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Comparison of the in vitro and in vivo degradations of silk fibroin scaffolds from mulberry and nonmulberry silkworms
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015003 (http://iopscience.iop.org/1748-605X/10/1/015003) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 193.255.248.150 This content was downloaded on 12/01/2015 at 06:03
Please note that terms and conditions apply.
IOP Publishing
Biomed. Mater. 10 (2015) 015003
doi:10.1088/1748-6041/10/1/015003
Paper
received
8 July 2014
Comparison of the in vitro and in vivo degradations of silk fibroin scaffolds from mulberry and nonmulberry silkworms
re vised
13 November 2014 accep ted for publication
20 November 2014 published
22 December 2014
Renchuan You1, Yamei Xu1, Yi Liu2, Xiufang Li1 and Mingzhong Li1 1
National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No 199 Ren’ai Road, Industrial Park, Suzhou 215123, People’s Republic of China 2 Center of Burns and Plastic Surgery of CPLA, Lanzhou General Hospital of Lanzhou Command, 333 Binhe Road, Lanzhou 730050, People’s Republic of China E-mail: [email protected] Keywords: silk fibroin, Bombyx mori silks, nonmulberry silks, scaffolds, degradation
Abstract Degradation behavior is very important in the field of silk-based biomaterials. Mulberry and nonmulberry silk fibroins are structurally and functionally distinguishable; however, no studies have examined the differences in the degradation behaviors of silk materials from various silkworm species. In this study, Ca(NO3)2 was used as a uniform solvent to obtain regenerated mulberry and nonmulberry (Antheraea pernyi and Antheraea yamamai) silk fibroin (SF) solutions, and the degradation behaviors of various SF scaffolds were examined. In vitro and in vivo results demonstrated that regenerated mulberry SF scaffolds exhibited significantly higher mass loss and free amino acid content release than did nonmulberry SF scaffolds. The differences in the primary structures and condensed structures between mulberry and nonmulberry SF contributed to the significant difference in degradation rates, in which the characteristic (–Ala–)n repeats, compact crystal structure and high α-helix and β-sheet contents make nonmulberry SF more resistant than mulberry SF to enzymatic degradation. Moreover, the Antheraea pernyi and Antheraea yamamai SFs possess similar primary structures and condensed structures, although a slight difference in degradation was observed; this difference might depend on the differences in molecular weight following the regeneration process. The results indicate that the original sources of SF significantly influence the degradation rates of SF-based materials; therefore, the original sources of SF should be fully considered for preparing tissue engineering scaffolds with matched degradation rates.
1. Introduction Silk fibroin (SF) is a promising biomaterial for tissue engineering and regenerative medicine applications due to its abundance, mechanical robustness, biocompatibility, and tunable biodegradability [1–4]. Silks can be classified as mulberry and nonmulberry, which are produced by domesticated Bombyx mori (Bombycidae family) and wild silkworm species, respectively. Bombyx mori silk-based biomaterials have been extensively used for tissue engineering scaffolds [5, 6], biomedical devices [7, 8] and for drug release [9]. Of the various wild silkworms, Antheraea pernyi and Antheraea yamamai are relatively common species that belong to the Saturniidae family (order Lepidoptera, phylum Arthropoda). Antheraea pernyi is massproduced in northeast China for the production of silk © 2015 IOP Publishing Ltd
fiber, and Antheraea yamamai is primarily produced in East Asia, such as in China, Japan and Korea [10, 11]. To date, nonmulberry silk has mainly been used in high-quality clothing. Recently, the nonmulberry silks (Antheraea pernyi, Antheraea yamamai and Antheraea mylitta) have attracted considerable interest for biomedical applications because they contain an abundance of Arg–Gly–Asp (RGD) sequences, which are known to function as integrin receptors [12–16]. Mulberry and nonmulberry silks are structurally and functionally distinguishable. Bombyx mori SF (BmSF) is composed of a heavy (H) chain and a light (L) chain linked by a disulfide bond [2]. H-chains, L-chains and a P25 glycoprotein are assembled in a 6 : 6 : 1 ratio in Bombycidae [17]. The P25 protein is believed to play a significant role in maintaining the integrity of the complex, which is non-covalently linked to these chains [2].
IOP Publishing
Biomed. Mater. 10 (2015) 015003
R You et al
Nonmulberry SF lacks both an L-chain and the P25 protein [18]. Moreover, the abundant (–Ala–)n polypeptide sequences in nonmulberry silks are more hydrophobic than the (–Gly–Ala–)n repeats in mulberry silks [18]. The poly(–Ala–) β-sheets impart a higher binding energy than do poly(–Gly–Ala–) β-sheets, which make nonmulberry SF more resistant to dissolution in salt solvents in comparison to mulberry SF [18–20]. Furthermore, mulberry and nonmulberry SFs also exhibit different bioactivities for cell adhesion, proliferation and differentiation. The presence of abundant RGD sequences in nonmulberry SF leads to enhanced cell adhesion and proliferation [21]. A comparative study on osteochondral repair demonstrated that Antheraea mylitta SF scaffolds are more chondroinductive, whereas mulberry SF scaffolds are more osteoinductive [22]. Degradation behavior is very important in the fields of biomaterials and regenerative medicine; ideally, the degradation rate of scaffolds should match the tissue regeneration rate [4]. SF, like most proteins, can be catalytically hydrolyzed by proteinases in vitro and in vivo [23–29], and the degradation products of SF are soluble peptides and free amino acids, which are easily metabolized and absorbed by the body [30]. The type of enzyme plays a key role in the degradation of SF materials. α-Chymotrypsin can digest the less crystalline regions of SF but does not degrade the β-sheet crystals, leading to a weaker degradability [31], whereas protease XIV and collagenase IA were found to be more aggressive to regenerated mulberry and nonmulberry SF materials [23, 24]. The degradation rates of SF-based materials are correlated with the β-sheet content, in which a low β-sheet content contributes to easier enzymatic degradation [3, 4, 31–33]. Natural SF fibers contain abundant β-sheet crystallites after natural spinning, whereas such high β-sheet content appears impossible to achieve in regenerated SF-based materials. Therefore, regenerated SF materials, such as films, nanofibers and porous scaffolds, are more readily degraded compared with natural SF fibers [24, 26, 28]. Moreover, the fabrication process and pore structure of silk-based materials also affect the degradation rate. The scaffold obtained using an aqueous process exhibited more rapid in vivo degradation than hexafluoroisopropanol-derived scaffolds, and a higher initial SF concentration and smaller pore size led to lower levels of tissue ingrowth and degradation [27, 34]. However, none of these studies examined the differences in the degradation behaviors of SF materials from different silkworm species in great detail. The structural and physico-chemical diversity of polymers significantly influence the degradation behaviors of polymers [35, 36]; therefore, it is of interest to compare the degradation behaviors of mulberry and nonmulberry SF biomaterials. In this study, three races of SF were selected: Bm-SF from mulberry silkworms and Antheraea pernyi (Ap-SF) and Antheraea yamamai SF (Ay-SF) from nonmulberry silkworms. Porous scaffolds derived from the three races of silkworms were used to investigate the differences in the in vitro and in vivo degradation behaviors. 2
2. Materials and methods 2.1. Preparation of regenerated SF scaffolds Bombyx mori raw silks (Huzhou, Zhejiang, China) were boiled three times in a 0.05% Na2CO3 solution for 30 min to remove sericin. Antheraea pernyi raw silks (Dandong, Liaoning, China) and Antheraea yamamai cocoons (Changsha, Hunan, China) were boiled three times in a 0.25% Na2CO3 solution for 30 min. After thoroughly rinsing, the extracted fibers were air dried at 60 °C and dissolved in molten Ca(NO3)2 at 105 °C ± 2 °C for 3 h. The mixtures were dialyzed against distilled water (MWCO 9–12 kDa) in cellulose tubes for 4 d. The resulting SF solution was stored at 4 °C after filtration. The SF solutions derived from the three races of silkworms were diluted to 1.5%, and then 2-morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) (Sigma-Aldrich) were added to the SF solutions at 20%, 10% and 20% by SF weight, respectively. The mixtures were poured into stainless steel vessels and frozen at −40 °C for 6 h, followed by lyophilization for 48 h using a Virtis Genesis 25-LE Freeze Dryer to obtain porous scaffolds. 2.2. In vitro enzymatic degradation The SF scaffolds were first cut into samples with approximately equivalent sizes (3 × 3 cm2). Then, the scaffolds were weighed and incubated at 37 °C in a phosphate-buffered saline solution (PBS; 0.05 M, pH 7.4) that contained 1.0 U ml−1 collagenase IA (from Clostridium histolyticum, EC 3.4.24.3, Sigma-Aldrich). The samples were incubated in enzyme solution (bath ratio 1 : 50) for 1, 3, 6, 12 and 18 d under slow shaking, and samples without enzyme but in PBS served as controls. For each type of scaffold, at least three samples were used to obtain statistically significant data. The degradation solution was replaced with fresh enzyme solution every 3 d. At the designated time points, the degradation products and residues were collected for analysis after rinsing and lyophilizing for morphological observations and structural analyses. To calculate the mass loss, the remaining scaffolds were rinsed with deionized water and then dried at 105 °C to a constant weight, and the remaining ratio was expressed as the percentage of retained dry weight relative to the initial dry weight. 2.3. Morphological observation and structural analysis The morphological changes of the SF scaffolds were observed by scanning electron microscopy (SEM; S-570, Hitachi, Japan) after gold sputtering. Fourier transform infrared (FTIR) spectroscopy analysis of the SF scaffolds was performed to determine the conformational changes. The scaffolds were cut into microparticles with a size of less than 40 μm, and then the samples were prepared in KBr pellets. FTIR spectra were recorded using a Nicolet 5700 spectrometer
IOP Publishing
Biomed. Mater. 10 (2015) 015003
R You et al
(Thermo Scientific, USA). To quantify the secondary structures, Fourier self-deconvolution of the amide I region (1595–1705 cm−1) was performed using Opus 6.5 software (Bruker, Germany), and the Fourier selfdeconvolution spectra were curve-fitted to measure the relative areas of the amide I region components [37]. Furthermore, x-ray diffraction (XRD) was used to determine the crystal structures of the scaffolds using an x-ray diffractometer (X′Pert-Pro MPD, PANalytical B.V. Holland) with Cu Kα radiation at 40 kV and 30 mA and with a scan rate of 0.6 min−1. 2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The regenerated SF solutions and degradation products at 3, 6 and 18 d were examined using SDSPAGE according to our previous report [4]. In brief, the samples were run on polyacrylamide gel in running buffer (0.25M Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol and 5% 2-mercaptoethanol, pH 8.3). The stacking gel contained 5% acrylamide, 0.1% ammonium persulfate and 0.1% SDS in 1.0M Tris-HCl buffer (pH 6.8), and the separating gel contained 8–12% acrylamide, 0.1% ammonium persulfate and 0.1% SDS in 1.5M Tris-HCl buffer (pH 8.8). Pre-stained protein served as the molecular weight (MW) marker. 2.5. Amino acid analysis The free amino acids of the degradation products were detected using an amino acid analyzer (L-8800, Hitachi, Japan). An equal volume of 8% sulfosalicylic acid solution was added to the degradation solution and then centrifuged at 15,000 rpm for 15 min [4]. The supernatant was diluted with 0.02N HCl and filtered with 0.22 μm syringe filters, and then the filtrate was analyzed using an amino acid analyzer. To measure the amino acids contents in the SF scaffolds and degradation residues, the samples were hydrolyzed in 6N HCl at 110 °C for 24 h and then analyzed using an amino acid analyzer (L-8800, Hitachi, Japan). 2.6. In vivo degradation All animal experiments were conducted in accordance with the Management Ordinance of Experimental Animal of China ([2001] No 545) and were approved by the Jiangsu Province according to the experimental animals management rules ([2008] No 26). The SF scaffolds (approximately 20 × 20 mm2) were implanted into the backs of SD rats (180–200 g, SPF grade, male), with 5 rats in each group. Pentobarbital sodium (30 mg kg−1 body weight) was administered prior to surgery. Full-thickness wounds were created on the upper back of each rat, and the scaffolds were implanted as dermal substitutes into the defect sites, followed by covering with thin split-thickness skin grafts. The wounds were then closed with 6-0 silk sutures and covered by Vaseline carbasus and dry carbasus. Specimens were harvested at 28 d, and the harvested samples were immediately fixed in 4% formaldehyde 3
Figure 1. SDS-PAGE of the SF from (a) Bombyx mori, (b) Antheraea yamamai and (c) Antheraea pernyi, where (M1) is the molecular weight markers. The concentration of the separating gel was 8%.
in PBS at room temperature and embedded in paraffin to cut tissue sections. The sections were stained with hematoxylin and eosin (H & E) and were observed under an optical microscope (Olympus BH-2, Japan). The degradation ratio of the scaffolds was approximately calculated using Image-Pro Plus 6.0 software (Media Cybernetics Inc., USA). In a typical tissue, nuclei are stained by hematoxylin and show dispersive blue dots, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Silk fibroin that is negatively charged will react with the positively charged hematoxylin through electrostatic interactions, resulting in a light blue–purple strip and sheet-like staining. In H&E pictures, the blue–purple area was highlighted by enhancing the contrast, and then the highlighted area was captured as remaining scaffolds to calculate the remaining area after filtering the cell nuclei staining area. The degradation ratio was expressed as the percentage of reduced area relative to the initial area before implantation (n =5 for each sample). Statistical comparisons were performed using SPSS version 16.0 software (SPSS Inc., Chicago, Illinois). The deductive statistics (t-test, ANONA) were conducted and the data were expressed as mean ± standard deviation, and p
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My IOPscience
Comparison of the in vitro and in vivo degradations of silk fibroin scaffolds from mulberry and nonmulberry silkworms
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015003 (http://iopscience.iop.org/1748-605X/10/1/015003) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 193.255.248.150 This content was downloaded on 12/01/2015 at 06:03
Please note that terms and conditions apply.
IOP Publishing
Biomed. Mater. 10 (2015) 015003
doi:10.1088/1748-6041/10/1/015003
Paper
received
8 July 2014
Comparison of the in vitro and in vivo degradations of silk fibroin scaffolds from mulberry and nonmulberry silkworms
re vised
13 November 2014 accep ted for publication
20 November 2014 published
22 December 2014
Renchuan You1, Yamei Xu1, Yi Liu2, Xiufang Li1 and Mingzhong Li1 1
National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No 199 Ren’ai Road, Industrial Park, Suzhou 215123, People’s Republic of China 2 Center of Burns and Plastic Surgery of CPLA, Lanzhou General Hospital of Lanzhou Command, 333 Binhe Road, Lanzhou 730050, People’s Republic of China E-mail: [email protected] Keywords: silk fibroin, Bombyx mori silks, nonmulberry silks, scaffolds, degradation
Abstract Degradation behavior is very important in the field of silk-based biomaterials. Mulberry and nonmulberry silk fibroins are structurally and functionally distinguishable; however, no studies have examined the differences in the degradation behaviors of silk materials from various silkworm species. In this study, Ca(NO3)2 was used as a uniform solvent to obtain regenerated mulberry and nonmulberry (Antheraea pernyi and Antheraea yamamai) silk fibroin (SF) solutions, and the degradation behaviors of various SF scaffolds were examined. In vitro and in vivo results demonstrated that regenerated mulberry SF scaffolds exhibited significantly higher mass loss and free amino acid content release than did nonmulberry SF scaffolds. The differences in the primary structures and condensed structures between mulberry and nonmulberry SF contributed to the significant difference in degradation rates, in which the characteristic (–Ala–)n repeats, compact crystal structure and high α-helix and β-sheet contents make nonmulberry SF more resistant than mulberry SF to enzymatic degradation. Moreover, the Antheraea pernyi and Antheraea yamamai SFs possess similar primary structures and condensed structures, although a slight difference in degradation was observed; this difference might depend on the differences in molecular weight following the regeneration process. The results indicate that the original sources of SF significantly influence the degradation rates of SF-based materials; therefore, the original sources of SF should be fully considered for preparing tissue engineering scaffolds with matched degradation rates.
1. Introduction Silk fibroin (SF) is a promising biomaterial for tissue engineering and regenerative medicine applications due to its abundance, mechanical robustness, biocompatibility, and tunable biodegradability [1–4]. Silks can be classified as mulberry and nonmulberry, which are produced by domesticated Bombyx mori (Bombycidae family) and wild silkworm species, respectively. Bombyx mori silk-based biomaterials have been extensively used for tissue engineering scaffolds [5, 6], biomedical devices [7, 8] and for drug release [9]. Of the various wild silkworms, Antheraea pernyi and Antheraea yamamai are relatively common species that belong to the Saturniidae family (order Lepidoptera, phylum Arthropoda). Antheraea pernyi is massproduced in northeast China for the production of silk © 2015 IOP Publishing Ltd
fiber, and Antheraea yamamai is primarily produced in East Asia, such as in China, Japan and Korea [10, 11]. To date, nonmulberry silk has mainly been used in high-quality clothing. Recently, the nonmulberry silks (Antheraea pernyi, Antheraea yamamai and Antheraea mylitta) have attracted considerable interest for biomedical applications because they contain an abundance of Arg–Gly–Asp (RGD) sequences, which are known to function as integrin receptors [12–16]. Mulberry and nonmulberry silks are structurally and functionally distinguishable. Bombyx mori SF (BmSF) is composed of a heavy (H) chain and a light (L) chain linked by a disulfide bond [2]. H-chains, L-chains and a P25 glycoprotein are assembled in a 6 : 6 : 1 ratio in Bombycidae [17]. The P25 protein is believed to play a significant role in maintaining the integrity of the complex, which is non-covalently linked to these chains [2].
IOP Publishing
Biomed. Mater. 10 (2015) 015003
R You et al
Nonmulberry SF lacks both an L-chain and the P25 protein [18]. Moreover, the abundant (–Ala–)n polypeptide sequences in nonmulberry silks are more hydrophobic than the (–Gly–Ala–)n repeats in mulberry silks [18]. The poly(–Ala–) β-sheets impart a higher binding energy than do poly(–Gly–Ala–) β-sheets, which make nonmulberry SF more resistant to dissolution in salt solvents in comparison to mulberry SF [18–20]. Furthermore, mulberry and nonmulberry SFs also exhibit different bioactivities for cell adhesion, proliferation and differentiation. The presence of abundant RGD sequences in nonmulberry SF leads to enhanced cell adhesion and proliferation [21]. A comparative study on osteochondral repair demonstrated that Antheraea mylitta SF scaffolds are more chondroinductive, whereas mulberry SF scaffolds are more osteoinductive [22]. Degradation behavior is very important in the fields of biomaterials and regenerative medicine; ideally, the degradation rate of scaffolds should match the tissue regeneration rate [4]. SF, like most proteins, can be catalytically hydrolyzed by proteinases in vitro and in vivo [23–29], and the degradation products of SF are soluble peptides and free amino acids, which are easily metabolized and absorbed by the body [30]. The type of enzyme plays a key role in the degradation of SF materials. α-Chymotrypsin can digest the less crystalline regions of SF but does not degrade the β-sheet crystals, leading to a weaker degradability [31], whereas protease XIV and collagenase IA were found to be more aggressive to regenerated mulberry and nonmulberry SF materials [23, 24]. The degradation rates of SF-based materials are correlated with the β-sheet content, in which a low β-sheet content contributes to easier enzymatic degradation [3, 4, 31–33]. Natural SF fibers contain abundant β-sheet crystallites after natural spinning, whereas such high β-sheet content appears impossible to achieve in regenerated SF-based materials. Therefore, regenerated SF materials, such as films, nanofibers and porous scaffolds, are more readily degraded compared with natural SF fibers [24, 26, 28]. Moreover, the fabrication process and pore structure of silk-based materials also affect the degradation rate. The scaffold obtained using an aqueous process exhibited more rapid in vivo degradation than hexafluoroisopropanol-derived scaffolds, and a higher initial SF concentration and smaller pore size led to lower levels of tissue ingrowth and degradation [27, 34]. However, none of these studies examined the differences in the degradation behaviors of SF materials from different silkworm species in great detail. The structural and physico-chemical diversity of polymers significantly influence the degradation behaviors of polymers [35, 36]; therefore, it is of interest to compare the degradation behaviors of mulberry and nonmulberry SF biomaterials. In this study, three races of SF were selected: Bm-SF from mulberry silkworms and Antheraea pernyi (Ap-SF) and Antheraea yamamai SF (Ay-SF) from nonmulberry silkworms. Porous scaffolds derived from the three races of silkworms were used to investigate the differences in the in vitro and in vivo degradation behaviors. 2
2. Materials and methods 2.1. Preparation of regenerated SF scaffolds Bombyx mori raw silks (Huzhou, Zhejiang, China) were boiled three times in a 0.05% Na2CO3 solution for 30 min to remove sericin. Antheraea pernyi raw silks (Dandong, Liaoning, China) and Antheraea yamamai cocoons (Changsha, Hunan, China) were boiled three times in a 0.25% Na2CO3 solution for 30 min. After thoroughly rinsing, the extracted fibers were air dried at 60 °C and dissolved in molten Ca(NO3)2 at 105 °C ± 2 °C for 3 h. The mixtures were dialyzed against distilled water (MWCO 9–12 kDa) in cellulose tubes for 4 d. The resulting SF solution was stored at 4 °C after filtration. The SF solutions derived from the three races of silkworms were diluted to 1.5%, and then 2-morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) (Sigma-Aldrich) were added to the SF solutions at 20%, 10% and 20% by SF weight, respectively. The mixtures were poured into stainless steel vessels and frozen at −40 °C for 6 h, followed by lyophilization for 48 h using a Virtis Genesis 25-LE Freeze Dryer to obtain porous scaffolds. 2.2. In vitro enzymatic degradation The SF scaffolds were first cut into samples with approximately equivalent sizes (3 × 3 cm2). Then, the scaffolds were weighed and incubated at 37 °C in a phosphate-buffered saline solution (PBS; 0.05 M, pH 7.4) that contained 1.0 U ml−1 collagenase IA (from Clostridium histolyticum, EC 3.4.24.3, Sigma-Aldrich). The samples were incubated in enzyme solution (bath ratio 1 : 50) for 1, 3, 6, 12 and 18 d under slow shaking, and samples without enzyme but in PBS served as controls. For each type of scaffold, at least three samples were used to obtain statistically significant data. The degradation solution was replaced with fresh enzyme solution every 3 d. At the designated time points, the degradation products and residues were collected for analysis after rinsing and lyophilizing for morphological observations and structural analyses. To calculate the mass loss, the remaining scaffolds were rinsed with deionized water and then dried at 105 °C to a constant weight, and the remaining ratio was expressed as the percentage of retained dry weight relative to the initial dry weight. 2.3. Morphological observation and structural analysis The morphological changes of the SF scaffolds were observed by scanning electron microscopy (SEM; S-570, Hitachi, Japan) after gold sputtering. Fourier transform infrared (FTIR) spectroscopy analysis of the SF scaffolds was performed to determine the conformational changes. The scaffolds were cut into microparticles with a size of less than 40 μm, and then the samples were prepared in KBr pellets. FTIR spectra were recorded using a Nicolet 5700 spectrometer
IOP Publishing
Biomed. Mater. 10 (2015) 015003
R You et al
(Thermo Scientific, USA). To quantify the secondary structures, Fourier self-deconvolution of the amide I region (1595–1705 cm−1) was performed using Opus 6.5 software (Bruker, Germany), and the Fourier selfdeconvolution spectra were curve-fitted to measure the relative areas of the amide I region components [37]. Furthermore, x-ray diffraction (XRD) was used to determine the crystal structures of the scaffolds using an x-ray diffractometer (X′Pert-Pro MPD, PANalytical B.V. Holland) with Cu Kα radiation at 40 kV and 30 mA and with a scan rate of 0.6 min−1. 2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The regenerated SF solutions and degradation products at 3, 6 and 18 d were examined using SDSPAGE according to our previous report [4]. In brief, the samples were run on polyacrylamide gel in running buffer (0.25M Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol and 5% 2-mercaptoethanol, pH 8.3). The stacking gel contained 5% acrylamide, 0.1% ammonium persulfate and 0.1% SDS in 1.0M Tris-HCl buffer (pH 6.8), and the separating gel contained 8–12% acrylamide, 0.1% ammonium persulfate and 0.1% SDS in 1.5M Tris-HCl buffer (pH 8.8). Pre-stained protein served as the molecular weight (MW) marker. 2.5. Amino acid analysis The free amino acids of the degradation products were detected using an amino acid analyzer (L-8800, Hitachi, Japan). An equal volume of 8% sulfosalicylic acid solution was added to the degradation solution and then centrifuged at 15,000 rpm for 15 min [4]. The supernatant was diluted with 0.02N HCl and filtered with 0.22 μm syringe filters, and then the filtrate was analyzed using an amino acid analyzer. To measure the amino acids contents in the SF scaffolds and degradation residues, the samples were hydrolyzed in 6N HCl at 110 °C for 24 h and then analyzed using an amino acid analyzer (L-8800, Hitachi, Japan). 2.6. In vivo degradation All animal experiments were conducted in accordance with the Management Ordinance of Experimental Animal of China ([2001] No 545) and were approved by the Jiangsu Province according to the experimental animals management rules ([2008] No 26). The SF scaffolds (approximately 20 × 20 mm2) were implanted into the backs of SD rats (180–200 g, SPF grade, male), with 5 rats in each group. Pentobarbital sodium (30 mg kg−1 body weight) was administered prior to surgery. Full-thickness wounds were created on the upper back of each rat, and the scaffolds were implanted as dermal substitutes into the defect sites, followed by covering with thin split-thickness skin grafts. The wounds were then closed with 6-0 silk sutures and covered by Vaseline carbasus and dry carbasus. Specimens were harvested at 28 d, and the harvested samples were immediately fixed in 4% formaldehyde 3
Figure 1. SDS-PAGE of the SF from (a) Bombyx mori, (b) Antheraea yamamai and (c) Antheraea pernyi, where (M1) is the molecular weight markers. The concentration of the separating gel was 8%.
in PBS at room temperature and embedded in paraffin to cut tissue sections. The sections were stained with hematoxylin and eosin (H & E) and were observed under an optical microscope (Olympus BH-2, Japan). The degradation ratio of the scaffolds was approximately calculated using Image-Pro Plus 6.0 software (Media Cybernetics Inc., USA). In a typical tissue, nuclei are stained by hematoxylin and show dispersive blue dots, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Silk fibroin that is negatively charged will react with the positively charged hematoxylin through electrostatic interactions, resulting in a light blue–purple strip and sheet-like staining. In H&E pictures, the blue–purple area was highlighted by enhancing the contrast, and then the highlighted area was captured as remaining scaffolds to calculate the remaining area after filtering the cell nuclei staining area. The degradation ratio was expressed as the percentage of reduced area relative to the initial area before implantation (n =5 for each sample). Statistical comparisons were performed using SPSS version 16.0 software (SPSS Inc., Chicago, Illinois). The deductive statistics (t-test, ANONA) were conducted and the data were expressed as mean ± standard deviation, and p
