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Improved Hemocompatibility and Endothelialization of Vascular Grafts by Covalent Immobilization of Sulfated Silk Fibroin on Poly(lactic-co-glycolic acid) Scaffolds Haifeng Liu,* Xiaoming Li, Xufeng Niu, Gang Zhou, Ping Li, and Yubo Fan* Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, People’s Republic of China ABSTRACT: Endothelialization of vascular grafts prior to implantation has been investigated widely to enhance biocompatibility and antithrombogenicity. Thrombosis of artificial vessels is typically caused by platelet adhesion and agglomeration following endothelial cells detachment when exposed to the shear stress of blood circulation. The present study thus aimed at preventing platelet adhesion and aggregation onto biomaterials before the endothelial confluence is fully achieved. We report this modification of poly(lactic-co-glycolic acid) (PLGA) scaffolds, both to impart hemocompatibility to prevent platelet adhesion and aggregation before the endothelial confluence is fully achieved and to support EC growth to accelerate endothelialization. The modification was achieved by covalent immobilization of sulfated silk fibroin on PLGA scaffolds using γ irradiation. Using phosphate-buffered saline (PBS) as an aging medium, it was demonstrated that the scaffolds prepared by γ irradiation had a good retention of sulfated silk fibroin. The systematic in vitro hemocompatibility evaluation revealed that sulfated silk fibroin covalently immobilized PLGA (S-PLGA) scaffolds-reduced platelet adhesion and activation, prolonged whole blood clotting time, activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). To evaluate further in vitro cytocompatibility of the scaffolds, we seeded vascular ECs on the scaffolds and cultured them for 2 weeks. The ECs were seen to attach and proliferate well on S-PLGA scaffolds, forming cell aggregates that gradually increased in size and fused with adjacent cell aggregates to form a monolayer covering the scaffold surface. Moreover, it was demonstrated through the gene transcript levels and the protein expressions of EC-specific markers that the cell functions of ECs on S-PLGA scaffolds were better preserved than those on PLGA scaffolds. Therefore, this study has described the generation of a vascular graft that possesses the unique ability to display excellent hemocompatibility while simultaneously supporting extensive endothelialization.
1. INTRODUCTION Vascular disease is the leading cause of mortality in today’s world, necessitating surgical interventions including smalldiameter (inner diameter 0.05). However, the cell viabilities of ECs on S-PLGA scaffolds were significantly higher than those on PLGA scaffolds after 7 days (p < 0.05) (Figure 8B). 3.7. Transcript levels of endothelial ECM genes. The expressions of EC-specific genes at the mRNA level were examined for ECs cultured on the scaffolds using real-time RTPCR (Figure 9). The results showed obvious gene expression profile differences between PLGA and S-PLGA groups. The transcript levels of PECAM-1, CD146, and VE-C of ECs cultured on PLGA scaffolds did not change significantly during the 2 weeks in culture (p > 0.05). In addition, there were no significant differences between PLGA and S-PLGA groups at day 3 (p > 0.05). However, the transcript levels of PECAM-1, CD146, and VE-C of ECs cultured on S-PLGA scaffolds were significantly upregulated after the first week in culture (p < 0.05). 3.8. Expression of Endothelial ECM Proteins. Western blot results indicated that ECs cultured on PLGA and S-PLGA scaffolds for 7 and 14 days expressed EC-specific proteins (Figure 10). In addition, the positive staining became darker with culture time. The histogram of densitometric data showed the overall results of the relative optical density of each lane expressed in Western blot membranes. To compare the results 2919
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Figure 7. SEM photomicrographs showing adherence and proliferation of ECs cultured on PLGA scaffolds (A F) and on S-PLGA scaffolds (G L) for 1, 7, and 14 days. (A C,G I) Scale bars = 50 μm. (D F,J L) Scale bars = 10 μm. The arrows indicate ECs growing on the scaffolds.
Figure 8. DNA content (A) and metabolism (B) of ECs on PLGA and S-PLGA scaffolds. (Data in mean ( SD, n = 6, * significant difference between PLGA and S-PLGA scaffolds at p < 0.05 at same time points).
Figure 9. Transcript levels of EC-specific genes by ECs cultured on PLGA and S-PLGA scaffolds for 3, 7, and 14 days. Levels, quantified using real-time RT-PCR, are normalized to the housekeeping gene, GAPDH. * Significant difference between two groups at each time point (p < 0.05).
from different samples, the responses of ECs cultured on PLGA scaffolds for 7 days were standardized to 100. In comparison with PLGA scaffolds, the expressions of PECAM-1, CD146, and VE-C on S-PLGA scaffolds showed significant increases at the protein
level (p < 0.05). To determine the localization of EC-related ECM proteins, ECs cultured on the scaffolds were immunostained with antibodies against PECAM-1, CD146, and VE-C (Figure 11). The results of immunocytochemistry staining were 2920
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Figure 10. Western blot analysis of endothelial ECM proteins by ECs cultured on PLGA and S-PLGA scaffolds for 7 and 14 days. To compare results from different samples, the responses of ECs cultured on PLGA scaffolds for 7 days were standardized to 100. The responses of ECs cultured on PLGA scaffolds for 14 days and on S-PLGA scaffolds for 7 and 14 days were then determined on a relative basis. Data are shown as means ( standard deviation from three samples. * Significant difference between two groups at each time point (p < 0.05).
Figure 11. Immunocytochemistry staining of ECs on PLGA and S-PLGA scaffolds at days 7 and 14. Scale bars are 100 μm.
consistent with those of Western blots, which showed that ECs stained positively for their related markers on both PLGA and S-PLGA scaffolds.
4. DISCUSSION In blood vessels, the endothelium is an essential modulator of vascular tone and thrombogenicity that forms a critical barrier
between the vessel wall and blood components.36 One of the major downfalls of small-diameter vascular grafts is the inability to obtain a confluent endothelium on the luminal surface. EC detachment in flow leaves large parts of the scaffold surface exposed to blood, which can lead to thrombus formation and graft failure via platelet adhesion and aggregation. Therefore, a successful vascular graft must have the two fundamental biological qualities: one is to express hemocompatibility until the EC 2921
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Biomacromolecules lining is fully achieved, and the other is to support cell growth and expansion for accelerating endothelialization.37 In this study, our goal was to improve simultaneously the hemocompatibility and endothelialization of vascular grafts by covalent immobilization of sulfated silk fibroin on a porous PLGA scaffold using γ irradiation. It has been demonstrated that incorporation of sulfate and sulfonate groups into polymers can impart anticoagulant activity on them.38 In addition, we have shown the good anticoagulant activity of sulfated silk fibroin nanofibrous scaffolds in our previous study.18 Reitz et al.39 first reported the sulfation of silk fibroin with chlorosulphonic acid in pyridine. They studied the chemistry of the reaction with emphasis on the identification of the amino acid side groups taking part in the reaction and demonstrated that aliphatic and phenolic hydroxyl, thiol, amine, guanidyl, and indole groups were able to be transformed into sulfates and sulfamates. In the current study, the incorporation of sulfate groups in silk fibroin molecules was identified by FTIR spectroscopy (Figure 1). Surface morphology of PLGA scaffolds before and after immobilization was observed by SEM, as shown in Figure 2. It was found that after immobilization with sulfated silk fibroin the surface morphology of PLGA scaffold changed from a smooth surface to a rough surface with layers of flake attached evenly on the surface. The XPS result further confirmed the successful introduction of sulfate groups onto the PLGA scaffold (Figure 3 and Table 1). More importantly, the concentration of sulfated silk fibroin remained at a high level even after 30 days of immersion in PBS in dynamic state with only 10% of sulfated silk fibroin being released into the medium (Figure 4). The stable covalently immobilized sulfated silk fibroin surface is the guarantee for hemocompatibility and cytocompatibility of the S-PLGA scaffolds. Platelet adhesion and activation are thought to be a major mechanism by which biomaterial thrombogenicity is transduced.33 Therefore, our in vitro hemocompatibility tests have typically involved measurement of platelet adhesion and granule release. The number of platelets adhered onto the scaffolds was quantified by measuring LDH activity. The sensitivity and reliability of the LDH method for counting platelets has been confirmed by some researchers, revealing a linear correlation between LDH activity and platelet number.40,41 As shown in Figure 5A, a significantly lower number of platelets adhered on S-PLGA scaffolds as compared with PLGA scaffolds (p < 0.05). Upon activation, platelets undergo an obvious change in cell shape, promoting platelet platelet contact and adhesion, which leads to the release of their intracellular granular contents including P-selectin.42 P-selectin is translocated from the Rgranules of platelets to the membrane surface upon activation.43 Moreover, this translocation accounts only for the activation of platelets adhered to the scaffolds. Therefore, a soluble form of P-selectin found in the plasma can serve as a reliable marker of platelet activation.44,45 The ELISA result showed that (Figure 5B) exposure to PLGA scaffolds resulted in the release of significantly more soluble P-selectin as compared with S-PLGA scaffolds (p < 0.05), indicating that the platelets exposed to PLGA scaffolds were more activated. There are many potential reasons why grafts fail, but one of the main problems is occlusion due to blood coagulation, particularly under conditions of relatively low flow.46 In this study, the quantity of hemoglobin entrapped by fibrin was used to assess the whole blood clotting times. At each time point measured, blood incubated with S-PLGA scaffolds had a significantly higher
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absorbance than PLGA scaffolds (p < 0.05), indicating that it clotted more slowly (Figure 6). The blood coagulation cascade includes intrinsic pathways, extrinsic pathways, and common pathways.47 APTT is used to evaluate the intrinsic factors such as VIII, IX, XI, and XII, common pathways, or both.48 PT is used to evaluate the extrinsic factors such as V, VII, and X.49 TT is a test of fibrin formation, which is directly induced by the addition of thrombin.50 The interaction of S-PLGA scaffold surface with plasma proteins, as reflected through the significant prolongation in plasma coagulation times (APTT, PT, and TT), suggested the effective inhibition of intrinsic factors, extrinsic factors, and thrombin (Table 2). These findings underscore the importance of using various assays that assess the contributions of different blood components when evaluating the hemocompatibility of a vascular graft. All of the above-mentioned results indicated that the S-PLGA scaffold developed in this study displayed promising hemocompatibility. It is likely that any eventual engineered vascular tissue will require an intact, functional endothelium to function well over the long-term, and thus endothelialization is critical to the success of this field. Therefore, the assessment of adherence, growth, and function of ECs cultured on S-PLGA scaffold in vitro will provide initial confirmation of the utility of this scaffold. The morphologies of ECs cultured on the scaffolds were revealed by SEM photomicrographs (Figure 7). The ECs were seen to attach and proliferate well on S-PLGA scaffolds, forming cell aggregates that gradually increased in size and fused with adjacent cell aggregates to form a monolayer covering the scaffold surface. Compared with the cells cultured on S-PLGA scaffolds, ECs cultured on PLGA scaffolds proliferated much slower and did not form contiguous cell sheet even after 2 weeks in culture, which may be due to the lack of cell recognition sites on the hydrophobic PLGA scaffold surface. The growth and metabolic behavior of vascular cells on S-PLGA scaffolds were characterized by DNA content and MTT assays (Figure 8). The results showed that the cell numbers and cell viabilities of ECs on S-PLGA scaffold were significantly higher than those on PLGA scaffold after 7 days in culture (p < 0.05), which indicated that the S-PLGA scaffold was able to support ECs proliferation without producing toxic effects for at least 14 days. The immobilized sulfated silk fibroin provides stable EC anchorage through interactions with cell surface receptors and influences the proliferation, migration, and survival of ECs. After modification, the cells can now easily recognize the scaffold surfaces as biological components and are therefore likely to result in better cell interaction with the scaffold. The results in this study are consistent with those reported by Ma et al.51 which showed that sulfonated silk fibroin could promote the endothelium regeneration by improving cell proliferation. Tissue-engineered small-diameter vascular grafts should not only possess good cytocompatibility related to cell attachment, cell proliferation, and cell viability but also encourage cell functions of vascular cells, which can be assessed through gene and protein expression of cell specific makers. Therefore, for proper prediction of material efficacy and determination, if ECs still retain their phenotypes when cultured on S-PLGA scaffolds, gene and protein expression of universal EC markers, such as PECAM-1, CD146, and VE-C were examined using real-time RT-PCR, Western blotting, and immunocytochemistry in this study. It was noted that during 2 weeks of culture, the ECs on S-PLGA scaffolds notably modulated the expression of some important specific genes and proteins. The role of immobilized 2922
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Biomacromolecules sulfated silk fibroin obviously had an effect on the phenotypic status of the cells. The cell functions of ECs on S-PLGA scaffolds were better preserved as compared with PLGA scaffolds (Figures 9 11). Many factors, including the mechanics, biochemistry, and surface morphology, have been shown to govern the formation of a confluent and functional endothelial monolayer on a polymeric substrate.52,53 These assay results demonstrated that the S-PLGA scaffold possesses necessary biochemical properties to recruit ECs onto its surface and further provides a favorable biophysical environment for the endothelial regeneration. Taken together, the present data underlined that S-PLGA scaffolds not only showed good hemocompatibility but also showed a strong ability to promote ECs proliferation and did not adversely affect cellular functions.
5. CONCLUSIONS In this study, sulfated silk fibroin was covalently immobilized on a PLGA scaffold by γ irradiation for the improvement of both hemocompatibility and endothelialization of a vascular graft. The scaffold described here that can simultaneously inhibit the early thrombus formation and accelerate endothelialization could potentially improve the long-term potency of smalldiameter vascular grafts, which will be the focus of further work. Whereas the current work focused on immobilization of sulfated silk fibroin on PLGA scaffolds, this method can be extended to sulfated silk fibroin immobilized on other synthetic materials, fabricating vascular grafts with different mechanical properties. In vivo corroboration of the data would be another objective. To conclude, the present immobilization of sulfate silk fibroin may offer a potential improvement for small-diameter vascular grafts. ’ AUTHOR INFORMATION Corresponding Author
*(H.L.) Tel: 86-10-82338456. Fax: 86-10-82338456. E-mail:
[email protected]. Address: School of Biological Science and Medical Engineering, Beihang University, Xue Yuan Road No. 37, Haidian District, Beijing 100191, People’s Republic of China. (Y.F.) Tel: 86-10-82339428. Fax: 86-10-82339428. E-mail:
[email protected]. Address: School of Biological Science and Medical Engineering, Beihang University, Xue Yuan Road No. 37, Haidian District, Beijing 100191, People’s Republic of China.
’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (30900306, 11002016, 31000431, and 10925208), Program for New Century Excellent Talents in University from Ministry of Education of China, Fundamental Research Funds for the Central Universities, and Program of One Hundred Talented People from Beihang University. ’ REFERENCES (1) Soldani, G.; Losi, P.; Bernabei, M.; Burchielli, S.; Chiappino, D.; Kull, S. Biomaterials 2010, 31, 2592–2605. (2) Nerem, R. M.; Seliktar, D. Annu. Rev. Biomed. Eng. 2001, 3, 225–243. (3) Heyligers, J. M.; Verhagen, H. J.; Rotmans, J. I.; Weeterings, C.; de Groot, P. G.; Moll, F. L. J. Vasc. Surg. 2006, 43, 587–591. (4) Yang, Z. L.; Wang, J.; Luo, R. F.; Maitz, M. F.; Jing, F. J.; Sun, H.; Huang, N. Biomaterials 2010, 31, 2072–2083.
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