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Novel biomaterial from reinforced salmon collagen gel prepared by fibril formation and cross-linking
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 98, No. 1, 40–47. 2004
Novel Biomaterial from Reinforced Salmon Collagen Gel Prepared by Fibril Formation and Cross-Linking SHUNJI YUNOKI,1* NOBUHIRO NAGAI,1 TAKESHI SUZUKI,2 AND MASANOBU MUNEKATA1 Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-13, Nishi-8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan 1 and Ihara & Company Ltd., 3-263-23 Zenibako, Otaru, Hokkaido 047-0261, Japan 2 Received 5 March 2004/Accepted 22 April 2004
The improvement of the thermal stability of gel prepared from salmon atelocollagen (SC) was studied. The denaturation temperature (Td) of the SC solution was found to be 18.6°C. Neutral buffer including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was mixed with acidic SC solution at 4°C, resulting in the introduction of EDC cross-linking during fibril formation. The mechanical strength and thermal stability of the resultant cross-linked SC fibrillar gels reached maximum values at an EDC concentration of 50 mM (f-50 gel). In particular, the melting temperature of the f-50 gel was 47°C, much higher than that of the EDC cross-linked SC gel without fibril formation at the same EDC concentration. The proliferation rate of human periodontal ligament cells on the f-50 gel was higher than that of a porcine atelocollagen fibrillar gel. These results suggest that the gel employed for biomaterials can be fabricated from low Td fish collagen by EDC cross-linking during fibril formation. [Key words: salmon, fish collagen, biomaterial, fibril formation, fibrillar gel, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]
as a novel natural source for collagen fibrillar gels. In spite of such advantages, fish collagen fibrillar gels have not been extensively studied, with the exception shark collagen (10, 11), probably due to their low denaturation temperature (Td), which renders these materials difficult to handle. The Td of shark collagen solution is approximately 30°C (12), which results in the dissolution of the fibrillar gel of this collagen at 37°C (10). This indicates that the gel could not be practically used at the actual physical temperature of human. The Td of most fish collagens is under 30°C (13). The application of fish collagen fibrillar gels to biomaterials has thus been prevented by their relatively low level of thermal stability. Chum salmon has been caught in abundance as an edible fish, and a large amount of the skin of this fish has been produced as waste from the food industry. The Td (approximately 19°C) and the amino acid composition of acid-soluble salmon collagen have previously been reported (14, 15). Recently, we reported that collagen matrices fabricated from salmon atelocollagen (SC) can be stabilized by UV irradiation and dehydrothermal treatment, and it was found that their stability was comparable to that of bovine atelocollagen matrices (16). However, SC fibrillar gel can be unstable at the actual physical temperature of human. The fibril formation of collagen involves the aggregation and alignment of collagen molecules (17, 18), which, according to previous reports, improves thermal stability (11, 12). We hypothesized that the introduction of cross-linking among collagen fibrils during fibril formation would result in a further increase in the thermal stability of collagen
Type I collagen (abbreviated here as collagen) is widely spread in nature and therefore large amounts of collagen are easily obtained (1). Collagen molecules form fibrils or fibril bundles in tissues, and can be extracted by acid solubilization. Acid-soluble collagen molecules consist of a rod-like triple-helix of approximately 300 nm in length and 1.5 nm in diameter and short extra-helical telopeptides (2). Acidsoluble collagen molecules self-assemble and form fibrils under physiological conditions (3–5). Networks of collagen fibrils create a matrix gel. Since these telopeptides are antigenic (1), atelocollagen, the telopeptides of which are enzymatically digested, has been utilized primarily for cosmetics and biomaterials. Recently, atelocollagen fibrillar gels have been used for cellular matrices (6, 7) and tissue engineering (8, 9). In general, collagens for biomaterials are prepared from mammalian tissues, including bovine and porcine sources. However, mammalian tissues have recently been suspected as posing a large risk of pathogens such as bovine spongiform encephalopathy (BSE). Although fish skin is thought to be safe and a potentially large source of collagen, it has not yet been widely used. In addition, large quantities of fish skin are discarded as waste in the food industry. Collagens are easily extracted from wasted fish skins with high yield. Therefore, the practical use of fish collagens could possibly contribute to the recycling of unutilized resources. Due to such advantages, fish collagens have the potential to be used * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)11-706-6568 40
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fibrillar gel, thereby providing fibrillar gels as biomaterials from low-Td salmon collagen. In the present study, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a water soluble carbodiimide, was used as a cross-linking reagent for SC. EDC has recently become popular as a cross-linking reagent for collagen (19–22) due to ease of handling and potentially low cytotoxicity. Here, we show the increase in the thermal stability of SC resulting from fibril formation and cross-linking by EDC.
MATERIALS AND METHODS Preparation of SC SC was prepared from fresh skin of chum salmon (Oncorhynchus Keta) by a procedure reported previously (16) with slight modifications. The defatted salmon skin described above (127 g) was suspended in 3 l of 0.5 M acetic acid at 4°C. The following procedures were performed at 4°C. The solution was gently filtered with gauze, and the filtrate was centrifuged at 10,000 ´ g for 30 min to remove small 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. NaCl solution was added to the supernatant to a final concentration of 5%. After 24 h the precipitate was separated by centrifugation at 10,000´ g for 30 min, and re-dissolved in 1.5 l of 0.5 M acetic acid solution. The salting-out was repeated twice. The resultant solution of SC was filtered with membrane filter (0.45-mm pore size; Advantec, Tokyo) to remove micro-sized residues, dialyzed against deionized-water, and lyophilized. Denaturation temperature The Td of SC solution was measured by the method of Nomura et al. (12) with modifications. The optical rotation of 0.25% SC solution in pH 3 dilute HCl was measured with a polarimeter (SEPA-300; Horiba, Kyoto) by a stepwise increase in temperature. The temperature was held for 30 min at each step (1°C). The temperature at which the change in rotation was half the maximum value was taken as the Td. Fibril formation of SC The Fibril formation of SC was observed by a procedure as follows: 1 ml of neutral Na-phosphate buffer at a specific pH was added to a PMMA cuvette for spectrophotometer. The cuvette was set in a spectrophotometer (UV-mini 1240; Shimadzu, Kyoto) with a temperature control apparatus. After the temperature of the buffer was stabilized, 1 ml of the 0.5% (w/v) SC solution in pH 3 dilute HCl (the acidic SC solution) was added and immediately mixed. The resulting fibril formation was monitored by absorbance (ABS) at 310 nm as the turbidity change. The pH of the buffer was fixed at 5.5, 6.1, 6.8, 7.4, and 7.9. The buffer included NaCl at the concentration where the final concentration in the gels was to be 0, 35, 70, and 140 mM. Degree of fibril formation The SC fibrillar gel was prepared in a centrifugation tube by the procedure described above. After the 1-d incubation, the gel was centrifuged (10,000 ´g, 10 min) to precipitate the collagen fibrils. The protein content of the supernatant was determined by a ninhydrin reaction according to a previous method (23). SC fibrillar gels were also prepared in plastic petri dishes and glass tubes. The Na-phosphate buffer (20 ml) was added to the equal volume of the acidic SC solution with stirring. The mixture was immediately poured into plastic petri dishes (diameter: 35 mm) or glass tubes (diameter: 10 mm), and then incubated at 4°C for 24 h. Preparation of SC gel by introduction of EDC cross-linking during fibril formation ( f-gel) The introduction of EDC (Dojindo, Tokyo) cross-linking during fibril formation of SC was performed by mixture of acidic SC solution and EDC solution in pH 6.8, 30 mM Na-phosphate buffer including 70 mM NaCl. EDC
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was dissolved in the buffer to a concentration of 0, 20, 50, 100, and 200 mM. The EDC solution (20 ml) was added to the equal volume of the acidic SC solution with stirring. The mixture was immediately poured into the plastic petri dishes or the glass tubes. Following incubation at 4°C for 24 h provided gels at final EDC concenrations of 10, 25, 50, and 100 mM (f-0, -10, -25, -50, and -100 gels, respectively). Preparation of SC gel by introduction of EDC cross-linking without fibril formation (n-gel) The mixture of acidic SC solution and EDC solution in water provided EDC cross-linked SC gels without fibril formation. EDC was dissolved in deionizedwater to concentrations of 20, 50, 100, and 200 mM. The EDC solution (20 ml) was added to the equal volume of the acidic SC solution with stirring. The mixture was immediately poured into the plastic petri dishes or the glass tubes. Following incubation at 4°C for 24 h provided gels at final EDC concenrations of 10, 25, 50, and 100 mM (n-10, -25, -50, and -100 gels, respectively). Preparation of porcine atelocollagen fibrillar gel Porcine atelocollagen (PC) was purchased as a 0.3% (w/v) solution in pH 3 dilute HCl from Nitta Gelatin (Cellmatrix Type-P; Nitta Gelatin, Osaka). The PC fibrillar gel (PC gel) was prepared by the mixture of 0.3% (w/v) PC solution and one-sixth volume of Na-phosphate buffer (pH 6.8, 90 mM) including 210 mM NaCl at 4°C. The mixture was subsequently incubated at 37°C for 24 h, resulted in a PC gel. Microscopic observation The collagen fibrils were observed by high-resolution scanning electron microscopy (SEM; JSM6500F, JEOL, Tokyo). The preparation of the specimen was performed according to a previous report (10). Briefly, the gels were fixed by glutaraldehyde, and then dehydrated by ethanol and subsequent isoamyl acetate. The gels were subsequently subjected to a critical point drying. The dried gels were coated with Au using an ion coater (E-1010; Hitachi, Tokyo), and subjected to the SEM measurements. The SEM apparatus was operated at 5.0 kV and a magnification of 15,000. Thermal stability of gel The thermal stability of the gels was evaluated by the melting temperature (Tm). The gels were prepared in the glass tubes with the height of 40 ±3 mm by the procedures described above. The tubes were set in a water bath, and the temperature was stepwise increased from 18°C. The temperature was held for 30 min at each step (1°C/step). A temperature at which more than one half volume of the gel melted was taken as the Tm. Mechanical strength of gel The mechanical strength of the gels was measured by a rheometer (CD-200D; Sun Scientific, Tokyo). The gels were prepared in the plastic petri dish with the height of 5 ±1 mm by the procedures described above. The measurement of mechanical strength was performed using a circular probe (diameter: 20 mm) moving into the gel at a speed of 50 mm/min. The stress at a constant depth (1.3 mm) was defined as the mechanical strength of gel. Free amine group content The degree of cross-linking was estimated by the free amine group contents of collagen molecules in the collagen gels. The lyophilized collagen gels were washed five times in excessive volume of 20% (v/v) ethanol to remove the residual salts, EDC, and by-products, lyophilized again, and subjected to the determination of the free amine group contents. The free amine group content was determined spectrophotometrically after reaction of the free amine groups with 2,4,6-trinitrobenzenesulphonic acid (24) and was expressed as the retention of amine groups; the ratio (%) of the free amine group contents of crosslinked sample to that of the uncross-linked sample. Cell culture The cell proliferation on the collagen fibrillar gels was tested in vitro using the human periodontal ligament (PDL) cells. The f-50 gel and PC gel were used for the cultivation. The PDL cells were isolated by a method previously reported (25),
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and the cells less than passage 15 were used for the cell proliferation assay. The PDL cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical, Tokyo) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) at 37°C in air containing 5% CO2. The culture medium was changed every 2 d. At semi-confluent, PDL cells were subcultured in the same medium. The collagen gels were fabricated in non-coating plastic dishes (diameter of 20 mm; Nunc, Rochester, NY, USA). The collagen gels were rinsed four times with phosphate buffer saline (-), and further rinsed with DMEM once before the test. PDL cells were seeded on the collagen gels at density of 5´103 cells/cm2. The medium was changed every 2 d. At days 1, 3, and 7, the cells on the collagen gels were observed by an inverted light microscope (CK2; Olympus, Tokyo).
NaCl concentration. In a mixture including 140 mM of NaCl, the degree of fibril formation ranged from 67% to 82%. All of mixtures of SC solution and Na-phosphate buffer provided an SC fibrillar gel after incubation for 24 h at 4°C, with the exception of the mixture that contained no NaCl. The mixture including no NaCl showed fluidity after incubation, in spite of their high degree of fibril formation. The Tm of SC fibrillar gels ranged from 23°C to 28°C, which was higher than the Td of the SC solution, but was much lower than the actual physical temperature of human (approximately 37°C). Effect of EDC on fibril formation Fibril formation of SC in the presence of EDC was monitored by turbidity
RESULTS Denaturation temperature The specific rotation of SC was measured at various temperatures (Fig. 1) according to a previous method (12). The Td value of SC was found to be 18.6°C. The absolute value of the specific rotation of SC (approximately 390°) drastically decreased at around the Td due to increases in temperature, and then leveled off to the value of gelatin (i.e., approximately 125°). Fibril formation of SC The fibril formation of SC was monitored by a turbidity change observed at 310 nm (Fig. 2). The pH and NaCl concentration as a measure of ionic strength exerted an effect on increases in turbidity. A rapid rise in turbidity was observed in the mixture of SC solution and Na-phosphate buffer at pH from 6.1 to 7.9 including no NaCl, where the entire increase in turbidity was observed within 1 min at all pH levels. The rise in turbidity tended to be suppressed with increases in the NaCl concentration (Fig. 2). In a mixture including 140 mM of NaCl, the collagen did not attain the entire increase in turbidity within 7 min. Turbidity increases were not observed at pH 5.5, irrespective of the NaCl concentration (data not shown). The degree of fibril formation in the mixture of SC solution and Na-phosphate buffer was measured as the ratio (%) of collagen participating in the fibril formation to the total collagen (Fig. 3). In a mixture including no NaCl, the degree of fibril formation exceeded 83% at all the pH. The degree of fibril formation decreased with an increase in the
FIG. 1. Denaturation curve of SC as measured by specific rotation. The denaturation temperature of SC was found to be 18.6°C. SC, Salmon atelocollagen.
FIG. 2. Effect of pH and NaCl concentration on the fibril formation of SC. Na-phosphate buffer with various pH (closed triangles, 6.1; closed squares, 6.8; closed circles, 7.1; open triangles, 7.4; open squares, 7.9) was added to the equal volume of the acidic SC solution at 4°C, and the resultant fibril formation was observed as a rising of turbidity at 310 nm. The concentration of NaCl in the figure indicates the final concentration in the mixture of the SC solution and Na-phosphate buffer. SC, Salmon atelocollagen.
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FIG. 3. Effect of pH and NaCl concentration on degree of fibril formation in SC fibrillar gels. The degree of fibril formation indicates the ratio (%) of collagen participating in the fibril formation to the total collagen. The concentration of NaCl in the figure indicates the final concentration in the SC fibrillar gels. SC, Salmon atelocollagen.
FIG. 4. Effect of EDC concentration on fibril formation of SC. Na-phosphate buffer (pH 6.8; NaCl, 70 mM) including various concentrations of EDC was added to the equal volume of the acidic SC solution at 4°C, and the resultant fibril formation was observed as a rising of turbidity at 310 nm. The final concentration of EDC in the mixtures was 0 mM (closed triangles), 10 mM (closed circles), 25 mM (open circles), 50 mM (closed squares), or 100 mM (open squares). EDC, 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen.
change in the same manner as fibril formation without EDC (Fig. 4). The rise in turbidity was suppressed by the increase in the EDC concentration. Fibril formation was not recognized when the final EDC concentration in the mixture reached 100 mM. Preparation of SC gel by introduction of EDC crosslinking during fibril formation ( f-gel) The mixture of the acidic SC solution and neutral Na-phosphate buffer including EDC caused the introduction of EDC cross-linking during fibril formation, resulted in the fibrillar gels after a 24-h incubation period (f-gels). Collagen molecules were successfully cross-linked by EDC in all of the f-gels except for f-0, as described below. The appearance of f-0, -10, -25, -50, and -100 gels is shown in Fig. 5. The appearance of f-10, -25, and -50 gels was turbid, as also shown in the case of f-0, an SC fibrillar gel without EDC. In contrast, the appearance of f-100 gel was transparent and colorless. Preparation of SC gel by introduction of EDC crosslinking without fibril formation (n-gel) The mixture of the acidic SC solution and EDC solution in water caused the
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FIG. 5. Appearance of SC gels. n-50 indicates SC gel prepared by introduction of EDC cross-linking without fibril formation at an EDC concentration of 50 mM. f-0, -10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC cross-linking during fibril formation at an EDC concentration of 0, 10, 25, 50, and 100 mM, respectively. SC, Salmon atelocollagen; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The appearance of f-10, -25, and -50 gels was turbid, as also shown in the case of f-0, an SC fibrillar gel without EDC. In contrast, the appearance of f-100 gel was transparent and colorless, as also shown in the case of n-50 gel without fibril formation.
introduction of EDC cross-linking to the collagen molecules without fibril formation, resulted in the gels without collagen fibrils after a 24-h incubation period (n-gels). Collagen molecules were successfully cross-linked by EDC in all of the n-gels as described below. The EDC cross-linked SC gels, n-10, -25, -50, and -100 gels, were without exception transparent and colorless, as shown in Fig. 5 (n-50 gel), indicating that fibril formation did not occur. The gel at an EDC concentration below 10 mM showed fluidity. Fibril structure of gels The fibril structure of SC gels was observed by SEM (Fig. 6). Well-developed fibril networks were observed on the f-0, -10, -25, and -50 gels, the appearance of which was turbid. The fibril structure of these gels was only slightly affected by the EDC concentration in the gels. On the other hand, collagen fibrils were not observed in the f-100 gel or in any of the n-gels, the appearance of which was transparent and colorless. Thermal stability of gel The Tm of the f- and n-gels was measured as described above (Table 1). The Tm value of f-0 gel was 28°C. The Tm values of the f-gels increased with increases in the EDC concentration in the gels. Surprisingly, the Tm value of the f-50 gel was 47°C, which was much higher than that of all the n-gels (around 39°C). In contrast, the Tm value of the f-100 gel (39°C) was lower than that of the f-50 gel, in spite of its higher concentration of EDC in the gel. Mechanical strength of gel The mechanical strength of the f-gels is shown in Fig. 7, as compared with those of the n-gels. All of the n-gels were brittle. In contrast, the f-gels showed flexibility, except for the brittle f-100 gel. The strength of the f-gels increased with an increase in the EDC concentration in the gels, and reached a maximum value at 50 mM. However, the strength of the f-100 gel was lower than that of the f-50 gel. The strength of the n-gels also increased with increases in the EDC concentration in the gels, and the strength was almost saturated at an EDC concentration of 25 mM. The strength of the f-gels was comparable to that of the n-gels at the same EDC concentration, with the
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FIG. 6. SEM images of collagen fibrils in collagen gels. n-50 indicates SC gel prepared by introduction of EDC cross-linking without fibril formation at an EDC concentration of 50 mM. f-0, -10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC cross-linking during fibril formation at an EDC concentration of 0, 10, 25, 50, and 100 mM, respectively. PC indicates porcine atelocollagen fibrillar gel without EDC cross-linking. SC, Salmon atelocollagen; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Well-developed collagen fibrils were observed in f-0, -10, -25, -50 and PC gels, while not in n-50 and f-100 gels.
exception of gels at 100 mM. It was then determined that the f-100 gel was less strong than the n-100 gel. Free amine group content The degree of cross-linking was estimated by measurement of the free amine group contents (Fig. 8). The free amine group contents in the f-gels decreased from 100% to 84% at an EDC concentration of 10 mM, and then leveled off. On the other hand, the drastic decrease in free amine group contents was shown in the n-gels, i.e., a decrease from 100% to 25% was observed at an EDC concentration of 25 mM, and then the decrease leveled off. Cell proliferation on f-gel The PDL cells on the f-50
gel was observed on days 1, 3, and 7, and this proliferation was compared with that on the PC gel (Fig. 9). Cell proliferation on the f-50 gel proceeded faster than on the PC gel. Interestingly, the cells assumed a specific appearance on PC gel that differed from that on the f-50 gel. The cells on the f50 gel were flat in appearance. On the other hand, the morphology of the cells on PC gel was slim and rod-like. DISCUSSION Atelocollagen fibrillar gels, as well as atelocollagen sponges, have been very important for the development of
TABLE 1. Effect of the EDC concentration on the thermal stability of EDC cross-linked SC fibrillar gels n-10 n-25 n-50 n-100 f-0 f-10 f-25 f-50 f-100 PC gel Tm (°C) 37 39 39 39 28 32 37 47 37 47 SC, Salmon atelocollagen; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; PC, porcine atelocollagen. The thermal stability of the gels was evaluated by the Tm. n-10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC cross-linking without fibril formation at an EDC concentration of 10, 25, 50, and 100 mM, respectively. f-0, -10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC crosslinking during fibril formation at an EDC concentration of 0, 10, 25, 50, and 100 mM, respectively. PC gel indicates PC fibrillar gel without EDC cross-linking.
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cellular matrices (6, 7) and tissue engineering (8, 9). However, the potential use of fibrillar gel from low-Td fish collagen as a collagen-based biomaterial has not been sufficiently investigated to date, probably due to the handling difficulty resulting from the low Td of this type of gel. The present study defined that the thermal stability of SC fibrillar gel was extensively improved by introduction of crosslinking during fibril formation of collagen, and the gel would have a potential to be utilized for the development of cellular matrices and tissue engineering. Nomura et al. (10) has reported that the optical density from porcine collagen fibrils gradually decreased at 10°C, indicating that porcine collagen cannot form fibrils at such a low temperature. The fibril formation is considered to be an aggregation of collagen molecules caused by the effects of hydrophobic and electrostatic interactions (17, 18). The low temperature discourages hydrophobic interactions, resulting in the suppression of the fibril formation. However, the active fibril formation of SC was observed at 4°C, a temperature at which porcine collagen could not form fibrils. The
FIG. 7. Effect of EDC concentration on mechanical strength of SC gels. The EDC concentration in the figure indicates the final concentration in the gels. Closed bars, SC gels prepared by introduction of EDC cross-linking during fibril formation (f-gels); open bars, SC gels prepared by introduction of EDC cross-linking without fibril formation (n-gels); EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen.
FIG. 8. Effect of EDC concentration on free amine group content of collagen in SC gels. The EDC concentration in the figure indicates the final concentration in the gels. Closed circles, SC gels prepared by introduction of EDC cross-linking during fibril formation (f-gels); open circles, SC gels prepared by introduction of EDC cross-linking without fibril formation (n-gels); EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen.
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ability of SC to form fibrils at such a low temperature seems to be characteristic of low-Td fish collagen. Fish collagen fibrillar gel has been thoroughly investigated using the example of acid-soluble shark collagen (10, 11, 26). According to a previous report (13), the Td of a shark collagen solution (28°C) was shown to be relatively high among those of fish collagens; however, it was found that shark collagen fibrillar gel gradually melts at a temperature of 37°C (10). These findings indicate that the thermal stability of shark collagen-derived gel renders it insufficient to serve as a collagen-based biomaterial. The Tm of all of the SC fibrillar gels was within the range of 23–28°C, which was in accord with the Td of SC solution (18.6°C). This result indicates that it may be necessary to improve Tm by cross-linking in order to use SC fibrillar gel as a collagenbased biomaterial. The solution in collagen fibrillar gels is restricted by collagen fibril networks; this feature renders it difficult to exchange the solution with a solution containing cross-linking reagents. Furthermore, cross-linking should be introduced only at the surface of collagen fibrils. The Tm of the collagen fibrillar gel is known to be higher than the Td of the collagen solution (11). In our investigations, the Tm of all of the SC fibrillar gels (23–28°C) was higher than the Td of the SC solution (18.6°C), indicating that the aggregation of collagen molecules participates somewhat in the thermal stability of collagen. We hypothesized that the introduction of cross-linking during fibril formation resulted in a further increase in the Tm of the collagen gel. Recently, EDC has become popular as a cross-linking reagent for collagen-based biomaterials (19–22). EDC is watersoluble, and can be applied for use in a collagen solution. EDC cross-links collagen molecules by the formation of isopeptides without being incorporated itself, thus precluding depolymerization and possible release of potentially cytotoxic reagents (27). Furthermore, the by-product of the crosslinking reaction is urea (28), which has no cytotoxicity and can be easily removed during routine rinsing of the matrices. We selected EDC as a cross-linking reagent for the SC gel. The fibril formation rate of SC was suppressed with an increase in the EDC concentration. The introduction of EDC cross-linking is rapid, thereby over half of the introduction of cross-linking is achieved within 10 min (29). The mixture of SC solution and neutral buffer, including EDC, did not provide collagen fibrils at an EDC concentration of 100 mM, at which the cross-linking of collagen molecules is thought to prevent subsequent fibril formation. A high concentration of NaCl (i.e., a final concentration of 140 mM) suppressed the fibril formation rate of SC, where the mixture of SC solution and buffer did not lead to the formation of collagen fibrils at a lower EDC concentration (50 mM) at any pH (data not shown). Based on these results, it appeared that a buffer that would enable a faster fibril formation rate would be desirable. It is possible that the mixture of acidic SC solution and an equal volume of Na-phosphate buffer (pH 6.8, NaCl 70 mM), including various concentrations of EDC, provides one of the most desirable conditions for simultaneous EDC cross-linking and fibril formation. According to a previous report, EDC was sufficiently stable and ac-
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FIG. 9. Cell proliferation on PC gel and EDC cross-linked SC fibrillar gel. The human periodontal ligament cells were cultured on the gels. f-50 gel, SC gels prepared by introduction of EDC cross-linking during fibril formation at an EDC concentration of 50 mM; PC gel, PC fibrillar gel without EDC cross-linking. EDC, 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen; PC, porcine atelocollagen. Cell proliferation on the f-50 gel proceeded faster than on the PC gel. Appearance of the cells on PC gel differed from that on the f-50 gel, in particular, on days 1 and 3.
tive under such conditions (30). At EDC concentrations of 0, 10, 25, and 50 mM, the resultant gels (f-0, -10, -25, and -50 gels, respectively) were turbid. Networks of collagen fibrils were observed in the gels. The quantity of free amine groups of the collagen molecules in the gels was lowered due to the presence of EDC. These results indicate that gels (f-gels) were successfully fabricated by introduction of EDC cross-linking during fibril formation. The degree of cross-linking of the f-gels was much lower than that of the EDC cross-linked SC gels without fibril formation at the same EDC concentration (n-gels). The mixture of acidic SC solution and EDC solution in water had an approximate pH of 4, where collagen molecules are stable. The aggregation of collagen molecules is thought to prevent the introduction of cross-linking on the f-gels. The degree of amide formation derived from EDC at a pH 6.8 is known to be lower than that at pH 4 (30), which would also be expected to result in a lower degree of cross-linking in the f-gels. The mechanical strength and thermal stability of the f-gels increased with an increase in the EDC concentration in the gels to 50 mM, whereas the degree of cross-linking remained almost constant. These results suggest that the properties of the f-gels were affected by the pattern of introduction of the cross-linking, rather than by the degree of cross-linking. At a low EDC concentration, large clusters of uncross-linked collagen fibrils should be formed due to the predominant fibril formation over the introduction of cross-linking by EDC. In contrast, the EDC cross-linking should be relatively uniformly introduced in the collagen fibrils at the EDC concentration of 50 mM because of the low fibril formation rate, resulting in the higher mechanical strength and thermal stability. In spite of the lower degree of cross-linking in the f-gels, the mechanical strength was comparable to that of the n-gels at the same EDC concentration (i.e., 10, 25, and 50 mM), in-
dicating that the fibril formation participates in the determination of mechanical strength. At an EDC concentration of 50 mM, the introduction of EDC cross-linking during fibril formation participated in an increase in the thermal stability of the gel more than cross-linking without fibril formation does. This conclusion would support our hypothesis that EDC cross-linking and the aggregation and arrangement of collagen molecules synergistically improve the thermal stability of collagen gels. Fibril formation did not occur in the f-100 gel, similar in the n-gels, because the high concentration of EDC completely prevented the fibril formation. The mechanical strength and thermal stability of the f-100 gel was lower than that of the n-100 gel in spite of their same EDC concentration, probably owing to the lower degree of cross-linking and loss of fibril formation. In this study, only one condition was shown to provide a synergistic effect of cross-linking and fibril formation, leading to an improvement in the thermal stability of the collagen gel. Other conditions and water-soluble cross-linking reagents should be considered in future studies. In addition, the pattern of introduction of cross-linking during fibril formation cannot be understood in this study, which should also be considered to understand the mechanism of increase in thermal stability. The active proliferation of human PDL cells was observed on the f-50 gel, suggesting the potential of this EDC cross-linked SC fibrillar gel for use in collagen-based biomaterials. Although the reason for the more rapid proliferation of these cells on the f-50 gel than that observed on the PC gel was not clarified by our data analysis, it is possible that the activation of this proliferation on the f-50 gel was due to a higher concentration of fibril networks and/or to the partial denaturation of collagen molecules. The fibril network in the PC gel was rougher than that in the f-50 gel, probably owing to the lower fibril growth rate of PC (data not shown). In our previous report, it was shown that col-
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lagen molecules in SC sponges are partially denatured at the actual physical temperature, suggesting the partial denaturation of collagen molecules in the cultivated f-50 gels. PDL cells are fibroblastic multipotent cells (31). On the PC gel, these cells appeared as a slim and assumed a rod-like form; this morphology has been reported in the case of human dermal fibroblasts on collagen fibril-coated dishes (32). On the other hand, these cells appeared flat on the f-50 gel, which was similar to the previously described appearance of fibroblasts on a plane dish (32). It is possible that the appearance of these cells on the f-50 gel differs from their appearance as it is generally observed on fibrillar gel. In conclusion, we successfully improved the thermal stability of a gel fabricated from SC, the Td of which is typical among fish collagens. The Tm of the SC gel was synergistically improved by the introduction of EDC cross-linking during fibril formation. The human cells used here were highly proliferative on this EDC cross-linked SC fibrillar gel, suggesting its potential to be utilized for the development of cellular matrices and tissue engineering. REFERENCES 1. Stenzel, K. H., Miyata, T., and Rubin, A. L.: Collagen as a biomaterial. Annu. Rev. Biophys. Bioeng., 3, 231–253 (1974). 2. Kadler, K. E., Holmes, D. F., Trotter, J. A., and Chapman, J. A.: Collagen fibril formation. Biochem. J., 316 (Pt 1), 1–11 (1996). 3. Gross, J. and Kirk, D.: The heat precipitation of collagen from neutral salt solutions: some rate-regulating factors. J. Biol. Chem., 233, 355–360 (1958). 4. Williams, B. R., Gelman, R. A., Poppke, D. C., and Piez, K. A.: Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results. J. Biol. Chem., 253, 6578– 6585 (1978). 5. Helseth, D. L., Jr. and Veis, A.: Collagen self-assembly in vitro. Differentiating specific telopeptide-dependent interactions using selective enzyme modification and the addition of free amino telopeptide. J. Biol. Chem., 256, 7118–7128 (1981). 6. Uchio, Y., Ochi, M., Matsusaki, M., Kurioka, H., and Katsube, K.: Human chondrocyte proliferation and matrix synthesis cultured in Atelocollagen gel. J. Biomed. Mater. Res., 50, 138–143 (2000). 7. Iwasa, J., Ochi, M., Uchio, Y., Katsube, K., Adachi, N., and Kawasaki, K.: Effects of cell density on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Artif. Organs, 27, 249–255 (2003). 8. Katsube, K., Ochi, M., Uchio, Y., Maniwa, S., Matsusaki, M., Tobita, M., and Iwasa, J.: Repair of articular cartilage defects with cultured chondrocytes in atelocollagen gel. Comparison with cultured chondrocytes in suspension. Arch. Orthop. Trauma Surg., 120, 121–127 (2000). 9. Ochi, M., Uchio, Y., Tobita, M., and Kuriwaka, M.: Current concepts in tissue engineering technique for repair of cartilage defect. Artif. Organs, 25, 172–179 (2001). 10. Nomura, Y., Toki, S., Ishii, Y., and Shirai, K.: Improvement of the material property of shark type I collagen by composing with pig type I collagen. J. Agric. Food Chem., 48, 6332– 6336 (2000). 11. Nomura, Y., Toki, S., Ishii, Y., and Shirai, K.: The physicochemical property of shark type I collagen gel and membrane. J. Agric. Food Chem., 48, 2028–2032 (2000). 12. Nomura, Y., Yamano, M., and Shirai, K.: Renaturation of a1 chains from shark skin collagen type I. J. Food Sci., 60, 1233–1236 (1995).
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13. Burjanadze, T. V.: New analysis of the phylogenetic change of collagen thermostability. Biopolymers, 53, 523–528 (2000). 14. Kimura, S., Zhu, X.-P., Matsui, R., Shijoh, M., and Takamizawa, S.: Characterization of fish muscle type I collagen. J. Food Sci., 53, 1315–1318 (1988). 15. Matsui, R., Ishida, M., and Kimura, S.: Characterization of an a3 chain from the skin type I collagen of chum salmon (Oncorhynchus Keta). Comp. Biochem. Physiol. B, 99, 171– 174 (1991). 16. Yunoki, S., Suzuki, T., and Takai, M.: Stabilization of low denaturation temperature collagen from fish by physical crosslinking methods. J. Biosci. Bioeng., 96, 575–577 (2003). 17. Piez, K. A.: Structure and assembly of the native collagen fibril. Connect. Tissue Res., 10, 25–36 (1982). 18. Veis, A.: Collagen fibrillogenesis. Connect. Tissue Res., 10, 11–24 (1982). 19. 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). 20. Hafemann, B., Ghofrani, K., Gattner, H. G., Stieve, H., and Pallua, N.: Cross-linking by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) of a collagen/elastin membrane meant to be used as a dermal substitute: effects on physical, biochemical and biological features in vitro. J. Mater. Sci. Mater. Med., 12, 437–446 (2001). 21. Taguchi, T., Ikoma, T., and Tanaka, J.: An improved method to prepare hyaluronic acid and type II collagen composite matrices. J. Biomed. Mater. Res., 61, 330–336 (2002). 22. Park, S. N., Park, J. C., Kim, H. O., Song, M. J., and Suh, H.: Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking. Biomaterials, 23, 1205–1212 (2002). 23. Mandl, I., Macrennan, J. D., and Howes, E. L.: Isolation and characterization of proteinase and collagenase from Cl. Histolyticum. J. Clin. Invest., 32, 1323–1329 (1953). 24. Olde Damink, L. H., Dijkstra, P. J., van Luyn, M. J., van Wachem, P. B., Nieuwenhuis, P., and Feijen, J.: Glutaraldehyde as a crosslinking agent for collagen-based biomaterials. J. Mater. Sci., 6, 460–472 (1994). 25. Nohutcu, R. M., McCauley, L. K., Koh, A. J., and Somerman, M. J.: Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro. J. Periodontol., 68, 320–327 (1997). 26. Nomura, Y., Yamano, M., Hayakawa, C., Ishii, Y., and Shirai, K.: Structural property and in vitro self-assembly of shark type I collagen. Biosci. Biotechnol. Biochem., 61, 1919– 1923 (1997). 27. Osborne, C. S., Reid, W. H., and Grant, M. H.: Investigation into the biological stability of collagen/chondroitin-6-sulphate gels and their contraction by fibroblasts and keratinocytes: the effect of crosslinking agents and diamines. Biomaterials, 20, 283–290 (1999). 28. Khor, E.: Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials, 18, 95–105 (1997). 29. Olde Damink, L. H., Dijkstra, P. J., van Luyn, M. J., van Wachem, P. B., Nieuwenhuis, P., and Feijen, J.: Cross-linking of dermal sheep collagen using a water-soluble carbodiimide. Biomaterials, 17, 765–773 (1996). 30. Nakajima, N. and Ikada, Y.: Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media. Bioconjug. Chem., 6, 123–130 (1995). 31. McCulloch, C. A. and Bordin, S.: Role of fibroblast subpopulations in periodontal physiology and pathology. J. Periodontal Res., 26, 144–154 (1991). 32. Yoshizato, K., Taira, T., and Shioya, N.: Collagen-dependent growth suppression and changes in the shape of human dermal fibroblasts. Ann. Plast. Surg., 13, 9–14 (1984).
Novel Biomaterial from Reinforced Salmon Collagen Gel Prepared by Fibril Formation and Cross-Linking SHUNJI YUNOKI,1* NOBUHIRO NAGAI,1 TAKESHI SUZUKI,2 AND MASANOBU MUNEKATA1 Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-13, Nishi-8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan 1 and Ihara & Company Ltd., 3-263-23 Zenibako, Otaru, Hokkaido 047-0261, Japan 2 Received 5 March 2004/Accepted 22 April 2004
The improvement of the thermal stability of gel prepared from salmon atelocollagen (SC) was studied. The denaturation temperature (Td) of the SC solution was found to be 18.6°C. Neutral buffer including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was mixed with acidic SC solution at 4°C, resulting in the introduction of EDC cross-linking during fibril formation. The mechanical strength and thermal stability of the resultant cross-linked SC fibrillar gels reached maximum values at an EDC concentration of 50 mM (f-50 gel). In particular, the melting temperature of the f-50 gel was 47°C, much higher than that of the EDC cross-linked SC gel without fibril formation at the same EDC concentration. The proliferation rate of human periodontal ligament cells on the f-50 gel was higher than that of a porcine atelocollagen fibrillar gel. These results suggest that the gel employed for biomaterials can be fabricated from low Td fish collagen by EDC cross-linking during fibril formation. [Key words: salmon, fish collagen, biomaterial, fibril formation, fibrillar gel, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]
as a novel natural source for collagen fibrillar gels. In spite of such advantages, fish collagen fibrillar gels have not been extensively studied, with the exception shark collagen (10, 11), probably due to their low denaturation temperature (Td), which renders these materials difficult to handle. The Td of shark collagen solution is approximately 30°C (12), which results in the dissolution of the fibrillar gel of this collagen at 37°C (10). This indicates that the gel could not be practically used at the actual physical temperature of human. The Td of most fish collagens is under 30°C (13). The application of fish collagen fibrillar gels to biomaterials has thus been prevented by their relatively low level of thermal stability. Chum salmon has been caught in abundance as an edible fish, and a large amount of the skin of this fish has been produced as waste from the food industry. The Td (approximately 19°C) and the amino acid composition of acid-soluble salmon collagen have previously been reported (14, 15). Recently, we reported that collagen matrices fabricated from salmon atelocollagen (SC) can be stabilized by UV irradiation and dehydrothermal treatment, and it was found that their stability was comparable to that of bovine atelocollagen matrices (16). However, SC fibrillar gel can be unstable at the actual physical temperature of human. The fibril formation of collagen involves the aggregation and alignment of collagen molecules (17, 18), which, according to previous reports, improves thermal stability (11, 12). We hypothesized that the introduction of cross-linking among collagen fibrils during fibril formation would result in a further increase in the thermal stability of collagen
Type I collagen (abbreviated here as collagen) is widely spread in nature and therefore large amounts of collagen are easily obtained (1). Collagen molecules form fibrils or fibril bundles in tissues, and can be extracted by acid solubilization. Acid-soluble collagen molecules consist of a rod-like triple-helix of approximately 300 nm in length and 1.5 nm in diameter and short extra-helical telopeptides (2). Acidsoluble collagen molecules self-assemble and form fibrils under physiological conditions (3–5). Networks of collagen fibrils create a matrix gel. Since these telopeptides are antigenic (1), atelocollagen, the telopeptides of which are enzymatically digested, has been utilized primarily for cosmetics and biomaterials. Recently, atelocollagen fibrillar gels have been used for cellular matrices (6, 7) and tissue engineering (8, 9). In general, collagens for biomaterials are prepared from mammalian tissues, including bovine and porcine sources. However, mammalian tissues have recently been suspected as posing a large risk of pathogens such as bovine spongiform encephalopathy (BSE). Although fish skin is thought to be safe and a potentially large source of collagen, it has not yet been widely used. In addition, large quantities of fish skin are discarded as waste in the food industry. Collagens are easily extracted from wasted fish skins with high yield. Therefore, the practical use of fish collagens could possibly contribute to the recycling of unutilized resources. Due to such advantages, fish collagens have the potential to be used * Corresponding author. e-mail: [email protected] phone/fax: +81-(0)11-706-6568 40
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fibrillar gel, thereby providing fibrillar gels as biomaterials from low-Td salmon collagen. In the present study, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a water soluble carbodiimide, was used as a cross-linking reagent for SC. EDC has recently become popular as a cross-linking reagent for collagen (19–22) due to ease of handling and potentially low cytotoxicity. Here, we show the increase in the thermal stability of SC resulting from fibril formation and cross-linking by EDC.
MATERIALS AND METHODS Preparation of SC SC was prepared from fresh skin of chum salmon (Oncorhynchus Keta) by a procedure reported previously (16) with slight modifications. The defatted salmon skin described above (127 g) was suspended in 3 l of 0.5 M acetic acid at 4°C. The following procedures were performed at 4°C. The solution was gently filtered with gauze, and the filtrate was centrifuged at 10,000 ´ g for 30 min to remove small 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. NaCl solution was added to the supernatant to a final concentration of 5%. After 24 h the precipitate was separated by centrifugation at 10,000´ g for 30 min, and re-dissolved in 1.5 l of 0.5 M acetic acid solution. The salting-out was repeated twice. The resultant solution of SC was filtered with membrane filter (0.45-mm pore size; Advantec, Tokyo) to remove micro-sized residues, dialyzed against deionized-water, and lyophilized. Denaturation temperature The Td of SC solution was measured by the method of Nomura et al. (12) with modifications. The optical rotation of 0.25% SC solution in pH 3 dilute HCl was measured with a polarimeter (SEPA-300; Horiba, Kyoto) by a stepwise increase in temperature. The temperature was held for 30 min at each step (1°C). The temperature at which the change in rotation was half the maximum value was taken as the Td. Fibril formation of SC The Fibril formation of SC was observed by a procedure as follows: 1 ml of neutral Na-phosphate buffer at a specific pH was added to a PMMA cuvette for spectrophotometer. The cuvette was set in a spectrophotometer (UV-mini 1240; Shimadzu, Kyoto) with a temperature control apparatus. After the temperature of the buffer was stabilized, 1 ml of the 0.5% (w/v) SC solution in pH 3 dilute HCl (the acidic SC solution) was added and immediately mixed. The resulting fibril formation was monitored by absorbance (ABS) at 310 nm as the turbidity change. The pH of the buffer was fixed at 5.5, 6.1, 6.8, 7.4, and 7.9. The buffer included NaCl at the concentration where the final concentration in the gels was to be 0, 35, 70, and 140 mM. Degree of fibril formation The SC fibrillar gel was prepared in a centrifugation tube by the procedure described above. After the 1-d incubation, the gel was centrifuged (10,000 ´g, 10 min) to precipitate the collagen fibrils. The protein content of the supernatant was determined by a ninhydrin reaction according to a previous method (23). SC fibrillar gels were also prepared in plastic petri dishes and glass tubes. The Na-phosphate buffer (20 ml) was added to the equal volume of the acidic SC solution with stirring. The mixture was immediately poured into plastic petri dishes (diameter: 35 mm) or glass tubes (diameter: 10 mm), and then incubated at 4°C for 24 h. Preparation of SC gel by introduction of EDC cross-linking during fibril formation ( f-gel) The introduction of EDC (Dojindo, Tokyo) cross-linking during fibril formation of SC was performed by mixture of acidic SC solution and EDC solution in pH 6.8, 30 mM Na-phosphate buffer including 70 mM NaCl. EDC
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was dissolved in the buffer to a concentration of 0, 20, 50, 100, and 200 mM. The EDC solution (20 ml) was added to the equal volume of the acidic SC solution with stirring. The mixture was immediately poured into the plastic petri dishes or the glass tubes. Following incubation at 4°C for 24 h provided gels at final EDC concenrations of 10, 25, 50, and 100 mM (f-0, -10, -25, -50, and -100 gels, respectively). Preparation of SC gel by introduction of EDC cross-linking without fibril formation (n-gel) The mixture of acidic SC solution and EDC solution in water provided EDC cross-linked SC gels without fibril formation. EDC was dissolved in deionizedwater to concentrations of 20, 50, 100, and 200 mM. The EDC solution (20 ml) was added to the equal volume of the acidic SC solution with stirring. The mixture was immediately poured into the plastic petri dishes or the glass tubes. Following incubation at 4°C for 24 h provided gels at final EDC concenrations of 10, 25, 50, and 100 mM (n-10, -25, -50, and -100 gels, respectively). Preparation of porcine atelocollagen fibrillar gel Porcine atelocollagen (PC) was purchased as a 0.3% (w/v) solution in pH 3 dilute HCl from Nitta Gelatin (Cellmatrix Type-P; Nitta Gelatin, Osaka). The PC fibrillar gel (PC gel) was prepared by the mixture of 0.3% (w/v) PC solution and one-sixth volume of Na-phosphate buffer (pH 6.8, 90 mM) including 210 mM NaCl at 4°C. The mixture was subsequently incubated at 37°C for 24 h, resulted in a PC gel. Microscopic observation The collagen fibrils were observed by high-resolution scanning electron microscopy (SEM; JSM6500F, JEOL, Tokyo). The preparation of the specimen was performed according to a previous report (10). Briefly, the gels were fixed by glutaraldehyde, and then dehydrated by ethanol and subsequent isoamyl acetate. The gels were subsequently subjected to a critical point drying. The dried gels were coated with Au using an ion coater (E-1010; Hitachi, Tokyo), and subjected to the SEM measurements. The SEM apparatus was operated at 5.0 kV and a magnification of 15,000. Thermal stability of gel The thermal stability of the gels was evaluated by the melting temperature (Tm). The gels were prepared in the glass tubes with the height of 40 ±3 mm by the procedures described above. The tubes were set in a water bath, and the temperature was stepwise increased from 18°C. The temperature was held for 30 min at each step (1°C/step). A temperature at which more than one half volume of the gel melted was taken as the Tm. Mechanical strength of gel The mechanical strength of the gels was measured by a rheometer (CD-200D; Sun Scientific, Tokyo). The gels were prepared in the plastic petri dish with the height of 5 ±1 mm by the procedures described above. The measurement of mechanical strength was performed using a circular probe (diameter: 20 mm) moving into the gel at a speed of 50 mm/min. The stress at a constant depth (1.3 mm) was defined as the mechanical strength of gel. Free amine group content The degree of cross-linking was estimated by the free amine group contents of collagen molecules in the collagen gels. The lyophilized collagen gels were washed five times in excessive volume of 20% (v/v) ethanol to remove the residual salts, EDC, and by-products, lyophilized again, and subjected to the determination of the free amine group contents. The free amine group content was determined spectrophotometrically after reaction of the free amine groups with 2,4,6-trinitrobenzenesulphonic acid (24) and was expressed as the retention of amine groups; the ratio (%) of the free amine group contents of crosslinked sample to that of the uncross-linked sample. Cell culture The cell proliferation on the collagen fibrillar gels was tested in vitro using the human periodontal ligament (PDL) cells. The f-50 gel and PC gel were used for the cultivation. The PDL cells were isolated by a method previously reported (25),
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and the cells less than passage 15 were used for the cell proliferation assay. The PDL cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical, Tokyo) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) at 37°C in air containing 5% CO2. The culture medium was changed every 2 d. At semi-confluent, PDL cells were subcultured in the same medium. The collagen gels were fabricated in non-coating plastic dishes (diameter of 20 mm; Nunc, Rochester, NY, USA). The collagen gels were rinsed four times with phosphate buffer saline (-), and further rinsed with DMEM once before the test. PDL cells were seeded on the collagen gels at density of 5´103 cells/cm2. The medium was changed every 2 d. At days 1, 3, and 7, the cells on the collagen gels were observed by an inverted light microscope (CK2; Olympus, Tokyo).
NaCl concentration. In a mixture including 140 mM of NaCl, the degree of fibril formation ranged from 67% to 82%. All of mixtures of SC solution and Na-phosphate buffer provided an SC fibrillar gel after incubation for 24 h at 4°C, with the exception of the mixture that contained no NaCl. The mixture including no NaCl showed fluidity after incubation, in spite of their high degree of fibril formation. The Tm of SC fibrillar gels ranged from 23°C to 28°C, which was higher than the Td of the SC solution, but was much lower than the actual physical temperature of human (approximately 37°C). Effect of EDC on fibril formation Fibril formation of SC in the presence of EDC was monitored by turbidity
RESULTS Denaturation temperature The specific rotation of SC was measured at various temperatures (Fig. 1) according to a previous method (12). The Td value of SC was found to be 18.6°C. The absolute value of the specific rotation of SC (approximately 390°) drastically decreased at around the Td due to increases in temperature, and then leveled off to the value of gelatin (i.e., approximately 125°). Fibril formation of SC The fibril formation of SC was monitored by a turbidity change observed at 310 nm (Fig. 2). The pH and NaCl concentration as a measure of ionic strength exerted an effect on increases in turbidity. A rapid rise in turbidity was observed in the mixture of SC solution and Na-phosphate buffer at pH from 6.1 to 7.9 including no NaCl, where the entire increase in turbidity was observed within 1 min at all pH levels. The rise in turbidity tended to be suppressed with increases in the NaCl concentration (Fig. 2). In a mixture including 140 mM of NaCl, the collagen did not attain the entire increase in turbidity within 7 min. Turbidity increases were not observed at pH 5.5, irrespective of the NaCl concentration (data not shown). The degree of fibril formation in the mixture of SC solution and Na-phosphate buffer was measured as the ratio (%) of collagen participating in the fibril formation to the total collagen (Fig. 3). In a mixture including no NaCl, the degree of fibril formation exceeded 83% at all the pH. The degree of fibril formation decreased with an increase in the
FIG. 1. Denaturation curve of SC as measured by specific rotation. The denaturation temperature of SC was found to be 18.6°C. SC, Salmon atelocollagen.
FIG. 2. Effect of pH and NaCl concentration on the fibril formation of SC. Na-phosphate buffer with various pH (closed triangles, 6.1; closed squares, 6.8; closed circles, 7.1; open triangles, 7.4; open squares, 7.9) was added to the equal volume of the acidic SC solution at 4°C, and the resultant fibril formation was observed as a rising of turbidity at 310 nm. The concentration of NaCl in the figure indicates the final concentration in the mixture of the SC solution and Na-phosphate buffer. SC, Salmon atelocollagen.
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FIG. 3. Effect of pH and NaCl concentration on degree of fibril formation in SC fibrillar gels. The degree of fibril formation indicates the ratio (%) of collagen participating in the fibril formation to the total collagen. The concentration of NaCl in the figure indicates the final concentration in the SC fibrillar gels. SC, Salmon atelocollagen.
FIG. 4. Effect of EDC concentration on fibril formation of SC. Na-phosphate buffer (pH 6.8; NaCl, 70 mM) including various concentrations of EDC was added to the equal volume of the acidic SC solution at 4°C, and the resultant fibril formation was observed as a rising of turbidity at 310 nm. The final concentration of EDC in the mixtures was 0 mM (closed triangles), 10 mM (closed circles), 25 mM (open circles), 50 mM (closed squares), or 100 mM (open squares). EDC, 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen.
change in the same manner as fibril formation without EDC (Fig. 4). The rise in turbidity was suppressed by the increase in the EDC concentration. Fibril formation was not recognized when the final EDC concentration in the mixture reached 100 mM. Preparation of SC gel by introduction of EDC crosslinking during fibril formation ( f-gel) The mixture of the acidic SC solution and neutral Na-phosphate buffer including EDC caused the introduction of EDC cross-linking during fibril formation, resulted in the fibrillar gels after a 24-h incubation period (f-gels). Collagen molecules were successfully cross-linked by EDC in all of the f-gels except for f-0, as described below. The appearance of f-0, -10, -25, -50, and -100 gels is shown in Fig. 5. The appearance of f-10, -25, and -50 gels was turbid, as also shown in the case of f-0, an SC fibrillar gel without EDC. In contrast, the appearance of f-100 gel was transparent and colorless. Preparation of SC gel by introduction of EDC crosslinking without fibril formation (n-gel) The mixture of the acidic SC solution and EDC solution in water caused the
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FIG. 5. Appearance of SC gels. n-50 indicates SC gel prepared by introduction of EDC cross-linking without fibril formation at an EDC concentration of 50 mM. f-0, -10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC cross-linking during fibril formation at an EDC concentration of 0, 10, 25, 50, and 100 mM, respectively. SC, Salmon atelocollagen; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The appearance of f-10, -25, and -50 gels was turbid, as also shown in the case of f-0, an SC fibrillar gel without EDC. In contrast, the appearance of f-100 gel was transparent and colorless, as also shown in the case of n-50 gel without fibril formation.
introduction of EDC cross-linking to the collagen molecules without fibril formation, resulted in the gels without collagen fibrils after a 24-h incubation period (n-gels). Collagen molecules were successfully cross-linked by EDC in all of the n-gels as described below. The EDC cross-linked SC gels, n-10, -25, -50, and -100 gels, were without exception transparent and colorless, as shown in Fig. 5 (n-50 gel), indicating that fibril formation did not occur. The gel at an EDC concentration below 10 mM showed fluidity. Fibril structure of gels The fibril structure of SC gels was observed by SEM (Fig. 6). Well-developed fibril networks were observed on the f-0, -10, -25, and -50 gels, the appearance of which was turbid. The fibril structure of these gels was only slightly affected by the EDC concentration in the gels. On the other hand, collagen fibrils were not observed in the f-100 gel or in any of the n-gels, the appearance of which was transparent and colorless. Thermal stability of gel The Tm of the f- and n-gels was measured as described above (Table 1). The Tm value of f-0 gel was 28°C. The Tm values of the f-gels increased with increases in the EDC concentration in the gels. Surprisingly, the Tm value of the f-50 gel was 47°C, which was much higher than that of all the n-gels (around 39°C). In contrast, the Tm value of the f-100 gel (39°C) was lower than that of the f-50 gel, in spite of its higher concentration of EDC in the gel. Mechanical strength of gel The mechanical strength of the f-gels is shown in Fig. 7, as compared with those of the n-gels. All of the n-gels were brittle. In contrast, the f-gels showed flexibility, except for the brittle f-100 gel. The strength of the f-gels increased with an increase in the EDC concentration in the gels, and reached a maximum value at 50 mM. However, the strength of the f-100 gel was lower than that of the f-50 gel. The strength of the n-gels also increased with increases in the EDC concentration in the gels, and the strength was almost saturated at an EDC concentration of 25 mM. The strength of the f-gels was comparable to that of the n-gels at the same EDC concentration, with the
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FIG. 6. SEM images of collagen fibrils in collagen gels. n-50 indicates SC gel prepared by introduction of EDC cross-linking without fibril formation at an EDC concentration of 50 mM. f-0, -10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC cross-linking during fibril formation at an EDC concentration of 0, 10, 25, 50, and 100 mM, respectively. PC indicates porcine atelocollagen fibrillar gel without EDC cross-linking. SC, Salmon atelocollagen; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Well-developed collagen fibrils were observed in f-0, -10, -25, -50 and PC gels, while not in n-50 and f-100 gels.
exception of gels at 100 mM. It was then determined that the f-100 gel was less strong than the n-100 gel. Free amine group content The degree of cross-linking was estimated by measurement of the free amine group contents (Fig. 8). The free amine group contents in the f-gels decreased from 100% to 84% at an EDC concentration of 10 mM, and then leveled off. On the other hand, the drastic decrease in free amine group contents was shown in the n-gels, i.e., a decrease from 100% to 25% was observed at an EDC concentration of 25 mM, and then the decrease leveled off. Cell proliferation on f-gel The PDL cells on the f-50
gel was observed on days 1, 3, and 7, and this proliferation was compared with that on the PC gel (Fig. 9). Cell proliferation on the f-50 gel proceeded faster than on the PC gel. Interestingly, the cells assumed a specific appearance on PC gel that differed from that on the f-50 gel. The cells on the f50 gel were flat in appearance. On the other hand, the morphology of the cells on PC gel was slim and rod-like. DISCUSSION Atelocollagen fibrillar gels, as well as atelocollagen sponges, have been very important for the development of
TABLE 1. Effect of the EDC concentration on the thermal stability of EDC cross-linked SC fibrillar gels n-10 n-25 n-50 n-100 f-0 f-10 f-25 f-50 f-100 PC gel Tm (°C) 37 39 39 39 28 32 37 47 37 47 SC, Salmon atelocollagen; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; PC, porcine atelocollagen. The thermal stability of the gels was evaluated by the Tm. n-10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC cross-linking without fibril formation at an EDC concentration of 10, 25, 50, and 100 mM, respectively. f-0, -10, -25, -50, and -100 indicate SC gels prepared by introduction of EDC crosslinking during fibril formation at an EDC concentration of 0, 10, 25, 50, and 100 mM, respectively. PC gel indicates PC fibrillar gel without EDC cross-linking.
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cellular matrices (6, 7) and tissue engineering (8, 9). However, the potential use of fibrillar gel from low-Td fish collagen as a collagen-based biomaterial has not been sufficiently investigated to date, probably due to the handling difficulty resulting from the low Td of this type of gel. The present study defined that the thermal stability of SC fibrillar gel was extensively improved by introduction of crosslinking during fibril formation of collagen, and the gel would have a potential to be utilized for the development of cellular matrices and tissue engineering. Nomura et al. (10) has reported that the optical density from porcine collagen fibrils gradually decreased at 10°C, indicating that porcine collagen cannot form fibrils at such a low temperature. The fibril formation is considered to be an aggregation of collagen molecules caused by the effects of hydrophobic and electrostatic interactions (17, 18). The low temperature discourages hydrophobic interactions, resulting in the suppression of the fibril formation. However, the active fibril formation of SC was observed at 4°C, a temperature at which porcine collagen could not form fibrils. The
FIG. 7. Effect of EDC concentration on mechanical strength of SC gels. The EDC concentration in the figure indicates the final concentration in the gels. Closed bars, SC gels prepared by introduction of EDC cross-linking during fibril formation (f-gels); open bars, SC gels prepared by introduction of EDC cross-linking without fibril formation (n-gels); EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen.
FIG. 8. Effect of EDC concentration on free amine group content of collagen in SC gels. The EDC concentration in the figure indicates the final concentration in the gels. Closed circles, SC gels prepared by introduction of EDC cross-linking during fibril formation (f-gels); open circles, SC gels prepared by introduction of EDC cross-linking without fibril formation (n-gels); EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen.
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ability of SC to form fibrils at such a low temperature seems to be characteristic of low-Td fish collagen. Fish collagen fibrillar gel has been thoroughly investigated using the example of acid-soluble shark collagen (10, 11, 26). According to a previous report (13), the Td of a shark collagen solution (28°C) was shown to be relatively high among those of fish collagens; however, it was found that shark collagen fibrillar gel gradually melts at a temperature of 37°C (10). These findings indicate that the thermal stability of shark collagen-derived gel renders it insufficient to serve as a collagen-based biomaterial. The Tm of all of the SC fibrillar gels was within the range of 23–28°C, which was in accord with the Td of SC solution (18.6°C). This result indicates that it may be necessary to improve Tm by cross-linking in order to use SC fibrillar gel as a collagenbased biomaterial. The solution in collagen fibrillar gels is restricted by collagen fibril networks; this feature renders it difficult to exchange the solution with a solution containing cross-linking reagents. Furthermore, cross-linking should be introduced only at the surface of collagen fibrils. The Tm of the collagen fibrillar gel is known to be higher than the Td of the collagen solution (11). In our investigations, the Tm of all of the SC fibrillar gels (23–28°C) was higher than the Td of the SC solution (18.6°C), indicating that the aggregation of collagen molecules participates somewhat in the thermal stability of collagen. We hypothesized that the introduction of cross-linking during fibril formation resulted in a further increase in the Tm of the collagen gel. Recently, EDC has become popular as a cross-linking reagent for collagen-based biomaterials (19–22). EDC is watersoluble, and can be applied for use in a collagen solution. EDC cross-links collagen molecules by the formation of isopeptides without being incorporated itself, thus precluding depolymerization and possible release of potentially cytotoxic reagents (27). Furthermore, the by-product of the crosslinking reaction is urea (28), which has no cytotoxicity and can be easily removed during routine rinsing of the matrices. We selected EDC as a cross-linking reagent for the SC gel. The fibril formation rate of SC was suppressed with an increase in the EDC concentration. The introduction of EDC cross-linking is rapid, thereby over half of the introduction of cross-linking is achieved within 10 min (29). The mixture of SC solution and neutral buffer, including EDC, did not provide collagen fibrils at an EDC concentration of 100 mM, at which the cross-linking of collagen molecules is thought to prevent subsequent fibril formation. A high concentration of NaCl (i.e., a final concentration of 140 mM) suppressed the fibril formation rate of SC, where the mixture of SC solution and buffer did not lead to the formation of collagen fibrils at a lower EDC concentration (50 mM) at any pH (data not shown). Based on these results, it appeared that a buffer that would enable a faster fibril formation rate would be desirable. It is possible that the mixture of acidic SC solution and an equal volume of Na-phosphate buffer (pH 6.8, NaCl 70 mM), including various concentrations of EDC, provides one of the most desirable conditions for simultaneous EDC cross-linking and fibril formation. According to a previous report, EDC was sufficiently stable and ac-
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FIG. 9. Cell proliferation on PC gel and EDC cross-linked SC fibrillar gel. The human periodontal ligament cells were cultured on the gels. f-50 gel, SC gels prepared by introduction of EDC cross-linking during fibril formation at an EDC concentration of 50 mM; PC gel, PC fibrillar gel without EDC cross-linking. EDC, 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide; SC, salmon atelocollagen; PC, porcine atelocollagen. Cell proliferation on the f-50 gel proceeded faster than on the PC gel. Appearance of the cells on PC gel differed from that on the f-50 gel, in particular, on days 1 and 3.
tive under such conditions (30). At EDC concentrations of 0, 10, 25, and 50 mM, the resultant gels (f-0, -10, -25, and -50 gels, respectively) were turbid. Networks of collagen fibrils were observed in the gels. The quantity of free amine groups of the collagen molecules in the gels was lowered due to the presence of EDC. These results indicate that gels (f-gels) were successfully fabricated by introduction of EDC cross-linking during fibril formation. The degree of cross-linking of the f-gels was much lower than that of the EDC cross-linked SC gels without fibril formation at the same EDC concentration (n-gels). The mixture of acidic SC solution and EDC solution in water had an approximate pH of 4, where collagen molecules are stable. The aggregation of collagen molecules is thought to prevent the introduction of cross-linking on the f-gels. The degree of amide formation derived from EDC at a pH 6.8 is known to be lower than that at pH 4 (30), which would also be expected to result in a lower degree of cross-linking in the f-gels. The mechanical strength and thermal stability of the f-gels increased with an increase in the EDC concentration in the gels to 50 mM, whereas the degree of cross-linking remained almost constant. These results suggest that the properties of the f-gels were affected by the pattern of introduction of the cross-linking, rather than by the degree of cross-linking. At a low EDC concentration, large clusters of uncross-linked collagen fibrils should be formed due to the predominant fibril formation over the introduction of cross-linking by EDC. In contrast, the EDC cross-linking should be relatively uniformly introduced in the collagen fibrils at the EDC concentration of 50 mM because of the low fibril formation rate, resulting in the higher mechanical strength and thermal stability. In spite of the lower degree of cross-linking in the f-gels, the mechanical strength was comparable to that of the n-gels at the same EDC concentration (i.e., 10, 25, and 50 mM), in-
dicating that the fibril formation participates in the determination of mechanical strength. At an EDC concentration of 50 mM, the introduction of EDC cross-linking during fibril formation participated in an increase in the thermal stability of the gel more than cross-linking without fibril formation does. This conclusion would support our hypothesis that EDC cross-linking and the aggregation and arrangement of collagen molecules synergistically improve the thermal stability of collagen gels. Fibril formation did not occur in the f-100 gel, similar in the n-gels, because the high concentration of EDC completely prevented the fibril formation. The mechanical strength and thermal stability of the f-100 gel was lower than that of the n-100 gel in spite of their same EDC concentration, probably owing to the lower degree of cross-linking and loss of fibril formation. In this study, only one condition was shown to provide a synergistic effect of cross-linking and fibril formation, leading to an improvement in the thermal stability of the collagen gel. Other conditions and water-soluble cross-linking reagents should be considered in future studies. In addition, the pattern of introduction of cross-linking during fibril formation cannot be understood in this study, which should also be considered to understand the mechanism of increase in thermal stability. The active proliferation of human PDL cells was observed on the f-50 gel, suggesting the potential of this EDC cross-linked SC fibrillar gel for use in collagen-based biomaterials. Although the reason for the more rapid proliferation of these cells on the f-50 gel than that observed on the PC gel was not clarified by our data analysis, it is possible that the activation of this proliferation on the f-50 gel was due to a higher concentration of fibril networks and/or to the partial denaturation of collagen molecules. The fibril network in the PC gel was rougher than that in the f-50 gel, probably owing to the lower fibril growth rate of PC (data not shown). In our previous report, it was shown that col-
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lagen molecules in SC sponges are partially denatured at the actual physical temperature, suggesting the partial denaturation of collagen molecules in the cultivated f-50 gels. PDL cells are fibroblastic multipotent cells (31). On the PC gel, these cells appeared as a slim and assumed a rod-like form; this morphology has been reported in the case of human dermal fibroblasts on collagen fibril-coated dishes (32). On the other hand, these cells appeared flat on the f-50 gel, which was similar to the previously described appearance of fibroblasts on a plane dish (32). It is possible that the appearance of these cells on the f-50 gel differs from their appearance as it is generally observed on fibrillar gel. In conclusion, we successfully improved the thermal stability of a gel fabricated from SC, the Td of which is typical among fish collagens. The Tm of the SC gel was synergistically improved by the introduction of EDC cross-linking during fibril formation. The human cells used here were highly proliferative on this EDC cross-linked SC fibrillar gel, suggesting its potential to be utilized for the development of cellular matrices and tissue engineering. REFERENCES 1. Stenzel, K. H., Miyata, T., and Rubin, A. L.: Collagen as a biomaterial. Annu. Rev. Biophys. Bioeng., 3, 231–253 (1974). 2. Kadler, K. E., Holmes, D. F., Trotter, J. A., and Chapman, J. A.: Collagen fibril formation. Biochem. J., 316 (Pt 1), 1–11 (1996). 3. Gross, J. and Kirk, D.: The heat precipitation of collagen from neutral salt solutions: some rate-regulating factors. J. Biol. Chem., 233, 355–360 (1958). 4. Williams, B. R., Gelman, R. A., Poppke, D. C., and Piez, K. A.: Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results. J. Biol. Chem., 253, 6578– 6585 (1978). 5. Helseth, D. L., Jr. and Veis, A.: Collagen self-assembly in vitro. Differentiating specific telopeptide-dependent interactions using selective enzyme modification and the addition of free amino telopeptide. J. Biol. Chem., 256, 7118–7128 (1981). 6. Uchio, Y., Ochi, M., Matsusaki, M., Kurioka, H., and Katsube, K.: Human chondrocyte proliferation and matrix synthesis cultured in Atelocollagen gel. J. Biomed. Mater. Res., 50, 138–143 (2000). 7. Iwasa, J., Ochi, M., Uchio, Y., Katsube, K., Adachi, N., and Kawasaki, K.: Effects of cell density on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Artif. Organs, 27, 249–255 (2003). 8. Katsube, K., Ochi, M., Uchio, Y., Maniwa, S., Matsusaki, M., Tobita, M., and Iwasa, J.: Repair of articular cartilage defects with cultured chondrocytes in atelocollagen gel. Comparison with cultured chondrocytes in suspension. Arch. Orthop. Trauma Surg., 120, 121–127 (2000). 9. Ochi, M., Uchio, Y., Tobita, M., and Kuriwaka, M.: Current concepts in tissue engineering technique for repair of cartilage defect. Artif. Organs, 25, 172–179 (2001). 10. Nomura, Y., Toki, S., Ishii, Y., and Shirai, K.: Improvement of the material property of shark type I collagen by composing with pig type I collagen. J. Agric. Food Chem., 48, 6332– 6336 (2000). 11. Nomura, Y., Toki, S., Ishii, Y., and Shirai, K.: The physicochemical property of shark type I collagen gel and membrane. J. Agric. Food Chem., 48, 2028–2032 (2000). 12. Nomura, Y., Yamano, M., and Shirai, K.: Renaturation of a1 chains from shark skin collagen type I. J. Food Sci., 60, 1233–1236 (1995).
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13. Burjanadze, T. V.: New analysis of the phylogenetic change of collagen thermostability. Biopolymers, 53, 523–528 (2000). 14. Kimura, S., Zhu, X.-P., Matsui, R., Shijoh, M., and Takamizawa, S.: Characterization of fish muscle type I collagen. J. Food Sci., 53, 1315–1318 (1988). 15. Matsui, R., Ishida, M., and Kimura, S.: Characterization of an a3 chain from the skin type I collagen of chum salmon (Oncorhynchus Keta). Comp. Biochem. Physiol. B, 99, 171– 174 (1991). 16. Yunoki, S., Suzuki, T., and Takai, M.: Stabilization of low denaturation temperature collagen from fish by physical crosslinking methods. J. Biosci. Bioeng., 96, 575–577 (2003). 17. Piez, K. A.: Structure and assembly of the native collagen fibril. Connect. Tissue Res., 10, 25–36 (1982). 18. Veis, A.: Collagen fibrillogenesis. Connect. Tissue Res., 10, 11–24 (1982). 19. 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). 20. Hafemann, B., Ghofrani, K., Gattner, H. G., Stieve, H., and Pallua, N.: Cross-linking by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) of a collagen/elastin membrane meant to be used as a dermal substitute: effects on physical, biochemical and biological features in vitro. J. Mater. Sci. Mater. Med., 12, 437–446 (2001). 21. Taguchi, T., Ikoma, T., and Tanaka, J.: An improved method to prepare hyaluronic acid and type II collagen composite matrices. J. Biomed. Mater. Res., 61, 330–336 (2002). 22. Park, S. N., Park, J. C., Kim, H. O., Song, M. J., and Suh, H.: Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking. Biomaterials, 23, 1205–1212 (2002). 23. Mandl, I., Macrennan, J. D., and Howes, E. L.: Isolation and characterization of proteinase and collagenase from Cl. Histolyticum. J. Clin. Invest., 32, 1323–1329 (1953). 24. Olde Damink, L. H., Dijkstra, P. J., van Luyn, M. J., van Wachem, P. B., Nieuwenhuis, P., and Feijen, J.: Glutaraldehyde as a crosslinking agent for collagen-based biomaterials. J. Mater. Sci., 6, 460–472 (1994). 25. Nohutcu, R. M., McCauley, L. K., Koh, A. J., and Somerman, M. J.: Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro. J. Periodontol., 68, 320–327 (1997). 26. Nomura, Y., Yamano, M., Hayakawa, C., Ishii, Y., and Shirai, K.: Structural property and in vitro self-assembly of shark type I collagen. Biosci. Biotechnol. Biochem., 61, 1919– 1923 (1997). 27. Osborne, C. S., Reid, W. H., and Grant, M. H.: Investigation into the biological stability of collagen/chondroitin-6-sulphate gels and their contraction by fibroblasts and keratinocytes: the effect of crosslinking agents and diamines. Biomaterials, 20, 283–290 (1999). 28. Khor, E.: Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials, 18, 95–105 (1997). 29. Olde Damink, L. H., Dijkstra, P. J., van Luyn, M. J., van Wachem, P. B., Nieuwenhuis, P., and Feijen, J.: Cross-linking of dermal sheep collagen using a water-soluble carbodiimide. Biomaterials, 17, 765–773 (1996). 30. Nakajima, N. and Ikada, Y.: Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media. Bioconjug. Chem., 6, 123–130 (1995). 31. McCulloch, C. A. and Bordin, S.: Role of fibroblast subpopulations in periodontal physiology and pathology. J. Periodontal Res., 26, 144–154 (1991). 32. Yoshizato, K., Taira, T., and Shioya, N.: Collagen-dependent growth suppression and changes in the shape of human dermal fibroblasts. Ann. Plast. Surg., 13, 9–14 (1984).