- Email: [email protected]
Blood Compatibility Evaluation of Elastic Gelatin Gel from Salmon Collagen
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 106, No. 4, 412–415. 2008 DOI: 10.1263/jbb.106.412
© 2008, The Society for Biotechnology, Japan
Blood Compatibility Evaluation of Elastic Gelatin Gel from Salmon Collagen Nobuhiro Nagai,1* Ryosuke Kubota,2 Ryohei Okahashi,2 and Masanobu Munekata2 Creative Research Initiative “Sousei” (CRIS), Hokkaido University, N21-W10, Kita-ku, Sapporo, Hokkaido 001-0021, Japan1 and Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan2 Received 7 May 2008/Accepted 23 June 2008
Blood compatibility of a novel elastic gelatin gel (e-gel) from salmon collagen was evaluated. After a 10-min incubation of the e-gel with rat whole blood, there was a macroscopically small thrombus formation on the e-gel. Microscopic observation revealed that few platelets had adhered to the e-gel. Furthermore, the platelet adhesion rate was markedly lower on the e-gel compared to collagen-coated and fibrinogen-coated surfaces. Comparable results were obtained with re-crosslinked e-gel. In conclusion, the e-gel demonstrated good blood compatibility. [Key words: blood compatibility, elastic gel, platelet adhesion, salmon collagen]
biodegradability, and produces a minimal inflammatory response (7). Owing to the mechanical and biodegradable properties of the r-e-gel, it could potentially be used to engineer blood vessels in vivo. However, the blood compatibility of the r-e-gel has not been investigated. In this study, the interactions of the r-e-gel and the e-gel with rat whole blood and plasma were investigated to assess their compatibility for use in vascular-tissue engineering. The e-gel was prepared by a previously reported method (6). SC was kindly supplied by Ihara & Company (Hokkaido). An SC solution (0.5% w/v), in dilute HCl (pH 3), was mixed with a 25 mM sodium phosphate (NaP) buffer (pH 6.8), 58.3 mM NaCl, and 83.3 mM water-soluble carbodiimide (WSC; Dojindo, Tokyo) at 4°C. The final concentrations of SC, NaP, NaCl, and WSC in the mixture were 0.2% (w/v), 15 mM, 35 mM, and 60 mM, respectively. The mixture was immediately transferred into a 6-well plate (Asahi Techno Glass, Tokyo). After incubation at 4°C for 24 h, the resulting SC gel was incubated in a water bath at 60°C for 5 min to produce the e-gel. To produce the r-e-gel, the e-gel was immersed in 70% (v/v) ethanol at 4°C for 24 h, and the dehydrated e-gel was immersed in 70% (v/v) ethanol, that included 1% (w/v) WSC, at 4°C for 24 h. The r-e-gel was washed five times with phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4). To prepare the argatroban-coated samples, the r-e-gel and the e-gel (circular shape, diameter of 8 mm) were immersed in 1 mg/ml argatroban solution in methanol (Mitsubishi Chemical, Tokyo) overnight at 4°C (9). To prepare collagenand gelatin-coated control plates, 200 μl of 3 mg/ml collagen solution (from porcine skin; Nitta Gelatin, Osaka) and 3 mg/ml gelatin solution (from porcine skin, Nitta Gelatin) were added to 48-well microplates (Asahi Techno Glass) and ventilation-dried at room temperature (10). As an alternative to Dacron and ePTFE, non-treated, polystyrene-sur-
Synthetic materials such as polyethylene terephthalate (Dacron) and expanded polytetrafluoroethylene (ePTFE) have been clinically applied as vascular grafts for a long time and satisfactory results have been obtained when these grafts were used to replace or bypass large-diameter blood vessels. However, when used in small-diameter blood vessels (inner diameter < 6 mm), the patency rates are poor compared to autologous vein grafts. These failures are due to early thrombosis and gradual neointimal hyperplasia, and the pathological changes occurred due to the lack of blood or mechanical compatibility of the synthetic grafts (1). To address this problem, tissue engineering approach is promising. A variety of biodegradable polymers and scaffolds have been evaluated to develop a tissue-engineered vascular graft (2–5). These approaches depend on either the in vitro or in vivo cellular remodeling of a polymeric scaffold. For successful in vivo cellular remodeling, the biocompatibility, biodegradability, and mechanical properties of the scaffold must be suitable to the dynamic environment of the blood vessel. Therefore, the ideal scaffold should employ a biocompatible and biodegradable polymer with elastic properties that interact favorably with cells and blood. Recently, we have developed a novel elastic gelatin gel (e-gel) from salmon collagen (SC) (6). The e-gel was prepared during collagen fibrillogenesis by introducing chemical crosslinks and subsequently heating the resulting collagen gel at 60°C. Among all collagen-based materials, the e-gel is the first material that demonstrates excellent rubberlike mechanical properties. It has been investigated for use in regenerative medicine as an artificial vascular graft (7) and a stretching-culture scaffold (8). The e-gel reinforced by a subsequent re-crosslinking treatment (r-e-gel) exhibits suitable mechanical properties for arterial grafting, excellent * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)11-706-9296 412
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FIG. 1. Macroscopic views of the samples subjected to the blood coagulation tests. The samples were photographed immediately after dropping the whole blood samples (A) and 10 min after dropping the blood followed by rinsing (B). At+, with argatroban treatment; At−, without argatroban treatment; coll, collagen-coated plate; gel, gelatin-coated plate; non, non-treated plate.
faced plates were used as a synthetic polymer surface (10). All experimental animals received humane care in accordance with the Principles of Laboratory Animal Care (formulated by the National Institutes of Health, publication no. 56-23, received 1985). The research protocol was approved by the Ethics Committee. Blood (ca. 8 ml) was collected from a Wistar rat (10 weeks of age, male, 250 g) into a conical tube and then immediately dropped on the gel samples (10 μl/sample). After a 10-min incubation at room temperature, the samples were rinsed three times with PBS. The samples were photographed and subjected to scanning electron microscopy (SEM). The gels were fixed using 2.5% glutaraldehyde and then dehydrated with a sequential immersion in 50%, 75%, 85%, 95%, and 99% ethanol. The immersion in 99% ethanol was repeated three times. The ethanol in the dehydrated samples was thoroughly exchanged with isoamyl acetate, and the samples were subjected to critical point drying. The dried samples were coated with Au using an ion coater (E-1010; Hitachi, Tokyo) and then observed under SEM (JSM-6500F; Jeol, Tokyo). The SEM apparatus was operated at 5.0 kV. Blood was collected from a Wistar rat (10 weeks of age, male, 250 g) into a tube containing 3.8% trisodium citrate (VP-C050K; Terumo, Tokyo). Whole blood (ca. 8 ml) was centrifuged at 220×g for 20 min at room temperature. The top two-third of the platelet-rich supernatant (plasma) was collected and gently suspended 1 : 4 in 0.9% NaCl. To increase platelet adhesion to the collagen and fibrinogen, the platelet-rich plasma (PRP) was supplemented with MgCl2 to a final concentration of 5 mM (11). The r-e-gels coated with argatroban were prepared as described above. To prepare a fibrinogen-coated control plate, 200 μl of 10 mg/ml fibrinogen solution (bovine, Wako Pure Chemical Industries, Osaka) was added to a 48-well microplate (Asahi Techno Glass) and ventilation-dried at room temperature. The plates used were collagen-coated, gelatin-coated, and non-treated. The coated-plates and gels were washed twice in 0.9% NaCl. Immediately after washing, 100 μl of PRP solution was added to each sample. The samples were incubated for 1 h at room temperature and then washed twice in 0.9% NaCl. Then 300 μl of substrate solution, containing 1 mg/ml p-nitrophenyl phosphate (Sigma Aldrich, Tokyo) dissolved in 0.1 M sodium citrate/0.1 M citric acid and 0.1% Triton-X-100 (pH 5.4), was rapidly added to each sample. To prepare a stand-
ard curve, PRP was diluted 1 : 2, 1 : 4, 1 : 8, and 1 : 16 with 0.9% NaCl, and each PRP dilution (100 μl) was mixed with 300 μl of substrate solution. After a 40-min incubation, the enzymatic reaction was stopped by adding 50 μl of 2 N NaOH to each sample. The colored p-nitrophenol produced by the acid-phosphatase reaction of the platelets was measured with a microplate reader (model 680; BioRad, Tokyo) at an absorbance of 405 nm. The percentage of adherent platelets was calculated using the formula: (Abssample − Absblank) / (Abstotal − Absblank) × 100. Figure 1 shows the gross appearance of the samples subjected to the blood coagulation tests. Irrespective of the anticoagulant coating, the r-e-gel and the e-gel demonstrated little thrombus formation. In contrast, there was a large amount of clotted blood on the surfaces of the collagen-coated and non-treated plates. On the surface of gelatin-coated plates, the amount of the clotting was intermediate as compared to the other plates. The experiments were repeated 4 times and the results were reproducible. These results indicate that both the r-e-gel and the e-gel had anticoagulant ability, and that the re-crosslinking treatment had no discernable effect on the coagulant properties. Figure 2 shows the SEM images of the r-e-gel and the e-gel subjected to the blood coagulation tests. The SEM images showed that there were few platelets on both gels irrespective of the anticoagulant coating and re-crosslinking treatments. Furthermore, the adherent platelets maintained round shapes, indicating that they were not activated, and no platelets had spread throughout the entire area of the samples (arrows in Fig. 2B, C). These results demonstrate that the surfaces of the r-e-gel and the e-gel prevented the adherence of platelets and inactivated the platelet reaction. Figure 3 shows the number of platelets that adhered to the samples. The platelet number was estimated from the acid phosphatase reaction (10). There was a linear relationship (R2 = 0.99) between the PRP concentration and the absorbance values at 405 nm, indicating that the acid phosphatase reaction of the platelets may be considered a reliable indicator of platelet number (data not shown). The results demonstrate that the platelet adhesion rates were markedly low on the r-e-gel irrespective of the anticoagulant coating when compared to the collagen-coated and fibrinogen-coated surfaces. The e-gel also showed an adhesion rate as low as the
414
J. BIOSCI. BIOENG.,
NAGAI ET AL.
FIG. 2. SEM images of samples subjected to blood coagulation tests in Fig. 1. After a 10-min incubation of whole blood with the samples followed by a PBS rinse, the samples were subjected to SEM. Panels show the r-e-gels with argatroban treatment (A), without argatroban (B), e-gels with argatroban treatment (C), without argatroban (D), and non-treated r-e-gel (E). Arrows indicate platelets that adhered to the samples. Bar: 10 μm.
FIG. 3. Platelet adhesion rates on the r-e-gels and the control samples. Results are expressed as mean ±SD. At, argatroban treatment. * Significant differences from collagen and fibrinogen at p <0.05 by Student’s t-test (n = 6).
r-e-gel (data not shown). On the other hand, the platelet adhesion to the gelatin-coated surface was as low as to the r-e-gel. The experiments were repeated 4 times and the results were reproducible. These results were in agreement with the macroscopic coagulation tests shown in Fig. 1. This report demonstrated that the r-e-gel and the e-gel had anticoagulant ability without the anticoagulant coating. Various anticoagulants, including argatroban and heparin, have been used for vascular grafting to prevent acute thrombogenicity of grafts during the early stages of implantation. However, due to the gradual release of anticoagulants from the graft surface, their anticoagulation activity is not permanent, which leads to a recurrence of thrombogenicity. Although the mechanism underlying the anticoagulant ability of the r-e-gel and the e-gel is unclear, we have shown that they have excellent compatibility with blood and would appear to be useful for the development of vascular grafts. In fact, in our previous study, we successfully implanted the r-e-gel into a rat-abdominal aorta for 1 month (7). Similar to the in vitro results here, there was little thrombus formation in vivo. Considering that the platelets adhered better to the
collagen-coated than to the gelatin-coated surface, the anticoagulant ability of the r-e-gel and the e-gel may have been due to heat denaturation. The e-gel was prepared by heat treatment of the SC gel at 60°C resulting in collagen denaturation (gelatinization). In fact, the random-coiled structure of gelatin is considered to provide the rubber-like stretchability of the e-gel. In agreement with our results, PolanowskaGrabowska and coworkers reported that the platelet adhesion rate on a gelatin-coated surface was lower than on collagen-coated or fibrinogen-coated surfaces (12). However, blood coagulation is known to depend on material properties, such as surface-free energy, surface charge, and wettability; these properties govern protein adsorption involving platelet adhesion (13, 14). Experiments using human whole blood are needed to test the clinical applications of the gels. Further examinations are necessary to ensure the blood compatibility of the r-e-gel and the e-gel. In conclusion, unlike established collagen-based materials, the r-e-gel and the e-gel are novel materials with excellent blood compatibility. The feasibility of preparing gel grafts as a replacement for native vascular tissue will be reported in the near future. This work was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT).
REFERENCES 1. Isenberg, B. C., Williams, C., and Tranquillo, R. T.: Smalldiameter artificial arteries engineered in vitro. Circ. Res., 98, 25–35 (2006). 2. Iwai, S., Sawa, Y., Ichikawa, H., Taketani, S., Uchimura, E., Chen, G., Hara, M., Miyake, J., and Matsuda, H.: Biodegradable polymer with collagen microsponge serves as a new bioengineered cardiovascular prosthesis. J. Thorac. Cardiovasc. Surg., 128, 472–479 (2004). 3. Lepidi, S., Grego, F., Vindigni, V., Zavan, B., Tonello, C., Deriu, G. P., Abatangelo, G., and Cortivo, R.: Hyaluronan biodegradable scaffold for small-caliber artery grafting: preliminary results in an animal model. Eur. J. Vasc. Endovasc.
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Surg., 32, 411–417 (2006). 4. Shum-Tim, D., Stock, U., Hrkach, J., Shinoka, T., Lien, J., Moses, M. A., Stamp, A., Taylor, G., Moran, A. M., Landis, W., Langer, R., Vacanti, J. P., and Mayer, J. E., Jr.: Tissue engineering of autologous aorta using a new biodegradable polymer. Ann. Thorac. Surg., 68, 2298–2304; discussion 2305 (1999). 5. Wildevuur, C. R., van der Lei, B., and Schakenraad, J. M.: Basic aspects of the regeneration of small-calibre neoarteries in biodegradable vascular grafts in rats. Biomaterials, 8, 418– 422 (1987). 6. Yunoki, S., Mori, K., Suzuki, T., Nagai, N., and Munekata, M.: Novel elastic material from collagen for tissue engineering. J. Mater. Sci. Mater. Med., 18, 1369–1375 (2007). 7. Nagai, N., Nakayama, Y., Zhou, Y. M., Takamizawa, K., Mori, K., and Munekata, M.: Development of salmon collagen vascular graft: mechanical and biological properties and preliminary implantation study. J. Biomed. Mater. Res. B. Appl. Biomater., doi: 10.1002/jbm.b.31121 (May 13, 2008). (published online) 8. Kanayama, T., Nagai, N., Mori, K., and Munekata, M.: Application of elastic salmon collagen gel to uniaxial stretching culture of human umbilical vein endothelial cells. J. Biosci. Bioeng., 105, 554–557 (2008).
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9. Sato, S., Nakayama, Y., Miura, Y., Okamoto, Y., Asano, H., Ishibashi-Ueda, H., Zhou, Y. M., Hayashida, K., Matsuhashi, T., Seiji, K., Sato, A., Yamada, T., Takahashi, S., and Ishibashi, T.: Development of self-expandable covered stents. J. Biomed. Mater. Res. B. Appl. Biomater., 83, 345–353 (2007). 10. Eriksson, A. C. and Whiss, P. A.: Measurement of adhesion of human platelets in plasma to protein surfaces in microplates. J. Pharmacol. Toxicol. Methods., 52, 356–365 (2005). 11. Whiss, P. A. and Andersson, R. G.: Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression. Blood Coagul. Fibrinolysis, 13, 407–416 (2002). 12. Polanowska-Grabowska, R., Simon, C. G., Jr., and Gear, A. R.: Platelet adhesion to collagen type I, collagen type IV, von Willebrand factor, fibronectin, laminin and fibrinogen: rapid kinetics under shear. Thromb. Haemost., 81, 118–123 (1999). 13. Kulik, E. and Ikada, Y.: In vitro platelet adhesion to nonionic and ionic hydrogels with different water contents. J. Biomed. Mater. Res., 30, 295–304 (1996). 14. Lee, J. H., Khang, G., Lee, J. W., and Lee, H. B.: Platelet adhesion onto chargeable functional group gradient surfaces. J. Biomed. Mater. Res., 40, 180–186 (1998).
© 2008, The Society for Biotechnology, Japan
Blood Compatibility Evaluation of Elastic Gelatin Gel from Salmon Collagen Nobuhiro Nagai,1* Ryosuke Kubota,2 Ryohei Okahashi,2 and Masanobu Munekata2 Creative Research Initiative “Sousei” (CRIS), Hokkaido University, N21-W10, Kita-ku, Sapporo, Hokkaido 001-0021, Japan1 and Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan2 Received 7 May 2008/Accepted 23 June 2008
Blood compatibility of a novel elastic gelatin gel (e-gel) from salmon collagen was evaluated. After a 10-min incubation of the e-gel with rat whole blood, there was a macroscopically small thrombus formation on the e-gel. Microscopic observation revealed that few platelets had adhered to the e-gel. Furthermore, the platelet adhesion rate was markedly lower on the e-gel compared to collagen-coated and fibrinogen-coated surfaces. Comparable results were obtained with re-crosslinked e-gel. In conclusion, the e-gel demonstrated good blood compatibility. [Key words: blood compatibility, elastic gel, platelet adhesion, salmon collagen]
biodegradability, and produces a minimal inflammatory response (7). Owing to the mechanical and biodegradable properties of the r-e-gel, it could potentially be used to engineer blood vessels in vivo. However, the blood compatibility of the r-e-gel has not been investigated. In this study, the interactions of the r-e-gel and the e-gel with rat whole blood and plasma were investigated to assess their compatibility for use in vascular-tissue engineering. The e-gel was prepared by a previously reported method (6). SC was kindly supplied by Ihara & Company (Hokkaido). An SC solution (0.5% w/v), in dilute HCl (pH 3), was mixed with a 25 mM sodium phosphate (NaP) buffer (pH 6.8), 58.3 mM NaCl, and 83.3 mM water-soluble carbodiimide (WSC; Dojindo, Tokyo) at 4°C. The final concentrations of SC, NaP, NaCl, and WSC in the mixture were 0.2% (w/v), 15 mM, 35 mM, and 60 mM, respectively. The mixture was immediately transferred into a 6-well plate (Asahi Techno Glass, Tokyo). After incubation at 4°C for 24 h, the resulting SC gel was incubated in a water bath at 60°C for 5 min to produce the e-gel. To produce the r-e-gel, the e-gel was immersed in 70% (v/v) ethanol at 4°C for 24 h, and the dehydrated e-gel was immersed in 70% (v/v) ethanol, that included 1% (w/v) WSC, at 4°C for 24 h. The r-e-gel was washed five times with phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4). To prepare the argatroban-coated samples, the r-e-gel and the e-gel (circular shape, diameter of 8 mm) were immersed in 1 mg/ml argatroban solution in methanol (Mitsubishi Chemical, Tokyo) overnight at 4°C (9). To prepare collagenand gelatin-coated control plates, 200 μl of 3 mg/ml collagen solution (from porcine skin; Nitta Gelatin, Osaka) and 3 mg/ml gelatin solution (from porcine skin, Nitta Gelatin) were added to 48-well microplates (Asahi Techno Glass) and ventilation-dried at room temperature (10). As an alternative to Dacron and ePTFE, non-treated, polystyrene-sur-
Synthetic materials such as polyethylene terephthalate (Dacron) and expanded polytetrafluoroethylene (ePTFE) have been clinically applied as vascular grafts for a long time and satisfactory results have been obtained when these grafts were used to replace or bypass large-diameter blood vessels. However, when used in small-diameter blood vessels (inner diameter < 6 mm), the patency rates are poor compared to autologous vein grafts. These failures are due to early thrombosis and gradual neointimal hyperplasia, and the pathological changes occurred due to the lack of blood or mechanical compatibility of the synthetic grafts (1). To address this problem, tissue engineering approach is promising. A variety of biodegradable polymers and scaffolds have been evaluated to develop a tissue-engineered vascular graft (2–5). These approaches depend on either the in vitro or in vivo cellular remodeling of a polymeric scaffold. For successful in vivo cellular remodeling, the biocompatibility, biodegradability, and mechanical properties of the scaffold must be suitable to the dynamic environment of the blood vessel. Therefore, the ideal scaffold should employ a biocompatible and biodegradable polymer with elastic properties that interact favorably with cells and blood. Recently, we have developed a novel elastic gelatin gel (e-gel) from salmon collagen (SC) (6). The e-gel was prepared during collagen fibrillogenesis by introducing chemical crosslinks and subsequently heating the resulting collagen gel at 60°C. Among all collagen-based materials, the e-gel is the first material that demonstrates excellent rubberlike mechanical properties. It has been investigated for use in regenerative medicine as an artificial vascular graft (7) and a stretching-culture scaffold (8). The e-gel reinforced by a subsequent re-crosslinking treatment (r-e-gel) exhibits suitable mechanical properties for arterial grafting, excellent * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)11-706-9296 412
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FIG. 1. Macroscopic views of the samples subjected to the blood coagulation tests. The samples were photographed immediately after dropping the whole blood samples (A) and 10 min after dropping the blood followed by rinsing (B). At+, with argatroban treatment; At−, without argatroban treatment; coll, collagen-coated plate; gel, gelatin-coated plate; non, non-treated plate.
faced plates were used as a synthetic polymer surface (10). All experimental animals received humane care in accordance with the Principles of Laboratory Animal Care (formulated by the National Institutes of Health, publication no. 56-23, received 1985). The research protocol was approved by the Ethics Committee. Blood (ca. 8 ml) was collected from a Wistar rat (10 weeks of age, male, 250 g) into a conical tube and then immediately dropped on the gel samples (10 μl/sample). After a 10-min incubation at room temperature, the samples were rinsed three times with PBS. The samples were photographed and subjected to scanning electron microscopy (SEM). The gels were fixed using 2.5% glutaraldehyde and then dehydrated with a sequential immersion in 50%, 75%, 85%, 95%, and 99% ethanol. The immersion in 99% ethanol was repeated three times. The ethanol in the dehydrated samples was thoroughly exchanged with isoamyl acetate, and the samples were subjected to critical point drying. The dried samples were coated with Au using an ion coater (E-1010; Hitachi, Tokyo) and then observed under SEM (JSM-6500F; Jeol, Tokyo). The SEM apparatus was operated at 5.0 kV. Blood was collected from a Wistar rat (10 weeks of age, male, 250 g) into a tube containing 3.8% trisodium citrate (VP-C050K; Terumo, Tokyo). Whole blood (ca. 8 ml) was centrifuged at 220×g for 20 min at room temperature. The top two-third of the platelet-rich supernatant (plasma) was collected and gently suspended 1 : 4 in 0.9% NaCl. To increase platelet adhesion to the collagen and fibrinogen, the platelet-rich plasma (PRP) was supplemented with MgCl2 to a final concentration of 5 mM (11). The r-e-gels coated with argatroban were prepared as described above. To prepare a fibrinogen-coated control plate, 200 μl of 10 mg/ml fibrinogen solution (bovine, Wako Pure Chemical Industries, Osaka) was added to a 48-well microplate (Asahi Techno Glass) and ventilation-dried at room temperature. The plates used were collagen-coated, gelatin-coated, and non-treated. The coated-plates and gels were washed twice in 0.9% NaCl. Immediately after washing, 100 μl of PRP solution was added to each sample. The samples were incubated for 1 h at room temperature and then washed twice in 0.9% NaCl. Then 300 μl of substrate solution, containing 1 mg/ml p-nitrophenyl phosphate (Sigma Aldrich, Tokyo) dissolved in 0.1 M sodium citrate/0.1 M citric acid and 0.1% Triton-X-100 (pH 5.4), was rapidly added to each sample. To prepare a stand-
ard curve, PRP was diluted 1 : 2, 1 : 4, 1 : 8, and 1 : 16 with 0.9% NaCl, and each PRP dilution (100 μl) was mixed with 300 μl of substrate solution. After a 40-min incubation, the enzymatic reaction was stopped by adding 50 μl of 2 N NaOH to each sample. The colored p-nitrophenol produced by the acid-phosphatase reaction of the platelets was measured with a microplate reader (model 680; BioRad, Tokyo) at an absorbance of 405 nm. The percentage of adherent platelets was calculated using the formula: (Abssample − Absblank) / (Abstotal − Absblank) × 100. Figure 1 shows the gross appearance of the samples subjected to the blood coagulation tests. Irrespective of the anticoagulant coating, the r-e-gel and the e-gel demonstrated little thrombus formation. In contrast, there was a large amount of clotted blood on the surfaces of the collagen-coated and non-treated plates. On the surface of gelatin-coated plates, the amount of the clotting was intermediate as compared to the other plates. The experiments were repeated 4 times and the results were reproducible. These results indicate that both the r-e-gel and the e-gel had anticoagulant ability, and that the re-crosslinking treatment had no discernable effect on the coagulant properties. Figure 2 shows the SEM images of the r-e-gel and the e-gel subjected to the blood coagulation tests. The SEM images showed that there were few platelets on both gels irrespective of the anticoagulant coating and re-crosslinking treatments. Furthermore, the adherent platelets maintained round shapes, indicating that they were not activated, and no platelets had spread throughout the entire area of the samples (arrows in Fig. 2B, C). These results demonstrate that the surfaces of the r-e-gel and the e-gel prevented the adherence of platelets and inactivated the platelet reaction. Figure 3 shows the number of platelets that adhered to the samples. The platelet number was estimated from the acid phosphatase reaction (10). There was a linear relationship (R2 = 0.99) between the PRP concentration and the absorbance values at 405 nm, indicating that the acid phosphatase reaction of the platelets may be considered a reliable indicator of platelet number (data not shown). The results demonstrate that the platelet adhesion rates were markedly low on the r-e-gel irrespective of the anticoagulant coating when compared to the collagen-coated and fibrinogen-coated surfaces. The e-gel also showed an adhesion rate as low as the
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J. BIOSCI. BIOENG.,
NAGAI ET AL.
FIG. 2. SEM images of samples subjected to blood coagulation tests in Fig. 1. After a 10-min incubation of whole blood with the samples followed by a PBS rinse, the samples were subjected to SEM. Panels show the r-e-gels with argatroban treatment (A), without argatroban (B), e-gels with argatroban treatment (C), without argatroban (D), and non-treated r-e-gel (E). Arrows indicate platelets that adhered to the samples. Bar: 10 μm.
FIG. 3. Platelet adhesion rates on the r-e-gels and the control samples. Results are expressed as mean ±SD. At, argatroban treatment. * Significant differences from collagen and fibrinogen at p <0.05 by Student’s t-test (n = 6).
r-e-gel (data not shown). On the other hand, the platelet adhesion to the gelatin-coated surface was as low as to the r-e-gel. The experiments were repeated 4 times and the results were reproducible. These results were in agreement with the macroscopic coagulation tests shown in Fig. 1. This report demonstrated that the r-e-gel and the e-gel had anticoagulant ability without the anticoagulant coating. Various anticoagulants, including argatroban and heparin, have been used for vascular grafting to prevent acute thrombogenicity of grafts during the early stages of implantation. However, due to the gradual release of anticoagulants from the graft surface, their anticoagulation activity is not permanent, which leads to a recurrence of thrombogenicity. Although the mechanism underlying the anticoagulant ability of the r-e-gel and the e-gel is unclear, we have shown that they have excellent compatibility with blood and would appear to be useful for the development of vascular grafts. In fact, in our previous study, we successfully implanted the r-e-gel into a rat-abdominal aorta for 1 month (7). Similar to the in vitro results here, there was little thrombus formation in vivo. Considering that the platelets adhered better to the
collagen-coated than to the gelatin-coated surface, the anticoagulant ability of the r-e-gel and the e-gel may have been due to heat denaturation. The e-gel was prepared by heat treatment of the SC gel at 60°C resulting in collagen denaturation (gelatinization). In fact, the random-coiled structure of gelatin is considered to provide the rubber-like stretchability of the e-gel. In agreement with our results, PolanowskaGrabowska and coworkers reported that the platelet adhesion rate on a gelatin-coated surface was lower than on collagen-coated or fibrinogen-coated surfaces (12). However, blood coagulation is known to depend on material properties, such as surface-free energy, surface charge, and wettability; these properties govern protein adsorption involving platelet adhesion (13, 14). Experiments using human whole blood are needed to test the clinical applications of the gels. Further examinations are necessary to ensure the blood compatibility of the r-e-gel and the e-gel. In conclusion, unlike established collagen-based materials, the r-e-gel and the e-gel are novel materials with excellent blood compatibility. The feasibility of preparing gel grafts as a replacement for native vascular tissue will be reported in the near future. This work was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT).
REFERENCES 1. Isenberg, B. C., Williams, C., and Tranquillo, R. T.: Smalldiameter artificial arteries engineered in vitro. Circ. Res., 98, 25–35 (2006). 2. Iwai, S., Sawa, Y., Ichikawa, H., Taketani, S., Uchimura, E., Chen, G., Hara, M., Miyake, J., and Matsuda, H.: Biodegradable polymer with collagen microsponge serves as a new bioengineered cardiovascular prosthesis. J. Thorac. Cardiovasc. Surg., 128, 472–479 (2004). 3. Lepidi, S., Grego, F., Vindigni, V., Zavan, B., Tonello, C., Deriu, G. P., Abatangelo, G., and Cortivo, R.: Hyaluronan biodegradable scaffold for small-caliber artery grafting: preliminary results in an animal model. Eur. J. Vasc. Endovasc.
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