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Stabilization of low denaturation temperature collagen from fish by physical cross-linking methods
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 96, No. 6, 575–577. 2003
Stabilization of Low Denaturation Temperature Collagen from Fish by Physical Cross-Linking Methods SHUNJI YUNOKI,1* TAKESHI SUZUKI,2 AND MITSUO TAKAI1 Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-13, Nishi-8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan1 and Ihara & Company Ltd., 3-263-23 Zenibako, Otaru, Hokkaido 047-0261, Japan2 Received 4 August 2003/Accepted 9 September 2003
Collagen matrices were prepared from atelo salmon collagen (SC). SC has a lower denaturation temperature (19°C) than mammalian collagen. SC matrices were successfully stabilized by ultraviolet irradiation and dehydrothermal treatment, and their optimum conditions were determined. By sponging at 37°C, partial denaturation of the collagen molecules resulted in shrinkage of the matrices. [Key words: fish collagen, denaturation, collagen matrix, cross-linking, dehydrothermal treatment, UV irradiation]
Atelo salmon collagen (SC) was prepared from fresh skin of chum salmon (Oncorhynchus keta). The skin without muscle and scales was cut into small pieces (approximately 3 ´ 3 cm) and defatted 3 times with MeOH/CHCl3. The deffated skin was suspended in 0.5 M acetic acid at 4°C. The following procedures were performed at 4°C. The suspension was centrifuged (10,000´g, 30 min) to remove any residues. For the digestion of telopeptides, powdered pepsin from porcine gastric mucosa (art. 7185; Merck, Darmstadt, Germany) was added to the supernatant to a concentration of 30 mg/l, and gently stirred for 2 d. Collagen in the solution was precipitated by salting-out twice with 5% NaCl. The resultant precipitate was dissolved in 0.5 M acetic acid and ultracentrifuged (100,000´g, 60 min) to remove microsized residues, dialyzed against deionized-water, and lyophilized. Atelo bovine collagen (BC) was purchased as a solution from Koken (Tokyo). The collagen matrices and films were prepared from 0.5 (w/v)% collagen in dilute HCl by lyophilization and air-drying at 4°C, respectively. The collagen matrices and films were cross-linked by UV irradiation or DHT treatment. UV irradiation was carried out with a 15W UV lamp (SUV-16 254 nm; As One, Osaka) at 4°C. Collagen matrices were cross-linked by severe dehydration according to a previous report (4). Collagen matrices were heated to 90–160°C for 12–120 h in vacuo. The stability of the collagen matrices was assessed by their solubility in phosphate buffer saline (PBS)(-) at 37°C. The vials and PBS(-) were sterilized to avoid the proliferation of bacteria. Collagen matrices of approximately 10 mg (n = 3 per group) were precisely weighed, and suspended in 10 ml of PBS(-) at 37°C for 5 d. The suspension was filtered with a membrane filter (0.45-mm pore size) and the protein content of the filtrate was measured by absorbance using a spectrophotometer (U-2000A; Hitachi, Tokyo). The compositions of aromatic residues are very small in type I collagens, therefore the measurements were performed at 230 nm assigned to the peptide bonds.
Numerous attempts have recently been made to use type I collagen (abbreviated as collagen) for biomaterials. The cross-linking methods for stabilization of collagen are divided into physical treatments such as ultraviolet (UV) irradiation (1) and dehydrothermal (DHT) treatment (1–5), and treatments involving chemicals such as glutaraldehyde (6) and carbodiimide (5). Chemical treatments confer remarkably high strength and stability to the collagen matrix, but may also result in potential cytotoxicity or poor biocompatibility (7), while physical treatments have no potential cytotoxicity and can provide sufficient stability (3). In the above studies, mammalian collagen has been used as a base for the collagen matrices. There have been few reports on the application of fish collagen to biomaterials. Most fish collagen solutions have a denaturation temperature (Td) under 30°C, and the matrices are expected to be less stable under actual physical conditions. However, we think that fish collagens have various advantages as described below. In our experience, collagen is more easily extracted in higher yield from fish skin than mammalian skin. Fish collagens should have a relatively low risk of possessing unknown pathogens such as bovine spongiform encephalopathy (BSE). Denatured collagen matrices show better tissue regeneration than native collagen matrices (3). Cross-linked fish collagen matrices are expected to possess partially denatured structures under actual physical conditions because of their low Td. Such structures will be advantageous for tissue regeneration and, simultaneously, sufficient stability under actual physical conditions. We studied the low Td collagen from the skin of chum salmon. The Td has been reported to be about 19°C (8). We attempted to determine the optimum cross-linking conditions and properties of low Td collagen matrices stabilized by physical cross-linking methods compared to bovine collagen matrices. * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)11-706-6568 575
576
J. BIOSCI. BIOENG.,
YUNOKI ET AL.
The denaturation of collagen molecules by cross-linking was estimated by Fourier transform-infrared spectroscopy (FT-IR) analysis. Collagen films (thickness < 10 mm) were cross-linked by two physical methods: UV irradiation at a dose of 16 J/cm2 on one side; and DHT treatment at 90°C for 5 d, at 110°C for 3 d, at 130°C for 1 d, or at 160°C for 1 d. The collagen films were measured by FT-IR (FT/IR 350; JASCO, Tokyo) to estimate the denaturation of the collagen helix according to a previous report (9). The content of collagen helix H (%) was estimated from the ratio of two mid-infrared bands R (= A1235/A1450) by setting the R equivalent to H= 100 (%) for an untreated collagen film and to H= 0 (%) for an untreated gelatin film. The thermal stability of the cross-linked collagen matrices was estimated with a differential scanning calorimeter (DSC) (DSC200; Seiko Instruments, Chiba). The matrices (2–5 mg) were sufficiently dried in vacuo and placed in an aluminum cell (f5 mm). The DSC measurements were carried out in the temperature range 20–180°C at a scanning rate of 10°C/min. The helix-coil transition temperatures (Tt) were measured at the top of the transition peaks. The denaturation by sponging was estimated by FT-IR analysis. The cross-linked collagen films were put between two pieces of Teflon filter and suspended in water in a 37°C incubator. After 24 h the water was removed and dried in vacuo. The H (%) of the film was estimated by FT-IR according to the method described above. Susceptibility to collagenase was assayed as solubility in collagenase solution at 37°C. Collagen matrices of approximately 5 mg (n = 3 per group) were precisely weighed, incubated in 2 ml of 0.1 M Tris–HCl buffer (pH 7.4) containing 0.05 M CaCl2 for 1 h at 37°C, and then 2 ml of the same buffer containing 100 unit/ml of powdered collagenase from Clostridium histolyticum (EC 3.4.24.3; Wako Pure Chemical Industries, Osaka) was added. After 24 h the undigested matrices were removed by filtration and the filtrates were hydrolyzed with 6 M HCl for 24 h at 110°C. The hydrolysates were analyzed for hydroxyproline according to a previous method (10). Collagen matrices can be stabilized by UV irradiation (UV-C; around 250 nm wavelength). The solubility of SC matrices was dose-dependently decreased by UV irradiation on one side (Fig. 1). The stability was poorer than that of the
FIG. 1. Stabilization of collagen matrices and films by UV irradiation. The solubility values are the mean ± SD (n = 3). One, One side irradiation; both, both sides irradiated with each side receiving a halfdose.
TABLE 1. Denaturation of collagen helix by UV irradiation and DHT treatment H (%) DHT 90°C 110°C 130°C 160°C BC 95 (84) 91 89 (73) 82 61 SC 102 (40) 91 87 (16) 67 39 The values in parentheses indicate H (%) after sponging. UV
cross-linked films, and was improved by irradiation of a half-dose to both sides (Fig. 1). These results were probably due to the limitation of UV light attenuation. The denaturation and fragmentation of collagen by UV irradiation has been reported (1). We investigated the denaturation of the UV cross-linked collagens by FT-IR and DSC. No significant changes of H (%) were recognized by UV irradiation (16 J/cm2) (Table 1), indicating that the threedimensional structure of the triple-helix was little changed. The Tt (°C) of BC and SC matrices was slightly decreased by UV irradiation (both sides; 16 J/cm2) from 101°C to 92°C and from 94°C to 83°C, respectively. The partial fragmentations of the collagen peptides, which have no effect on the observed IR bands, probably caused the decrease in thermal stability. The optimum dose for SC matrices was determined to be 16 J/cm2 on both sides in order to achieve a higher stability and simultaneously avoid further damage, according to the stabilization and denaturation (Fig. 1 and Table 1). SC matrices could also be cross-linked by DHT treatment. Figure 2 shows the temperature-dependent and duration-dependent suppression in solubility of SC matrices by DHT treatment. No effects were observed at 90°C, although significant suppressions in solubility were observed at temperatures above 110°C (Fig. 2). At 160°C, the solubility went down rapidly, and leveled off in as little as 12 h (Fig. 2). The denaturation of collagen by DHT treatment has been reported (1, 2). The temperature-dependent decrease in H (%) by DHT treatment was estimated (Table 1). The Tt (°C) of BC and SC matrices was slightly decreased by DHT treatment at 110°C for 3 d from 101°C to 96°C and from 94°C to 87°C, respectively, similar to UV irradiation. These results indicate that the thermal stability and three-dimensional structure of the collagen triple-helix were slightly
FIG. 2. Stabilization of collagen matrices by DHT treatment. The solubility values are the mean ±SD (n = 3). The temperatures indicate the DHT treatment temperatures.
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The partially denatured structure of the SC matrices may cause poorer stability in vivo because the collagen triplehelix resists general proteases. However, it has also been reported that denatured collagen matrices show better cellular responses than native collagen matrices (3), suggesting that the partially denatured structure may contribute to tissue regeneration. These advantages and disadvantages of the SC matrices for artificial organs and regenerative medicine should be elucidated by in vivo experiments. FIG. 3. Collagenase susceptibility of collagen matrices. UV, Crosslinked by UV irradiation under the optimal condition; DHT, crosslinked by DHT treatment under the optimal condition; untreated, not cross-linked. The solubility values are the mean ± SD (n = 3).
affected by DHT treatment. Although, to the best of our knowledge, there are no reports that the collagen molecules are damaged by excessive treatment duration, DHT treatment should be stopped when the stabilization is saturated. According to the stabilization and denaturation (Fig. 2 and Table 1), it was determined that the optimum treatment condition for the SC matrices was 110°C for 3 d in order to achieve higher stability and avoid further damage. Assays for collagenase susceptibility have been widely applied to assess the biological stability of collagen matrices. SC and BC matrices were stabilized by two physical crosslinking methods under the optimum conditions for SC matrix (described above) and then subjected to the collagenase assay (Fig. 3). The resistance of SC matrices to collagenase digestion was comparable to those of the cross-linked BC matrices (Fig. 3). This result suggests that cross-linked SC matrices have potential for use as a substitute of the implantable collagen matrices. However, an unexpected problem was also encountered. The cross-linked SC matrices showed significant shrinkage by suspension in 37°C PBS(-) as follows: UV cross-linked, from f3.0 mm to f1.9 mm; DHT cross-linked; from f3.0 mm to f2.3 mm. On the other hand, cross-linked BC matrices showed little shrinkage. In order to determine the cause of the shrinkage, the alteration of H (%) by sponging in water at 37°C was estimated. The results were shown in the parentheses in Table 1. The H (%) of cross-linked SC was extensively reduced, but that of cross-linked BC was reduced only slightly. It appears that denaturation of the collagen molecules are responsible for the shrinkage.
REFERENCES 1. Weadock, K. S., Miller, E. J., Bellincampi, L. D., Zawadsky, J. P., and Dunn, M. G.: Physical crosslinking of collagen fibers: comparison of ultraviolet irradiation and dehydrothermal treatment. J. Biomed. Mater. Res., 29, 1373–1379 (1995). 2. Gorham, S. D., Light, N. D., Diamond, A. M., Willins, M. J., Bailey, A. J., Wess, T. J., and Leslie, N. J.: Effect of chemical modifications on the susceptibility of collagen to proteolysis. II. Dehydrothermal crosslinking. Int. J. Biol. Macromol., 14, 129–138 (1992). 3. Koide, M., Osaki, K., Konishi, J., Oyamada, K., Katakura, T., Takahashi, A., and Yoshizato, K.: A new type of biomaterial for artificial skin: dehydrothermally crosslinked composites of fibrillar and denatured collagens. J. Biomed. Mater. Res., 27, 79–87 (1993). 4. Wang, M. C., Pins, G. D., and Silver, F. H.: Collagen fibres with improved strength for the repair of soft tissue injuries. Biomaterials, 15, 507–512 (1994). 5. Pieper, J. S., Oosterhof, A., Dijkstra, P. J., Veerkamp, J. H., and Van Kuppevelt, T. H.: Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate. Biomaterials, 20, 847–858 (1999). 6. White, M. J., Kohno, I., Rubin, A. L., Stenzel, K. H., and Miyata, T.: Collagen films: effect of cross-linking on physical and biological properties. Biomater. Med. Dev. Art. Org., 1, 703–715 (1973). 7. Huang-Lee, L. L. H., Cheung, D. T., and Nimni, M. E.: Biochemical changes and cytotoxicity associated with the degradation of polymeric glutaraldehyde derived crosslinks. J. Biomed. Mater. Res., 24, 1185–1201 (1990). 8. Kimura, S., Zhu, X., Matsui, R., Shijoh, M., and Takamizawa, S.: Characterization of fish muscle type I collagen. J. Food Sci., 53, 1315–1318 (1988). 9. Gordon, P. L., Huang, C., Load, R. C., and Yannas, I. V.: The far-infrared spectrum of collagen. Macromolecules, 7, 954–956 (1974). 10. Woessner, J. F., Jr.: The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys., 93, 440–447 (1961).
Stabilization of Low Denaturation Temperature Collagen from Fish by Physical Cross-Linking Methods SHUNJI YUNOKI,1* TAKESHI SUZUKI,2 AND MITSUO TAKAI1 Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-13, Nishi-8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan1 and Ihara & Company Ltd., 3-263-23 Zenibako, Otaru, Hokkaido 047-0261, Japan2 Received 4 August 2003/Accepted 9 September 2003
Collagen matrices were prepared from atelo salmon collagen (SC). SC has a lower denaturation temperature (19°C) than mammalian collagen. SC matrices were successfully stabilized by ultraviolet irradiation and dehydrothermal treatment, and their optimum conditions were determined. By sponging at 37°C, partial denaturation of the collagen molecules resulted in shrinkage of the matrices. [Key words: fish collagen, denaturation, collagen matrix, cross-linking, dehydrothermal treatment, UV irradiation]
Atelo salmon collagen (SC) was prepared from fresh skin of chum salmon (Oncorhynchus keta). The skin without muscle and scales was cut into small pieces (approximately 3 ´ 3 cm) and defatted 3 times with MeOH/CHCl3. The deffated skin was suspended in 0.5 M acetic acid at 4°C. The following procedures were performed at 4°C. The suspension was centrifuged (10,000´g, 30 min) to remove any residues. For the digestion of telopeptides, powdered pepsin from porcine gastric mucosa (art. 7185; Merck, Darmstadt, Germany) was added to the supernatant to a concentration of 30 mg/l, and gently stirred for 2 d. Collagen in the solution was precipitated by salting-out twice with 5% NaCl. The resultant precipitate was dissolved in 0.5 M acetic acid and ultracentrifuged (100,000´g, 60 min) to remove microsized residues, dialyzed against deionized-water, and lyophilized. Atelo bovine collagen (BC) was purchased as a solution from Koken (Tokyo). The collagen matrices and films were prepared from 0.5 (w/v)% collagen in dilute HCl by lyophilization and air-drying at 4°C, respectively. The collagen matrices and films were cross-linked by UV irradiation or DHT treatment. UV irradiation was carried out with a 15W UV lamp (SUV-16 254 nm; As One, Osaka) at 4°C. Collagen matrices were cross-linked by severe dehydration according to a previous report (4). Collagen matrices were heated to 90–160°C for 12–120 h in vacuo. The stability of the collagen matrices was assessed by their solubility in phosphate buffer saline (PBS)(-) at 37°C. The vials and PBS(-) were sterilized to avoid the proliferation of bacteria. Collagen matrices of approximately 10 mg (n = 3 per group) were precisely weighed, and suspended in 10 ml of PBS(-) at 37°C for 5 d. The suspension was filtered with a membrane filter (0.45-mm pore size) and the protein content of the filtrate was measured by absorbance using a spectrophotometer (U-2000A; Hitachi, Tokyo). The compositions of aromatic residues are very small in type I collagens, therefore the measurements were performed at 230 nm assigned to the peptide bonds.
Numerous attempts have recently been made to use type I collagen (abbreviated as collagen) for biomaterials. The cross-linking methods for stabilization of collagen are divided into physical treatments such as ultraviolet (UV) irradiation (1) and dehydrothermal (DHT) treatment (1–5), and treatments involving chemicals such as glutaraldehyde (6) and carbodiimide (5). Chemical treatments confer remarkably high strength and stability to the collagen matrix, but may also result in potential cytotoxicity or poor biocompatibility (7), while physical treatments have no potential cytotoxicity and can provide sufficient stability (3). In the above studies, mammalian collagen has been used as a base for the collagen matrices. There have been few reports on the application of fish collagen to biomaterials. Most fish collagen solutions have a denaturation temperature (Td) under 30°C, and the matrices are expected to be less stable under actual physical conditions. However, we think that fish collagens have various advantages as described below. In our experience, collagen is more easily extracted in higher yield from fish skin than mammalian skin. Fish collagens should have a relatively low risk of possessing unknown pathogens such as bovine spongiform encephalopathy (BSE). Denatured collagen matrices show better tissue regeneration than native collagen matrices (3). Cross-linked fish collagen matrices are expected to possess partially denatured structures under actual physical conditions because of their low Td. Such structures will be advantageous for tissue regeneration and, simultaneously, sufficient stability under actual physical conditions. We studied the low Td collagen from the skin of chum salmon. The Td has been reported to be about 19°C (8). We attempted to determine the optimum cross-linking conditions and properties of low Td collagen matrices stabilized by physical cross-linking methods compared to bovine collagen matrices. * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)11-706-6568 575
576
J. BIOSCI. BIOENG.,
YUNOKI ET AL.
The denaturation of collagen molecules by cross-linking was estimated by Fourier transform-infrared spectroscopy (FT-IR) analysis. Collagen films (thickness < 10 mm) were cross-linked by two physical methods: UV irradiation at a dose of 16 J/cm2 on one side; and DHT treatment at 90°C for 5 d, at 110°C for 3 d, at 130°C for 1 d, or at 160°C for 1 d. The collagen films were measured by FT-IR (FT/IR 350; JASCO, Tokyo) to estimate the denaturation of the collagen helix according to a previous report (9). The content of collagen helix H (%) was estimated from the ratio of two mid-infrared bands R (= A1235/A1450) by setting the R equivalent to H= 100 (%) for an untreated collagen film and to H= 0 (%) for an untreated gelatin film. The thermal stability of the cross-linked collagen matrices was estimated with a differential scanning calorimeter (DSC) (DSC200; Seiko Instruments, Chiba). The matrices (2–5 mg) were sufficiently dried in vacuo and placed in an aluminum cell (f5 mm). The DSC measurements were carried out in the temperature range 20–180°C at a scanning rate of 10°C/min. The helix-coil transition temperatures (Tt) were measured at the top of the transition peaks. The denaturation by sponging was estimated by FT-IR analysis. The cross-linked collagen films were put between two pieces of Teflon filter and suspended in water in a 37°C incubator. After 24 h the water was removed and dried in vacuo. The H (%) of the film was estimated by FT-IR according to the method described above. Susceptibility to collagenase was assayed as solubility in collagenase solution at 37°C. Collagen matrices of approximately 5 mg (n = 3 per group) were precisely weighed, incubated in 2 ml of 0.1 M Tris–HCl buffer (pH 7.4) containing 0.05 M CaCl2 for 1 h at 37°C, and then 2 ml of the same buffer containing 100 unit/ml of powdered collagenase from Clostridium histolyticum (EC 3.4.24.3; Wako Pure Chemical Industries, Osaka) was added. After 24 h the undigested matrices were removed by filtration and the filtrates were hydrolyzed with 6 M HCl for 24 h at 110°C. The hydrolysates were analyzed for hydroxyproline according to a previous method (10). Collagen matrices can be stabilized by UV irradiation (UV-C; around 250 nm wavelength). The solubility of SC matrices was dose-dependently decreased by UV irradiation on one side (Fig. 1). The stability was poorer than that of the
FIG. 1. Stabilization of collagen matrices and films by UV irradiation. The solubility values are the mean ± SD (n = 3). One, One side irradiation; both, both sides irradiated with each side receiving a halfdose.
TABLE 1. Denaturation of collagen helix by UV irradiation and DHT treatment H (%) DHT 90°C 110°C 130°C 160°C BC 95 (84) 91 89 (73) 82 61 SC 102 (40) 91 87 (16) 67 39 The values in parentheses indicate H (%) after sponging. UV
cross-linked films, and was improved by irradiation of a half-dose to both sides (Fig. 1). These results were probably due to the limitation of UV light attenuation. The denaturation and fragmentation of collagen by UV irradiation has been reported (1). We investigated the denaturation of the UV cross-linked collagens by FT-IR and DSC. No significant changes of H (%) were recognized by UV irradiation (16 J/cm2) (Table 1), indicating that the threedimensional structure of the triple-helix was little changed. The Tt (°C) of BC and SC matrices was slightly decreased by UV irradiation (both sides; 16 J/cm2) from 101°C to 92°C and from 94°C to 83°C, respectively. The partial fragmentations of the collagen peptides, which have no effect on the observed IR bands, probably caused the decrease in thermal stability. The optimum dose for SC matrices was determined to be 16 J/cm2 on both sides in order to achieve a higher stability and simultaneously avoid further damage, according to the stabilization and denaturation (Fig. 1 and Table 1). SC matrices could also be cross-linked by DHT treatment. Figure 2 shows the temperature-dependent and duration-dependent suppression in solubility of SC matrices by DHT treatment. No effects were observed at 90°C, although significant suppressions in solubility were observed at temperatures above 110°C (Fig. 2). At 160°C, the solubility went down rapidly, and leveled off in as little as 12 h (Fig. 2). The denaturation of collagen by DHT treatment has been reported (1, 2). The temperature-dependent decrease in H (%) by DHT treatment was estimated (Table 1). The Tt (°C) of BC and SC matrices was slightly decreased by DHT treatment at 110°C for 3 d from 101°C to 96°C and from 94°C to 87°C, respectively, similar to UV irradiation. These results indicate that the thermal stability and three-dimensional structure of the collagen triple-helix were slightly
FIG. 2. Stabilization of collagen matrices by DHT treatment. The solubility values are the mean ±SD (n = 3). The temperatures indicate the DHT treatment temperatures.
VOL. 96, 2003
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The partially denatured structure of the SC matrices may cause poorer stability in vivo because the collagen triplehelix resists general proteases. However, it has also been reported that denatured collagen matrices show better cellular responses than native collagen matrices (3), suggesting that the partially denatured structure may contribute to tissue regeneration. These advantages and disadvantages of the SC matrices for artificial organs and regenerative medicine should be elucidated by in vivo experiments. FIG. 3. Collagenase susceptibility of collagen matrices. UV, Crosslinked by UV irradiation under the optimal condition; DHT, crosslinked by DHT treatment under the optimal condition; untreated, not cross-linked. The solubility values are the mean ± SD (n = 3).
affected by DHT treatment. Although, to the best of our knowledge, there are no reports that the collagen molecules are damaged by excessive treatment duration, DHT treatment should be stopped when the stabilization is saturated. According to the stabilization and denaturation (Fig. 2 and Table 1), it was determined that the optimum treatment condition for the SC matrices was 110°C for 3 d in order to achieve higher stability and avoid further damage. Assays for collagenase susceptibility have been widely applied to assess the biological stability of collagen matrices. SC and BC matrices were stabilized by two physical crosslinking methods under the optimum conditions for SC matrix (described above) and then subjected to the collagenase assay (Fig. 3). The resistance of SC matrices to collagenase digestion was comparable to those of the cross-linked BC matrices (Fig. 3). This result suggests that cross-linked SC matrices have potential for use as a substitute of the implantable collagen matrices. However, an unexpected problem was also encountered. The cross-linked SC matrices showed significant shrinkage by suspension in 37°C PBS(-) as follows: UV cross-linked, from f3.0 mm to f1.9 mm; DHT cross-linked; from f3.0 mm to f2.3 mm. On the other hand, cross-linked BC matrices showed little shrinkage. In order to determine the cause of the shrinkage, the alteration of H (%) by sponging in water at 37°C was estimated. The results were shown in the parentheses in Table 1. The H (%) of cross-linked SC was extensively reduced, but that of cross-linked BC was reduced only slightly. It appears that denaturation of the collagen molecules are responsible for the shrinkage.
REFERENCES 1. Weadock, K. S., Miller, E. J., Bellincampi, L. D., Zawadsky, J. P., and Dunn, M. G.: Physical crosslinking of collagen fibers: comparison of ultraviolet irradiation and dehydrothermal treatment. J. Biomed. Mater. Res., 29, 1373–1379 (1995). 2. Gorham, S. D., Light, N. D., Diamond, A. M., Willins, M. J., Bailey, A. J., Wess, T. J., and Leslie, N. J.: Effect of chemical modifications on the susceptibility of collagen to proteolysis. II. Dehydrothermal crosslinking. Int. J. Biol. Macromol., 14, 129–138 (1992). 3. Koide, M., Osaki, K., Konishi, J., Oyamada, K., Katakura, T., Takahashi, A., and Yoshizato, K.: A new type of biomaterial for artificial skin: dehydrothermally crosslinked composites of fibrillar and denatured collagens. J. Biomed. Mater. Res., 27, 79–87 (1993). 4. Wang, M. C., Pins, G. D., and Silver, F. H.: Collagen fibres with improved strength for the repair of soft tissue injuries. Biomaterials, 15, 507–512 (1994). 5. Pieper, J. S., Oosterhof, A., Dijkstra, P. J., Veerkamp, J. H., and Van Kuppevelt, T. H.: Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate. Biomaterials, 20, 847–858 (1999). 6. White, M. J., Kohno, I., Rubin, A. L., Stenzel, K. H., and Miyata, T.: Collagen films: effect of cross-linking on physical and biological properties. Biomater. Med. Dev. Art. Org., 1, 703–715 (1973). 7. Huang-Lee, L. L. H., Cheung, D. T., and Nimni, M. E.: Biochemical changes and cytotoxicity associated with the degradation of polymeric glutaraldehyde derived crosslinks. J. Biomed. Mater. Res., 24, 1185–1201 (1990). 8. Kimura, S., Zhu, X., Matsui, R., Shijoh, M., and Takamizawa, S.: Characterization of fish muscle type I collagen. J. Food Sci., 53, 1315–1318 (1988). 9. Gordon, P. L., Huang, C., Load, R. C., and Yannas, I. V.: The far-infrared spectrum of collagen. Macromolecules, 7, 954–956 (1974). 10. Woessner, J. F., Jr.: The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys., 93, 440–447 (1961).