Abstract
Silk fibroin fiber from Bombyx mori has the advantages of superior tensile strength, non-toxicity, and good biocompatibility. However, its dense crystal structure makes implanted silk fibroin fiber resistant to biodegradation in the body. In the work described herein, silk fibroin fiber was treated with nine different dosages (0−3000 kGy) of Cobalt-60 (60Co) γ-rays, and the degradation performances of the irradiated silk fibroin fibers in collagenase IA (digestion for 30 days) were investigated. The results showed that the degradation leaching rate increased by 25, 35, and 58% at doses of 100, 200, and 500 kGy, respectively, and the diameter was about 62−65% compared with that in the control group. At a dose of 100 kGy, clear degradation stripes appeared on the surface of the silk fibroin fibers, and significantly increased at a dose of 200 kGy. With further increases of 60Co dose, the stripes deepened and turned into cracks. Degradation increased in the amorphous regions of silk fibroin fiber after treatment with γ-rays. With increasing doses of 60Co, degradation also occurred in crystalline areas and the silk II structure was partly converted into silk I. Our studies have shown that appropriate doses (such as 30−500 kGy) of γ-ray pre-treatment on silk fibroin fiber can promote its biological degradation.
Silk fibroin fiber from Bombyx mori is a natural polymer which has the advantages of superior tensile strength, non-toxicity, and good biocompatibility, and it has been used for surgical sutures for more than a hundred years. With a larger application scale than collagen sutures, silk fibroin fiber is the most widely used natural surgical suture. 1 Over the past decade, research on fibroin as a biomedical material has received ever increasing attention. Regenerated fibroin has good biocompatibility, no toxicity or immunogenicity, and it can support the growth of various kinds of cells, including corneal cells, fibroblasts, vascular endothelial cells, osteoblasts, chondrocytes, nerve cells, and activate the adhesion, proliferation, and differentiation of bone mesenchymal stem cells. Many medical and biomaterial professionals have devoted their research efforts towards stem cell‐silk fibroin scaffolds for the repair of bone tissues, ligaments, cartilage, connective tissues, blood vessels, and nerve tissues.2–8
As a medical material, it should be biodegradable and have an appropriate degradation rate after implantation. Silk fibroin fiber is a crystalline polymer, and the dense anti-parallel β-sheet of the silk II crystal structure imparts the fiber with resistance to biodegradation after implantation. In general, silk fibroin fiber loses most of its tensile strength only a year after being implanted into living organisms and the sample becomes undetectable two years after implantation. 9 According to the definition of the United States Pharmacopoeia (USP), a degradable material should lose most of its tensile strength within 60 days of implantation, on which basis silk fibroin fiber would be classified as a nondegradable material. 10 In fact, silk fibroin fiber can be degraded, since it is a protein and will be hydrolyzed by proteases. Greenwald et al. 11 showed that six weeks after implantation of silk fibroin fibers into rat skin tissue as surgical sutures, there was a 55% decrease in tensile strength and a 16% decrease in modulus of elasticity. A survey by Postlethwait 12 revealed that after implantation of silk fibroin sutures into rat subcutaneous tissue, their tensile strength had decreased by 29% after 10 days, by 73% after 30 days, and by 83% after 70 days. Four weeks after implantation in rabbits, it was found that the number of fibers had decreased and that the fibers had fractured. An 80% loss of tensile strength was noted after 12 weeks, and complete loss of strength occurred after 2 years. Salthouse et al. 13 investigated the degradation of silk sutures implanted in rabbit ocular tissues and indicated that the volume and diameter of the sutures were significantly reduced at 42 days after implantation, and that the sutures were completely absorbed after 90 days. Therefore, it is clear that silk fibroin fiber is a degradable biological material, albeit one with a slow degradation rate. Horan et al. 14 simulated the enzymatic degradation system, whereby silk threads were treated with 1 mg/mL of protease XIV at 37 °C, and found that debris particles could be isolated from the samples within 7 days and that the diameter of the silk fiber was reduced to 66% of the original after 10 days, while the quality was reduced to 50% of the original after 42 days. Mandal et al. 15 investigated the degradation of silk fiber substrate collected manually by a spinning approach from the mouths of silkworms; the fibers were treated with 1 U/mL of protease XIV, whereupon the solid residuals were 84, 70, and 46 wt% after 7, 14, and 21 days of degradation, respectively. Yang et al. 16 manufactured nerve graft materials with silk fibroin fiber as the filling. After 24 weeks of protease degradation, 79% of the fibers had been degraded. These studies indicated that silk fibroin fiber with the β-sheet structure is degraded at a slow rate, such that it would be likely to cause organism rejection in vivo upon long-term implantation13,17 and induce potentially life-threatening infections.
The degradation rate of a protein is related to its aggregation structure and the number of enzyme binding sites in the peptide chain. The crystalline structure composed of dense β-sheet accounts for about 55% of silk fibroin fiber, and the binding force is strong between folded chains and micro-fibrils with a compact structure. As a result, the enzyme molecules are hampered in accessing and acting on the silk fibroin chains. γ-Rays have high-intensity penetrability and can stimulate molecular groups and influence molecular bond energies, and so are widely used in the aggregation or degradation of polymer materials. Kojthung et al. 18 proposed that irradiation could improve the biodegradability of silk fibroin fiber. In the present study, 60Co γ-rays have been selected to irradiate silk fibroin fibers, and we have studied the enzymatic degradation performance and variation characteristics of silk fibroin fiber at different doses with the intention of devising a new and effective approach to improve and regulate the biological degradation of silk fibroin fiber, and to further expand the applications of silk fibroin fiber in the field of textile biotechnology.
Materials and methods
Material preparation
Raw silk from silk worms treated three times in 0.06 wt% Na2CO3 solution at 98–100°C for 30 min to remove sericin. After drying, the silk fibroin fibers were sealed in a container and irradiated with 60Co γ-rays at different doses, specifically 0 (control group), 30, 50, 100, 200, 500, 1000, 2000, and 3000 kGy, respectively. The silk fibers were irradiated in vacuo and not under any tension.
Enzymatic degradation
The irradiated silk fibroin samples were weighed and immersed in aliquots of 0.36 mg/mL collagen IA solution at 37°C for 30 days, with stirring. The solution was replaced with fresh collagen IA solution every three days. The degradation products were washed with deionized water and centrifuged, and this process was repeated three times to remove residual collagenase. The collected material was then oven dried and the dry weight was recorded to calculate the leaching rate. Treating with phosphate buffered saline (PBS, pH = 7.4) was selected as the control.
Measurement
The surface morphology of silk fibroin fiber was observed and the fiber diameter was determined by cold field emission scanning electron microscopy (SEM, Hitachi S-4700, Japan). The crystal structure of silk fibroin fiber was determined with an X-ray diffractometer (XRD, Mercury CCD, Japan) at a tube voltage of 40 kV, a tube current of 40 mA, and a scanning speed of 2°/min, and the diffraction intensity curve was recorded by scanning the 2θ range 5‐50°. DSC analysis of samples was performed with a Perkin-Elmer Diamond thermal analyzer (Q600, USA).
Results and discussion
Appearance of irradiated fiber after degradation
In terms of morphology, upon irradiation with low doses (0‐200 kGy) of 60Co, the fibers remained intact after 30 days of enzymatic degradation (Figure 1, a–e). Breakage and powder appeared in the degraded fibers at doses greater than 200 kGy, and more powder appeared with increasing 60Co dose (Figure 1, f and g). The residues consisted only of powder after 30 days of degradation at doses greater than 2000 kGy (Figure 1, h and i).
Appearance of irradiated fiber after degradation. a) 0 kGy, b) 30 kGy, c) 50 kGy, d) 100 kGy, e) 200 kGy, f) 500 kGy, g) 1000 kGy, h) 2000 kGy, i) 3000 kGy.
Varying degrees of cracks and ravines appeared on the surfaces of the irradiated fibers after degradation (Figure 2A). The surface of unirradiated silk fibroin fiber remained smooth after 30 days of enzymatic degradation (Figure 2A, a). At 60Co doses of 30‐100 kGy, slight degradation stripes appeared on the surface (Figure 2A, b–d). At a 60Co dose of 200 kGy, the fiber surface was covered with degradation stripes (Figure 2A, e), which deepened into cracks at doses of up to 500 kGy (Figure 2A, f). During the irradiation, 60Co γ-rays penetrated towards the interior of the silk fibroin fibers, weakening hydrogen bonds and intermolecular forces between the peptide chains and the micro-fibrils, thereby opening access to the enzyme and increasing its contact area, thus resulting in degradation and cracking of the fibers. Degradation occurred first in non-crystalline regions, so that rough stripes appeared on the fiber surface. With increasing 60Co dose, the penetrating force of the rays was enhanced, fiber degradation splits appeared between internal micro-fibrils or fibrils, the degradation was accelerated, the crack area and depth increased (Figure 2A, g–i), and long fibers were broken into shorter ones, ultimately leading to the powder form.
Longitudinal surface (A) and diameter (B) of irradiated silk fibroin fiber after enzymatic degradation. a) 0 kGy, b) 30 kGy, c) 50 kGy, d) 100 kGy, e) 200 kGy, f) 500 kGy, g) 1000 kGy, h) 2000 kGy, i) 3000 kGy. Data of the fiber diameters were mean ± standard deviation from ten samples.
Compared with the control group, the diameter of the irradiated fibers was significantly reduced after degradation (Figure 2A, 2B). The diameter was 13.78 µm for unirradiated silk fibroin fiber after 30 days of degradation. The fiber diameter was 11.51 µm after degradation at a 60Co dose of 30 kGy, and there was a 14.46% decrease in diameter compared with the control group. With increasing 60Co dose, the diameter of the fibers after degradation tapered off. At a 60Co dose of 200 kGy, the fiber diameter after degradation was 62.6% of that for the control group. After irradiation and fiber degradation at higher doses of 60Co (≥500 kGy), the structure became loose due to the splits between the micro-fibrils or fibrils, so the fiber diameter did not significantly decrease but slightly increased.
Leaching rate of enzymatic degradation in irradiated fiber
At a 60Co dose of 30 kGy, there was no significant difference in the weight loss from silk fibroin fiber compared with that from the control group after 30 days of collagenase IA degradation, these weight losses amounting to 14.13 and 14.99%, respectively. At 60Co doses of 50‐500 kGy, the degradation weight loss showed a linear increase. At doses of 100, 200, and 500 kGy, the weight losses were 17.44, 19 and 22.53%, respectively, and the leaching rates of degradation were increased by 25, 35 and 58%, respectively, compared with the control group. At 60Co doses of greater than 500 kGy, the degradation weight loss significantly increased. After 2000 and 3000 kGy of radiation, the degradation weight losses from silk fibroin fibers were 38.88% and 52.83%, respectively (Figure 3).
Weight loss of of irradiated silk fibroin fiber after enzymatic degradation. a) in collagenase IA, b) in PBS.
At 60Co doses of less than 500 kGy, there was no difference in weight loss between the silk fibroin fiber in PBS and the control group, whereas at doses of more than 500 kGy the leaching rate from the silk fibroin fiber increased significantly with increasing 60Co dose. Because high doses of 60Co γ-rays have strong penetrating power, which can destroy the peptide bonds of the silk fibroin fiber, cleavage of the peptide chains occurs and the crystalline region is damaged. The silk fibroin fiber was evidently quite easily broken, which contributed to its dissolution. At a 60Co dose of 2000 kGy, the breaking strength and elongation rate of the silk fibroin fiber were only 1.19 CN/tex and 2.39%, respectively. At a 60Co dose of 3000 kGy, the silk fibroin fiber showed complete loss of its mechanical properties. 19
XRD of irradiated and degraded fibers
XRD can reveal the crystal structure of crystalline polymers. Figure 4 indicates that all of the samples showed characteristic absorption peaks at around 20.6° and 9.1°, which can apparently be ascribed to the silk II structure. The absorption peak intensity of the irradiated silk fibroin fiber at around 24.1° decreased after degradation, and migrated to the vicinity of 24.7°, indicating that silk II has a tendency to convert to silk I.
X-ray diffraction curves of irradiated silk fibroin fiber after enzymatic degradation. a) 0 kGy, b) 30 kGy, c) 50 kGy, d) 100 kGy, e) 200 kGy, f) 500 kGy, g) 1000 kGy, h) 2000 kGy, i) 3000 kGy.
Crystallinity of irradiated silk fibroin fiber after enzymatic degradation (%)
Thermal properties of irradiated fiber after the degradation
After γ-irradiation from 60Co, the thermal stability of silk fibroin fibers decreases.
18
After degradation, the thermal stability of the irradiated silk fibroin fibers also decreased to an extent that depends on the radiation dose. At 60Co doses of up to 200 kGy, there was little difference in the apparent thermal cracking temperature compared with the control group (Figure 5, a–e). At 60Co doses greater than 200 kGy, the apparent thermal cracking temperature gradually decreased (Figure 5, f–i). Although the crystallinity increased after degradation, it only indicated that the crystal form of the fibroin protein did not change. At high doses, we thought the chemical bonds within the peptide chains and the chains of silk fibroin fiber were attacked, the bond energy was decreased, and the chains were broken, cleaving the silk fibroin fiber and forming a powdery crystalline material composed of β-sheets with a weak binding force.
DSC curves of irradiated silk fibroin fiber after enzymatic degradation. a) 0 kGy, b) 30 kGy, c) 50 kGy, d) 100 kGy, e) 200 kGy, f) 500 kGy, g) 1000 kGy, h) 2000 kGy, i) 3000 kGy.
Conclusions
γ-Ray pre-treatment of silk fibroin fibers can improve their biodegradability. At a 60Co dose of 30 kGy, there was no significant change in the diameter or leaching rate of silk fibroin fiber compared with the unirradiated fibers. At 60Co doses of 100, 200, and 500 kGy, the diameters of the degraded fibers were 62‐65% of those of unirradiated fibers, and the leaching rates were 17.44%, 19%, and 22.53%, respectively, representing increases of 25‐58%. At 60Co doses of 30‐200 kGy, varying degrees of degradation stripes appeared on the fiber surfaces. At 60Co dose of 500 kGy and above, the organizational structure within the fiber became loose, and there was fission between micro-fibrils and fibrils. The degradation stripes on the fiber surface deepened and changed into cracks, and the leaching rate significantly increased. Collagenase degradation firstly occurred in the amorphous regions of the silk fibroin fibers. With increasing 60Co dose, the crystallinity of the degraded silk fibroin fibers gradually increased, and the amorphous region content gradually decreased. Upon treatment with a high dose of 60Co, the crystalline regions of the silk fibroin fibers were also degraded, while part of the silk II changed to silk I.
Footnotes
Funding
This work was supported by the National Natural Science Foundation of China (grant numbers 51075422, 51173125); the Natural Science Foundation of Jiangsu Province of China (grant numbers BK2009147, BK2010253); the Society Development Foundation of Suzhou City of China (grant number SYG201001); and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).