PEI, and (rhCol III/PDA-PEI)_n samples. After 1 day and 3 days of coculture, the RAW 264.7 morphology was evaluated by FDA staining, and the cell viability was determined with a CCK-8 assay. Furthermore, adherent RAW 264.7 macrophages on the surface of the samples were fixed to characterize the M1 and M2 phenotypes of macrophages by immunofluorescence staining (CD68, CD86, and CD206). Enzyme-linked immunosorbent assay (ELISA) (Jingmei Biotechnology) was utilized to detect the release of IL-10 and IL-23 in the supernatant after the macrophages were cultured with samples for 3 d.

In vivo study

In vivo assessment of subcutaneous implantation

For in vivo experiments, all animals were purchased from Chengdu Dossy Experimental Animals Co., Ltd. Male Sprague Dawley rats (SD rats) weighing approximately 300 g were used to assess the subcutaneous implantation. The uncoated, PDA-PEI- and (rhCol III/PDA-PEI)_n-coated PLA sheets were carefully implanted into the subcutaneous tissue of adult SD rats. The methods of implantation and staining were described in Method S6.

In vivo stent implantation

Before and after immobilization with RAPA and (rhCol III/PDA-PEI)₂ coatings, the vascular stents were implanted into the abdominal aortic vessels of the rabbits after anesthesia. After 1 and 3 months of implantation, the stents encased in the neointimal tissue were harvested and fixed with 4% paraformaldehyde for 2 days. The detailed procedures of in vivo stent implantation were described in Method S7.

Subsequently, coronary stent implantation in miniature pigs (approximately weighing 35 kg) was conducted to explore the safety and effectiveness of the (rhCol III/PDA-PEI)₂ coating in vivo and the potential for clinical application. All three stent types (bare CoCr alloy stent, RAPA-modified, and (rhCol III/PDA-PEI)₂-modified CoCr stents) were implanted in the three coronary arteries (right coronary artery, left anterior descending artery, and left circumflex artery) of each pig, and a total of 3 miniature pigs (n = 3 pigs/stent group) were employed. After 3 months of implantation, HE staining was performed to evaluate the hyper proliferation of the intima of different coatings. Furthermore, coronary arteriography was conducted to observe and record the patency of the animal models.

Statistical analysis

The number of biological replicates (n) and technical replicates were described for all experiments in the article, and the analyzed data were expressed as the mean ± s.d. GraphPad Prism was employed for the statistical analysis. One-way analysis of variance (ANOVA) was used for the comparison of more than two groups. Two-way ANOVA was applied to determine the differences between the two groups. The probability value was represented by P, and P < 0.05 values were considered statistically significant.

Data and materials availability

The main data supporting the results of this study are available within the paper and its Supplementary Information. The raw and analyzed datasets generated during the study are available for research purposes from the corresponding authors upon reasonable request.

Acknowledgments

We thank Shanxi Jinbo for assisting us in designing tailored recombinant humanized collagen type III (as-designed rhCol III) together, Fei Chen, Chunjuan Bao, and Ji Bao for helping with the histological staining processing. We acknowledge funding support for the research described in this study from the National Natural Science Foundation of China (32101107), CAMS Innovation Fund for Medical Sciences (2021-I2M-5-013) and National Key Research and Development Programs, China (2022YFB3807303).

Author contributions

H.W., L.Y., and Y.W. designed the study and wrote the paper. R.F. provided technical support and data analysis. K.H. contributed to the stent modification. X.Y. and Y.Q. contributed to the stent deployment and surgical support.

Competing interests

The authors declare that they have no competing interests.

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There is NO Competing Interest.

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One Produces Multi: A Drug-free Cardiovascular Stent Functionalized with Tailored Collagen Supports in-situ Healing of Vascular Tissues

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One Produces Multi: A Drug-free Cardiovascular Stent Functionalized with Tailored Collagen Supports in-situ Healing of Vascular Tissues

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Abstract

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Main

RESULTS AND DISCUSSION

Construction and characterization of (rhCol III/PDA-PEI)_n coatings

Hemocompatibility test

Cell proliferation and morphology of vascular cells

Conclusion

Methods

Materials

Fabrication of the (rhCol III/PDA-PEI)n coatings

Characterization of the (rhCol III/PDA-PEI)_n coatings

Real-time monitoring of the construction process using quartz crystal microbalance-dissipation (QCM-D)

Stability tests

Platelet adhesion test

Anti-thrombogenicity evaluation by ex vivo arteriovenous shunt assay

Cell proliferation and morphology assay

EC proliferation

RNA extraction and high-throughput RNA sequencing of ECs

SMC proliferation

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

One Produces Multi: A Drug-free Cardiovascular Stent Functionalized with Tailored Collagen Supports in-situ Healing of Vascular Tissues

Status:

Journal Publication

Version 1

Abstract

Figures

Main

RESULTS AND DISCUSSION

Construction and characterization of (rhCol III/PDA-PEI)n coatings

Hemocompatibility test

Cell proliferation and morphology of vascular cells

Conclusion

Methods

Materials

Fabrication of the (rhCol III/PDA-PEI)n coatings

Characterization of the (rhCol III/PDA-PEI)n coatings

Real-time monitoring of the construction process using quartz crystal microbalance-dissipation (QCM-D)

Stability tests

Platelet adhesion test

Anti-thrombogenicity evaluation by ex vivo arteriovenous shunt assay

Cell proliferation and morphology assay

EC proliferation

RNA extraction and high-throughput RNA sequencing of ECs

SMC proliferation

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Construction and characterization of (rhCol III/PDA-PEI)_n coatings

Characterization of the (rhCol III/PDA-PEI)_n coatings