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Supercritical CO2 fluid-assisted cross-linking of porcine acellular dermal matrix by ethylene glycol diglycidyl ether
Journal of CO₂ Utilization 25 (2018) 264–274
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Journal of CO2 Utilization journal homepage: www.elsevier.com/locate/jcou
Supercritical CO2 fluid-assisted cross-linking of porcine acellular dermal matrix by ethylene glycol diglycidyl ether
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Yining Chena,b, Nianhua Dana,b, , Weihua Dana,b, Guofei Yua a b
Key Laboratory for Leather Chemistry and Engineering of the Education Ministry, Sichuan University, Chengdu, Sichuan, 610065, China Research Center of Biomedical Engineering, Sichuan University, Chengdu, Sichuan, 610065, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Porcine acellular dermal matrix Epoxy compound Cross-linking Supercritical CO2 fluid Wound healing
In addition to the cross-linker, which has an impact on the properties of the materials being cross-linked, the reaction media can impact the reaction’s effectiveness. In this study, porcine acellular dermal matrix (pADM), an aggregation of collagen fibers, was cross-linked by ethylene glycol diglycidyl ether (EGDE) in supercritical CO2 fluid (sc-CO2) reaction medium (EGDE-pADM). And pADM cross-linked in ethyl alcohol medium (EtOH-EGDEpADM) was used as control. The physicochemical properties and biocompatibility of the cross-linked pADM were evaluated. The results showed that EGDE-pADM had improved thermal stability, hydrophilicity, mechanical strength, resistance to enzymatic degradation and pore structure compared with the uncross-linked pADM and EtOH- EGDE-pADM. And the improvement of scaffolds’ properties was related to the pressure of sc-CO2. The EGDE-pADM had excellent biocompatibility both in vitro and in vivo; it improved the adhesion and proliferation of L929 fibroblast cells, especially when compared with EtOH-EGDE-pADM using organic solvent, and expedited the wound healing process. These findings indicate that the reaction medium had a certain influence on the properties and cytotoxicity of the cross-linked pADM, sc-CO2 fluid was proven to be a safe and reliable crosslinking reaction medium for collagen.
1. Introduction Collagen, the main structural protein in connective tissue, is involved in various cellular processes, for instance, cell proliferation and differentiation [1–3]. Collagen has many advantages, such as low immunogenicity and irritation, and good bioactivity, biocompatibility and biodegradability. Collagenous materials have been widely used in biomaterials and biotechnology fields [4–6]. Porcine acellular dermal matrix material (pADM) is an ensemble of collagen fibers, mainly type-I collagen, that are arranged in a three-dimensional network structure [7]. However, inherent defects, including poor mechanical property, low thermal and structural stability, and low resistance to enzymatic degradation, have reportedly limited the applications of collagen matrix to a large extent. In general, modification of collagen, i.e., by the physical and chemical methods, is the most commonly used method for improving the physicochemical properties and reducing antigenicity of collagen [8,9]. However, the physical methods usually makes little contribution to the physicochemical property of collagen compared with chemical methods. The chemical method is a more efficient approach for collagen cross-linking in biomedical applications compared with the physical modification [10].
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In the past decades, various cross-linking reagents have been extensively used, to modify collagenous materials and increase their effectiveness [11–14]. In collagen modification, aqueous buffered solution is generally used as a reaction medium, where by collagen is modified by the chemicals; it is thus important that the cross-linker is water soluble. Although some collagen modifiers have excellent properties, their ineffectiveness in aqueous reaction media may inevitably affect the properties of collagen to some extent. Therefore, suitable reaction media can be considered as important as suitable cross-linkers. For instance, epoxy compound (EC), one of the most commonly used cross-linker [15,16], has been used in the modifications of heart valves [17], tendons [18], and dermis [19]. The epoxy groups in EC form covalent bonds with available reactive groups in collagen (e.g. amino groups). The number of epoxy groups has been found to closely link with the cross-linking efficiency–the higher the number of epoxy groups, the higher the cross-linking efficiency. Nonetheless, low water solubility of EC leads to low reaction rate and efficiency. Ethylene glycol diglycidyl ether (EGDE) is a typical bifunctional molecule that comprises of two epoxy groups, which is hard to dissolve in aqueous solution. When an organic solvent is used as a reaction medium, in which EGDE is highly soluble, the solvent is not suitable, causing a
Corresponding author at: Department of Biomass Chemistry and Engineering, Sichuan University, Chengdu, Sichuan, 610065, China. E-mail address: [email protected] (N. Dan).
https://doi.org/10.1016/j.jcou.2018.03.017 Received 17 February 2018; Received in revised form 14 March 2018; Accepted 20 March 2018 2212-9820/ © 2018 Published by Elsevier Ltd.
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2.3. Measurement of shrinkage temperature (Ts)
detrimental effect to the cross-linked collagen. Therefore, to tackle such problems, another solvent system is worth trying. Supercritical CO2 fluid (sc-CO2) is a special fluid, whose temperature and pressure are above its critical points; it is generally characterized by properties similar to those of gas and liquid. The sc-CO2 has been widely used in many areas, such as printing, dyeing, and cleaning [20–23]. It has been reported to significantly promote the penetration, distribution, homogeneity, and reactivity of chemical reagents [24,25]. Additionally, owing to its high compatibility and dispersion, and low viscosity, sc-CO2 can effectively control reactivity and selectivity of a chemical reaction [26], and has therefore been extensively utilized. Moreover, because CO2 is inert, it is feasible for the materials to maintain their good biocompatibility. Thus, sc-CO2 is a highly potential reaction medium, which may promote mass transfer and improve reaction rate and efficiency. In this work, pADM was cross-linked by EGDE in sc-CO2 reaction medium. The effects of pressure on the cross-linking reaction were analyzed based on thermal stability and fixation index. The effects of cross-linking on the structure of pADM were investigated by Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). The mechanical properties, hydrophilicity, and collagenase degradation were also studied. Furthermore, in vitro and in vivo biocompatibilities of cross-linked pADM were examined in L929 fibroblast cells and skin-wounded rats, respectively.
The native and cross-linked pADM were cut into 5 mm × 70 mm rectangles, and subsequently placed in a shrinkage temperature tester (SW-II, Changchun Hardware Tools Factory, China). Five specimen of each type were used and the Ts values were averaged and expressed as mean ± standard deviation. 2.4. Measurement of fixation index The number of free amino groups in the specimens was determined by ninhydrin colorimetric method [9,27]. The number of free amino groups, after heating in ninhydrin, is proportional to the optical absorbance of the solution. The absorbance was measured using a UV–vis spectrophotometer (UV751GD, Shanghai, China). The degree of crosslinking, expressed as mean ± standard deviation (n = 3), was calculated by following equation:
Fixation index (%) =
(NH2, before)−(NH2, after ) × 100 NH2,before
where NH2, before is the number of free amino groups in native pADM (before cross-linking) and NH2, after is the number of free amino groups in cross-linked pADM (after cross-linking). 2.5. Measurement of FTIR spectra
2. Experimental
The specimens mixed with KBr were pressed into tablets with ratios of 1 to 100–150. Fourier transform infrared (FTIR) spectra were measured from the tablets using a FTIR spectrophotometer (Nicolet iS10, Thermo Scientific CO., America). The spectra were collected from 4000 to 400 cm−1 at a rate of 32 scans per spectrum and a resolution of 4 cm−1 at room temperature with a humidity of around 65%.
2.1. Materials Porcine acellular dermal matrix (pADM) was exclusively provided by the JiangyinBenshine Biological Technology CO.Ltd., according to the authorization of our corresponding patent (ZL200410022506.9). Ethylene glycol diglycidyl ether (EGDE) was purchased from TCI (Shanghai) Chemical Industry CO.Ltd. Bacterial collagenase type I, RPMI 1640 medium, antibiotic solution, calf serum, trypsin, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT), tetraethyl rhodamine isothiocyanate-phalloidin, and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Unless otherwise indicated, other chemicals and reagents were purchased from Kelong Chemical Reagent Co., Ltd. (Chengdu, China).Mouse fibroblastL929 was purchased from Huaxi State Key Laboratory of Biotherapy (Chengdu, China).
2.6. Characterization by SEM The morphologies of native and cross-linked pADM were characterized by scanning electron microscopy (JSM-7500F, JEOL, Japan). The specimens were sputter-coated with gold, and the images were acquired at an accelerating voltage of 5 kV. 2.7. Measurement of hydrophilicity The hydrophilicities of native and cross-linked pADM were measured in terms of water contact angle using a goniometer (dataphysics OCA-H200, Germany) at room temperature. In brief, 5 μl (per drop) of distilled water was carefully dropped onto the surface of the specimens, and the angles in five different regions were then measured and averaged.
2.2. Cross-linking of pADM Porcine acellular dermal matrix was first cut into rectangles with dimensions of 40 mm × 70 mm and approximate weights of 1 g, and then soaked in sodium bicarbonate-sodium hydroxide buffer, pH 10.4 for pretreatment. After that, the pre-treated pADM was placed in a reaction unit of the in-house designed sc-CO2 equipment containing EGDE, where the cross-linking reaction was allowed to take place. The sc-CO2 equipment was operated at temperatures between −20 and 300 °C. Scheme.1 schematically shows the sc-CO2 reaction equipment. The maximum operating pressure and effective working volume of the equipment were 20 MPa and 1000 mL, respectively. Considering the critical temperature and pressure of 31 °C and 7.38 MPa respectively [26], the reaction pressure was controlled at 7.5, 8.5, and 9.5 MPa, and the dosage of 4% was used. The temperature and time were 37 °C and 12 h, respectively. The pADM cross-linked with EGDE was denoted as EGDE-pADM. In the studies of cross-linking effect and biocompatibility, pADM cross-linked with 4% EGDE in ethyl alcohol medium at 37 °C for 48 h, which was further washed and lyophilized, was used as a control, denoted as EtOH-EGDE-pADM.
2.8. Measurement of mechanical properties The samples’ mechanical properties, including tensile strength and elongation at break, were determined at a strain rate of 100 mm/min using a universal testing machine (AI-7000S, Gotech, China). The samples were cut into bone-shaped specimens with dimensions of length × width = 20 mm × 4 mm. The measurements were carried out at 25 °C using five specimens of each sample type; the values were averaged and expressed as mean ± standard deviation. 2.9. Degradation properties test To characteristic the degradation properties of the material, dry pADM scaffolds were cut into quarter-sized pieces, weighed (denoted as W1), then soaked in collagenase type I/PBS solution (1 U/ml, 3 ml/mg sample) for 7 days at 37 °C. The collagenase/PBS solution was changed 265
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Scheme 1. Schematic outline of the process of sc-CO2-assisted cross-linking of pADM by using EGDE.
every two days. To measure the degradation amount, samples were removed from the collagenase/PBS solution at days 1, 2, 4 and 7, and rinsed with PBS followed by distilled water. The samples were lyophilized and weighed (denoted W2). Degradation amount was calculated using the formula:
Degradation amount (%) =
2.10. Evaluation of in vitro biocompatibility The biocompatibilities of native and cross-linked pADM were investigated in L929 fibroblast cells using cell proliferation assay and confocal laser scanning microscopy (CLSM). Briefly, the samples were sealed and sterilized using a 60Co irradiation, and thereafter were soaked in 9.3 mL of culture media and incubated at 37 °C for 24 h, to prepare sample solutions. In the meantime, L929 fibroblast cells were cultured in RPMI 1640 medium supplemented with 1% (v/v) antibiotic
W1−W2 × 100 W1
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Fig. 1. The effect of reaction media to the Ts and Fixation index of EGDEpADM.
solution and 10% calf serum in 96-well plates, for 24 h at 37 °C in 5% CO2 atmosphere. After that, the culture medium was replaced with the sample solution, and then further incubated for additional 1, 3, and 5 days at 37 °C in 5% CO2 atmosphere. At each time point, 20 μl of 3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) was added, and incubated at 37 °C for 4 h to allow the formation of formazan crystals. Subsequently, DMSO (200 μl/well) was added to dissolve the formazan crystals, and the optical density (OD) at 492 nm was then measured using a microplate reader (Model 550, Bio Rad Corp. USA). The biocompatibility of pADM was further evaluated and observed by CLSM. Briefly, L929 fibroblasts were cultured on pADM scaffolds for 3 days at 37 °C in 5% CO2 atmosphere. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After the cells were washed with PBS they were stained with tetraethyl rhodamine isothiocyanate-phalloidin and 4,6-diamidino-2-phenylindole (DAPI), and then visualized by a confocal laser scanning microscope (CLSM;TCS SPII, Leica).
Fig. 2. FTIR spectra of EGDE-pADM treated with EGDE under different reaction media.
3. Results and discussion 3.1. Thermal stability and fixation index analysis The thermal stability of cross-linked pADM is a characteristic parameter determining the effectiveness of the cross-linking reaction. Thermal-induced structural transitions lead to denaturation of collagen fibrous network, in which the collagen fibers undergo hydrothermal contraction, the intermolecular and intramolecular forces (i.e. hydrogen bonding, hydrophobic bonding, and crosslink bridges) were broken down, resulting in the weakening or dislocation of the triple helix [28]. Increasing resistance against hydrothermal stress is one of the important aspects in the stabilization of collagen matrix [16]. The temperature at which initiates the denaturation and shrinkage (under a constant load) is called “shrinkage temperature’’ or “Ts” [29]; it is an indicator of strength of tissues. On the other hand, fixation index is another parameter widely used to evaluate the degree of cross-linking. Epoxide generally reacts with amino, carboxyl, and hydroxyl groups. However, in biological tissues, lysyl ε-amino has been reported to be the most reactive functional group for epoxide [30]. In addition, when pADM, an aggregation of collagenous fibers, was immersed in alkaline phosphate buffer solution, its amino groups became more reactive. Thus, the fixation index, which can be calculated based on the number of free amino groups, reflects the degree of cross-linking [27]. The effects of reaction media and reaction pressure on Ts and fixation index were illustrated in Fig. 1, which showed that the Ts and fixation index of cross-linked pADM (in sc-CO2 reaction medium) were greater than those of native pADM. The shrinkage temperature of 9.5 MPa-EGDEpADM was 73.4 °C, which is about 13.3 °C higher than that of native pADM. This indicates that cross-linked pADM has improved thermal stability compared with its uncross-linked counterpart. Although the Ts and fixation index of EtOH-EGDE-pADM were higher than those of pADM, they were considerably lower than those of EGDE-pADM. The pressure used in the experiment was higher than that of a critical pressure of CO2 (7.38 MPa) [26], which indicates that in sc-CO2 reaction medium, pADM molecules was cross-linked via covalent bonds between epoxy and amino groups. It is noteworthy that the crosslinking process in sc-CO2 reaction medium only takes 12 h, which needed at least 48 h under the condition of ethanol, and the crosslinking effectiveness was better in sc-CO2 reaction medium. It can be asserted that better results and higher efficiency came into being in the sc-CO2 reaction medium. The thermal stability of collagen depends on
2.11. Evaluation of in vivo biocompatibility Sprague-Dawley rats with weighs of 300–350 g were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (1 mL/kg). Under an aseptic condition, approximately 10 mm × 20 mm fullthickness skin excision wounds were created on both sides (left and right) of the dorsal surface of the rats (two injuries/rat). The EGDEpADM scaffolds were cut into 10 mm × 20 mm rectangles and then sterilized with 60Co irradiation. The scaffold was implanted on rat’s right-sided wound and then covered with a bandage; the left-sided wound was used as a control. Penicillin was injected intramuscularly for 3 days to prevent the wound from infection. The animals were sacrificed at 1, 2, and 3 weeks post-surgery. The wound and surrounding tissues were removed from animals for further analysis. The samples were fixed in 10% buffered formalin, dehydrated using a gradient of ethanol, and then cleared with xylene. Paraffin-embedded tissue samples were sectioned into 5-μm thick slides. In histological studies, hematoxylin and eosin (H&E) staining, and immunohistological staining were used according to standard protocols. The studies were carried out to observe the expressions of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). All experimental animals were handled according to the guidelines for human use and care of laboratory animals, set out by the National Institutes of Health (NIH). The procedures performed on animals were approved by the Animal Care and Use Committee of Sichuan University.
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Fig. 3. SEM micrographs of pADM treated with EGDE under different reaction media (100×, 5000×), (A) pADM, (B)7.5 MPa-EGDE-pADM, (C) 8.5 MPa-EGDEpADM (D) 9.5 MPa-EGDE-pADM (E) EtOH-EGDE-pADM.
pADM, and EtOH-EGDE-pADM. The FTIR peaks associated with amides I, II and III are known to be directly related to the shape of the polypeptide chain [31] The characteristic amide I peak observed at around 1640 cm−1is due to the stretching vibrations of carbonyl groups (C]O bond) in the polypeptide chain; it is a sensitive marker for secondary structure of peptide. The amide II and III bands observed at approximately 1540 and 1238 cm−1 were assigned to the NeH bending vibrations coupled with CeN stretching vibrations. The NeH group stretching vibration attributed to the amide A and amide B frequencies at 3420 and 2927 cm−1, respectively [7, 32]. The FTIR spectra further showed that the peaks associated with amino groups were nearly unchanged, indicating that the secondary structure of native pADM is nearly unaffected by the cross-linker. The peaks observed at a frequency range of 1100 –1000 cm−1 in cross-linked EGDE-pADM were stronger compared with that of pADM; this is possibly due to –C-O-C- motif introduced by EGDE. The results indicated that pADM molecules were cross-linked by EGDE, and sc-CO2 is a beneficial reaction medium that does not interfere with the structure of collagen backbone.
the degree of cross-linking. Indicated by improved thermal stability, pADM molecules were effectively cross-linked by EGDE in sc-CO2. The improved thermal stability of pADM may be the results of covalent bonds between the epoxy groups of EGDE and the available amino groups of pADM. Moreover, the Ts and fixation index of EGDE-pADM gradually increased with increasing pressure, suggesting that the supercritical fluids with low viscosity and high diffusivity can lead to higher reaction rate in extreme conditions. Furthermore, higher fixation index often implies that the number of free amino groups remained in the tissues is lower. It is known that reducing the number of free amino groups in the biological tissue can diminish its antigenicity. 3.2. FTIR analysis Fourier transform infrared (FTIR) is a preferred method in investigating the secondary structure of collagen, given that the FTIR spectra of protein molecules are directly correlated to their backbone conformation [14]. Fig. 2 shows the FTIR spectra of pADM, EGDE268
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Fig. 7. Proliferation of L929 fibroblasts cultured in the extraction liquid of pADM treated with EGDE under different reaction media. Fig. 4. Water contant angle of pADM treated with EGDE under different reaction media.
3.3. SEM analysis The morphologies of native and cross-linked pADM (EGDE-pADM and EtOH-EGDE-pADM) were visualized by SEM. As illustrated in Fig. 3, pADM was composed of collagen fibers bundles, which were stacked and tightly wound together, forming a unique three-dimensional network structure. Moreover, the morphology of EGDE-pADM assembled that of the native pADM, and has a large number of pore structures with uniform pore size. Comparing the morphologies of pADM cross-linked in different reaction media, the pore structure of EGDE-pADM cross-linked in sc-CO2 reaction medium was more specific. The pore size of EGDE-pADM increased with increasing reaction pressure, and was larger than that of pADM and EtOH-EGDE-pADM. The observations can be discussed in two aspects: (i) sc-CO2 may fill in the gaps between collagen fibers during the reaction process, causing the loosening of the mesh structure; and (ii) the cross-linked structure may be restrained by the cross-linker EGDE, resulting in a relatively rigid structure. Protein cross-linking is an effective strategy that improves the structural stability by forming stabilizing bridges between collagen fibers or protein molecules. Certain pore size and porosity are beneficial for cell growth and proliferation [33]. The results indicated that after being cross-linked in the supercritical reaction medium, EGDE-pADM had an excellent three-dimensional network structure, which is a suitable scaffold for cell growth. Compared with ethanol, sc-CO2 could improve the pore structures of the scaffolds, thus providing additional advantages.
Fig. 5. Tensile strength and longation at break of pADM treated with EGDE under different reaction media.
3.4. Hydrophilicity The hydrophilicity of EGDE-pADM was analyzed using water contact angle (WCA). Fig. 4 shows the WCA values of native pADM and pADM cross-linked in different reaction media. The WCA value reflects the hydrophobic-hydrophilic properties of a material–high WCA value indicates that the material is more hydrophobic, while low WCA value indicates that the material is more hydrophilic [27]. The WCA value of EGDE-pADM was considerably smaller than that of pADM. The WCA of 8.5/9.5 MPa-EGDE-pADM was reduced by as much as ∼18°, indicating that it is hydrophilic. The surface properties of biomaterials are important–biomaterials with hydrophilic surfaces are favorable for cell adhesion and proliferation, which can be beneficial to wound healing [34]. Epoxy compounds formed cross-linked bridges with amino, carboxyl, and hydroxyl groups, and created hydroxyl group-bearing molecules, causing the EGDE-pADM scaffolds to be more hydrophilic, which is a more favorable surface propertyof biomaterial. That is because sc-CO2 is more conducive to the reaction.
Fig. 6. Degradation properties of pADM treated with EGDE under different reaction media.
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of all 7.5 MPa-EGDE-pADM, EtOH-EGDE-pADM and native pADM were not significantly different; however, the elongation at break of 7.5 MPaEGDE-pADM and EtOH-EGDE-pADM were slightly higher than that of native pADM. Under higher pressure, the 8.5 MPa-EGDE-pADM and 9.5 MPa-EGDE-pADM had considerably higher tensile strength but moderately lower elongation at break compared to native pADM. According to the results (3.1), it was apparent that EGDE had high reactivity towards pADM in sc-CO2, leading to high cross-linking efficiency. The cross-linker forms bonds, acting as a bridge, between collagen fibers, which can help stabilize the overall structure and improve the stability of collagen; thus the tensile strength of the crosslinked pADM was increased compared with native pADM. Moreover, the cross-linker can promote a relatively rigid structure of collagen, which can better with stand the external force. However, the rigid structure of cross-linked collagen fibers may suffer from stress concentration due to stretch/elongation, and such stress can lead to brittleness, causing lower elongation at break of the cross-linked collagen [35,36]. It was apparent that the cross-linking process affected the mechanical properties of collagen matrix. Proper mechanical strength could ensure that the collagen matrix has the ability to avoid deformation due to extrusion and stretch during its uses. 3.6. Degradation properties For collagen scaffolds, the ability to resist enzyme degradation has an impact on the maintenance of its stability. The stability of scaffolds is very important for cell seeding in vitro and implantation in vivo. The aggregate structure of pADM and triple helix of collagen provides stability against degradation to some extent by enzymes other than collagenases. Therefore, the enzymatic degradation properties of the scaffolds were studied by incubating with bacterial type I collagenase. The degradation of samples to collagenase at various time points was determined and is displayed in Fig. 6. All samples showed some degree of degradation in the collagenase solution. A significant reduction in the degradation of pADM was observed which showed zero resistance against collagenase after 4 days of treatment. EtOH-EGDE -pADM had a certain degree of resistance to enzyme degradation, showing little difference with 7.5 MPa-EGDE-pADM. Conversely, 8.5 MPa-EGDE-pADM and 9.5 MPa-EGDE-pADM scaffolds demonstrated a significant improved resistance to collagenase, at greater than 75% (i.e. less than 25% degradation) after 7 days of incubation. The enzymatic stability of the EGDE-pADM was distinctly enhanced compared with pADM scaffold, due to the effectiveness of cross-linking in sc-CO2. 3.7. In vitro biocompatibility Good biocompatibility is the most basic and important requirement for biomedical materials. We evaluated the cytotoxicity of the materials using MTT assay and CLSM, and the corresponding results were shown in Figs. 7 and 8, respectively. As shown in Fig. 7, the optical density (OD) value for each group increased with increasing culture time, indicating that the cell growth is rather normal. Comparing each time point, the OD value of EGDE-pADM was greater than that of the blank and the native pADM, and the OD value of EtOH-EGDE-pADM was slightly lower than that of the blank. Collagen, the main component of pADM, is well-known for its biological activity that is beneficial to cell adhesion and proliferation [37,38]. The EtOH-EGDE-pADM exhibited some cytotoxicity, which may be due to the remaining solvents used in the cross-linking reaction and/or the unreacted cross-linking agent. When the cross- linking reaction was carried out in supercritical CO2 medium, it is possible that the unreacted cross-linking agent was discharged with the CO2 medium. Therefore, there were no reactive medium molecule and/or cross-linking agent remaining in the sample used for cytotoxicity assay. Thus, the EGDE-pADM exhibited no toxicity to the cells tested, which suggests that it is biocompatible (in addition to its hydrophilicity that is favorable for cell growth and proliferation).
Fig. 8. CLSM observation of L929 fibroblasts cultured for 3 days on pADM treated with EGDE under different reaction media (20×, 40×). (A) pADM, (B) 7.5 MPa-EGDE-pADM, (C) 8.5 MPa-EGDE-pADM (D) 9.5 MPa-EGDE-pADM (E) EtOH-EGDE-pADM.
3.5. Mechanical properties Tensile testing was used to determine the mechanical properties of EGDE-pADM, and the results were shown in Fig. 5. The tensile strength 270
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Fig. 9. Images demonstrating the healing pattern of wounds over time (A: 0 week, B: 1 week; C: 2 weeks; D: 3 weeks, left: blank, right: specimen).
Fig. 10. H&E staining of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
The results illustrated that the CO2 supercritical fluid is a more suitable reaction medium for the modification of biological materials. Fig. 8 shows the images of L929 fibroblast cells cultured on different pADM scaffolds for 3 days. The cells had a typical spindle shape except for those grown on EtOH-EGDE-pADM scaffold. The morphology of the cells grown on EGDE-pADM was uniform, indicating that the cells were able to grow very well on the cross-linked matrix. Because cells are highly sensitive the biocompatibility of the matrix, the results, showing that the cells were able to grow well with proper morphology on the
EGDE-pADM scaffold, prompted us to conclude that the EGDE-pADM has good biocompatibility [39,40]. The observations by CLSM were in accordance with MTT assay, which demonstrated the pADM crosslinked in CO2 supercritical medium had good biocompatibility. Crosslinking reaction media have great influences on the cytotoxicity of the cross-linked scaffolds. The sc-CO2 is a safe and reliable reaction medium that has the advantage of supercritical fluid compared to other types of solvents or solution systems.
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Fig. 11. bFGF immunohistochemical analysis of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
Fig. 12. VEGF immunohistochemical analysis of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
more clearly-formed skin, the epidermis of the new skin became thicker, and the collagen fibers in the dermis were more orderly arranged and tightly wound together. The formation of epithelium in wound treated with 8.5 MPa-EGDE-pADM group was more robust than that treated with control, indicating that the cross-linked EGDE-pADM improves the wound healing efficiency. The skin wound healing process is regulated by various growth factors, which are important for the cell growth and tissue repair. Fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF) are known to promote cell proliferation, capillaries formation, epidermal regeneration, and wound healing [42]. Immunohistochemistry is a specific and sensitive tool that has been used to study the expression level of bFGF, VEGF, and PDGF through their specific staining [4]. The immunohistochemical results were shown in Figs. 11–13. Positive expressions of bFGF, VEGF, and PDGF of rats in the experimental group were significantly higher than that of the control group. In addition, the expression region of rats in the experimental group was also larger than that in the blank group. These demonstrated that EGDE-pADM was able to promote tissue repair, an indication of its
3.8. In vivo biocompatibility The in vivo biocompatibility of EGDE-pADM was evaluated through its wound healing ability. As shown in Fig. 8, the ability of EGDE-pADM (Fig. 9, right) to heal wounded rats was compared with that of control (vaseline gauze; Fig. 9, left). The healing of the right-sided wound was significantly faster than that of the left-sided wound. Two weeks after the surgery, the wound-healing rate of the 8.5 MPa-EGDE-pADM was greater than 70%, which was far greater than that of the control. The wound was completely healed after 3 weeks post-surgery, and wet environment was likely to promote wound healing. Additionally, the cross-linked EGDE-pADM, which was found to be hydrophilic and highly biocompatible, may promote the migration of epidermal cell, and induce the restoration of skins, thus expediting the wound healing process. Fig. 10 shows the H&E-stained wounds at different time points. The images showed that at two weeks post-operation, the epidermis began to develop, the dermis was partly repaired, and the newly formed collagen fibers were loosely warped around each other [41]. At three weeks post-operation, would treated with 8.5 MPa-EGDE-pADM has a 272
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Fig. 13. PDGF immunohistochemical analysis of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
a certain influence on the cytotoxicity of the cross-linked pADM, sc-CO2 fluid was, however, proven to be a safe medium for the cross-linking reaction of collagen. The in vivo biocompatibility evaluation showed that the EGDE-pADM promoted wound repair while speed up the healing process. Overall, the cross-linked EGDE-pADM had good physicochemical properties and biological performance, and sc-CO2, a reliable cross-linking reaction medium, can potentially be used and applied in collagen cross-linking in biomedical field.
potential uses as skin wound healing biomaterials. 3.9. Advantages of sc-CO2 Supercritical fluid has the characteristics of both liquid and gas, but is neither liquid nor gas [26]. The supercritical carbon dioxide fluid was used as the reaction medium for the cross-linking reaction between epoxy compound and collagen matrix, and revealed many advantages over other solvent media. Firstly, many reactants are highly soluble in sc-CO2 fluid. Epoxy compounds, which generally have low solubility in aqueous medium, had improved solubility in sc-CO2 fluid; thus was able to effectively cross-link biomaterials in non-aqueous and non-organic solvent reactions. Secondly, sc-CO2 is non-toxic and safe. Because CO2 is volatile, residual solvents can be avoided. By adjusting the post-reaction temperature and pressure, it could be recycled after each crosslinking reaction. These factors are important for collagenous materials to maintain their good biocompatibility. Thirdly, supercritical fluid, which has good fluidity and permeability, and higher kinetic energy than liquid, can enters into the porous matrix of collagen (it has a behavior of gas). These characteristics can help improve mass transfer, increasing the reaction rate while improving the degree of cross-linking. Fourthly, as a reaction medium, sc-CO2 can be completely removed after the cross-linking reaction. The entire process can be complete without additional aqueous solution or organic solvents, and the collagen matrix was able to maintain its original structure. In addition, after the entire process is completed, sc-CO2 can be reintroduced into the reaction by extraction to further remove the residual cross-linking agent. Moreover, sc-CO2 can improve the properties of scaffolds, such as increasing the porosity and pore size.
Acknowledgements This research was supported by the National Natural Science Foundation of China (contract grant number 51473001), and key technology research and development program of Jiangyin city (JYKJ3369). References [1] J.L. Sun, K. Jiao, L.N. Niu, Y. Jiao, Q. Song, L.J. Shen, F.R. Tay, J.H. Chen, Biomaterials 113 (2017) 203–216. [2] X. Liu, N. Dan, W. Dan, J. Gong, Int. J. Biol Macromol. (2015), http://dx.doi.org/ 10.1016/j.ijbiomac.2015.11.015. [3] K. Gelse, Adv. Drug Deliv. Rev. 55 (2003) 1531–1546. [4] Y. Hu, W. Dan, S. Xiong, Y. Kang, A. Dhinakar, J. Wu, Z. Gu, Acta Biomater. 47 (2017) 135–148. [5] C.A. Carruthers, C.L. Dearth, J.E. Reing, C.R. Kramer, D.H. Gagne, P.M. Crapo, O. Garcia Jr, A. Badhwar, J.R. Scott, S.F. Badylak, Tissue Eng. Part A 21 (2015). [6] S. Chattopadhyay, R.T. Raines, Biopolymers 101 (2014) 821–833. [7] X. Liu, N. Dan, W. Dan, Int. J. Biol. Macromol. 88 (2016) 179–188. [8] S. Zhu, Z. Gu, Y. Hu, W. Dan, S. Xiong, J. Appl. Polym. Sci. (2016) 133. [9] Y. Hu, L. Liu, W. Dan, N. Dan, Z. Gu, X. Yu, Int. J. Biol. Macromol. 55 (2013) 221–230. [10] Y. Xu, L. Li, X. Yu, Z. Gu, X. Zhang, Carbohydr. Polym. 87 (2012) 1589–1595. [11] Z. Tian, K. Wu, W. Liu, L. Shen, G. Li, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 140 (2015) 356. [12] C. Alliraja, J.R. Rao, T. Palanisamy, RSC Adv. 5 (2015) 20939–20944. [13] S. Xiao, W. Dan, N. Dan, RSC Adv. 5 (2015) 88324–88330. [14] Y. Hu, L. Liu, Z. Gu, W. Dan, N. Dan, X. Yu, Carbohydr. Polym. 102 (2014) 324–332. [15] R. Zeeman, P.J. Dijkstra, P.B. van Wachem, M.J. van Luyn, M. Hendriks, P.T. Cahalan, J. Feijen, J. Biomed. Mater. Res. A 46 (2015) 424–433. [16] M. Sato, Y. Hiramatsu, S. Matsushita, S. Sato, Y. Watanabe, Y. Sakakibara, J. Artif. Organs 17 (2014) 265–271. [17] A. Soda, R. Tanaka, K. Takashima, T. Hirayama, M. Umezu, Y. Yamane, Am. Soc. Artif. Intern. Organs J. 55 (2009) 13–18. [18] H.W. Sung, C.S. Hsu, Y.S. Lee, D.S. Lin, J. Biomed. Mater. Res. 31 (1996) 511–518. [19] R. Zeeman, P.J. Dijkstra, P.B.V. Wachem, M.J.A.V. Luyn, M. Hendriks, P.T. Cahalan, J. Feijen, Biomaterials 20 (1999) 921–931. [20] J.H. Jun, K. Sawada, T. Takagi, G.B. Kim, C.H. Park, M. Ueda, Color. Technol. 121 (2005) 25–28.
4. Conclusions We explored the possibility of using supercritical fluid as a reaction medium in the cross-linking reaction of collagen. Porcine acellular dermal matrix (pADM) was effectively cross-linked by epoxy compound in sc-CO2 reaction medium. Compared with its native form and the pADM cross-linked in ethyl alcohol, the thermal stability of the pADM cross-linked in sc-CO2 was significantly improved, and the fixation index increased with increasing reaction pressure. In addition, the cross-linked pADM had tensile strength, appeared to be hydrophilic and showed resistance to enzymatic degradation. Despite the evaluation of in vitro biocompatibility, which indicated that the reaction medium had 273
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[33] H.M. Powell, S.T. Boyce, Biomaterials 27 (2006) 5821. [34] G.D. Winter, Nature 193 (1962) 293. [35] B. Balakrishnan, M. Mohanty, P.R. Umashankar, A. Jayakrishnan, Biomaterials 26 (2005) 6335. [36] B. Balakrishnan, A. Jayakrishnan, Biomaterials 26 (2005) 3941–3951. [37] K.M. Pawelec, S.M. Best, R.E. Cameron, J. Mater. Chem. B Mater. Biol. Med. 4 (2016) 6484–6496. [38] C. Mu, K. Zhang, W. Lin, D. Li, J. Biomed. Mater. Res. A 101 (2013) 385. [39] Y. Hu, Y. Zhu, X. Zhou, C. Ruan, H. Pan, J.M. Catchmark, J. Mater. Chem. B 4 (2016) 1235–1246. [40] Z. Yu, Q. Xu, C. Dong, S.S. Lee, L. Gao, Y. Li, M. D’Ortenzio, J. Wu, Curr. Pharm. Des. 21 (2015) 4342–4354. [41] T. Liu, W. Dan, N. Dan, X. Liu, X. Liu, X. Peng, Mater. Sci. Eng. C Mater. Biol. Appl. 77 (2017) 202–211. [42] B. Wang, Y. Han, Q. Lin, H. Liu, C. Shen, K. Nan, H. Chen, J. Mater. Chem. B 4 (2016) 1853–1861.
[21] R. Manfred, W. Eckhard, J. Björn, G. Helmut, J. Supercrit. Fluids 66 (2012) 291–296. [22] E. Onem, G. Gulumser, M. Renner, O. Yesil-Celiktas, J. Supercrit. Fluids 104 (2015) 259–264. [23] C.H. Lo, Y. Chao, J. Mater. Sci. Chem. Eng. 05 (2017) 1–8. [24] A. Marsal, P.J. Celma, J. Cot, M. Cequier, J. Supercrit. Fluids 16 (2000) 217–223. [25] Q. Yang, S. Qin, J. Chen, W. Ni, Q. Xu, J. Appl. Polym. Sci. 113 (2009) 4015–4022. [26] J.A. Behles, J.M. Desimone, Pure Appl. Chem. 73 (2001) 1281–1285. [27] Y. Chen, N. Dan, L. Wang, X. Liu, W. Dan, RSC Adv. 6 (2016) 38052–38063. [28] Y. Di, R.J. Heath, Polym. Degrad. Stab. 94 (2009) 1684–1692. [29] N.N. Fathima, M. Baias, B. Blumich, T. Ramasami, Int. J. Biol. Macromol. 47 (2010) 590–596. [30] R. Tu, S.-H. Shen, D. Lin, C. Hata, K. Thyagarajan, Y. Noishiki, R.C. Quijano, J. Biomed. Mater. Res. 28 (1994) 677. [31] X. Liu, W. Dan, H. Ju, N. Dan, J. Gong, RSC Adv. 5 (2015) 52079–52087. [32] Y. Hu, L. Liu, W. Dan, N. Dan, Z. Gu, J. Appl. Polym. Sci. 130 (2013) 2245–2256.
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Journal of CO2 Utilization journal homepage: www.elsevier.com/locate/jcou
Supercritical CO2 fluid-assisted cross-linking of porcine acellular dermal matrix by ethylene glycol diglycidyl ether
T
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Yining Chena,b, Nianhua Dana,b, , Weihua Dana,b, Guofei Yua a b
Key Laboratory for Leather Chemistry and Engineering of the Education Ministry, Sichuan University, Chengdu, Sichuan, 610065, China Research Center of Biomedical Engineering, Sichuan University, Chengdu, Sichuan, 610065, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Porcine acellular dermal matrix Epoxy compound Cross-linking Supercritical CO2 fluid Wound healing
In addition to the cross-linker, which has an impact on the properties of the materials being cross-linked, the reaction media can impact the reaction’s effectiveness. In this study, porcine acellular dermal matrix (pADM), an aggregation of collagen fibers, was cross-linked by ethylene glycol diglycidyl ether (EGDE) in supercritical CO2 fluid (sc-CO2) reaction medium (EGDE-pADM). And pADM cross-linked in ethyl alcohol medium (EtOH-EGDEpADM) was used as control. The physicochemical properties and biocompatibility of the cross-linked pADM were evaluated. The results showed that EGDE-pADM had improved thermal stability, hydrophilicity, mechanical strength, resistance to enzymatic degradation and pore structure compared with the uncross-linked pADM and EtOH- EGDE-pADM. And the improvement of scaffolds’ properties was related to the pressure of sc-CO2. The EGDE-pADM had excellent biocompatibility both in vitro and in vivo; it improved the adhesion and proliferation of L929 fibroblast cells, especially when compared with EtOH-EGDE-pADM using organic solvent, and expedited the wound healing process. These findings indicate that the reaction medium had a certain influence on the properties and cytotoxicity of the cross-linked pADM, sc-CO2 fluid was proven to be a safe and reliable crosslinking reaction medium for collagen.
1. Introduction Collagen, the main structural protein in connective tissue, is involved in various cellular processes, for instance, cell proliferation and differentiation [1–3]. Collagen has many advantages, such as low immunogenicity and irritation, and good bioactivity, biocompatibility and biodegradability. Collagenous materials have been widely used in biomaterials and biotechnology fields [4–6]. Porcine acellular dermal matrix material (pADM) is an ensemble of collagen fibers, mainly type-I collagen, that are arranged in a three-dimensional network structure [7]. However, inherent defects, including poor mechanical property, low thermal and structural stability, and low resistance to enzymatic degradation, have reportedly limited the applications of collagen matrix to a large extent. In general, modification of collagen, i.e., by the physical and chemical methods, is the most commonly used method for improving the physicochemical properties and reducing antigenicity of collagen [8,9]. However, the physical methods usually makes little contribution to the physicochemical property of collagen compared with chemical methods. The chemical method is a more efficient approach for collagen cross-linking in biomedical applications compared with the physical modification [10].
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In the past decades, various cross-linking reagents have been extensively used, to modify collagenous materials and increase their effectiveness [11–14]. In collagen modification, aqueous buffered solution is generally used as a reaction medium, where by collagen is modified by the chemicals; it is thus important that the cross-linker is water soluble. Although some collagen modifiers have excellent properties, their ineffectiveness in aqueous reaction media may inevitably affect the properties of collagen to some extent. Therefore, suitable reaction media can be considered as important as suitable cross-linkers. For instance, epoxy compound (EC), one of the most commonly used cross-linker [15,16], has been used in the modifications of heart valves [17], tendons [18], and dermis [19]. The epoxy groups in EC form covalent bonds with available reactive groups in collagen (e.g. amino groups). The number of epoxy groups has been found to closely link with the cross-linking efficiency–the higher the number of epoxy groups, the higher the cross-linking efficiency. Nonetheless, low water solubility of EC leads to low reaction rate and efficiency. Ethylene glycol diglycidyl ether (EGDE) is a typical bifunctional molecule that comprises of two epoxy groups, which is hard to dissolve in aqueous solution. When an organic solvent is used as a reaction medium, in which EGDE is highly soluble, the solvent is not suitable, causing a
Corresponding author at: Department of Biomass Chemistry and Engineering, Sichuan University, Chengdu, Sichuan, 610065, China. E-mail address: [email protected] (N. Dan).
https://doi.org/10.1016/j.jcou.2018.03.017 Received 17 February 2018; Received in revised form 14 March 2018; Accepted 20 March 2018 2212-9820/ © 2018 Published by Elsevier Ltd.
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2.3. Measurement of shrinkage temperature (Ts)
detrimental effect to the cross-linked collagen. Therefore, to tackle such problems, another solvent system is worth trying. Supercritical CO2 fluid (sc-CO2) is a special fluid, whose temperature and pressure are above its critical points; it is generally characterized by properties similar to those of gas and liquid. The sc-CO2 has been widely used in many areas, such as printing, dyeing, and cleaning [20–23]. It has been reported to significantly promote the penetration, distribution, homogeneity, and reactivity of chemical reagents [24,25]. Additionally, owing to its high compatibility and dispersion, and low viscosity, sc-CO2 can effectively control reactivity and selectivity of a chemical reaction [26], and has therefore been extensively utilized. Moreover, because CO2 is inert, it is feasible for the materials to maintain their good biocompatibility. Thus, sc-CO2 is a highly potential reaction medium, which may promote mass transfer and improve reaction rate and efficiency. In this work, pADM was cross-linked by EGDE in sc-CO2 reaction medium. The effects of pressure on the cross-linking reaction were analyzed based on thermal stability and fixation index. The effects of cross-linking on the structure of pADM were investigated by Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). The mechanical properties, hydrophilicity, and collagenase degradation were also studied. Furthermore, in vitro and in vivo biocompatibilities of cross-linked pADM were examined in L929 fibroblast cells and skin-wounded rats, respectively.
The native and cross-linked pADM were cut into 5 mm × 70 mm rectangles, and subsequently placed in a shrinkage temperature tester (SW-II, Changchun Hardware Tools Factory, China). Five specimen of each type were used and the Ts values were averaged and expressed as mean ± standard deviation. 2.4. Measurement of fixation index The number of free amino groups in the specimens was determined by ninhydrin colorimetric method [9,27]. The number of free amino groups, after heating in ninhydrin, is proportional to the optical absorbance of the solution. The absorbance was measured using a UV–vis spectrophotometer (UV751GD, Shanghai, China). The degree of crosslinking, expressed as mean ± standard deviation (n = 3), was calculated by following equation:
Fixation index (%) =
(NH2, before)−(NH2, after ) × 100 NH2,before
where NH2, before is the number of free amino groups in native pADM (before cross-linking) and NH2, after is the number of free amino groups in cross-linked pADM (after cross-linking). 2.5. Measurement of FTIR spectra
2. Experimental
The specimens mixed with KBr were pressed into tablets with ratios of 1 to 100–150. Fourier transform infrared (FTIR) spectra were measured from the tablets using a FTIR spectrophotometer (Nicolet iS10, Thermo Scientific CO., America). The spectra were collected from 4000 to 400 cm−1 at a rate of 32 scans per spectrum and a resolution of 4 cm−1 at room temperature with a humidity of around 65%.
2.1. Materials Porcine acellular dermal matrix (pADM) was exclusively provided by the JiangyinBenshine Biological Technology CO.Ltd., according to the authorization of our corresponding patent (ZL200410022506.9). Ethylene glycol diglycidyl ether (EGDE) was purchased from TCI (Shanghai) Chemical Industry CO.Ltd. Bacterial collagenase type I, RPMI 1640 medium, antibiotic solution, calf serum, trypsin, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT), tetraethyl rhodamine isothiocyanate-phalloidin, and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Unless otherwise indicated, other chemicals and reagents were purchased from Kelong Chemical Reagent Co., Ltd. (Chengdu, China).Mouse fibroblastL929 was purchased from Huaxi State Key Laboratory of Biotherapy (Chengdu, China).
2.6. Characterization by SEM The morphologies of native and cross-linked pADM were characterized by scanning electron microscopy (JSM-7500F, JEOL, Japan). The specimens were sputter-coated with gold, and the images were acquired at an accelerating voltage of 5 kV. 2.7. Measurement of hydrophilicity The hydrophilicities of native and cross-linked pADM were measured in terms of water contact angle using a goniometer (dataphysics OCA-H200, Germany) at room temperature. In brief, 5 μl (per drop) of distilled water was carefully dropped onto the surface of the specimens, and the angles in five different regions were then measured and averaged.
2.2. Cross-linking of pADM Porcine acellular dermal matrix was first cut into rectangles with dimensions of 40 mm × 70 mm and approximate weights of 1 g, and then soaked in sodium bicarbonate-sodium hydroxide buffer, pH 10.4 for pretreatment. After that, the pre-treated pADM was placed in a reaction unit of the in-house designed sc-CO2 equipment containing EGDE, where the cross-linking reaction was allowed to take place. The sc-CO2 equipment was operated at temperatures between −20 and 300 °C. Scheme.1 schematically shows the sc-CO2 reaction equipment. The maximum operating pressure and effective working volume of the equipment were 20 MPa and 1000 mL, respectively. Considering the critical temperature and pressure of 31 °C and 7.38 MPa respectively [26], the reaction pressure was controlled at 7.5, 8.5, and 9.5 MPa, and the dosage of 4% was used. The temperature and time were 37 °C and 12 h, respectively. The pADM cross-linked with EGDE was denoted as EGDE-pADM. In the studies of cross-linking effect and biocompatibility, pADM cross-linked with 4% EGDE in ethyl alcohol medium at 37 °C for 48 h, which was further washed and lyophilized, was used as a control, denoted as EtOH-EGDE-pADM.
2.8. Measurement of mechanical properties The samples’ mechanical properties, including tensile strength and elongation at break, were determined at a strain rate of 100 mm/min using a universal testing machine (AI-7000S, Gotech, China). The samples were cut into bone-shaped specimens with dimensions of length × width = 20 mm × 4 mm. The measurements were carried out at 25 °C using five specimens of each sample type; the values were averaged and expressed as mean ± standard deviation. 2.9. Degradation properties test To characteristic the degradation properties of the material, dry pADM scaffolds were cut into quarter-sized pieces, weighed (denoted as W1), then soaked in collagenase type I/PBS solution (1 U/ml, 3 ml/mg sample) for 7 days at 37 °C. The collagenase/PBS solution was changed 265
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Scheme 1. Schematic outline of the process of sc-CO2-assisted cross-linking of pADM by using EGDE.
every two days. To measure the degradation amount, samples were removed from the collagenase/PBS solution at days 1, 2, 4 and 7, and rinsed with PBS followed by distilled water. The samples were lyophilized and weighed (denoted W2). Degradation amount was calculated using the formula:
Degradation amount (%) =
2.10. Evaluation of in vitro biocompatibility The biocompatibilities of native and cross-linked pADM were investigated in L929 fibroblast cells using cell proliferation assay and confocal laser scanning microscopy (CLSM). Briefly, the samples were sealed and sterilized using a 60Co irradiation, and thereafter were soaked in 9.3 mL of culture media and incubated at 37 °C for 24 h, to prepare sample solutions. In the meantime, L929 fibroblast cells were cultured in RPMI 1640 medium supplemented with 1% (v/v) antibiotic
W1−W2 × 100 W1
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Fig. 1. The effect of reaction media to the Ts and Fixation index of EGDEpADM.
solution and 10% calf serum in 96-well plates, for 24 h at 37 °C in 5% CO2 atmosphere. After that, the culture medium was replaced with the sample solution, and then further incubated for additional 1, 3, and 5 days at 37 °C in 5% CO2 atmosphere. At each time point, 20 μl of 3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) was added, and incubated at 37 °C for 4 h to allow the formation of formazan crystals. Subsequently, DMSO (200 μl/well) was added to dissolve the formazan crystals, and the optical density (OD) at 492 nm was then measured using a microplate reader (Model 550, Bio Rad Corp. USA). The biocompatibility of pADM was further evaluated and observed by CLSM. Briefly, L929 fibroblasts were cultured on pADM scaffolds for 3 days at 37 °C in 5% CO2 atmosphere. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After the cells were washed with PBS they were stained with tetraethyl rhodamine isothiocyanate-phalloidin and 4,6-diamidino-2-phenylindole (DAPI), and then visualized by a confocal laser scanning microscope (CLSM;TCS SPII, Leica).
Fig. 2. FTIR spectra of EGDE-pADM treated with EGDE under different reaction media.
3. Results and discussion 3.1. Thermal stability and fixation index analysis The thermal stability of cross-linked pADM is a characteristic parameter determining the effectiveness of the cross-linking reaction. Thermal-induced structural transitions lead to denaturation of collagen fibrous network, in which the collagen fibers undergo hydrothermal contraction, the intermolecular and intramolecular forces (i.e. hydrogen bonding, hydrophobic bonding, and crosslink bridges) were broken down, resulting in the weakening or dislocation of the triple helix [28]. Increasing resistance against hydrothermal stress is one of the important aspects in the stabilization of collagen matrix [16]. The temperature at which initiates the denaturation and shrinkage (under a constant load) is called “shrinkage temperature’’ or “Ts” [29]; it is an indicator of strength of tissues. On the other hand, fixation index is another parameter widely used to evaluate the degree of cross-linking. Epoxide generally reacts with amino, carboxyl, and hydroxyl groups. However, in biological tissues, lysyl ε-amino has been reported to be the most reactive functional group for epoxide [30]. In addition, when pADM, an aggregation of collagenous fibers, was immersed in alkaline phosphate buffer solution, its amino groups became more reactive. Thus, the fixation index, which can be calculated based on the number of free amino groups, reflects the degree of cross-linking [27]. The effects of reaction media and reaction pressure on Ts and fixation index were illustrated in Fig. 1, which showed that the Ts and fixation index of cross-linked pADM (in sc-CO2 reaction medium) were greater than those of native pADM. The shrinkage temperature of 9.5 MPa-EGDEpADM was 73.4 °C, which is about 13.3 °C higher than that of native pADM. This indicates that cross-linked pADM has improved thermal stability compared with its uncross-linked counterpart. Although the Ts and fixation index of EtOH-EGDE-pADM were higher than those of pADM, they were considerably lower than those of EGDE-pADM. The pressure used in the experiment was higher than that of a critical pressure of CO2 (7.38 MPa) [26], which indicates that in sc-CO2 reaction medium, pADM molecules was cross-linked via covalent bonds between epoxy and amino groups. It is noteworthy that the crosslinking process in sc-CO2 reaction medium only takes 12 h, which needed at least 48 h under the condition of ethanol, and the crosslinking effectiveness was better in sc-CO2 reaction medium. It can be asserted that better results and higher efficiency came into being in the sc-CO2 reaction medium. The thermal stability of collagen depends on
2.11. Evaluation of in vivo biocompatibility Sprague-Dawley rats with weighs of 300–350 g were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (1 mL/kg). Under an aseptic condition, approximately 10 mm × 20 mm fullthickness skin excision wounds were created on both sides (left and right) of the dorsal surface of the rats (two injuries/rat). The EGDEpADM scaffolds were cut into 10 mm × 20 mm rectangles and then sterilized with 60Co irradiation. The scaffold was implanted on rat’s right-sided wound and then covered with a bandage; the left-sided wound was used as a control. Penicillin was injected intramuscularly for 3 days to prevent the wound from infection. The animals were sacrificed at 1, 2, and 3 weeks post-surgery. The wound and surrounding tissues were removed from animals for further analysis. The samples were fixed in 10% buffered formalin, dehydrated using a gradient of ethanol, and then cleared with xylene. Paraffin-embedded tissue samples were sectioned into 5-μm thick slides. In histological studies, hematoxylin and eosin (H&E) staining, and immunohistological staining were used according to standard protocols. The studies were carried out to observe the expressions of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). All experimental animals were handled according to the guidelines for human use and care of laboratory animals, set out by the National Institutes of Health (NIH). The procedures performed on animals were approved by the Animal Care and Use Committee of Sichuan University.
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Fig. 3. SEM micrographs of pADM treated with EGDE under different reaction media (100×, 5000×), (A) pADM, (B)7.5 MPa-EGDE-pADM, (C) 8.5 MPa-EGDEpADM (D) 9.5 MPa-EGDE-pADM (E) EtOH-EGDE-pADM.
pADM, and EtOH-EGDE-pADM. The FTIR peaks associated with amides I, II and III are known to be directly related to the shape of the polypeptide chain [31] The characteristic amide I peak observed at around 1640 cm−1is due to the stretching vibrations of carbonyl groups (C]O bond) in the polypeptide chain; it is a sensitive marker for secondary structure of peptide. The amide II and III bands observed at approximately 1540 and 1238 cm−1 were assigned to the NeH bending vibrations coupled with CeN stretching vibrations. The NeH group stretching vibration attributed to the amide A and amide B frequencies at 3420 and 2927 cm−1, respectively [7, 32]. The FTIR spectra further showed that the peaks associated with amino groups were nearly unchanged, indicating that the secondary structure of native pADM is nearly unaffected by the cross-linker. The peaks observed at a frequency range of 1100 –1000 cm−1 in cross-linked EGDE-pADM were stronger compared with that of pADM; this is possibly due to –C-O-C- motif introduced by EGDE. The results indicated that pADM molecules were cross-linked by EGDE, and sc-CO2 is a beneficial reaction medium that does not interfere with the structure of collagen backbone.
the degree of cross-linking. Indicated by improved thermal stability, pADM molecules were effectively cross-linked by EGDE in sc-CO2. The improved thermal stability of pADM may be the results of covalent bonds between the epoxy groups of EGDE and the available amino groups of pADM. Moreover, the Ts and fixation index of EGDE-pADM gradually increased with increasing pressure, suggesting that the supercritical fluids with low viscosity and high diffusivity can lead to higher reaction rate in extreme conditions. Furthermore, higher fixation index often implies that the number of free amino groups remained in the tissues is lower. It is known that reducing the number of free amino groups in the biological tissue can diminish its antigenicity. 3.2. FTIR analysis Fourier transform infrared (FTIR) is a preferred method in investigating the secondary structure of collagen, given that the FTIR spectra of protein molecules are directly correlated to their backbone conformation [14]. Fig. 2 shows the FTIR spectra of pADM, EGDE268
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Fig. 7. Proliferation of L929 fibroblasts cultured in the extraction liquid of pADM treated with EGDE under different reaction media. Fig. 4. Water contant angle of pADM treated with EGDE under different reaction media.
3.3. SEM analysis The morphologies of native and cross-linked pADM (EGDE-pADM and EtOH-EGDE-pADM) were visualized by SEM. As illustrated in Fig. 3, pADM was composed of collagen fibers bundles, which were stacked and tightly wound together, forming a unique three-dimensional network structure. Moreover, the morphology of EGDE-pADM assembled that of the native pADM, and has a large number of pore structures with uniform pore size. Comparing the morphologies of pADM cross-linked in different reaction media, the pore structure of EGDE-pADM cross-linked in sc-CO2 reaction medium was more specific. The pore size of EGDE-pADM increased with increasing reaction pressure, and was larger than that of pADM and EtOH-EGDE-pADM. The observations can be discussed in two aspects: (i) sc-CO2 may fill in the gaps between collagen fibers during the reaction process, causing the loosening of the mesh structure; and (ii) the cross-linked structure may be restrained by the cross-linker EGDE, resulting in a relatively rigid structure. Protein cross-linking is an effective strategy that improves the structural stability by forming stabilizing bridges between collagen fibers or protein molecules. Certain pore size and porosity are beneficial for cell growth and proliferation [33]. The results indicated that after being cross-linked in the supercritical reaction medium, EGDE-pADM had an excellent three-dimensional network structure, which is a suitable scaffold for cell growth. Compared with ethanol, sc-CO2 could improve the pore structures of the scaffolds, thus providing additional advantages.
Fig. 5. Tensile strength and longation at break of pADM treated with EGDE under different reaction media.
3.4. Hydrophilicity The hydrophilicity of EGDE-pADM was analyzed using water contact angle (WCA). Fig. 4 shows the WCA values of native pADM and pADM cross-linked in different reaction media. The WCA value reflects the hydrophobic-hydrophilic properties of a material–high WCA value indicates that the material is more hydrophobic, while low WCA value indicates that the material is more hydrophilic [27]. The WCA value of EGDE-pADM was considerably smaller than that of pADM. The WCA of 8.5/9.5 MPa-EGDE-pADM was reduced by as much as ∼18°, indicating that it is hydrophilic. The surface properties of biomaterials are important–biomaterials with hydrophilic surfaces are favorable for cell adhesion and proliferation, which can be beneficial to wound healing [34]. Epoxy compounds formed cross-linked bridges with amino, carboxyl, and hydroxyl groups, and created hydroxyl group-bearing molecules, causing the EGDE-pADM scaffolds to be more hydrophilic, which is a more favorable surface propertyof biomaterial. That is because sc-CO2 is more conducive to the reaction.
Fig. 6. Degradation properties of pADM treated with EGDE under different reaction media.
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of all 7.5 MPa-EGDE-pADM, EtOH-EGDE-pADM and native pADM were not significantly different; however, the elongation at break of 7.5 MPaEGDE-pADM and EtOH-EGDE-pADM were slightly higher than that of native pADM. Under higher pressure, the 8.5 MPa-EGDE-pADM and 9.5 MPa-EGDE-pADM had considerably higher tensile strength but moderately lower elongation at break compared to native pADM. According to the results (3.1), it was apparent that EGDE had high reactivity towards pADM in sc-CO2, leading to high cross-linking efficiency. The cross-linker forms bonds, acting as a bridge, between collagen fibers, which can help stabilize the overall structure and improve the stability of collagen; thus the tensile strength of the crosslinked pADM was increased compared with native pADM. Moreover, the cross-linker can promote a relatively rigid structure of collagen, which can better with stand the external force. However, the rigid structure of cross-linked collagen fibers may suffer from stress concentration due to stretch/elongation, and such stress can lead to brittleness, causing lower elongation at break of the cross-linked collagen [35,36]. It was apparent that the cross-linking process affected the mechanical properties of collagen matrix. Proper mechanical strength could ensure that the collagen matrix has the ability to avoid deformation due to extrusion and stretch during its uses. 3.6. Degradation properties For collagen scaffolds, the ability to resist enzyme degradation has an impact on the maintenance of its stability. The stability of scaffolds is very important for cell seeding in vitro and implantation in vivo. The aggregate structure of pADM and triple helix of collagen provides stability against degradation to some extent by enzymes other than collagenases. Therefore, the enzymatic degradation properties of the scaffolds were studied by incubating with bacterial type I collagenase. The degradation of samples to collagenase at various time points was determined and is displayed in Fig. 6. All samples showed some degree of degradation in the collagenase solution. A significant reduction in the degradation of pADM was observed which showed zero resistance against collagenase after 4 days of treatment. EtOH-EGDE -pADM had a certain degree of resistance to enzyme degradation, showing little difference with 7.5 MPa-EGDE-pADM. Conversely, 8.5 MPa-EGDE-pADM and 9.5 MPa-EGDE-pADM scaffolds demonstrated a significant improved resistance to collagenase, at greater than 75% (i.e. less than 25% degradation) after 7 days of incubation. The enzymatic stability of the EGDE-pADM was distinctly enhanced compared with pADM scaffold, due to the effectiveness of cross-linking in sc-CO2. 3.7. In vitro biocompatibility Good biocompatibility is the most basic and important requirement for biomedical materials. We evaluated the cytotoxicity of the materials using MTT assay and CLSM, and the corresponding results were shown in Figs. 7 and 8, respectively. As shown in Fig. 7, the optical density (OD) value for each group increased with increasing culture time, indicating that the cell growth is rather normal. Comparing each time point, the OD value of EGDE-pADM was greater than that of the blank and the native pADM, and the OD value of EtOH-EGDE-pADM was slightly lower than that of the blank. Collagen, the main component of pADM, is well-known for its biological activity that is beneficial to cell adhesion and proliferation [37,38]. The EtOH-EGDE-pADM exhibited some cytotoxicity, which may be due to the remaining solvents used in the cross-linking reaction and/or the unreacted cross-linking agent. When the cross- linking reaction was carried out in supercritical CO2 medium, it is possible that the unreacted cross-linking agent was discharged with the CO2 medium. Therefore, there were no reactive medium molecule and/or cross-linking agent remaining in the sample used for cytotoxicity assay. Thus, the EGDE-pADM exhibited no toxicity to the cells tested, which suggests that it is biocompatible (in addition to its hydrophilicity that is favorable for cell growth and proliferation).
Fig. 8. CLSM observation of L929 fibroblasts cultured for 3 days on pADM treated with EGDE under different reaction media (20×, 40×). (A) pADM, (B) 7.5 MPa-EGDE-pADM, (C) 8.5 MPa-EGDE-pADM (D) 9.5 MPa-EGDE-pADM (E) EtOH-EGDE-pADM.
3.5. Mechanical properties Tensile testing was used to determine the mechanical properties of EGDE-pADM, and the results were shown in Fig. 5. The tensile strength 270
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Fig. 9. Images demonstrating the healing pattern of wounds over time (A: 0 week, B: 1 week; C: 2 weeks; D: 3 weeks, left: blank, right: specimen).
Fig. 10. H&E staining of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
The results illustrated that the CO2 supercritical fluid is a more suitable reaction medium for the modification of biological materials. Fig. 8 shows the images of L929 fibroblast cells cultured on different pADM scaffolds for 3 days. The cells had a typical spindle shape except for those grown on EtOH-EGDE-pADM scaffold. The morphology of the cells grown on EGDE-pADM was uniform, indicating that the cells were able to grow very well on the cross-linked matrix. Because cells are highly sensitive the biocompatibility of the matrix, the results, showing that the cells were able to grow well with proper morphology on the
EGDE-pADM scaffold, prompted us to conclude that the EGDE-pADM has good biocompatibility [39,40]. The observations by CLSM were in accordance with MTT assay, which demonstrated the pADM crosslinked in CO2 supercritical medium had good biocompatibility. Crosslinking reaction media have great influences on the cytotoxicity of the cross-linked scaffolds. The sc-CO2 is a safe and reliable reaction medium that has the advantage of supercritical fluid compared to other types of solvents or solution systems.
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Fig. 11. bFGF immunohistochemical analysis of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
Fig. 12. VEGF immunohistochemical analysis of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
more clearly-formed skin, the epidermis of the new skin became thicker, and the collagen fibers in the dermis were more orderly arranged and tightly wound together. The formation of epithelium in wound treated with 8.5 MPa-EGDE-pADM group was more robust than that treated with control, indicating that the cross-linked EGDE-pADM improves the wound healing efficiency. The skin wound healing process is regulated by various growth factors, which are important for the cell growth and tissue repair. Fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF) are known to promote cell proliferation, capillaries formation, epidermal regeneration, and wound healing [42]. Immunohistochemistry is a specific and sensitive tool that has been used to study the expression level of bFGF, VEGF, and PDGF through their specific staining [4]. The immunohistochemical results were shown in Figs. 11–13. Positive expressions of bFGF, VEGF, and PDGF of rats in the experimental group were significantly higher than that of the control group. In addition, the expression region of rats in the experimental group was also larger than that in the blank group. These demonstrated that EGDE-pADM was able to promote tissue repair, an indication of its
3.8. In vivo biocompatibility The in vivo biocompatibility of EGDE-pADM was evaluated through its wound healing ability. As shown in Fig. 8, the ability of EGDE-pADM (Fig. 9, right) to heal wounded rats was compared with that of control (vaseline gauze; Fig. 9, left). The healing of the right-sided wound was significantly faster than that of the left-sided wound. Two weeks after the surgery, the wound-healing rate of the 8.5 MPa-EGDE-pADM was greater than 70%, which was far greater than that of the control. The wound was completely healed after 3 weeks post-surgery, and wet environment was likely to promote wound healing. Additionally, the cross-linked EGDE-pADM, which was found to be hydrophilic and highly biocompatible, may promote the migration of epidermal cell, and induce the restoration of skins, thus expediting the wound healing process. Fig. 10 shows the H&E-stained wounds at different time points. The images showed that at two weeks post-operation, the epidermis began to develop, the dermis was partly repaired, and the newly formed collagen fibers were loosely warped around each other [41]. At three weeks post-operation, would treated with 8.5 MPa-EGDE-pADM has a 272
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Fig. 13. PDGF immunohistochemical analysis of blank group (a) and 8.5 MPa-EGDE-pADM group (b) (a1, b1 : 1 week, a2, b2: 2 weeks, a3, b3: 3 weeks).
a certain influence on the cytotoxicity of the cross-linked pADM, sc-CO2 fluid was, however, proven to be a safe medium for the cross-linking reaction of collagen. The in vivo biocompatibility evaluation showed that the EGDE-pADM promoted wound repair while speed up the healing process. Overall, the cross-linked EGDE-pADM had good physicochemical properties and biological performance, and sc-CO2, a reliable cross-linking reaction medium, can potentially be used and applied in collagen cross-linking in biomedical field.
potential uses as skin wound healing biomaterials. 3.9. Advantages of sc-CO2 Supercritical fluid has the characteristics of both liquid and gas, but is neither liquid nor gas [26]. The supercritical carbon dioxide fluid was used as the reaction medium for the cross-linking reaction between epoxy compound and collagen matrix, and revealed many advantages over other solvent media. Firstly, many reactants are highly soluble in sc-CO2 fluid. Epoxy compounds, which generally have low solubility in aqueous medium, had improved solubility in sc-CO2 fluid; thus was able to effectively cross-link biomaterials in non-aqueous and non-organic solvent reactions. Secondly, sc-CO2 is non-toxic and safe. Because CO2 is volatile, residual solvents can be avoided. By adjusting the post-reaction temperature and pressure, it could be recycled after each crosslinking reaction. These factors are important for collagenous materials to maintain their good biocompatibility. Thirdly, supercritical fluid, which has good fluidity and permeability, and higher kinetic energy than liquid, can enters into the porous matrix of collagen (it has a behavior of gas). These characteristics can help improve mass transfer, increasing the reaction rate while improving the degree of cross-linking. Fourthly, as a reaction medium, sc-CO2 can be completely removed after the cross-linking reaction. The entire process can be complete without additional aqueous solution or organic solvents, and the collagen matrix was able to maintain its original structure. In addition, after the entire process is completed, sc-CO2 can be reintroduced into the reaction by extraction to further remove the residual cross-linking agent. Moreover, sc-CO2 can improve the properties of scaffolds, such as increasing the porosity and pore size.
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4. Conclusions We explored the possibility of using supercritical fluid as a reaction medium in the cross-linking reaction of collagen. Porcine acellular dermal matrix (pADM) was effectively cross-linked by epoxy compound in sc-CO2 reaction medium. Compared with its native form and the pADM cross-linked in ethyl alcohol, the thermal stability of the pADM cross-linked in sc-CO2 was significantly improved, and the fixation index increased with increasing reaction pressure. In addition, the cross-linked pADM had tensile strength, appeared to be hydrophilic and showed resistance to enzymatic degradation. Despite the evaluation of in vitro biocompatibility, which indicated that the reaction medium had 273
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