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Int. J. Mol. Sci. 2014, 15(2), 2142-2156; doi:10.3390/ijms15022142
Published: 29 January 2014
Abstract: The bonding behavior between hydrophobically modified alkaline-treated gelatin (hm-AlGltn) films and porcine blood vessels was evaluated under wet conditions. Hexanoyl (Hx: C6), decanoyl (Dec: C10), and stearyl (Ste: C18) chlorides were introduced into the amino groups of AlGltn to obtain HxAlGltn, DecAlGltn, and SteAlGltn, respectively, with various modification percentages. The hm-AlGltn was fabricated into films and thermally crosslinked to obtain water-insoluble films (t-hm-AlGltn). The 42% modified t-HxAlGltn (t-42HxAlGltn) possessed higher wettability than the 38% modified t-DecAlGltn (t-38DecAlGltn) and the 44% modified t-SteAlGltn (t-44SteAlGltn) films, and the t-42HxAlGltn film showed a high bonding strength with the blood vessel compared with all the hm-AlGltn films. Histological observations indicated that t-42HxAlGltn and t-38DecAlGltn remained on the blood vessel even after the bonding strength measurements. From cell culture experiments, the t-42HxAlGltn films showed significant cell adhesion compared to other films. These findings indicate that the Hx group easily interpenetrated the surface of blood vessels and effectively enhanced the bonding strength between the films and the tissue.
Wound closure is one of the fundamental requirements in surgical operations. A suture is usually employed to close the wounded region. However, the use of sutures prolongs operation time and is not suitable for some complicated areas such as the junction site of a blood vessel and a lung. Therefore, tissue adhesives have been developed and used in the clinical field for shortening the operation time and for the closure of wounds with complicated structures . However, these adhesives still possess some disadvantages in terms of bonding strength and biocompatibility [2–11]. For example, TachoSil® (CSL Behring K.K., Tokyo, Japan) is a common clinically available adhesive composed of equine collagen and fibrin; it, however, has several shortcomings. Xenogeneic product collagen and allogeneic fibrin may result in infection, and the fibrin-induced adhesivity is not very strong. Especially in terms of bonding strength, adhesives bonded on soft tissues are exposed to adverse environments because about 70% of our body is made of water. In addition, body fluids such as blood plasma and lymph fluid spill out from the wound area after a surgical operation.
To overcome these obstacles, molecular design of tissue adhesive materials that show good bonding behavior even on moisture-containing tissues is required. We recently developed a hydrophobically modified gelatin (hm-Gltn)-based adhesive and showed that the resulting hm-Gltn adhesive with a low modification percentage formed a stronger bond on porcine arterial media as compared to the original Gltn-based adhesives [12–16]. Our results suggest that films composed of hm-Gltn have the ability to bond to soft tissues even under wet conditions. However, the detailed adhesive properties of these hm-Gltn films have not yet been clarified.
For this purpose, hydrophobically modified alkali-treated Gltns (hm-AlGltns) were prepared by the modification of the amino groups of AlGltn with fatty acid chlorides with various chain lengths, including hexanoyl (Hx: C6) chloride, decanoyl (Dec: C10) chloride, and stearyl (Ste: C18) chloride. We selected those fatty acid chlorides because our previous research showed that a longer hydrophobic group such as Ste resulted in good adhesivity to tunica media [13,15]. Therefore, the longest fatty acid is Ste in this research, and together we used a tunica blood vessel as an adherend, so, the fatty acid chlorides with different chain lengths could be compared with Ste. The obtained hm-AlGltns with various modification percentages were cast to fabricate films; these were then thermally crosslinked to prepare water-insoluble hm-AlGltn (t-hm-AlGltn) films. Using the t-hm-AlGltn films, surface wettability and bonding behavior on porcine blood vessels were evaluated.
2. Results and Discussion
2.1. Synthesis and Characterization of hm-AlGltns
As shown in Figure 1A, three different fatty acid chlorides, hexanoyl (Hx) chloride, decanoyl (Dec) chloride, and stearyl (Ste) chloride, were reacted with the amino groups of the AlGltn molecules by nucleophilic reactions to obtain HxAlGltn, DecAlGltn, and SteAlGltn.
Table 1 notes the characteristics of the hm-AlGltns after the nucleophilic substitution reactions of the amino groups of AlGltn with the fatty acid chlorides. The substituted quantities of amino groups with fatty acid chlorides were determined by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) method [13–17]. Through this reaction, each hydrophobic group (Hx, Dec, Ste) was successfully introduced into each AlGltn with amide bonds to form hm-AlGltns, whose modification percentages ranged from 10% to 44%. Table 1 also lists the independent thermal denaturation temperature (Td) of each hm-AlGltn. The value of Td of the 30% hm-AlGltns was lower than that of both the 10% and 40% hm-AlGltns. In addition, the hm-AlGltns with longer side chains exhibited a lower Td. The result may indicate that interaction between and coagulation of the introduced hydrophobic side chains together with the modest introduction ratio for crystallization, affected each Td.
Figure 1C shows the 1H-NMR spectra of the hydrophobic group of the molecule on the AlGltn or hm-AlGltns. The peak of the alkyl chain appears at 1.3 ppm. The peak intensities of the original AlGltn, 32HxAltn, 24DecAlGltn, and 26SteAlGltn at 1.3 ppm were 0.14, 1.48, 1.98, and 1.69. Therefore, each hydrophobic group was successfully introduced into each hm-AlGltn. On the other hand, the hm-AlGltns with longer side chains showed sharper 1H-NMR peaks.
The hydrated carbons of the longer side chains were easily detectable. Therefore, the 44SteAlGltn showed the sharpest peak as compared with other hm-AlGltns.
Weight losses of AlGltn, 42HxAlGltn, 38DecAlGltn, and 44SteAlGltn under heating from 25–300 °C were compared in Figure 1D. 42HxAlGltn showed the highest weight loss from 35 to 115 °C indicating that there is more bound water in 42HxAlGltn than AlGltn, 38DecAlGltn, and 44SteAlGltn. The higher weight loss means a looser network structure, each (hm-)AlGltn resulting from the AlGltn molecule of (hm-)AlGltn has higher flexibility.
2.7. Cell Adhesion onto t-hm-AlGltn Films
To compare cell adhesivity onto the films, L929 cells were seeded onto t-AlGltn, t-42HxAlGltn, t-38DecAlGltn, and t-44SteAlGltn films. After being cultured for 5 min, the number of adhered cells was counted. Also, the morphology of the adhered cells was observed using a scanning electron microscope (SEM).
Figure 5 shows SEM images of L929 cells adhered onto t-hm-AlGltn films or culture plate dishes after being cultured for 5 min. The cells on the tissue culture plate show a spherical shape (Figure 5e). However, the cells on each t-(hm-)AlGltn surface, especially on t-42HxAlGltn surface, spread more extensively on the t-42HxAlGltn surface (Figure 5a–d), indicating that short side chains such as Hx could easily interact with the cell membrane.
Many more L929 cells adhered on t-42HxAlGltn than on t-AlGltn, t-38DecAlGltn, and t-44SteAlGltn (data is not shown). The obvious difference may come from the easy Hx interpenetration into the cell membrane due to its low melting point.
The results indicate that the introduced hydrophobic group Hx interacts with the tissue components, including collagen and the cell membrane. This means that the Hx group penetrated into the hydrophobic domains of the tissue, such as the hydrophobic amino acid residue and the phospholipids of the cell membrane. Figure 6 shows the effect of the chain length and the density on the ability of t-hm-AlGltn molecules to bond onto a blood vessel. When long chains such as those of the Ste and Dec groups are introduced into the AlGltn molecules, the resulting hm-AlGltn cannot easily interact with collagen molecules on the surface of the blood vessel because of their higher melting points and volume exclusion. Also, longer chains with lower mobility can hardly interact with hydrophobic groups because of hydrophobic amino acids in blood vessels. On the other hand, t-42HxAlGltn molecules with dense and short chains with a lower melting point easily interact with blood vessels and form stronger bonds.
In this study, t-24DecAlGltn and t-38DecAlGltn were employed for comparison. If DecAlGltns with a slightly higher introduction ratio like t-30DecAlGltn and t-40DecAlGltn were compared, the bonding strength would be weaker than t-24DecAlGltn and t-38DecAlGltn. The hypothesis is from our previous data that hm-Gltns with a much higher introduction ratio, over 50% introduction ratios, bonded more weakly than t-hm-Gltns with appropriate introduction ratios. The higher amount of alkyl chains can agglomerate by hydrophobic interaction in the film, therefore, there will be less hydrophobic group which can interact with the hydrophobic group in the ECM of the blood vessel.
3. Experimental Section
BeMatrix™, an alkaline-treated gelatin (AlGltn) derived from porcine skin, was kindly donated by Nitta Gelatin Inc. (Osaka, Japan). Ethanol (EtOH), 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), dimethylsulfoxide (DMSO), triethylamine (TEA), 2,4,6-trinitrobenzoylsulfonic acid (TNBS), hydrochloric acid (HCl), sodium dodecyl sulfide (SDS), calcium chloride, 10% formalin neutral buffer solution, 4′,6-diamidino-2-phenylindole (DAPI), tris(hydroxymethyl)aminomethane, tert-butylalcohol, and glycine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Hexanoyl (Hx: C6) chloride, decanoyl (Dec: C10) chloride, and stearyl (Ste: C18) chloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). A porcine aorta was purchased from Funakoshi Corporation (Tokyo, Japan). L929 cells were purchased from (RIKEN Bio Resource Center Cell Bank, RBRC-RCB2619, Ibaraki, Japan). All chemicals were used without further purification.
3.2. Synthesis of hm-AlGltns
Based on former reports [13–16], hm-AlGltns with various chain lengths and densities were prepared by the reaction between fatty acid chlorides and primary amino groups of AlGltn. The employed fatty acid chlorides were hexanoyl (Hx: C6), decanoyl (Dec: C10), and stearyl (Ste: C18) chloride. First, AlGltn (10 g) was fully dissolved into 99 mL of dried DMSO at 80 °C. Then, one mL of TEA was added into the AlGltn/DMSO solution to obtain 100 mL of 10 w/v% AlGltn/DMSO solution under a dry N2 atmosphere. The fatty acid chloride was subsequently added to the AlGltn solution and stirred for 17 h at room temperature. The resulting hm-AlGltn/DMSO solution was then poured into 300 mL of cold EtOH and stirred for 1 h. Subsequently, the precipitate of hm-AlGltn was washed twice with 300 mL of cold EtOH followed by evaporation under vacuum to leave a white cake of which the yield was calculated.
3.3. Characterization of hm-AlGltns
The modification percentages of the hydrophobic groups in AlGltn were quantified by the method previously reported using TNBS [13–17]. Briefly, each hm-AlGltn and the original AlGltn were dissolved in DMSO to obtain 0.05 w/v % solutions. Then, 100 μL of 0.1 v/v % TEA/DMSO, 50 μL of 0.1 w/v % SDS/DMSO, and 100 μL of 0.1 w/v % TNBS/DMSO were added to 100 μL of each (hm-) AlGltn/DMSO solution, followed by incubation at 37 °C for 2 h under light-shielding conditions. Then, 50 μL of the 2 N-HCl/DMSO solution was added to stop the reaction. Finally, the intensity of light absorbance was measured spectrophotometrically at 340 nm using a microplate reader (GENios A-5082, Tecan Japan, Kanagawa, Japan). The substitution percentage of amino groups with the fatty acid chlorides was then calculated from the intensities of hm-AlGltn compared with the original AlGltn.
The modification of the fatty acid in AlGltn was confirmed by 1H-NMR (AL300, JEOL, Tokyo, Japan) and FT-IR (FTIR-8400S, Shimadzu, Kyoto, Japan) measurements. The typical peaks were found at 2357 cm−1 (C=O bond of long-chain fatty acids) and 2332–2323 cm−1 (C–N bond of amino bonding between fatty acids and the amino groups of the AlGltn molecules).
Thermogravimetry (TG) analysis was executed to analyze thermal behavior of obtained hm-AlGltns (TG8120, Rigaku, Tokyo, Japan). Heating was conducted from 30 to 300 °C at a heating rate of 10 °C/min. Aluminium oxide was employed as control.
The thermal behavior of the hm-AlGltn solution was also analyzed by differential thermal analysis (DSC) (DSC8230, Rigaku, Tokyo, Japan). The hm-AlGltns were dissolved in ultrapure water (Merck Millipore, Tokyo, Japan) to prepare 70 w/v% samples. Heating was conducted from 0 to 100 °C at a heating rate of 5 °C/min under nitrogen atmosphere.
3.8. Measurement of Bonding Strength
There was no existing protocol in place to evaluate the bonding strength between the tissue surface and the film; therefore, the following measurement method was applied. The porcine blood vessel was dissected with a dermal punch into disks 4 mm in diameter. The dissected blood vessel was bonded onto a probe with GelBoy. The t-hm-AlGltn films were also punched out into 7 mm diameter disks and placed on a heated plate at 37 °C. They were fixed to the heated plate with scotch tape (3M, Tokyo, Japan) with a hole 4 mm in diameter. The bonding strength was then measured using a Texture Analyzer (TA-XT2i, Stable Micro Systems, Godalming, UK) (n = 3) with the following conditions: 180 s contact time, 20 g/mm2 applied force, and 10 mm/min tracking speed.
3.9. Observation of t-hm-AlGltn Film–Blood Vessel Interfaces
After the bonding strength measurement, each sample was fixed with a 10% formalin neutral buffer solution followed by hematoxylin and eosin (HE) staining. Cross sections of the stained samples were observed with an optical microscope (BX51, Olympus, Tokyo, Japan).
3.10. Cell Adhesion onto t-hm-AlGltn Film
A mouse fibroblast cell line, L929, was used to evaluate cell adhesion onto the t-hm-AlGltn films. The L929s were first cultured in a medium (RPMI-1640 (R8758, Sigma-Aldrich, St. Louis, MO, USA)) containing 2 v/v % fetal bovine serum. The t-hm-AlGltn films were placed on 24-well plates and a glass ring was put on each film. L929 cells (5.0 × 104 cells) were seeded onto each film for 5 min and the films were rinsed with 2 mL of phosphate buffered saline (PBS, pH 7.4). Then the cells were fixed with 10% formalin neutral buffer solution for 60 min and permeabilized in 0.2 v/v % Triton-X 100 in PBS for 2 min followed by 0.1% DAPI in PBS for 10 min in light-shielding conditions at room temperature. The adhered cells were observed with an IX81 inverted fluorescence microscope (Olympus Co. Ltd., Tokyo, Japan). The counted number of adhered cells was calculated from the area of the microscopic field (n = 3).
The cells were then observed with a scanning electron microscope (SEM). In brief, the cells were gradually dehydrated with a 50–99 v/v % ethanol/water solution. Then, the cells were immersed in tert-butylalcohol twice followed by freeze drying at −80 °C. The cells were then observed by SEM.
3.11. Statistical Analysis
Statistical analysis was carried out using Student’s t-test with Microsoft Excel software. Statistically significant differences were accepted when p <0.05. The data are shown as mean ± standard deviation (S.D.).
Thermally crosslinked film adhesives composed of hydrophobically modified AlGltn with Hx (C6), Dec (C10), or Ste (C18) were fabricated and their bonding behaviors on porcine blood vessels were evaluated. The t-42HxAlGltn film with short and dense hydrophobic groups showed higher wettability, lower water content, and stronger bonding to the blood vessel compared to the other t-hm-AlGltn films.
The t-42HxAlGltn and t-38DecAlGltn films remaining after bonding strength measurement were confirmed by histological observation. L929 cells rapidly adhered and extended onto the t-42HxAlGltn film compared with other films. These results indicate that the t-42HxAlGltn film has potential for biomedical applications as a film adhesive.
We thank Ayako Tsubokawa and Tomoka Kojima of the International Center for Materials Nanoarchitectonics (MANA) and the National Institute for Materials Science (NIMS) for their technical support. This work was supported financially in part by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)”, initiated by the Council for Science and Technology Policy (CSTP), JSPS Grant-in-Aid for Scientific Research (B) (KAKENHI Grant Number 25289254), and the World Premier International Research Center (WPI) Initiative on Materials Nanoarchitectonics (MANA), MEXT, Japan.
Conflicts of Interest
The authors declare no conflict of interest.
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|Table 1. Hydrophobically modified alkaline-treated gelatin films (hm-AlGltns) with various modification ratios.|
|Abbreviation||Number of carbons||Fatty acid chloride||Modification (%)||Yield (%)||Td (ºC)|
|in feed (μL)||in amino groups of AlGltn (%)|
The prefixes of the abbreviations indicate the reacted ratio of amino group of AlGltn molecule with fatty acid chloride and the introduced hydrophobic group (Hx, Dec, or Ste): 12HxAlGltn means that 12% of amino group of AlGltn molecule was reacted with Hx chloride.
|Table 2. T-hm-AlGltns with various modification percentages.|
|Abbreviation||Number of carbons||Amino groups used for thermal crosslinking (%)||Stiffness in dried state (MPa)|
|t-AlGltn||0||14.9 ± 6.3||4.66 ± 0.22|
|t-12HxAlGltn||6||30.7 ± 5.9||5.39 ± 0.05|
|t-32HxAlGltn||6||11.0 ± 4.9||4.39 ± 0.20|
|t-42HxAlGltn||6||10.1 ± 1.9||3.68 ± 0.60|
|t-10DecAlGltn||10||20.0 ± 2.6||5.06 ± 0.11|
|t-24DecAlGltn||10||3.5 ± 0.5||4.24 ± 0.29|
|t-38DecAlGltn||10||0.2 ± 2.0||3.06 ± 0.06|
|t-10SteAlGltn||18||2.9 ± 0.9||4.01 ± 0.11|
|t-26SteAlGltn||18||7.2 ± 0.1||3.49 ± 0.42|
|t-44SteAlGltn||18||3.5 ± 1.4||2.19 ± 0.24|
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