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sodium alginate composite porous materials with controllable degradation
Journal Pre-proofs Silk fibroin/sodium alginate composite porous materials with controllable degradation Yiyu Wang, Sisi Fan, Yuwei Li, Chunqing Niu, Xiang Li, Yajin Guo, Junhua Zhang, Jian Shi, Xinyu Wang PII: DOI: Reference:
S0141-8130(19)36013-1 https://doi.org/10.1016/j.ijbiomac.2019.10.141 BIOMAC 13642
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
1 August 2019 15 October 2019 15 October 2019
Please cite this article as: Y. Wang, S. Fan, Y. Li, C. Niu, X. Li, Y. Guo, J. Zhang, J. Shi, X. Wang, Silk fibroin/ sodium alginate composite porous materials with controllable degradation, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.141
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Silk fibroin/sodium alginate composite porous materials with controllable degradation
Yiyu Wang3, Sisi Fan3, Yuwei Li3, Chunqing Niu3, Xiang Li 3, Yajin Guo1,2, Junhua Zhang3, Jian Shi4 and Xinyu Wang1,2 * 1
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing,
Wuhan University of Technology, Wuhan 430070, People’s Republic of China 2
Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan
University of Technology, Wuhan 430070, Republic of China 3
Hubei Province Research Center of Engineering Technology for Utilization of Botanical
Functional Ingredients, Hubei Engineering University, Xiaogan 432000, People’s Republic of China. 4
Department of Machine Intelligence and Systems Engineering, Faculty of Systems Science
and Technology, Akita Prefectural University, Akita 015-0055, Japan
E-mail: [email protected] (X. Wang)
Abstract: In this study, silk fibroin (SF)/sodium alginate (SA) porous materials (PMs) with different blend ratios were generated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as crosslinking agent by a simple freeze-dried method. Degradation experiment of SF/SA PMs have been systematically investigated up to 18 days in Collagenase IA solution at 37 °C, Phosphate buffer saline (PBS) solution without enzyme was used as a control. The results showed SF/SA 50/50 PMs exhibited a lowest rate of weight loss, about 68% of the weight retained within 18 d in Collagenase IA solution. SEM images indicated Collagenase IA can degrade fibroin leading to collapse of the pure SF PMs, while SF/SA 50/50 PMs still possessed integrity of pore structure during enzyme degradation with increasing exposure time. The crystalline structure of the SF in the SF/SA PMs changed to silk II after degradation for 18 d. Furthermore, the results of the in vivo degradation by subcutaneous implantation in rats showed that all PMs can be degraded at different levels, and exhibited good subcutaneous histocompatibility to the host animals. The degradability was strongly correlated to the blend ratios in a series of SF/SA composite PMs, and insights gained in this study can serve as a guide to match desired degradation behavior with specific applications for the SF/SA composite PMs.
Keywords: Silk fibroin, sodium alginate, porous materials, degradation
1. Introduction Many biodegradable porous materials (PMs) systems based on naturally derived and biocompatible polymers have been studied and proven to be wide applications in tissue engineering and biomedicine [1-3]. For these applications purpose, the PMs should have biocompatibility and porous structural properties similar to the specific tissues to maximally mimic the native extracellular matrix (ECM) characteristics. What's more, the PMs should be biodegradable at proper degradation rate to maintain their structure integraty before the new tissue form and degradation products should exhibit no toxic to host [4]. Therefore, it is a key issue to control the degradation rate of PMs. Among all the natural materials used, silk fibroin (SF) is from silkworms, Bombyx mori cocoons, have attracted wide attention due to its low immunogenicity, impressive mechanical properties, biocompatibility and easy fabrication [5-7]. In addition, the regenerated SF biomaterials with different formation were proven to be degradable [8]. SF, as a protein, can be catalytically degraded into amino acids under the action of proteolytic enzymes, which would be able to be absorbed and metabolized by human [9]. Zhou et al. investigated the degradation behavior of the electrostatic spinning SF scaffolds by protease XIV in vitro and in vivo, the results showed about 65% of the electrostatic spinning SF scaffolds were degraded within 24 d in protease XIV, and the scaffolds were completely degraded in vivo after implantation for 8 weeks and well tolerated by the host animals [10]. Li et al. studied the enzymatic degradation behavior of the SF conduits with silk I structure, revealing that the degradation rate could be effectively regulated by molecular weight of SF [11]. The degradation rate of crosslinked silk fibroin films can be regulating by changing the genipin crosslinking degree [12]. Sodium alginate (SA) is another prominent biopolymers currently under study for the fabrication of porous scaffolds or hydrogels [13, 14]. It is extracted from native brown seaweed (Phaeophyceae) and composed of two uronic acids, β (1-4) linked D-mannuronic
acid (M) and α (1-4) linked L-guluronic acid (G) [15, 16]. Although SA is a popular biomaterials for biomedical applications, and it maintains stability and hardens simply by exposure to calcium chloride [17], the slow and uncontrollable degradation rate of SA crosslinked by calcium ions limit its application in the field of tissue engineering [18]. Thus, blending and changing the crosslinking method were adopted to solve this problem. SF/SA blended
porous
scaffolds
have
been
fabricated
successfully
using
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) as crosslinking agent in the previous studies, their stability and good cell biocompatibility also have been confirmed [19]. The combination of a protein and a polysaccharide leads to the formation of a hybrid material, which can provide a novel structure organization and superior properties that greatly expand the scope of their application in several areas of tissue engineering [20]. What's more,
degradation
behavior of silk based hybrid materials were studied according to previous reports, such as chitosan-poly(p-dioxanone)/silk fibroin porous conduits [21], Silk fibroin/gelatin/nanohydrox -yapatite (SF/GE/nHAp) composite scaffolds [22], and silk fibroin/chitosan scaffold [23]. Therefore, the purpose of this study was to obtain preliminary insight into the in vitro and in vivo degradation behavior of the SF/SA PMs. Herein, SF/SA composite PMs were prepared using EDC as a crosslinking agent by a freezedried method. In this study, enzymatic degradation behavior of these materials with Collagenase IA in vitro were investigated. The quantitative, morphological and structural changes of the SF/SA composite PMs after incubated in Collagenase IA were evaluated. The materials incubated in PBS were also investigated as a control. The blend ratios played an important role in regulating the degradation rate. Furthermore, subcutaneous implantation in rats was implemented to study the in vivo degradation behavior of PMs. Histology observation was performed to evaluate their morphological changes and tissue responses. 2. Materials and methods 2.1 Preparation of SF/SA porous materials
A SF solution was prepared using a chemical degumming method as described previously before dissolution and dialysis [24]. Bombyx mori raw silk fibers (Huzhou, China) were treated three times in 0.205 wt% Na2CO3 solutions at 98 ± 2 °C for 30 min respectively to remove sericin. After being air-dried, the refined silks were dissolved in ternary solvent CaCl2:CH3CH2OH:H2O (mole ratio= 1:2:8) at 72 ± 2 °C for 1 h. Then the mixed solution was dialyzed in deionized water for 4 days to get fibroin solution with concentration of about 3 wt%. A 2 wt% SA stock solution was obtained by dissolving SA powder in deionized water at 60 °C for 1 h. Similarly, the SF solution was diluted to 2 wt% with deionized water. Blending was performed by mixing the SF and SA stock solutions with the same concentration (2 wt%) but different volumes in a glass beaker. The final weight ratios of SF/SA in mixed solution were 100/0, 75/25, 50/50, 25/75, respectively, and these products were recorded as 100Fc, 75Fc, 50Fc, 25Fc. According to the previous [25], the EDC, NHS and MES were added into the solution to account for 20%, 10% and 20% weight ratio against the total weight of SF and SA in solution, respectively. The mixed solution was stirred gently at 4 °C for 30 minutes and then poured into stainless steel dish, frozen at −40 °C for 8 h, followed by lyophilization for 48 h. All kinds of SF/SA PMs were stored in the refrigerator at 4 °C for further study. 2.2 In vitro enzymatic degradation Before degradation experiment, all materials were cut into the 2 × 2 cm2 square, and the thickness of the material was 2~3mm. All materials were immersed with deionized water for 24h to remove residual reagent, and then after freeze-drying for this experiment. The treated materials were weighted and incubated at 37 °C in a PBS solution (0.05 M, pH =7.4) containing 0.5 U/mL Collagenase IA (from Clostridium histolyticum, EC 3.4.24.3, Sigmae-Aldrich). The samples (n=3 per time point) were incubated in enzyme solution (bath ratio 1:50) for 1, 3, 6, 12 and 18 days under slow shaking, and the another group were immersed in PBS without enzyme under similar conditions were used as a control. The
Collagenase IA solution and PBS solution was replaced freshly every 24 h. At the designated time points, the degradation remains were rinsed via distilled water for 6 times (each for 60 s) and collected for analysis. The remaining samples were dried at 60 °C to constant weight. The percentage of mass retention (MR) of scaffolds after different period of degradation was calculated using the equation (1): MR / %
W1 100 W0
(1)
where W0 represents the initial weight of the sample (mg) and W1 represents the final weight of the sample (mg). 2.3 The morphological and structural analyse The morphologies of initial PMs and after degradation for 6 and 18 days were observed by scanning electron microscopy (SEM, S-4800, Hitachi, Japan). For the molecular conformation measurements, Fourier-transform infrared (FTIR) spectroscopy analysis was performed using a Nicolet 5700 spectrometer (Thermo Scientific, USA). Furthermore, X-ray diffraction (XRD) was performed to investigate the changes in the crystal structure of the samples using an Xray diffractometer (Bruker D8 Advance X-Ray Diffractometer, Germany) with CuKα radiation with a wavelength of 1.5406 Å was used. The scanning speed was 2 °/min. 2.4 In vivo degradation experiment The in vivo degradation of the PMs was carried out by subcutaneous implantation in rats. Male SD rats (about 150 g) were used, and their use was approved by Animal Management Centre, National Institute for the Control of Pharmaceutical & Biological Products. 3% pentobarbital was given to the rats by a peritoneal injection. Then the PMs (length × width × thickness of ~ 6 mm × 5 mm × 2mm) were implanted in the back of the SD rats under aseptic condition. The rats were sacrificed to retrieve the residual materials for analysis at 21 d after implantation. The remained PMs with tissues were harvested and washed in PBS, fixed in 10% paraformaldehyde, dehydrated through a series of graded alcohols, embedded in paraffin
and sectioned at a thickness of 5 μm slices. Sections were then deparaffinized, rehydrated through a series of graded alcohols. And then Stained with hematoxylin and eosin (H&E) and Masson. All the sections were observed by fluorescence microscope (Olympus, IX71, Japan). Area retention ratio is defined by the ratio of the distribution area of the residual material to the original area of the material. And area of the residual material was calculated by Image J software according to the HE stains morphologies. 3. Results and discussion 3.1 Weight loss of the composite porous materials during enzyme degradation in vitro As a inducing biomaterial for repairing defective tissue, the degradation property is an important issue in the evaluation system. The degradation rate matching the tissue regeneration rate can effectively repair the tissue and eliminate the trouble of removing the biomaterial thus it is more conducive to tissue regeneration. In this study, the stable SF/SA PMs combined their salient features were prepared successfully through freeze dried method by using EDC as a mild and safe crosslinking agent. We used the Collagenase IA solution to simulate the physiological environment in vivo, and the blank PBS solution as a control. After incubated in Collagenase IA solution (0.5 U mL-1) at 37 °C for 24 h, the four groups were intact and no cracks. After 6 days, most of the materials kept complete shape, except the 25Fc groups, cracks began to appear at the edge of the materials. After 12 days, the 50Fc still kept morphology intact, while the other groups degraded obviously, significant cracks appeared and more small fragments were observed. After 18 days, 100Fc, 75Fc, and 25Fc groups degraded into small pieces, but the 50Fc group just turned to be thinner, slightly broken, and only a small number of fragments was visible in the Collagenase IA solution. In the control system, the four groups still kept morphology complete after 12 days in the PBS solution, and crack and collapse of the 25Fc and 75Fc appeared relatively later than that in the Collagenase IA solution. After 18 days in the PBS solution, The 25Fc scaffold appeared
fragmentation and the 75Fc scaffold began to crack, while the morphology of the100Fc and 50Fc scaffolds remained best. Fig. 1 show the mass retention percentage of different groups at different time points in Collagenase IA solution and blank PBS solution respectively. It can be observed from Fig. 1(A) that after incubated Collagenase IA solution 18 days, the mass retention percentage of 50Fc was approximately 68% while that in PBS was 74%. The 25Fc degraded fastest during degradation process, and the mass retention percentage was only approximately 31% in Collagenase IA solution. The degradation rates of 100Fc and 75Fc were similar, but incubated Collagenase IA solution for 12 days, the degradation rate of 75Fc accelerated, making the mass retention percentage of 75Fc lower than 100Fc. In summary, the mass retention percentage of PMs from high to low is 50Fc > 100Fc > 75Fc > 25Fc. In the PBS solution, the mass loss of all scaffolds were significantly lower than that in the Collagenase IA solution, the smallest mass loss was about 26% assigned to 50Fc, and the largest mass loss was about 52%, assigning to 25Fc. It can be seen that the degradation rate of SF/SA PMs with different blend ratios were different in each designed time period. The degradation behavior of different groups are related to the SF/SA blend ratio and EDC crosslinking effect. Because SA is a linear polysaccharide, the intermolecular covalent bonds and hydrogen bonds in the macromolecule make it form macromolecular network structure in aqueous solution, so when SF/SA scaffold is incubated in solution, it will act as molecular skeleton. SF is susceptible to enzyme degradation in Collagenase IA solution, and the degraded SF turned to be some peptides and amino acids, resulting in collapse and fragmentation. SA is not degraded by collagenase, so adding a certain amount of SA can improve the antienzyme performance of PMs. Another reason for the difference in degradation rates between different samples is related to the EDC crosslinking effect between SF and SA macromolecules. EDC mainly reacts with the free carboxyl group and amino group in the side chain group of the molecule to form an amide
bond to crosslink the macromolecules [26]. In this degradation experiment, the EDC crosslinking reaction between SF and SA improved the stability of the composite PMs. The 50Fc is the most resistant to collagenase degradation, which indicate that when the blend ratio of SF and SA is 1:1, the covalent crosslinking react more completely, resulting in more stable amide bonds formed in the macromolecular network structure. Therefore, the degradation rate of the 50Fc was the slowest. While degradation rate of 25Fc is the fastest, because the SF content reduce greatly, leading to the amino groups available for the cross-linking reaction also reduced, thereby inhibiting the EDC cross-linking reaction and ultimately reducing the stability of the PMs. Consequently, the SF/SA blend ratios have obvious effect on EDC crosslinking reaction, in turn regulating the degradation rate of the composite SF/SA PMs. 3.2 Morphology changes during degradation. The changes of the micro-morphology of the SF/SA during degradation process can be more clearly observed by SEM. Fig. 2 and Fig. 3 show the pore structure images of the 100Fc, 75Fc and 50Fc incubated the Collagenase IA solution and the PBS solution in different days. Since the 25Fc group degrades rapidly, no further analysis of its morphology has been made. From Fig. 2, it can be seen that uniform pores are distributed in different SF/SA PMs before degradation, and the internal pores are irregular long spindles with average pore diameter of 120 μm. Different groups showed different morphological changes in the Collagenase IA solution as a function of exposure time. After 6 days, they still keep their original shape and pore structure, but the pore size of 100Fc decrease and the internal pore partially collapse, while the pores of 75Fc showed shrinkage and many small holes appeared in hole walls, the pore size of the 50Fc increased. This may be due to the scaffold swelling obviously with the increase of the SA content. After 18 days, it can be seen clearly that the 100Fc didn't keep its original morphology and collapsed, totally losing shape. Although the pore wall of the 75Fc began to collapse, but did not completely collapse into powders due to the presence of some amount of SA. The pore wall of the 50Fc did not significantly collapse, but the pore size
became larger, there appeared more micropores in the 50Fc from the 6th day to the 18th day of degradation (Fig. S1). Fig. 3 shows the morphology changes of different PMs before and after the degradation in PBS as a control. The pore size of all scaffolds became larger, but the pores still kept their original morphology. Fig. S2 implied there were no obvious microporous structures can be observed in these three samples during degradation in PBS from the 6th day to the 18th day This may be due to the lack of enzymatic hydrolysis of collagenase in PBS solution, the main effect on the material degradation is dissolution. From above results, it was found that Collagenase IA had a significant effect on SF degradation, while Collagenase IA could not degrade SA. The SA macromolecule remained in the blend scaffolds played a skeleton supporting role, and the 50Fc can maintain a threedimensional porous structure within three weeks. These SEM results also suggested that Collagenase IA can degrade fibroin leading to collapse of 100Fc and formation of micropores on the pore wall of SF/SA composite PMs. These collapses and micropores appeared in PMs were probably correlated with the degradation in the amorphous regions. The structural changes during degradation were further analyzed in vitro to elucidate these morphological changes. 3.3 Secondary structure changes during the degradation Process 100Fc represents the structural changes of fibroin in pure SF PMs, while 50Fc, as the representative of SF/SA composite PMs, can explain the structural changes of fibroin in SF/SA composite PMs during degradation process. Hence, secondary structure of 100Fc and 50Fc in the different degradation time points were determined by FTIR and XRD to reveal the crystalline structure changes of SF during degradation process. Fig. 4 shows FTIR spectra of ECD crosslinked 100Fc before and after degradation in Collagenase IA solution and PBS solution. In the Collagenase IA solution, the characteristic peak of SF before degradation appeared mainly around 1648, 1545 and 1245 cm-1, which
indicated that the structure of 100Fc was mainly composed of random coil and silk I prior to degradation [27]. The FTIR spectra of the 100Fc after degradation by Collagenase IA and PBS for the same time exhibited subtle difference. After a period of degradation, the peak at 1648, 1545 and 1245 cm-1 shifted continuously toward lower wavenumbers, and the characteristic peaks at 1627 cm-1 were assigned to silk II structure. This result showed that the structure of 100Fc changed from random coil and silk I to silk II during the degradation process. There were distinct characteristic peak at 1627 cm-1 as shown in the lines of 100Fc degraded for 6 days and 18 days in Collagenase IA solution (Fig. 4(A)). The similar trend of wavenumber shift occurred in the PBS solution, but the characteristic peak shift was not significant at 1648 cm-1.
Fig. 5 shows FTIR spectra of EDC crosslinked 50Fc before and after degradation in Collagenase IA solution and PBS solution. It can be seen that final degradation products still had the characteristic peaks of SF and SA in the both of degradation systems [19]. This indicated that the blend scaffolds after the EDC cross-linking had good compatibility and the mass loss of the both components occurred simultaneously during the degradation process. The peaks at 1653, 1545 and 1245 cm−1 shifted toward the 1627, 1529, and 1234 cm−1 respectively over degradation time in the Collagenase IA and PBS. The results showed that the secondary structure of SF in the blend scaffolds undergone a transition from random coil and silk I to the silk II. Previous report suggested there were two kinds of crystalline structure of SF, silk I and silk II, with the typical diffraction peaks at 19.7(s), 24.7 (m) and 20.9 (vs), respectively [9]. The X-ray diffraction curve can also reflect the secondary structure changes of SF. Fig.6 shows the XRD pattern of EDC crosslinked 100Fc before and after degradation in Collagenase IA solution and PBS solution. Compared to the XRD pattern of a pure SF scaffold with complete
structure (Fig. S3), the pattern of 100Fc without
degradation exhibited slight sharper around 20°, which implied structure . The XRD of the 100Fc after degradation in Collagenase IA and PBS exhibited similar trend over time. With prolonged degradation time, diffraction peaks at 20.9° gradually increased, and the peak shape became sharp-pointed, which indicating silk II structure appeared. The diffraction peak at 24.7° appeared after degradation 6 days, which also confirmed appearance of silk II. In the PBS solution, XRD pattern showed silk II structure increased gradually with the extension of time. Fig. 7 shows the XRD pattern of EDC crosslinked 50Fc before and after degradation in Collagenase IA solution and PBS solution. Similarly, the characteristic peaks of SF can be seen in the XRD curves. However, SA has no regular crystal structure in the composite PMs, so there is not obvious characteristic peak observed for SA. With the degradation time increased, the peak at 19.7° shifted to 20.9° in the Collagenase IA (Fig. 7 A). In addition, a weaker diffraction peak at 24.7° and 27.2° appeared, which also indicated that the relative content of the silk II structure in the 50Fc increased, while the content of the amorphous structure and the silk I structure decreased. During degradation process in the PBS, the secondary structure of SF in the 50Fc scaffold exhibited similar tendency to that Collagenase IA with increasing exposure time, except after degradation 6 days, the curves appeared an obvious peak at 12.6°, indicating that unstable crystal (silk I) and crystal (silk II) were coexisting in the SF of 50Fc. The sharp peaks appearing at 2θ about 32° after degradation 18 days may be artificial peaks originating from the remnant salts. With the extension of degradation time, the PMs will adsorb more and more inorganic salts from the solution, so XRD can detect these residues obviously on the 18th day of degradation, similar phenomena appeared in SFCS scaffolds after different period of degradation [23]. As reported in many other studies [9, 28-30], the possible degradation mechanism for the SF scaffolds can be speculated as three steps: first, hydrophilic region of the SF molecules
disclosed under the action of enzyme; then, the amorphous structure was digested; third, many fragments with crystalline structure were became free particles, then moved, rather than degraded. The degradation mechanism may be used to explain the degradation behavior for most of the SF materials. However, the degradation behavior of SF/SA composite PMs would be different because of addition of SA content and formation of covalent bond of SF and SA crosslinked by EDC. Firstly, EDC crosslinked 100Fc PMs exhibited more obvious the silk II structure with increasing exposure time in enzyme solution. This may be ascribed to the role of enzymes in the process of degradation that is to destroy the crosslinking site (amide bond), so SF molecular chain can form the stable silk II structure through self-assembly and hydrophobic effect. While in PBS, amide bond among SF molecular chains have not been destroyed, made 100Fc insoluble in the aqueous solution, and these sites partly hindering the self-assembly of SF molecules chains, so the silk II structure of 100Fc in the PBS was relatively weak. And more noncrystal and unstable crystal (silk I) structures of fibroin degraded in Collagenase IA solution in the first step, also resulting in the increase of crystal structure content (silk II) in the degraded samples. Secondly, the structural changes of fibroin in 50Fc PMs in enzyme solution were similar to that in PBS, and silk II structure in PBS exhibited slightly more obvious on the 18th day of degradation. The reason for this difference may be that EDC crosslinking reaction generated sites not only exists in among SF macromolecule, and still exist between SF and SA macromolecules, making weaken the enzymatic degradation effect on the crosslinking sites. And the enzymes only digest SF macromolecule, according to the mechanism of SF degradation, the amorphous structure gradually reduce due to digest firstly, leading to silk II structure increase, and after 18 d of degradation, crystalline structure will become small particles and then moved out, finally cut silk II structure. In PBS, as a result of EDC crosslinking effect, 50Fc had good stability, only a fraction of fibroin and sodium alginate dissolved, and dissolve part SF belongs to the hydrophilic amorphous structure, the
amorphous structure of SF reduced result in silk II structure increases. What's more, in warm conditions, SF macromolecule are inclined to form silk II structure through self assembled. This finding suggests that SF/SA composite PMs would be degraded through protease. Furthermore, it indicated that the SF/SA PMs with different SA content would degrade in a different rate because of the different crosslinking effect on the PMs. 3.4 In vivo degradation performance In order to detect the in vivo degradation behavior of these SF/SA PMs, the materials were implanted subcutaneously in SD rats, and the remained materials and immune system response of host animals to the materials were observed based on gross analysis as well as histological images. After implantation of the PMs in the subcutaneous tissue for 21 days, materials were tolerated well by the host animals and no abnormal conditions were occurred in the implanted sites. Fig. 8 shows HE images of the PMs implanted
for 21
days. Almost no projections of the implanted material were seen from 75Fc group (Fig 8 (B) insert picture). While there were visible protrusions in the implanted position of 100Fc group (Fig 8 (A) insert picture). From the HE images, it can be seen clearly that after 21 days of subcutaneous implantation, all the PMs were infiltrated and largely lost their structural integrity. The pores in the PMs have been filled with tissue, a large number of cells, extracellular matrix and blood vessels. Materials were scattered around the original implant site. Area retention ratio of the residual material was further estimated roughly by the Image J solfware (Table S1). The size of the residual material of 75Fc was much smaller than that of 100Fc, and the area retention ratio of 50Fc was in the middle. The macroscopic observation results and HE stained pictures indicated that the PMs had excellent subcutaneous histocompatibility and exhibited different residual area in vivo. Fig. 9 shows Masson stained images of subcutaneous tissue with the PMs implanted subcutaneously for 21 days, in which the materials were dyed purple-red or light blue, the collagen bundle was dyed dark blue, and the nuclei were dyed magenta. It can be seen that
after degradation for 21 days, a large amount of collagen fiber bundles and fibroblasts were infiltrated into the PMs. Among them, the collagen fibers in the 50Fc were relatively long and distributed in large amounts, followed by the 100Fc, and the collagen fiber content was the smallest assigned to 75Fc. This result also confirmed that the pores of the PMs were filled with the extracellular matrix, and the fibroblasts in the pores of the PMs exhibited normal metabolic functions, secreting a large amount of collagen uniformly distributed in the pores of the materials. 4. Conclusions To assess the degradation behavior of the EDC crosslinked SF/SA PMs, these blend materials with different blend ratios were immersed in the Collagen IA solution in vitro. The weight of the SF/SA PMs immersed in Collagenase IA decreased as the degradation time increased. The degradation rate of the composite SF/SA PMs could be regulated through changing the blend ratios. What's more, only 50Fc kept integrate of pore structure after degrading in the Collagenase IA solution for 18 days. Better crosslinking efficiency of 50Fc led to greater resistance to enzyme degradation. The overall crystalline structure of SF whether in 100Fc or 50Fc changed to silk II from random coil and silk I during the degradation. Furthermore, more than half of the SF/SA PMs were degraded after subcutaneous implantation in SD rats for 3 weeks, meanwhile, these materials were well tolerated by the host animals. Because degradation behavior of PMs need to be studied deeply in the future. These results indicate that the in vivo behavior of the SF/SA PMs can be predicted and thus controlled to match the diverse needs for the engineering and repair of various tissues with specific functional requirements, repair characteristics, and repair rates. The information obtained in the present study is important for the further investigation of this SF based PMs, which have shown potential in a wide variety of tissue engineering and medical applications.
This work was supported by the Major Special Projects of technological innovation of Hubei Province, China (No. 2017ACA168); the National Key R&D Program of China (2017YFC11 03800); the financial supports from Research Project of Hubei Provincial Department of Education (Q20182701).
[1] X. Liu, J.M. Holzwarth, P.X. Ma, Functionalized synthetic biodegradable polymer scaffolds for tissue engineering, Macromol. Biosci. 12(7) (2012) 911-919. [2] I. Armentano, M. Dottori, E. Fortunati, S. Mattioli, J. Kenny, Biodegradable polymer matrix nanocomposites for tissue engineering: a review, Polym. Degrad. Stab. 95(11) (2010) 2126-2146. [3] N. Bhardwaj, S.C. Kundu, Silk fibroin protein and chitosan polyelectrolyte complex porous scaffolds for tissue engineering applications, Carbohydr. Polym. 85(2) (2011) 325-333. [4] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym. Sci. 32(8-9) (2007) 762-798. [5] F.G. Omenetto, D.L. Kaplan, New opportunities for an ancient material, Science 329(5991) (2010) 528-531. [6] B. Kundu, R. Rajkhowa, S.C. Kundu, X. Wang, Silk fibroin biomaterials for tissue regenerations, Adv. Drug Del. Rev. 65(4) (2013) 457-470. [7] N. Kasoju, U. Bora, Silk Fibroin in Tissue Engineering, Advanced Healthcare Materials 1(4) (2012) 393-412. [8] Y. Cao, B. Wang, Biodegradation of silk biomaterials, International journal of molecular sciences 10(4) (2009) 1514-1524. [9] M. Li, M. Ogiso, N. Minoura, Enzymatic degradation behavior of porous silk fibroin sheets, Biomaterials 24(2) (2003) 357-365. [10] J. Zhou, C. Cao, X. Ma, L. Hu, L. Chen, C. Wang, In vitro and in vivo degradation behavior of aqueous-derived electrospun silk fibroin scaffolds, Polym. Degrad. Stab. 95(9) (2010) 1679-1685. [11] X. Li, L. Wang, L. Li, Z. Luo, S. Yan, Q. Zhang, R. You, Water-stable silk fibroin nerve conduits with tunable degradation prepared by a mild freezing-induced assembly, Polym. Degrad. Stab. 164 (2019) 61-68. [12] R. You, Y. Xu, G. Liu, Y. Liu, X. Li, M. Li, Regulating the degradation rate of silk fibroin films through changing the genipin crosslinking degree, Polym. Degrad. Stab. 109 (2014) 226-232. [13] M.A. de Moraes, M.F. Silva, R.F. Weska, M.M. Beppu, Silk fibroin and sodium alginate blend: miscibility and physical characteristics, Materials Science and Engineering: C 40 (2014) 85-91. [14] X. Gong, C. Branford-White, L. Tao, S. Li, J. Quan, H. Nie, L. Zhu, Preparation and characterization of a novel sodium alginate incorporated self-assembled Fmoc-FF composite hydrogel, Materials Science and Engineering: C 58 (2016) 478-486. [15] J. Venkatesan, I. Bhatnagar, P. Manivasagan, K.-H. Kang, S.-K. Kim, Alginate composites for bone tissue engineering: a review, Int. J. Biol. Macromol. 72 (2015) 269-281. [16] K.Y. Lee, D.J. Mooney, Alginate: properties and biomedical applications, Prog. Polym. Sci. 37(1) (2012) 106-126. [17] P. Smrdel, M. Bogataj, F. Podlogar, O. Planinšek, N. Zajc, M. Mazaj, V. Kaučič, A. Mrhar, Characterization of calcium alginate beads containing structurally similar drugs, Drug Dev. Ind. Pharm. 32(5) (2006) 623-633.
[18] F. Pati, J. Jang, D.-H. Ha, S. Won Kim, J.-W. Rhie, J.-H. Shim, D.-H. Kim, D.-W. Cho, Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink, Nature Communications 5 (2014) 3935-3945. [19] Y. Wang, X. Wang, J. Shi, R. Zhu, J. Zhang, Z. Zhang, D. Ma, Y. Hou, F. Lin, J. Yang, A biomimetic silk fibroin/sodium alginate composite scaffold for soft tissue engineering, Scientific reports 6 (2016) 39477. [20] X. Jia, K.L. Kiick, Hybrid Multicomponent Hydrogels for Tissue Engineering, Macromol. Biosci. 9(2) (2010) 140-156. [21] Y. Wan, J. Zhang, Y. Luo, T. Zhou, H. Wu, Preparation and degradation of chitosan-poly (p-dioxanone)/silk fibroin porous conduits, Polym. Degrad. Stab. 119 (2015) 46-55. [22] A. Teimouri, M. Azadi, R. Emadi, J. Lari, A.N. Chermahini, Preparation, characterization, degradation and biocompatibility of different silk fibroin based composite scaffolds prepared by freeze-drying method for tissue engineering application, Polym. Degrad. Stab. 121 (2015) 18-29. [23] Z. She, B. Zhang, C. Jin, Q. Feng, Y. Xu, Preparation and in vitro degradation of porous three-dimensional silk fibroin/chitosan scaffold, Polymer Degradation & Stability 93(7) (2008) 1316-1322. [24] S. Yan, Q. Zhang, J. Wang, Y. Liu, S. Lu, M. Li, D.L. Kaplan, Silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds for dermal tissue reconstruction, Acta Biomater. 9(6) (2013) 6771-6782. [25] Q. Lv, K. Hu, Q. Feng, F. Cui, Fibroin/collagen hybrid hydrogels with crosslinking method: preparation, properties, and cytocompatibility, Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 84(1) (2008) 198-207. [26] C.R. Cammarata, M.E. Hughes, C.M. Ofner III, Carbodiimide induced cross-linking, ligand addition, and degradation in gelatin, Mol. Pharm. 12(3) (2015) 783-793. [27] Q. Lu, B. Zhang, M. Li, B. Zuo, D.L. Kaplan, Y. Huang, H. Zhu, Degradation mechanism and control of silk fibroin, Biomacromolecules 12(4) (2011) 1080-1086. [28] A. Vasconcelos, G. Freddi, A. Cavaco-Paulo, Biodegradable materials based on silk fibroin and keratin, Biomacromolecules 9(4) (2008) 1299-1305. [29] Y. Wang, D.D. Rudym, A. Walsh, L. Abrahamsen, H.-J. Kim, H.S. Kim, C. Kirker-Head, D.L. Kaplan, In vivo degradation of three-dimensional silk fibroin scaffolds, Biomaterials 29(24-25) (2008) 3415-3428. [30] G. Forlani, A.M. Seves, O. Ciferri, A bacterial extracellular proteinase degrading silk fibroin, Int. Biodeterior. Biodegrad. 46(4) (2000) 271-275.
Figure Captions: Fig. 1. Degradation behaviour of different PMs: (A) in the PBS solution containing Collagenase IA; (B) in the PBS solution. Fig. 2. SEM images of the different PMs during degradation in Collagenase IA solution for 0, 6 and 18 days. Scale bar=500 μm. Fig. 3. SEM images of the different PMs during degradation in PBS solution for 0, 6 and 18 days. Scale bar=500 μm.
Fig. 4. FTIR spectra of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 5. FTIR spectra of 50Fc incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 6. XRD data of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 7. XRD data of 50Fc scaffold incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 8. H&E stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days (A-C), and images D, E, and F are the enlarged views of the area in images A, B and C, respectively. (A) 100Fc, (B) 75Fc, (C) 50Fc. Red arrows = remaining scaffolds. Fig. 9. Masson stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days. (A) 100Fc, (B) 75Fc, (C) 50Fc.
Fig. 1. Degradation behaviour of different PMs: (A) in the PBS solution containing Collagenase IA; (B) in the PBS solution.
Fig. 2. SEM images of the different PMs during degradation in Collagenase IA solution for 0, 6 and 18 days. Scale bar=500 μm.
Fig. 3. SEM images of the different PMs during degradation in PBS solution for 0, 6 and 18 days. Scale bar=500 μm.
Fig. 4. FTIR spectra of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 5. FTIR spectra of 50Fc incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 6. XRD data of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 7. XRD data of 50Fc scaffold incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 8. H&E stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days (A-C), and images D, E, and F are the enlarged views of the area in images A, B and C, respectively. (A) 100Fc, (B) 75Fc, (C) 50Fc. Red arrows = remaining scaffolds.
Fig. 9. Masson stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days. (A) 100Fc, (B) 75Fc, (C) 50Fc.
Highlights:
SF/SA
SF/SA 50/50 porous materials can maintain relatively intact three-dimensional porous
porous materials can be fabricated by a facile
structure within three weeks during enzyme degradation.
porous materials
SF/SA
porous materials were well tolerated by the host animals.
Supplementary Materials：
Silk fibroin/sodium alginate composite porous materials with controllable degradation
Yiyu Wang3, Sisi Fan3, Yuwei Li3, Chunqing Niu3, Xiang Li 3, Yajin Guo1,2, Junhua Zhang3, Jian Shi4 and Xinyu Wang1,2 * 1
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing,
Wuhan University of Technology, Wuhan 430070, People’s Republic of China 2
Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan
University of Technology, Wuhan 430070, Republic of China 3
Hubei Province Research Center of Engineering Technology for Utilization of Botanical
Functional Ingredients, Hubei Engineering University, Xiaogan 432000, People’s Republic of China. 4
Department of Machine Intelligence and Systems Engineering, Faculty of Systems Science
and Technology, Akita Prefectural University, Akita 015-0055, Japan *
Correspondence and requests for materials should be addressed to X. W.
([email protected])
Figure S1. Magnifying SEM images of the 50Fc during degradation in Collagenase IA solution for 6 and 18 days.
Figure S2. Magnifying SEM images of the 100Fc, 75Fc and 50Fc during degradation in PBS solution for 6 and 18 days.
S F s c a ffo ld w ith o u t tre a tm e n t
Diffraction intensity(cps)
21 .7
10
20
30
40
50
Diffraction angle (2θ) Figure. S3. A XRD pattern of SF scaffold through freeze-drying method without any treatment
Table S1 Area retention ratios of PMs in vivo Sample
100Fc
75Fc
50Fc
Area retention ratios (%)
60.13
11.82
39.58
S0141-8130(19)36013-1 https://doi.org/10.1016/j.ijbiomac.2019.10.141 BIOMAC 13642
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
1 August 2019 15 October 2019 15 October 2019
Please cite this article as: Y. Wang, S. Fan, Y. Li, C. Niu, X. Li, Y. Guo, J. Zhang, J. Shi, X. Wang, Silk fibroin/ sodium alginate composite porous materials with controllable degradation, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.141
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© 2019 Published by Elsevier B.V.
Silk fibroin/sodium alginate composite porous materials with controllable degradation
Yiyu Wang3, Sisi Fan3, Yuwei Li3, Chunqing Niu3, Xiang Li 3, Yajin Guo1,2, Junhua Zhang3, Jian Shi4 and Xinyu Wang1,2 * 1
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing,
Wuhan University of Technology, Wuhan 430070, People’s Republic of China 2
Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan
University of Technology, Wuhan 430070, Republic of China 3
Hubei Province Research Center of Engineering Technology for Utilization of Botanical
Functional Ingredients, Hubei Engineering University, Xiaogan 432000, People’s Republic of China. 4
Department of Machine Intelligence and Systems Engineering, Faculty of Systems Science
and Technology, Akita Prefectural University, Akita 015-0055, Japan
E-mail: [email protected] (X. Wang)
Abstract: In this study, silk fibroin (SF)/sodium alginate (SA) porous materials (PMs) with different blend ratios were generated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as crosslinking agent by a simple freeze-dried method. Degradation experiment of SF/SA PMs have been systematically investigated up to 18 days in Collagenase IA solution at 37 °C, Phosphate buffer saline (PBS) solution without enzyme was used as a control. The results showed SF/SA 50/50 PMs exhibited a lowest rate of weight loss, about 68% of the weight retained within 18 d in Collagenase IA solution. SEM images indicated Collagenase IA can degrade fibroin leading to collapse of the pure SF PMs, while SF/SA 50/50 PMs still possessed integrity of pore structure during enzyme degradation with increasing exposure time. The crystalline structure of the SF in the SF/SA PMs changed to silk II after degradation for 18 d. Furthermore, the results of the in vivo degradation by subcutaneous implantation in rats showed that all PMs can be degraded at different levels, and exhibited good subcutaneous histocompatibility to the host animals. The degradability was strongly correlated to the blend ratios in a series of SF/SA composite PMs, and insights gained in this study can serve as a guide to match desired degradation behavior with specific applications for the SF/SA composite PMs.
Keywords: Silk fibroin, sodium alginate, porous materials, degradation
1. Introduction Many biodegradable porous materials (PMs) systems based on naturally derived and biocompatible polymers have been studied and proven to be wide applications in tissue engineering and biomedicine [1-3]. For these applications purpose, the PMs should have biocompatibility and porous structural properties similar to the specific tissues to maximally mimic the native extracellular matrix (ECM) characteristics. What's more, the PMs should be biodegradable at proper degradation rate to maintain their structure integraty before the new tissue form and degradation products should exhibit no toxic to host [4]. Therefore, it is a key issue to control the degradation rate of PMs. Among all the natural materials used, silk fibroin (SF) is from silkworms, Bombyx mori cocoons, have attracted wide attention due to its low immunogenicity, impressive mechanical properties, biocompatibility and easy fabrication [5-7]. In addition, the regenerated SF biomaterials with different formation were proven to be degradable [8]. SF, as a protein, can be catalytically degraded into amino acids under the action of proteolytic enzymes, which would be able to be absorbed and metabolized by human [9]. Zhou et al. investigated the degradation behavior of the electrostatic spinning SF scaffolds by protease XIV in vitro and in vivo, the results showed about 65% of the electrostatic spinning SF scaffolds were degraded within 24 d in protease XIV, and the scaffolds were completely degraded in vivo after implantation for 8 weeks and well tolerated by the host animals [10]. Li et al. studied the enzymatic degradation behavior of the SF conduits with silk I structure, revealing that the degradation rate could be effectively regulated by molecular weight of SF [11]. The degradation rate of crosslinked silk fibroin films can be regulating by changing the genipin crosslinking degree [12]. Sodium alginate (SA) is another prominent biopolymers currently under study for the fabrication of porous scaffolds or hydrogels [13, 14]. It is extracted from native brown seaweed (Phaeophyceae) and composed of two uronic acids, β (1-4) linked D-mannuronic
acid (M) and α (1-4) linked L-guluronic acid (G) [15, 16]. Although SA is a popular biomaterials for biomedical applications, and it maintains stability and hardens simply by exposure to calcium chloride [17], the slow and uncontrollable degradation rate of SA crosslinked by calcium ions limit its application in the field of tissue engineering [18]. Thus, blending and changing the crosslinking method were adopted to solve this problem. SF/SA blended
porous
scaffolds
have
been
fabricated
successfully
using
1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) as crosslinking agent in the previous studies, their stability and good cell biocompatibility also have been confirmed [19]. The combination of a protein and a polysaccharide leads to the formation of a hybrid material, which can provide a novel structure organization and superior properties that greatly expand the scope of their application in several areas of tissue engineering [20]. What's more,
degradation
behavior of silk based hybrid materials were studied according to previous reports, such as chitosan-poly(p-dioxanone)/silk fibroin porous conduits [21], Silk fibroin/gelatin/nanohydrox -yapatite (SF/GE/nHAp) composite scaffolds [22], and silk fibroin/chitosan scaffold [23]. Therefore, the purpose of this study was to obtain preliminary insight into the in vitro and in vivo degradation behavior of the SF/SA PMs. Herein, SF/SA composite PMs were prepared using EDC as a crosslinking agent by a freezedried method. In this study, enzymatic degradation behavior of these materials with Collagenase IA in vitro were investigated. The quantitative, morphological and structural changes of the SF/SA composite PMs after incubated in Collagenase IA were evaluated. The materials incubated in PBS were also investigated as a control. The blend ratios played an important role in regulating the degradation rate. Furthermore, subcutaneous implantation in rats was implemented to study the in vivo degradation behavior of PMs. Histology observation was performed to evaluate their morphological changes and tissue responses. 2. Materials and methods 2.1 Preparation of SF/SA porous materials
A SF solution was prepared using a chemical degumming method as described previously before dissolution and dialysis [24]. Bombyx mori raw silk fibers (Huzhou, China) were treated three times in 0.205 wt% Na2CO3 solutions at 98 ± 2 °C for 30 min respectively to remove sericin. After being air-dried, the refined silks were dissolved in ternary solvent CaCl2:CH3CH2OH:H2O (mole ratio= 1:2:8) at 72 ± 2 °C for 1 h. Then the mixed solution was dialyzed in deionized water for 4 days to get fibroin solution with concentration of about 3 wt%. A 2 wt% SA stock solution was obtained by dissolving SA powder in deionized water at 60 °C for 1 h. Similarly, the SF solution was diluted to 2 wt% with deionized water. Blending was performed by mixing the SF and SA stock solutions with the same concentration (2 wt%) but different volumes in a glass beaker. The final weight ratios of SF/SA in mixed solution were 100/0, 75/25, 50/50, 25/75, respectively, and these products were recorded as 100Fc, 75Fc, 50Fc, 25Fc. According to the previous [25], the EDC, NHS and MES were added into the solution to account for 20%, 10% and 20% weight ratio against the total weight of SF and SA in solution, respectively. The mixed solution was stirred gently at 4 °C for 30 minutes and then poured into stainless steel dish, frozen at −40 °C for 8 h, followed by lyophilization for 48 h. All kinds of SF/SA PMs were stored in the refrigerator at 4 °C for further study. 2.2 In vitro enzymatic degradation Before degradation experiment, all materials were cut into the 2 × 2 cm2 square, and the thickness of the material was 2~3mm. All materials were immersed with deionized water for 24h to remove residual reagent, and then after freeze-drying for this experiment. The treated materials were weighted and incubated at 37 °C in a PBS solution (0.05 M, pH =7.4) containing 0.5 U/mL Collagenase IA (from Clostridium histolyticum, EC 3.4.24.3, Sigmae-Aldrich). The samples (n=3 per time point) were incubated in enzyme solution (bath ratio 1:50) for 1, 3, 6, 12 and 18 days under slow shaking, and the another group were immersed in PBS without enzyme under similar conditions were used as a control. The
Collagenase IA solution and PBS solution was replaced freshly every 24 h. At the designated time points, the degradation remains were rinsed via distilled water for 6 times (each for 60 s) and collected for analysis. The remaining samples were dried at 60 °C to constant weight. The percentage of mass retention (MR) of scaffolds after different period of degradation was calculated using the equation (1): MR / %
W1 100 W0
(1)
where W0 represents the initial weight of the sample (mg) and W1 represents the final weight of the sample (mg). 2.3 The morphological and structural analyse The morphologies of initial PMs and after degradation for 6 and 18 days were observed by scanning electron microscopy (SEM, S-4800, Hitachi, Japan). For the molecular conformation measurements, Fourier-transform infrared (FTIR) spectroscopy analysis was performed using a Nicolet 5700 spectrometer (Thermo Scientific, USA). Furthermore, X-ray diffraction (XRD) was performed to investigate the changes in the crystal structure of the samples using an Xray diffractometer (Bruker D8 Advance X-Ray Diffractometer, Germany) with CuKα radiation with a wavelength of 1.5406 Å was used. The scanning speed was 2 °/min. 2.4 In vivo degradation experiment The in vivo degradation of the PMs was carried out by subcutaneous implantation in rats. Male SD rats (about 150 g) were used, and their use was approved by Animal Management Centre, National Institute for the Control of Pharmaceutical & Biological Products. 3% pentobarbital was given to the rats by a peritoneal injection. Then the PMs (length × width × thickness of ~ 6 mm × 5 mm × 2mm) were implanted in the back of the SD rats under aseptic condition. The rats were sacrificed to retrieve the residual materials for analysis at 21 d after implantation. The remained PMs with tissues were harvested and washed in PBS, fixed in 10% paraformaldehyde, dehydrated through a series of graded alcohols, embedded in paraffin
and sectioned at a thickness of 5 μm slices. Sections were then deparaffinized, rehydrated through a series of graded alcohols. And then Stained with hematoxylin and eosin (H&E) and Masson. All the sections were observed by fluorescence microscope (Olympus, IX71, Japan). Area retention ratio is defined by the ratio of the distribution area of the residual material to the original area of the material. And area of the residual material was calculated by Image J software according to the HE stains morphologies. 3. Results and discussion 3.1 Weight loss of the composite porous materials during enzyme degradation in vitro As a inducing biomaterial for repairing defective tissue, the degradation property is an important issue in the evaluation system. The degradation rate matching the tissue regeneration rate can effectively repair the tissue and eliminate the trouble of removing the biomaterial thus it is more conducive to tissue regeneration. In this study, the stable SF/SA PMs combined their salient features were prepared successfully through freeze dried method by using EDC as a mild and safe crosslinking agent. We used the Collagenase IA solution to simulate the physiological environment in vivo, and the blank PBS solution as a control. After incubated in Collagenase IA solution (0.5 U mL-1) at 37 °C for 24 h, the four groups were intact and no cracks. After 6 days, most of the materials kept complete shape, except the 25Fc groups, cracks began to appear at the edge of the materials. After 12 days, the 50Fc still kept morphology intact, while the other groups degraded obviously, significant cracks appeared and more small fragments were observed. After 18 days, 100Fc, 75Fc, and 25Fc groups degraded into small pieces, but the 50Fc group just turned to be thinner, slightly broken, and only a small number of fragments was visible in the Collagenase IA solution. In the control system, the four groups still kept morphology complete after 12 days in the PBS solution, and crack and collapse of the 25Fc and 75Fc appeared relatively later than that in the Collagenase IA solution. After 18 days in the PBS solution, The 25Fc scaffold appeared
fragmentation and the 75Fc scaffold began to crack, while the morphology of the100Fc and 50Fc scaffolds remained best. Fig. 1 show the mass retention percentage of different groups at different time points in Collagenase IA solution and blank PBS solution respectively. It can be observed from Fig. 1(A) that after incubated Collagenase IA solution 18 days, the mass retention percentage of 50Fc was approximately 68% while that in PBS was 74%. The 25Fc degraded fastest during degradation process, and the mass retention percentage was only approximately 31% in Collagenase IA solution. The degradation rates of 100Fc and 75Fc were similar, but incubated Collagenase IA solution for 12 days, the degradation rate of 75Fc accelerated, making the mass retention percentage of 75Fc lower than 100Fc. In summary, the mass retention percentage of PMs from high to low is 50Fc > 100Fc > 75Fc > 25Fc. In the PBS solution, the mass loss of all scaffolds were significantly lower than that in the Collagenase IA solution, the smallest mass loss was about 26% assigned to 50Fc, and the largest mass loss was about 52%, assigning to 25Fc. It can be seen that the degradation rate of SF/SA PMs with different blend ratios were different in each designed time period. The degradation behavior of different groups are related to the SF/SA blend ratio and EDC crosslinking effect. Because SA is a linear polysaccharide, the intermolecular covalent bonds and hydrogen bonds in the macromolecule make it form macromolecular network structure in aqueous solution, so when SF/SA scaffold is incubated in solution, it will act as molecular skeleton. SF is susceptible to enzyme degradation in Collagenase IA solution, and the degraded SF turned to be some peptides and amino acids, resulting in collapse and fragmentation. SA is not degraded by collagenase, so adding a certain amount of SA can improve the antienzyme performance of PMs. Another reason for the difference in degradation rates between different samples is related to the EDC crosslinking effect between SF and SA macromolecules. EDC mainly reacts with the free carboxyl group and amino group in the side chain group of the molecule to form an amide
bond to crosslink the macromolecules [26]. In this degradation experiment, the EDC crosslinking reaction between SF and SA improved the stability of the composite PMs. The 50Fc is the most resistant to collagenase degradation, which indicate that when the blend ratio of SF and SA is 1:1, the covalent crosslinking react more completely, resulting in more stable amide bonds formed in the macromolecular network structure. Therefore, the degradation rate of the 50Fc was the slowest. While degradation rate of 25Fc is the fastest, because the SF content reduce greatly, leading to the amino groups available for the cross-linking reaction also reduced, thereby inhibiting the EDC cross-linking reaction and ultimately reducing the stability of the PMs. Consequently, the SF/SA blend ratios have obvious effect on EDC crosslinking reaction, in turn regulating the degradation rate of the composite SF/SA PMs. 3.2 Morphology changes during degradation. The changes of the micro-morphology of the SF/SA during degradation process can be more clearly observed by SEM. Fig. 2 and Fig. 3 show the pore structure images of the 100Fc, 75Fc and 50Fc incubated the Collagenase IA solution and the PBS solution in different days. Since the 25Fc group degrades rapidly, no further analysis of its morphology has been made. From Fig. 2, it can be seen that uniform pores are distributed in different SF/SA PMs before degradation, and the internal pores are irregular long spindles with average pore diameter of 120 μm. Different groups showed different morphological changes in the Collagenase IA solution as a function of exposure time. After 6 days, they still keep their original shape and pore structure, but the pore size of 100Fc decrease and the internal pore partially collapse, while the pores of 75Fc showed shrinkage and many small holes appeared in hole walls, the pore size of the 50Fc increased. This may be due to the scaffold swelling obviously with the increase of the SA content. After 18 days, it can be seen clearly that the 100Fc didn't keep its original morphology and collapsed, totally losing shape. Although the pore wall of the 75Fc began to collapse, but did not completely collapse into powders due to the presence of some amount of SA. The pore wall of the 50Fc did not significantly collapse, but the pore size
became larger, there appeared more micropores in the 50Fc from the 6th day to the 18th day of degradation (Fig. S1). Fig. 3 shows the morphology changes of different PMs before and after the degradation in PBS as a control. The pore size of all scaffolds became larger, but the pores still kept their original morphology. Fig. S2 implied there were no obvious microporous structures can be observed in these three samples during degradation in PBS from the 6th day to the 18th day This may be due to the lack of enzymatic hydrolysis of collagenase in PBS solution, the main effect on the material degradation is dissolution. From above results, it was found that Collagenase IA had a significant effect on SF degradation, while Collagenase IA could not degrade SA. The SA macromolecule remained in the blend scaffolds played a skeleton supporting role, and the 50Fc can maintain a threedimensional porous structure within three weeks. These SEM results also suggested that Collagenase IA can degrade fibroin leading to collapse of 100Fc and formation of micropores on the pore wall of SF/SA composite PMs. These collapses and micropores appeared in PMs were probably correlated with the degradation in the amorphous regions. The structural changes during degradation were further analyzed in vitro to elucidate these morphological changes. 3.3 Secondary structure changes during the degradation Process 100Fc represents the structural changes of fibroin in pure SF PMs, while 50Fc, as the representative of SF/SA composite PMs, can explain the structural changes of fibroin in SF/SA composite PMs during degradation process. Hence, secondary structure of 100Fc and 50Fc in the different degradation time points were determined by FTIR and XRD to reveal the crystalline structure changes of SF during degradation process. Fig. 4 shows FTIR spectra of ECD crosslinked 100Fc before and after degradation in Collagenase IA solution and PBS solution. In the Collagenase IA solution, the characteristic peak of SF before degradation appeared mainly around 1648, 1545 and 1245 cm-1, which
indicated that the structure of 100Fc was mainly composed of random coil and silk I prior to degradation [27]. The FTIR spectra of the 100Fc after degradation by Collagenase IA and PBS for the same time exhibited subtle difference. After a period of degradation, the peak at 1648, 1545 and 1245 cm-1 shifted continuously toward lower wavenumbers, and the characteristic peaks at 1627 cm-1 were assigned to silk II structure. This result showed that the structure of 100Fc changed from random coil and silk I to silk II during the degradation process. There were distinct characteristic peak at 1627 cm-1 as shown in the lines of 100Fc degraded for 6 days and 18 days in Collagenase IA solution (Fig. 4(A)). The similar trend of wavenumber shift occurred in the PBS solution, but the characteristic peak shift was not significant at 1648 cm-1.
Fig. 5 shows FTIR spectra of EDC crosslinked 50Fc before and after degradation in Collagenase IA solution and PBS solution. It can be seen that final degradation products still had the characteristic peaks of SF and SA in the both of degradation systems [19]. This indicated that the blend scaffolds after the EDC cross-linking had good compatibility and the mass loss of the both components occurred simultaneously during the degradation process. The peaks at 1653, 1545 and 1245 cm−1 shifted toward the 1627, 1529, and 1234 cm−1 respectively over degradation time in the Collagenase IA and PBS. The results showed that the secondary structure of SF in the blend scaffolds undergone a transition from random coil and silk I to the silk II. Previous report suggested there were two kinds of crystalline structure of SF, silk I and silk II, with the typical diffraction peaks at 19.7(s), 24.7 (m) and 20.9 (vs), respectively [9]. The X-ray diffraction curve can also reflect the secondary structure changes of SF. Fig.6 shows the XRD pattern of EDC crosslinked 100Fc before and after degradation in Collagenase IA solution and PBS solution. Compared to the XRD pattern of a pure SF scaffold with complete
structure (Fig. S3), the pattern of 100Fc without
degradation exhibited slight sharper around 20°, which implied structure . The XRD of the 100Fc after degradation in Collagenase IA and PBS exhibited similar trend over time. With prolonged degradation time, diffraction peaks at 20.9° gradually increased, and the peak shape became sharp-pointed, which indicating silk II structure appeared. The diffraction peak at 24.7° appeared after degradation 6 days, which also confirmed appearance of silk II. In the PBS solution, XRD pattern showed silk II structure increased gradually with the extension of time. Fig. 7 shows the XRD pattern of EDC crosslinked 50Fc before and after degradation in Collagenase IA solution and PBS solution. Similarly, the characteristic peaks of SF can be seen in the XRD curves. However, SA has no regular crystal structure in the composite PMs, so there is not obvious characteristic peak observed for SA. With the degradation time increased, the peak at 19.7° shifted to 20.9° in the Collagenase IA (Fig. 7 A). In addition, a weaker diffraction peak at 24.7° and 27.2° appeared, which also indicated that the relative content of the silk II structure in the 50Fc increased, while the content of the amorphous structure and the silk I structure decreased. During degradation process in the PBS, the secondary structure of SF in the 50Fc scaffold exhibited similar tendency to that Collagenase IA with increasing exposure time, except after degradation 6 days, the curves appeared an obvious peak at 12.6°, indicating that unstable crystal (silk I) and crystal (silk II) were coexisting in the SF of 50Fc. The sharp peaks appearing at 2θ about 32° after degradation 18 days may be artificial peaks originating from the remnant salts. With the extension of degradation time, the PMs will adsorb more and more inorganic salts from the solution, so XRD can detect these residues obviously on the 18th day of degradation, similar phenomena appeared in SFCS scaffolds after different period of degradation [23]. As reported in many other studies [9, 28-30], the possible degradation mechanism for the SF scaffolds can be speculated as three steps: first, hydrophilic region of the SF molecules
disclosed under the action of enzyme; then, the amorphous structure was digested; third, many fragments with crystalline structure were became free particles, then moved, rather than degraded. The degradation mechanism may be used to explain the degradation behavior for most of the SF materials. However, the degradation behavior of SF/SA composite PMs would be different because of addition of SA content and formation of covalent bond of SF and SA crosslinked by EDC. Firstly, EDC crosslinked 100Fc PMs exhibited more obvious the silk II structure with increasing exposure time in enzyme solution. This may be ascribed to the role of enzymes in the process of degradation that is to destroy the crosslinking site (amide bond), so SF molecular chain can form the stable silk II structure through self-assembly and hydrophobic effect. While in PBS, amide bond among SF molecular chains have not been destroyed, made 100Fc insoluble in the aqueous solution, and these sites partly hindering the self-assembly of SF molecules chains, so the silk II structure of 100Fc in the PBS was relatively weak. And more noncrystal and unstable crystal (silk I) structures of fibroin degraded in Collagenase IA solution in the first step, also resulting in the increase of crystal structure content (silk II) in the degraded samples. Secondly, the structural changes of fibroin in 50Fc PMs in enzyme solution were similar to that in PBS, and silk II structure in PBS exhibited slightly more obvious on the 18th day of degradation. The reason for this difference may be that EDC crosslinking reaction generated sites not only exists in among SF macromolecule, and still exist between SF and SA macromolecules, making weaken the enzymatic degradation effect on the crosslinking sites. And the enzymes only digest SF macromolecule, according to the mechanism of SF degradation, the amorphous structure gradually reduce due to digest firstly, leading to silk II structure increase, and after 18 d of degradation, crystalline structure will become small particles and then moved out, finally cut silk II structure. In PBS, as a result of EDC crosslinking effect, 50Fc had good stability, only a fraction of fibroin and sodium alginate dissolved, and dissolve part SF belongs to the hydrophilic amorphous structure, the
amorphous structure of SF reduced result in silk II structure increases. What's more, in warm conditions, SF macromolecule are inclined to form silk II structure through self assembled. This finding suggests that SF/SA composite PMs would be degraded through protease. Furthermore, it indicated that the SF/SA PMs with different SA content would degrade in a different rate because of the different crosslinking effect on the PMs. 3.4 In vivo degradation performance In order to detect the in vivo degradation behavior of these SF/SA PMs, the materials were implanted subcutaneously in SD rats, and the remained materials and immune system response of host animals to the materials were observed based on gross analysis as well as histological images. After implantation of the PMs in the subcutaneous tissue for 21 days, materials were tolerated well by the host animals and no abnormal conditions were occurred in the implanted sites. Fig. 8 shows HE images of the PMs implanted
for 21
days. Almost no projections of the implanted material were seen from 75Fc group (Fig 8 (B) insert picture). While there were visible protrusions in the implanted position of 100Fc group (Fig 8 (A) insert picture). From the HE images, it can be seen clearly that after 21 days of subcutaneous implantation, all the PMs were infiltrated and largely lost their structural integrity. The pores in the PMs have been filled with tissue, a large number of cells, extracellular matrix and blood vessels. Materials were scattered around the original implant site. Area retention ratio of the residual material was further estimated roughly by the Image J solfware (Table S1). The size of the residual material of 75Fc was much smaller than that of 100Fc, and the area retention ratio of 50Fc was in the middle. The macroscopic observation results and HE stained pictures indicated that the PMs had excellent subcutaneous histocompatibility and exhibited different residual area in vivo. Fig. 9 shows Masson stained images of subcutaneous tissue with the PMs implanted subcutaneously for 21 days, in which the materials were dyed purple-red or light blue, the collagen bundle was dyed dark blue, and the nuclei were dyed magenta. It can be seen that
after degradation for 21 days, a large amount of collagen fiber bundles and fibroblasts were infiltrated into the PMs. Among them, the collagen fibers in the 50Fc were relatively long and distributed in large amounts, followed by the 100Fc, and the collagen fiber content was the smallest assigned to 75Fc. This result also confirmed that the pores of the PMs were filled with the extracellular matrix, and the fibroblasts in the pores of the PMs exhibited normal metabolic functions, secreting a large amount of collagen uniformly distributed in the pores of the materials. 4. Conclusions To assess the degradation behavior of the EDC crosslinked SF/SA PMs, these blend materials with different blend ratios were immersed in the Collagen IA solution in vitro. The weight of the SF/SA PMs immersed in Collagenase IA decreased as the degradation time increased. The degradation rate of the composite SF/SA PMs could be regulated through changing the blend ratios. What's more, only 50Fc kept integrate of pore structure after degrading in the Collagenase IA solution for 18 days. Better crosslinking efficiency of 50Fc led to greater resistance to enzyme degradation. The overall crystalline structure of SF whether in 100Fc or 50Fc changed to silk II from random coil and silk I during the degradation. Furthermore, more than half of the SF/SA PMs were degraded after subcutaneous implantation in SD rats for 3 weeks, meanwhile, these materials were well tolerated by the host animals. Because degradation behavior of PMs need to be studied deeply in the future. These results indicate that the in vivo behavior of the SF/SA PMs can be predicted and thus controlled to match the diverse needs for the engineering and repair of various tissues with specific functional requirements, repair characteristics, and repair rates. The information obtained in the present study is important for the further investigation of this SF based PMs, which have shown potential in a wide variety of tissue engineering and medical applications.
This work was supported by the Major Special Projects of technological innovation of Hubei Province, China (No. 2017ACA168); the National Key R&D Program of China (2017YFC11 03800); the financial supports from Research Project of Hubei Provincial Department of Education (Q20182701).
[1] X. Liu, J.M. Holzwarth, P.X. Ma, Functionalized synthetic biodegradable polymer scaffolds for tissue engineering, Macromol. Biosci. 12(7) (2012) 911-919. [2] I. Armentano, M. Dottori, E. Fortunati, S. Mattioli, J. Kenny, Biodegradable polymer matrix nanocomposites for tissue engineering: a review, Polym. Degrad. Stab. 95(11) (2010) 2126-2146. [3] N. Bhardwaj, S.C. Kundu, Silk fibroin protein and chitosan polyelectrolyte complex porous scaffolds for tissue engineering applications, Carbohydr. Polym. 85(2) (2011) 325-333. [4] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym. Sci. 32(8-9) (2007) 762-798. [5] F.G. Omenetto, D.L. Kaplan, New opportunities for an ancient material, Science 329(5991) (2010) 528-531. [6] B. Kundu, R. Rajkhowa, S.C. Kundu, X. Wang, Silk fibroin biomaterials for tissue regenerations, Adv. Drug Del. Rev. 65(4) (2013) 457-470. [7] N. Kasoju, U. Bora, Silk Fibroin in Tissue Engineering, Advanced Healthcare Materials 1(4) (2012) 393-412. [8] Y. Cao, B. Wang, Biodegradation of silk biomaterials, International journal of molecular sciences 10(4) (2009) 1514-1524. [9] M. Li, M. Ogiso, N. Minoura, Enzymatic degradation behavior of porous silk fibroin sheets, Biomaterials 24(2) (2003) 357-365. [10] J. Zhou, C. Cao, X. Ma, L. Hu, L. Chen, C. Wang, In vitro and in vivo degradation behavior of aqueous-derived electrospun silk fibroin scaffolds, Polym. Degrad. Stab. 95(9) (2010) 1679-1685. [11] X. Li, L. Wang, L. Li, Z. Luo, S. Yan, Q. Zhang, R. You, Water-stable silk fibroin nerve conduits with tunable degradation prepared by a mild freezing-induced assembly, Polym. Degrad. Stab. 164 (2019) 61-68. [12] R. You, Y. Xu, G. Liu, Y. Liu, X. Li, M. Li, Regulating the degradation rate of silk fibroin films through changing the genipin crosslinking degree, Polym. Degrad. Stab. 109 (2014) 226-232. [13] M.A. de Moraes, M.F. Silva, R.F. Weska, M.M. Beppu, Silk fibroin and sodium alginate blend: miscibility and physical characteristics, Materials Science and Engineering: C 40 (2014) 85-91. [14] X. Gong, C. Branford-White, L. Tao, S. Li, J. Quan, H. Nie, L. Zhu, Preparation and characterization of a novel sodium alginate incorporated self-assembled Fmoc-FF composite hydrogel, Materials Science and Engineering: C 58 (2016) 478-486. [15] J. Venkatesan, I. Bhatnagar, P. Manivasagan, K.-H. Kang, S.-K. Kim, Alginate composites for bone tissue engineering: a review, Int. J. Biol. Macromol. 72 (2015) 269-281. [16] K.Y. Lee, D.J. Mooney, Alginate: properties and biomedical applications, Prog. Polym. Sci. 37(1) (2012) 106-126. [17] P. Smrdel, M. Bogataj, F. Podlogar, O. Planinšek, N. Zajc, M. Mazaj, V. Kaučič, A. Mrhar, Characterization of calcium alginate beads containing structurally similar drugs, Drug Dev. Ind. Pharm. 32(5) (2006) 623-633.
[18] F. Pati, J. Jang, D.-H. Ha, S. Won Kim, J.-W. Rhie, J.-H. Shim, D.-H. Kim, D.-W. Cho, Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink, Nature Communications 5 (2014) 3935-3945. [19] Y. Wang, X. Wang, J. Shi, R. Zhu, J. Zhang, Z. Zhang, D. Ma, Y. Hou, F. Lin, J. Yang, A biomimetic silk fibroin/sodium alginate composite scaffold for soft tissue engineering, Scientific reports 6 (2016) 39477. [20] X. Jia, K.L. Kiick, Hybrid Multicomponent Hydrogels for Tissue Engineering, Macromol. Biosci. 9(2) (2010) 140-156. [21] Y. Wan, J. Zhang, Y. Luo, T. Zhou, H. Wu, Preparation and degradation of chitosan-poly (p-dioxanone)/silk fibroin porous conduits, Polym. Degrad. Stab. 119 (2015) 46-55. [22] A. Teimouri, M. Azadi, R. Emadi, J. Lari, A.N. Chermahini, Preparation, characterization, degradation and biocompatibility of different silk fibroin based composite scaffolds prepared by freeze-drying method for tissue engineering application, Polym. Degrad. Stab. 121 (2015) 18-29. [23] Z. She, B. Zhang, C. Jin, Q. Feng, Y. Xu, Preparation and in vitro degradation of porous three-dimensional silk fibroin/chitosan scaffold, Polymer Degradation & Stability 93(7) (2008) 1316-1322. [24] S. Yan, Q. Zhang, J. Wang, Y. Liu, S. Lu, M. Li, D.L. Kaplan, Silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds for dermal tissue reconstruction, Acta Biomater. 9(6) (2013) 6771-6782. [25] Q. Lv, K. Hu, Q. Feng, F. Cui, Fibroin/collagen hybrid hydrogels with crosslinking method: preparation, properties, and cytocompatibility, Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 84(1) (2008) 198-207. [26] C.R. Cammarata, M.E. Hughes, C.M. Ofner III, Carbodiimide induced cross-linking, ligand addition, and degradation in gelatin, Mol. Pharm. 12(3) (2015) 783-793. [27] Q. Lu, B. Zhang, M. Li, B. Zuo, D.L. Kaplan, Y. Huang, H. Zhu, Degradation mechanism and control of silk fibroin, Biomacromolecules 12(4) (2011) 1080-1086. [28] A. Vasconcelos, G. Freddi, A. Cavaco-Paulo, Biodegradable materials based on silk fibroin and keratin, Biomacromolecules 9(4) (2008) 1299-1305. [29] Y. Wang, D.D. Rudym, A. Walsh, L. Abrahamsen, H.-J. Kim, H.S. Kim, C. Kirker-Head, D.L. Kaplan, In vivo degradation of three-dimensional silk fibroin scaffolds, Biomaterials 29(24-25) (2008) 3415-3428. [30] G. Forlani, A.M. Seves, O. Ciferri, A bacterial extracellular proteinase degrading silk fibroin, Int. Biodeterior. Biodegrad. 46(4) (2000) 271-275.
Figure Captions: Fig. 1. Degradation behaviour of different PMs: (A) in the PBS solution containing Collagenase IA; (B) in the PBS solution. Fig. 2. SEM images of the different PMs during degradation in Collagenase IA solution for 0, 6 and 18 days. Scale bar=500 μm. Fig. 3. SEM images of the different PMs during degradation in PBS solution for 0, 6 and 18 days. Scale bar=500 μm.
Fig. 4. FTIR spectra of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 5. FTIR spectra of 50Fc incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 6. XRD data of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 7. XRD data of 50Fc scaffold incubated in (A) Collagenase IA solution, (B) PBS for different days. Fig. 8. H&E stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days (A-C), and images D, E, and F are the enlarged views of the area in images A, B and C, respectively. (A) 100Fc, (B) 75Fc, (C) 50Fc. Red arrows = remaining scaffolds. Fig. 9. Masson stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days. (A) 100Fc, (B) 75Fc, (C) 50Fc.
Fig. 1. Degradation behaviour of different PMs: (A) in the PBS solution containing Collagenase IA; (B) in the PBS solution.
Fig. 2. SEM images of the different PMs during degradation in Collagenase IA solution for 0, 6 and 18 days. Scale bar=500 μm.
Fig. 3. SEM images of the different PMs during degradation in PBS solution for 0, 6 and 18 days. Scale bar=500 μm.
Fig. 4. FTIR spectra of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 5. FTIR spectra of 50Fc incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 6. XRD data of 100Fc incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 7. XRD data of 50Fc scaffold incubated in (A) Collagenase IA solution, (B) PBS for different days.
Fig. 8. H&E stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days (A-C), and images D, E, and F are the enlarged views of the area in images A, B and C, respectively. (A) 100Fc, (B) 75Fc, (C) 50Fc. Red arrows = remaining scaffolds.
Fig. 9. Masson stains morphologies of subcutaneously implanted SF/SA PMs in SD rats at 21 days. (A) 100Fc, (B) 75Fc, (C) 50Fc.
Highlights:
SF/SA
SF/SA 50/50 porous materials can maintain relatively intact three-dimensional porous
porous materials can be fabricated by a facile
structure within three weeks during enzyme degradation.
porous materials
SF/SA
porous materials were well tolerated by the host animals.
Supplementary Materials：
Silk fibroin/sodium alginate composite porous materials with controllable degradation
Yiyu Wang3, Sisi Fan3, Yuwei Li3, Chunqing Niu3, Xiang Li 3, Yajin Guo1,2, Junhua Zhang3, Jian Shi4 and Xinyu Wang1,2 * 1
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing,
Wuhan University of Technology, Wuhan 430070, People’s Republic of China 2
Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan
University of Technology, Wuhan 430070, Republic of China 3
Hubei Province Research Center of Engineering Technology for Utilization of Botanical
Functional Ingredients, Hubei Engineering University, Xiaogan 432000, People’s Republic of China. 4
Department of Machine Intelligence and Systems Engineering, Faculty of Systems Science
and Technology, Akita Prefectural University, Akita 015-0055, Japan *
Correspondence and requests for materials should be addressed to X. W.
([email protected])
Figure S1. Magnifying SEM images of the 50Fc during degradation in Collagenase IA solution for 6 and 18 days.
Figure S2. Magnifying SEM images of the 100Fc, 75Fc and 50Fc during degradation in PBS solution for 6 and 18 days.
S F s c a ffo ld w ith o u t tre a tm e n t
Diffraction intensity(cps)
21 .7
10
20
30
40
50
Diffraction angle (2θ) Figure. S3. A XRD pattern of SF scaffold through freeze-drying method without any treatment
Table S1 Area retention ratios of PMs in vivo Sample
100Fc
75Fc
50Fc
Area retention ratios (%)
60.13
11.82
39.58