SEMA4D-heparin Complexes Immobilized on Titanium Surfaces Have

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

SEMA4D-heparin Complexes Immobilized on Titanium Surfaces Have Anticoagulant, Cell-Migration-Promoting, and Immunoregulatory Effects Yuanyuan Cui,† Feng Zhou,†,‡ Lihong Bai,§ Lai Wei,† Jianying Tan,† Zheng Zeng,† Qiang Song,† Junying Chen,*,† and Nan Huang† †

Key Laboratory of Advanced Technology of Materials, Ministry of Education, and §School of Humanity, Southwest Jiaotong University, Chengdu 610031, People’s Republic of China ‡ Institute of Aeronautics and Astronautics, University of Electronic Science and Technology of China, Chengdu 611731, People’s Republic of China ABSTRACT: Soluble semaphorin 4D (SEMA4D) is a 120 kDa transmembrane protein, which belongs to the semaphorin family of axon guidance molecules that act primarily axonal repellents. SEMA4D elicits its migration-promoting and immunomodulatory effects through activation of PLXNB1 and CD72, respectively. In this study, SEMA4D combined with heparin were adsorbed onto cationic surfaces. The biocompatibility evaluation results indicated that the SEMA4D-heparinmodified surfaces displayed less platelet adhesion and activation, prolonged activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) and reduced fibrinogen gamma chain (FGG) exposure and fibrinogen adhesion. Additionally, endothelial cells (ECs) showed improved adhesion density and proliferation activity on the SEMA4D-heparin-modified surfaces. Chemotactic and haptotaxis assays indicated a highly guided migration for ECs on the modified surfaces. The immunological tests revealed that the SEMA4D-heparin complexes had a positive immunomodulatory effect on macrophages and promoted macrophages polarization into M2 phenotypes. Overall, the results suggested that the SEMA4D-heparin complexes can be a potential therapeutic agent to promote tissue healing and accelerate in situ endothelialization with minimal side effects and positive immunomodulatory effect. KEYWORDS: heparin, SEMA4D, anticoagulation, migration-promoting, immunoregulatory Blood is the first element to come into contact with medical implants after material implantation. Besides releasing certain pro-healing factors, such as platelet-derived growth factor, C-XC motif chemokine 12, vascular endothelial growth factor and semaphorin-4D (SEMA4D),6,7 frequently activated platelets can also promote clotting formation through the release of proinflammatory biomolecules, including histamine, serotonin, thromboxane A2, platelet-activating factor and transforming growth factor beta,8,9 which is dangerous or even lethal for patients. Therefore, anticoagulation combined with platelet inhibition is usually chosen as an ideal strategy for designing blood-contact implants.10 Heparin, is widely used as an anticoagulant based on its ability to not only accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade,11 but also mediate various pathophysiological and physiological processes through both specific and nonspecific interactions with verious proteins.12

1. INTRODUCTION Biomaterial scaffolds are critically important in many tissue engineering and regenerative medicine strategies, as they can provide support for cell migration and cell adhesion as well as create a space for tissue repair/regeneration. However, biomaterial implantations inevitably result in injury and consequently in an innate and adaptive inflammatory response, which may directly influence tissue regeneration and biomaterial functions.1,2 For instance, the persistence of an inflammatory stimulus will slow down the tissue repair process or indicate a nonhealing wound.3 Accordingly, as in other areas of biology, biomaterial implants can be designed as tools to control biomolecules, tissue and cellular interactions that modulate immune cells in order to positively regulate the functioning of the immune system.4 In addition, migration, a highly integrated multistep process, is also a prominent component during the orchestration of tissue repair and regeneration.5 For example, the migration of pre-existent endothelial cells (ECs) toward the injury sites is crucially important for vascular healing. © XXXX American Chemical Society

Received: February 3, 2018 Accepted: March 13, 2018 Published: March 13, 2018 A

DOI: 10.1021/acsbiomaterials.8b00098 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Heparin is known to bind acute phase13 and complement proteins14 and this property contributes to its anti-inflammatory activity. The high affinity binding between heparin and antithrombin15,16 leads to thrombin inhibition and decreased fibrinogen formation, thus eventually results in less platelet activation.17 Semaphorins are soluble or membrane-bound proteins, which regulate multiple aspects of cellular function, as well as communication, differentiation and morphology.18 Together with their corresponding receptors, plexins, semaphorins have emerged as central regulators of diverse physiological and pathophysiological processes in various organs.19 Semaphorin 4D (SEMA4D), which belongs to class IV semaphorins, is a type I integral membrane protein that is also expressed in soluble form.20 PLXNB1, the high affinity receptor of SEMA4D, is widely expressed in the nervous system21 and endothelial cells (ECs).22 When activated by SEMA4D, PLXNB1 exerts a migration-promoting effect on ECs through the activation of PTK2B, SRC, and the PIK3R1−Akt pathway,23 and elicits a potent proangiogenic response,22 which is important for tissue healing. In addition, the low affinity receptor for SEMA4D, namely CD72, is mainly expressed on immune cells,24,25 and mediates the immunoregulatory effects of SEMA4D. Through interaction with CD72, SEMA4D has critical functions in regulating the humoral immune response.26 Additionally, it is reported SEMA4D that inhibits the spontaneous and MCP-3-induced migration of freshly isolated monocytes.27 Moreover, SEMA4D also enhances B cell responses by shutting off the negative signaling effects of CD72, and eventually generating a positive immune response.28 In this study, SEMA4D-heparin complexes were adsorbed onto cationic surfaces modified by poly L-lysine (PLL) and anticipated to promote ECs migration, and exert anticoagulant, as well positive immunomodulatory effects to promote the wound healing process. PLL, a strong positively charged polymer, in an aqueous solutions can be spontaneously adsorbed onto several metal oxide surfaces (e.g., TiO2, Si0.4Ti0.6O2 and Nb2O5).29 Heparin, having the highest negative charge density of any known biomolecules11 can associate with SEMA4D through ionic or electrostatic interactions to form complexes. These SEMA4D-heparin complexes can be adsorbed onto PLL-modified surfaces and immobilized by the bonding between sulfate groups from heparin and amine groups of PLL. Using different SEMA4D concentrations adsorbed onto the modified surfaces, the chemical composition, physical properties, and associated biological activities of SEMA4D-heparin complexes were evaluated.

2.2. Immobilization of SEMA4D-heparin Complexes on Titanium Surface. Titanium (Ti, Φ10 mm) plates were prepared according our previous study.30 Briefly, the prepared Ti plates were activated in a 2 M Sodium hydroxide (NaOH) solution at 80 °C for 12 h. Afterward, the NaOH activated Ti plates, denoted as Ti−OH, were immersed in a 2.5 mg/mL PLL solution (in PBS pH 7.4), incubated at 4 °C overnight, and denoted as PLL. Different concentrations (50, 100, and 200 ng/mL) of SEMA4D, in PBS pH 7.4, were separately mixed with 10 mg/mL heparin solution (in PBS pH7.4) in the same volume and then the complexes were incubated at 37 °C for 1 h. Subsequently, the PLL plates were coated with SEMA4D-heparin complexes by immersing in the above SEMA4D-heparin solution at 37 °C, for at least 3 h, and the plates were denoted as 4D25, 4D50, and 4D100. A detailed schematic diagram of the preparation of the SEMA4D-heparin complexes and immobilization is shown in Figure 1. A.

Figure 1. (A) Schematic diagram of the immobilization method of SEMA4D-heparin on the Titanium-based surface; (B) minimally invasive method of in vivo wire implantation. 2.3. X-ray Photoelectron Spectroscopy (XPS). The surface elemental composition of the modified plates were analyzed by X-ray photoelectron spectroscopy (XPS) analysis on an AXIS His spectrometer (Kratos Ltd., Manchester, UK) with a monochromatic Al Ka X-ray source (1486.6 eV photons, 150 W). 2.4. Atomic Force Microscopy (AFM) and Water Contact Angle (WCA). The surface topographies were characterized by atomic force microscopy (AFM) (Bruker Nano Surfaces, Santa Barbara, CA, USA) in PeakForce tapping mode, and processed by Nanoscope Analysis. The static water contact angle (WCA) were measured with a DSA100 Mk 2 goniometer (Krüss Optronic GmbH, Hamburg, Germany), and calculated using the circle segment function of the DSA 1.8 software. 2.5. Blood Compatibility Evaluation. 2.5.1. Platelets Adhesion and Activation. These experiments were performed by incubation with platelet-rich plasma (PRP).30 Fresh PRP (50 μL) was added to each plates and incubated for 1 h at 37 °C. After gently rinsing with a 0.9% NaCl solution, all plates were fixed in a 2.5% glutaraldehyde solution overnight. The plates were then examined and photographed. After critical point drying with CO2, the platelet morphology was evaluated by scanning electron microscopy (SEM). Also, the activeated platelets was identified by Selectin P (a wellknown marker of activated platelets).30 Briefly, PRP (50 μL) was added onto each plate, which was then rinsed after incubation (37 °C, 1h). After blocking with BSA, the primary antibody, mouse antihuman selectin P (1:800), was added and the plates were incubated for 1 h at 37 °C. After rinsing with PBS, the secondary antibody, horseradish peroxidase (HRP)-labeled goat antimouse IgG antibody, was added and incubated (37 °C, 1h). Finally, the color reaction was carried out by using TMB (chromogenic agent) and 1 M H2SO4(stop agent), and the reaction solution was measured at 450 nm.

2. MATERIALS AND METHODS 2.1. Materials and Reagents. Phosphate buffer saline (PBS, 0.067 M, pH 7.4), poly L-lysine (PLL, MW150, 000−300,000), Toluidine Blue O (TBO), and Acid Orange II (AO II) were purchased from Sigma−Aldrich (Saint Louis, MO, USA). Heparin (MW < 8000, potency >160 U mg−1) was purchased from Shanghai Bio Science& Technology Company (Shanghai, P.R. China). Soluble semaphorin 4D (SEMA4D, MW 78 kDa) was purchased from Pepro Tech Company (Rocky Hill, NJ, US). Mouse monoclonal anti human Pselectin antibody, rhodamine, 3,3′,5,5′-Tetramethylbenzidine (TMB) chromogenic agent, Horseradish peroxidase (HRP)-conjugated goat anti mouse IgG antibody were purchased from Sigma-Aldrich. Mouse antihuman fibrinogen γ chain (FGG) antibody and HRP-conjugated mouse antihuman fibrinogen antibody were purchase from BD Bioscience (Shanghai, P.R. China). B


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ACS Biomaterials Science & Engineering 2.5.2. Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT), and Thrombin Time (TT). Activated partial thromboplastin time (APTT) assay was carried out to determine the effect of the plate surfaces on the coagulation system. First, the fresh whole blood was centrifuged at 3000 rpm for 15 min to obtain the platelet-poor plasma (PPP).31 Then, 50 μL of PPP was added to each plate and the experimental plates coated with SEMA4D-heparin were incubated at 37 °C for 30 min. Finally, 100 μL of 0.025 M CaCl2 was added into above mixture and the clotting time was measured using an automatic blood coagulation analyzer (ACL-200, Beckman Coulter, Brea, CA, USA). PPP was incubated with 100 μL of APTT reagent at 37 °C for 3 min and used as control. For prothrombin time (PT) and thrombin time (TT), the tests were performed as follows. A 100 μL volume of PT or TT reagent was added into the test tubes, and then incubated with another 100 μL of tested PPP at 37 °C for 3 min, to be used as control. Thereafter, the clotting times were measured using the above-mentioned coagulation analyzer. 2.5.3. Fibrinogen Adsorption and Conformational Change. The fibrinogen adsorption was conducted according to our previous study.30 Brifely, PPP (50 μL) was added and incubated for 1 h. After blocking with BSA, HRP-conjugated mouse antihuman fibrinogen antibody was added and incubated for another 1 h. After rinsing, the color reaction was carried out using TMB and 1 M H2SO4 and the reaction solution was measured at 450 nm. The exposed FGG was like the fibrinogen adsorption assay. First, the primary antibody (mouse anti human fibrinogen-γ) was added and incubated. Then the secondary antibody (HRP-conjugated goat anti mouse IgG) was further needed to label the primary antibody. Finally, the color reaction and measurment were the same as the adsoption assay. 2.6. In Vitro Cell Proliferation. In vitro cell growth analysis was performed to evaluate the biocompatibility and behavior of cells on the SEMA4D-heparin surfaces. ECs were harvested from human umbilical vein according to Jaffe et al.32 Briefly, after washing with 0.9% NaCl, the umbilical vein was digested by type II collagenase (15 min at 37 °C). The HUVECs were harvested and cultured in DMEM-F12 medium. The seeded density of EC was 5 × 104 cells/cm2. 2.7. EC Migration Ability. 2.7.1. Haptotaxis Assay. Haptotaxis assay was performed accoding to Zhilu Y. et al.33 First, Ti samples were vertically folded. Then on the same one sample, half was modified with SEMA4D-heparin complexes and the other half was untreated. Cell culture was performed on the half of untreated Ti sample until a confluent monlayer of cell was obtained. At this time, the samples were turned over to allow cell migrate on the treated half. Finally, the fluorescence photographs of different samples were taken and the cell migration distances were evaluated. 2.7.2. Chemotaxis Assay. The Chemotaxis assay was conducted by using the Boyden chamber. Briefly, all plates together with a certain volume of DMEM/F-12 media (containing 10% FBS) were putted into 24-well plates. Then, serum deprived ECs (3 × 105 cell/ml) were added into upper Boyden chamber and cultured. After a determine period of time, the cells migrating through the film of Boyden chamber were evaluated. 2.8. Inflammatory Properties. The inflammatory effect was evaluated using the MA cell line RAW246.7 purchased from Southwest Medical University (Luzhou, P.R. China) and cultured in high glucose DMEM media with 10% FBS. After 1 day in culture, the supernatants of all plates were collected and conserved at −20 °C. The levels of IL10, IL6, and TNF were measured by ELISA analysis to further evaluate the MAs inflammatory properties. 2.9. In Vivo Implantation. Sprague−Dawley male rats (9 weeks old) were used in this experiment. Detailed implantation was presented in Figure 1B. Briefly, a syringe needle was first inserted into the rat abdominal aorta to create a implantion room for the wire sample. The wire samples (2−3 cm) were then implanted into the determined places through pricking into the needles. Three fixed points were chosen to ensure the wire samples contact with the aorta closely. Afer in vivo implantation (3 weeks), the aortas together with the wire samples were harvested and fixed at room temperature.

Hematoxylin and eosin (H&E) and immunofluorescence staining were conducted after the paraffin-section. Rabbit antirat platelet and endothelial cell adhesion molecule 1 (PECAM1, 1:20 abcam, Cambridge, UK) and rabbit actin, alpha 2, smooth muscle, aorta (ACTA2, 1:100, abcam) antibodies were used as the first antibody to lable the ECs and smooth muscle cells (SMCs) separately. 2.10. Statistic Analysis. At least three parallel independent experiments were carried out for each of the tests described above. The data were expressed as the mean ± standard deviation (SD). A probability value below 0.05 (P < 0.05) was considered significant.

3. RESULTS 3.1. XPS. The XPS wide scan spectra of the SEMA4Dheparin modified surfaces are shown in Figure 2, and the

Figure 2. XPS analysis of different SEMA4D-heparin-modified surfaces.

elemental composition of different sample surfaces is listed in Table 1. The results revealed that with the increase of the Table 1. Elemental Composition by XPS sample ID

C(%)

N(%)

O(%)

S(%)

PLL 4D25 4D50 4D100

47.52 48.41 48.29 48.75

7.05 7.60 7.28 6.90

45.43 42.77 42.53 42.26

0 1.22 1.90 2.09

SEMA4D concentration the elemental Nitrogen (N) content exhibits a downward trend. Elemental N mainly derived from PLL and also heparin as N elemental from SEMA4D would be covered by surface heparin. After SEMA4D-heparin modification, PLL loosely formed layer became more dense as SEMA4D-heparin complexes fill in the loose structure of PLL (see AFM results in Figure 3), leading to the elevated amount of surface elemental nitrogen. However, with the increase of SEMA4D, the large volume of SEMA4D protein will cover the PLL molecules, thus causing the decrease of elemental N content. Compared with PLL, a new peak, corresponding to S 2p derived from heparin, emerged at ∼168.8 eV. As the SEMA4D density rose, the elemental Sulfur (S) content increased, indicating that with the increase in SEMA4D the surface heparin is rising, further demonstrating that the increase in SEMA4D density promotes the loading of surface heparin. 3.2. AFM Images. AFM analysis was used to examine the surface topography before and after the immobilization of different concentrations of SEMA4D-heparin complexes (Figure 3). After NaOH activation, the roughness of Ti increased from 2.1 to 35.1 nm. Coating with PLL decreased the C


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groups, including hydroxyl, carboxyl, and sulfo groups. PLL and SEMA4D have a slight hydrophobic tendency due to their long hydrocarbon chain and essential hydrophobic power during protein folding.34,35 The WCA shows an increasing trend with the increase in SEMA4D, suggesting that SEMA4D is increasing on the modified sample surfaces. 3.4. Blood Compatibility Evaluation. 3.4.1. Platelets Adhesion and Activation. The platelet adhesion assay was carried out to determine blood compatibility of the modified surfaces (Figure 5). The amount of heparin on the plate

Figure 3. AFM images of the morphology of various SEMA4Dheparin complexes immobilized on the plate surfaces. A−F show the bare Ti, Ti−OH, PLL, 4D25, 4D50, and 4D100, respectively.

roughness to 33.7 nm. Immobilization of different amounts of SEMA4D-heparin complexes further changes the surface roughness of the plates. Except for the 4D50 plates, with the increase of the SEMA4D density, the surface roughness becomes smoother, further indicating that the corresponding SEMA4D-heparin complexes for 4D25 and 4D100 are becoming more uniformly distributed with the increase in the SEMA4D density. Because of the specific interaction between SEMA4D and heparin, the SEMA4D-heparin complexes of 4D50 formed particles that were not so uniform, thus leading to a rougher layer after after modification with SEMA4D-heparin complexes. Overall, the AFM results indicated that the interaction between heparin and SEMA4D at different density could lead to the formation of complexes of different sizes, thus further changing the roughness of the surface of the plates. 3.3. WCA. The alteration of the surface hydrophilicity was determined by measuring the WCA of different modified plates surfaces. As shown in Figure 4, bare Ti had a relatively highWCA of 70°, which dropped to 9.2° after NAOH activation. This can be explained by the introduction of large quantity of hydroxyl (OH) groups. Although it contains some hydrophilic groups, such as amine, PLL exerted a slight hydrophobic effect due to the large molecular weight of the hydrocarbon, reaching a contact angle of around 12°. Heparin is rich in hydrophilic

Figure 5. (A) Number of platelets adhering on the Ti and modified plates; (B) amount of selectin P for activated platelets for all plates; (C) immunofluorescence images (first row) and SEM images (second and third row) of adherent platelets on different plate surfaces. (mean ± SD, n = 4; *p < 0.05 indicates significant difference compared with Ti).

surfaces can reduce fibrin-induced platelet adhesion. On the other hand, the adhered platelets were increased with the increase of SEMA4D, partly due to the increase of SEMA4D binding on the plate surfaces (Figure 5A). SEM analysis clearly showed the platelet morphologies on the different sample surfaces (Figure 5C). The platelets on the Ti were aggregated and mainly appeared spread out or round but stretched out with many dendritic or pseudopodia-like protrusions. Meanwhile, the platelets on the modified plates were almost round. According to Malara A. et al.,36 different morphologies indicate different level of platelet activation. Indeed, the amount of selectin P, a marker of activated platelets, agrees well with the SEM data. Ti produces the highest level of platelet activation. The amount of selectin P is increased with the increase of the SEMA4D density, due to the platelet pro-activating effect of SEMA4D.37 The number and morphology of adhered platelets indicated significant activation and poor hemocompatibility for Ti and minor activation and excellent anticoagulant property for the modified plates. 3.4.2. APTT, PT, and TT. Although it is well-established that the intrinsic (mainly contact activation) and extrinsic (tissue factor) coagulation pathway are interconnected in vivo,38 the plasma coagulation cascade is commonly divided into the two pathways for the convenience of discussion and coagulopathy assessment.39 Generally, APTT indicate deficits or defects in the intrinsic and common coagulation pathwaysand PT indicate deficits or defects in the in extrinsic and common coagulation

Figure 4. Water contact angle after immobilization of SEMA4Dheparin complexes (mean ± SD, n = 4, * P < 0.05 indicates significant difference compared with Ti). D


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promote thrombus growth, as well as accounting for important clot viscoelastic properties, fibrin(ogen) participates in platelet adhesion and aggregation through integrin ITGA2B, triggering lamellipodial extension and platelet spreading.44,45 In addition, fibrin(ogen) can also bind leukocytes through integrin ITGB2, which is a high affinity receptor on stimulated monocytes and neutrophils, contributing to the subsequent inflammatory response.46 Accordingly, the anticoagulant property of both the amount of fibrinogen and FGG on different plate surfaces was investigated. The results revealed that the amounts of fibrinogen and FGG on the SEMA4D-heparin modified plates were significantly reduced compared with those with Ti, as shown in Figure 6C, D. Additionally, with the increase in the SEMA4D density, the concentration of surface heparin is increased (discussed in the XPS results). The increased heparin concentration contributes to the inhibition of the coagulation pathway, thus accounting for reduced fibrinogen adhesion and activation on the modified surfaces. These results suggested that surfaces with immobilized SEMA4D-heparin complexes significantly prevented fibrinogen adsorption and the FGG conformational change, providing evidence for the anticoagulant property observed in the platelet adhesion and activation assays. 3.5. EC Proliferation Assay. ECs were directly cultured on plate surfaces to investigate the effect of SEMA4D-heparin complexes on cell adhesion and proliferation behavior. As the results in Figure 7A, B illustrate, during the first day, even

pathways, while TT is always used to discriminate between problems in thrombin generation (normal TT) and the inhibition of thrombin activity (abnormal TT). Heparin, which contains sulfur-ionizable groups, binds to the enzyme inhibitor antithrombin III (AT), leading to a conformational change in AT and eventually causing its activation.16 The activated AT then inactivates thrombin and other proteases involved in blood clotting, specifically it inhibits the activation of factor Xa, a key initial factor in the common pathway.40,41 Accordingly, heparin can influence the coagulation pathway via activation of AT. The results shown in Figure 6. A reveal that the APTT of plasma incubated on the SEMA4D-heparin modified plate

Figure 6. Clotting time for (A) APTT, (B) PT, and (C) TT. (D) Fibrinogen adhesion amount and (E) FGG exposure amount for different plate surfaces (mean ± SD, n = 4, *p < 0.05 indicates significant difference; APTT (25−32), PT (11.0−13.5), TT (>20) for a healthy).

surfaces was significantly prolonged compared with those in the Ti and PPP blank controls. The APTT exhibited a decreasing trend with the increase of SEMA4D. This result showed that the increase of SEMA4D results in poor blood compatibility. Although the APTT of 4D100 was the lowest among all the modified plates, it was still prolonged by approximately 15 and 17 s compared with the blank control PPP and Ti, respectively, indicating a significant improvement of the anticoagulant property of the SEMA4D-heparin modified plates. However, the effect of the modified plates on the PT (Figure 6B) is rather weak, with only about 0.2−0.3 s prolongation on the modified plate surfaces. These results indicated that the extrinsic pathway is almost absent from the anticoagulant effect of the modified plate surfaces. The TT value (Figure 6C) indicates that its duration was significantly extended by ∼40−60s on the modified plate surfaces, suggesting that the immobilized SEMA4D-heparin complexes on the surfaces act against blood clotting by inhibiting the generation of thrombin.41 3.4.3. Fibrinogen Adhesion and Conformational Change. Fibrinogen adhesion and conformational change (e.g., FGG exposure) are generally considered to play critical a role in platelet activation and aggregation. The target of the coagulation pathway is the activation of fibrinogen and the polymerization of fibrin. Fibrinogen activation is achieved through the exposure of FGG in the C-terminal region of each fibrinogen molecule via allosteric effect. FGG participates in fibrin polymerization and cross-linking, as well as in binding and regulation of factor XIII activity.42,43 Besides its primary role of providing a scaffolding for the intravascular thrombus to

Figure 7. (A) EC proliferation results after 1 day and 3 days in culture and proliferation rate at day 3; (B) immunofluorescence images of ECs after 3 days in culture; (C) EC haptotaxis assay on differently modified plates; (D) the results of EC chemotaxis assay using the Boyden chamber and corresponding immunofluorescence image of the nucleus (mean ± SD, n = 4; *P < 0.05 indicates significant difference compared with Ti).

though the cell density showed no significant difference, more cells adhered on the modified surfaces with the increase of the SEMA4D concentration. In addition, PLXNB1, the receptor of SEMA4D, which can specifically bind with SEMA4D at a relatively high binding affinity (Kd ≈ 1 × 10−9 M),47 was extensively expressed on the ECs surface. It is this specified ligand−receptor interaction that promoted the ECs adhesion during the first day. After another 2 days in culture, the cell density showed a decline in proliferation with the continuous increase of SEMA4D. The 4D25 group, modified with the minimum density of SEMA4D-heparin complexes, showed the highest proliferation rate. This could be explained by the high amount of heparin on the interfaces where cells directly adhere. The proliferation of ECs could be promoted at a moderate density (∼3−7 μg/cm−1) of heparin, but the outcome is poor E


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ACS Biomaterials Science & Engineering when the local heparin concentration is high.31 Compared with the Ti blank groups, the cell proliferation rate for all modified plates was improved to different extents. 3.6. Haptotaxis Assay. Haptotaxis refers to the movement of cells in response to chemical stimulus from adherent molecules. While chemotaxis relates to cells moving with directional preference according to soluble molecules.48 ECs migrating directly on different samples were used to evaluate the haptotaxis (Figure 7C). Haptotaxis of ECs on modified slide surfaces is a key process for endothelium regeneration. After 1 day in culture, the migration distances of cell migrating on modified slide surfaces were much larger than those of cells on the blank Ti groups, indicating that the SEMA4D-heparin coating provides a haptotaxis surface for endothelial cells, and can promote ECs attachment, spreading and migration. It is well-known that SEMA4D combined with its receptor PLXNB1 is involved in cell adhesion and exerts a pro-angiogenic effect.21,23 Our results indicate that as the SEMA4D density increases, more ECs can be polarized and migrate a longer distance. Notably, compared with ECs assayed on 4D25 and 4D100 plates, ECs on 4D50 plates migrated a shorter distance, specifically, 445 μm compared with 486 and 572 μm for 4D25 and 4D100, respectively. This may be due to the complicated interactions between heparin and proteins. Heparin has high negative charge density and conformational flexibility. Protein side chains also possess high degree of conformational flexibility.49 When heparin comes into contact with SEMA4D, conformational changes will happen in the SEMA4D structure. Many factors can influence these changes, such as pH, temperature, ionic density, and heparin or SEMA4D concentration.49 The overall conformational change will further influence the ligand−receptor recognition on ECs cells. The detail mechanism underlying the interaction of proteins with heparin and controlling protein conformational change toward a favorable state still require further investigation. 3.7. Boyden Chamber Assay. The Boyden chamber assay was used to measure ECs chemotaxis at a population level (Figure 7. D and E). Compared with the Ti control, ECs showed a significant chemotaxis response to the SEMA4Dheparin modified surfaces. Additionally, except for the 4D50 groups, with the increase in the SEMA4D concentration, the EC movement toward the modified surfaces increased, suggesting that SEMA4D acts as a potential chemoattractant for ECs. This phenomenon was also reported by J. R. Basile et al.21 The number of migrating cells was not strictly positively correlated with the SEMA4D concentration, considering that more cells migrated through Boyden membranes in the 4D25 group than those in the 4D50 group. A similar phenomenon was observed in the haptotaxis assays. This can be explained by unfavorable SEMA4D conformational changes caused by heparin binding, which can lead to a worse ligand−receptor recognition on the ECs. In the Ti group, sedimentation due to gravity is considered as the primary motion type for cells in the upper chamber, thus inevitably leading to the accumulation of cells in the suspension as well as in the membranes.50 In modified groups, the soluble SEMA4D acted as a chemoattractant and directly induced cells to migrate though the membranes. 3.8. Inflammatory Effect. Macrophages (MAs) are recognized as central immune effector cells that besides playing defensive and hemostatic roles, are also involved in inflammatory reactions, tissue hemostasis, tissue development, and tissue repair. According to varying environmental cues,

MAs can become polarized or activated with different functions. Two main types of MA polarization have been described, namely the M1 and M2MAs. The M1MAs possess a killing/ inflammation-promoting activaty while the M2MAs have the repair/growth-promoting functions.51,52 M1MAs are efficient producers of agents with cytotoxic effect, such as reactive oxygen/nitrogen species, as well as various pro-inflammatory cytokines, such as IL12, IL23, IL1B, IL6, and TNF.52 M2MAs are stimulated by anti-inflammatory mediators, such as glucocorticoids, TGFB1, IL4, and IL10,53 and they secrete the growth-promoting molecule, ornithine, as well as antiinflammatory cytokines, such as TGFB1 and IL10.53 M2MAs mainly take part in the dampening of inflammation, as well as in angiogenesis, tissue remodeling, and immunoregulation.54 This part of the study was undertaken to evaluate the inflammatory effects of the SEMA4D-heparin modified surfaces. After 24 h in culture, the number of MAs adhered on the modified plate surfaces was significantly lower than that on the Ti plate surfaces. With the continuous increase of the SEMA4D concentration, the number of adherent MAs was gradually increased (Figure 8A, E). This is attributed to the

Figure 8. (A) Macrophage number after 1 day in culture on different plates. (B) TNF, (C) IL6, and (D) IL10 concentration in the media after 1 day in culture of macrophage. (E)Immunofluorescence images.

binding between SEMA4D and its specific receptor on MAs, CD72.24,25,55 The secreted cytokines were measured to establish the polarization of the MAs on the plates. As shown in Figure 8B−D, the pro-inflammatory cytokine TNF, is mainly secreted by M1MAs, and its expression was significantly increased as the MAs contacted the surfaces of the nonmodified Ti groups. With the increase in the SEMA4D density, TNF exhibited a declining trend, and its concentration was the lowest in the 4D100 groups. IL6, another pro-inflammatory cytokine, also showed a similar trend like TNF. However, the situation was different when it came to IL10. MAs on the SEMA4D-heparin-modified surfaces produced an increasing amount of the anti-inflammatory cytokine IL10 with the increase in the SEMA4D density. On the other hand, the MAs on the Ti blank group secreted a relatively high amount of proinflammatory molecules and the lowest amount of antiinflammatory factors. All these results indicated that the SEMA4D-heparin-modified surfaces can exert anti-inflammatory effects. 3.9. In Vivo Assay. In humans, the endothelium of blood vessels performs functions that inhibit the processes of intimal hyperplasia, thrombosis and calcification. Thus, it is not surprising that research in blood−contact equipment is directed toward imitating the function of the natural endothelium. In this study, in vivo wire implantation was performed to evaluate in situ the endothelialization degree on the surfaces of the SEMA4D-heparin modified wires. After in vivo implantation, all wires were surrounded by neo-tissues with different intimal F


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ACS Biomaterials Science & Engineering hyperplasia degree (Figure 9A, B). Ti had the highest intimal hyperplasia level of 8.5%, while the SEMA4D-heparin modified

Figure 10. (A) Neo-tissues were additionally immunofluorescently stained with antibodies against ACTA2 and PECAM1 (* indicates the implantation sites for wire samples), and(B) their corresponding expression amount.

Figure 9. Neo-tissues formed around the wires after in vivo implantation (3 weeks), (A) H&E staining images shows the morphologies of neo-tissues (* indicates theimplanted sites for wires); (B) shows the hyperblastosis percent and spreading rate of the neo-tissues (* denotes the P < 0.05 compare Ti with other samples).

endothelialization and were composed of an extremely small amount of ECs, but with a large amounts of SMCs. After being modified by SEMA4D-heparin complexes, the amount of SMCs decreased, whereas the ECs became more continuous and confluent, indicating the improvement of endothelization in the surfaces modified by SEMA4D-heparin complexes. However, with the increase in the SEMA4D density, the 4D50 samples, instead of the 4D100, showed the best endothelialization with the highest number of ECs and the lowest number of SMAs, further suggesting that the appropriate endothelialization depended on adequate density of biofactors modified on the implantations. Noteworthy, it has been reported that under physiological conditions the density of SEMA4D released from activated platelets is around 65 ng/mL,37 which is similar density to that of the group 4D50, thus the optimal endothelization in vivo occurred on the 4D50 group.

surfaces have a relatively small hyperblastosis percentage between 3.5−4.7%. Noteworthy, with the increase of the density of modified SEMA4D, the neo-tissues around the modified surfaces show a gradual increasing trend. Except for the hyperblastosis volumes, the spreading areas of neo-tissues on the vascular walls, were also significantly different between the Ti and modified surfaces (Figure 9A, B). The implanted pure Ti wires promoted the neo-tissues to form a large area of spreading (30.7%) on the vascular walls, suggesting that a large extent of the natural endothelium of the vessel walls was disturbed after the Ti wires implantation. However, the wires modified with SEMA4D and heparin had a smaller influence (8−12%) on the normal vascular endothelium with a slight tendency of increasing the spreading area with the increase of the SEMA4D concentration. The neo-tissues were further analyzed by immunofluorescence staining (Figure 10A). ECs and SMCs were identified by PECAM1 and ACTA2, respectively. After in vivo implantation, the neo-tissue for the Ti group had the highest expression of the SMCs marker ACTA2 (44.2%) and the lowest expression of the ECs marker PECAM1 (0.3%). In the modified groups, the neo-tissue displayed a low expression of the SMCs marker ACTA2 (28.0, 20.2, and 23.6% for the 4D25, 4D50, and 4D100 groups, respectively) and increasing expression of the ECs marker PECAM1 (0.3, 1.0, and 0.8% for 4D25, 4D50. and 4D100 groups, respectively) (Figure 10B). The lower number of SMCs also indicates the low risk of intimal hyperplasia.56,57 A continuous endothelium provides an effective protection for the lumen to prevent hayperplasia and thrombus formation, also serves as a strong indication of the completion of luminal endothelialization.30 With the increase in the SEMA4D content, the number of SMCs was decreased and showed its lowest number in the 4D50 modified surfaces. The neo-tissues surrounding the Ti had the lowest level of

4. DISCUSSION Heparin, with the highest negative charge density of any known biomolecules,11 can interact with proteins in a nonspecific way,11,58 and thus variably binds to specific basic amino acid domains. These basic amino acids consist of either a linear sequence or spatially close domains within the final folding of the proteins.59 SEMA4D (PI = 8.5), which has a positive charge density, can electrostatically interact with heparin at physiological pH. PLL, a cationic polyelectrolyte, has a strong positive charge and is frequently used as a carrier for loading negative biomolecules, such as DNA and heparin.31,60,61 Thus, an anionrich metal surface modified by PLL can easily adsorb and immobilize the SEMA4D-heparin complexes through strong intermolecular electrostatic interaction between PLL and exposed heparin. In this study, the successful immobilization of SEMA4D-heparin complexes was monitored by XPS spectra analysis, revealing that as the SEMA4D increased, the elemental S content increased. G


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ACS Biomaterials Science & Engineering The nonselective bindings between heparin and proteins will cause conformational changes in the proteins, resulting in protein activation/inactivation, or other advantageous/disadvantageous changes in protein biological functions. The changes are strictly environmentally dependent, factors like heparin/protein concentration, pH and ionic density, can be involved. Accordingly, the SEMA4D conformation will be changed by binding with heparin. Such conformational change will in turn expose considerable amounts of the protein internal architecture, such as nonpolar residues,61 and reduce the surface free energy (a higher contact angle) of the SEMA4Dheparin complexes, thus increasing the WCAs of the modified plate surfaces as the SEMA4D density increases. AFM analysis revealed the surface topography before and after the modification with SEMA4D-heparin, suggesting that the interaction between the same amount of heparin and different density of SEMA4D would result in different conformational changes of the SEMA4D molecule and distinct size of SEMA4D-heparin, and as a result different morphology on the plate surfaces. Heparin is well-known for its anticoagulant property and is commonly used in the clinic for the treatment of thrombophlebitis, thrombosis, and embolism.62 The binding of heparin to antithrombin induces a global conformational change and activation of antithrombin. Thrombin activation is rapidly inhibited by forming an antithrombin−thrombin−heparin ternary complex, leading to a decrease in the coagulation reaction rate.16 The blood compatibility results correlated directly with the heparin amounts on the sample surfaces. Although the amount of surface heparin was increased by increasing the amount of modified SEMA4D, the strong interactions between SEMA4D and heparin would impact the subsequent binding between antithrombin and heparin. The 4D25 surface with a small density of SEMA4D provided a larger amount of effective heparin, thus resulting in the smallest number of adherent platelets, and the maximum level of prolongation of the APTT, TT and PT. This is mainly because heparin not only inhibits the function of specific common factors, such as thrombin (FIIa), FXa, and fibrinogen, but also blocks the function of intrinsic factors, such as FIXa and FXIa63,64 (Figure 11). In addition, the number of adherent and activated platelets exhibited an increasing trend with the increase of surface SEMA4D. The Ti groups showed the largest number of adherent and activated platelets. Taking the wettability into consideration, the hydrophobic surfaces of Ti exhibited a less-favorable interaction between water and protein,65 and attracted increased fibrinogen adhesion.66,67 The binding of platelets to fibrinogen via the receptor ITGA2B leads to the exposure of thrombin-susceptible cleavage sites and causes further propagation of coagulation,17 thus leading to a poor blood compatibility. Compared with the Ti groups, the modified surfaces showed fewer adherent platelets and a small degree of platelet activation, indicating the excellent anticoagulant activity of the SEMA4D-heparin surfaces. The results of the fibrinogen adhesion and FGG exposure analysis further showed the poor hemocompatibility of the Ti group and the improved hemocompatibility for SEMA4D-heparin-modified surfaces. Cell migration is essential for fast tissue repair and regeneration. The migration of preexisting ECs to the vascular damage sites is a critical process for vascular repair. The repair process requires a substrate that not only exerts a chemotactic effect for these noncontiguous ECs, but also a haptotaxis effect on ECs in direct contact with implants. Actually, similar

Figure 11. SEMA4D-heparin modified biomaterial exerts an anticoagulant and anti-inflammatory effect and promote EC migration and proliferation. The released heparin can activate antithrombin (AT), which thus inactivates the thrombin (IIa) more than 1000-fold, leading to a coagulation inhibition.16,83 At the same time, heparin can inhibit other coagulation factors, such as IXa, Xa, and XIa64,63 to further inhibit the coagulation process. The SEMA4D on the modified surfaces promote the polarization of MAs into M2 phenotype MAs, which secrete the anti-inflammatory factor IL10 and enhance the wound healing process. Also, SEMA4D inhibits the polarization of MAs into the M1 pro-inflammation phenotype, and suppresses the secretion of pro-inflammation factors, such as IL6 and TNF.81,84 After binding with SEMA4D, PLXNB1 can directly activate some EC memberane-bound receptors, such as Met, EGFR, and HERBB2, those tyrosine kinases receptors then activate microtubules and actin dynamics through the AKT pathway, and finally cause EC migration and proliferation.72,22,82 PLXNB1, activated by SEMA4D can also inactivate CRMPs through inhibition of phosphoinositide 3-kinase (PI3K) and AKT activation that leads to GSK3B activation and as a result to the inactivation of CRMPs (indicated in green).70,71 Also, PLXNB1, after binding with SEMA4D, causes the recruitment and activation of PDZ-RhoGEF and leukemia-associated RhoGEF through association mediated by their PDZ domains, leading to downstream assembly of myosin/actin stress fibers through subsequent activation of Rho, ROCK and cofilin. The assembly of stress fibers generate contraction tensions and promote the focal adhesion through integrin activation, which can subsequently activate nonreceptor tyrosine kinases, such as PTK2B and SRC. PTK2B activation results in the phosphorylation of phosphatidylinositol 3-kinase and the activation of the Akt and Erk1/2 pathways, leading to increased EC migration and proliferation.68,69 PLXNB1 activated by SEMA4D can also regulate Rho activity and ROCK through p190-RhoGAP to influence cytoskeletal dynamics.71,72

chemotactic migration with SEMA4D have been reported by Paolo Conrotto et al.22 However, to the best of our knowledge, no studies have been reported on the chemotactic properties of SEMA4D-heparin complexes immobilized on materials. In our studies, both the results of the chemotactic and haptotaxis assays indicated that the SEMA4D-heparin complexes promote directional migration of ECs, a common feature for all proangiogenic biomolecules. SEMA4D acts through its receptors PLXNB1, which is highly expressed on ECs, to elicit a migration-promoting effect that involves the activation of the Rho, Met, and AKT pathway22,68−72 (Figure 11). High ECs motility induced by SEMA4D/PLXNB1 are achieved by promoting the formation of focal adhesion complexes, stress fibers, and the phosphorylation of the myosin light chain.23,21,73 However, the chemotactic and haptotaxis results were not H


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vascular injury, activated platelets would shed SEMA4D from the platelet membrane. Soluble SEMA4D causes actin reorganization, cell migration and tube formation by endothelial cells21 and immunoregulatory effect by DCs, B cells, T cells and MAs via its specific receptor CD72. These responses indicate a new pathway whereby platelets can promote wound healing and the growth of new vessels.37 However, although ECs migrated the longest distance and proliferated at the fastest rate when the dosage of SEMA4D was high, the in situ endothelialization was not the most optimal. This is because in situ endothelialization depends not only on the anticoagulation process and the EC migration and proliferation, but also on the immunoregulatory effect of the biomaterial itself and the immune response from the body. Our data revealed that the effect of immunoregulation on in situ endothelialization is more important than the anticoagulant effect and the EC migration and proliferation. Besides, the immunoregulatory effect of SEMA4D is extremely dosedependent,82 only the density is adequate and at similar amount with the physiological density (65 ng/mL) the in situ endothelialization could achieve an optimal level.

positively correlated with the SEMA4D concentration. The 4D50 group with the moderate amount of SEMA4D quantity showed a worse chemotactic and haptotaxis effects compared with those of the 4D25 and 4D100 groups. Generally, nonpolar portions of a ligand protein preferentially interact with nonpolar regions of a receptor. This effect is known as “hydrophobic effect”.35 It is this “hydrophobic effect” that enhances the recognition of receptor-SEMA4D and promotes ECs migration during the haptotaxis assay. Regarding the WCA, the surfaces of 4D50 (see WCA results) showed a relatively small WCA, indicating a more hydrophilic property and maybe a smaller nonpolar residues exposure of the SEMA4D molecules. This is a slight disadvantage for ligand− receptor recognition and resulted in shorter migration distances (see 4D50 in Figure 7. C). The data for 4D25 in the chemotactic/haptotaxis assays and the WCA measurements suggested that the SEMA4D-heparin complexes, instead of SEMA4D alone, play a migration-promoting function by acting on the receptor PLXNB1 on ECs, and heparin could modulate the biological activities of SEMA4D by influencing its conformations. The engineered biomaterial implantation always elicits an adaptive immune reaction that influences the host response to the material component and directly regulates the process of wound healing. The interaction of materials with immunocompetent cells is one important approach to evaluate the immune reaction against implanted materials. MAs are regarded as an important part of the immune system that play a critical role in wound healing and tissue regeneration. After interaction with the SEMA4D-heparin modified surfaces, MAs were activated and polarized to different extents by all samples. The secreted chemokines illustrated an obviously elevated antiinflammatory function on the modified surfaces and the dominant presence of MAs with the M2 phenotype. The proinflammatory factors, IL6 and TNF, showed a decrease trend as the SEMA4D density rose. IL10, a very potent immunosuppressive cytokine,74,75 exhibited an increasing potency with the increase of the SEMA4D concentration, with the largest increase in the 4D50 groups. The combination of heparin and SEMA4D did not induce the MAs to react in a strict SEMA4D dose-dependent manner. Although heparin has a certain anti-inflammatory function,76,77 the data of the secreted inflammatory cytokines indicated that the anti-inflammatory effect were mainly exerted by SEMA4D (Figure 11). Through the interaction with its receptor CD72, SEMA4D exerts critical regulatory effects on the humoral immune response, but the immunomodulatory effect of SEMA4D is completely dependent on the biological context. SEMA4D has been reported to enhance proliferation and survival of B cells,78 promote antigen-specific antibody responses of B cells79 and inhibit migration of monocytes and immature dendritic cells (DCs).27,80 It has also been reported that SEMA4D promotes the polarization of peripheral blood monocytes into M2phenotype MAs.81 Here, after binding with heparin, SEMA4D tended to exert anti-inflammatory and M2-polarizationpromoting effects, indicating a positive immunoregulatory effect. Endothelialization is critically important for wound healing and tissue regeneration. The in vivo wire implantation indicated that compared with bare Ti the plates modified with SEMA4D and heparin could promote in situ endothelialization through a combined anticoagulant and anti-inflammatory effect, EC migration and proliferation (Figure 11). In the context of

5. CONCLUSION In conclusion, SEMA4D combined with heparin was adsorbed onto cationic surfaces. According to the biocompatibility evaluation results, the SEMA4D-heparin modified surfaces displayed less platelet adhesion and activation, prolonged APTT, PT, and TT values and caused less fibrinogen adhesion and FGG exposure, leading to an improved anticoagulant property. Furthermore, the adhesion density and proliferation activity of ECs were improved on SEMA4D-heparin modified surfaces. Chemotactic and haptotaxis assays indicated a highly directed migration for ECs on the modified surfaces. The immune tests showed that the SEMA4D-heparin had a positive immunomodulatory effect on MAs, and promoted MAs polarization into M2 phenotype MAs. Overall, the results suggested that the SEMA4D-heparin complexes could potentially be used as a therapeutic agent to promote tissue healing and accelerate in situ endothelialization with low side effects and a positive immunomodulatory effect.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86−28-87634148. Fax: +8628-87600625. ORCID

Yuanyuan Cui: 0000-0003-0157-7736 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (31470921 and 3177040158). REFERENCES

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